Scientific book Vectormune ND by clarisse

SCIENTIFIC INFORMATION

VECTORMUNE ND 速

SCIENTIFIC INFORMATION

VECTORMUNE ND ®

e l i F c fi i t n e i Sc VECTORMUNE® ND

TABLE OF CONTENTS INTRODUCTION.............................................................................................................................................6 PRESENTATION OF VECTORMUNE® ND .........................................................................................11 1• Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Fabienne Rauw, Yannick Gardin, Vilmos Palya, Sofia Anbari, Sophie Lemaire, Marc Boschmans, Thierry van den Berg, Bénédicte Lambrecht. Vaccine 28 (2010), pp. 823–833................................................................. 12 2• Distribution and humoral immunity induced by a rHVT-ND vaccine in SPF chickens. Rauw F, Gardin Y, Vilmos P, Ngabirano E, van Borm S, van den Berg T, Lambrecht B, 2012. Proceedings of the 9th International Symposium on Marek’s disease and Avian Herpesviruses, June 24-28, Berlin, Germany............. 23 3• Protection and Antibody Response Caused by Turkey Herpesvirus Vector Newcastle Disease Vaccine. Motoyuki Esaki, Alecia Godoy, Jack K. Rosenberger, Sandra C. Rosenberger, Yannick Gardin, Atsushi Yasuda, and Kristi Moore Dorsey. Avian Diseases 57, pp. 750–755, 2013............................................................ 24 4• High level of protection achieved by vaccination with a recombinant HVT NDV vector vaccine against different genotypes of Newcastle disease virus present in Latin America. V. Palya, T. Tatár-Kis, T. Mató, B. Felföldi, E. Kovács, and Y. Gardin. Proceedings of the sixty third western poultry disease conference, April 1 to 5, 2014, Puerto Vallarta, Jalisco, Mexico............................................................. 30 5• Comparative efficacy of several vaccination programs including or not including recombinant ND vaccine against challenge with Mexican Chimalhuacan NDV strain. Palya V. et al., 2008. Proceedings of the 57th WPDC/XXXIII ANECA conference, April 9-12, Jalisco, Mexico, pp. 36-38 ............................................................................................................................... 34 6• Efficacy of a recombinant Newcastle disease vaccine (Vectormune® ND) based on clinical protection and shedding of challenge virus in broiler chickens. Palya V. et al., 2011. Proceedings of the XVIIth World Veterinary Poultry Association congress, August 14-18, Cancun, Mexico, pp. 757-763...................................................................................................................... 37 7• Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions. Palya V. et al., 2012a. Avian Diseases 56, pp. 282-287...................................................................................................... 44 8• Safety assessment of a turkey herpes virus vector Newcastle disease vaccine in chickens. Xiao-Hui Yu, Yuan – Yuan Zhang, Na Tang, Li-Li Zhao and Guo-Zhong Zhang. Journal of Animal and veterinary advances 13 (14), pp. 879-900, 2014............................................................................ 50 9• The combination of attenuated Newcastle disease (ND) vaccine with rHVT-ND vaccine at 1 day-old is more protective against ND virus challenge than when combined with inactivated ND vaccine. F. Rauw, Y. Gardin, V. Palya, T. van den Berg & B. Lambrecht, Avian Pathology, 2014, Vol. 43, No. 1, pp. 26–36............ 54 10• Efficacy of several vaccination programs against Newcastle disease challenge. Satra J. et al., 2011. Proceedings of the XVIIth World Veterinary Poultry Association congress, August 14-18, Cancun, Mexico, pp. 909-914.................................................................................................................... 65 11• Simultaneous administration of rHVT-F (Newcastle Disease) and rHVT-H5 (Avian Influenza) vector vaccines reduces, but does not prevent, development of immunity against both diseases. Fabienne Rauw, Yannick Gardin, Vilmos Palya, Timea Tata-Kis, Kristi Moore Dorsey, Thierry van den Berg, Bénédicte Lambrecht. WVPAC2013 - 19-23 august 2013 - Nantes FRANCE................................................................. 71

12• Lack of interference between rHVT-H5 and rHVT-F vaccines administrated simultaneously to day-old chickens and efficacy against AI and ND challenges performed at 4 or 8 weeks of age. F. Rauw, V. Palya, T. Tatar-Kis, K. Moore Dorsey, T. van den Berg, B. Lambrecht and Y. Gardin. Proc. of the 8th AI symposium, London............................................................................................................................ 72 13• Compatibility of vectored LT vaccines and Vectormune® ND. Godoy A. et al., 2011. Proceedings of the American Association of Avian Pathologists annual meeting, July 16-19, St. Louis, Missouri......................................................................................................................................... 73 14• Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers. Vilmos Palya, Tímea Tatár-Kis, Tamás Mató, Balázs Felföldi, Edit Kovács, Yannick Gardin. Veterinary Immunology and Immunopathology 158 (2014), pp. 105–115....................................................................... 74 15• Effect of experimental Newcastle disease challenge of laying hens receiving vaccination program that includes recombinant HVT NDV. R. Merino, 62nd Western Poultry Disease Conference 2013, pp. 17-19......................................................................... 85 16• Markedly different onset of protection elicited by two different HVT based recombinant Newcastle Disease Vaccines. PALYA Vilmos, TATÁR-KIS Tímea, MATÓ Tamás, WALKÓNÉ KOVÁCS Edit, FELFÖLDI Balázs, HOMONNAY Zalán Gábor, GARDIN Yannick. WVPAC2013 - 19-23 august 2013 - Nantes FRANCE............................... 88 17• The use of rHVT-F vector vaccine improves chicken flocks immunity against the Newcastle Disease under field conditions. GARDIN Yannick, GACAD Emilio, UMANDAL Mel, PANIAGO Marcelo, LOZANO Fernando, EL-ATTRACHE John. VPAC2013 - 19-23 august 2013 - Nantes FRANCE.......................................................................................................... 89 18• Monitoring the vaccination with a rHVT-NDV vectored vaccine in commercial broilers through serological methods. Palya V. et al., 2012b. Proceedings of the American Association of Avian Pathologists annual meeting, August 4-7, San Diego, California.................................................................................................................................... 90 19• Field safety and efficacy of a vector Marek’s / Newcastle disease vaccine (rHVT-NDV) as assessed by clinical and productive performance in a large population of commercial broilers. L. Sesti, C. Kneipp, R. Paranhos, P. Paulet, and C. Cazaban. WVPAC2013 - 19-23 august 2013 - Nantes FRANCE..... 91 20• Assessment of a vector Marek’s / Newcastle vaccine through clinical and productive performance of commercial broilers in two different epidemiological situations of Newcastle Disease challenge. Luiz Sesti, Yesenia Vega, Jorge Cortegana, Pascal Paulet, Marcelo Paniago, Fernando Lozano. WVPAC2013 - 19-23 august 2013 - Nantes FRANCE...................................................................................................... 94 21• New approaches in the prevention of velogenic Newcastle disease in Mexico. Lechuga M. et al., 2012. Proceedings of the 61st Western Poultry Disease Conference, April 2-4, Scottsdale, Arizona.......................................................................................................................................... 95 22• Evaluacion de una vacuna vectorizada contra la enfermedad de Newcastle como parte de un programa de prevencion. Duenas D. et al., 2012. Proceedings of the XXXVII ANECA conference, May 2-6, Puerto Vallarta, Mexico, pp. 105-110............................................................................................................................... 97 23• Nuevos enfoques en la prevencion de la enfermedad de Newcastle velogenica. Higuera S. et al., 2012. Proceedings of the XXXVII ANECA conference, May 2-6, Puerto Vallarta, Mexico, pp. 169-172............................................................................................................................. 107 24• Onset of Newcastle disease immunity in turkeys vaccinated with Vectormune HVT NDV. John K. Rosenberger and Sandra C. Rosenberger. AAAP 2012.................................................................................... 111 / 5

INTRODUCTION

e l i F c fi i t n e i Sc VECTORMUNE® ND

SCIENTIFIC INFORMATION

VECTORMUNE® ND

espite many years of enforcement of international and national trading regulations, introduction of the biosecurity concept, harmonization and spreading of laboratory diagnostic and monitoring techniques, implementation of vaccination programs, Newcastle Disease (ND) is still listed among the most damaging poultry diseases considering both clinical and economical consequences. Some regions or countries like Western Europe, the USA, Brazil, Chile, etc. have been successful in lowering or even eliminating the incidence of the disease so that ND is nowadays considered by them only as an epizootic risk and vaccination programs they are applying, if existing, are always of the light type. On the contrary, many countries from Latin America, Eastern Europe, Africa, Middle East, and Asia are still living with the enzootic form of the disease with continuous waves of unavoidable pressure regularly plaguing them. In these countries, vaccination is considered as a routine obligation guided by modest ambitions that are simply restricted to ensuring clinical and economical protection in case of challenge. Wild birds population that play an important role of reservoir of Newcastle Disease Virus (NDV), backyard and small scale farming operations that maintain the infection, the tradition of live birds markets that ensure its spreading are important factors explaining from where the disease comes from and how it circulates. But ND is also seen, even today, in poultry producing operations that are much better organized, following stringent biosecurity rules, and often applying sound and very heavy vaccination programs. In fact, this has been a frustrating paradox that has really drawn the attention of veterinarians and production managers until the concept of flock monitoring and flock profiling have become popular. Then, NDV antibody testing conducted on samples taken from broilers at the

end of the growing period, or layers in production, has revealed the frequent presence of low antibody positive, or even fully antibody negative flocks in spite of sometimes very intensive vaccination programs including one or more inactivated and several live attenuated ND (good quality and effective) vaccines. Further investigations have then highlighted the facts that: • In endemic countries, day old chicks are carrying high levels of maternally derived antibodies that are interfering with live as well as killed ND vaccines given at the hatchery up to a point where neutralization is complete and prevent any vaccine take. • Because of a lack of education of the workers, too much time required as well as various practical constraints including drinking water quality and residues of sanitizer, improper use of sprayers, vaccinations conducted at farm, either to avoid this early interference, or boost previous immunizations, are very often very poorly conducted and really unreliable. • Live attenuated vaccines that are the backbone of a broiler ND vaccination program are responsible for lesions of the upper respiratory tract, post vaccination and rolling reactions that slow down the growth and leave the chickens susceptible to other pathogens necessitating costly and unwanted antibiotic treatments. For these reasons, as soon as the ND pressure is believed as going away, farmers prefer to go back to “easy and light” vaccination programs, which open the door again to ND. These limitations explain why it is really not uncommon to discover unprotected flocks in endemic countries, and why severe clinical outbreaks are often reported, despite “good vaccination!” They also explain why, until recently, the general opinion was that ND would stay for long in most of ND endemic countries and why vaccination was, at best, considered as an aid to maintain poultry production, but definitely far from being an effective tool of an eradication program. This was the situation when rHVT-ND vector vaccines got out of the development phase and became available for experiments and investigations. At Ceva, this rHVT-ND vaccine was Vectormune® ND. There was certainly hope but there were also many questions, uncertainties and doubts, and because of that, a very significant extra investment was made to know and understand the potentialities of this new product. Scientific work inside as well as outside the company, in collaboration with independent research centers, was designed, organized and conducted… and what we harvested in terms of information regarding vaccine induced immunity as well as tangible protection results far exceeded our expectations.

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SCIENTIFIC INFORMATION

VECTORMUNE ND ®

We now know that Vectormune® ND is much more than just a new ND vaccine, and it is the objective of this scientific information booklet to present some studies that helped us to realize how innovative and potent this product is. But before going ahead with the reading of these studies, it is probably important to briefly present the product and summarize the most striking associated features and benefits. Vectormune® ND is a recombinant vector vaccine, using HVT as vector in the genome of which, the “F” gene extracted from a genotype I NDV has been inserted. The HVT strain used (FC 126), its origin, the low number of passage it has undergone, the “F” insert, the insertion site, the promoter selected to ensure the expression of the F gene, the terminal sequence, etc. are all key elements explaining the efficacy of this vaccine with most of them patented and proprietary to Ceva Animal Health so that Vectormune® ND is actually unique and cannot be compared to other rHVT-ND vaccines. The “F” (for “fusion”) protein is the epitope present on the surface of NDV, allowing it to attach and penetrate target cells. It is at the same time a key factor of virulence of the virus as well as a key protective antigen. One can easily understand that if immunity is built up against “F”, then NDV can no longer infect cells and create damage, and this is probably explaining the incredibly high efficacy of Vectormune® ND. Vaccinated chickens are not only protected against clinical and economical consequences of infection, but replication of NDV inside the chicken’s body is also hampered as indicated by a reduction of the shedding of the challenge virus as well as a more limited increase in antibody titer following infection. The HVT strain used to carry and express the “F” gene is known for decades as a very safe and very stable virus, used worldwide to vaccinate chickens against Marek’s disease. The particular strain and passage level used for the construction of Vectormune® ND replicates actively in the chicken and this may explain why protection against ND appears so quickly. Antibody response to Vectormune® ND (including IgG, IgM and IgA) can be detected in SPF chickens as soon as 9-12 days post vaccination (Rauw et al. 2012). Immune response to Vectormune® ND is not only composed of circulating antibodies but also includes local antibodies. It is not only humoral but also involves cellular immunity (Rauw et al. 2010). Antibody response to Vectormune ® ND can be detected using the Haemagglutination Inhibition (HI) test. Following vaccination at day one, in the presence of passive immunity, antibody response can be clearly differentiated from the controls at around 21-28 days of age. On the reverse, this detection is

e l i F c fi i t n e i Sc VECTORMUNE® ND

not possible with commercial ELISA kits (Idexx, Biochek). If no live vaccine is used, this can be a simple serological method to differentiate vaccinated only from vaccinated and infected or simply infected animals (DIVA procedure). Duration of immunity is probably the most impressive feature of this vaccine. Following a single injection of Vectormune® ND on day of hatch, layers are totally protected against clinical signs, mortality and drop in egg production until at least 72 weeks of age (Palya et al., 2012c). This cannot be compared to any existing ND vaccination conventional program where a minimum of 2 to 4 killed and 5 to 8 live vaccinations would be necessary to achieve acceptable (but not comparable) level of protection. Vectormune® ND is provided with a broad spectrum of efficacy. Perfect protection has been demonstrated against challenges conducted with high doses of various NDV strains belonging to diverse genotypes including genotype II (Texas GB strain, B1B1 strain), genotype IV (Herts 33 strain), genotype V (Mexican Chimalhuacan strain), and genotype VII (Malaysian isolate). Since protection with Vectormune® ND requires replication of HVT vector, and in spite of a rather early protection, the first days / weeks of life are not covered by Vectormune® ND. For this reason, in ND endemic countries, vaccine strategy with Vectormune® ND requires to pay attention to the quality (level and homogeneity) of maternally derived antibodies and to ensure early protection by application of a live attenuated ND vaccine to be given by spray on the first day of age in the hatchery. In order to avoid damages to the trachea, which are detrimental to both the growth and the respiratory tract integrity of the chickens, it is strongly advisable to use a vaccine based on an apathogenic enterotropic NDV strain like the Phy.LMV.42 NDV strain present in Cevac® Vitapest L (ND only) or Cevac® Vitabron L (combination of ND + IB). In countries where ND is only an epizootic risk, it is advisable to remove any live ND vaccine from the program. The combination of a reliable ND passive immunity together with Vectormune® ND induced active immunity will ensure a very significant level of protection. Following several years of investigation work with Vectormune® ND, we do believe that this vaccine is more a revolution than a simple evolution. Vectormune® ND will most probably change the approach of Newcastle disease prevention in the field and soon be considered as a strong tool toward the longterm control of this important poultry disease. Yannick Gardin Director Innovation Strategy Department Ceva Animal Health, Libourne, France.

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SCIENTIFIC INFORMATION

VECTORMUNE ND ®

e l i F c fi i t n e i Sc VECTORMUNE® ND

PRESENTATION OF VECTORMUNE® ND Vectormune® ND is a genetically-engineered live vaccine based on the serotype 3 Marek’s disease virus, herpesvirus of turkeys (HVT) strain. This virus has been inserted with the gene that is encoding for the F (fusion) protein of the Newcastle disease virus, D26 strain. This vaccine is intended for use in commercial chickens when administered by the in ovo route at 18 to 19 days of incubation or by the subcutaneous route to healthy day-of-age chickens. It is used as an aid in the prevention of Newcastle disease (ND) and Marek’s disease (MD). Some of the advantages to using Vectormune® ND over conventional Newcastle disease virus (NDV) vaccines are the increased safety, long duration of immunity, ability to overcome maternal antibodies and lack of interference with other vaccines. There are several factors that make Vectormune® ND safer than conventional live NDV vaccines. First, the bird will not actually receive the whole live NDV therefore there will be no risk of disease or respiratory reactions commonly seen when using conventional NDV vaccines (Alexander and Gough, 2003). Furthermore the lack of live NDV means there is no risk of NDV spreading to other birds (Alexander and Gough, 2003; Bell et al, 1991). For these reasons the vector vaccine could be considered a safer choice for a vaccine strategy. Another advantage to utilizing Vectormune® ND is the long duration of immunity provided by the turkey herpesvirus (HVT) vector system (Jackwood, 1999). HVT continues to replicate in the chicken providing continued protection against both ND and MD. Additionally Vectormune® ND may be administered via the in ovo route allowing for a relatively easy and cost effective method of vaccination. This provides chickens that are protected at hatch even in the face of maternal antibodies which is a common problem faced when vaccinating with conventional NDV vaccines (Alexander and Gough, 2003; Bell et al, 1991; Westbury et al, 1984). Finally, Vectormune® ND will not interfere with other Marek’s disease virus (MDV serotypes 1 or 2) or other live virus vaccinations. Vectormune® ND is an efficacious and safe alternative to using conventional NDV vaccines. Source: TECHNOLOGY AND CONSTRUCTION OF VECTOR VACCINES; Kristi Moore Dorsey, Motoyuki Esaki, and Lauren Noland

This scientific file summarizes several articles that have already been published in Congresses, Seminars and scientific journals. Note: the former product’s trade name used to be Vectormune® HVT-NDV. It can be found in some graphs, and it is actually the same product.

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Scientific File • N°1

EXTRACT OF VACCINE 28 (ELSEVIER, 2010) PP 823 - 833 824

VECTORMUNE® ND Vaccine 28 (2010) 823–833 Vaccine 28 (2010) 823–833

Contents lists available at ScienceDirect Contents lists available at ScienceDirect

Vaccine Vaccine journal homepage: www.elsevier.com/locate/vaccine journal homepage: www.elsevier.com/locate/vaccine

Improved Improved vaccination vaccination against against Newcastle Newcastle disease disease by by an an in in ovo ovo recombinant recombinant HVT-ND combined with an adjuvanted live vaccine at day-old HVT-ND combined with an adjuvanted live vaccine at day-old Fabienne Rauw a,∗ , Yannick Gardin b , Vilmos Palya cc , Soﬁa Anbari aa , Sophie Lemaire aa , Fabienne Rauw a,∗a, Yannick Gardin b , Vilmos Palya , Soﬁa Anbari , Sophie Lemaire , Marc Boschmans a , Thierry van den Berg aa , Bénédicte Lambrecht aa Marc Boschmans , Thierry van den Berg , Bénédicte Lambrecht a Avian a b

Virology and Immunology Unit, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, 1180 Ukkel (Brussels), Belgium Avian Virology and Immunology Unit, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, 1180 Ukkel (Brussels), Belgium CEVA Santé Animale, BP 126, 33501 Libourne, France b c CEVA Santé Animale, BP 126, 33501 Libourne, France CEVA Phylaxia, Szallas utca 5, H-1107 Budapest, Hungary c CEVA Phylaxia, Szallas utca 5, H-1107 Budapest, Hungary

a r t i c l e a r t i c l e

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Article history: Article history: Received 17 August 2009 Received August form 2009 1 October 2009 Received 17 in revised Received in 1 October 2009 Accepted 12revised Octoberform 2009 Accepted 12 October 2009 2009 Available online 29 October Available online 29 October 2009 Keywords: Keywords: Newcastle Disease Virus Newcastle Disease Virus Recombinant turkey herpesvirus Recombinant turkey herpesvirus Chitosan Chitosan Vaccination Vaccination Immunity Immunity

a b s t r a c t a b s t r a c t The continuous outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks demonstrate The outbreaks of fatal are Newcastle (ND)and in commercial poultry flocks demonstrate that continuous current vaccination strategies not fullydisease efficacious should be improved by new generation that current In vaccination strategies are not fully efficacious and should be improved new generation of vaccines. this context, maternally immune conventional layer chickens wereby vaccinated in ovo of vaccines. this context, maternallyexpressing immune conventional were vaccinated indayovo with a turkeyInherpesvirus recombinant the fusion (F)layer gene chickens of NDV (rHVT-ND) and/or at with a turkey herpesvirus recombinant expressing the fusion (F) gene of NDV (rHVT-ND) and/or at dayold with an apathogenic enterotropic live ND vaccine co-administrated or not with chitosan by oculoold with an The apathogenic enterotropic live responses ND vaccine co-administrated or notagainst with chitosan by oculonasal route. induced vaccinal immune and conferred protection a challenge with a nasal route.NDV The velogenic induced vaccinal immune responses and conferred protectionrHVT-ND/live against a challenge with a circulating viscerotropic strain were evaluated. The innovative ND-chitosan circulating velogenic viscerotropic strain were evaluated. The innovative rHVT-ND/live ND-chitosan vaccinationNDV regimen provided the best protection against mortality and morbidity as well as the strongest vaccination provided thecould best protection mortality and morbidity well as response the strongest reduction ofregimen virus shedding that be related against to the higher measured cellular as immune and reduction of virus shedding that could be related to the higher measured cellular immune response and digestive antibody-mediated immunity. digestive antibody-mediated immunity. © 2009 Elsevier Ltd. All rights reserved. © 2009 Elsevier Ltd. All rights reserved.

F. Rauw et al. / Vaccine 28 (2010) 823–833

approach is the well defined and controlled in ovo application of live ND vaccines, when live vaccines are not or less sensitive to passive immunity [13,14], or the use of recombinant vector that are less sensitive to the interference of MDA, like the recombinant herpesvirus of turkey (HVT) [15–19] or the recombinant fowl poxvirus (FPV) [20–25]. Recombinant HVT (rHVT) vaccines have several advantages [18]: the herpesvirus induce a strong cellmediated immunity (CMI) in mammals and are fully safe for in ovo administration [19,26]. As far as efficacy is concerned, the choice of the gene(s) inserted in these recombinants is of great importance. Among NDV antigens expressed by these vectors, the fusion (F) and/or the haemagglutinin-neuraminidase (HN) envelope glycoproteins have been used successfully to protect chickens against virulent NDV challenge [27]. Nevertheless, although both F and HN glycoproteins elicit neutralizing antibodies, only antibody to F protein can promote prevent cell-to-cell spreading of the virus. Moreover, the use of the F gene represents a potential method of differentiating infected from vaccinated animals (DIVA), such immunized birds developing no haemagglutination inhibition antibody. The F gene was therefore preferentially inserted alone or together with the HN gene in recombinant vectors. Finally, the development of safe and strong adjuvants is necessary to maximize the efficacy of the vaccination and to increase the local immune response induced in chickens by new and/or conventional live vaccines administered through the mucosal route. In this context, cholera toxin [10], stimulatory CpG oligodeoxynucleotides [28,29], avian cytokines [30,31], chitosan [31,32] and other adjuvant [33,34] have been successfully investigated for ND vaccination. We propose a new ND vaccination regimen, including in ovo injection of a rHVT-ND vaccine combined with a day-old application by mucosal route of a live apathogenic enterotropic ND vaccine associated or not with an adjuvant, the chitosan. The aim of this study was the evaluation of induced immunity, protection and the establishment of correlation between them. 2. Materials and methods 2.1. Chickens

1. Introduction 1. Introduction Newcastle Disease Virus (NDV), also known as avian ParamyxNewcastle Disease Virus (NDV), also known as avianrespiratory Paramyxovirus type-1 (AMPV-1), is an economically important ovirus type-1 (AMPV-1), is an economically important respiratory pathogen of poultry. In addition to good biosecurity practices, conpathogen of poultry. In addition good biosecurity control of Newcastle disease (ND)to primarily consists practices, in preventive trol of Newcastle disease (ND) primarily consists preventive vaccination of flocks and culling of infected and atin risk of being vaccination of flocks and culling infected and at riskprograms of being infected birds (protection zone). ofCurrent vaccination infected birds (protection zone). Current vaccination programs include the use of attenuated (live) vaccines based on lentogenic include the use of attenuated (live) based on lentogenic NDV strains followed, in case of longvaccines living laying birds, by inactiNDV strains followed, in case of long living laying birds, byperiod, inactivated (killed) vaccines before the start of the egg production vated (killed) vaccines before the start of the egg production period, in order to induce a good protective immunity while producing in order adverse to induce a good protective while producing minimal effects in birds. Both immunity vaccines have their advanminimal adverse effects in birds. Both vaccines have their advantages and disadvantages, which have been reviewed previously tages and disadvantages, which have been reviewed previously [1,2]. Nevertheless, the current vaccines and vaccination strategies [1,2]. Nevertheless, the current strategies protect against morbidity and vaccines mortalityand andvaccination significantly reduce protect against morbidity and mortality and significantly reduce but do not stop the infection and the viral excretion, which is critibut do controlling not stop thethe infection thedisease viral excretion, which is critical for spreadand of the [3]. Another limitation cal for controlling spread of the disease [3]. Another to their efficacy isthe that the current ND vaccines mightlimitation induce a to theirprotection efficacy isagainst that the current ND vaccines induce a better viruses isolated in past might epizootics than better protection against viruses isolated in past epizootics than against more recent circulating viscerotropic viruses [3,4]. Finally, against moreof recent circulating viscerotropic viruses [3,4]. Finally, the presence maternally derived antibody (MDA) interferes with the presence of maternally derived antibody (MDA) interferes with ∗ Corresponding author. Tel.: +32 02 379 13 21; fax: +32 02 379 13 37. ∗ Corresponding author. Tel.: +32 02 379 13 21; fax: +32 02 379 13 37. E-mail address: farau@var.fgov.be (F. Rauw). E-mail address: farau@var.fgov.be (F. Rauw).

the establishment of a persisting good protective immunity after a the establishment of a persisting single day-old vaccination [5–7]. good protective immunity after a single day-old vaccination [5–7]. Different approaches have been investigated in conventional Different approaches have been investigated in conventional chicks provided with MDA to overcome the limitations of current chicks provided with to overcome the limitations ofand current vaccination. Firstly, theMDA development of improved vaccines vacvaccination. Firstly, the development of improved vaccines and vaccination strategies that induce a better protection against infection cination that induce betterisprotection against infection and limitstrategies the transmission ofavirus needed. Although a good and limit the transmission ofimmunity virus is needed. Although a good correlation between humoral and protection has been correlation between humoral immunity and protection has demonstrated [3,4,8], cell- [9] and local antibody-mediated been [10] demonstrated [9] and local antibody-mediated immunities are[3,4,8], knowncellto play an important role to decrease[10] the immunities are known to play an important role tothe decrease the excretion and dissemination of the virus. Therefore, improved excretion and dissemination of the virus. Therefore, the improved vaccination strategies should presumably target the induction of vaccination should presumably targetthe thespread induction of both cellularstrategies and humoral immunity to inhibit of the both cellular and humoral immunity to inhibit the spread of the virus in the field, as well as respiratory and digestive immunity virus in theorfield, as well respiratory immunity to prevent decrease theasduration and and leveldigestive of virus shed from to prevent or decrease the duration and level of virus shed from mucosa. Secondly, it was suggested to privilege the vaccination mucosa. Secondly, it was suggested to privilege the vaccination with an enterotropic NDV strain in order to induce a stronger local with an enterotropic NDV strain in order to induce stronger local immunity in the digestive tract [11] and to reduce athe virus shedimmunity in the digestive tract [11] and to reduce the virus shedding, considering that the majority of the velogenic NDV lineages ding, considering the majority the world velogenic lineages currently causing that outbreaks aroundofthe are NDV viscerotropic currently causing outbreaks around the world are viscerotropic [12]. Thirdly, there is a need to prime an immune response as [12]. Thirdly, there is a need primewhere an immune responseand as soon as possible, especially in to regions ND is enzootic soon possible, especially inpressure regions where ND is enzootic and whereasthere is a high disease in the fields. To overcome where there is a of high disease pressureeither in thea fields. overcome the interference passive immunity, boosterTovaccination the interference of passive immunity, either a booster vaccination is required at 2–3 weeks of age according to the MDA decline or is 2–3 weeksisof age according to days the MDA decline or therequired primaryat vaccination delayed until 7–10 of age. Another the primary vaccination is delayed until 7–10 days of age. Another

Conventional Isa Brown layer chickens (SSL: sex sale linked) were hatched from eggs provided by Wijverkens hatchery (Halle, Belgium). The conventional layer eggs were obtained from 38- and 40-week-old breeder ﬂocks which had received the following ND vaccination regimens: a live ND vaccine (Nobilis Clone 30, Intervet) at 4 and 8 weeks of age followed by a booster vaccination at 16 weeks of age with an inactivated ND vaccine (Nobilis Newcavac, Intervet). After hatching, all birds were kept in biosecurity level 3 (BSL-3) isolators and animal experiments were conducted under the authorization and supervision of the Biosafety and Bioethics Committees of the Veterinary and Agrochemical Research Institute (VAR), following national and European regulations. 2.2. Vaccines, adjuvant and challenge strain The live ND vaccine (Cevac Vitapest L) was provided by Ceva Santé Animale (Production and R&D center of Ceva-Phylaxia, Budapest, Hungary). The vaccine is based on the apathogenic enterotropic PHY.LMV.42 strain [11,35,36] (ICPI range between 0.00 and 0.16; IVPI = 0.00; MDT > 168), belonging to genotype I of NDV [37]. The vaccine was reconstituted in phosphate-buffered saline (PBS) to get 1 dose in 50 l, which corresponds to ≈106 EID50 /dose. The chitosan (chitosan hydrochloride) adjuvant was provided by Ceva-Phylaxia. It is a chloride salt of an unbranched binary heteropolysaccharide consisting of the two units N-acetyl-d-

glucosamine and d-glucosamine. Chitosan was dissolved in PBS at a final concentration of 0.5% (w/v) and was used to reconstitute and dilute the vaccine to the final concentration. The cryopreserved cell-associated rHVT expressing the F protein of the avirulent D26 NDV strain [38] (rHVT-ND, Vectomune® ND) was produced by Ceva-Biomune (USA). The recombinant vaccine was diluted in corresponding vaccine diluent (Ceva-Biomune, USA) to get 1 dose (3000 pfu/dose) in 100 l and was inoculated in ovo on the 18th embryonation day (ED) with manual injection as previously described [14]. The velogenic viscerotropic Chimalhuacan NDV strain used for challenge was isolated in Mexico [39] and belongs to Class II genotype V of NDV (ICPI = 1.89). Oculo-nasal inoculation of 105 EID50 of this strain induces 100% of mortality within 3–6 days in SPF and commercial broiler chickens challenged at 3–6 weeks of age (personal observations). 2.3. Mitogens and NDV antigens The mitogens phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono) were purchased from Sigma (Belgium). NDV recall antigens were prepared from NDV La Sota strain as previously described [11,40] and named NDV all proteins (prot-NDV). 2.4. Measurement of NDV-specific cell-mediated immunity The induction of NDV-specific CMI was evaluated by the production of ChIFN after ex vivo antigen-activation of splenocytes and peripheral blood lymphocytes (PBLs), as previously described [11,32]. Briefly, spleens were removed aseptically from chickens and heparinized blood samples were layered by sedimentation to isolate splenocytes and PBL, respectively. Immune cells were then activated by mitogens (PMA/Iono, 1 g/ml), as positive control of ex vivo activability of lymphocytes, and by NDV recall antigens (protNDV, 1 g/ml). ChIFN production was measured by capture ELISA. Cellular immune responses were expressed as stimulation indices (S.I.) that were calculated for each bird by dividing the optical density (O.D.) values of mitogen- and antigen-activated lymphocytes by the O.D. of non-activated lymphocytes [11] and the S.I. per group were calculated. The chicken having an O.D. < 0.1 or a S.I. < 2 for mitogen-activation was excluded from further antigen-activation analysis. An O.D. ≥ 0.1 and a S.I. ≥ 2 for NDV-activation were considered evidence of NDV-specific CMI. 2.5. Measurement of the NDV-specific humoral and local antibody-mediated immunity NDV-specific humoral immunity was evaluated by haemagglutination inhibition (HI) test and NDV-specific IgG ELISA [11]. Local antibody-mediated immunity to NDV was measured at inoculation and preferential replication sites of the vaccine by NDV-specific IgG ELISA on tears and supernatant of ex vivo duodenum tissue culture, respectively, as previously described [11]. 2.6. Measurement of virus shedding via oropharyngeal and cloacal routes after challenge The oropharyngeal and cloacal cotton swabs were immersed in 1 ml of sterile Brain Heart Infusion sampling buffer (37 g dissolved in 1 l of distilled water) (Becton Dickinson Benelux, Belgium) supplemented with antibiotics (10,000 IU/ml of penicillin, 2 mg/ml of streptomycin, 1 mg/ml of gentamycin and 650 g/ml of kanamycin). The swabs were stored at −80 ◦ C until further analysis. RNA was extracted from 50 l of immersed swabs using the MagMAX 96 AI/ND viral RNA Ambion kit (Applied Biosystems, Lennik,

0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.10.049 doi:10.1016/j.vaccine.2009.10.049

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F. Rauw et al. / Vaccine 28 (2010) 823–833 Table 1 Protection against mortality after challenge with Chimalhuacan NDV strain on vaccinated conventional layer chickens.

Moreover, the quality of the sample and the RNA extraction procedure were validated using avian -actin as previously described [41].

Age of challenge (week)a

Groups

Negative Live ND rHVT-ND rHVT-ND/live ND rHVT-ND/live ND-chitosan

3 (Exp II)

5 (Exp I)

6 (Exp II)

2.7. Experimental design

20% N.D. N.D. 100% 100%

0% 90% 70% 100% N.D.

0% N.D. N.D. 70% 90%

Two studies were conducted on conventional layer chickens with different experimental designs. In the first experiment, the conventional layer eggs were obtained from 38-week-old breeder flock and were assigned to two groups at 18 ED. One group was vaccinated in ovo with rHVT-ND vaccine and the other group was left untreated. On the day of hatching, the chicks were moved to BSL3 isolators and each of both groups was further divided into two groups of 30 birds: groups 1 and 2 originated from the non-treated chicks while groups 3 and 4 originated from the in ovo vaccinated animals. Chicks in groups 2 and 4 were vaccinated with the live ND vaccine by oculo-nasal route and identified as live ND and rHVTND/live ND group, respectively. The group 3 included the rHVT-ND vaccinated chicks and the birds in group 1 served as unvaccinated negative controls. At day-old, serum of 10 unvaccinated birds was sampled to determine the MDA level. Tears, serum, spleen, and duodenum samples were collected (n = 5) at 2, 3, 4 and 5 weeks post-vaccination (pv). At 5 weeks pv, 10 chickens from each group were individually identified and challenged with 105 EID50 /200 l of Chimalhuacan NDV strain by oculo-nasal route. After challenge, chickens were monitored daily for clinical symptoms (swelling of the head, depression, prostration and nervous signs) and mortality over a 2-week period. Birds that showed the clinical signs typical of ND or died were considered as unprotected. Oropharyngeal and cloacal swabs were taken at 2, 4, 7 and 10 days post-challenge (pch). Spleen, serum and duodenum samples were taken from the surviving chickens at 2 weeks pch. In the second experiment, the conventional layer eggs were obtained from 40-week-old breeder flocks and assigned to two groups at 18 ED. One group was vaccinated in ovo with rHVTND vaccine and the other group was not treated. On the day of hatching, the chicks were moved to BSL-3 isolators and the in ovo vaccinated group was further divided into two groups of 60 birds:

N.D. = not determined (see Section 2.7 for details). a Data represent survival rate 2 weeks after challenge by oculo-nasal route with Chimalhuacan NDV strain.

Belgium) on a KingFisher magnetic particle processor (Thermo Scientific) according to manufacturer’s instructions. Purified RNA was eluted in 50 l of elution buffer. The quantification of the Chimalhuacan NDV challenge virus in the oropharyngeal and cloacal swabs was done by quantitative realtime reverse transcription-polymerase chain reaction (QRRT-PCR). The number of matrix (M) gene copies was measured as described previously [11] with minor modifications related to NDV challenge strain. Briefly, after an initial reverse transcription step and a hot start Taq-activation step, 50 cycles (95 ◦ C for 15 s, 54 ◦ C for 34 s, 72 ◦ C for 10 s) were performed on an Applied Biosystems 7500 realtime PCR cycler. Primers (M+4100 and M-4220) and MGB-Taqman probes (M+4169FAM-TAMRA) worked on the M gene and were synthesized by Eurogentec (Liège, Belgium). The virus titre of each sample was determined relative to a standard curve [11] consisting of total viral RNA of Chimalhuacan NDV strain. This standard curve was included in each run. The sensitivity threshold of NDV QRRTPCR to Chimalhuacan NDV strain (R2 = 0.998, efficiency = 94.17%) was determined at 10 EID50 per reaction, based on results of standard curve. It meant that QRRT-PCR reactions were completely comparable above 102.7 EID50 per ml of swabs and quantitative comparisons possible. The results were expressed as the titre of challenge strain per ml of swabs.

Groups

Negative

Cloaca

4 bA

7/10 3.74 ± 0.76c A A

9/9 4.54 ± 0.50A AB

N.D. –

1/10 2.79A

6/10 3.99 ± 0.68A

1/9 4.65

0/9 <2.70

rHVT-ND

4/10A 3.29 ± 0.57A

7/10AB 4.00 ± 0.67A

3/7A 3.52 ± 0.84

0/7A <2.70

rHVT-ND/live ND

3/10A 3.42 ± 0.77A

5/10B 3.72 ± 0.69A

1/10A 4.27

0/10A <2.70

Negative

1/10A 3.57

9/9A 5.11 ± 0.78A

N.D. –

Live ND

0/10A <2.70

8/10AB 3.72 ± 0.63B

1/9A 3.47

0/9A <2.70

rHVT-ND

0/10A <2.70

5/10B 4.26 ± 0.83AB

0/7A <2.70

rHVT-ND/live ND

0/10A <2.70

4/10B 3.68 ± 0.75B

1/10A 3.52

Live ND

2.8. Statistical analysis Statistical analyses of data were performed using Minitab 13 and STATA 10 software (statistical programmes for Windows 2000) and differences were considered as signiﬁcant at P < 0.05. The analysis of the ChIFN production, the antibody level, the titre of viral excretion and the qualitative criterion “positive QRRT-PCR reaction” were carried out in order to compare the groups as previously described [11]. The qualitative criterion “positive cell activation” was analysed by the Fisher’s exact test [11]. The immune response

was compared before and after challenge by the Student t-test in order to underline a boosting effect due to the challenge. 3. Results 3.1. Protection against morbidity, mortality and virus shedding after challenge In the first experiment, the efficacy of the rHVT-ND and live ND vaccines used alone or in combination was evaluated and compared for protection against challenge with the velogenic viscerotropic Chimalhuacan NDV strain in conventional layer chickens at 5 weeks of age. After challenge, the unvaccinated birds showed the first clinical signs at 3 days pch, and all had died by 6 days, while the birds vaccinated with the rHVT-ND/live ND combination were fully protected, showing neither morbidity nor mortality (Table 1). Single live ND vaccination resulted in 80 and 90% protection against clinical symptoms and mortality, respectively, while the rHVT-ND administrated alone provided 70% protection. Viral shedding via oropharyngeal and cloacal routes were observed from the 2nd and 4th day pch, respectively (Table 2A). At 4 days pch, the number of chickens excreting virus via the oropharynx was significantly decreased (P < 0.05) in the rHVT-ND/live ND group but not the excreted titres, when compared to the unvaccinated group. On the

Table 2B Shedding of Chimalhuacan NDV strain after challenge of vaccinated conventional layer chickens at 3- and 6-week-old (second experiment). Age

Oropharyngeal swabs 3 weeks

Age

Groups

Days post-challengea 2

Negative

10/10b A 5.31 ± 0.70c A

9/9A 6.76 ± 0.85A

5/5A 5.85 ± 0.81A

3/3A 3.88 ± 0.11A

rHVT-ND/live ND

9/10A 3.92 ± 0.70B

9/10A 5.03 ± 0.88B

6/10A 3.76 ± 0.81B

2/10B 2.98 ± 0.09B

rHVT-ND/live ND-chitosan

7/10A 3.59 ± 0.54B

8/10A 3.92 ± 0.98C

6/10A 3.98 ± 0.68B

2/10B 3.15 ± 0.08B

Negative

8/10A 3.96 ± 0.92A

10/10A 6.74 ± 0.56A

N.D. –

rHVT-ND/live ND

2/10B 3.18 ± 0.29A

10/10A 5.75 ± 0.87B

8/8A 4.56 ± 0.78A

6/7A 3.48 ± 0.77A

rHVT-ND/live ND-chitosan

4/10AB 4.00 ± 1.40A

9/10A 4.16 ± 1.08C

5/10B 4.60 ± 1.66A

4/10A 3.35 ± 0.64A

Groups

Days post-challenge 2

Negative

3/10A 4.22 ± 1.24

9/9A 6.40 ± 1.05A

5/5A 5.44 ± 0.59A

0/3A <2.70

rHVT-ND/live ND

0/10A <2.70

6/10AB 5.45 ± 0.44B

6/10A 3.30 ± 0.44B

0/10A <2.70

rHVT-ND/live ND-chitosan

1/10A 2.92

5/10B 4.48 ± 0.52C

5/10B 4.66 ± 0.64A

1/10A 3.86

Negative

2/10A 4.49 ± 0.30

6/10A 5.30 ± 0.98AB

N.D. –

0/7A <2.70

rHVT-ND/live ND

1/10A 3.51

6/10A 5.75 ± 0.87A

4/8A 4.56 ± 0.78A

3/7A 3.48 ± 0.77A

0/10A <2.70

rHVT-ND/live ND-chitosan

0/10A <2.70

5/10A 3.56 ± 0.78B

3/10A 3.98 ± 0.93A

1/10A 3.52A

a Data are determined by QRRT-PCR on 1 ml of swabs taken at specified time pch. Different uppercase superscript letters indicate a significant (P < 0.05) difference between the groups (per column). The cut-off of QRRT-PCR specific to Chimalhuacan NDV strain was determined at 102.7 EID50 per ml swabs. The total numbers of chickens tested were reduced with time because of specific mortality (see Section 3 for details). N.D. = not determined, due to specific mortality. b Data represent frequency (number positive/total tested) of virus detection in 1 ml swabs. c Data represent mean ± standard deviation of log10 EID50 of NDV in 1 ml swabs of positive chickens.

14 /

one group of chicks was vaccinated by oculo-nasal route with the live ND vaccine alone while the second group received the live ND vaccine co-administrated with chitosan. These groups were identiﬁed as rHVT-ND/live ND and rHVT-ND/live ND-chitosan group, respectively. The untreated group served as unvaccinated negative controls. At day-old, serum of 10 unvaccinated birds was sampled to determine the MDA level. Tears, serum, spleen, and duodenum samples were collected (n = 5) at 3, 5, 6 and 8 weeks pv. Heparinized blood was taken weekly from 3 to 8 weeks pv. At weeks 3 and 6, 10 chickens per group were challenged and protection and virus shedding were evaluated as described above. Spleen, serum and duodenum samples were taken from the surviving chickens at 2 weeks pch.

Days post-challengea 2

Oropharynx

F. Rauw et al. / Vaccine 28 (2010) 823–833

6 weeks

Table 2A Shedding of Chimalhuacan NDV strain after challenge of vaccinated conventional layer chickens at 5-week-old (ﬁrst experiment). Swabs

826

Cloacal swabs 3 weeks

6 weeks

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reverse, on the same day, the number of excreting birds as well as the excreted titres via the cloaca were both significantly reduced (P < 0.05) in the rHVT-ND/live ND group compared to the negative one. When the rHVT-ND or live ND vaccines were administrated alone, no reduction of virus shedding was observed. Since lower protection and lower effect on virus shedding were observed with these two vaccines if given separately, these vaccination regimens were not further included in the second experiment described below. In the second experiment, the effects of chitosan adjuvant on the protective immunity induced by rHVT-ND/live ND vaccination regimen on one hand, and of the time between vaccination and challenge on the other hand, were evaluated. When the challenge was performed at 3 weeks of age, only 80% of unvaccinated birds died within the 2 weeks observation period (Table 1) but each negative animal showed clinical signs. On the contrary, chickens vaccinated with the rHVT-ND/live ND and rHVT-ND/live ND-chitosan regimens were fully protected. Moreover, the amount of virus excreted via the oropharyngeal route was significantly lower (P < 0.05) in the vaccinated birds at each tested day pch compared to unvaccinated group (Table 2B). Interestingly, at day 4, it was significantly lower (P < 0.05) in the rHVT-ND/live ND-chitosan group, when compared to the rHVT-ND/live ND group. Excretion via the cloacal route was also significantly lower (P < 0.05) at day 4 pch in both rHVT-ND/live ND and rHVT-ND/live ND-chitosan groups when compared to the unvaccinated birds. On the same day, the cloacal excretion in the rHVT-ND/live ND-chitosan group was significantly lower (P < 0.05), when compared to the rHVT-ND/live ND vaccinated chickens, as previously mentioned for oropharyngeal excretion. When the challenge was performed at 6 weeks of age, all unvaccinated chickens died by 6 days pch, while 70 and 90% of rHVT-ND/live ND and rHVT-ND/live ND-chitosan vaccinated birds, respectively, survived the challenge (Table 1). Interestingly, only 50% of the rHVT-ND/live ND group was protected against clinical signs while the rHVT-ND/live ND-chitosan vaccination regimen provided a higher clinical protection (80%). At 4 days pch, the amount of excreted virus via oropharyngeal route was significantly reduced (P < 0.05) by both vaccination regimens (Table 2B) compared to unvaccinated group. Again, at this time, the chickens vaccinated with the rHVT-ND/live ND-chitosan combination excreted significantly less virus (P < 0.05) by the oropharyngeal route than the rHVT-ND/live ND group. In the same way, the amount of excreted virus via cloacal route was significantly lower (P < 0.05) on day 4 in the rHVT-ND/live ND-chitosan group, when compared to the rHVT-ND/live ND vaccinated chickens. 3.2. Humoral immunity The mean of HI antibody titres at day-old of 10 unvaccinated conventional layer chickens were equal to 6.0 and 9.2 log2 in the first and second flock, respectively. Then, the profile of the NDVspecific IgG (Fig. 1A and B) and HI (Fig. 2A and B) antibody titres indicates a decline of passive maternally derived immunity until the 4th week of age. At that time, a progressive active vaccineinduced primary immune response was already detectable in some of the vaccinated groups. Considering the first experiment, the chickens which received the live ND vaccine alone or in combination with the rHVT-ND elicited a significant specific IgG response (P < 0.05) from 4 weeks pv, while the birds vaccinated with the rHVT-ND alone showed an significant rise in titre (P < 0.05) only 1 week later, at 5 weeks pv (Fig. 1A). At that time, there was no meaningful difference between the titres of the three vaccinated groups. Considering the HI test results, only the two groups having received the live ND or the rHVT-ND/live ND vaccination regimen showed an increase in titre (P < 0.05) at 4 weeks pv (Fig. 2A). Inter-

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The NDV-specific CMI in the spleen was not boosted by the challenge in the rHVT-ND, rHVT-ND/live ND and rHVT-ND/live NDchitosan groups, contrary to the live ND vaccinated birds as showed by the statistical comparison of results before and after challenge. 3.3.2. As measured on PBL As further investigation, the peripheral CMI was also evaluated in the second experiment. The PBL activation with PMA/Iono mitogens showed that 80% of conventional layer chickens were able to elicit peripheral white blood cells activability (O.D. > 0.1 and S.I. > 2) after 3 weeks of age (Table 4). Animals having an O.D. < 0.1 or a S.I. < 2 for mitogen-activation were excluded from further PBL NDV-activation analysis. A NDV-specific peripheral CMI was noted (P < 0.05) in both rHVT-ND/live ND and rHVT-ND/live ND-chitosan groups from the 4th to 7th weeks pv. At week 5, this peripheral cellular response was significantly higher (P < 0.05) in rHVT-ND/live ND-chitosan vaccinated chickens, when compared to the rHVTND/live ND group. As observed in the spleen, the NDV-specific CMI in the blood was not boosted by the challenge of the chickens in the rHVT-ND/live ND and rHVT-ND/live ND-chitosan groups. 3.4. Local antibody-mediated immunity

Fig. 1. Serum NDV-specific IgG antibody titre of conventional layer chickens vaccinated with the rHVT-ND and/or the live ND and the chitosan according to different vaccination regimens. Chickens were challenged at 5 weeks of age (A) in the first experiment, and at 3 or 6 weeks (B) in the second experiment, with 105 EID50 of the Chimalhuacan NDV strain by oculo-nasal route. Data represent mean ± standard deviation of absorbance values determined on serum diluted 1:100 by ELISA at specified time pv (n = 5). Means ± standard deviations with no common letters differ significantly (P < 0.05).

estingly, 1 week later, only the rHVT-ND/live ND group showed positive titre. In the second experiment, a significant increase of IgG titres (P < 0.05) was observed as soon as 3 weeks pv in both rHVT-ND/live ND and rHVT-ND/live ND-chitosan groups, compared to unvaccinated group (Fig. 1B). This difference remained significant at 6 and 8 weeks pv. At 5 weeks pv, significant higher (P < 0.05) IgG titre was observed in the rHVT-ND/live ND-chitosan group, when compared to the rHVT-ND/live ND group. The HI titres of the rHVT-ND/live ND-chitosan group were significantly superior to the controls at 5, 6 and 8 weeks pv (Fig. 2B). This was also observed for the rHVTND/live ND group at 5 weeks pv but not at 6 and 8 weeks pv. There was no significant difference between HI titres of the rHVT-ND/live ND and rHVT-ND/live ND-chitosan groups at any tested age. The humoral immunity of the vaccinated groups in both experiments was boosted significantly (P < 0.05) by the challenge, as showed by the statistical comparison of results before and after challenge. 3.3. Cell-mediated immunity 3.3.1. As measured on splenocytes In all experiments, the results obtained at the different sampling time points on splenocytes activation with PMA/Iono mito-

Fig. 2. Serum HI antibody titre of conventional layer chickens vaccinated with the rHVT-ND and/or the live ND and the chitosan according to different vaccination regimens. Chickens were challenged at 5 weeks of age (A) in the first experiment, and at 3 or 6 weeks (B) in the second experiment, with 105 EID50 of the Chimalhuacan NDV strain by oculo-nasal route. Data represent mean ± standard deviation of HI antibody titres at specified time pv (n = 5), which corresponds to the last dilution showing an inhibition of haemagglutination of 4 haemagglutination units of NDV La Sota strain. The HI geometric mean titres were expressed as reciprocal log2 and titres >3 were considered positive. Means ± standard deviations with no common letters differ significantly (P < 0.05).

gens showed that all conventional layer chickens were similarly immunocompetent (O.D. > 0.1 and S.I. > 2), validating the spleen cells activability (data not shown). In the first experiment (Table 3A), the rHVT-ND and live ND vaccines administered alone or in combination induced a NDV-specific CMI starting from 3 weeks pv. At 4 weeks pv, a NDV-specific cellular immune response was measurable in more than 80% of vaccinated chickens, regardless to the vaccination regimen applied. The level of the NDV-specific CMI was significant (P < 0.05) in the rHVT-ND/live ND group, when compared to the unvaccinated group. At 5 weeks pv, both the number of vaccinated chickens showing specific CMI and the levels of their responses were reduced. No statistical difference could be observed between the three vaccination regimens. In the second experiment (Table 3B), NDV-specific CMI was measurable already at 3 weeks pv in both the rHVT-ND/live ND and rHVT/live ND/chitosan groups. However, the difference between the vaccinated and unvaccinated chickens was only significant for the rHVT-ND/live ND-chitosan group. At 4 weeks pv, both vaccinated groups showed significant cellular response, without any difference between vaccination regimens. The duration of NDV-specific CMI was shorter in the rHVT-ND/live ND-chitosan vaccinated chickens, considering its decline at the 8th week, when compared to the rHVT-ND/live ND group (P < 0.05).

3.4.1. As measured in the tears Lachrymal NDV-specific IgG antibody of maternal origin was detected in unvaccinated conventional layer chickens until the 4th week of age (Fig. 3A and B). In the first experiment (Fig. 3A), the chickens which received the live ND vaccine alone or in combination with the rHVT-ND elicited a significant lachrymal antibody-mediated immunity (P < 0.05) from 4 weeks pv, while the birds vaccinated with the rHVT-ND alone showed positive response (P < 0.05) a week later. At week 5, there was no meaningful difference between the titres in these three vaccinated groups. The profile of this lachrymal NDV-specific IgG antibody detection was similar to the one observed in the serum. In the second experiment (Fig. 3B), both rHVT-ND/live ND and rHVT-ND/live ND-chitosan groups showed an increase in IgG titres (P < 0.05) from the 3rd to the 8th week pv. There was no significant difference between these two vaccination regimens. 3.4.2. As measured in the duodenum The profile of the duodenal NDV-specific IgG corresponded to the decline of passive maternally derived immunity and the primary progressive vaccine-induced immune response (Fig. 4A and B). In the first experiment (Fig. 4A), the antibody-mediated immunity in the digestive tract showed significant increase (P < 0.05) from the 4th week pv only in the rHVT-ND/live ND vaccinated chickens. The duodenal antibody response of the three rHVT-ND, live ND and rHVT-ND/live ND groups was boosted significantly (P < 0.05) by the challenge. In the second experiment (Fig. 4B), the digestive antibodymediated immunity increased significantly (P < 0.05) from the 6th week pv only in chickens vaccinated with the rHVT-ND/live NDchitosan combination. This group differed significantly (P < 0.05) from the rHVT-ND/live ND group. Interestingly, the duodenal antibody-mediated immunity was not boosted in the rHVT-ND/live ND-chitosan group when challenge was done at 6 weeks of age, as showed by the statistical comparison of results before and after challenge. 4. Discussion The continuous outbreaks of fatal ND in commercial poultry flocks in many part of the world indicate that routine vaccination in the field often fails to induce sufficiently high levels of immunity to

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Table 4 NDV-speciﬁc peripheral cell-mediated immunity of vaccinated conventional layer chickens (second experiment).

Table 3A NDV-speciﬁc splenic cell-mediated immunity of vaccinated conventional layer chickens (ﬁrst experiment). Groupsa

Weeks post-vaccination

830

Mitogens and antigens

Weeks post-vaccination

Groupsa Negative

rHVT-ND/live ND

rHVT-ND/live ND-chitosan

PMA/Iono

<2.00b A 0/5c A

<2.00A 0/5A

9.36 ± 0.09A 5/5A

4.14 ± 3.42A 4/5A

10.25 ± 8.88A 5/5A

w/o challenge

4.80 ± 2.27A 5/5A

2.63 ± 1.11A 4/5A

6.03 ± 4.19A 5/5A

2 weeks pch

13.45 ± 18.98A 2/3A

7.43 ± 4.58A 5/5A

20.35 ± 10.49A 5/5A

13.09 ± 9.02A 4/5A

9.73 ± 5.51A 5/5A

8.82 ± 6.81A 5/5A

3.01 ± 1.41A 4/5A

8.96 ± 7.44A 4/5A

5.05 ± 3.86A 5/5A

w/o challenge

9.07 ± 10.08A 4/5A

7.10 ± 4.63A 5/5A

6.54 ± 4.82A 4/5A

2 weeks pch

S.M. –

2.26 ± 0.62A 3/5A

5.73 ± 3.21A 4/5A

N.D. –

<2.00d B 0/5e A

3.79 ± 1.34A 2/4A

4.20A 1/5A

w/o challenge

<2.00B 0/5B

<2.00B 0/4B

3.61 ± 0.85A 4/5A

2 weeks pch

<2.00B 0/2A

3.08 ± 1.01AB 2/5A

13.25 ± 9.10A 3/5A

<2.00B 0/4A

17.26 ± 9.03A 2/5A

8.55 ± 4.70AB 3/5A

<2.00B 0/4A

3.16 ± 0.90A 3/4A

4.42 ± 3.21A 2/5A

w/o challenge

<2.00A 0/4A

<2.00A 0/5A

<2.00A 0/4A

2 weeks pch

S.M. –

<2.00A 0/3A

<2.00A 0/4A

Negative

rHVT-ND

Live ND

rHVT-ND/live ND

<2.00b A 0/5c A

<2.00A 0/5A

<2.00B 0/5B

30.60 ± 16.45A 4/5AB

32.81 ± 16.45A 5/5A

34.78 ± 14.52A 5/5A

<2.00B 0/5B

33.86 ± 38.42AB 5/5A

36.13 ± 21.61AB 4/5AB

47.92 ± 38.98A 5/5A

<2.00A 0/5B

16.54 ± 16.14A 5/5A

15.70 ± 10.62A 3/5AB

13.33 ± 16.74A 2/5AB

S.M. –

27.56 ± 17.33A 5/5A

51.32 ± 16.52A 3/5A

34.53 ± 5.67A 2/5A

2 weeks pch

S.M. = specific mortality. a Different uppercase superscript letters indicate a significant (P < 0.05) difference between the groups (per line). b Data represent mean ± standard deviation of S.I. that was calculated for each antigen-responding bird by dividing the O.D. values of antigen-activated splenocytes (if equal or greater than 0.1) by the O.D. of non-activated splenocytes. An O.D. and a S.I. equal to or greater than 0.1 and 2, respectively, were considered as evidence of specific antigen-activation. c Data represent frequency (number antigen-positive/mitogen-positive chickens) of splenic cellular response to antigen-activation.

control ND [3]. Especially, in region where ND is enzootic and where there is high pressure from the field, the need for very early immunization is hampered by the interference of vaccines with a usually high level of passive immunity. In addition, current vaccination strategies can be effective in preventing serious illness and death of infected birds, but prevent neither infection nor shedding of the virus. The goal of the present study was to determine if an innovative vaccination strategy could increase protection and reduce viral shedding, and presumably, the spreading and the transmission of the virus. Therefore, a recent viscerotropic NDV field strain (Chimalhuacan, Mexico) was used to challenge conventional chickens provided with MDA. This strain induced 100% mortality within 6 days when inoculated at 5 weeks of age, after MDA have waned, thus validating the challenge. At 3 weeks of age, mortality induced by the same challenge was reduced to 80%, indicating the presence and likely interference of some residual passive immunity. In the first experiment, the protection afforded by the rHVTND/live ND combination was better than the single vaccination with the rHVT-ND or the live ND vaccine, confirming previous results [42]. Moreover, a direct correlation was shown between

the measured immune response induced by these different vaccination regimens and the protection. Indeed, the higher immunity was observed with the application of the combined rHVT-ND/live ND vaccination regimen, compared to this induced by the rHVTND or the live ND vaccines given alone, reﬂecting the combined advantages of each vaccine. The live ND vaccine replicates actively [1,2,11] and induces therefore a broad immune response directed against different viral polypeptides, such as the HN protein, the phosphoprotein (P), the nucleoprotein (NP) and the M protein. Such live ND vaccines induce a high humoral and local antibodymediated immunity as well as a strong CMI [11]. Nevertheless, their application at day-old is known to be sensitive to the interference of MDA [5–7]. As HVT is poorly sensitive to maternal immunity and passive antibody are not fully absorbed yet at 18 ED, the in ovo application of rHVT-ND allows to overcome this passive immunity and primes an immune response at very early age [19,26]. Moreover, the HVT replicates in a highly cell-associated manner in lymphocytes and it is therefore suggested that this delivery system would induce a great degree of cell-mediated immune mechanisms [43]. It is also well known that HVT establishes a persistent viremia in

Table 3B NDV-speciﬁc splenic cell-mediated immunity of vaccinated conventional layer chickens (second experiment). Groupsa

Weeks post-vaccination

Negative

rHVT-ND/live ND

rHVT-ND/live ND-chitosan

<2.00b B 0/5c B

30.26 ± 24.62AB 5/5A

32.15 ± 28.41A 5/5A

w/o challenge

<2.00B 0/5A

16.95 ± 8.27A 4/5A

15.33 ± 7.99A 3/5A

2 weeks pch

2.13 ± 0.17A 2/3A

4.83 ± 2.21A 2/5A

2.25 ± 0.09A 2/5A

<2.00B 0/5B

22.20 ± 21.23A 4/5AB

16.46 ± 12.11AB 5/5A

w/o challenge

<2.00B 0/5A

20.48 ± 11.62A 2/5A

6.20 ± 5.53B 3/5A

2 weeks pch

S.M. –

4.06 ± 0.37A 2/5A

3.85 ± 2.01A 4/5A

3 5

6 8

18 /

Prot-NDV

N.D. = not determined due to no peripheral cellular response to mitogen-activation; S.M. = specific mortality. a Different uppercase superscript letters indicate a significant (P < 0.05) difference between the groups (per line). b Data represent mean ± standard deviation of S.I. that was calculated for each bird by dividing the O.D. values of mitogen-activated PBL (if equal or greater than 0.1) by the O.D. of non-activated PBL. An O.D. and a S.I. equal to or greater than 0.1 and 2, respectively, were considered as evidence of mitogen-activation. c Data represent frequency (number mitogen-positive/total tested chickens) of peripheral cellular response to mitogen-activation (positive control). d Data represent mean ± standard deviation of S.I. that was calculated for each mitogen-responding bird by dividing the O.D. values of antigen-activated PBLs (if equal or greater than 0.1) by the O.D. of non-activated PBL. An O.D. and a S.I. equal to or greater than 0.1 and 2, respectively, were considered as evidence of specific antigen-activation. e Data represent frequency (number antigen-positive/mitogen-positive chickens) of peripheral cellular response to antigen-activation.

chickens for at least 8 weeks following vaccination [19,44], offering the advantage of delivering foreign antigens to the immune system of vaccinated birds during an extended period of time [45] and is therefore expected to induce a longer lasting immunity. Here, for the ﬁrst time, it was shown that the in ovo vaccination with the rHVT-ND results in the induction of cell-mediated immune response to the F-NDV antigen from the 3rd to 5th week of age, as tested in our conditions. This indicates also that the expression of the recombinant F protein in the host and/or the immunogenic nature of the recombinant protein in rHVT-ND vector are high. It would therefore be interesting to further follow up the CMI response beyond 6 weeks of age. In our study, the single in ovo vaccination with the rHVT-ND vaccine alone did not provide full protection against challenge with the Chimalhuacan NDV strain, conﬁrming previous results [42]. Indeed, it was previously demonstrated that the infection with rHVT is not associated with local antibody-mediated immunity and protection of the upper respiratory tract [15,19]. In our hands, a lower humoral and lachrymal antibody-mediated immunity was shown after rHVT-ND vacci-

nation, when compared to the live ND vaccination. In addition, no antibody-mediated immune response could be measured in the digestive tract during the ﬁrst 5 weeks of life. This could be explained by the exclusive antibody response limited to the F protein after the in ovo inoculation of the rHVT-ND vaccine, compared to the live ND vaccine, and is an indication of a need for an additional vaccination with a live ND vaccine to increase local immunity in the digestive tract in order to circumvent viral shedding. In the second experiment, the protection afforded by the rHVTND/live ND combination in the case of challenge at 6 weeks of age was different than previously observed [42,46,47]. This difference may be explained by the higher MDA level at day-old and the in ovo route of rHVT-ND inoculation. Indeed, the site of injection of the embryonated egg is of paramount importance to achieve adequate replication of the vaccine virus [48,49] and manual injection of embryo is more variable than the subcutaneous inoculation of chicks at day-old with an automatic injector. Secondly, we investigated whether protection could be improved if in ovo vaccination with the rHVT-ND was combined at day-old with the apathogenic

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active immunity induced after vaccination with the ND live vaccine and the adjuvant effect of chitosan on NDV-specific CMI. Therefore, in addition to the combined vaccination against Marek’s disease, this rHVT-ND/live ND-chitosan vaccination program points out a more efficient vaccination strategy of poultry that can be more easily and more effectively applied at the hatchery, ensuring an improved controlled vaccine uptake and earlier induction of immunity. Further investigations, regarding the duration of immunity and the induced respiratory immunity, are, however, necessary. Acknowledgement The authors gratefully acknowledge C. Delgrange for qualified technical assistance in bird experiments (animal handling and sampling assistance). References

Fig. 3. Lachrymal NDV-specific IgG antibody titre of conventional layer chickens vaccinated with the rHVT-ND and the live ND and the chitosan according to different vaccination regimens. Chickens were challenged at 5 weeks of age (A) in the first experiment, and at 3 or 6 weeks (B) in the second experiment, with 105 EID50 of the Chimalhuacan NDV strain by oculo-nasal route. Data represent mean ± standard deviation of absorbance values determined by ELISA at specified time pv (n = 5). Tears samples were diluted 1:4. Means ± standard deviations with no common letters differ significantly (P < 0.05).

enterotropic live ND vaccine co-administrated with the chitosan adjuvant. The analysis of the immune response induced by the rHVT-ND/live ND-chitosan vaccination regimen revealed an earlier and higher antibody-mediated immunity in the digestive tract as well as an increased cell-mediated immune response in the spleen and blood during the first 5 weeks following vaccination. This increased antigen-specific CMI could be related to the adjuvant effect of the chitosan, as previously shown in SPF chickens [32] and mice [50]. Conversely, after this time, the lower splenic and peripheral NDV-specific CMI induced by this rHVT-ND/live ND-chitosan combination could indicate a lower proportion of effector/memory T-lymphocytes in the spleen and blood. This could be related to the elimination by apoptosis of most of these cells after pathogen clearance, to reach a stable pool of memory lymphocytes maintaining homeostasis [51,52]. Although not exclusive, a second explanation would be an earlier T-cells homing in the peripheral lymphoid organs [53,54], including the gut-associated lymphoid tissues (GALT) [50,55]. The immune mechanisms underlying the adjuvant effect of the chitosan on the CMI in the digestive tract [32] and the antibody-mediated immune response in the trachea [56–58] and lung [11] should be investigated further. Surprisingly, no improvement of the humoral and lachrymal antibody-mediated immunity was observed as compared to the other investigated ND vaccination regimens. Nevertheless, a chitosan effect on the dura-

20 /

Fig. 4. Duodenal NDV-specific IgG antibody titre of conventional layer chickens vaccinated with the rHVT-ND and the live ND and the chitosan according to different vaccination regimens. Chickens were challenged at 5 weeks of age (A) in the first experiment, and at 3 or 6 weeks (B) in the second experiment, with 105 EID50 of the Chimalhuacan NDV strain by oculo-nasal route. Data represent mean ± standard deviation of absorbance values determined by ELISA at specified time pv (n = 5). The Ig response was measured in undiluted supernatants of ex vivo duodenal tissues cultures. Means ± standard deviations with no common letters differ significantly (P < 0.05).

tion of humoral and lachrymal antibody-mediated immunity could not be excluded and should be investigated further. There is therefore a direct correlation between the higher measured immune response induced by this rHVT-ND/live ND-chitosan vaccination regimen and the protection, as demonstrated by a better resistance against challenge performed at 3 and 6 weeks of age and a stronger reduction of virus shedding, as measured in oropharyngeal and cloacal swabs. Additionally, after the challenge, no booster effect on cell- and digestive antibody-mediated immunity was observed in this vaccinated group, suggesting a lower challenge virus replication and consequently an improved local protection. This confirms previous results demonstrating that the antigenspecific CMI is essential for NDV clearance [9] and is combined with the specific antibody detected in the mucus of the digestive tract. However, the decreased protection observed at 6 weeks of age indicates the likely need for a boosting vaccination at 3 or 4 weeks of age, especially for long living birds placed in region where infection pressure is high. In summary, the present investigations revealed a new distinct and interesting feature. The rHVT-ND/live ND-chitosan vaccination regimen presented here combines the individual advantages of its three components: the definite advantage of overcoming the MDA in neonatal chickens by the in ovo vaccination with a rHVT-ND, the

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[19] Reddy SK, Sharma JM, Ahmad J, Reddy DN, McMillen JK, Cook SM, et al. Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek’s diseases in specific-pathogen-free chickens. Vaccine 1996;14(6):469–77. [20] Boursnell MEG, Green PF, Samson ACR, Campbell JI, Deuter A, Peters RW, et al. A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV. Virology 1990;178:297–300. [21] Taylor J, Edbauer C, Rey-Senelonge A, Bouquet JF, Norton E, Goebel S, et al. Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens. J Virol 1990;64:1441–50. [22] Iritani Y, Aoyama S, Takigami S, Hayashi Y, Ogawa R, Yanagida N, et al. 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N°1 • Scientific File

PROCEEDINGS OF THE 9TH INTERNATIONAL SYMPOSIUM ON MAREK’S DISEASE AND AVIAN HERPESVIRUSES, 2012.

VECTORMUNE® ND F. Rauw et al. / Vaccine 28 (2010) 823–833 [44] Sharma JM, Lee LF, Wakenell PS. Comparative viral, immunologic, and pathologic responses of chickens inoculated with herpesvirus of turkeys as embryos or at hatch. Am J Vet Res 1982;45(8):1619–23. [45] Tsukamoto K, Saito S, Saeki S, Sato T, Tanimura N, Isobe T, et al. Complete, longlasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. J Virol 2002;76(11):5637–45. [46] Cambre JFR, Hein RG, Dominguez V, Aguilera A. The effect of using a HVT/ND+SB-1 recombinant vaccine on the reisolation of Newcastle disease virus Chimalhuacan strain. In: 57th western poultry disease conference & XXXIII annual ANECA convention. 2008. p. 35. [47] Hein RG, Rios JF, Aguilera A, Domhof J. Newcastle disease (ND) efﬁcacy in broilers vaccinated at one day of age with the recombinant HVT/F(ND):innovacND-SB challenged with the Mexican vND virus isolate Chimalhuacan. In: 57th western poultry disease conference & XXXIII annual ANECA convention. 2008. p. 32–3. [48] Johnston PA, Liu H, O’Connell T, Phelps P, Bland M, Tyczkowki J, et al. Applications in in ovo technology. Poultry Sci 1997;76(1):165–78. [49] Jochemsen P, Jeurissen SHM. The localization and uptake of in ovo injection soluble and particulate substances in the chicken. Poultry Sci 2002;81: 1811–7. [50] McNeela EA, O’Connor D, Jabbal-Gill I, Illum L, Davis SS, Pizza M, et al. A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM197) of

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[51] [52] [53]

[54] [55] [56]

[57]

[58]

833

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Scientific File • N°3

EXTRACT FROM “AVIAN DISEASES”, 2013 (PP. 750–755).

VECTORMUNE® ND AVIAN DISEASES 57:750–755, 2013 AVIAN DISEASES 57:750–755, 2013 AVIAN DISEASES 57:750–755, 2013

Turkey herpesvirus vector Newcastle disease vaccine

Protection and Antibody Response Caused by Turkey Herpesvirus Vector Newcastle Protection Caused by Protection and and Antibody Antibody Response ResponseDisease CausedVaccine by Turkey Turkey Herpesvirus Herpesvirus Vector Vector Newcastle Newcastle Disease Vaccine Disease Vaccine AB A C C D B Motoyuki Esaki, AB Alecia Godoy,A Jack K. Rosenberger,C Sandra C. Rosenberger,C Yannick Gardin,D Atsushi Yasuda,B and AB Alecia Godoy,A Jack K. Rosenberger,C Sandra C. Rosenberger, C Yannick Gardin,D Atsushi Yasuda,B and Motoyuki Esaki, AE Motoyuki Esaki, Alecia Godoy, Jack K. Rosenberger, SandraDorsey C. Rosenberger, Yannick Gardin, Atsushi Yasuda, and Kristi Moore AE Kristi Kristi Moore Moore Dorsey DorseyAE A ACeva Animal Health (Biomune Campus), 8901 Rosehill Road, Lenexa, KS 66215 B ACeva Animal Health (Biomune Campus), 8901 Rosehill Road, Lenexa, KS 66215 CevaAnimal AnimalHealth Health(Japan (Biomune Campus), 8901 RosehillYokohama, Road, Lenexa, KS 66215 Campus), 1-6 Suehiro-cho, 230-0045, Japan BCeva Ceva Animal Health (Japan Campus), 1-6 Suehiro-cho, Yokohama, 230-0045, Japan B C Ceva Animal Health (Japan Campus),Way, 1-6 Suehiro-cho, Yokohama, LLC, 1 Innovation Suite 1000, Newark, DE230-0045, 19711 Japan CAviServe LLC, 1 Innovation Way, Suite 1000, Newark, DE 19711 CAviServe D AviServe LLC, 1 Innovation Way, 1000, Newark,France DE 19711 Animal Health, BP 126,Suite 33501 Libourne, DCeva DCeva Animal Health, BP 126, 33501 Libourne, France

Ceva Animal Health, BP 126, 33501 Libourne, Received 3 April 2013; Accepted 15 July 2013; Published ahead Received 3 April 2013; Accepted 15 July 2013; Published ahead Received 3 April 2013; Accepted 15 July 2013; Published ahead

France of print 31 July 2013 of print 31 July 2013 of print 31 July 2013

SUMMARY. Newcastle disease (ND) is prevalent worldwide and causes significant clinical and economic losses to the poultry SUMMARY. Newcastle disease (ND) is prevalent worldwide and causes significant clinical and economic losses to the poultry SUMMARY. diseaseusing (ND) prevalent vaccines worldwide causes significant clinical and economic losses to with the distinct poultry industry. Current Newcastle vaccine programs liveisattenuated andand inactivated vaccines have limitations, and new vaccines industry. Current vaccine programs using live attenuated vaccines and inactivated vaccines have limitations, and new vaccines with distinct industry.are Current vaccine programs using livesolution attenuated vaccinesND, and inactivated vaccines have limitations, and new vaccines distinct features needed. To offer an alternative to control a turkey herpesvirus vector Newcastle disease vaccine with (HVT/ND) features are needed. To offer an alternative solution to control ND, a turkey herpesvirus vector Newcastle disease vaccine (HVT/ND) features arethe needed. an alternative solution to control ND, developed. a turkey herpesvirus vector Newcastle vaccine expressing fusion To geneoffer of Newcastle disease virus (NDV) has been First, immunogenicity of thedisease HVT/ND was (HVT/ND) evaluated in expressing the fusion gene of Newcastle disease virus (NDV) has been developed. First, immunogenicity of the HVT/ND was evaluated in expressing the fusion gene Newcastle disease virus (NDV) hasovo been developed. First, immunogenicity the HVT/ND was to evaluated in specific-pathogen-free layerofchickens after vaccination by the in route to 18-day-old embryos or by theofsubcutaneous route 1-day-old specific-pathogen-free layer chickens after vaccination by the in ovo route to 18-day-old embryos or by the subcutaneous route to 1-day-old specific-pathogen-free layerNDV chickens vaccination by the route to 18-day-old embryos or by the subcutaneous routeassay to 1-day-old chicks. Antibodies against wereafter detected at 24 days of in ageovo using a commercial NDV enzyme-linked immunosorbent (ELISA) chicks. Antibodies against NDV were detected at 24 days of age using a commercial NDV enzyme-linked immunosorbent assay (ELISA) chicks. against NDV were detected 24 days using awere commercial enzyme-linked immunosorbent assay (ELISA) kit and Antibodies the hemagglutination inhibition test. Atatleast 90%ofofage chickens protectedNDV against challenge with velogenic neurotropic NDV kit and the hemagglutination inhibition test. At least 90% of chickens were protected against challenge with velogenic neurotropic NDV kit andGB thestrain hemagglutination At leastat90% werenone protected challengechallenged with velogenic neurotropic NDV Texas (genotype II;inhibition pathotypetest. velogenic) 4 wkofofchickens age, while of the against nonvaccinated, controls were protected Texas GB strain (genotype II; pathotype velogenic) at 4 wk of age, while none of the nonvaccinated, challenged controls were protected Texaschallenge. GB strainSecond, (genotype at chicken 4 wk of elicits age, while none of the nonvaccinated, protected from theII; agepathotype at which velogenic) a vaccinated an immunologic response to the challenged HVT/ND controls preparedwere for this study, from challenge. Second, the age at which a vaccinated chicken elicits an immunologic response to the HVT/ND prepared for this study, fromthus challenge. Second, agevirus, at which a vaccinated chicken elicits anchickens immunologic to the HVT/ND prepared for this study, and is protected fromthe ND was assessed in commercial broiler after inresponse ovo vaccination of 18-day-old embryos. Challenge and thus is protected from ND virus, was assessed in commercial broiler chickens after in ovo vaccination of 18-day-old embryos. Challenge and thus is protected ND virus,NDV was assessed in commercial broiler chickens aftervia inthe ovorespiratory vaccinationtract of 18-day-old was conducted using afrom low-virulence strain (genotype II; pathotype lentogenic) each week embryos. between 1Challenge and 5 wk was conducted using a low-virulence NDV strain (genotype II; pathotype lentogenic) via the respiratory tract each week between 1 and 5 wk wasage, conducted a low-virulence NDV (genotype II; pathotype lentogenic) the respiratory tract each week between and mild 5 wk of in orderusing to mimic the situation in strain areas where virulent NDV strains do notvia normally exist and low-virulence strains 1cause of age, in order to mimic the situation in areas where virulent NDV strains do not normally exist and low-virulence strains cause mild of age, in order to mimic the to situation in losses. areas where virulent strains do not normally exist and low-virulence strains causeswabs mild respiratory symptoms leading economic Protection wasNDV evaluated by the presence or absence of isolated virus from tracheal respiratory symptoms leading to economic losses. Protection was evaluated by the presence or absence of isolated virus from tracheal swabs respiratory symptoms leading economicwas losses. Protection evaluated by the presence or absence of isolated virus fromFull tracheal swabs at 5 days postchallenge. Partialtoprotection observed at 3 was wk of age, when 6 out of 10 (60%) chickens were protected. protection at 5 days postchallenge. Partial protection was observed at 3 wk of age, when 6 out of 10 (60%) chickens were protected. Full protection at days postchallenge. observed 3 wk and of age, out(100%) of 10 (60%) chickens were protected. Full protection was5 obtained at 4 and 5Partial wk ofprotection age, whenwas 9 out of 10 at (90%) 10 when out of610 chickens were protected, respectively. Finally, was obtained at 4 and 5 wk of age, when 9 out of 10 (90%) and 10 out of 10 (100%) chickens were protected, respectively. Finally, was obtained at 4 challenge and 5 wkwith of age, whenTexas 9 outGB ofstrain 10 (90%) out ofevaluated 10 (100%) chickens were protected, respectively. Finally, protection against virulent at 19 and wk of10age was in commercial female layer chickens vaccinated at protection against challenge with virulent Texas GB strain at 19 wk of age was evaluated in commercial female layer chickens vaccinated at protection challenge with virulent Texas GBchickens strain atwere 19 wk of age was evaluated in commercial femalesuccumbed layer chickens vaccinated at 1 day of ageagainst with HVT/ND. All of the vaccinated protected, while all of the challenge controls to the challenge. 1 day of age with HVT/ND. All of the vaccinated chickens were protected, while all of the challenge controls succumbed to the challenge. 1 day of age with HVT/ND. All of the vaccinated chickenswere weremaintained protected, through while all 50 of the Furthermore, anti-NDV antibodies measured by ELISA wk challenge of age. controls succumbed to the challenge. Furthermore, anti-NDV antibodies measured by ELISA were maintained through 50 wk of age. Furthermore, anti-NDV antibodies measured by ELISA were maintained through 50 wk of age. RESUMEN. Proteccioń y respuesta de anticuerpos por una vacuna contra la enfermedad de Newcastle con un herpesvirus de los RESUMEN. Proteccio´´ n y respuesta de anticuerpos por una vacuna contra la enfermedad de Newcastle con un herpesvirus de los RESUMEN. Proteccion y respuesta de anticuerpos por una vacuna contra la enfermedad de Newcastle con un herpesvirus de los pavos como vector. pavos como vector. pavos como vector.de Newcastle (ND) es prevalente en todo el mundo y causa pe´rdidas clıńicas y econo´micas significativas para la La enfermedad La enfermedad de Newcastle (ND) es prevalente en todo el mundo y causa pe´´rdidas clı´ńicas y econo´´ micas significativas para la ´ n actuales La enfermedad de Newcastle es prevalente en todo vacunas el mundo y causa perdidas clınicasinactivadas y economicas significativas paray se la industria avıćola. Los programas (ND) de vacunacio vivas atenuadas y vacunas tienen limitaciones, ´ n actuales con industria avı´ćola. Los programas de vacunacio con vacunas vivas atenuadas y vacunas inactivadas tienen limitaciones, y se ´sticas ´diferentes. ´ n alternativa industria cola. Los programas de vacunacio n actuales con vacunas vivas atenuadas y vacunas inactivadas tienen limitaciones, y de se necesitan avı nuevas vacunas con caracterı Para ofrecer una solucio para controlar a la enfermedad necesitan nuevas vacunas con caracterı´´sticas diferentes. Para ofrecer una solucio´´ n alternativa para controlar a la enfermedad de ´ unacon necesitan vacunas caracterı sticas diferentes. Para ofrecer una solucioanun alternativa para controlar la enfermedad de Newcastle,nuevas se desarrollo vacuna contra la enfermedad de Newcastle utilizando herpesvirus de pavo comoa vector (HVT/ND) Newcastle, se desarrollo´´ una vacuna contra la enfermedad de Newcastle utilizando a un herpesvirus de pavo como vector (HVT/ND) ´ lacomo Newcastle, una´ nvacuna contra enfermedadde deNewcastle Newcastle(NDV). utilizando un herpesvirus pavo vector (HVT/ND) que expresaseeldesarrollo gene de fusio del virus de lalaenfermedad Ena primer lugar, sede evaluo inmunogenicidad de la que expresa el gene de fusio´´ n del virus de la enfermedad de Newcastle (NDV). En primer lugar, se evaluo´´ la inmunogenicidad de la ´ficos (NDV). ´ n seinevaluo que expresa el geneen de aves fusiode n del virus de la enfermedad de especı Newcastle primer lugar, de la vacuna HVT/ND postura libres de pato´genos despue´s En de la vacunacio ovo a la losinmunogenicidad 18 dıás de desarrollo ´ genos especı´ficos despue´s de la vacunacioń in ovo a los 18 dıás de desarrollo vacuna HVT/ND en´ aves de postura libres de pato vacuna HVT/ND deńea postura libresdedeunpato especı despueánticuerpos s de la vacunacio ovo a los 18 dıás de desarrollo embrionario, o por en vıá aves subcuta en pollos dı´´ágenos de edad. Se´ficos detectaron contra´ nlain enfermedad de Newcastle a los 24 embrionario, o por vıá subcuta´ńea en pollos de un dıá de edad. Se detectaron anticuerpos contra la enfermedad de Newcastle a los 24 ás de edad con ´ n con enzimas embrionario, o por a subcuta nea en pollos de undedıinmunoabsorcio a de edad. Se detectaron anticuerpos la enfermedad de Newcastle a los 24 dı unvıestuche comercial del ensayo ligadascontra (ELISA) para Newcastle y por la prueba de dı´ás de edad con un estuche comercial del ensayo de inmunoabsorcio´´ n con enzimas ligadas (ELISA) para Newcastle la prueba de ó con layy por ´ n decon ´ n. Al del ńica dı as de edad un estuche comercial ensayo de inmunoabsorcio nestuvieron con enzimas ligadas (ELISA) para Newcastle por laveloge prueba de inhibicio la hemaglutinacio menos el 90% de los pollos protegidos contra el desafı cepa inhibicioń de la hemaglutinacio´´ n. Al menos el 90% de los pollos estuvieron protegidos contra el desafı´ó con la cepa veloge´ńica ńico) inhibicio n deGB la hemaglutinacio menos el 90% de los protegidos contraque el desafı o con velogenica neurotro´pica Texas (genotipon.II;Alpatotipo veloge a laspollos cuatroestuvieron semanas de edad, mientras ninguna de la las cepa aves control no neurotro´pica GB Texas (genotipo II; patotipo veloge´ńico) a las cuatro semanas de edad, mientras que ninguna de las aves control no ó. En ´ la edad neurotro´picaestuvo GB Texas (genotipo patotipo veloge nico) lugar, a las cuatro semanas de edad, mientras que ninguna de las aves controluna no vacunadas, protegida ante II; el desafı segundo se evaluo a la que los pollos vacunados desarrollaban vacunadas, estuvo protegida ante el desafı´ó. En segundo lugar, se evaluo´´ la edad a la que los pollos vacunados desarrollaban una ´ n contra vacunadas, estuvo ´protegida ante el desafıHVT/ND o. En segundo lugar, se evaluo a lala que los pollos vacunados respuesta inmunolo gica contra la vacuna para este estudio y porla loedad tanto proteccio el virus desarrollaban de Newcastle una fue respuesta inmunolo´´ gica contra la vacuna HVT/ND para este estudio y por lo tanto´ la proteccio´´ n contra el virus de Newcastle fue ´ n in ovo ´ a cabo fue respuesta inmunolo gica contracomerciales la vacuna HVT/ND este estudio y por lo 18 tanto proteccio n contra el virus Se dellevo Newcastle evaluada en pollos de engorde despue´s depara la vacunacio a los dıaslade desarrollo embrionario. un evaluada en pollos de engorde comerciales despue´´s de la vacunacio´´ n in ovo a los 18 dı´ás de desarrollo embrionario. Se llevo´´ a cabo un ó usando ás respiratorias ńico) evaluada en pollos decepa engorde comerciales despue s de II; la vacunacio n inóvo a los a18 dıáss de las desarrollo embrionario. Sesemana llevo a entre cabo un desafı una de baja virulencia (genotipo patotipo lento ge trave vı cada las desafı´ó usando una cepa de baja virulencia (genotipo II; patotipo lento´´ ge´ńico) a trave´´s de las vı´ás respiratorias cada semana entre las ´ n en a´reas desafı o usando una cepa de baja (genotipo II; patotipo lento a traves de lasno vıasexisten respiratorias cada semana entre las semanas una y cinco de edad, convirulencia el fin de imitar la situacio enge lasnico) que normalmente cepas virulentas de Newcastle ´ ´ semanas una y cinco de edad, con el fin de imitar la situacio´ n en a´reas en las que normalmente no existen cepas virulentas de Newcastle ´ n se de ´ por la una cinco de edad, causan con el fin de imitar la situacionleves en aque reas conducen en las que anormalmente no´ micas. existenLa cepas virulentas Newcastle ysemanas las cepas deybaja virulencia sıńtomas respiratorios pe´rdidas econo proteccio evaluo ´rdidas econo´micas. La proteccioń se evaluo´ por la y las cepas de baja virulencia causan sı´ńtomas respiratorios leves que conducen a pe ´rdidas ás ´s del´ micas. ´ n´parcial ypresencia las cepasode baja virulencia causanviral sıntomas leves que conducen n se evaluo por la ausencia de aislamiento a partirrespiratorios de hisopos traqueales a los cincoadıpe despueecono desafı´ó.La Seproteccio observo´´´proteccio ´ ´ ´ presencia o ausencia de aislamiento viral a partir de hisopos traqueales a los cinco dıás despue´s del desafıó. Se observo´ proteccio´ n parcial ´ ndesafı presencia o ausencia aislamiento viral a partir deseis hisopos traqueales a los cinco dıas despue s del Se observo n parcial a las tres semanas de de edad, cuando se protegieron de diez (60%) pollos. Se obtuvo proteccio totalo. a las cuatro yproteccio cinco semanas de a las tres semanas de edad, cuando se protegieron seis de diez (60%) pollos. Se obtuvo proteccio´´ n total a las cuatro y cinco semanas de a las tres semanas de edad, cuando seis(100%) de diez pollos (60%) estuvieron pollos. Se obtuvo proteccio n total a las cuatro cincosesemanas edad, cuando nueve de diez (90%)se yprotegieron diez de diez protegidos, respectivamente. Por uýltimo, evaluo´ de la ´ ´ edad, cuando nueve de diez (90%) y diez de diez (100%) pollos estuvieron protegidos, respectivamente. Por u´ ltimo, se evaluo´ ´la ó con ´ n contra edad, cuando nueve de diez (90%) y diez de diez (100%) pollos estuvieron protegidos, respectivamente. Por ultimo, se evaluo laa proteccio el desafı la cepa virulenta GB Texas a las 19 semanas de edad en gallinas de postura comerciales vacunadas al dı proteccio´´ n contra el desafı´ó con la cepa virulenta GB Texas a las 19 semanas de edad en gallinas de postura comerciales vacunadas al dıá ó murieron proteccio n contra el desafı o con la cepa virulenta Texas a estaban las 19 semanas de edad en gallinas de postura comerciales vacunadas al dıá de edad con la vacuna HVT/ND. Todas las avesGB vacunadas protegidas, mientras que todos los controles de desafı ´ de edad con la vacuna HVT/ND. Todas las aves vacunadas estaban protegidas, mientras que todos los controles de desafıó murieron ´ n. Adema ´s, los anticuerpos de edad con la vacuna HVT/ND. Todas lascontra aves vacunadas protegidas, todos los controles de semanas desafıo murieron ante la exposicio Newcastleestaban determinados por mientras ELISA seque mantuvieron hasta las 50 de edad. ante la exposicio´´ n. Adema´´s, los anticuerpos contra Newcastle determinados por ELISA se mantuvieron hasta las 50 semanas de edad. ante la exposicio n. Ademadisease, s, los anticuerpos contra Newcastle por ELISA se mantuvieron hasta las 50 semanas de edad. Key words: Newcastle turkey herpesvirus, vector determinados vaccine, protection, immunity Key words: Newcastle disease, turkey herpesvirus, vector vaccine, protection, immunity Key words: Newcastle disease, turkey herpesvirus, vector vaccine, protection, Abbreviations: BCIP/NBT 5 5-bromo-4-chloro-3-indolyl-phosphate/nitro blueimmunity tetrazolium; CEF 5 chicken embryo fibroblasts; Abbreviations: BCIP/NBT 5 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; CEF 5 chicken embryo fibroblasts; Abbreviations: BCIP/NBT 5 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; chicken embryo fibroblasts; dose F 5 fusion; CEF HI 55hemagglutination inhibition; EID 50 5 embryo infectious 50; ELISA 5 enzyme-linked immunosorbent assay; EID 5 embryo infectious dose50; ELISA 5 enzyme-linked immunosorbent assay; F 5 fusion; HI 5 hemagglutination inhibition; embryo infectious dose50; ELISA immunosorbent assay; F5 fusion; HIvector 5 hemagglutination inhibition; EID50 HN 55 hemagglutinin-neuraminidase; HVT55enzyme-linked turkey herpesvirus; HVT/ND 5 turkey herpesvirus Newcastle disease vaccine; 50 HN 5 hemagglutinin-neuraminidase; HVT 5 turkey herpesvirus; HVT/ND 5 turkey herpesvirus vector Newcastle disease vaccine; HN 5 hemagglutinin-neuraminidase; HVT 5NDV turkey5herpesvirus; HVT/ND 5 turkey vector Newcastle disease kDa kilodalton; ND 5 Newcastle disease; Newcastle disease virus; PVDFherpesvirus 5 polyvinylidenedifluoride; SPF 5vaccine; specific kDa 5 kilodalton; ND 5 Newcastle disease; NDV 5 Newcastle disease virus; PVDF 5 polyvinylidenedifluoride; SPF 5 specific kDa 5 kilodalton; ND 5 Newcastle pathogen free; UL 5 unique long disease; NDV 5 Newcastle disease virus; PVDF 5 polyvinylidenedifluoride; SPF 5 specific pathogen free; UL 5 unique long pathogen free; UL 5 unique long E ECorresponding ECorresponding

author. E-mail: kristi.moore@ceva.com author. E-mail: kristi.moore@ceva.com Corresponding author. E-mail: kristi.moore@ceva.com

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Newcastle disease (ND) is one of the most important global poultry diseases, and it causes significant clinical and economic losses to the poultry industry (1). ND is caused by avian paramyxovirus type I, also known as Newcastle disease virus or NDV, with a nonsegmented, negative-sense RNA genome. The clinical expression of ND in chickens varies greatly depending on NDV strains. The NDV strains have been classified into five pathotypes based on the clinical signs observed in infected chickens. These pathotypes are 1) viscerotropic velogenic strains that cause an acute, lethal infection accompanying hemorrhagic intestinal lesions, 2) neurotropic velogenic strains that cause an acute, lethal infection with respiratory and neurologic signs, 3) mesogenic strains that cause respiratory signs with low mortality, 4) lentogenic strains that cause mild or subclinical respiratory infection, and 5) asymptomatic-enteric strains with subclinical enteric infection (17). Although all NDV strains belong to a single serotype, NDV can also be divided into several genotypes based on molecular characterization (2). Whether this genetic diversity among NDV strains leads to inadequate vaccine efficacy is a matter of debate (5,8,12,13). In many countries, prophylactic vaccination is applied to control ND (22). Inactivated vaccines and live attenuated vaccines, including those using lentogenic strains such as LaSota and Hitchner B1, are commercially available. Although these vaccines have contributed greatly to the control of ND over the years, they also have a number of drawbacks. First, live vaccines can cause subclinical or acute respiratory disease, especially with the presence of complicating infections, resulting in production losses in some parts of the world, including the United States (1,22). Also, maternally derived antibodies may adversely affect effectiveness of live and inactivated vaccines. Despite extensive vaccination with prophylactic vaccines, outbreaks have been reported in vaccinated populations in many parts of the world (5), indicating that there is room for improvement in the current vaccine programs. Van Boven et al. stress the importance of providing uniform protection to a flock of chickens (26); however, this cannot be easily achieved with current vaccination procedures involving spray or drinking water vaccination using live attenuated vaccines, occasionally followed by needle injection of inactivated vaccines to individual chickens. Various vector vaccines expressing NDV antigen genes such as the fusion (F) gene and the hemagglutinin-neuraminidase (HN) gene have been developed to overcome the issues related to the current vaccines and offer more beneficial characteristics (3,14,20,23,24). Of those possible vectors, turkey herpesvirus (HVT) appears to have uniquely advantageous characteristics. It is an extremely safe vaccine that has been used to prevent Marek’s disease over 30 yr (21), and it can be applied either in ovo or to 1-day-of-age chicks at the hatchery. It is also known to elicit strong cell-mediated immunity along with humoral immunity (7,9,19). Furthermore, since HVT persists in inoculated chickens (27), a longer period of protection against infection can be expected. Also, the efficacy of HVT vector vaccines does not appear to be excessively affected by the presence of maternally derived antibodies, probably because HVT replicates in a cell-associated manner (14). Therefore, we developed a HVT vector vaccine containing the NDV F gene between unique long (UL) genes 45 and UL46. This noncoding intergenic region between UL45 and UL46 does not affect virus replication (6). This HVT vector vaccine expressing the F protein has been shown to induce both humoral and cell-mediated immunity, protect chickens from lethal NDV challenge with genotype V viscerotropic velogenic strains, and reduce virus shedding after challenge (18,19). In research described here, the initiation of protection induced by the turkey herpesvirus vector Newcastle disease vaccine (HVT/ND) was

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examined by investigating its ability to hinder replication of lowvirulence NDV in tracheas. We also investigated protection conferred by HVT/ND at 19 wk of age after vaccination to commercial layer chickens at 1 day of age. We also monitored humoral immunity through 50 wk of age. MATERIALS AND METHODS Viruses. Turkey herpesvirus vector Newcastle disease vaccine VECTORMUNEH HVT NDV (Ceva Biomune, Lenexa, KS) contains the F gene of lentogenic NDV D26 strain (genotype I) (16,28). The HVT/ND virus was propagated in chicken embryo fibroblasts (CEF). Marek’s disease vaccine serotype 2, SB-1, and parental HVT FC-126 strain were also propagated in CEF. The genotype II neurotropic velogenic Texas GB strain of NDV and genotype II lentogenic B1 strain of NDV were propagated in the allantoic fluid of 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. The titers of the specific viruses used for vaccination of chickens in the present study were determined and are provided in the following sections with the description of each part of the study. Expression of NDV F protein by HVT/ND. Antiserum against the NDV F protein was produced in rabbits by immunization with purified F protein produced in Escherichia coli. The rabbit anti-NDV F serum was used in western blot analysis to confirm expression of the NDV F protein by HVT/ND. Briefly, lysates of CEF infected with HVT/ND for 3 days and lysates of NDV B1 strain harvested from the allantoic fluid of embryonated chicken eggs were separated on 8% acrylamide gel and transferred to polyvinylidenedifluoride (PVDF) membrane (Millipore Corporation, Billerica, MA). The membrane was then incubated with the rabbit anti-NDV F serum and alkaline phosphatase-labeled anti-rabbit IgG antibody (Bethyl Laboratories, Montgomery, TX), and then developed with 5-bromo-4-chloro-39-indolyphosphate/nitro-blue tetrazolium (BCIP/NBT; Bio-Rad Laboratories, Hercules, CA). NDV serology. Anti-NDV antibody levels were determined by using a commercial NDV enzyme-linked immunosorbent assay (ELISA) kit (Idexx Laboratories, Westbrook, ME). Log 10 antibody titers were calculated according to the manufacturer’s instruction. The positive/ negative cutoff titer value was 396. The hemagglutination inhibition (HI) test was conducted using the LaSota strain as antigen at 10 hemagglutinating units. Briefly, twofold serial dilutions (50 ml) of chicken sera were made with phosphate buffered saline, and diluted antigen containing 10 hemagglutinating units was added. After 45 min of incubation, 50-ml aliquots of 0.5% chicken red blood cells were added, and hemagglutination was scored following another 45 min of incubation. Efficacy of HVT/ND in SPF chickens against velogenic neurotropic NDV challenge. The HVT/ND prepared specifically for this study and mixed with Marek’s disease virus serotype 2, SB-1 strain, was administered in ovo to SPF embryos (Charles River Laboratories, Wilmington, MA) at 18 days of incubation or subcutaneously in the neck of 1-day-of-age SPF chicks at HVT/ND and SB-1 titers specific to this study. One group was mock-vaccinated with vaccine diluent by the in ovo route and served as a challenge control group. Chickens were bled at 24 days of age to examine anti-NDV antibodies. At 28 days of age, chickens were challenged intramuscularly with 104.0 embryo infectious dose50 (EID50)/dose of the NDV Texas GB strain and observed daily for 14 days for neurologic signs typical of neurotropic ND, such as tremors, loss of coordination, paralysis, or death. Age at which protection is induced by HVT/ND. Commercial broiler chickens (Ross708 3 Ross708) with NDV maternal antibodies were used in this study. Embryos at 18 days of incubation were vaccinated with either HVT/ND prepared specifically for this study and mixed with SB-1 at titers specific to this study, or with HVT parental FC-126 mixed with SB-1, both at the same target doses, as a control. Each week between 1 and 5 wk of age, 10 chickens from each group were inoculated with 103.5 EID50 per bird of lentogenic B1 strain of NDV by the ocular route. Five days postinoculation, tracheal swabs were / 25

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Fig. 1. Expression of NDV F protein by HVT/ND was confirmed by western blot analysis using rabbit anti-NDV F serum. NDV B1 strain (lane 1), CEF infected with HVT/ND (lane 2), and CEF infected with parental HVT FC126 (lane 3) were lysed and separated on 8% acrylamide gel. Separated proteins were blotted on PVDF membrane and reacted with the rabbit anti-NDV F serum, alkaline phosphatase– labeled anti-rabbit IgG antibody, and then developed with BCIP/NBT. M: Precision Plus Protein Standards (Bio-Rad Laboratories). Arrow indicates the 60-kDa NDV F0 protein, and asterisk indicates the 50kDa NDV F1 protein. taken individually, and virus isolation was attempted by inoculating 9to 11-day-old SPF chicken embryos into the allantoic sac. Inoculated embryos were monitored for 7 days for mortality. Allantoic fluids were collected from dead embryos and tested for hemagglutination (HA) activity. After 7 days, allantoic fluids were harvested from the remaining live embryos and tested for HA activity. Embryos were also observed for lesions such as acute hemorrhage and congestion. Chickens were considered protected if embryos inoculated with tracheal swabs were free from mortality, HA activity, or lesions. Efficacy of HVT/ND in commercial layer chickens against velogenic neurotropic NDV challenge at 19 wk postimmunization. The HVT/ND mixed with SB-1 at titers specific to this study was administered subcutaneously to 1-day-of-age female layer chicks obtained from a commercial source. One group was left unvaccinated and served as a challenge control. Chickens were bled at 5, 10, and 15 wk of age to examine anti-NDV antibodies. At 19 wk of age, chickens were challenged intramuscularly with 104.0 EID50/dose of the NDV Texas GB strain and observed daily for 14 days for neurologic signs typical of neurotropic ND, such as tremors, loss of coordination, paralysis, and death.

RESULTS

Expression of NDV F protein by HVT/ND. Expression of NDV F protein by HVT/ND was confirmed by western blot assay

Turkey herpesvirus vector Newcastle disease vaccine

using rabbit anti-F serum. The 60-kilodalton (kDa) F0 precursor protein was observed with CEF lysates infected with HVT/ND, while the 50-kDa F1 protein was observed with NDV B1 strain harvested from embryonated eggs (Fig. 1). This was expected because F0 protein of avirulent NDV strains is cleaved by proteases in embryonated eggs, but not in cell culture (15). No band was observed in the HVT parent strain control. Efficacy of HVT/ND in SPF chickens against velogenic neurotropic NDV challenge. Efficacy of HVT/ND against genotype II velogenic neurotropic NDV was evaluated in SPF chickens. The HVT/ND mixed with SB-1 virus was administered either in ovo to SPF embryos at 18 days of incubation or subcutaneously to 1-day-of-age SPF chicks. Detectable anti-NDV antibodies were observed at 24 days of age using a commercial NDV ELISA kit as well as by the HI test (Table 1). Chickens were challenged with neurotropic velogenic NDV Texas GB strain at 28 days of age. All chickens (30/30) in the subcutaneous group and 97% (29/30) of chickens in the in ovo group did not show any clinical signs of ND, and therefore were considered protected (Table 1). One chicken in the in ovo group showed neurologic clinical signs of NDV and died. All chickens in the mock-vaccinated challenge control group showed neurologic clinical signs of NDV and died within a week. Age at which protection is induced by HVT/ND. The age at which protection is induced by HVT/ND, as prepared for this study, was evaluated in commercial broiler chickens by its ability to hinder replication of a low-virulence NDV in tracheas. Broiler chickens with maternal antibodies to NDV (geometric mean HI titer of 5.36 log 2 at 1 day of age) were vaccinated in ovo with HVT/ND mixed with SB-1 and challenged each week between 1 and 5 wk of age with a low-virulence B1 strain of NDV by the ocular route. No evident clinical signs were observed after inoculation in any chickens in the vaccinated group or the parental HVT–vaccinated, positive control group. Protection from local NDV replication in the respiratory tract was evaluated by ND virus isolation from tracheal swabs. Little protection was observed at 1 and 2 wk of age, when 0% and 10% of the chickens vaccinated with HVT/ND were protected (Table 2). Partial protection (60%) was observed at 3 wk of age. Protection increased to 90% at 4 wk of age and 100% at 5 wk of age. ND virus was isolated from all chickens in the parental HVT–vaccinated, positive control group between 1 and 5 wk of age. Efficacy of HVT/ND in commercial layer chickens against velogenic neurotropic NDV challenge at 19 wk postimmunization. To investigate whether or not HVT/ND offers protection 19 wk after immunization, female layer chickens from a commercial source were vaccinated at 1 day of age with HVT/ND mixed with SB-1 and raised until 19 wk of age. Chickens were bled at 5, 10, and 15 wk of age, and anti-NDV antibodies were measured using a commercial NDV ELISA kit. Positive anti-NDV antibodies were detected at 5 wk of age and maintained until 15 wk of age (Fig. 2). These levels of ELISA titers (1500–3000) were maintained through 50 wk of age in another study also using commercial layer chickens

Table 2. Initiation of protection by HVT/ND evaluated by respiratory challenge with a low-virulence NDV strain. % ProtectionA Group

1 wk

2 wk

3 wk

4 wk

5 wk

1: Parental HVT + SB-1 2: HVT/ND + SB-1

0 0

0 10

0 60

0 90

0 100

A Chickens were considered protected if embryos inoculated with tracheal swabs were negative for mortality, HA activity, and lesions.

(data not shown). At 19 wk of age, chickens were challenged with NDV Texas GB strain. All 30 vaccinated chickens were protected, while all chickens in the challenge control group showed neurologic clinical signs of NDV and died. These results demonstrated that HVT/ND provided protection against velogenic neurotropic NDV at 19 wk of age in commercial layer chickens following vaccination at 1 day of age. We also showed that humoral immunity against NDV was detected until 50 wk of age. DISCUSSION

Newcastle disease continues to be one of the major problems for the poultry industry despite extensive use of vaccines such as live attenuated and inactivated vaccines. There is no doubt that existing live attenuated and inactivated vaccines have contributed greatly to control ND. However, they also have a number of drawbacks, including vaccine reactions from live attenuated vaccines, susceptibility to maternally derived antibodies, and difficulties in uniformly applying vaccines. In order to overcome these issues and offer a better alternative solution to control ND, we developed a turkey herpesvirus vector Newcastle disease vaccine, in which we inserted the F gene from lentogenic NDV D26 strain (genotype I) into the noncoding region between the UL45 and UL46 genes of HVT. We chose the NDV F gene as an insert over the HN gene because, although both proteins are known to elicit neutralizing antibodies and to be protective antigens, research suggests that the F protein is the superior protective antigen of the two (10,11). This F-proteinexpressing HVT/ND vaccine has been shown to induce both

753

humoral and cell-mediated immunity in vaccinated chickens, protect chickens from lethal NDV challenge with genotype V viscerotropic velogenic strains, and reduce virus shedding after challenge (18,19). In research described here, the initiation of protection induced by the HVT/ND prepared for this study was examined by investigating the vaccine’s ability to hinder replication of low-virulence NDV in tracheas. We also investigated protection provided by HVT/ND in vaccinated chickens at 19 wk postimmunization and monitored humoral immunity against NDV through 50 wk of age. In the first part of the study, immunogenicity of this vaccine was confirmed in SPF chickens. After in ovo vaccination to 18-day-old embryos or subcutaneous vaccination to 1-day-of-age chicks, chickens developed immunity against NDV, as measured with a commercial NDV ELISA, in which the plate was coated with ND virus particles, and a HI assay using inactivated NDV LaSota as an antigen. Challenge was conducted at 28 days of age by neurotropic velogenic Texas GB strain (genotype II) of NDV, and excellent protection was observed with both the in ovo and subcutaneous groups. This vaccine contains the F gene of genotype I D26 strain, and it offered protection against neurotropic velogenic genotype II strain in this study and viscerotropic velogenic genotype V strain in a previous study (18,19). Additionally, this vaccine provides protection against various other NDV strains, including genotype VII strains (V. Palya and Y. Gardin, pers. comm.), indicating that this vaccine offers a broad range of protection. Palya et al. reported that birds vaccinated with this HVT/ND developed HI antibodies despite the fact that the HVT/ND contains only the F gene, but not the HN gene (18). We also observed increased HI titers in chickens vaccinated with the HVT/ND in this study. It may be due to steric hindrance of the hemagglutination activity of the HN protein caused by attachment of anti-F antibodies to the surface of virus envelopes. Kumar et al. also reported increased HI titers by NDV F protein in a study using avian paramyxovirus serotype 3 vector NDV F virus and suggested also that interaction of the F protein with HN protein might be required for HA activity (10). In the second part of the study, which addressed the age at which protection is induced by the HVT/ND prepared for the study, challenge was conducted using a low-virulence NDV strain via the

Table 1. Efficacy of HVT/ND in SPF chickens against velogenic neurotropic NDV. NDV ELISA Group

Vaccine route

% positive

1: HVT/ND + SB-1 2: HVT/ND + SB-1 3: Mock-vaccinated controls

In ovo SQ Mock

93 87 0

A B

Geometric mean HI Geometric mean titers titer (log 2)

1169 1484 1

4.4 6 0.97 4.1 6 0.82 1.0

NDV challengeA (positive/totalB)

% Protection

1/30a 0/30a 10/10b

97 100 0

Positive chickens showed neurologic signs typical of neurotropic ND, such as tremors, loss of coordination, paralysis, or death. Lowercase letters indicate a significant difference among groups by Fisher’s exact test (P , 0.05).

26 /

Fig. 2. Geometric mean NDV ELISA titers in chickens vaccinated with HVT/ND. Chickens vaccinated at 1 day of age were bled at 5, 10, and 15 wk of age, and anti-NDV antibodies were measured using a commercial NDV ELISA kit (Idexx Laboratories).

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N°3 • Scientific File 754

M. Esaki et al.

respiratory tract, and protection was evaluated by the presence or absence of virus isolation from tracheal swabs at 5 days postchallenge. We chose this type of evaluation in order to mimic the situation in areas where virulent NDV strains do not normally exist and low-virulence vaccines or field strains cause mild respiratory symptoms often in conjunction with other organisms, which can lead to economic losses (22). Commercial broiler chickens with maternal antibodies to NDV were vaccinated in ovo, and challenge was conducted each week between 1 and 5 wk of age. Partial protection (60%) was observed at 3 wk of age, and full protection was obtained at 4 and 5 wk of age in the HVT/ND-vaccinated group, while NDV was isolated from all birds in the challenge controls. This result is consistent with the virulent NDV challenge conducted by Palya et al., where they saw partial protection at 20 days of age and full protection at 27 and 40 days of age (18). It appears that, after in ovo vaccination or subcutaneous vaccination to 1-day-of-age chicks, protective immunity starts to develop between 2 and 3 wk of age, and is complete after 4 wk of age. We also demonstrated that HVT/ND is capable of hindering replication of lentogenic NDV (genotype II) in tracheas, and therefore, vaccination with HVT/ND should provide protection against mild respiratory diseases associated with replication of low-virulence NDV, often in conjunction with other organisms. In the third part of the study, which examined protection of HVT/ ND in vaccinated chickens after 19 wk of immunization, commercial layer chickens were vaccinated subcutaneously at 1 day of age with HVT/ND mixed with Marek’s disease virus serotype 2, SB-1 strain. HVT is often combined with the SB-1 strain and/or attenuated serotype 1 Marek’s disease virus to take advantage of the synergistic effect against Marek’s disease (21). Vaccinated chickens were protected against lethal NDV challenge at 19 wk of age. NDVspecific antibodies detected by commercial NDV ELISA were maintained through 15 wk of age in this study. These NDV-specific antibodies conferred by the HVT/ND were maintained through 50 wk of age in another study (data not shown). This long-lasting antibody response against NDV is probably due to the fact that HVT establishes a long duration of viremia at least up to 40 wk of age (4,25,27), and therefore, continuous stimulation of the immune system by foreign antigens takes place. To the best of our knowledge, this is the first report demonstrating protection conferred by HVT vector vaccines after as long as 19 wk postimmunization and humoral immunity against inserted antigens until 50 wk postimmunization. The present studies demonstrated that HVT/ND was capable of protecting vaccinated chickens against challenge with genotype II neurotropic velogenic NDV. We also demonstrated that replication of low-virulence NDV (genotype II; pathotype lentogenic) in tracheas was hindered in chickens vaccinated with HVT/ND prepared for this study. The initiation of protection appears to be between 2 and 3 wk of age, and full protection was observed by 4 wk of age. Furthermore, we showed that HVT/ND provided protection against velogenic neurotropic NDV (genotype II; pathotype velogenic) at 19 wk postimmunization in commercial layer chickens. Maternally derived antibodies to NDV did not appear to strongly interfere with development of humoral immunity against NDV provided by HVT/ND. In summary, turkey herpesvirus vector ND vaccine prepared for this study has the potential to be an excellent tool to control ND. REFERENCES 1. Alexander, D. J., and D. A. Senne. Newcastle disease. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald,

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L. K. Nolan, and D. E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 75–100. 2008. 2. Ballagi-Pordany, A., E. Wehmann, J. Herczeg, S. Bela´k, and B. Lomniczi. Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene. Arch. Virol. 141:243–261. 1996. 3. Boursnell, M. E., P. F. Green, J. I. Campbell, A. Deuter, R. W. Peters, F. M. Tomley, A. C. Samson, P. Chambers, P. T. Emmerson, and M. M. Binns. Insertion of the fusion gene from Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant. J. Gen. Virol. 71:621–628. 1990. 4. Calnek, B. W., W. R. Shek, and K. A. Schat. Latent infections with Marek’s disease virus and turkey herpesvirus. J. Natl. Cancer Inst. 66:585–590. 1981. 5. Dortmans, J. C., B. P. Peeters, and G. Koch. Newcastle disease virus outbreaks: vaccine mismatch or inadequate application Vet. Microbiol. 160:17–22. 2012. 6. Esaki, M., L. Noland, T. Eddins, A. Godoy, S. Saeki, S. Saitoh, A. Yasuda, and K. M. Dorsey. Safety and efficacy of a turkey herpesvirus vector laryngotracheitis vaccine for chickens. Avian Dis. 57:192–198. 2013. 7. Heller, E. D., and K. A. Schat. Enhancement of natural killer cell activity by Marek’s disease vaccines. Avian Pathol. 16:51–60. 1987. 8. Hu, S., H. Ma, Y. Wu, W. Liu, X. Wang, Y. Liu, and X. Liu. A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics. Vaccine 27:904–910. 2009. 9. Kitamoto, N., K. Ikuta, S. Kato, and S. Yamaguchi. Cell-mediated cytotoxicity of lymphocytes from chickens inoculated with herpesvirus of turkey against a Marek’s disease lymphoma cell line (MSB-1). Biken J. 22:11–20. 1979. 10. Kumar, S., B. Nayak, P. L. Collins, and S. K. Samal. Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens. J. Virol. 85:6521–6534. 2011. 11. Meulemans, G., M. Gonze, M. C. Carlier, P. Petit, A. Burny, and L. Long. Protective effects of HN and F glycoprotein-specific monoclonal antibodies on experimental Newcastle disease. Avian Pathol. 15:761–768. 1986. 12. Miller, P. J., C. Estevez, Q. Yu, D. L. Suarez, and D. J. King. Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses. Avian Dis. 53:39–49. 2009. 13. Miller, P. J., D. J. King, C. L. Afonso, and D. L. Suarez. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 25:7238–7246. 2007. 14. Morgan, R. W., J. Gelb Jr, C. R. Pope, and P. J. Sondermeijer. Efficacy in chickens of a herpesvirus of turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: onset of protection and effect of maternal antibodies. Avian Dis. 37:1032–1040. 1993. 15. Nagai, Y., H. D. Klenk, and R. Rott. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72:494–508. 1976. 16. Nagai, Y., T. Yoshida, M. Hamaguchi, H. Naruse, M. Iinuma, K. Maeno, and T. Matsumoto. The pathogenicity of Newcastle disease virus isolated from migrating and domestic ducks and the susceptibility of the viral glycoproteins to proteolytic cleavage. Microbiol. Immunol. 24: 173–177. 1980. 17. Office International des Epizootics [OIE]. Newcastle disease. In: Manual of diagnostic tests and vaccines for terrestrial animals 2012, Chapter 2.3.14. OIE, Paris, France. pp. 576–589. 2012. 18. Palya, V., I. Kiss, T. Tata´r-Kis, T. Mato´, B. Felfo¨ldi, and Y. Gardin. Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions. Avian Dis. 56:282–287. 2012. 19. Rauw, F., Y. Gardin, V. Palya, S. Anbari, S. Lemaire, M. Boschmans, T. van den Berg, and B. Lambrecht. Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine 28:823–833. 2010.

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20. Reddy, S. K., J. M. Sharma, J. Ahmad, D. N. Reddy, J. K. McMillen, S. M. Cook, M. A. Wild, and R. D. Schwartz. Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek’s diseases in specific-pathogen-free chickens. Vaccine 14:469–477. 1996. 21. Schat, K. A., and V. Nair. Marek’s disease. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 452–514. 2008. 22. Senne, D. A., D. J. King, and D. R. Kapczynski. Control of Newcastle disease by vaccination. Dev. Biol. (Basel) 119:165–170. 2004. 23. Sonoda, K., M. Sakaguchi, H. Okamura, K. Yokogawa, E. Tokunaga, S. Tokiyoshi, Y. Kawaguchi, and K. Hirai. Development of an effective polyvalent vaccine against both Marek’s and Newcastle diseases based on recombinant Marek’s disease virus type 1 in commercial chickens with maternal antibodies. J. Virol. 74:3217–3226. 2000. 24. Taylor, J., C. Edbauer, A. Rey-Senelonge, J. F. Bouquet, E. Norton, S. Goebel, P. Desmettre, and E. Paoletti. Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens. J. Virol. 64:1441–1450. 1990. 25. Tsukamoto, K., S. Saito, S. Saeki, T. Sato, N. Tanimura, T. Isobe, M. Mase, T. Imada, N. Yuasa, and S. Yamaguchi. Complete, long-lasting

protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. J. Virol. 76:5637–5645. 2002. 26. van Boven, M., A. Bouma, T. H. Fabri, E. Katsma, L. Hartog, and G. Koch. Herd immunity to Newcastle disease virus in poultry by vaccination. Avian Pathol. 37:1–5. 2008. 27. Witter, R. L., and L. Offenbecker. Duration of vaccinal immunity against Marek’s disease. Avian Dis. 22:396–407. 1978. 28. Yamane, N., T. Odagiri, J. Arikawa, M. Morita, N. Sukeno, and N. Ishida. Isolation of orthomyxoviruses from migrating and domestic ducks in northern Japan in 1976–1977. Jpn. J. Med. Sci. Biol. 31:407–415. 1978.

ACKNOWLEDGMENTS We thank Takanori Sato, Shuji Saitoh, Sakiko Saeki, Ayumi Fujisawa, Mayumi Kubomura, Lauren Jensen, David Hout, and Peter Flegg for their contribution to development of HVT/ND and to animal trials. We also thank Stacy Overman for her assistance with technical writing.

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Scientific File • N°4

PROCEEDINGS OF THE 63TH WESTERN POULTRY DISEASE CONFERENCE, 2014, PUERTO VALLARTA, JALISCO, MEXICO.

VECTORMUNE® ND

HIGH LEVEL OF PROTECTION ACHIEVED BY VACCINATION WITH RECOMBINANT HVT-NDV VECTOR BY VACCINE AGAINST HIGHALEVEL OF PROTECTION ACHIEVED VACCINATION DIFFERENT GENOTYPES OF NEWCASTLE DISEASE AGAINST VIRUS WITH A RECOMBINANT HVT-NDV VECTOR VACCINE PRESENT INOF LATIN AMERICA DIFFERENT GENOTYPES NEWCASTLE DISEASE VIRUS PRESENT IN LATIN AMERICA

NIVEL ALTO DE PROTECCIÓN ALCANZADO MEDIANTE LA UTILIZACIÓN DE UNA VACUNA RECOMBINANTE VECTOR HVT-NDV CONTRA NIVEL ALTOGENOTIPOS DE PROTECCIÓN ALCANZADO MEDIANTE LA DISTINTOS DE VIRUS DE LA ENFERMEDAD DEUTILIZACIÓN NEWCASTLE DE UNA VACUNA RECOMBINANTE VECTOR HVT-NDV CONTRA PRESENTES EN LATINOAMÉRICA DISTINTOS GENOTIPOS DE VIRUS DE LA ENFERMEDAD DE NEWCASTLE A LATINOAMÉRICA EN V. PalyaA, T.PRESENTES Tatár-KisA, T. Mató , B. FelföldiA, E. KovácsA, and Y. GardinB A

A A B Scientific Investigation Unit,ACeva-Phylaxia, Animal Health, 5 Szállás utca, V.Support PalyaA, and T. Tatár-Kis , T. Mató , B. FelföldiA, E.Ceva Kovács , and Y. Gardin Budapest, Hungary, H-1107 B A Ceva Animal Libourne,Ceva France Scientific Support and Investigation Unit,Health, Ceva-Phylaxia, Animal Health, 5 Szállás utca, Budapest, Hungary, H-1107 B Ceva AnimalRESUMEN Health, Libourne, France

La enfermedad de Newcastle (ND) es una de las enfermedades más importantes que causan grandes pérdidas a RESUMEN la industria avícola. En décadas pasadas, ha habido un cambio importante en los genotipos del virus de la ND (NDV) han sido de identificados como es losuna prevalentes en la avicultura. La vacunación papel importante Laque enfermedad Newcastle (ND) de las enfermedades más importantes que juega causanungrandes pérdidas a en la prevención de la enfermedad. Se probó la eficacia de una vacuna recombinante del herpesvirus de lapavos la industria avícola. En décadas pasadas, ha habido un cambio importante en los genotipos del virus de ND (HVT) que expresa el gen F de una cepa genotipo I del NDV contra cepas del NDV velogénicas que representan (NDV) que han sido identificados como los prevalentes en la avicultura. La vacunación juega un papel importantea los genotipos presentes en Latinoamérica (Por la ejemplo Genotipos V, VII recombinante y VIId). PollosdelSPF de un díadedepavos edad en la prevención de la enfermedad. Se probó eficacia de una vacuna herpesvirus fueron vacunados y desafiados a las 4 semanas de edad con una de las tres cepas velogénicas de NDV pertenecientes (HVT) que expresa el gen F de una cepa genotipo I del NDV contra cepas del NDV velogénicas que representan a alos(A) genotipopresentes V (muestra (B) genotipo VIIb Genotipos (muestra peruana) (C) genotipo VIId de (muestra genotipos en mexicana), Latinoamérica (Por ejemplo V, VII yoVIId). Pollos SPF un díaasiática). de edad La evaluación de la protecciónaselasrealizó en base a la presencia ausencia de signos clínicos la reducción de la fueron vacunados y desafiados 4 semanas de edad con una deolas tres cepas velogénicas de yNDV pertenecientes eliminación delVvirus. El usomexicana), de la vacuna losperuana) signos clínicos y redujo VIId de manera significativa a (A) genotipo (muestra (B) recombinante genotipo VIIbprevino (muestra o (C) genotipo (muestra asiática). tanto la eliminación nasal como la cloacal de todas cepas utilizadas ende el desafío. Se resultados detallados. La evaluación de la oro protección se realizó en base a la las presencia o ausencia signos clínicos y la reducción de la eliminación del virus. El uso de la vacuna recombinante previno los signos clínicos y redujo de manera significativa SUMMARY tanto la eliminación oro nasal como la cloacal de todas las cepas utilizadas en el desafío. Se resultados detallados. In the past decades, there has been a major shift in the genotypes of velogenic NDV strains responsible for SUMMARY severe epidemics in different parts of the world. Vaccination plays an important role in the prevention of the disease. Efficacy of apast recombinant turkeyhas herpesvirus (rHVT) vaccine F-gene of aNDV genotype I NDV strain was In the decades, there been a major shift in theexpressing genotypes the of velogenic strains responsible for tested against velogenic NDV strains the genotypes present inrole Middle South America (i.e.: severe epidemics in different parts of the representing world. Vaccination plays an important in theand prevention of the disease. genotype VII). SPFturkey chicksherpesvirus had been vaccinated at oneexpressing d old andthe challenged wk ofI NDV age with onewas of Efficacy ofVaand recombinant (rHVT) vaccine F-gene ofata four genotype strain three NDV strains belonging (A) genotypethe V (Mexican VIIbSouth (Peruvian isolate) or testedvelogenic against velogenic NDV strains torepresenting genotypesisolate), present(B) in genotype Middle and America (i.e.: (C) genotype VIId (Asian Level of protection evaluated on the basis of at clinical signs genotype V and VII). SPFisolate). chicks had been vaccinatedwas at one d old and challenged four wk of and age mortality with one as of well as reduction of challenge virus shedding. Vaccination with the recombinant vaccine prevented clinical signs three velogenic NDV strains belonging to (A) genotype V (Mexican isolate), (B) genotype VIIb (Peruvian isolate) or and reduced significantly oro-nasal cloacal shedding of all tested challenge (C) genotype VIId (Asianboth isolate). Leveland of protection was evaluated on the basis ofstrains. clinical signs and mortality as Velogenic strains of ND virus (NDV) cause a devastating disease of poultry in Middle and South America, well as reduction of challenge virus shedding. Vaccination with the recombinant vaccine prevented clinical signs Asia, Africa and Middle East, till today. Considerable genetic diversity has been shown among NDV strains. The and reduced significantly both oro-nasal and cloacal shedding of all tested challenge strains. genotype of velogenic NDV strains shows geographical region specific occurrence and temporal distribution with Velogenic strains of ND virus (NDV) cause a devastating disease of poultry in Middle and South America, apparent links well-defined epizootics (2,4). Genotype V,diversity VII andhas VIII areshown the predominant of Asia, Africa andtoMiddle East, till today. Considerable genetic been among NDVgenotypes strains. The velogenic NDV causingNDV the recent in poultry. region Amongspecific them genotype VII and is the most widespread, has genotype of velogenic strainsepidemics shows geographical occurrence temporal distributionitwith been associated to many outbreaks in Asia, Africa, Middle East countries (3).genotypes of apparent links to well-defined epizootics (2,4). Genotype V,and VIIsome and South VIII American are the predominant Velogenic strains from Mexico (1998-2006) characterized as members V (7), while the velogenic NDV NDV causing the recent epidemics in poultry.were Among them genotype VII is of thegenotype most widespread, it has strains detected to in many Venezuela and Peru belong to genotype VII.and Thesome Venezuelan isolates are grouping been associated outbreaks in Asia, Africa, Middle East South American countries (3).together with genotype VIId strains from from ChinaMexico (8), while the Peruvian ones are mostas closely related to a Malaysian strain Velogenic NDV strains (1998-2006) were characterized members of genotype V (7), while the BMYBU87078, described as lineage 5bbelong (genotype VIIb) by VII. Aldous al. (1). strains detected in Venezuela and Peru to genotype TheetVenezuelan isolates are grouping together with genotype VIId strains from China (8), while the Peruvian ones are most closely related to a Malaysian strain BMYBU87078, described as lineage 5b (genotype VIIb) by Aldous et al. (1). 63rd WPDC/XXXIX ANECA 30 /

63rd WPDC/XXXIX ANECA

Control of Newcastle disease, in addition to good biosecurity practices, primarily relies on preventive vaccination. Most vaccination programmes for ND include the use of live or inactivated vaccines. The continuous occurrence of ND outbreaks in commercial poultry flocks in many parts of the world indicates that routine vaccination in the field often fails to induce adequate immunity to control ND. The presence of maternally derived antibodies (MDA) also interferes with the establishment of an early and persisting immunity after single or even repeated vaccination during the first 2 to 3 weeks of life. Another option for vaccinal protection against ND is the application of recombinant HVT vaccines, expressing the F gene of NDV (rHVT-NDV vaccines). There are several advantages of this type of vaccines, such as safety (i.e. lack of post-vaccinal respiratory reactions, good hatchability after in ovo vaccination) efficacy even in the presence of MDA to NDV and life-long immunity with a single application (5,6). Efficacy of a rHVT-NDV vaccine, expressing F gene from a serotype I NDV strain was tested against three different NDV strains representing the genetic groups present in Latin America (ie. genotype V, VIIb and VIId) under controlled experimental conditions. MATERIALS AND METHODS Chickens. One day old SPF layer-type chickens were used for the experiment. The chicks were kept in isolated animal rearing house during the immunization period. Challenge infections and post-challenge observations were conducted in isolators. Vaccine and challenge viruses. The cryo-preserved cell-associated rHVT-NDV vaccine (Vectormune® ND, Ceva Animal Health) expressing the F protein of the avirulent D26/76 genotype I NDV strain was used in the study. The vaccine was diluted to dose in the corresponding diluent. The challenge virus strains were isolated from field ND cases and propagated in embryonated SPF hens’ eggs. All of them proved to be free from extraneous agents. The isolate representing genotype V originated from Mexico, 2008 (identification: D516/1 MX, GenBank accession number JQ002630, (5)). As the representative of genotype VIIb, a Peruvian NDV strain from 2005 (D575/6 PE) was selected. This challenge strain shows 98% nucleotide homology on the partial F gene sequence with the Poultry/Peru/1918-03/2008 strain (3). Genotype VIId strain used in the study, that represents the genotype reported from Venezuela, was a Chinese isolate from 2010 (D1500/2/1). The Venezuelan NDV isolate described by Perozo et al. (8) shows high homology with the genotype VIId isolate (BLAST Search) used in our experiment. Study design. One d old chicks were assigned to two groups: one was vaccinated with one dose of the rHVTNDV vaccine s.c., the other group remained untreated. At 28 d of age, 30 vaccinated and 30 control chickens were submitted to challenge infection (3 subgroups were set from both groups according to the challenge virus used; number of birds/subgroup was 10). Infection was performed with a dose of 5.0 log 10 ELD 50 /chicken via intra-nasal route. During the post-challenge observation period clinical signs and mortality were monitored daily. Oro-nasal (choanal slit) and cloacal swabs were collected at 2, 4, and 7 d post-challenge. Measurement of NDV RNA load in the swab samples. RNA was extracted from the swab samples by QIAxtractor Virus kit (Qiagen) according to the manufacturer’s instructions. Then quantitative real-time one-step RT-PCR, amplifying a fragment of M gene (TaqMan® NDV reagents and controls, Life TechnologiesTM) was performed. Internal positive control supplied by the manufacturer was included. Titer equivalent unit was calculated by extrapolation from sample Ct to the Ct of standard (total RNA extracted from tenfold dilution series of the relevant challenge virus strain with known ELD 50 titer). RESULTS All non-vaccinated control chickens died due to the challenge infection between 4 to 6 d post-infection, while all vaccinated chickens proved to be protected both against clinical signs and mortality attributable to challenge. Challenge virus RNA load measurement results are summarized in Table 1. NDV RNA load in the oro-nasal swabs of the non-vaccinated control birds was higher compared to the cloacal swabs at 2 days post-challenge (dpch), but the level of shedding via the two routes became comparable and very high at 4 dpch, when mortality started. No data available for the control group since all control birds died by 7 dpch. There was a very strong, significant effect of vaccination on the shedding of challenge virus. Only limited shedding was detected via the oro-nasal route of vaccinated chickens (3.5-5.5 log 10 reduction compared to the controls was achieved at 4 dpch). Cloacal shedding was more efficiently suppressed resulting in no detectable NDV RNA in the cloacal swabs in the great majority of samples (only a few samples contained very low level of NDV RNA at a single sampling date in the subgroup challenged with the genotype VIId strain). Challenge virus RNA load of the cloacal samples in the vaccinated subgroups was by 5.5-6.6 log 10 lower compared to the corresponding control subgroup. DISCUSSION

63rd WPDC/XXXIX ANECA

Efficacy of a recombinant HVT-NDV vaccine, expressing the F-gene of a NDV strain belonging to genotype I

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N°4 • Scientific File

cloacal samples in the vaccinated subgroups was by 5.5-6.6 log 10 lower compared to the corresponding control subgroup.

Table 1. ND challenge virus RNA load in the oro-nasal and cloacal swabs. titer equivalent (lg ELD 50 /0.1 ml; mean and STD or difference between the two groups) oro-nasal swab cloacal swab

DISCUSSION Efficacy of a recombinant HVT-NDV vaccine, expressing the F-gene of a NDV strain belonging to genotype I was tested against three different NDV challenge strains. The challenge strains represent all those genetic groups of velogenic NDV which are prevalent in Middle and South America. Although the insert in the vaccine was heterologous to the challenge strain (the donor strain belongs to a very distinct genotype), a single vaccination with one dose at day-old provided not only full protection against clinical signs and mortality, but strongly reduced (by the oro-nasal route) or even totally prevented (by the cloacal route) the shedding of challenge virus. The strong immunity which developed in the vaccinated birds was able to prevent the dissemination of challenge virus in the body (no cloacal shedding), and very strongly suppressed its local replication in the oro-nasal mucosa. Our results indicate that very high level of protection can be achieved with the tested rHVT-NDV vaccine against genetically divergent velogenic NDV strains circulating in Middle and South America. REFERENCES 1. Aldous, E. W., J. K. Mynn, J. Banks and D. J. Alexander. A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol. 32: 239-257. 2003. 2. Czeglédi, A., D. Ujvári, E. Somogyi, E. Wehmann, O. Werner, B. Lomniczi. Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications. Virus Res. 120: 36–48. 2006. 3. Diel, D. G., L. Susta, S. C. Garcia, M. L. Killian, C. C. Brown, P. J. Miller and C. L. Afonso. Complete genome and clinicopathological characterization of a virulent Newcastle disease virus isolate from South America. J. Clin. Microbiol. 50:378-387. 2012. 4. Miller, P. J., E. L. Decanini and C. L. Afonso. Newcastle disease: Evolution of genotypes and the related diagnostic challenges. Infect. Genet. Evol. 10:26–35. 2010. 5. Palya, V., I. Kiss, T. Tatár-Kis, T. Mató, B. Felföldi and Y. Gardin. Advancement in vaccination against Newcastle disease: Recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions. Avian Dis. 56:282–287. 2012. 6. Palya, V., T. Tatár-Kis, T. Mató, B. Felföldi, E. Kovács and Y. Gardin. Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers. Vet. Immunol. Immunpathol. In press, available online. 7. Perozo, F., R. Merino, C. L. Afonso, P. Villegas and N. Calderon. Biological and phylogenetic characterization of virulent Newcastle disease virus circulating in Mexico. Avian Dis. 52:472–479. 2008. 8. Perozo, F., R. Marcano and C. Afonso. Biological and phylogenetic characterization of a genotype VII Newcastle disease virus from Venezuela: Efficacy of field vaccination. J. Clin. Microbiol. 50: 1204-1208. 2012.

challenge virus

group

2 dpch b

vaccinated Genotype V (strain D516/1)

control reduction vaccination

Genotype VII PE (strain D575/6)

control reduction vaccination vaccinated

Genotype VIId (strain D1500)

control

7 dpch

2 dpch b

4 dpch b

7 dpch

0.8 ±0.9 3.9a ±0.6

1.3 ±1.3 6.8 a ±0.4

0.9 ±1.0 NA -

0.0 ±0.1 1.2 a ±1.2

0.0 ±0.0 6.4 a ±0.5

0.0 ±0.0 NA -

3.1

5.5

1.2

6.4

vaccinated

4 dpch

units

2.9 ±0.5 4.8 a ±0.3

3.5 ±0.5 7.0 a ±0.3

1.2 ±1.3 NA -

0.0 ±0.0 1.1 a ±1.5

0.0 ±0.0 6.6 a ±0.4

0.0 ±0.0 NA -

1.9

3.5

1.1

6.6

2.2 ±1.3 4.8 a ±0.5

1.8 ±1.4 5.8 a ±0.5

0.6 ±1.1 NA -

0.0 ±0.0 2.3 a ±0.6

0.2 ±0.5 5.7 a ±0.7

0.0 ±0.0 NA -

reduction by 2.6 4.0 NA 2.3 5.5 NA vaccination Different superscript letters indicate statistically significant difference between the vaccinated and the corresponding control group (Kruskal-Wallis test, p<0.05) Results from the same type of samples collected at the same date were compared NA: not applicable

PROCEEDINGS OF THE SIXTY-THIRD WESTERN POULTRY DISEASE CONFERENCE April 1 to 5, 2014 Puerto Vallarta, Jalisco, Mexico MEMORIAS DE LA XXXIX CONVENCIÓN ANUAL ASOCIACIÓN NATIONAL DE ESPECIALISTAS EN CIENCIAS AVÍCOLAS 1 al 5 de abril de 2014 Puerto Vallarta, Jalisco, México

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PROCEEDINGS OF THE 57TH WPDC/XXXIII ANECA CONFERENCE, 2012 JALISCO, MEXICO (PP. 36-38).

VECTORMUNE® ND

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Scientific File • N°6

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VECTORMUNE® ND

EFFICACY OF A RECOMBINANT NEWCASTLE DISEASE VACCINE (VECTORMUNE HVT NDV) BASED ON CLINICAL PROTECTION AND SHEDDING OF CHALLENGE VIRUS IN BROILER CHICKENS 1*

V. Palya , I. Kiss , T. Tatár-Kis , T. Mató , B. Felföldi , Y. Gardin 1 Ceva-Phylaxia Co. Ltd., Szállás u. 5., 1107 Budapest, Hungary 2 CEVA, Libourne, France *Corresponding author: Dr. Vilmos Palya E-mail: vilmos.palya@ceva.com Address: Ceva-Phylaxia Co. Ltd., Szállás u. 5., 1107 Budapest, Hungary Phone: +36 1 262 9505 Fax: +36 1 260 3889

Resumen. Debido a las deficiencias de su eficacia, se necesitan mejorar las actuales estrategias de vacunación contra la enfermedad de Newcastle (ND). Este estudio tuvo como objetivo evaluar una generación más nueva de vacunas ND para la protección clínica y el nivel de excreción del virus de desafío. Los pollos de engorda fueron vacunados in ovo o por vía subcutánea en el nacimiento con una vacuna recombinante basada en herpesvirus de pavo que expresa antígenos protectores clave del virus de la enfermedad de Newcastle (NDV). Los grupos de aves fueron desafiados en el día

Introduction Newcastle disease virus (NDV) causes disease in more than 250 species of birds and typically manifests in respiratory, gastrointestinal, and/or nervous system symptoms. The most severe form of Newcastle Disease (ND) can result in disease and mortality rates exceeding 90 percent in susceptible chickens (2). NDV is a type strain for avian paramyxoviruses and belong to the Mononegavirales order, Paramyxoviridae family, Paramyxovirinae subfamily, and Avulavirus genus (10). Two classification systems exist for NDV strains, with no consensus as to which is more appropriate (11). A system suggested by Aldous (1) classifies NDV into lineages and sublineages, while another scheme divides

20 (D20), D27, y D40 de edad con una cepa velogénica viscerotrópica NDV. La protección fue de 57 y 81%, 100 y 95%, y 100 y 100% después de los desafíos posteriores en las aves vacunadas in ovo y por vía subcutánea, respectivamente. La excreción de virus de desafío fue significativamente menor y se redujo gradualmente en las aves vacunadas en comparación con el grupo de control, considerando las rutas orofaríngeas y cloacales, y fue indetectable al D7 postdesafío 3, en el frotis cloacal. Key words. Newcastle disease, recombinant HVT-NDV vaccine

the strains into two classes then further into several genotypes within each classes (3; 5). The genetic variety of NDV probably reflects the diversity of its natural reservoir hosts species, the availability of a huge number of susceptible poultry populations, and the effect of live bird markets that promote virus transmission among multiple bird species. Current vaccines for ND are used widely in commercial poultry and protect the vaccinated birds from disease, but do not stop the virus from being spread from infected to healthy birds. Another limitation of the conventional ND vaccines is that they might induce a better protection against viruses isolated in past epizootics than against more recent circulating viscerotropic viruses (9, 17). Finally, the presence of maternally derived antibody

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XVII WVPA Congress. Cancun, Mexico

EFFICACY OF HVT NDV) B

Resumen.

Debido a las def necesitan mejora de vacunación Newcastle (ND). objetivo evaluar de vacunas ND p el nivel de excre Los pollos de en ovo o por vía su con una vacuna herpesvirus de pa protectores cla enfermedad de grupos de aves f

Introduction

Newcastle disea disease in more and typically gastrointestinal, symptoms. The Newcastle Disea disease and mo percent in suscep

NDV is a paramyxoviruses Mononegavirales / 37 Paramyx family, Avulavirus genu

N°6 • Scientific File

(MDA) interferes with the establishment of a persisting good protective immunity after a single day-old vaccination (4, 6, 18).

Newcastle

Considering the above mentioned, a vaccine that reduces both the shedding and transmission of the viruses among birds is very much needed by the poultry industry.

Measurement

In the presented study chickens were vaccinated in ovo or subcutaneously at the first day of age with a recombinant herpes virus of turkey (HVT) expressing the fusion protein of NDV. The construct, Vectormune HVT NDV, had already been reported to confer protection for layer chickens (12). Our study aimed to further investigate the vaccine in broilers concerning protective efficacy and capability of reducing challenge virus shedding.

supplemented

Chickens.18 days old embryonated eggs and day-old Ross 308 broiler chickens carrying maternally derived antibodies (MDA) to NDV were used in the study. Vaccine and challenge strain. The cryopreserved cell-associated Vectormune HVT NDV (Ceva-Biomune, USA) expressing the F protein of an avirulent NDV strain (14) was diluted in corresponding vaccine diluent (Ceva-Biomune, USA) to get 1 dose (3000 pfu) in 100µl and was inoculated in ovo on the 18th days of embryonation (ED), or subcutaneously at one day of age (200 µl/chicken) with manual injection. For challenge the Mexican velogenic viscerotropic Chimalhuacan NDV strain was used by oculo-nasal inoculation of 105 EID50 challenge virus. Measurement of humoral immune responses response

NDV.

The was

according

Antibody

Test

Kit)

the

manufacturer’s

virus

shedding

instructions. of

via

oropharyngeal and cloacal routes after challenge. The oropharyngeal and cloacal cotton swabs were immersed in sterile PBS with

antibiotics.

RNA

extraction and PCR amplification were carried out according to published method (7).The virus titre of each sample was

Table 1. Schematic presentation of the experimental design. D-3 VTM HVT NDV in ovo vaccination

VTM HVT NDV sc vaccination Controls (no vaccination)

D20

D27

D40

20 birds challenged with velogenic NDV

(CH1)

(CH2)

(CH3)

determined relative to a standard curve

Results

consisting

The mean of HI antibody titres at day-old of 10 unvaccinated broilers were 7.13 log2. Then the profile of the HI antibody titres indicated a decline of passive maternally derived immunity until the 4th week of age.

total viral

RNA

the

Chimalhuacan NDV strain. The sensitivity threshold

NDV

QRRT-PCR

Chimalhuacan NDV strain was determined at 10 EID50 virus per reaction. The results

At that time, a progressive vaccine-induced active immune response was already detectable in some of the vaccinated birds. By the sixth weeks of age the vaccinated groups showed significant increase of HI antibody titres.

were expressed as log EID50titre of the

Materials and methods

against

Disease

humoral measured

immune by

haemagglutination inhibition (HI) test and NDV-specific IgG ELISA (Biochek CK116

challenge virus per ml of swabs. Experimental design. Two groups of chickens were vaccinated either in ovo (100 µl/egg) with manual injection or subcutaneously (sc; 200 µl/chicken) at hatch with Vectormune HVT NDV (Table 1.). Twenty-twenty broilers from each group were challenged with velogenic NDV at 20, 5.0 27 and 40 days of age. 10 EID50 challenge virus/bird was administered by the oculonasal route. Pre-challenge sampling comprised serum samples from all birds submitted to challenge. Post-challenge samplings throughout the 14 days observation period included taking oropharyngeal and cloacal swabs at D3 and D7 after challenge, gross-pathology and histopathology from dead and sick birds at the end of the experiment. As far as protection parameters concerned chickens which died or showed clinical signs indicative of ND were considered unprotected while chickens which survived til the end of the 14-days post-challenge period without showing clinical signs indicative of ND were considered protected.

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Figure 1. Decay of MDA and detection of humoral immune response to Vectormune HVT NDV vaccination in commercial broilers by HI test.

Figure 2. Clinical protection against challenge with velogenic NDV.

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Figure 4. Quantitative detection of the challenge virus by RT-PCR from the oropharyngeal and cloacal swabs after successive challenges.

Figure 2. Clinical protection against challenge with velogenic NDV.

XVII WVPA Congress. Cancun, Mexico

XVII WVPA Congress. Cancun, Mexico The level of protection against NDV challenge carried out at 3 weeks of age was already reasonably good (57-81%), which improved further by the time of the second

and third challenge, when the protection reached 95-100% in both vaccinated groups.

and third challenge, when the protection The level of protection against NDV reached 95-100% in both vaccinated challenge carried out of at NDV 3 weeks of ageafter was consecutive Figure 3. Percentage shedders challenges. already reasonably good (57-81%), which groups. improved further by the time of the second Figure 3. Percentage of NDV shedders after consecutive challenges.

Figure 4. Quantitative detection of the challenge virus by RT-PCR from the oropharyngeal and cloacal swabs after successive challenges.

759 Reduction of challenge virus shedding was observed cloacalvirus swabs alreadywas at Reduction in of the challenge shedding seven days after the first challenge observed in the cloacal swabs already at regarding both after the percentage shedders seven days the first of challenge and the amount of excreted virus (Figures 3. regarding both the percentage of shedders and 4.). Afterwards, both the number of and the amount of excreted virus (Figures 3. shedders and the amount excreted NDV and 4.). Afterwards, both ofthe number of Discussion shedders and the amount of excreted NDV Discussion The continuous threat of ND outbreaks in commercial poultry flocks necessitate early The continuous threat of ND outbreaks in vaccination, which flocks is hampered by early the commercial poultry necessitate interference usually level of by passive vaccination, of which is high hampered the immunity with the vaccines. Current interference of usually high level of passive vaccination strategies ND Current can be immunity with the against vaccines. effective in preventing serious illness vaccination strategies against ND can and be death of infected birds, but prevent neither effective in preventing serious illness and infection nor shedding theprevent virus. The aim death of infected birds,ofbut neither of this study was to determine if an infection nor shedding of the virus. The aim innovative vaccine could increase protection of this study was to determine if an and reducevaccine viral shedding, and presumably, innovative could increase protection the spreading and the transmission of the and reduce viral shedding, and presumably, virus. A recombinant HVT-ND vaccine the spreading and the transmissionth of was the used applied either in ovo vaccine (18 daywas of virus. and A recombinant HVT-ND th embryonation) or at day in oldovo to (18 commercial used and applied either day of broilers carrying reasonably high level of embryonation) or at day old to commercial MDA to carrying NDV, bearing in mind thatlevel HVT of is broilers reasonably high hardly sensitive to maternal immunity, thus, MDA to NDV, bearing in mind that HVT is the recombinant HVT-ND is capable of hardly sensitive to maternal immunity, thus, priming an early immune response at a very the recombinant HVT-ND is capable of early age (13). Furthermore, since HVT priming an early immune response at a very replicates a highly cell-associated manner early agein (13). Furthermore, since HVT in lymphocytes, it is suggested that this replicates in a highly cell-associated manner delivery system itwould induce that a great in lymphocytes, is suggested this degree of cell-mediated immune response delivery system would induce a great (8). degree of cell-mediated immune response (8).

760

gradually decreased by the second and third challenges and at the third gradually decreased by D7 theafter second and challenge, only one and two birds fromthird the third challenges and at D7 after the vaccinated groups shedtwovirus challenge, only one and birdsfrom from the the oropharynx and no shedder by the cloacal vaccinated groups shed virus from the route was identified. oropharynx and no shedder by the cloacal route was identified. It has also been described that HVT establishes viremia inthat chickens It has alsoa persistent been described HVT for at least 8 weeks following vaccination establishes a persistent viremia in chickens (15), advantage delivering for atoffering least 8 the weeks followingofvaccination foreign antigens to the immune system of (15), offering the advantage of delivering vaccinated birds during extended period foreign antigens to the an immune system of of time (16) and is therefore expected to vaccinated birds during an extended period induce longer lasting immunity. of timea (16) and is therefore expected to induce a longer lasting immunity. Our results demonstrated that early vaccination HVT-ND provided very Our resultswith demonstrated that early good protection by the 4th weeks of age, vaccination with HVT-ND provided very regardless the administration route, and good protection by the 4th weeks of age, also, it efficiently reduced shedding of the regardless the administration route, and challenge virus,reduced thusshedding preventing also, it efficiently of the transmission of the infection. Interestingly, challenge virus, thus preventing the HI profileof of groups Interestingly, indicated a transmission thethe infection. vaccinal immune response while the the HI profile of the groups indicated a recombinant construct contains only thetheF vaccinal immune response while gene of NDV, i.e. contains not possessing recombinant construct only the F haemagglutinating acitivity. A plausible gene of NDV, i.e. not possessing explanation for this phenomenon could be haemagglutinating acitivity. A plausible a sphaeric interference of anti-F antibodies explanation for this phenomenon could be with the haemagglutinating activity of the a sphaeric interference of anti-F antibodies HN glycoprotein (Esaki, M. personal with the haemagglutinating activity of the communication). HN glycoproteinAltogether, (Esaki, the M. presented personal study revealed definite advantages of communication). Altogether, the presented Vectormune HVT NDV vaccine to traditional study revealed definite advantages of ND vaccines,HVT supporting and extending Vectormune NDV vaccine to traditional ND vaccines, supporting and extending

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previous findings obtained in layer chickens (12). In summary, the main benefits of the recombinant HVT-ND vaccine are: (i) no vaccine reaction as with most of the live ND vaccines; (ii) no local reaction/systemic distress as observed with inactivated vaccines and (iii) no interference with ND maternal antibodies. The recombinant vaccine also induces an extended duration of immunity, which reduces the need to administer additional live or inactivated ND vaccines.

Conclusions The presented experiment proved the efficacy of the Vectormune HVT NDV recombinant vaccine demonstrated by clinical protection parameters, and its efficiency in reducing challenge virus shedding. By applying this vaccine at the hatchery, controlled vaccine uptake and early and efficient immunity can be provided to the vaccinated flocks.

Acknowledgements Thanks are due to Gabriella Somfai and Magdolna Lénárt for the excellent technical assistance throughout the experiment.

References 1.

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Aldous, E. W., Mynn, J. K., Banks, J., and Alexander, D. J. (2003): A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene, Avian Pathol. 32, pp. 239–256. Alexander, D.J. (2004):Highly pathogenic avian influenza/Newcastle disease. In: OIE manual of diagnostic tests and vaccines for terrestrial animals, 5th ed., pp. 258–282. OIE, Paris, France. Ballagi-Pordány, A., Wehmann, E., Herczeg, J.,Belák, S., and Lomniczi, B.

(1996): Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene, Arch. Virol.141, pp. 243– 261. 4. Bell, G., Nicholls, P. J., Norman, C., Cooper, K. and Cross, G. M. (1991): The serological responses of chickens to mass vaccination with a live V4 Newcastle disease virus vaccine in the field and the laboratory. 1. Meat chickens, Aust Vet J, 68 (3), pp. 85–89. 5. Czeglédi, A., Ujvári, D., Somogyi, E., Wehmann, E., Werner, O., and Lomniczi, B. (2006):Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications, Virus Res. 120, pp. 36–48. 6. Czifra, G., Mészáros, J., Horváth, E., Moving, V., and Engström, B. E. (1998): Detection of NDV-specific antibodies and the level of protection provided by a single vaccination in young chickens, Avian Pathol 27 (6), pp. 562–565. 7. Farkas, T., Székely, E., Belák, S., Kiss, I. (2009): Real-time PCR-based pathotyping of Newcastle disease virus by use of TaqMan minor groove binder probes. J Clin Microbiol. 47(7):2114-23. 8. Heller, E. D. and Schat, K. A. (1987): Enhancement of natural killer cell activity by Marek's disease vaccines, Avian Pathol 16 (1), pp. 51–60. 9. Kapczynski, D. R., and King, D. J. (2005): Protection of chickens against overt clinical disease and determination of viral shedding following vaccination with commercially available Newcastle disease virus vaccines upon challenge with highly virulent virus from the California 2002 exotic Newcastle disease outbreak, Vaccine 23, pp. 3424– 3433. 10. Mayo, M. A. (2002): A summary of taxonomic changes recently approved by ICTV, Arch. Virol. 147, pp. 1655– 1656. 11. Miller, P. J., Decanini, E. L., and Afonso, C. L. (2010): Newcastle disease: Evolution of genotypes and the related diagnostic challenges. Infection, Genetics and Evolution. Volume 10, Issue 1, January 2010, Pages 26-35. 12. Rauw, F., Gardin, Y., Palya, V., Anbari, S., Lemaire, S., Boschmans, M., van den Berg, T., and Lambrecht, B. (2010): Improved vaccination against

XVII WVPA Congress. Cancun, Mexico

Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine, 28, (3): 823- 833. 13. Reddy, S. K., Sharma, J. M., Ahmad, J., Reddy, D. N., McMillen, J. K., and Cook, S. M. (1996):Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek's diseases in specificpathogen-free chickens, Vaccine 14 (6), pp. 469–477. 14. Sato, H., Oh-hira, M., Ishida, N., Imamura, Y., Hattori, S., and Kawakita, M. (1987): Molecular cloning and nucleotide sequence of P, M and F genes of Newcastle disease virus avirulent strain D26, Virus Res7 (3), pp. 241–255. 15. Sharma, J. M., Lee, L. F., and Wakenell, P. S. (1982): Comparative viral, immunologic, and pathologic responses of chickens inoculated with herpesvirus

of turkeys as embryos or at hatch, Am J Vet Res 45 (8), pp. 1619–1623. 16. Tsukamoto, K., Saito, S., Saeki, S., Sato, T., Tanimura, N., and Isobe, T. (2002): Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens, J Virol 76 (11), pp. 5637–5645. 17. Van Boven, M., Bouma, A., Fabri, T. H. F., Katsma, E., Hartog, L., and Koch, G. (2008): Herd immunity to Newcastle disease virus in poultry by vaccination, Avian Pathol 37 (1), pp. 1–5. 18. Westbury, H.A., Parsons, G., and Allan, W.H. (1984): Comparison of the immunogenicity of Newcastle disease virus strains V4, Hitchner B1 and LaSota in chickens. 2. Tests in chickens with maternal antibody to the virus, Aust Vet J,61, pp. 10–13.

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EXTRACT FROM « AVIAN DISEASES » (PP. 282-287).

VECTORMUNE® ND AVIAN DISEASES 56:282–287, 2012 Table 1.

AVIAN DISEASES 56:282–287, 2012

Advancement in Vaccination Against Newcastle Disease: Recombinant HVT NDV Provides High in Clinical Protection andNewcastle Reduces Challenge Virus Shedding with the Advancement Vaccination Against Disease: Recombinant HVT NDV Absenceand of Vaccine Provides High Clinical Protection ReducesReactions Challenge Virus Shedding with the Absence of Vaccine Reactions V. Palya,AC I. Kiss,A T. Tata´r-Kis,A T. Mato´,A B. Felfo¨ldi,A and Y. GardinB A A A ´r-Kis,Co. ´ ,A B. V.ACeva-Phylaxia Palya,AC I. Kiss, T. Tata T.Ltd., Mato Felfo¨ldi, Y.Hungary GardinB Veterinary Biologicals 1107 Budapest, Sza´lla´and s u. 5. B

Ceva Sante´ Animale, 10 Avenue de la Ballastie`re, Libourne, France A Ceva-Phylaxia Veterinary Biologicals Co. Ltd., 1107 Budapest, Sza´lla´s u. 5. Hungary B Received 16 September 4 December Published ahead of France print 5 December 2011 `re, Libourne, Ceva 2011; Sante´ Accepted Animale, 10 Avenue de2011; la Ballastie Received 16 September 2011; Accepted 4 December 2011; Published ahead of print 5 December 2011 SUMMARY. Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a SUMMARY. ND Newcastle is ainhighly contagious of chickens causing significant economic losses worldwide. new-generation vaccinedisease for its(ND) efficacy providing clinicaldisease protection and reducing virus shedding after challenge. Broiler Due to the limitation in their efficacy, current vaccination against ND need improvements. Thisvaccine study aimed to expressing evaluate a chickens were vaccinated in ovo or subcutaneously at hatchstrategies with a turkey herpesvirus-based recombinant (rHVT) ND vaccine for its efficacy in providing clinical and reducing shedding after challenge. Broiler anew-generation key protective antigen (F glycoprotein) of Newcastle disease virusprotection (NDV). Groups of birds virus were challenged at 20, 27, and 40 days chickens were vaccinated in ovo or subcutaneously at hatch with a turkey was herpesvirus-based vaccine expressing of age with a genotype V viscerotropic velogenic NDV strain. Protection 57% and 81%,recombinant 100% and 95%, and(rHVT) 100% and 100% aafter key the protective antigen (F glycoprotein) disease virusvaccinated (NDV). Groups of birds were challenged 20, 27, and 40 days subsequent challenges in the in of ovoNewcastle and subcutaneously chickens, respectively. Humoralatimmune response to of age with acould genotype V viscerotropic velogenic NDV strain. Protection was 57% 81%, and 95%, and 100% and 100% vaccination be detected from 3–4 wk of age. Challenge virus shedding wasand lower and100% gradually decreased over time in the after the subsequent challenges in unvaccinated the in ovo andcontrol subcutaneously respectively. Humoral immune response to vaccinated birds compared to the chickens. vaccinated In spite of chickens, the phylogenetic distance between the NDV F gene vaccination be detected wk of age.virus Challenge virusI and shedding was lower and decreased time ingood the inserted intocould the vector vaccinefrom and 3–4 the challenge (genotype V, respectively), the gradually rHVT NDV vaccineover provided vaccinated birds compared to the unvaccinated controlvirus chickens. In spite of the phylogenetic distance between the NDV F gene clinical protection and significantly reduced challenge shedding. inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good ´ n contra RESUMEN. Avances en la vacunacio la enfermedad de Newcastle: Una vacuna recombinante contra la enfermedad de clinical protection and significantly reduced challenge virus shedding. Newcastle con el vector HVT ofrece una alta proteccioń clıńica y reduce la eliminacioń del virus de desafıó con ausencia de RESUMEN. Avances en la vacunacioń contra la enfermedad de Newcastle: Una vacuna recombinante contra la enfermedad de reacciones a la vacuna. ńica y reduce ó con ausencia Newcastle con el vector HVT ofrece alta enfermedad proteccioń clı la eliminacio del virus de desafı de La enfermedad de Newcastle (ND)una es una altamente contagiosa de los´ npollos que causa importantes pe´rdidas reacciones la vacuna. ´ micasa en econo todo el mundo. Debido a las limitaciones en su eficacia, las actuales estrategias de vacunacioń contra dicha La enfermedad de mejorarse. Newcastle Este (ND) es una enfermedad altamente pollos que causa importantes pe´rdidas ´ n contra enfermedad necesitan estudio tuvo como objetivo evaluarcontagiosa una vacunadedelosnueva generacio la enfermedad de ´ micasenensu todo ´ n contra econo el para mundo. Debido a las´ nlimitaciones en sula eficacia, las´ nactuales de desafı vacunacio dicha ó. Pollos Newcastle eficacia conferir proteccio clıńica y reducir diseminacio del virusestrategias despue´s del de engorde enfermedad necesitan mejorarse. por Estelas estudio objetivo evaluar vacuna de nueva generacio n contra la enfermedad de ńea con fueron vacunados al nacimiento vıás intuvo ovo como o subcuta una una vacuna recombinante con un ´virus herpes de pavo como ó. Pollos de ´ n clıńica ´ nvirus ´s del desafı Newcastle en suque eficacia para conferir proteccio reducir la diseminacio del virus engorde ńa F) del vector (rHVT) expresaba un antı´geno protector clavey (la glicoproteı de ladespue enfermedad de Newcastle. Grupos de ńea fueron vacunados al nacimiento pory 40 las dı vı´ás subcuta unańica vacuna recombinante con unVvirus herpes pavo como ´ pica genotipo aves fueron desafiadas a los 20, 27 as in de ovo edado con una cepacon veloge viscerotro del virus de de Newcastle. La ´genoy protector ńa F) vector (rHVT) que57% expresaba clave (la glicoproteı virus deós la enfermedad delos Newcastle. Grupos de ´ n fue del ´s del proteccio y 81%,undeantı 100% 95%, y del 100% y 100% despue de los desafı posteriores en pollos vacunados in ´ pica aves a losrespectivamente. 20, 27 y 40 dıásLa derespuesta edad coninmune una cepahumoral velogeńica genotipo V del virus de Newcastle. ´ n pudo ovo ofueron por vı´desafiadas a subcutańea, a laviscerotro vacunacio ser detectada a partir de terceraLay ós posteriores ´ n fue del ´s de los´ desafı proteccio 57% yLa81%, de 100% y 95%, deldesafı 100% y 100% en losdelpollos vacunados in ó fue ´ n del cuarta semanas de edad. diseminacio virusyde menordespue y disminuyo gradualmente a lo largo tiempo en las aves á subcuta ńea,´ nrespectivamente. ´ nfilogene ovo o por vıen La respuesta inmune humoral pudo ser partir de tercera ´ticadetectada vacunadas comparacio con los pollos control no vacunados. A pesar adelalavacunacio distancia entre el agene F del virus dey óófue ´ n del ´ gradualmente cuarta semanas de edad. diseminacio menor yI disminuyo lo largo del tiempo las aves Newcastle insertado en laLa vacuna de vector y elvirus virusdededesafı desafı (genotipo y V, respectivamente), la avacuna rHVT contra en el virus de ´ n con vacunadas en comparacio los proteccio pollos control no yvacunados. A ´pesar de la distancia filogene´tica el gene F del ó.virus de ´ una ´ n clıńica ´ n entre Newcastle proporciono buena una reduccio n significativa en la eliminacio del virus de desafı Newcastle insertado en la vacuna de vector y el virus de desafıó (genotipo I y V, respectivamente), la vacuna rHVT contra el virus de Key words: Newcastle disease, vaccination, recombinant HVT NDV vaccine, protection, virus shedding Newcastle proporciono´ una buena proteccioń clıńica y una reduccioń significativa en la eliminacioń del virus de desafıó. Abbreviations: Ct 5 threshold cycle; EID50 5 50% egg infective dose; F 5 fusion protein; HN 5 hemagglutinin-neuraminidase Key words: Newcastle disease, vaccination, recombinant HVT NDV vaccine, protection, virus shedding protein; HVT 5 herpesvirus of turkeys; M 5 matrix protein; MDA 5 maternally derived antibodies; N 5 nucleoprotein; 5 50%virus; egg ORF infective dose;reading F 5 fusion protein; HN 5 hemagglutinin-neuraminidase Abbreviations: Ct 5 threshold EID50disease ND 5 Newcastle disease; NDV 5cycle; Newcastle 5 open frame; P 5 phosphoprotein; QRRT-PCR 5 quanprotein;real-time HVT 5reverse herpesvirus of turkeys; rHVT M 5 matrix protein;of MDA maternally derived antibodies; N 5 nucleoprotein; titative transcription-PCR; 5 herpesvirus turkeys5recombinant vaccine; SPF 5 specific-pathogen-free ND 5 Newcastle disease; NDV 5 Newcastle disease virus; ORF 5 open reading frame; P 5 phosphoprotein; QRRT-PCR 5 quantitative real-time reverse transcription-PCR; rHVT 5 herpesvirus of turkeys recombinant vaccine; SPF 5 specific-pathogen-free

Newcastle disease virus (NDV) causes disease in more than 250 species of birds and typically manifests in respiratory and Newcastle disease virus (NDV) disease in more The thanmost 250 gastrointestinal or nervous system causes (or both) symptoms. species of birds and typically manifests in respiratory and severe form of Newcastle disease (ND) can result in mortality rates gastrointestinal or nervous system (or both) symptoms. The most exceeding 90% in susceptible chicken flocks (2). severe form Newcastle can result in and mortality rates NDV is aoftype species disease of avian(ND) paramyxoviruses belongs to exceeding 90% in susceptible chicken flocks (2). the Mononegavirales order, Paramyxoviridae family, Paramyxovirinae NDV is and a type species genus of avian andsystems belongs to subfamily, Avulavirus (1).paramyxoviruses Two classification exist the Mononegavirales order, Paramyxoviridae family, Paramyxovirinae for NDV strains with no consensus as to which is more appropriate subfamily, and suggested Avulavirusby genus (1).(1) Two classification exist (13). A system Aldous classifies NDV systems into lineages for NDV strains with no consensus as to which is more appropriate and sublineages, while another scheme divides the strains into two (13). systemare suggested by Aldous (1) classifies NDVgenotypes into lineages classesAwhich then further separated into several (3). and sublineages, while another scheme divides the strains intooftwo The genetic variety of NDV probably reflects the diversity its classes which are then further separated into several genotypes (3). The genetic variety of NDV probably reflects the diversity of its C

Corresponding author. E-mail: vilmos.palya@ceva.com

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natural reservoir hosts species, the availability of a huge number of susceptible poultry populations, and the effect of live bird markets natural reservoir hosts species, theamong availability of abird hugespecies. numberThe of that promote virus transmission multiple susceptible poultry populations, and the effect of live bird markets enveloped NDV virion contains a single stranded, negative-sense that transmission among bird species.in The RNApromote genome virus of about 15 kilobases andmultiple replicates entirely the enveloped NDV virion contains a single stranded, negative-sense cytoplasm (11). The genome codes for six proteins: an RNARNA genome of about 15 kilobases and replicates entirely in the dependent RNA polymerase (L), hemagglutinin-neuraminidase cytoplasm (11).fusion The (F) genome for(M) six protein, proteins: an RNA(HN) protein, protein,codes matrix phosphoprodependent RNA polymerase (L), hemagglutinin-neuraminidase tein (P), and a nucleoprotein (N). The P gene is unique in that (HN) protein, fusion matrix (M) in protein, phosphoprotranscriptional editing(F) of protein, its mRNA results two nonstructural tein (P), and a nucleoprotein (N). The P gene is unique in thata proteins, V and a potential W (8,20,24). The V protein plays transcriptional editing of its and mRNA results two nonstructural direct role in virus replication in host rangeinrestriction as well as proteins, and a potential W (8,20,24). protein F0 plays serving as Va virulence factor (8). Cleavage ofThe the Vprecursor intoa direct role in virus replication and in host range restriction as well as the F1 and F2 products is necessary for viral spread to other cells. serving as a virulence factor (8). Cleavage of the precursor F0 into The F and HN surface glycoproteins are the principal antigens that the and F2 products necessary elicitF1protective immune is response (2).for viral spread to other cells. The F and HN surface glycoproteins are the principal antigens that elicit protective immune response (2).

Schematic presentation of the experimental design.A Day 23

Groups

rHVT NDV in ovo rHVT NDV subcutaneous Controls

Day 0

Vaccination Vaccination No vaccination

Day 20

20 birds challenged with velogenic NDV (CH1)

Day 27

Day 40

20 birds challenged with velogenic NDV (CH2)

20 birds challenged with velogenic NDV (CH3)

Day 0 is the day of hatch; CH 5 challenge.

Current ND vaccines widely used in commercial poultry can protect the vaccinated birds from disease and reduce virus shedding but cannot prevent vaccinated birds from being infected, subsequently shedding the virus, and potentially transmitting it to susceptible birds. A further consideration regarding conventional ND vaccines is that they might induce a better protection against viruses isolated in past epizootics than against the ones causing the recent outbreaks (9,26). Finally, the presence of maternally derived antibodies (MDA) interferes with the establishment of an early and persisting immunity after a single vaccination in 1-day-old chicks (4,5,27). Considering the abovementioned items, a newer NDV vaccine should not only protect birds against the disease but, preferably, also reduce the amount of virus shed by vaccinated birds to a level that will prevent transmission of virus from bird to bird in vaccinated flocks. In addition, a vaccine that lacks adverse reactions is also very much needed by the poultry industry. A promising approach to achieve the above goals was the development of vector vaccines using the herpesvirus of turkeys (HVT) as a vector, which contains and expresses protective antigens, typically the F and HN glycoprotein (or both) of NDV (15). As in the case of HVT itself (16), long-term virus persistence was also shown for rHVT in inoculated chickens (18) and, furthermore, the expression of the F gene was measurable even after 30 wk of a single subcutaneous inoculation of 1-day-old chickens (19). Additionally, the immune response evoked by the rHVT-F construct appeared to be less sensitive to interference with MDA, which adds a further useful characteristic to this vector vaccine (14). Beyond that, the application of this kind of vaccine proved to be safe because it did not have adverse effects on hatchability or the survival of in ovo and posthatch, vaccinated, specific-pathogen-free (SPF) chickens (15,18). In the presented study, an rHVT NDV vaccine expressing the F protein of NDV was investigated in broiler chickens vaccinated in ovo or subcutaneously at hatch for clinical protection and challenge virus shedding. For the latter purpose, a quantitative real-time reverse transcription-PCR (QRRT-PCR) assay was used in order to provide sensitivity and accuracy to measurements and to save the labor and time necessary to perform the investigations. MATERIALS AND METHODS Chickens. Eighteen-day-old embryonated eggs and 1-day-old Ross 308 broiler chickens carrying MDA to NDV (at an HI titer of 7.13 log 2) were used in the study. After hatching, all birds were kept in isolators and animal experiments were conducted following national and European regulations. Vaccines and challenge strain. The cryo-preserved cell-associated rHVT NDV vaccine (VectormuneH HVT NDV, Ceva-Biomune, Lenexa, Kansas) expressing the F protein of the avirulent D26/76 genotype I NDV strain (21) was used in the study (GenBank accession number M24692). The HVT backbone of this vaccine harbors the F gene of NDV under the control of a modified chicken beta-actin promoter and is inserted into a noncoding inter-open reading frame (ORF) region located between UL45 and UL46 of the herpesvirus

genome (19). The vaccine was diluted in 100 ml of corresponding vaccine diluent (Ceva-Biomune) to obtain 1 commercial dose for the in ovo inoculation on the 18th embryonation day or in 200 ml for the subcutaneous inoculation at the day of hatching (200 ml/chicken), by manual injection, as previously described (12). For challenge, a recent Mexican isolate of viscerotropic velogenic NDV strain (APMV1/chicken/Mexico/D516/1/2008; GenBank accession number JQ002630) was used which belongs to the Class II genotype V of NDV. Oculo-nasal inoculation of 105 egg infective dose (EID50) of this strain induced 100% mortality within 3–6 days in SPF layer-type and MDA-free commercial broiler chickens challenged at 3– 6 wk of age. Measurement of humoral immune responses to NDV. The humoral immune response was measured by the hemagglutinationinhibition (HI) test using the LaSota strain as antigen at four hemagglutinating units and by an NDV-specific IgG ELISA (CK116 Newcastle Disease Antibody Test Kit, BioCheck, Reeuwijk, Holland) according to the manufacturer’s instructions. Measurement of virus shedding after challenge. Virus shedding after challenge via oropharyngeal and cloacal routes was measured by QRRT-PCR. The oropharyngeal and cloacal cotton swabs were immersed in 400 ml of sterile phosphate-buffered saline. The swabs were stored at 280 C until further analysis. RNA extraction and the ensuing PCR amplifications were carried out in duplicates according to a published method (6), except for the PCR runs which were accomplished in a Rotor-Gene Q instrument (Qiagen, Hilden, Germany). Samples with a threshold cycle (Ct) .35 were considered negative. The virus titer of each sample was determined relative to a standard curve consisting of the tenfold dilution series of the total viral RNA of the challenge NDV strain having a titer of 9 log 10 EID50/0.2 ml, and this standard curve was included in each run. There was a firm inverse linear relationship between the QRRT-PCR Ct values and the virus titers, and a clear distinction between different virus titers could be made on the basis of their Ct values. This standard curve allowed the extrapolation of unknown virus titers based on their Ct values. The sensitivity threshold of NDV QRRT-PCR for the NDV challenge strain was determined as 10 EID50 per reaction. The results were expressed as log 10 EID50 titer of challenge strain per milliliter of swabs, i.e., the EID50 per reaction value provided by the software of the PCR instrument was multiplied by the dilution factors that occurred during sample processing (nucleic acid extraction and further operations in the RT-PCR). Experimental design. Two groups of chickens were vaccinated either in ovo (100 ml/egg) or subcutaneously (200 ml/chicken) at hatch with the rHVT NDV vaccine (Table 1). A third group of chickens from the same source as the vaccinated ones served as unvaccinated controls. Twenty broilers from each group were challenged with the velogenic genotype V NDV strain APMV1/chicken/Mexico/D516/1/2008 at 20, 27, and 40 days of age. The challenge virus was administered by the oculo-nasal route at 105.0 EID50/bird. Prechallenge sampling comprised serum samples from all birds submitted to challenge. Postchallenge samplings throughout the 14-day observation period included taking oropharyngeal and cloacal swabs at day 3 and day 7 after challenge (or on the day of death) from ten, randomly selected birds (without formal randomization). Gross pathology and histopathology from dead and sick birds was used to confirm the diagnosis of ND. Protection was evaluated based on mortality and appearance of clinical signs indicative of Newcastle disease. Statistical analysis. Viral shedding was analyzed by an ANOVA. Statistical significance was defined as P , 0.05.

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Fig. 2. Clinical protection against challenge with velogenic NDV.

day 7 postchallenge excreted a significantly higher amount of virus than at day 3 (Fig. 4). This may indicate that residual MDA to NDV, which might be still present at the time these challenges were performed, somehow delayed the replication of the challenge virus in these birds but could not prevent the development of clinical signs and later death. The route of vaccine application (in ovo and subcutaneous) did not influence the results of virus shedding, as no significant difference was measured between them in this respect. Peak titers (7.7–8.2 log 10 EID50/ml) in the vaccinated groups were detected at day 7 after the first challenge performed at 20 days of age while, in the control group, these values were always above or around 8.0 log 10 EID50/ml following the challenges carried out at different ages (Fig. 4). Noticeably, while there were typically one or two birds out of the ten sampled ones in the vaccinated groups falling in the peak titer range, the majority of the control birds (7–8 out of the ten sampled) shed virus over 8.0 log 10 EID50/ml at each sampling. In the vaccinated groups following the challenge at 4 wk of age, 40%–50% of the birds shed no detectable amount of virus and their ratio increased by the time of subsequent challenge at 6 wk

of age, while in the controls there were little differences between the maximum and minimum shed titer values following the challenges performed at different ages.

DISCUSSION

The continuous threat of ND outbreaks in commercial poultry flocks necessitates early vaccination, which poses safety issues and interference of MDA with conventional ND vaccines administered at a young age. Current vaccination strategies against ND prevent serious illness and mortality of infected birds, and decrease virus excretion, but preventing infection by significantly reduced or ceased challenge virus shedding still remains a critical parameter to control the spread of the disease. In a recent publication, an rHVT NDV vaccine expressing the F protein of NDV was shown to have no statistically significant effect on the reduction of virus shedding in conventional layer chickens, albeit it did provide clinical protection (17). There have been reports about the evaluation of an HVT-based ND vaccine that contained the F gene, integrated into the

Fig. 1. Decay of MDA and detection of humoral immune response to rHVT NDV vaccination in commercial broilers by (A) HI test and by (B) ELISA test. The positivity threshold (indicated by dashed lines on the graphs) was 2 log 2 and 3.06 log 10 titer for the HI and ELISA test, respectively. The error bars are the associated standard deviations. RESULTS

The hatchability after in ovo vaccination was 92.5%. The mean HI antibody titers to NDV of 10 unvaccinated broilers was 7.13 log 2 at 1 day old. In addition, the profile of the HI antibody titers indicated a decline of MDA until the third to fourth week of age in all groups. At 4 wk of age, a vaccine-induced active humoral immune response was already detectable by the HI test in some of the vaccinated birds. During the following weeks, the HI titers increased progressively, reaching a moderately high level by 6 wk of age. The antibody titers measured by ELISA showed correlation with the HI titers; however, the measured ELISA values remained below the positivity threshold given for the kit by the manufacturer except for those of the day 0 results (Fig. 1). The level of protection against ND following challenge carried out at 3 wk of age was already significant (57–81%) in the vaccinated chickens compared to the unvaccinated controls that had already proven to be fully susceptible (0% protection). Protection improved further by the time of the second (27 days of age) and third challenges (40 days of age) when it reached 95%–100% in both the in ovo and the subcutaneously vaccinated groups (Fig. 2).

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Figs. 3 and 4 demonstrate the tendency of virus shedding after the successive challenges. Both the percentage of shedders and the amount of virus shed decreased steadily by the age of the birds at challenge in the vaccinated groups, while the control birds shed the virus at practically the same high level via both the oropharyngeal and the cloacal routes after each challenge. The difference between the amount of virus shed by the vaccinated and control group was significant except for the oropharyngeal route following challenge 1 and 2 (this latter case occurred only in the subcutaneously vaccinated group) at day 3 postchallenge sampling. The differences between the vaccinated and the control groups were even more pronounced for the cloacal swabs, resulting in reduced number of shedders and significantly lower means of challenge virus shedding by the vaccinated chickens than by the nonvaccinated birds. Regarding the level of shedding, no real comparison between the vaccinated and control birds could be done at day 7 sampling, as most of the nonvaccinated chickens had died by this sampling date. Before death (typically on day 5–6 postchallenge), the control chickens shed an extremely high amount of virus (.8 log 10 EID50/ml). Noticeably, the control birds that survived in the first and second challenges until

Fig. 3. Percentage of NDV shedders after consecutive challenges (CH1, CH2, and CH3). D3 and D7 means day 3 and day 7 postchallenge. Following challenge 3, no bird was alive in the control group on day 7 postchallenge.

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Fig. 4. Quantitative detection of the challenge virus by RT-PCR from the oropharyngeal and cloacal swabs after successive challenges. The error bars represent minimum and maximum values measured in each group. Following challenge 3, no bird was alive in the control group on day 7 postchallenge.

interrupted gene homologue to the US10 protein of herpes simplex virus under the control of Rous sarcoma virus long terminal repeat promoter (14,15,23). The aim of the present study was to evaluate the onset of immunity and the level of protection against clinical disease and virus shedding afforded by a different rHVT-based ND vaccine (Vectormune HVT NDV, Ceva-Biomune) expressing the F gene in the noncoding inter-ORF region between UL45 and UL46 of the HVT genome, under the control of a chicken beta-actin promoter, when administered in the face of MDA to NDV. The vaccine was applied either in ovo (at 18th day of embryonation) or at 1 day old to commercial broilers carrying considerably high levels of MDA to NDV, bearing in mind that HVT is hardly sensitive to maternal antibodies (18). Furthermore, because HVT replicates in a highly cell-associated manner in lymphocytes, it is suggested that this delivery system would induce a great degree of cell-mediated immune response (7). It has also been described that HVT establishes a persistent viremia in chickens for at least 8 or even 30 wk following vaccination (19,22), offering the advantage of delivering foreign antigens to the immune system of vaccinated birds during an extended period of time (25) and is, therefore, expected to induce a long-lasting immunity. Selection of the F gene of NDV as an insert into the vector provides the construct good immunogenicity and protective characteristics, beyond that of the HN protein, as was demonstrated by Kumar et al. (10). Previous studies suggested that the delivery of the NDV F gene in an HVT vector does not necessarily stimulate local immunity in the respiratory tract, and it takes a longer time for protective immunity to develop than what is needed for conventional live or inactivated vaccines (14,15). However, the onset of protection and immune response induced by rHVT NDV vaccines substantially depends on the composition of the construct and the timing and route of application, among several other possible factors; therefore, comparison of two different recombinant constructs, even if they are based on the same vector, should be done in parallel animal trials. Our results demonstrated that an rHVT NDV vaccination in ovo or at hatch provided complete, or almost complete, clinical protection by the fourth week of age, regardless of the administration route. Additionally, rHVT NDV vaccination efficiently reduced the shedding of the challenge virus, thus significantly limiting the transmission of the infection. The highly efficient reduction of cloacal virus shedding could be attributed to a strong cell-mediated

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systemic immunity induced by the rHVT NDV vaccine, which had less effect on the oropharyngeal shedding where local immune mechanisms—not readily triggered by an HVT-based recombinant vaccine—play an important role (15,18). Interestingly, the vaccinated birds developed HI antibodies in spite of the fact that the recombinant construct contained only the F gene of NDV, i.e., was not expressing the HA protein of ND virus. A plausible explanation for this phenomenon could be a steric hindrance of anti-F antibodies with the hemagglutinating activity of the HN glycoprotein (M. Esaki, pers. comm.). Nevertheless, this finding was different from the one presented in a previous study (15), where there was practically no antibody response to vaccination detectable by HI testing. Because the differences between the two recombinant HVT-ND vaccines concerned primarily the insertion site and the promoter, these differences might have influenced their capability to induce antibodies detectable by HI testing. The presented study supported and extended previous findings regarding the safety of the rHVT NDV vaccine when administered in ovo or posthatch in commercial broiler chickens. The vaccine induced solid immunity in a reasonably short time after single administration in face of MDA to NDV and significantly reduced challenge virus shedding via both the oropharyngeal and cloacal routes as compared to the unvaccinated control animals. By applying this vaccine at the hatchery, controlled vaccine uptake and efficient homogenous immunity can be provided to the vaccinated broiler flocks. The safe administration of a uniform dose of this vector vaccine by an in ovo or subcutaneous route to a large number of birds, its resistance to interference with MDA, and the lack of horizontal spread of vaccine virus in the population makes it an appealing tool for the control of ND.

restriction site analysis of a region from the F gene. Arch. Virol. 141:243–261. 1996. 4. Bell, I. G., P. J. Nicholls, C. Norman, K. Cooper, and G. M. Cross. The serological responses of chickens to mass vaccination with a live V4 Newcastle disease virus vaccine in the field and in the laboratory. 1. Meat chickens. Aust. Vet. J. 68:85–89. 1991. 5. Czifra, G., J. Meszaros, E. Horvath, V. Moving, and B. E. Engstrom. Detection of NDV-specific antibodies and the level of protection provided by a single vaccination in young chickens. Avian Pathol. 27:562–565. 1998. 6. Farkas, T., E. Szekely, S. Belak, and I. Kiss. Real-time PCR-based pathotyping of Newcastle disease virus by use of TaqMan minor groove binder probes. J. Clin. Microbiol. 47:2114–2123. 2009. 7. Heller, E. D., and K. A. Schat. Enhancement of natural killer cell activity by Marek’s disease vaccines. Avian Pathol. 16:51–60. 1987. 8. Huang, Z., S. Krishnamurthy, A. Panda, and S. K. Samal. Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist. J. Virol. 77:8676–8685. 2003. 9. Kapczynski, D. R., and D. J. King. Protection of chickens against overt clinical disease and determination of viral shedding following vaccination with commercially available Newcastle disease virus vaccines upon challenge with highly virulent virus from the California 2002 exotic Newcastle disease outbreak. Vaccine 23:3424–3433. 2005. 10. Kumar, S., B. Nayak, P. L. Collins, and S. K. Samal. Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens. J. Virol. 85:6521–6534. 2011. 11. Lamb, R. A., and K. Daniel. Paramyxoviridae. In: Fields virology, 4th ed. D. M. Knipe and P. M. Howley, eds. Lippincott-Raven, Philadelphia, PA. pp. 1305–1334. 2001. 12. Mast, J., C. Nanbru, M. Decaesstecker, B. Lambrecht, B. Couvreur, G. Meulemans, and T. van den Berg. Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response. Vaccine 24:1756–1765. 2006. 13. Mayo, M. A. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147:1655–1663. 2002. 14. Morgan, R. W., J. Gelb Jr., C. R. Pope, and P. J. Sondermeijer. Efficacy in chickens of a herpesvirus of turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: onset of protection and effect of maternal antibodies. Avian Dis. 37:1032–1040. 1993. 15. Morgan, R. W., J. Gelb Jr., C. S. Schreurs, D. Lutticken, J. K. Rosenberger, and P. J. Sondermeijer. Protection of chickens from Newcastle and Marek’s diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein. Avian Dis. 36:858–870. 1992. 16. Purchase, H. G., W. Okazaki, and B. R. Burmester. Field trials with the herpes virus of turkeys (HVT) strain FC126 as a vaccine against Marek’s disease. Poult. Sci. 50:775–783. 1971.

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17. Rauw, F., Y. Gardin, V. Palya, S. Anbari, S. Lemaire, M. Boschmans, T. van den Berg, and B. Lambrecht. Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine 28:823–833. 2010. 18. Reddy, S. K., J. M. Sharma, J. Ahmad, D. N. Reddy, J. K. McMillen, S. M. Cook, M. A. Wild, and R. D. Schwartz. Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek’s diseases in specific-pathogen-free chickens. Vaccine 14:469–477. 1996. 19. Saitoh, S., Okuda, T., Kubomura, M., Dorsey, K. M., inventors; Zeon Corporation, Tokyo, Japan, assignee. 2002 January 31. Recombinant herpesvirus of turkeys and use thereof. U. S. Patent 6866852. 20. Samson, A. C., I. Levesley, and P. H. Russell. The 36K polypeptide synthesized in Newcastle disease virus-infected cells possesses properties predicted for the hypothesized ‘V’ protein. J. Gen. Virol. 72(Pt. 7):1709–1713. 1991. 21. Sato, H., M. Oh-hira, N. Ishida, Y. Imamura, S. Hattori, and M. Kawakita. Molecular cloning and nucleotide sequence of P, M and F genes of Newcastle disease virus avirulent strain D26. Virus Res. 7:241–255. 1987. 22. Sharma, J. M. Embryo vaccination of chickens with turkey herpesvirus: characteristics of the target cell of early viral replication in embryonic lung. Avian Pathol. 16:567–579. 1987. 23. Sondermeijer, P. J., J. A. Claessens, P. E. Jenniskens, A. P. Mockett, R. A. Thijssen, M. J. Willemse, and R. W. Morgan. Avian herpesvirus as a live viral vector for the expression of heterologous antigens. Vaccine 11:349–358. 1993. 24. Steward, M., A. C. Samson, W. Errington, and P. T. Emmerson. The Newcastle disease virus V protein binds zinc. Arch. Virol. 140:1321–1328. 1995. 25. Tsukamoto, K., S. Saito, S. Saeki, T. Sato, N. Tanimura, T. Isobe, M. Mase, T. Imada, N. Yuasa, and S. Yamaguchi. Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. J. Virol. 76:5637–5645. 2002. 26. van Boven, M., A. Bouma, T. H. Fabri, E. Katsma, L. Hartog, and G. Koch. Herd immunity to Newcastle disease virus in poultry by vaccination. Avian Pathol. 37:1–5. 2008. 27. Westbury, H. A., G. Parsons, and W. H. Allan. Comparison of the immunogenicity of Newcastle disease virus strains V4, Hitchner B1 and La Sota in chickens. 2. Tests in chickens with maternal antibody to the virus. Aust. Vet. J. 61:10–13. 1984.

ACKNOWLEDGMENTS We thank Gabriella Somfai and Magdolna Leńa´rt for the excellent technical assistance throughout the experiment. Dr. Zoltań Peńzes is acknowledged for his critical reading of the manuscript.

REFERENCES 1. Aldous, E. W., J. K. Mynn, J. Banks, and D. J. Alexander. A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol 32:239–256. 2003. 2. Alexander, D. J. Highly pathogenic avian influenza/Newcastle disease. In: OIE manual of diagnostic tests and vaccines for terrestrial animals. World Organisation for Animal Health, Paris, France. pp. 258–282. 2004. 3. Ballagi-Pordany, A., E. Wehmann, J. Herczeg, S. Belak, and B. Lomniczi. Identification and grouping of Newcastle disease virus strains by

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Avian Avian Pathology, Pathology, 2014 2014 Avianhttp://dx.doi.org/10.1080/03079457.2013.859655 Pathology, 2014 Vol. 43, Avian Pathology, 2014 Vol. 43, No. No. 1, 1, 26–36, 26–36, http://dx.doi.org/10.1080/03079457.2013.859655 Avian Pathology, 2014 Vol. 43, No. 1,Vol. 26–36, 43, http://dx.doi.org/10.1080/03079457.2013.859655 No. 1, 26–36, http://dx.doi.org/10.1080/03079457.2013.859655 Vol. 43, No. 1,VECTORMUNE® 26–36, http://dx.doi.org/10.1080/03079457.2013.859655 ND

EXTRACT FROM “AVIAN PATHOLOGY”, 2014, VOL. 43, NO. 1 (PP. 26–36).

Efficacy of rHVT-ND/live ND vaccination

The duration of immunity induced by the concurrent application of the aforementioned ND vaccines, adjuvanted or not with chitosan, was evaluated in the present study and compared with a classical vaccination programme, which included the combination of live and inactivated ND vaccine administration at 1-day-old. We present the results of challenge with a recent viscerotropic vNDV strain and investigations of the immune responses induced by these three vaccination schedules.

ORIGINAL ARTICLE ARTICLE ORIGINAL ORIGINAL ARTICLEARTICLE ORIGINAL ORIGINAL ARTICLE

The combination of attenuated Newcastle disease (ND) vaccine The combination of attenuated Newcastle disease (ND) vaccine The combination of attenuated Newcastle disease (ND) vaccine The combination of attenuated Newcastle disease (ND) vaccine with rHVT-ND vaccine at 1 day old is more protective against le disease (ND) vaccine with rHVT-ND vaccine atold 1 day old is more againstagainst with rHVT-ND vaccine at 1with day old isprotective more protective with rHVT-ND vaccine at 1 day is more protective against ND virus challenge than when combined inactivated ND more protective against virus thancombined when with inactivated ND ND NDchallenge virus than combined when with ND virusND challenge than challenge when withcombined inactivated NDinactivated vaccine ed with inactivated ND vaccinevaccine vaccine Materials and Methods 1* 1* F. Rauw , Y. Gardin22, V. Palya33, T. van den Berg11, and B. Lambrecht11

ambrecht

F. Rauw , Y. Gardin1*, V. Palya1*, 2T. van den23Berg , and3 B. Lambrecht 1 1 1 F. Rauw 2 ,F.Y.Rauw Gardin , V.Gardin Palya ,, V. T. van den andBerg B. Palya , T.Berg van ,den , and B. Lambrecht1 1* 3 , Y. 1 1 Lambrecht F. Rauw , Y. Gardin , V. Palya , T. van den Berg , and B. Lambrecht 1 2

Downloaded by [CEVA Phylaxia Biologicals Co Ltd], [Norbert Nemes] at 02:32 17 February 2014

Downloaded Downloaded Downloaded by [CEVA by by[CEVA [CEVA Phylaxia Phylaxia Phylaxia Biologicals Biologicals Biologicals Co Ltd], Co CoLtd], Ltd], [Norbert [Norbert [Norbert Nemes] Nemes] Nemes] at 02:32 atat02:32 02:32 17 February 17 17February February 20142014 2014

1Avian Virology and Immunology Unit, Veterinary and Agrochemical Research Centre (VAR), Brussels, Belgium, 2CEVA Santé Avian Virology and Immunology 3Unit, Veterinary and Agrochemical Research Centre (VAR), Brussels, Belgium, CEVA Santé 2 1 1 and 3CEVA Avian Virology Immunology Veterinary andVeterinary Agrochemical Research Centre (VAR),Centre Brussels, Belgium, CEVA Santé 2CEVA Animale, Libourne, France, and Phylaxia, Budapest, Hungary Avian Virology and3Unit, Immunology Unit, and Agrochemical Research (VAR), Brussels, Belgium, Animale, Libourne, France, and CEVA Phylaxia, Budapest, Hungary 1 Avian Virology 2and Immunology Unit, Veterinary and Agrochemical ResearchHungary Centre (VAR), Brussels, Belgium, 2CEVA Santé 3 Animale, Libourne, France, and CEVA Phylaxia, Budapest, Centre (VAR), Brussels, Belgium, CEVA Santé Animale, Libourne, France, and CEVA Phylaxia, Budapest, Hungary Animale, Libourne, France, and 3CEVA Phylaxia, Budapest, Hungary

Chickens. Commercial Isa Brown layer chickens (sex sale linked) were hatched from eggs supplied by Het Anker B.V. (Ochten, Belgium). After hatching, all birds were kept in biosecurity level 3 isolators and animal experiments were conducted under the authorization and supervision of the Santé Biosafety and Bioethics Committees of the Veterinary and Agrochemical Research Institute, following national and European regulations.

The The recurrent recurrent outbreaks outbreaks of of fatal fatal Newcastle Newcastle disease disease (ND) (ND) in in commercial commercial poultry poultry flocks flocks throughout throughout the the world world Vaccines, adjuvant and challenge strain. The live ND vaccine (Cevac The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks throughout world the world indicate that routine vaccinations are failing to sufficiently induce the high levels of immunity necessary to control The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks the throughout indicate that routine vaccinations are failing to sufficiently induce the high levels of immunity necessary to control Vitapest L) was provided by Ceva Santé Animale (Ceva-Phylaxia campus, The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks throughout the world thatvaccination routine vaccinations are that failing toare sufficiently the high levels immunity necessary tonecessary control to control cial poultry flocks throughout the world ND. There is aaindicate need for could be initiated at 1-day-old for mass application and indicate thatprogrammes routine vaccinations failing to induce sufficiently induce theof high levels of immunity Budapest, Hungary). This vaccine is based on the apathogenic enterotropic ND. There is need for vaccination programmes that could be initiated at 1-day-old for mass application and indicate that routine vaccinations are for failing to sufficiently induce the levels of immunity necessaryfor to control ND. There is a need programmes thathigh could be initiated at 1-day-old mass application gh levels of immunity necessary to control PHY.LMV.42 strain (Meszaros, 1991; Meszaros et al., 1992; Rauw et al., which would induce aa long-lasting immunity, with need for aa booster vaccination at aa later age. In this context, ND. There is vaccination a need forno vaccination programmes that could be initiated at 1-day-old for mass and application and which would induce long-lasting immunity, with no need for booster vaccination at later age. In this context, Thereapplication is awhich need would for vaccination programmesimmunity, that couldwith be no initiated at a1-day-old for mass application andIn this context, induce a long-lasting need for booster vaccination at a later age. 2009b) (intracerebral pathogenicity index (ICPI) range between 0.00 and ed at 1-day-oldND. for mass and the duration of immunity delivered by a vaccination programme including a recombinant herpesvirus of turkeys which would induce a long-lasting immunity, with no need for a booster vaccination at a later age. In this context, the duration of immunity deliveredimmunity, by a vaccination programme including a recombinant herpesvirus of context, turkeys would induce a long-lasting with no need for a booster vaccination at a later age. In this 0.16; intravenous pathogenicity index (IVPI) = 0.00; mean death time > the duration of virus immunity bylive a vaccination programme including a recombinant herpesvirus herpesvirus of turkeys of turkeys vaccination at awhich later age. In this context, expressing the F gene of ND (rHVT-ND) and ND vaccine at 1-day-old was compared with classical the duration ofdelivered immunity delivered by a vaccination programme including aaa recombinant expressing the F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-old was compared with classical 168), belonging to genotype I of NDV (Czegledi et al., 2006). The vaccine the duration of immunity delivered by a vaccination programme including a recombinant herpesvirus of turkeys expressing the F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-old was compared with a classical ding a recombinant herpesvirus of turkeysaaexpressing programme that included conventional and an inactivated ND vaccine at the same age in commercial thelive F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-oldlayer was compared with a classical programme that included conventional live and an inactivated ND vaccine at the same age in commercial layer was reconstituted in phosphate-buffered saline (PBS) to one dose in 50 µl, the programme Fa gene of ND virus (rHVT-ND) and livelive NDand vaccine at 1-day-old was compared with classical that included a local conventional an inactivated vaccine at the sameaage in same commercial layer 1-day-old was expressing comparedThe with classical chickens. humoral, cell-mediated and immunity were followed weekly and birds were challenged with aa age in commercial programme that included a conventional live and an ND inactivated ND vaccine at the layer which corresponds to approximately 106 egg infective dose that kills 50% of chickens. The humoral, cell-mediated and local immunity were followed weekly and birds were challenged with programme thatchickens. included a conventional live and an and inactivated ND vaccine atfollowed the sameweekly age inand commercial layer The humoral, cell-mediated local were birds were challenged a ccine at the same age in commercial layer eggs viscerotropic velogenic ND virus strain at 66 and 10 weeks of age. determined that immunity by chickens. The andWe local immunity were followedinduced weekly andthe birds werewith challenged with a (EID50)/dose, and was inoculated by the oculo-nasal route at 1-day-old. viscerotropic velogenic ND virus strain athumoral, andimmunity 10cell-mediated weeks of immunity age. We determined that immunity induced by the chickens. The humoral, cell-mediated and local were followed weekly and birds were challenged with a viscerotropic velogenic ND virus strain at 6 and 10 weeks of age. We determined that immunity induced by the The inactivated ND vaccine (Cevac Broiler ND K) was supplied by Ceva d weekly and birds were challenged with a vaccination programme involving the rHVT-ND vaccine was more protective than that provided by the viscerotropic velogenic ND virus strain at 6 and 10 weeks of age. We determined that immunity induced by the vaccination programme involving the atrHVT-ND vaccine protectivethat than that provided by the velogenic ND virus strain 6 and 10the weeks ofwas age. more We determined immunity induced by the vaccination programme involving rHVT-ND more protective than that provided the determined thatviscerotropic immunity induced by the conventional vaccine-based regime. This might be related to aa T-helper type 11was (Th1) cellular-driven vaccination programme involving the vaccine rHVT-ND vaccine was moreimmunological protective than thatbyprovided by Santé the Animale. This vaccine is a La Sota strain-based water-in-oil emulsion conventional vaccine-based regime. This might be related to T-helper type (Th1) cellular-driven immunological vaccine, and was inoculated subcutaneously in the neck at one dose per 100 vaccination programme involving the rHVT-ND vaccine was more protective than that provided by the conventional vaccine-based regime. This might be related to a T-helper type 1 (Th1) cellular-driven immunological e protective than that provided by to response, in T-helper type 22 (Th2) humoral-oriented immune response provided by current vaccine-based regime. This might be related to a T-helper cellular-driven immunological response, in contrast contrast tothethe theconventional T-helper type (Th2) humoral-oriented immune response providedtype by 1the the(Th1) current µl at 1-day-old. conventional vaccine-based regime. This might be related to2 a(Th2) T-helper type 1 (Th1) cellular-driven immunological response, in contrast to the T-helper type humoral-oriented immune response provided byprovided the current ype 1 (Th1) cellular-driven immunological conventional vaccine-based vaccination programmes. response, in contrast to the T-helper type 2 (Th2) humoral-oriented immune response by the current The cryopreserved cell-associated rHVT expressing the F protein of conventional vaccine-based vaccination programmes. to the vaccine-based T-helper type vaccination 2 (Th2) humoral-oriented immune response provided by the current conventional programmes. mmune responseresponse, providedinbycontrast the current the avirulent D26 NDV strain (Sato et al., 1987) (rHVT-ND, Vectomune® conventional vaccine-based vaccination programmes. conventional vaccine-based vaccination programmes. ND) was produced by Ceva-Biomune (Lenexa, KS, USA). One dose of recombinant vaccine was diluted in 100 µl of the corresponding

vaccine diluent (Ceva-Biomune) and inoculated subcutaneously in the neck A A variety variety of of approaches approaches have have been been investigated investigated in in at 1-day-old. Introduction Introduction A variety of approaches have been investigated in commercial chicks possessing maternally antibody A variety derived of approaches have been investigated in commercial chicks possessing have maternally derived antibody The chitosan (chitosan hydrochloride) adjuvant was provided by Ceva Santé Newcastle disease virus (NDV), also known as avian Introduction A variety of approaches been investigated in Newcastle disease virus (NDV), also known as avian commercial chicks possessing maternally derived antibody (MDA) against NDV NDV to to develop develop suitable vaccination of approaches have been investigated indisease virus (NDV), also known (MDA) commercial chicksvaccination possessing maternally derived antibody against aa suitable Newcastle as avian Animale. Chitosan is a chloride salt of an unbranched binary heteropolyparamyxovirus type-1, is an economically important pathoNewcastle disease virus (NDV), also known as avian commercial chicks possessing maternally derived antibody type-1, is an(NDV), economically important (MDA) NDVagainst toprotection develop vaccination programme that results inagainst long-lasting protection after hicks possessing paramyxovirus maternally disease derived antibody (MDA) NDV aafter tosuitable develop a suitable vaccination Newcastle virus also known as pathoavianimportant programme that results in aa long-lasting aa saccharide consisting of two N-acetyl-D-glucosamine and D-glucosamine units. paramyxovirus type-1, is an economically pathogen of poultry, which quickly assumes epizootic proportions paramyxovirus type-1, is an economically important patho(MDA) against NDV to develop a suitable vaccination gen ofapoultry, quickly assumes epizootic proportions programme that results a long-lasting after a single at It reported nst NDV to develop suitablewhich vaccination programme that results a protection long-lasting protection after a paramyxovirus type-1, iscontrol an economically important pathoChitosan was dissolved in PBS at a final concentration of 0.5% (w/v) and used single vaccination vaccination at the the hatchery. hatchery. It has has inbeen been reportedinthat that gen of poultry, which quickly assumes epizootic proportions if no strict and effective measures are implemented. programme that single results inand a long-lasting protection after a reported that gen of poultry, which quickly assumes epizootic proportions if no strict and effective control measures are implemented. vaccination at the hatchery. It has been the combination of live inactivated ND vaccines at to reconstitute and dilute the live ND vaccine to the final concentration. hat results in a long-lasting protection after a single vaccination at the hatchery. It has been reported that gen of poultry, which quickly assumes epizootic proportions the combination of live and inactivated ND vaccines at if no strict and effective control measures are implemented. In addition addition to to good good biosecurity biosecurity practices, controlcontrol of measures single vaccination the hatchery. Itlonger-lasting has thatND vaccines at strict and effective are implemented. In practices, control of theataacombination of live been and reported inactivated The viscerotropic Chimalhuacan vNDV strain belonging to class II 1-day-old induces higher and humoral ation at the hatchery. has been reported that iftono of live and inactivated ND vaccines at 1-day-old higher the andcombination longer-lasting humoral if noItstrict and effective control measures are of implemented. In (ND) addition good biosecurity practices, control of induces Newcastle disease (ND) primarily consists preventive the combination of live and inactivated ND vaccines at In addition to good biosecurity practices, control of 1-day-old induces a higher and longer-lasting humoral Newcastle disease primarily consists of preventive genotype V of NDV (ICPI = 1.89) used for challenge was isolated in Mexico immunity when when compared compared with with inactivated vaccine admiion of live and In inactivated ND vaccines at 1-day-old induces a higher and longer-lasting humoral immunity inactivated vaccine admiaddition to good biosecurity practices, control of Newcastle disease primarily consists of preventive 1-day-old a higher and1978; longer-lasting humoral vaccination of and the culling of infected birds and Newcastle (ND) primarily consistsalone ofinduces preventive (Calderon et al., 2005). Oculo-nasal inoculation of 105 EID50 of this strain immunity when compared with inactivated admi-vaccine admiof flocks flocks and primarily the culling(ND) of disease infected birds and nistered et al., Giambrone & duces a higher vaccination and longer-lasting humoral immunity when compared withvaccine inactivated nistered alone (Bennejean (Bennejean et al., 1978; Giambrone & Clay, Clay, Newcastle disease (ND) consists ofculling preventive vaccination of flocks and the of infected birds and induces 100% mortality within 3 to 6 days in specific pathogen free (SPF) immunity when compared with inactivated vaccine admibirds at risk of being infected (protection zone). Vaccination vaccination zone). of flocks and the culling of infected birds and alonenistered al., 1978; Giambrone Clay, birds at risk of of vaccine being infected (protection Vaccination 1986; al., This provides en compared with inactivated admialoneet(Bennejean et al., 1978;&Giambrone & Clay, 1986; Folitse Folitse et etnistered al., 1998). 1998). (Bennejean This programme programme provides vaccination flocks and the culling of prevailing infected birds and birds at risk of being infected (protection zone). Vaccination and commercial broiler chickens when challenged at 3 to 6 weeks of age nistered alone (Bennejean et al., 1978; Giambrone & Clay, programmes are tailored to suit the disease birds at risk of being infected (protection zone). Vaccination 1986; Folitse et al., 1998). programme programmes tailored to (protection suit the prevailing disease clinical protection against pneumotropic velogenic NDV e (Bennejean et al., 1978; Giambrone & Clay, 1986; Folitse et This al., 1998). This provides programme provides clinical protection against pneumotropic velogenic NDV birds at risk ofare being infected zone). Vaccination (personal observations). programmes are tailored to suit the prevailing disease 1986; Folitse et al., 1998). This programme provides situation and take into account other factors such as programmes are tailored to suit the prevailing disease clinical protection against pneumotropic velogenic NDV and are taketailored into account other factors such as (vNDV) isolated isolated in in past past epizootics epizootics (Bennejean et against al., 1978; 1978; e et al., 1998). situation This programme provides clinical protectionet pneumotropic velogenic NDV (vNDV) (Bennejean al., programmes to suit the prevailing disease situation and take into account other factors such as protection against pneumotropic velogenic NDV maternal immunity, additional vaccination programmes, situation and take into account clinical other factors as (vNDV) isolated in past epizootics (Bennejean al., 1978; et al., 1978; maternal immunity, additional vaccination programmes, Giambrone & such Clay, 1986; Folitse et isolated al., 1998; Chansirction against pneumotropic velogenic NDV (vNDV) in past epizooticset(Bennejean Giambrone & Clay, 1986; Folitse et al., 1998; Chansirsituation and take into account other factors such as programmes, maternal immunity, additional vaccination (vNDV) isolated in past epizootics (Bennejean et al., 1978; other pathogen circulation, types of flock, available maternal immunity, additional vaccination programmes, Giambrone & Clay, 1986; Folitse et al., 1998; Mitogens and Newcastle disease virus antigens. The mitogens phorbolother pathogen circulation, types of flock, available ipornchai & Sasipreeyajan, 2006) and has become routinely ted in past epizootics (Bennejean et al.,additional 1978; Giambrone Clay, routinely 1986; Folitse etChansiral., 1998; Chansiripornchai & Sasipreeyajan, 2006) and has & become maternal immunity, vaccination programmes, other pathogen circulation, types of & available Giambrone & available Clay, 1986; Folitse et al., problem. 1998; and Chansirlabour,etclimatic climatic conditions and inferred cost (Alexander (Alexander &flock, 12-myristate-13-acetate (PMA) and ionomycin (Iono) were purchased from ipornchai & Sasipreeyajan, 2006) has become routinely other pathogen circulation, typesused of flock, in countries where ND is a significant In this labour, conditions and inferred cost & Clay, 1986; Folitse al., 1998; Chansir& Sasipreeyajan, 2006) and has become routinely used in countries where ND isipornchai a significant problem. In this other pathogen circulation, types of outbreaks flock, available labour, climatic conditions and inferred cost (Alexander &a(Alexander ipornchai & new Sasipreeyajan, 2006) where and hasND become routinely Sigma (Diegem, Belgium). NDV recall antigens were prepared from the Senne, 2008). Nonetheless, the recurrent of fatal used in countries is a significant problem. In this labour, climatic conditions and inferred cost & context, ND vaccination regimen including the Senne, 2008). Nonetheless, the recurrent outbreaks of fatal Sasipreeyajan, 2006) and has become routinely used in countries where ND is a significant problem. In this context, a new ND vaccination regimen including the labour, climatic Senne, conditions andNonetheless, inferred cost (Alexander & NDV La Sota strain as previously described (Lambrecht et al., 2004; Rauw 2008). the recurrent outbreaks of fatal used in countries where ND is a significant problem. In this ND in commercial poultry flocks in many parts of the world context, a new ND vaccination regimen including the Senne, 2008). Nonetheless, the recurrent outbreaks concomitant useofof offatal recombinant herpesvirus of vaccination turkey in commercial poultry flocks in many parts of theofworld ries where ND is ND a significant problem. In this context,herpesvirus a new ND regimen including etthe concomitant use aa recombinant of turkey Senne, 2008). Nonetheless, the recurrent outbreaks fatal al., 2009b) and named inactivated NDV and all dissociated NDV proteins context, a new ND vaccination regimen including the ND in commercial poultry flocks in many parts of the world concomitant use of a recombinant herpesvirus of turkey indicate that routine vaccinations are failing to induce the ND in commercial poultry flocks in many parts of the world expressing the the F F gene gene of of NDV NDV (rHVT-ND)use andofaa alive live ND indicate that routine vaccinations are failing toofinduce the ew ND vaccination regimen including the concomitant recombinant herpesvirus of turkey expressing (rHVT-ND) and ND (prot-NDV). ND in commercial poultry flocks in many parts the world concomitant use of a recombinant herpesvirus of turkey indicate that indicate routine vaccinations are failing to are induce the expressing F2010a) gene ofor NDV (rHVT-ND) a live NDand a live ND high levels of immunity required to control ND Boven that routine vaccinations failing to induce the et vaccine adjuvanted (Rauw al., not al., use of a recombinant herpesvirus ofvaccinations turkey high levels immunity required toare control NDto(Van (Van Boven expressing the F (Palya gene ofet (rHVT-ND) vaccine adjuvanted (Rauw etthe al., 2010a) or not (Palya et NDV al., and indicate thatofroutine failing induce the ND (Van expressing the Fal., gene ofadjuvanted NDV (rHVT-ND) and a2010a) live was ND high levels of immunity required to control Boven vaccine (Rauw et al., or not (Palya al., 2008). Currently vaccines and vaccination strategies high levels of immunity required to control ND (Van Boven 2008; Rauw et 2010a) with chitosan at 1-day-old e F gene of NDVet (rHVT-ND) and a live ND vaccine adjuvanted (Rauwwas et al., 2010a) et or al., not (Palya et al., et al.,levels 2008). vaccines and vaccination strategies 2008; Rauw et al., 2010a) with chitosan at 1-day-old high of Currently immunity required to control NDdo (Van adjuvanted (Rauw et al., 2010a) or not (Palya et al., al., 2008). vaccines and vaccination strategies 2008; Rauw et al., 2010a) with chitosan at 1-day-old was Measurement of NDV-specific cell-mediated immunity in the spleen, the protect against morbidity andetCurrently mortality but notBoven stop al., 2008). Currently vaccines andvaccine vaccination strategies investigated. These vaccination programmes were shown to anted (Rauw et al., 2010a)against or not et (Palya et al., 2008; Rauw et al., 2010a) with chitosan at 1-day-old was protect morbidity and mortality but do not stop investigated. These vaccination programmes were shown to et al., viral 2008). Currently vaccines and vaccination strategies Rauw al., 2010a) with chitosan 1-day-old was were shown to protect against morbidity andmorbidity mortality butmortality do2008; not stop blood, and the digestive and respiratory tracts. The induction investigated. These vaccination programmes either infection or viral viral excretion. In addition, addition, in laying laying improve protection and immunity immunity againstatThese recent visceroprotect against and butprotection doetnot stop et al., 2010a) with chitosan at 1-day-old wasexcretion. investigated. vaccination programmes were shownperipheral to either viral infection or In in improve and against recent visceroprotect against morbidity and mortality butexcretion. doinactivated not In stop investigated. These vaccination programmes were shown to of NDV-specific cell-mediated immunity (CMI) was evaluated by the either viral infection or viral addition, in laying improve protection and immunity against recent viscerohens and breeders, a booster vaccination with an tropic vNDV during during the first first 6 6improve weeks of of life in in commercial commercial either viral with infection or viral excretion. In addition, in laying These vaccinationhens programmes wereashown tovaccination protection and immunity against recent visceroand breeders, booster an inactivated tropic vNDV the weeks life production of ChIFNγ after ex vivo antigen-activation of splenocytes, either viral infection or viral excretion. In addition, in laying improve and immunity against recent viscerohensat and breeders, a booster vaccination with an inactivated tropic vNDV during the first 6administered weeks of life in commercial vaccine is required the start of lay, in order to induce aavaccination chickens when compared with live vaccine hens and breeders, a booster with protection an inactivated ection and immunity against recent viscerotropic vNDV during the first 6 weeks of life in commercial vaccine is required at the start of lay, in order to induce chickens when compared with live vaccine administered peripheral blood lymphocytes (PBL), lamina propria lymphocytes of the hens and breeders, a booster vaccination with an inactivated tropic vNDV during the first 6 weeks of life in commercial is during required atlaying start ofatlay, order induce a to induce chickens compared live vaccine administered alone. protective immunity the period. vaccine isthe required theinstart of to lay, in order a when during the first 6good weeks of life invaccine commercial chickens whenwith compared with live vaccine administered alone. good protective immunity during the laying period. duodenum, tracheal lymphocytes and pulmonary lymphocytes as previously vaccine is required at the start of lay, in order to induce a chickens when compared with live vaccine administered good protective immunity during the laying period. en compared with live vaccine administered alone. good protective immunity during the laying period. alone. described (Rauw et al., 2009a, b, 2011). Briefly, spleens were removed alone. good protective immunity during the laying period.

Introduction Introduction

*To whom whom correspondence correspondence should should be be addressed. addressed. Tel: Tel: +32 +32 22 379 379 13 13 21. 21. Fax: Fax: +32 +32 2 2 379 379 13 13 37. 37. E-mail: E-mail: fabienne.rauw@coda-cerva.be fabienne.rauw@coda-cerva.be *To *To whom correspondence Tel:be+32 2 379 13 21.+32 Fax:2 +32 2 379 37.+32 E-mail: (Received accepted 23 September 2013) whom should correspondence should addressed. Tel: 379 13 21. 13 Fax: 2 379fabienne.rauw@coda-cerva.be 13 37. E-mail: fabienne.rauw@coda-cerva.be (Received 10 10 April April 2013; 2013; accepted 23*To September 2013)be addressed. *To2013 whom correspondence should be addressed. Tel: +32 2September 379 13 21.2013) Fax: +32 2 379 13 37. E-mail: fabienne.rauw@coda-cerva.be (Received 10 April 2013; accepted 23 © Houghton Trust Ltd (Received 10 April 2013; accepted 23 September 2013) mail: fabienne.rauw@coda-cerva.be © 2013 Houghton Trust Ltd (Received 10 April 2013; accepted 23 September 2013) © 2013 Houghton © Trust 2013 Ltd Houghton Trust Ltd 54 / © 2013 Houghton Trust Ltd

aseptically from chickens and heparinized blood samples were layered by sedimentation of red blood cells to isolate splenocytes and PBL, respectively (Rauw et al., 2009a, b). Lymphocytes from the digestive tract (Rauw et al., 2009a), the trachea and the lung (Rauw et al., 2011) were

isolated by enzymatic digestion. Immune cells were then activated by mitogens (PMA/Iono, 1 µg/ml), as a positive control of the ex vivo activability of lymphocytes, and by NDV recall antigens (prot-NDV, 1 µg/ml). ChIFNγ production was measured by capture enzyme-linked immunosorbent assay (ELISA). Cellular immune responses were expressed as stimulation indices (S.I.). These were calculated for each bird by dividing the optical density values of mitogen-activated and antigen-activated lymphocytes by the optical density of non-activated lymphocytes (Rauw et al., 2009b), and the S.I. per group were calculated.

Measurement of the NDV-specific humoral and local antibodymediated immunity. NDV-specific humoral immunity was evaluated by the haemagglutination inhibition (HI) test and NDV-specific IgG, IgM and IgA ELISA. HI tests and NDV-specific IgG ELISA were performed as previously described (Rauw et al., 2009b). For detection of NDV-specific IgM and IgA, MaxiSorp Nunc-Immuno F96 microwell plates (International Medical, Watermaal, Belgium) were coated overnight at 4°C with mouse antibody directed against chicken IgM or IgA (SouthernBiotech, Brussels, Belgium), respectively, diluted at 5 µg/ml in carbonate/bicarbonate pH 9.6 buffer. The following day, plates were washed three times with PBS supplemented with 0.1% Tween 80. Plates were blocked for 30 min at 37°C with PBS containing 3% bovine serum albumin (BSA) and then incubated with diluted samples, as specified afterwards, in PBS containing 0.1% Tween 80, 5% NaCl and 4% BSA for 1 h at room temperature. Inactivated NDV diluted at 1 µg/ml and biotin-labelled mouse antibody 4D6 directed against HN protein (Mast et al., 2006) were then added for 1 h at room temperature. Plates were incubated with streptavidin–horseradish peroxidase conjugate (Biosource Europe, Nivelles, Belgium) for 1 h at room temperature. After six washings, peroxidase activity was revealed by adding 100 µl tetra-methyl benzidine peroxidase substrate (Thermo Fisher Scientific, Erembodegem, Belgium) for 15 min in darkness, before stopping the reaction with 1 M H3PO4 buffer. Optical density was determined at 450 to 560 nm with an ELISA reader. Local antibody-mediated immunity to NDV was measured at preferential replication sites of the vaccine by NDV-specific IgG, IgM and IgA ELISA on lung washings and supernatant of ex vivo duodenum, and trachea tissue culture. The protocols for collection of lung washings and ex vivo duodenal tissue culture were described previously (Rauw et al., 2009b). The culture of tracheal tissue was based on paper by Zoth et al. (2008) with minor modifications. After collection of trachea, connective tissue, blood and fat in serosa were removed in PBS with 5% (w/v) gentamicin and most of the mucus was discarded by gently rubbing in one direction over the outside of the tubular part of the trachea. Tracheal tissue was then opened with scissors and cut into 1 cm length slices before washing. During this washing procedure, the tissue strips were pelleted by centrifugation for 5 min at 300 × g. Subsequently the tissue was re-suspended in 5 ml RPMI 1640 medium containing 10% FCSi. After incubation at 39°C for 48 h, the supernatant was collected by centrifugation and frozen at –20°C until the time of assay.

Measurement of virus shedding via oropharyngeal and cloacal routes after challenge. The quantification of Chimalhuacan NDV challenge virus in oropharyngeal and cloacal swabs was performed by quantitative real-time reverse transcription-polymerase chain reaction (QRRT-PCR) targeting the matrix (M) gene, as previously described (Rauw et al., 2010a). The sensitivity threshold of this NDV QRRT-PCR (R2 = 0.998, efficiency = 94.17%) was determined at 1 EID50 per reaction (102.7 EID50/ml swabs), based on standard curve data. For statistical analysis, undetermined samples were considered negative and received a value of 10 EID50/ml swabs. Results were expressed as the titre of challenge strain per millilitre of swabs (log10). In addition, quality of the sample and RNA extraction procedure were validated using avian β-actin as described previously (Van Borm et al., 2007).

Experimental design. Commercial layer chickens were assigned into four groups and vaccinated or not at 1-day-old. The first group was vaccinated with the rHVT-ND and live ND vaccines, while in the second group the live ND vaccine was co-administrated with chitosan. These were designated as the “rHVT-ND/live ND” and “rHVT-ND/live ND-Chitosan” groups, respectively. The third group was vaccinated with both live and inactivated ND vaccines, and designated the “inact ND/live-ND” group. The last group

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Efficacy of rHVT-ND/live ND vaccination

was left untreated and served as unvaccinated negative controls. At 1-dayold, the serum of 10 unvaccinated birds was sampled to determine the MDA level. Serum, blood, spleen, trachea, lung and duodenum samples were collected (n = 5) at 3, 4, 5, 6, 8, 10 and 12 weeks post vaccination. At 6 and 10 weeks post vaccination, 10 chickens from each group were individually tagged and challenged with 105 EID50/200 µl Chimalhuacan NDV strain by the oculo-nasal route. After challenge, chickens were monitored daily for clinical signs (swelling of the head, depression, prostration and nervous signs) and mortality over a 2-week period. Birds that showed the clinical signs typical of ND or died were considered unprotected. Oropharyngeal and cloacal swabs were taken at 2, 4, 7 and 10 days post infection (d.p.i.).

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Statistical analysis. Statistical analyses of data were performed using Minitab 13 (Minitab Ltd, Coventry, UK) and STATA 10 (Stata Corp LP, Texas, USA) software (statistical programmes for Windows 2000) and differences were considered significant at P < 0.05. The analyses of the ChIFNγ production, the antibody level and the titre of viral excretion were carried out to compare the groups by one-way analysis of variance and Turkey’s pair-wise comparison tests or by the non-parametric Kruskal– Wallis test, as previously described (Rauw et al., 2009b). The qualitative criteria “positive QRRT-PCR reaction” and “positive cell activation” were analysed by Fisher’s exact test, using the Bonferroni method to adjust the risk α (Rauw et al., 2009b).

Results Vaccination schedules including the rHVT-ND vaccine provided a greater and longer protection against clinical signs, mortality and virus shedding after NDV challenge. After challenge with the viscerotropic Chimalhuacan vNDV strain at 6 and 10 weeks of age, unvaccinated chickens showed typical clinical signs of ND, including swelling of the head, depression, prostration and nervous signs, from 3 d.p.i. Birds started dying on 5 d.p.i. and all birds were found dead by 6 d.p.i., which validated the challenge. Neither clinical signs nor mortality were observed among the rHVTND/live ND and the rHVT-ND/live ND-Chitosan vaccinated groups following either of the two challenge dates (Table 1). The chickens vaccinated with the inact ND/live ND combination were also fully protected at 6 weeks of age. At 10 weeks of age, protection against clinical signs and mortality was reduced to 70% and 90%, respectively, but the difference was not statistically significant. Table 1. Protection against morbidity and mortality after challenge with the Chimalhuacan vNDV strain of commercial layer chickens vaccinated at 1-day-old with the live ND and the rHVT-ND or the inact ND vaccines, according to different vaccination regimens.

Protection against Age of challenge 6 weeks

10 weeks

Group Negative rHVT-ND/live ND rHVT-ND/live NDChitosan Inact ND/live ND Negative rHVT-ND/live ND rHVT-ND/live NDChitosan Inact ND/live ND

Morbidity (%)

Mortality (%)

0 100 100

100 0 100 100

Data represent survival rate and clinical protection 2 weeks after challenge by the oculo-nasal route with Chimalhuacan NDV strain. 56 /

After challenge at 6 weeks, each of the three vaccination schedules significantly reduced challenge virus shedding at 4 d.p.i. by the oropharyngeal route (Table 2). Excretion stopped at 10 d.p.i. in both groups receiving the vaccination schedules which included the rHVT-ND vaccine. Similarly, the cloacal excretion was significantly reduced at 4 d.p.i. in each of the three investigated vaccination schedules and fewer than 40% of birds were found positive. At this age of challenge, there was no difference in virus shedding between the three vaccination programmes. Following challenge carried out at 10 weeks of age, each of the three investigated vaccination schedules significantly reduced challenge virus shedding at 4 d.p.i. by the oropharyngeal route. Interestingly, this reduction was significantly stronger at 4 and 7 d.p.i. in the rHVT-ND/live ND and rHVT-ND/ live ND-Chitosan groups in comparison with the inact ND/ live ND group. Moreover, both vaccination programmes including the rHVT-ND vaccine fully protected against cloacal excretion, while excreting chickens were observed at 4, 7 and 10 d.p.i. in the inact ND/live ND group.

The rHVT-ND/live ND vaccination schedule maintained a measurable peripheral CMI during the 12-week period of observation, as well as a greater and longer CMI in the digestive and respiratory tracts. NDVspecific peripheral CMI remained above the threshold of positivity during the whole 12-week observation period in the rHVT-ND/live ND group (Figure 1a), and was significantly different from the unvaccinated group at weeks 5, 6 and 12. This CMI was positive only at 4, 5, 8, 10 and 12 weeks of age in the inact ND/live ND group, with significant difference from unvaccinated group arising during the last week of observation. The peripheral cellular immune response in the rHVT-ND/live ND-Chitosan group was detected only during the first 5 weeks of age. All three vaccination schedules induced NDV-specific CMI in the digestive tract from the third week (Figure 1b). The duodenal CMI in the rHVT-ND/live ND group increased rapidly during the first 6 weeks to reach a peak level that was significantly higher at 6 weeks of age, compared with the other vaccinated and unvaccinated groups. Both vaccination programmes including the rHVT-ND vaccine were able to maintain a digestive antigen-specific cellular immunity during the 12 weeks of observation, while the digestive CMI in the inact ND/live ND group had waned after 8 weeks of age. The rHVT-ND/live ND combination induced NDV-specific CMI in lung from 6 to 10 weeks of age and the rHVTND/live ND-Chitosan vaccination regimen did from 8 to 10 weeks of age (Figure 1c). Indeed, although difference was not significant when compared with the unvaccinated group, positive CMI was detected in the lung of 40 to 80% of vaccinated chickens. The inact ND/live ND group remained negative during the whole experiment. No NDVspecific CMI could be observed in the trachea during the 12-week period of this experiment (data not shown). Large standard deviations and variations between experimental days were observed for CMI responses. These observations could be explained by biological variations between birds, especially in conventional animals, as observed after mitogenic activation of lymphocytes (data not shown). Experimental design with more than five chickens per group at each experimental day could prob‐ ably reduce the standard deviation but would be difficult to manage with four groups to evaluate on the same day.

Table 2. Shedding after challenge at 6 and 10 weeks of age with the Chimalhuacan vNDV strain on commercial layer chickens vaccinated at 1-day-old with the live ND and rHVT-ND or the inact ND vaccines according to different vaccination regimens.

Age

Group

Oropharyngeal swabsa 6 weeks Negative rHVT-ND/live ND rHVT-ND/live ND-Chitosan Inact ND/live ND 10 weeks

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Negative rHVT-ND/live ND rHVT-ND/live ND-Chitosan Inact ND/live ND

Cloacal swabs 6 weeks

Negative rHVT-ND/live ND rHVT-ND/live ND-Chitosan Inact ND/live ND

10 weeks

Negative rHVT-ND/live ND rHVT-ND/live ND-Chitosan Inact ND/live ND

2 d.p.i.

4 d.p.i.

7 d.p.i.

10 d.p.i.

3.71 ± 1.69bA 8/10cA 1.75 ± 1.21B 3/10AB 2.19 ± 1.56AB 4/10AB 1.48 ± 1.02B 2/10B 4.34 ± 0.75A 10/10A 2.22 ± 1.65BC 4/10AB 1.41 ± 0.87C 2/10B 3.49 ± 1.01AB 9/10AB

6.77 ± 0.23A 10/10A 3.03 ± 1.82B 6/10A 3.47 ± 2.06B 7/10A 3.40 ± 2.16B 6/10A 6.36 ± 0.41A 10/10A 2.07 ± 1.30C 4/10AB 2.05 ± 2.22C 2/10B 4.25 ± 1.52B 9/10AB

N.D.

1.92 ± 1.89A 3/10A 3.04 ± 2.61A 4/10A 2.35 ± 1.70A 4/10A N.D.

1.09 ± 0.27A 0/10A 1.03 ± 0.10A 0/10A 1.23 ± 0.72A 1/10A N.D.

1.00 ± 1.00B 0/10B 2.72 ± 2.28B 4/10AB 4.99 ± 2.44A 8/9A

1.00 ± 0.00A 0/10A 1.00 ± 0.00A 0/10A 1.81 ± 1.65A 2/9A

1.45 ± 0.96A 1/10A 1.18 ± 0.58A 1/10A 1.00 ± 0.00A 0/10A 1.00 ± 0.00A 0/10A 1.50 ± 1.05A 2/10A 1.00 ± 0.00A 0/10A 1.00 ± 0.00A 0/10A 1.00 ± 0.00A 0/10A

6.52 ± 0.65A 10/10A 1.43 ± 1.35B 1/10B 1.00 ± 0.00B 0/10B 1.00 ± 0.00B 0/10B 6.34 ± 0.46A 10/10A 1.00 ± 0.00B 0/10B 1.00 ± 0.00B 0/10B 1.92 ± 2.65B 2/10B

N.D.

2.03 ± 1.54A 3/10A 2.10 ± 1.97A 3/10A 2.74 ± 1.76A 4/10A N.D.

1.78 ± 1.46A 3/10A 1.93 ± 1.32A 2/10A 1.79 ± 1.43A 2/10A N.D.

1.00 ± 0.00B 0/10B 1.00 ± 0.00B 0/10B 2.89 ± 1.97A 5/9A

1.00 ± 0.00A 0/10A 1.00 ± 0.00A 0/10A 1.78 ± 2.35A 1/9A

Data were determined by QRRT-PCR on 1 ml swabs taken at specified d.p.i. Different uppercase superscript letters indicate a significant difference (P < 0.05) between the groups (per column). The cut-off value of QRRT-PCR specific to the Chimalhuacan NDV strain was determined at 102.7 EID50/ml swabs. The total numbers of chickens tested were reduced over time because of specific mortality (see Results section). N.D., not determined, due to specific mortality. bData represent mean ± standard deviation of log10 EID50 of NDV in 1 ml swabs. For statistical analysis, samples with data value < 2.7 log10 EID50/ml were considered negative for virus while undetected samples were considered negative and received a value of 1 log10 EID50/ml swabs for statistical purposes. cData represent frequency (number positive/total tested chickens) of virus detection in 1 ml swabs.

Each vaccination schedule induced a high CMI in the spleen during the first 6 weeks of age. The NDV-specific CMI in the spleen was significantly higher from the third to the sixth week of age in the three vaccinated groups when compared with unvaccinated birds (Figure 1d). The splenic CMI induced by the vaccination regimen including the inact ND vaccine tended to be the highest one at weeks 3, 4, 6 and 12. This superiority was significant at week 3 in comparison with the rHVT-ND/live ND-Chitosan group, and at week 12 in comparison with both vaccinated groups including the rHVT-ND vaccine. At 8 weeks of age, the NDV-specific CMI in the spleen from the rHVT-ND/live ND-Chitosan group remained at a constant and significantly higher level, when compared with the unvaccinated group, while it decreased in the two other vaccinated groups.

The inact ND/live ND vaccination regimen may induce a higher systemic antibody-mediated immunity. The mean HI antibody titre at 1 day old was 9.2 ± 1.2 log2 (data not

shown). Subsequently, HI titres indicated a decline of passive, maternally-derived, immunity until the fourth week of age (Figure 2). An active primary immune response was detected by HI tests in the three vaccinated groups starting at 3 weeks of age and was significantly higher than the unvaccinated group from the fourth week of age. The HI titres in the inact ND/live ND vaccinated chickens were significantly higher at week 4 than groups that received the vaccination schedules including the rHVT-ND vaccine. This superiority was maintained at weeks 6, 8 and 10, although not significantly. A similar tendency was observed by NDVspecific IgG ELISA during the whole experiment (data not shown). NDV-specific IgM was detected in the inact ND/ live ND vaccinated chickens between 3 and 5 weeks of age and the level was significantly higher than in the unvaccinated group (Figure 3). IgM appeared later, between the age of 5 and 8 weeks, in the rHVT-ND/live ND group with a significant difference with the unvaccinated group at 5 and 6 weeks of age. IgM was detected only at 5 weeks in the rHVT-ND/live ND-Chitosan group. No NDV-specific IgA / 57

N°9 • Scientific File F. Rauw et al.

Efficacy of rHVT-ND/live ND vaccination

Figure 1. NDV-speciﬁc cell-mediated immunity in the peripheral blood (1a), in the digestive tract (1b), in the lung (1c) and in the spleen (1d) of commercial layer chickens vaccinated at 1-day-old with the live ND and the rHVT-ND or inact ND vaccines, according to different vaccination regimens. Lymphocytes were stimulated with prot-NDV recall antigen (1 µg/ml), and supernatants of stimulated cells were harvested after 72 h of activation. ChIFNγ production was determined by the ChIFNγ capture ELISA. The results correspond to the mean ± standard deviation of stimulation indices at each time point (n = 5). Mean ± standard deviation at time points with no common uppercase letters differ signiﬁcantly (P < 0.05). S.I., stimulation indices. (Continued on next page)

was detected in the sera from any of the vaccinated groups throughout the duration of this experiment (data not shown).

The inact ND/live ND vaccination regimen may induce a higher local antibody-mediated immunity. The three vaccination regimens induced IgG in the digestive tract from 3 weeks of age (Figure 4). When compared with the unvaccinated group, the level of this antibody-mediated immunity was significantly higher from the fourth week in the inact ND/live ND group and 1 week later in the rHVT-

58 /

ND/live ND and rHVT-ND/live ND-Chitosan groups. The inact ND/live ND vaccination regimen tended to induce higher IgG levels in the duodenum until 6 weeks of age, but the difference was only significant at 4 weeks of age, compared with the two other vaccination schedules. NDVspecific IgA and IgM were detected in the digestive tract of 40% of inact ND/live ND vaccinated birds at 5 weeks of age, while the two other vaccinated groups that included the rHVT-ND vaccine remained negative (data not shown). All vaccination schedules induced secretion of IgG in the trachea from 3 weeks of age (Figure 5a). When compared with the unvaccinated group, the NDV-specific IgG level

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Figure 1.

(Continued)

was significantly higher from 4 weeks of age in the inact ND/live ND group, while this difference was observed one week later in the two other vaccinated groups. The tracheal IgG response of the inact ND/live ND vaccinated chickens was higher at weeks 4, 5 and 6 in comparison with the two other vaccinated groups, and the difference was significant at 4 weeks of age. The inact ND/live ND vaccination regimen also induced a significantly higher level of tracheal IgG at weeks 5 and 6 in comparison with the rHVT-ND/live ND-Chitosan vaccination programme, and at week 10 in comparison with the rHVT-ND/live ND vaccination regimen. The inact ND/live ND group showed a peak level of tracheal IgA (Figure 5b) and IgM (Figure 5c) at 4 and 5 weeks of age that was significantly higher compared with the unvaccinated group and the other vaccinated groups. No immunoglobulin could be detected in lung washings during the experiment (data not shown).

Discussion In regions where ND is enzootic and the field pressure of ND virus is high, there is much competition between field viruses and vaccine development. Therefore it is very important to induce an active immune response by vaccination as soon as possible. In addition, the typically high levels of passive immunity interfere with the efficacy of very early immunization by the commonly available ND vaccines. Moreover, in many countries, local customs or situations result in insufficient vaccination or poor timing of vaccination, which have serious consequences (Alexander & Senne, 2008). Thus, there is a tendency to provide very early (as soon as at 1-day-old) and frequent vaccinations of progeny with live ND vaccines. Although these vaccines in optimum conditions induce good protection related to a strong humoral immunity and CMI, they are known to be sensitive to MDA at 1-day-old (Rauw et al., 2009b), There / 59

N°9 • Scientific File Efficacy of rHVT-ND/live ND vaccination

F. Rauw et al.

Figure 2. Serum HI antibody titres after vaccination of 1-day-old commercial layer chickens with the live ND and rHVT-ND or inact ND vaccines according to different vaccination regimens. Data represent mean ± standard deviation of HI antibody titres at speciﬁed ages (n = 5), which corresponds to the last dilution showing an inhibition of haemagglutination of 4 haemagglutination units of NDV La Sota strain. The HI geometric mean titres were expressed as reciprocal log2, and titres > 3 log2 were considered positive (this cut-off value is indicated by the dotted line). Means ± standard deviations with no common uppercase letters differ signiﬁcantly (P < 0.05).

is a clear need for improving vaccination programmes that could be used at 1-day-old for mass application and induce a long protective immunity that requires fewer booster vaccinations at later ages. In this context, enterotropic live ND vaccine combined with either inactivated ND or rHVT-ND vaccines and adjuvanted, or not, with chitosan was used to immunize 1-day-old commercial layer chicks. The efficacy of these three vaccination schedules was then investigated. The clinical protection obtained at 6 weeks by the inact ND and live ND vaccines administrated simultaneously at 1 day old was more enhanced than previously described (Bennejean et al., 1978). The inact ND/live ND vaccination programme including a live ND vaccine with enterotropic tropism was more

protective against a viscerotropic vNDV at 4 weeks, in comparison with a tracheotropic live strain (Chansiripornchai & Sasipreeyajan, 2006). This protection against more recent viscerotropic vNDV strains may be partly explained by the use of an enterotropic strain as live ND vaccine, which has been reported to induce a higher antibody-mediated immunity in the digestive tract during the first weeks of life when inoculated at 1-day-old in SPF chickens (Rauw et al., 2009b). However, the inact ND/live ND vaccination schedule did not provide 100% clinical protection at 10 weeks of age and the reduction of viral excretion following challenge was moderate. On the contrary, the rHVT-ND/live ND and rHVT-ND/live ND-Chitosan vaccine combinations afforded complete

Figure 3. NDV-specific IgM measured by ELISA after vaccination of 1-day-old commercial layer chickens with the live ND and the rHVTND or inact ND vaccines according to different vaccination regimens. Data represent mean ± standard deviation of absorbance values determined by ELISA at specified time of ages (n = 5). Serum samples were diluted 1:100. Mean ± standard deviation at time points with no common uppercase letters differ significantly (P < 0.05). O.D., optical density. 60 /

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Figure 4. Duodenal NDV-specific IgG antibody titre of commercial layer chickens vaccinated at 1-day-old with the live ND and the rHVTND or inact ND vaccines according to different vaccination regimens. Data represent mean ± standard deviation of absorbance values determined by ELISA at specified ages (n = 5). The immunoglobulin response was measured in 1:2 diluted supernatants of ex vivo duodenal tissues cultures. Means ± standard deviations with no common uppercase letters differ significantly (P < 0.05). O.D., optical density.

protection against mortality and clinical signs after challenge at 10 weeks of age as well as prevention of viral shedding by cloacal route and a reduction in the duration of the oropharyngeal excretion. Previous studies have shown that 1-day-old subcutaneous inoculation of rHVT-ND, compared with the in ovo route, improved the protection in commercial layer chickens with the same MDA level (Rauw et al., 2010a). Indeed, the subcutaneous inoculation of chicks at 1-day-old appears to be less variable than an injection in ovo, as the site of injection in the embryonated egg is of crucial importance to achieve adequate replication of the vaccine virus (Johnston et al., 1997; Jochemsen & Jeurissen, 2002; Wakenell et al., 2002). To explain the differences observed in protection against mortality, morbidity and viral shedding, the active immune response profiles afforded by these three vaccination programmes was analysed. Because all vaccinated groups received the same live vaccine, the higher systemic and local antibody-mediated immunity observed in the inact ND/live ND combination must be related to the inact ND vaccine, which promotes a Th2-oriented immune response. The IgG antibodies are mainly transferred from the serum to the trachea (Zoth et al., 2008) and the duodenum (Muir, 1998), which may explain their elevated levels in the respiratory and digestive tracts, respectively, while IgA and IgM are produced locally. The water-in-oil emulsion used in the inact ND vaccine is known to retain the killed antigens at the injection site and to release it progressively, thus triggering a local inflammatory response and stimulating the recruitment of antigen-presenting cells (APC) and lymphocytes (Aucouturier et al., 2001; Degen et al., 2003; Jansen et al., 2006, 2007). After antigen phagocytosis, APC travel from the injection site to secondary lymphoid organs in order to interact with naïve T and B lymphocytes (Kaspers et al., 2008). The sustained release of nonreplicating antigens is expected to maintain a high level of antibody production by repeated exposure of B cells to antigens or persistence of long-lived plasma cells (Degen et al., 2003; Jansen et al., 2006), explaining the higher humoral immunity in the inact ND/live ND group. In the

case of combined vaccination with live ND vaccine, this antigen-presenting cell migration from injection site to secondary lymphoid organs allows the enhancement of the local IgA and IgM production, as observed in the trachea and duodenum at 4 and 5 weeks of age. This local response was primed by the live ND vaccine administrated simultaneously at 1-day-old and is known to replicate during the first week in the trachea and duodenum (Rauw et al., 2009b). In addition, the persistent specific ex vivo production of ChIFNγ by lymphocytes from the peripheral blood and the digestive tract during 12 weeks and the detection of pulmonary CMI in the rHVT-ND/live ND group suggest that the rHVT-ND vaccine promotes a Th1-oriented immune response. Owing to the persistent viraemia of HVT in lymphocytes for at least 30 weeks (Tsukamoto et al., 2002), the HVT-ND is thought to circulate throughout the body by infected PBL, which migrate to the lymphoid tissues of the digestive and respiratory tracts. The HVT-ND then delivers foreign antigens locally to the immune system over an extended period of time. Moreover, the HVT replicates in a highly cell-associated manner in lymphocytes, suggesting that this delivery system induces a high degree of CMI (Heller & Schat, 1987). These properties could explain the higher and longer-lasting Th1-oriented CMI observed in peripheral blood, duodenum and lungs of chickens vaccinated with the rHVT-ND/live ND combination, whereas the rHVT-ND vaccine enhances the cellular immune response primed by the live ND vaccine administrated simultaneously at 1-day-old (Rauw et al., 2009b). The absence of CMI in the trachea could be explained by the absence of organized lymphoid structure (Kothlow & Kaspers, 2008) and/or of vaccinal antigen in this organ at these times to stimulate local immune response (Rauw et al., 2009a). Surprisingly, the positive effect of chitosan adjuvant on antigen-specific CMI in the spleen and peripheral blood following vaccination at 1 day old with the live ND vaccine (Rauw et al., 2010b) was not detected when using the rHVT-ND/live ND-Chitosan vaccination schedule. However, a faster cellular immune response in the / 61

N°9 • Scientific File F. Rauw et al.

Efficacy of rHVT-ND/live ND vaccination

Figure 5. Tracheal NDV-specific IgG (5a), IgA (5b) and IgM (5c) antibody titre of commercial layer chickens vaccinated at 1-day-old with the live ND and the rHVT-ND or inact ND vaccines according to different vaccination regimens. Data represent mean ± standard deviation of absorbance values determined by ELISA at specified ages (n = 5). The immunoglobulin response was measured in 1:2 diluted supernatants of ex vivo tracheal tissue cultures. Means ± standard deviations with no common uppercase letters differ significantly (P < 0.05). O.D., optical density. (Continued on next page)

peripheral blood was observed, confirming previous findings (Rauw et al., 2010a). It is anticipated that the beneficial effect of this adjuvant on immunity induced by a live ND vaccine is less clear when a second vaccine also promoting a Th1-oriented immune response is used simultaneously, at 1-day-old. The differences observed in protection at 10 weeks of age indicate that the CMI in the respiratory and digestive tracts is required to efficiently reduce viral shedding. T cells, and especially CD8+ probably cytotoxic T lymphocytes (CTL), have been shown to be essential for NDV clearance from the Harderian gland, but not B cells (Cannon & Russell, 1986; Russell et al., 1997). CTL are most probably also involved in the viral clearance from the respiratory and digestive tracts, explaining the reduction of viral shedding, which could be stimulated more by the rHVT-ND/live ND than the inact ND/live ND combination. 62 /

In conclusion, the main advantage of the rHVT-ND/live ND over the inact ND/live ND vaccination programme is the induction of a longer-lasting protection against mortality and morbidity, as well as a stronger inhibition of viral shedding, by combining the advantages of the live ND and rHVT-ND vaccines. This study also shows that, in the case of ND, a strong Th1-mediated immune response combined to a humoral response is more protective than a Th2-oriented response. The addition of chitosan adjuvant in this rHVT/ND/live ND combination had no clear benefit. Given the known long-lasting persistency of HVT in vaccinated birds, protection induced by the rHVT-ND/live ND combination will last longer than that induced by the inact ND/live ND combination, and will better protect long-living chickens such as layers. However, further investigations are necessary to confirm this.

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Figure 5.

(Continued)

Acknowledgements The authors gratefully acknowledge J. F. Pirlot and S. Anbari for their technical contributions to this work and C. Delgrange and M. Vandenbroeck for bird handling and sampling assistance.

References Alexander, D.J. & Senne, D.A. (2008). Newcaslte disease, other avian Paramyxovirus, and Pneumovirus infections. In Y.M. Saif, A.M. Fadly, J.R. Glisson, L.R. McDougald, L.K. Nolan, & D.E. Swayne (Eds.). Diseases of Poultry 12th edn (pp. 75–115). Ames, Iowa, USA: Blackwell Publishing. Aucouturier, J., Dupuis, L. & Ganne, V. (2001). Adjuvants designed for veterinary and human vaccines. Vaccine, 19, 2666–2672. Bennejean, G., Guittet, M., Picault, J. P., Bouquet, J.F., Devaux, B., Gaudry, D. & Moreau, Y. (1978). Vaccination of one-day-old chicks against Newcastle disease using inactivated oil adjuvant vaccine and/or live vaccine. Avian Pathology, 7, 15–27. Calderon, N.L., Galindo-Muniz, F., Ortiz, M., Lomniczi, B., Fehervari, T. & Paasch, L.H. (2005). Thrombocytopenia in Newcastle disease: haematological evaluation and histological study of bone marrow. Acta Veterinaria Hungarica, 53, 507–513. Cannon, M.J. & Russell, P.H. (1986). Secondary in vitro stimulation of specific cytotoxic cells to Newcastle disease virus in chickens. Avian Pathology, 15, 731–740. Chansiripornchai, N. & Sasipreeyajan, J. (2006). Efficacy of live B1 or Ulster 2C Newcastle disease vaccines simultaneously vaccinated with inactivated oil adjuvant vaccine for protection of Newcastle disease virus in broiler chickens. Acta Veterinaria Scandinavica, 48, 1–4. Czegledi, A., Ujvari, D., Somogyi, E., Wehmann, E., Werner, O. & Lomniczi, B. (2006). Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications. Virus Research, 120, 36–48. Degen, W.G.J., Jansen, T. & Schijns, V.E. (2003). Vaccine adjuvant technology: from mechanistic concepts to practical applications. Expert Review of Vaccines, 2, 327–335. Folitse, R., Halvorson, D.A. & Sivanandan, V. (1998). Efficacy of combined killed-in-oil emulsion and live Newcastle disease vaccines in chickens. Avian Diseases, 42, 173–178. Giambrone, J.J. & Clay, R.P. (1986). Vaccination of day-old broiler chicks against Newcastle disease and infectious bursal disease using commercial live and/or inactivated vaccines. Avian Diseases, 30, 557–561. Heller, E.D. & Schat, K.A. (1987). Enhancement of natural killer cell activity by Marek’s disease vaccines. Avian Pathology, 16, 51–60.

Jansen, T., Hofmans, M.P.M., Theelen, M.J.G., Manders, F. & Schijns, V.E. J.C. (2006). Structure- and oil type-based efficacy of emulsion adjuvants. Vaccine, 24, 5400–5405. Jansen, T., Hofmans, M.P., Theelen, M.J., Manders, F.G. & Schijns, V.E. (2007). Dose and timing requirements for immunogenicity of viral poultry vaccine antigen: investigations of emulsion-based depot function. Avian Pathology, 36, 361–365. Jochemsen, P. & Jeurissen, S.H.M. (2002). The localization and uptake of in ovo injected soluble and particulate substances in the chicken. Poultry Science, 81, 1811–1817. Johnston, P.A., Liu, H., O’Connell, T., Phelps, P., Bland, M., Tyczkowski, J., Kemper, A., Harding, T., Avakian, A., Haddad, E., Whitfill, C., Gildersleeve, R. & Ricks, C.A. (1997). Applications in in ovo technology. Poultry Science, 76, 165–178. Kaspers, B., Kothlow, S. & Butter, C. (2008). Avian antigen presenting cells. In F. Davison, B. Kaspers, & K.A. Schat (Eds.). Avian Immunology 1st edn (pp. 183–202). London: Academic Press. Kothlow, S. & Kaspers, B. (2008). The avian respiratory immune system. In F. Davison, B. Kaspers, & K.A. Schat (Eds.). Avian Immunology 1st edn (pp 273–288). London: Academic Press. Lambrecht, B., Gonze, M., Meulemans, G. & van den Berg, T. (2004). Assessment of the cell-mediated immune response in chickens by detection of chicken interferon-γ in response to mitogen and recall Newcastle disease viral antigen stimulation. Avian Pathology, 33, 343–350. Mast, J., Nanbru, C., Decaesstecker, M., Lambrecht, B., Couvreur, B., Meulemans, G. & van den Berg, T. (2006). Vaccination of chickens embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response. Vaccine, 24, 1756–1765. Meszaros, J. (1991). Aerosol-vaccination against Newcastle disease. Deutsche Tierarztliche Wochenschrift, 98, 117–164. Meszaros, J., Szemeredi, M. & Tamasi, G. (1992). Immunization of day-old chickens against Newcastle disease. Acta Veterinaria Hungarica, 40, 121–127. Muir, W.I. (1998). Avian intestinal immunity: basic mechanism and vaccine design. Poultry and Avian Biology Reviews, 9, 87–106. Palya, V., Penzes, Z., Horváth, T., Kardi, V., Dorsey Moore, K. & Gardin, Y. (2008). Comparative efficacy of several vaccination programmes including or not recombinant HVT-ND vaccine against challenge with mexican Chimalhuacan NDV strain. Proceedings of the 57th Western Poultry Disease Conference & XXXIII Annual ANECA convention (p. 36). Puerto Vallarta, Mexico. Rauw, F., Anbari, S., van den Berg, T. & Lambrecht, B. (2009a). New tools to measure peripheral and local NDV cell-mediated immunity in chickens. Proceedings of the 3rd European Veterinary Immunology Workshop (EVIW) (p. 30). Berlin, Germany. Rauw, F., Gardin, Y., Palya, V., Van Borm, S., Gonze, M., Lemaire, S., van den Berg, T. & Lambrecht, B. (2009b). Humoral, cell-mediated and

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mucosal immunity induced by oculo-nasal vaccination of one-day-old SPF and conventional layer chicks with two different live Newcastle disease vaccines. Vaccine, 27, 3631–3642. Rauw, F., Gardin, Y., Palya, V., Anbari, S., Lemaire, S., Boschmans, M., van den Berg, T. & Lambrecht, B. (2010a). Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine, 28, 823–833. Rauw, F., Gardin, Y., Palya, V., Van Borm, S., Gonze, M., Lemaire, S., van den Berg, T. & Lambrecht, B. (2010b). The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chicken’s vaccination with live Newcastle disease vaccine. Veterinary Immunology and Immunopathology, 134, 249–258. Rauw, F., Anbari, S., van den Berg, T. & Lambrecht, B. (2011). Measurement of systemic and local respiratory cell-mediated immunity after influenza infection in chickens. Veterinary Immunology and Immunopathology, 143, 27–37. Russell, P.H., Dwivedi, P.N. & Davison, T.F. (1997). The effect of cyclosporin A and cyclophosphamide on the populations of B and T cells and virus in the Harderian gland of chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus. Veterinary Immunology and Immunopathology, 60, 171–185. Sato, H., Oh-hira, M., Ishida, N., Imamura, Y., Hattori, S. & Kawakita, M. (1987). Molecular cloning and nucleotide sequence of P, M and F genes

of Newcastle disease virus avirulent strain D26. Virus Research, 7, 241–255. Tsukamoto, K., Saito, S., Saeki, S., Sato, T., Tanimura, N., Isobe, T., Mase, M., Imada, T., Yuasa, N. & Yamaguchi, S. (2002). Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. Journal of Virology, 76, 5637–5645. Van Borm, S., Steensels, M., Ferreira, H.L., Boschmans, M., De Vriese, J., Lambrecht, B. & van den Berg, T. (2007). A universal avian endogenous real-time reverse transcriptase-polymerase chain reaction control and its application to avian influenza diagnosis and quantification. Avian Diseases, 51, 213–220. Van Boven, M., Bouma, A., Fabri, T.H.F., Katsma, E., Hartog, L. & Koch, G. (2008). Herd immunity to Newcastle disease virus in poultry by vaccination. Avian Pathology, 37, 1–5. Wakenell, P.S., Bryan, T., Schaefer, A.E., Avakian, A., Williams, C. & Whitfill, C. (2002). Effect of in ovo vaccine delivery route on herpesvirus of turkeys-SB-1 efficacy and viremia. Avian Diseases, 46, 274–280. Zoth, S.C., Gomez, E., Carrillo, E. & Berinstein, A. (2008). Locally produced mucosal IgG in chickens immunized with conventional vaccines for Newcastle disease virus. Brazilian Journal of Medical and Biological Research, 41, 318–323.

EFFICACY OF SEVERAL VACCINATION PROGRAMS AGAINST NEWCASTLE DISEASE EFFICACY OF SEVERAL VACCINATION PROGRAMS AGAINST NEWCASTLE DISEASE CHALLENGE. 1

CHALLENGE.

Satra, J. , Trakarnrungsee, S. , Chanthaworn, T. , Thaopeth, W , Paniago, M.T (), Turblin, V. 1 1 1 .1 .2 2 Satra, J. , Trakarnrungsee, S. , Chanthaworn, T. , Thaopeth, W , Paniago, M.T (), Turblin, V. 1Veterinary Biologics Assay Division, Bureau of Quality Control of Livestock Products, Department of 1Veterinary Biologics Division, 1212 Bureau of Quality Control of Livestock Products, Department of Livestock Assay Development. Pakchong, Nakhonratchasima 30130. Thailand

foreign antigens related to vaccine-induced

climatic conditions, past performance of

study, the Herpes Virus of Turkey expresses Malaysia

vaccination programs and others (1, 2, 3, 7).

the F gene of the Newcastle Disease virus.

RESUMEN However, despite all these RESUMEN variables, the use of a live ND vaccine in Seis grupos de pollitas comerciales DOC through spray in the hatcheries is Seis de grupos pollitas comerciales de un día edaddefueron colocadas en commonly done in several parts of the en de un día de edad fueron colocadas aisladores. En el primer día, los grupos A, B, world. In endemic regions, oil-emulsion aisladores. En elND primer día, los grupos C, D fueron vacunados contra A, B, la inactivated vaccines have also been la C, D fueron vacunados contra enfermedad de Newcastle con la vacuna successfully usedde in DOC (1) and theirlamajor enfermedad Newcastle vacuna vector HVT-NDV, vacuna decon cepa entérica advantages are the very low level of adverse vector HVT-NDV, vacuna de cepa entérica apatógena viva, cepa entérica apatógena reactions in vaccinated birds and extremely apatógena viva, cepa entérica apatógena viva + vacuna vector HVT-NDV y cepa high levels protective antibodies of long viva + of vacuna vector HVT-NDV y cepa entérica apatógena viva + vacuna ND duration that can be achieved (2).ND entérica apatógena viva + vacuna inactivada, respectivamente. Los grupos E y Furthermore, special inactivated vaccines inactivada, respectivamente. Losservir grupos Ey F no fueron vacunados para como have been developed for day-old or early Fcontroles. no fueron vacunados para servir como agecontroles. application by either subcutaneous (SC)

This vaccine can be administered by SC En el día 14, los grupos A, B, C, D y route to DOC injection En elor díaby 14,“in-ovo” los grupos A, B, C,atD y E tuvieron tasas thde protección de 90, 90, 90, around the 18 de day of embryonic E protección 95tuvieron y 90%, tasas respectivamente. Ende el 90, día90, 21,90, la development and its major advantage is to la 95 y 90%, respectivamente. En el día 21, tasa de protección fue de 100, 75, 95, 90 y avoid the protección interference MDA , in NDV tasa fuewith de 100, 75, 70%, de respectivamente. Finalmente, a95,los9028y addition to eliciting a high and long lasting 70%, respectivamente. Finalmente, los 55, 28 días, la tasa de protección fue de a100, immunity against ND. días, la ytasa derespectivamente. protección fue de 100, 55, 100, 90 10%, 100, 90 y 10%, respectivamente. However, as the protection against En general, el mejor tipo de ND depends the replication of the vector de Enonfue general, el mejor protección inducida por la tipo vacuna virus, it takes several days before it reaches protección fue inducida por la vacuna vector HVT-NDV, con o sin el uso a sufficient immune stimulation. Therefore, vector HVT-NDV, o enterotrópica sin el uso concomitante de la con vacuna in concomitante order to compensate for this delay in de la vacuna enterotrópica viva. onset viva.of immunity and ensure an optimal protection of the chickensDisease, during vaccination the first Key words: Newcastle words: Newcastle Disease, vaccination fewKey weeks of life, a vaccination programme programs, vector HVT-NDV vaccine, live

vaccination

equipment,

route

Livestock Development. 1212 Pakchong, Nakhonratchasima 30130. Thailand immunity against poultry diseases (11). In administration, quality and health of the 2Ceva Animal Health Asia Pacific, 3.06, Level 3, Wisma Academy, Lot 4A, Jln 19/1 46300 Kuala Lumpur, the case ofLot ND4A, vaccine in Kuala the present day-old chicks sizePacific, of the 2Ceva Animal(DOC), Health Asia 3.06,flock, Level 3, Malaysia Wisma Academy, Jln 19/1used 46300 Lumpur,

or intramuscular routes. area much Los desafíos se They hicieron los días Los desafíos se hicieron a los more concentrated than conventional oilydías 14, 21 y 28 días de edad con un virus ND 5 un

14, 21used y 28for días deflocks. edadde con10 EID virus por ND vaccines layer velogénico en una dosis 50 5 velogénico en una dosis de 10 EID50 por ave por vía intramuscular. Thevía intramuscular. association of live and ave por inactivated ND vaccines in DOC has been INTRODUCTION extensively investigated and the results INTRODUCTION show that Newcastle the HI titers, protection disease (ND) against is caused Newcastle disease (ND) is challenge and persistence of immunity are by specified viruses of the caused avian by specified viruses of the avian better achieved when the combination of of paramyxovirus type I (APMV-I) serotype

type I vaccines (APMV-I) of liveparamyxovirus inactivated isserotype used, theand genus Avulavirus belonging to the the genus Avulavirus belonging to the compared to live or inactivated alone (5,family 6, subfamily Paramyxovirinae, subfamily family 9, 15). The benefitParamyxovirinae, of the combination of Paramyxoviridae (10). Since its recognition Paramyxoviridae (10). Since its recognition liveinand killed in the hatchery is 1926, ND vaccine is regarded as being endemic

in NDcountries isinregarded endemic particularly a context strong viral in 1926, manyclear andasofbeing its prevention in many countries itsof control: prevention pressure as three it strengthens and prolongs the (a) includes differentand levels

includes differentthe levels control: (a) protection combining localof immunity control by atthree International Level, which means control at International Level, which means provided by of live vaccine definition theattenuated disease, import andwith export definition of the disease, import and export humoral immunity (circulating restrictions, notification to antibodies) international restrictions, notification to of international conferred by like inactivated agencies, OIE, andvaccines. control movement agencies, like OIE, and control of movement of personnel, (b) control at National Level, Recently, a new generation of of personnel, (b) control at National Level,

64 /

PROCEEDINGS OF THE 17TH WORLD VETERINARY POULTRY ASSOCIATION CONGRESS, 2011 AUGUST 14-18, CANCUN, MEXICO (PP. 909-914).

Scientific File • N°10

N°9 • Scientific File

programs, vector HVT-NDV vaccine, combining a vector HVT-NDV vaccine withlive apathogenic enteric strain vaccine, apathogenic enteric strain vaccine, inactivated ND ND vaccine a live attenuated or ND/IB vaccine has inactivated ND vaccine been recommended by the manufacturers for regions with high disease pressure.

which includes quarantine, monitoring and The objective of this trial was toand which includes quarantine, reporting system, slaughter monitoring and vaccination compare the system, level of slaughter protectionand induced by reporting vaccination policies and, at last, (c) control at Farm several different vaccination programs policies and, means at last,mainly (c) control at Farm Level, which biosecurity and against a challenge with a virulent NDand Level, which means mainly biosecurity vaccination (4). strain. vaccination (4). Despite being a small part of the Despite a small partplays of the global control ofbeing ND, vaccination an global control ofinND, an important roleMETHODS thevaccination preventionplays of this MATERIAL AND important role in the of this disease. However, an prevention ideal vaccination disease. However, idealdefined vaccination Day-old chicks program cannot be aneasily as it

program cannot defined inasthe it would depend on be leveleasily of challenge Four hundred and of fifty (450) daywould depend on level challenge in the field, disease control polices in the country, oldfield, pullets, Lohmann were divided controlbreed, polices in the country, level disease of maternally derived antibodies intolevel six groups. The groups A, B, C and D maternally againstof NDV (MDANDVderived ), type antibodies of birds contained of 100 chicks each, the group E against (MDANDV ), type of birds (broiler NDV or layers), vaccine strain, the and(broiler F hador 40layers), chicks vaccine and 10 strain, chicks,the

respectively. vaccines using the recombinant technology XVII WVPA Congress. Cancun, Mexico has been developed, among which the XVII WVPA Congress. Cancun, Mexico

/ 65

909

N°10 • Scientific File foreign antigens related to vaccine-induced

The groups A, B, C and D were

administration, quality and health of the

immunity against poultry diseases (11). In

vaccinated at day-old as described here

day-old chicks (DOC), size of the flock,

the case of ND vaccine used in the present

below and were kept in isolators.

climatic conditions, past performance of

study, the Herpes Virus of Turkey expresses

vaccination programs and others (1, 2, 3, 7).

the F gene of the Newcastle Disease virus.

vaccination

equipment,

However,

route

despite

all

these

variables, the use of a live ND vaccine in DOC through spray in the hatcheries is commonly done in several parts of the world. In endemic ND regions, oil-emulsion inactivated

vaccines

have

also

been

successfully used in DOC (1) and their major

route to DOC or by “in-ovo” injection at around

the

18th

day

the

antibodies was assessed by HI test using 4

vaccine by eye drop (ED) route (0.03 ml per

development and its major advantage is to addition to eliciting a high and long lasting

Group C: Live apathogenic enteric strain

immunity against ND.

(ED) + Vector HVT-NDV vaccine (SC);

reactions in vaccinated birds and extremely

ND depends on the replication of the vector

(ED) + Inactivated ND vaccine (SC, 0.1 ml

high levels of protective antibodies of long

virus, it takes several days before it reaches

per chick).

duration

(2).

a sufficient immune stimulation. Therefore,

Furthermore, special inactivated vaccines

in order to compensate for this delay in

have been developed for day-old or early

onset of immunity and ensure an optimal

age application by either subcutaneous (SC)

protection of the chickens during the first

or intramuscular routes. They are much

few weeks of life, a vaccination programme

more concentrated than conventional oily

combining a vector HVT-NDV vaccine with

vaccines used for layer flocks.

a live attenuated ND or ND/IB vaccine has

association

achieved

live

and

inactivated ND vaccines in DOC has been

Group E: Positive Control (unvaccinated but

show that the HI titers, protection against

compare the level of protection induced by

challenge and persistence of immunity are

several

better achieved when the combination of

against a challenge with a virulent ND

live and inactivated vaccines is used,

strain.

different

vaccination

pressure as it strengthens and prolongs the protection by combining the local immunity provided by live attenuated vaccine with humoral immunity (circulating antibodies) conferred by inactivated vaccines. Recently,

a new generation of

vaccines using the recombinant technology

vaccination, 20 birds per group were sampled and the sera were also tested after each challenge, blood samples were taken from each of the survivors in all groups. For easier interpretation and to

Group F: Negative Control (unvaccinated

and Elisa tests at once in all sera.

frozen till the last sampling, to perform HI

The results of the protection after challenge

strain (Lopburi strain – ICPI 1.86) was used

at 3, 7 and 10 days post-vaccination are summarized in the Table 1.

as a challenge virus for the groups A, B, C, D

programs

and E. The challenge was carried out by 5

intramuscular inoculation at a dosage of 10

EID50 per chick. Day-old chicks from group F were

MATERIAL AND METHODS

not

challenged

serve

days

post-

vaccination: All groups showed a very good level of protection varying between 90-95% and

observed during 14 days and clinical signs and mortality recorded.

old pullets, Lohmann breed, were divided

challenged

(negative controls). The chicks were kept in isolators,

Four hundred and fifty (450) day-

Birds

unvaccinated and unchallenged controls

Day-old chicks

without any major differences among them. This is probably due to a high residual amount of maternal immunity.

into six groups. The groups A, B, C and D contained of 100 chicks each, the group E

Birds

and F had 40 chicks and 10 chicks, 1

recombinant HVT (rHVT) is considered as Groups design

XVII WVPA Congress. Cancun, Mexico

Vectormune HVT-NDV – Ceva Santé Animale - France 2 Cevac® Vitapest L – Ceva Santé Animale France 3 Cevac® Broiler ND K – Ceva Santé Animale France

respectively.

has been developed, among which the the most potent vector for expressing

strain and a commercial Elisa test kit

challenged with virulent NDV)

The reference Thai NDV challenge

9, 15). The benefit of the combination of particularly clear in a context of strong viral

HAU of Newcastle disease virus, Lopburi

RESULTS

compared to live or inactivated alone (5, 6, live and killed vaccine in the hatchery is

derived

reduce variability, all the samples were kept

Challenge

for regions with high disease pressure. The objective of this trial was to

maternally

and unchallenged)

been recommended by the manufacturers

extensively investigated and the results

the

using both HI and Elisa test. Finally, 14 days

Group D: Live apathogenic enteric strain

can

At 14, 21 and 28 days post-

However, as the protection against

that

level

chick);

avoid the interference with MDANDV, in

advantages are the very low level of adverse

The

66 /

Group A: Vector HVT-NDV vaccine by SC injection (0.2 ml per chick) Group B: Live apathogenic enteric strain ND

embryonic

At day of age, before the beginning of the trial, 20 surplus birds were bled and

This vaccine can be administered by SC

Serology

910

challenged

days

post-

vaccination: 4

Newcastle Disease Antibody Test Kit, BioChek, Product Code CK119, Serial No. FS5010, Reader ELx800, Bio-Tek instrument INC.

XVII WVPA Congress. Cancun, Mexico

911 / 67

NÂ°10 â&#x20AC;˘ Scientific File At three weeks after vaccination, some differences programs

among start

the be

vaccination noticed.

At day 21, both serological tests indicate that the chicks from group D

The

started to show a better seroconversion as

unvaccinated control group still showed

compared to other groups. In fact, the HI

70% and this result was very similar to the

test results showed that group A chicks also

one obtained by the group B, which was

had a better seroconversion as compared to

vaccinated only with a live apathogenic

groups B, C and E.

enteric strain. The groups A, C and D showed a very high level of protection, particularly the group A which achieved 100% of protection.

These results are in accordance with

challenged

of a live ND live vaccine at day of age induced

days

post-

vaccination: At

four

weeks

the

protection rate of the unvaccinated group was only 10%. The group vaccinated with

of the serology are similar to control group.

birds from groups A, which were vaccinated

These results suggest that other immune mechanisms such as local and cellular immunity are involved.

live and inactivated vaccines presented the best seroconversion. Chickens from the

The

only the live apathogenic enteric strain (group B) achieved 55% while groups A, C and D had very high levels of protection. Once again, only the groups vaccinated with vector HVT-NDV reached 100% of protection.

live and vector HVT-NDV and group C, vaccinated with a live apathogenic strain had

reasonably

good

had a strong seroconversion reaching a geometrical mean titer (GMT) of 6001 while groups A and C achieved titers of 1196 and 1537, respectively. The groups B and E were

negative, titers ranging from 828 and 1158, suspect and titers above 1159 are positive.

stimulus of

defense

ND vaccine by eye-drop and (c) live ND vaccine by eye drop were challenged at 3, 4, 5 and 6 weeks of age. The vector HVT-NDV + a live ND combined vaccination regimen

Under the conditions in which this trial was carried out, the best protection

stimulation of the active immunity while the

rate was induced by the vector ND vaccine,

passive immunity declines and the immune

with or without the concomitant use of the

system reaches full competence (5, 6, 15).

live apathogenic enteric vaccine. Despite

hampered

the

interference of vaccines with a usually high level of passive immunity (13). In fact, it is

and Elisa tests obtained 14 days after each

live NDV vaccination (5, 8, 12, 14) impairing

passive immunity against ND suggests that

challenge are summarized in the Table 3.

the capacity of conventional ND vaccine to induce protection.

When the challenge was carried out 14 days after vaccination, the results of both

In this context, the use of vector ND

At 14 days of age, both tests

serological tests are similar among all the

vaccines, which escape the interference with

showed the expected decrease in the

groups. However, when the challenge was

MDANDV. At this point, no vaccination

MDANDV, becomes even more interesting.

done at 21 and 28 days post-vaccination, HI

Under the conditions in which this trial was

regimen was able to induce a measurable

and

the

carried out, the groups vaccinated with

active

considerably

seroconversion of the chickens in the

vector HVT-NDV vaccine, associated or not

different from the unvaccinated control

groups A and C tends to be lower than the

with a live ND vaccine, induced the best

group.

other groups.

results, reaching full protection.

912

CONCLUSIONS

these circumstances, there is a progressive

well-known that MDANDV can interfere with

XVII WVPA Congress. Cancun, Mexico

through in-ovo route associated with live

mechanism and disperse antigen slowly. In

The serological results from the HI

immunity

through in-ovo route, (b) Vector HVT-NDV

weeks of age.

was nearly 7log2 and 8767 in the HI and

humoral

HVT-NDV

full protection against challenge from 3 to 6

Indeed,

Elisa tests, respectively. This high level of

that

Vector

protection

enzootic and where there is high pressure

Serological results after challenge

show

(a)

inactivated ND vaccines induced a good

immunization

results

with

program tested in the trial as it provided

challenge.

live

from the field, the need for very early

Elisa

vaccinated

and

against

However, in regions where ND is

summarized in the Table 2.

68 /

combination

adjuvant acts

The HI and Elisa test results are

breeder flock.

commercial broilers. Groups of chicks were

was superior to any other vaccination

immunity as live vaccines (6) because the oil

show that only chickens from the group D

the DOC came from a well vaccinated

induced by different vaccination regimes in

to be as adversely affected by maternal

seroconversion. The results from Elisa test

guidelines, titers below 827 are considered

The level of MDANDV at day of age

the virulent Mexican Chimalhuacan strain

inactivated oil-emulsion vaccines seem not

negative. According to the manufacturer

Serological results after vaccination

protection

unvaccinated group even though the results

it is rather clear in the HI test results that

alone,

better

weeks of age when compared to the

Finally, at 28 days after vaccination,

groups B, vaccinated with a combination of post-vaccination,

significantly

against challenges carried out at 3 and 4

with vector HVT-NDV vaccine, and group D,

protection against mucosal challenge with

This trial results show that the use

which were vaccinated simultaneously with Birds

Palya et al (2008), who compared the

DISCUSSION

the maternal immunity managed to protect the progeny for the first 2-3 weeks, it did not prevent the vector HVT-NDV vaccine take at all. The association of a live and inactivated ND vaccines also induced strong level of protection.

REFERENCES 16. Alexander, D.J and Jones, R.C. Newcastle Disease. In: F.T.W Jordan (Ed.) Poultry Diseases, 5th Edition, WB Sanders, 2001. 17. Alexander, D.J and Jones, R.C. Newcastle Disease, Other Avian Paramyxovirus, and Pneumovirus Infections. In: Y.M. Saif (Ed.) Diseases of Poultry, 11th Edition, p. 63-92. Iowa State Press, 2003.

XVII WVPA Congress. Cancun, Mexico

913 / 69

Scientific File • N°11

N°10 • Scientific File

PROCEEDINGS OF THE 17TH WORLD VETERINARY POULTRY ASSOCIATION CONGRESS, 2011 AUGUST 14-18, CANCUN, MEXICO (PP. 909-914).

VECTORMUNE® ND

18. Al-Garib, S.O., Gielkens, A.L.J., Gruys, E. and Koch,G. Review of Newcastle disease virus with particular references to immunity and vaccination. World’s Poultry Science Journal, V. 59, p. 185200, 2003. 19. Bennejean, G. Newcastle Disease: Control Policies In: D.J Alexander (ed.). Newcastle Disease. Kluwer Academy Publishers. 318322, 1988.

20. Bennejean, G., Guittet, M., Picault, J.P., Bouquet, J.F., Devaux, B., Gaudry, D. Moreau, Y. Vaccination of day-old chicks against Newcastle Disease using inactivated oil adjuvant vaccine and/or live vaccine. Avian Pathology, v. 7, n.1, p. 15-27, 1978. 21. Box, P.G., Furminger, I.G.S., Robertson, W.W, Warden, D. The effect of Marek’s Disease vaccination on immunity of day-old chicks against Newcastle Disease, using B1 and oil emulsion vaccine. Avian Pathology, v.5, p. 299305, 1976. 22. Butcher, G.D., Miles, R.D., Nilipour, A.H. Newcastle and Infectious Bronchitis Vaccine Reactions in Commercial Broilers. University of Florida. (http://edis.ifas.ufl.edu/BODY_VM097). Accessed in 20/07/2004. 23. Ganapathy, K., Todd, V., Cargill, P.W., Montiel. E., Jones, R.C. Interaction between a live avian pneumovirus vaccine and two different Newcastle disease virus vaccines in broiler chickens with maternal antibodies to Newcastle disease virus. Avian Pathology, v.35, n.6, p.429–434, 2006. 24. Giambrone, J.J and Clay, R.P. Vaccination of Day-old Broiler Chicks Against Newcastle Disease and Infectious Bursal Disease Using Commercial Live and/or Inactivated Vaccines. Avian Diseases, v. 30, p. 557561, 1986.

70 /

25. Office International Des Epizooties (OIE). Newcastle Disease. In : OIE Terrestrial Manual, Chapter 2.3.14, p. 576-589, 2009. 26. Palya, V., Penzes, Z., Horvath, T., Kardi, V., Dorsey Moore, K., Gardin, Y. Comparative efficacy of several vaccination programs including or not including recombinant HVT-NDV vaccine against challenge with Mexican Chimalhuacan NDV strain. In: Proceedings of the Fifty-seventh Western Poultry Disease Comference. Jalisco, Mexico. April 9-12, 2008. 27. Rauw, F., Gardin, Y., Palya, V., van Borm, S., Gonze, M., Lemaire, S., van den Berg, T., Lambrecht, B. Humoral, cellmediated and mucosal immunity induced by oculo-nasal vaccination of one-day-old SPF and conventional layer chicks with two different live Newcastle disease vaccines. Vaccine, v. 27 p. 3631– 3642, 2009. 28. Rauw, F., Gardin, Y., Palya, V., Anbari, S., Lemaire, S., Boschmans, M., van den Berg, T., Lambrecht. Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine, v. 28, p. 823–833, 2010. 29. Russell, P.H. Newcastle disease virus: virus replication in the Harderian gland stimulates lacrimal IgA; the yolk sac provides early lacrimal IgG. Veterinary Immunology and Immunopathololy, v. 37, p. 151–163, 1993. 30. Warden, D. Furminger, I.G.S., Robertson, W.W. Immunizing Chicks against Newcastle Disease by Concurrent Inactivated Oil-emulsion and Live B1 Vaccines. Veterinary Record, v.18, p. 6566, 1975.

Simultaneous administration of rHVT-F (Newcastle Disease) and rHVT-H5 (Avian Influenza) vector vaccines reduces, but does not prevent, development of immunity against both diseases. Fabienne Rauw, Yannick Gardin, Vilmos Palya, Timea Tata-Kis, Kristi Moore Dorsey, Thierry van den Berg, Bénédicte Lambrecht Newcastle Disease and Avian Influenza are two major poultry diseases severely affecting production of many countries in Asia, Africa and Central America. As a consequence, in these regions, prevention programs routinely include vaccinations against both threats. Because of their capacity to overcome passive immunity and induce a more reliable, broader spectrum and longer lasting immunity than classical, live or inactivated, vaccines (1, 2), the recently developed rHVT-F (Newcastle Disease) and rHVT-H5 (Avian Influenza) vector vaccines are more and more frequently used in the field. Since both vaccines, are indicated for in-ovo or subcutaneous injection at the hatchery, questions regarding the possibility of simultaneous application have arisen. Previous experiments demonstrated lower protections when a rHVT-F (Newcastle Disease) and a rHVT-VP2 (Infectious Bursal Disease), or a rHVT-F and a rHVT-LT (Infectious LaryngoTracheitis) were administered simultaneously to commercial broilers (3, 4). On the reverse, other experiments conducted in SPF chickens have shown no or little interference between a rHVT-F and a rHVT-H5 administered together (5). The aim of this experiment was to assess the consequences of simultaneous administration of a rHVT-F (Vectormune ND – Ceva) and a rHVT-H5 (Vectormune AI – Ceva) vector vaccines to day-old, maternally immune or not, commercial layer pullets on various aspects of vaccines induced immunity. Investigations included: (i) protection against mortality, morbidity and viral shedding against ND and AI challenges applied at 4, 7 and 10 weeks of age, (ii) development of NDV and AIV specific HI, IgG, IgM and IgA antibody mediated immunity in various compartments of the chicken body including serum, digestive and respiratory tracts, (iii) development of NDV and AIV specific cell mediated immunity in the blood and in the spleen. The results indicated that maternally derived antibodies delayed, but did not prevent, onset of immunity. This interference is lower than the one observed with classical vaccines. The simultaneous application of the two vector vaccines did not add extra delay to the development of immune responses, but reduced the values of some of the parameters investigated. It was concluded that the observed interferences were acceptable, but should be taken into consideration depending on local epidemiological conditions. Keywords : Newcastle Disease, Avian Influenza, rHVT vector vaccines References : 1. Rauw et al, (2010). Vaccine, 28, 823-833 2. Rauw et al. (2011). Vaccine, 29, 2590-2600 3. Godoy et al. (2008). Proc. of the 145th AAAP meeting, New Orleans, July 19-23, p.76. 4. Godoy et al. (2011). Proc. of the 148th AAAP meeting, Saint Louis, July 16-19, p.33. 5. Rauw et al. (2012). Proc. of the 8th AI symposium, London. / 71

Scientific File • N°12

PROCEEDINGS OF THE 8TH AI SYMPOSIUM, LONDON, ENGLAND.

VECTORMUNE® ND

Scientific File • N°13

PROCEEDINGS OF THE AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS ANNUAL MEETING, 2011, ST. LOUIS, MISSOURI.

VECTORMUNE® ND

Lack of interference between rHVT-H5 and rHVT-F vaccines administrated simultaneously to day-old chickens and efficacy Lack of interference between rHVT-H5 and rHVT-F vaccines against AI and ND challenges performed at 4 or 8 weeks of age

administrated simultaneously to day-old chickens and efficacy CODA Veterinary CODA CERVA CERVA Veterinary and and Agrochemical Agrochemical Research Research Center Center -- Groeselenberg Groeselenberg,,99 B-1180 BRUSSELS BRUSSELS against AI and ND challenges performed at 4 or998B-1180 weeks of age phone : +32(0)2 379 04 00 BELGIUM phone : +32(0)2 379 www.var.fgov.be 04 00 www.var.fgov.be

CODA CODA CERVA CERVA

Veterinary Veterinary and and Agrochemical Agrochemical Research Research Center Center -- Groeselenberg Groeselenberg,,99 99

B-1180 B-1180 BRUSSELS BRUSSELS

AUTHORS: F. Rauw1, V. Palya2, T. Tatar-Kis2, K. Moore Dorsey3, T. van den Berg1, B. Lambrecht1 and Y. Gardin3*. 1

Avian Virology & Immunology ;

phone : +32(0)2 379 04 Lenexa, 00 BELGIUM phone +32(0)2 379 www.var.fgov.be 04 www.var.fgov.be Ceva Phylaxia, Budapest, Hungary ; 3 Ceva: Biomune, USA00 ; 4 Ceva Santé Animale, Libourne, France

2, T. Tatar-Kis2, K. Moore Dorsey3, T. van den Berg 1, B. Lambrecht 1 and Y. Gardin3*. ABSTRACT Experimental design AUTHORS: F. Rauw1, V. Palya 1

Avian Virology & Immunology ;

Ceva Phylaxia, Budapest, Hungary ;

Ceva Biomune, Lenexa, USA ;

The recombinant turkey herpesvirus vaccine expressing the H5 gene from a clade 2.2 H5N1 HPAIV strain (rHVT-H5) inoculated subcutaneously at day-old afforded a good protection of broiler SPF chickens against challenge with two antigenically highly ABSTRACT divergent Egyptian H5N1 clade 2.2.1 HPAIV [1]. This vaccination was also proved to be highly effective in broilers with H5N1 maternally derived antibodies (MDA). The rHVT The recombinant expressing the H5subcutaneously gene from a at clade 2.2 expressingturkey the F herpesvirus gene from vaccine NDV (rHVT-F) inoculated day-old H5N1 HPAIV strain (rHVT-H5) inoculated subcutaneously at day-old afforded a good afforded a good protection against challenge with velogenic NDV strain in layers with protection ofMDA broiler NDV [2]. SPF chickens against challenge with two antigenically highly objective of this trial 2.2.1 was toHPAIV evaluate if the rHVT-H5 and rHVT-F could divergentThe Egyptian H5N1 clade [1]. Thistwo vaccination was also vaccines proved to be be inoculated simultaneous at day-old without havingantibodies any impact(MDA). on immunity and highly effective in broilers with H5N1 maternally derived The rHVT efficacy (protection and virus shedding) of each vaccine. SPF chickens immunized with expressing the F gene from NDV (rHVT-F) inoculated subcutaneously at day-old vaccines showed an excellent protection against either Egyptian H5N1 clade 2.2.1 afforded both a good protection against challenge with velogenic NDV strain in layers with HPAIV or a velogenic Malaysian NDV challenges performed at 4 or 8 weeks of age. No NDV MDAsignificant [2]. difference of both AI and ND-specific humoral, cell-mediated and digestive The objective of this trial was to evaluate the two rHVT-H5 rHVT-F vaccines could antibody-mediated immunity was ifobserved between and rHVT-H5/rHVT-F vaccinated be inoculated simultaneous day-oldvaccinated without birds, havingrespectively. any impact immunity and chickens and rHVT-H5 at or rHVT-F Ouron study demonstrate efficacy (protection virus shedding) of the eachtwo vaccine. chickens immunized with the absence and of interference between vaccinesSPF when administrated together in both vaccines showed conditions. an excellent protection inoculation against either Egyptian H5N1 clade 2.2.1 our laboratory Simultaneous of both rHVT vaccines in hatchery might allow simultaneous protection against Avian Influenza and Newcastle Disease. HPAIV or a velogenic Malaysian NDV challenges performed at 4 or 8 weeks of age. No

Ceva Santé Animale, Libourne, France

Experimental design

significant difference of both AI and ND-specific humoral, cell-mediated and digestive antibody-mediated immunity was observed between rHVT-H5/rHVT-F vaccinated Results and discussion chickens and rHVT-H5 or rHVT-F vaccinated birds, respectively. Our study demonstrate the absence of interference between the two vaccines when administrated together in Protection against mortality our laboratory conditions. Simultaneous inoculation of both rHVT vaccines in hatchery Challenge might allow simultaneous protection against Avian Influenza and Newcastle Disease. Group NDV challenge AIV challenge timing Group 4 weeks 8 weeks 4 weeks 8 weeks Negative Negative

rHVT-‐AI N.D. N.D. 100% Protection against mortality rHVT-‐ND 100% 100% N.D. rHVT-‐AI/rHVT-‐ND 100% NDV challenge

Group

0% 100%

Results and discussion rHVT-‐AI Excretion after AIV challenge

N.D.

100% AIV 100% challenge 90%

When administrated separately by subcutaneous route at day-old, the rHVT-AI and the rHVT4 w100% eeks protection 8 weeks against 4 weeks 8 weeks ND afforded mortality and morbidity against challenge at 4 and 8 AIV et NDV0% was also Excretion observed when both vaccines were after Negative weeks. Complete 0% protection 0% against0% inoculated simultaneously, except after AIV challenge done 8 weeks when a somewhat AIVatchallenge rHVT-‐AI lower protection N.D. by 10 % N.D. could be 100% observed. 100%

rHVT-‐ND

100%

N.D.

100%

90%

NDV-specific humoral immunity

rHVT-‐AI/rHVT-‐ND

HI (La Sota)

AIV-specific humoral immunity HI (Egypt H5N1 2008)

When administrated separately by subcutaneous route at day-old, the rHVT-AI and the rHVTND afforded 100% protection against mortality and morbidity against challenge at 4 and 8 weeks. Complete protection against AIV et NDV was also observed when both vaccines were inoculated simultaneously, except after AIV challenge done at 8 weeks when a somewhat lower protection by 10 % could be observed. NDV-specific humoral immunity

HI (Egypt H5N1 2008)

HI (La Sota) IgM

IgM

AIV-specific humoral immunity

IgG

HI (Hungary H5N1 2006)

HI (Hungary H5N1 IgG 2006)

4 weeks

Cloaca D10

3/3

D10 2/10

3/3

7/10

3/10

0/10

2/10

rHVT-‐ND

N.D.

rHVT-‐AI/rHVT-‐ND

6/10

7/10

3/10

0/10

Negative Challenge

timing rHVT-‐AI

8 weeks

Oropharynx D2

Group

9/9

Oropharynx

4/10

9/9

N.D.

Cloaca

2/10 0/10 N.D. D2N.D. D4 N.D.

1/10

D7 N.D.

0/10

1/10

rHVT-‐ND

Negative rHVT-‐AI/rHVT-‐ND

7/10 3/3 9/10

1/10

0/10

3/3 1/10

0/10

2/10

D10 N.D.

D2 N.D.

D4 N.D.

N.D.D7

D10

rHVT-‐AI with rHVT-AI 7/10vaccine 7/10 allowed 3/10 3/10 0/10 2/10 of 2/10 2/10 The the reduction viral shedding 4 wvaccination eeks rHVT-‐NDand cloacaN.D. N.D. after N.D. AIV N.D.challenge, N.D. N.D. N.D. N.D. by oropharynx routes as shown by the comparison of the viral excretion titres3/10 (data0/10 not shown) and 0/10 the number of rHVT-‐AI/rHVT-‐ND 6/10 7/10 0/10 0/10 0/10 excreting birds with the unvaccinated groups. This reduction of shedding Negative 9/9 9/9 induced by the administration at day-old of the rHVT-AI vaccine was not rHVT-‐AI 4/10 2/10 0/10 1/10 0/10 0/10 0/10 1/10 affected 8 weeksby the simultaneous inoculation of the rHVT-ND vaccine. N.D. NDV N.D. challenge N.D. N.D. N.D. N.D. N.D. Analysis ofrHVT-‐ND viral excretion after are inN.D. progress. rHVT-‐AI/rHVT-‐ND 7/10 9/10 2/10 1/10 0/10 1/10 0/10 0/10 NDV and AIV-specific cell-mediated immunity

The vaccination with rHVT-AI vaccine allowed the reduction of viral shedding

L o woropharynx N D V - s p e cand i f i c cloaca C h I F Nroutes γ by after AIV challenge, as shown by the NDV-specific ChIFNγ production by the PBLviral could be comparison of excretion titres (data not shown) and production by PBL the number of m e a s u r e d b y E L I S A a f t e r excreting birds with the ndunvaccinated groups. This reduction of shedding to antigenic recall from the 2 induced by the administration at day-old of the rHVT-AI vaccine was not the 4th week of age in the rHVTaffected by the simultaneous inoculation of the rHVT-ND vaccine. ND vaccinated group only. No Analysis of viral excretion C h I FNγ producti on coul dafter be NDV challenge are in progress. measured on antigen-activated splenocytes due aspecific NDVtoand AIV-specific cell-mediated immunity background in broiler chickens. L oAIV-specific w N D V - sCMI p e was c i f iobserved c ChIFNγ No NDV-specific ChIFNγ after rHVT-AI vaccination (data be production by PBL could production by PBL not m eshown). asured by ELISA after

antigenic recall from the 2nd to the Local 4th week of age in the rHVTNDV and AIV-specific antibody-mediated immunity in the ND vaccinated group only. No tract digestive C h I FNγ producti on coul d be Comparable NDV-specific IgGs IgG measured on antigen-activated specific to were detected in the digestive splenocytes due to aspecific NDV tract from the second week in background in broiler chickens. both rHVT-ND and rHVT-AI/rHVTNo AIV-specific CMI was observed ND groups. Neither IgM nor IgA after rHVT-AI vaccination (data were present. not shown). No AIV-specific Ig was detected

by our home ELISA in the digestive tract of AIH5

vaccinated groups.immunity in the Local NDV and AIV-specific antibody-mediated digestive tract IgA

IgG

IgA

72 /

NDV-specific humoral immunity (IgG and HI) was IgG positive from the 2nd week of age in the rHVT-ND group while AIV-specific humoral immunity appeared one week later in the rHVT-AI group. These specific humoral immunities tended to be higher in groups vaccinated with one rHVT when compared with the group inoculated with the both vaccines, although the difference was not always significant (P < 0.05). NDV-specific IgM and IgA were measured in sera from the 2nd to the 4th week. No AIVspecific IgM and IgA could be detected in sera by our home ELISAs.immunity (IgG and HI) NDV-specific humoral

was positive from the 2nd week of age in the rHVT-ND group while AIV-specific humoral immunity appeared one week later in the

Our study demonstrated the absence of interference between Comparable NDV-specific IgGs IgG the two rHVT-AI and rHVT-ND vaccines on protection against AI specific to detected in the digestive and NDNDV infection when administrated together were at day-old. tract from the second week in Nevertheless, the specific humoral and cell-mediated and rHVT-AI/rHVTimmunities tended to be lower in comparison both with rHVT-ND those groups. Neither IgM nor IgA induced by the vaccine inoculated alone. Further ND experiments will be therefore needed, especially in chickens with maternally were present. derived antibody (in progress). No AIV-specific Ig was detected

References

by our home ELISA in the digestive tract of AIH5 vaccinated groups.

[1] Rauw et al., (2011). Vaccine, 29, 2590-2600 [2] Rauwstudy et al., (2010). Vaccine, 28, 823-833. Our demonstrated the absence

of interference between the two rHVT-AI and rHVT-ND vaccines on protection against AI and ND infection when administrated together at day-old. Nevertheless, the specific humoral and cell-mediated

/ 73

EXTRACT FROM “VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY” 158, 2014 (PP. 105–115).

Scientific File • N°14 VECTORMUNE® ND Veterinary Immunology and Immunopathology 158 (2014) 105–115

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm

Research paper

Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers Vilmos Palya a,∗ , Tímea Tatár-Kis a , Tamás Mató a , Balázs Felföldi a , Edit Kovács a , Yannick Gardin b a b

Scientiﬁc Support and Investigation Unit, Ceva Sante Animale, 5 Szállás utca, 1107 Budapest, Hungary Innovation Strategy Department, Ceva Sante Animale, 10, avenue de la Ballastière, 33500 Libourne, France

a r t i c l e Keywords: Newcastle disease Vaccination Recombinant vaccine rHVT-ND vaccine

i n f o

a b s t r a c t The onset and duration of immunity provided by a recombinant ND vaccine using HVT virus as vector (rHVT-ND) was followed up to 72 weeks of age in commercial layer chickens after single application or as part of two different vaccination regimes including conventional live and killed ND vaccines. Efficacy of the different vaccination programmes was checked, from 3 to 72 weeks of age, by serology as well as by challenges with a recent velogenic NDV isolate belonging to genotype VII. Assessment of protection was done based on the prevention of clinical signs and reduction of challenge virus shedding via the oro-nasal and cloacal routes. Single vaccination with the rHVT-ND vaccine at one day of age provided complete or almost complete (95–100%) clinical protection against NDV challenges from 4 weeks of age up to 72 weeks of age when the latest challenge was done. Shedding of challenge virus both by the oro-nasal and cloacal route was significantly reduced compared to the controls. Booster vaccination of rHVT-ND vaccinated birds with conventional ND vaccines significantly increased the level of anti-NDV serum antibodies and further reduced the oro-nasal excretion of challenge virus. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Newcastle disease (ND) is one of the most important diseases of poultry and other bird species and is a global threat

Abbreviations: APMV-1, avian paramyxovirus serotype 1; dpch, days post-challenge; dpi, days post-infection; EDS, egg drop syndrome; ELD50 , median embryo lethal dose; FP, fowl pox; HAU, haemagglutinating unit; HI, haemagglutination inhibition; HVT, Turkey herpesvirus/herpesvirus of turkeys; IB, infectious bronchitis; IBD, infectious bursal disease; MDA, maternally derived antibodies; ND, Newcastle disease; NDV, Newcastle disease virus; p.v., post-vaccination; rHVT-ND vaccine, recombinant HVT ND vaccine; rHVT/F, recombinant HVT vaccine expressing F gene of NDV; SPF, speciﬁed pathogen free. ∗ Corresponding author at: Ceva-Phylaxia Co. Ltd., 5 Szállás utca, 1107 Budapest, Hungary. Tel.: +36 1 434 4103; fax: +36 1 260 3889. E-mail address: vilmos.palya@ceva.com (V. Palya). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.11.008

74 /

to commercial poultry production. Velogenic strains of ND virus (NDV) cause a devastating disease of poultry throughout Asia, Africa, Middle East, Central and South America till today. NDV, also known as avian paramyxovirus serotype 1 (APMV-1) virus, is a member of the genus Avulavirus in the Paramyxoviridae family. NDV strains are classiﬁed into velogenic (highly virulent), mesogenic (medium virulent) and lentogenic/apathogenic (mild or non-virulent) pathotypes on the basis of their pathogenicity for chickens (Cattoli et al., 2011). The molecular basis for pathogenicity of NDV is mainly determined by the amino acid sequence of the protease cleavage site of the F protein, but other proteins (e.g., V and HN) also contribute to the determination of virulence (Dortmans et al., 2011). The principal antigens that elicit protective immune response are HN and F (Kumar et al., 2011).

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Considerable genetic diversity has been detected among NDV strains, but viruses sharing geographical and/or epidemiological relations tend to fall into specific lineages or clades. Phylogenetic analysis revealed that two major separations occurred during the history of ND. An ancient division in the original reservoir (wild waterfowl species) led to two basal sister classes, class I and II. Ancestors of only class II viruses colonized the chicken populations and subsequently converted to virulent forms. Division continued to occur in the secondary host (chicken) resulting in the branching-off class II viruses, compromising the recent velogenic genotypes (Czeglédy et al., 2006). Different genotypes of class II show geographical region specific occurrence and temporal distribution with apparent links to well defined epizootics (Miller et al., 2010). In the past decades, there has been a major shift in the genotypes of NDV strains that have been identified as prevalent in poultry. Control of Newcastle disease, in addition to good biosecurity practices, primarily relies on preventive vaccination of flocks and culling of infected and at risk of being infected birds (protection zone). Since all ND viruses belong to a single serotype, thus by definition any NDV strain utilized to prepare a vaccine should induce protection against morbidity and mortality following challenge with any virulent NDV strains (Bwala et al., 2009; Perozo et al., 2012). Most countries, where poultry is raised commercially and where the disease is endemic, rely on vaccination to keep the disease under control. At the present time, most vaccination programmes for ND include the use of live (containing lentogenic or apathogenic NDV strains) or inactivated (killed) vaccines in order to induce a good protective immunity while producing minimal adverse effects in the birds. Both types of vaccine have their advantages and disadvantages, but the occurrence of continuous ND outbreaks in commercial poultry flocks in many part of the world indicate that routine vaccination in the field often fails to induce sufficiently high levels of immunity to control ND. Current ND vaccines widely used in commercial poultry can protect the vaccinated birds from disease and reduce virus shedding, but cannot prevent vaccinated birds from being infected and subsequently shedding the virus, and potentially transmitting it to susceptible birds (Dortmans et al., 2012). The presence of maternally derived antibodies (MDA) also interferes with the establishment of an early and persisting immunity after single or even repeated vaccination during the first 2–3 weeks of life. A further consideration regarding conventional ND vaccines is that they might induce a better protection against viruses isolated in past epizootics than against the ones causing the recent outbreaks (Hu et al., 2009; Kapczynski and King, 2005; Miller et al., 2009, 2007; van Boven et al., 2008). The newly emerging virulent NDV strains (genogroup V and VII) have been suggested to have the ability to overcome vaccination barriers. While the causes of the apparent vaccine failures in the field have not clearly been identified in most cases, the efficacy of available conventional vaccines is being questioned based on the findings of the above referred papers. On the contrary, results of Dortmans et al. (2012) indicated that poor vaccination practices and or concurrent infection with

immunosuppressive pathogens rather than antigenic variation may be responsible for poor immunity levels. The shortcomings faced when current ND vaccines and vaccination schemes are used necessitated the search for more potent vaccines, which can be applied more efficiently in the control of ND. A promising approach to achieve the above goals is the development of vector vaccines. The first and foremost advantage for using a vector vaccine is its safety. Some live vaccines used in the poultry industry have some undesirable side-effects, such as horizontal transmission, reversion to virulence and vaccine reactions, any of which may result in disease or production loss (Alexander, 2008). With a vector vaccine, the gene(s) of the donor pathogen is inserted into a ‘safe’ vector, thus separating the key protective antigen from the live donor organism and its undesirable side-effects. Herpesvirus of turkeys (HVT) has already been used worldwide both as live vaccine and as vector for recombinant polyvalent vaccine in poultry. Recombinant vaccines against ND using the herpesvirus of turkeys (rHVT) as vector contain and express the protective antigens, typically the F and/or HN glycoprotein (Morgan et al., 1992). HVT-based recombinant vaccine containing the F protein (rHVT/F) elicited immune response and provided protection against lethal challenge with a velogenic strain of NDV (Morgan et al., 1992). As in case of HVT itself (Purchase et al., 1971), long term virus persistence was shown for rHVT also in inoculated chickens (Reddy et al., 1996), and furthermore, the expression of the F gene was measurable even after 30 weeks of a single s.c. inoculation of day-old chickens (Saitoh et al., 2003). Additionally, the immune response evoked by the rHVT/F construct appeared to be less sensitive to interference with MDA, which adds further useful characteristic to this vector vaccine (Morgan et al., 1993). Beyond that, the application of this kind of vaccine proved to be safe since it did not have adverse effects on hatchability or the survival of in-ovo and post-hatch vaccinated specified-pathogen-free (SPF) chickens (Morgan et al., 1992; Reddy et al., 1996). Efficacy of a commercialized rHVT-ND vaccine (Saitoh et al., 2003) expressing the F protein of the avirulent D26/76 genotype I NDV strain (Sato et al., 1987, GenBank accession number: M24692) has already been shown in SPF layers and in commercial broilers with MDA in previous publications (Rauw et al., 2010; Palya et al., 2012). The aim of the study presented here was to evaluate the onset and duration of immunity of the same vaccine in commercial layers up to 72 weeks of age after single ND vaccination at day old or as a component in ND vaccination programmes including conventional live and killed vaccines. 2. Materials and methods 2.1. Chickens Layers (Lohmann, Brown Lite) with MDA to NDV and MDV were purchased from a commercial hatchery. Chickens were randomly assigned to four groups according to their vaccination programme against ND. Number of day-old birds was 296, 280, 180 and 223 in groups 1, 2, 3 and 4, respectively. All groups were kept in isolated / 75

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lab-scale experimental poultry houses on deep litter. Groups 1 and 4 were kept in the same house sharing the same air space with physical separation to avoid direct contact with the drinkers, feeders and the litter of the other group. The other two groups (groups 2 and 3) were reared in isolated experimental poultry houses at two different locations (more than 10,000 m from each other) attended by different animal keepers. Biosecurity measures were in place to prevent transmission of live ND vaccine-viruses to the environment. Water and feed was provided ad libitum. Lighting parameters were set according to the management manual for Brown Lite breed provided by Lohmann. Required number of nests was introduced from the start of lay. Preventive vaccinations of all birds against coccidiosis, infectious bronchitis (IB), infectious bursal disease (IBD), fowl pox (FP) and egg drop syndrome (EDS) were performed using commercial vaccines. Immunization against Marek’s disease with Rispens strain was applied for all groups while the rHVT-ND vaccine was used for all groups except the control group. The animal study was conducted according to the national and European regulations. 2.2. Vaccines tested One dose of the cell-associated rHVT-ND vaccine (Vectormune® ND, Ceva Sante Animale) was applied with its special diluent subcutaneously at the day of hatch for all ND vaccinated groups. The vaccine was back-titrated at the end of immunization procedure, when 2500 pfu/dose was measured. Live vaccine containing an apathogenic strain (Cevac® Vitapest L, Ceva Sante Animale) was applied at day old via the intra-ocular route for groups 2 and 3. Booster vaccination of group 3 at six weeks of age was performed with another live conventional vaccine based on the lentogenic LaSota strain (Cevac® New L, Ceva Sante Animale) using coarse spray application. Booster vaccination before lay (at 15 weeks of age) with an oil-emulsion trivalent killed vaccine containing ND, IB and EDS antigens (Cevac® ND IB EDS K, Ceva Sante Animale) was done subcutaneously. All conventional vaccines (live and killed) were applied in one dose according to the manufacturer’s instruction for use. 2.3. Trial design Treatment (ND vaccinations) of the different groups and date of ND challenges is summarized in Table 1. Chicks in group 1 received only a single dose of rHVT-ND vaccine at day-old, while the chicks in group 2 were vaccinated with rHVT-ND and live vaccine at day-old, followed by booster vaccination with a killed vaccine at 15 weeks of age. The chicks in group 3 received rHVT-ND and live vaccine at hatch, then a booster vaccination with the LaSota vaccine at 6 weeks of age, followed by a second booster vaccination with a killed vaccine at 15 weeks of age. 2.4. NDV challenge and post-challenge samplings Consecutive NDV challenges were performed from 3 to 72 weeks of age to check the onset and duration of

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immunity induced by the different vaccination programmes. Groups 1, 2 and 4 were included in all challenges (at 3, 4, 6, 10, 15, 25, 33, 40, 55 and 72 weeks of age), while group 3 was checked only at selected dates (at 10 weeks of age, i.e., 4 weeks after booster vaccination with the live vaccine; and at 55 weeks of age). At the earliest two challenge dates (i.e. 3 and 4 weeks of age) SPF controls of the same age were also included to validate the challenge. The challenge strain used (D1524/1/1,2/MY/10) was isolated from Malaysia in 2010. The classification of this isolate as genotype VII strain was based on the analysis of the partial F-gene nucleotide sequence (Lomniczi et al., 1998). This strain belongs to the viscerotropic–velogenic pathotypes based on the mortality, clinical signs and gross-lesions induced in chickens. The strain caused 100% mortality in the SPF layers by 4–5 days post-infection (dpi), while in the non-immune commercial layers used in the study (group 4) the mortality reached 100% between 4 to 7 dpi (usually 5–6 dpi) after intra-nasal infection with a dose of 5.0 log10 median embryo lethal dose (ELD50 )/bird. All challenges were performed via the intra-nasal route with a dose of 5.0 log10 ELD50 . Twenty chickens from each vaccinated group and twenty or ten control birds (in case of the challenges up to 15 weeks of age 20 chickens, later 10 hens) were submitted to challenge. The same dose of the same virus-suspension was applied for challenge of 10 SPF chickens at 3 and 4 weeks of age. Post-challenge observation period lasted for 14 days, during which all animals were observed for clinical signs and mortality daily. Clinical signs including listlessness, weakness, increased respiration, oedema around the eyes and head, green diarrhoea, muscular tremors, paralysis of legs and wings, and torticollis were considered as specific signs of ND. Birds dying due to aspecific causes were excluded from the evaluation. Clinical protection was calculated as the ratio of birds without ND specific clinical signs or mortality. Swab samples were taken for the measurement of challenge virus replication and shedding from the choanal slit (oro-nasal swabs) and from the cloaca. Samplings were performed at 3 and 7 days post-challenge (dpch) after challenges at 3, 4, 6, 10, 40, 55 and 72 weeks of age. Head of the cotton swabs (EUROTUBO® collection swab, reference: 300203, Deltalab, Spain) were cut into an Eppendorf tube containing phosphate buffered saline (PBS) supplemented with a mixture of antibiotics (0.1 mg/ml gentamicinsulphate, 0.1 mg/ml colistin-sulphate and 0.05 mg/ml norfloxacin; Sigma–Aldrich) right after sampling. The swabs were stored at −75 ◦ C until further analysis. Protection against egg drop after NDV challenge was tested only at 33 weeks of age (at the peak of egg production). Hens (30–30 birds from groups 1, 2 and 4) were transported to the challenge site at 32 weeks of age. Rearing conditions and feed was the same as in case of prechallenge rearing sites. After an acclimatization period of 8 days, the follow up of egg production showed no persisting effect of transportation. Challenge infection was performed in groups 1 and 2, while 30 control birds of group 4 (to serve as control for the egg production) were mock-challenged and kept isolated from the vaccinated challenged groups. Further 10 hens from the control group (group 4) were transferred to another isolated site and were challenged

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Table 1 Trial design. Group

ND vaccination a

NDV challenge dates

Day-old

Booster (age in weeks)

1 (rHVT-ND)

rHVT-ND s.c.

–

2 (rHVT-ND&live(1)&killed)

rHVT-ND s.c., apathogenic live vaccine i.o. rHVT-ND s.c., apathogenic live vaccine i.o. –

Killed vaccine s.c. (15)

3 (rHVT-ND&live(1)&live(2)&killed) 4 (control)

3d , 4d , 6d , 10c , d , 15, 25d , 33b , 40d , 55c , d and 72d weeks of age

Lentogenic live vaccine spray (6) killed vaccine s.c. (15) –

a i.o. (intra-ocular) application was used for the conventional live ND vaccine and s.c. (subcutaneous) application for the vectored ND vaccine on the day of hatch. b This challenge was dedicated to the measurement of egg drop in the vaccinated groups after challenge. c Group 3 was challenged only at two dates: at 10 weeks of age (4 weeks post-vaccination) and at 55 weeks of age. d Challenges when shedding measurement was included.

with the same dose of NDV to validate the challenge system. Egg production was followed daily for 13 days in the challenged groups 1–2 and the non-challenged control group. Effect of NDV challenge was evaluated by the comparison of egg production data during the pre-challenge period (from the 2nd to the 8th day of acclimatization period) with the ones egg production during the post-challenge observation period (from the 1st to the 13th day post-challenge) for each group.

at the beginning of acclimatization period at 32 weeks of age. To detect antibodies to NDV in the serum samples, both ELISA (Newcastle Disease Antibody Test kit, BioChek B.V., Holland) and haemagglutination inhibition test (HI) were used. Haemagglutination inhibition (HI) test was performed using standard method against 4 haemagglutinating unit (HAU) of LaSota antigen. Positivity limit of HI titre was set to equal or higher than 2 log2 .

2.5. rHVT-ND vaccine virus detection

2.7. Challenge virus shedding measurement

Vaccine-take of rHVT-ND was checked from individual spleen samples by HVT specific real-time PCR method. Only groups 1 and 2 were sampled (group 2 was omitted from the samplings at 6 and 10 weeks of age). Number of birds included was 10/group at 2, 3, 4, 15, 19, 42 and 57 weeks of age, while at 74 weeks of age 20 hens in group 1 and 12 hens in group 2 were tested. At 6 and 10 weeks of age only 5 chickens from group 1 were tested. Samplings from 42 weeks of age onwards were performed post-challenge from the survived birds. Approximately 1 cm3 of spleen washomogenized by TissueLyser II (Qiagen) after the addition of 1 ml sterile PBS. DNA was purified from the organ homogenate by QIAmp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. HVT qPCR was performed by Rotor-Gene Probe PCR Kit (Qiagen) using 2 l purified DNA, 1 l HVT sorf 1 F3 5� -GGG TCC TCG ACT TGG AGT TT3� , 1 l R3 5� -GTG TAT TTG GCG ACG GAG AT-3� primers and 0.5 l HVT sorf 1 probe 3 5� (FAM)-TCA CAG GTG TTC GAT AGC GGG GA-(BHQ1)3� in 20 l final volume. Amplification and detection of specific products were done using RotorGene Q real-time PCR machine (Qiagen) with the following cycle profile: 1 cycle of 95 ◦ C for 10 min and 40 cycle of 95 ◦ C for 15 s and 60 ◦ C 45 s.

Virus shedding was tested by one step RT-real-time PCR. Swab samples were vortex mixed for 10 min to improve the elution of virus containing material from the swab. Resulted samples were clariﬁed by centrifugation (900 × g for 3 min at room-temperature). 200 l of the supernatant was used for RNA extraction by QIAxtractor Virus kit (Qiagen) according to the manufacturer’s instructions. Two microliter of RNA was used as a template for the realtime one-step RT-PCR, amplifying a fragment of M gene (TaqMan® NDV reagents and controls, Life TechnologiesTM ; Wise et al., 2004). Internal positive control supplied by the manufacturer was included. Ct value of the positive control in the different runs was set within a narrow range to ensure similar cut off in case of separate runs. Titre equivalent unit was calculated by extrapolation from sample Ct to the Ct of standard (total RNA extracted from tenfold dilution series of the challenge virus strain used in the trial with known titre determined by titration in embryonated SPF eggs). Results were expressed on log10 titre equivalent unit values (log10 ELD50 /0.1 ml).

2.6. Serology Serum samples were collected to measure the decay of MDA and the humoral immune response to vaccination at day-old, at 2 weeks of age and from 3 weeks of age at each challenge dates (pre-challenge samples). In case of the challenge performed at 33 weeks of age to check the effect on egg production, pre-challenge sampling was performed

2.8. Statistical analysis Statistical analysis was performed using Statgraphics Centurion software (StatPoint Technologies, Inc.). The following analyses were done: (i) HVT vaccine detection rate: number of birds positive and number of birds negative in groups 1 and 2 obtained at the different sampling dates was compared by Chi-square test; (ii) comparison of serological results obtained at the same age among the different groups: log10 ELISA titres and log2 HI titres were used for comparison of means by ANOVA, then 95% LSD test was

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used to find statistically homogeneous groups; (iii) pairused to find statistically homogeneous groups; (iii) pairwise comparison of serological results within the same wise comparison of serological results within the same group at two consecutive sampling dates: Mann–Whitney group at two consecutive sampling dates: Mann–Whitney test was used; (iv) clinical protection: distribution of inditest was used; (iv) clinical protection: distribution of individuals among the different categories (protected or not viduals among the different categories (protected or not protected) was compared by Fisher’s exact test; (v) chalprotected) was compared by Fisher’s exact test; (v) challenge virus shedding: log10 titre equivalent units in the lenge virus shedding: log10 titre equivalent units in the same type of swabs were compared between the different same type of swabs were compared between the different groups sampled at the same date by Kruskal–Wallis test groups sampled at the same date by Kruskal–Wallis test (homogenous groups were found using pair-wise compar(homogenous groups were found using pair-wise comparisons); (vi) pre- and post-challenge egg production within isons); (vi) pre- and post-challenge egg production within the same groups was compared by Mann–Whitney test. the same groups was compared by Mann–Whitney test. Trend of post-challenge egg production in the vaccinated Trend of post-challenge egg production in the vaccinated groups was compared with the mock-challenged control groups was compared with the mock-challenged control group by comparison of regression lines (ANOVA for the group by comparison of regression lines (ANOVA for the slopes). slopes). All statistical analyses were done at 95% confidence level All statistical analyses were done at 95% confidence level (p < 0.05). (p < 0.05). 3. Results 3. Results 3.1. Detection of rHVT-ND vaccine virus replication in 3.1. Detection of rHVT-ND vaccine virus replication in the spleen the spleen All tested non-vaccinated control animals proved to be All tested non-vaccinated control animals proved to be negative, with no detectable virus DNA. In the ratio of negative, with no detectable virus DNA. In the ratio of positives in the vaccinated groups 1 and 2 there was no positives in the vaccinated groups 1 and 2 there was no significant difference (p = 0.252). The overall ratio of posisignificant difference (p = 0.252). The overall ratio of positives (groups 1 and 2 together) showed fluctuation in the tives (groups 1 and 2 together) showed fluctuation in the range of 70–100% (except a single occasion at 3 weeks of range of 70–100% (except a single occasion at 3 weeks of age in group 1). Although the highest positivity ratio was age in group 1). Although the highest positivity ratio was achieved between 4 and 19 weeks of age, there was no clear achieved between 4 and 19 weeks of age, there was no clear tendency of decrease in the detection rate throughout the tendency of decrease in the detection rate throughout the tested period. Results are summarized in Table 2. tested period. Results are summarized in Table 2. 3.2. Humoral immune response to vaccination 3.2. Humoral immune response to vaccination Serological Serological Figs. 1 and 2. Figs. 1 and 2.

results results

obtained obtained

are are

summarized summarized

in in

3.2.1. HI test results 3.2.1. HI test results Level of anti-NDV MDA measured at day-old was Level of anti-NDV MDA measured at day-old was 5.5 ± 2.6 log2 , which decayed to a low level by 3 weeks of 5.5 ± 2.6 log2 , which decayed to a low level by 3 weeks of age and dropped below the positivity limit of the HI test age and dropped below the positivity limit of the HI test in the majority of control chickens by 4 weeks of age. No in the majority of control chickens by 4 weeks of age. No HI antibodies could be detected in the control birds from HI antibodies could be detected in the control birds from 6 weeks of age onwards.rHVT-ND vaccine alone induced 6 weeks of age onwards.rHVT-ND vaccine alone induced low level of antibodies during the first six weeks of life. low level of antibodies during the first six weeks of life. Although the HI test detected elevated antibody level in Although the HI test detected elevated antibody level in this group compared to the controls at 4 and 6 weeks of this group compared to the controls at 4 and 6 weeks of age, but the mean antibody level was very low, and some age, but the mean antibody level was very low, and some birds were still sero-negative at six weeks of age. However, birds were still sero-negative at six weeks of age. However, all of these birds proved to be protected against NDV chalall of these birds proved to be protected against NDV challenge (at 6 weeks of age) indicating protective immunity lenge (at 6 weeks of age) indicating protective immunity without the presence of detectable HI antibodies. Strong without the presence of detectable HI antibodies. Strong increase of HI titres was observed between 6 and 10 weeks increase of HI titres was observed between 6 and 10 weeks of age and the mean HI titre exceeded 5 log2 by 10 weeks of age and the mean HI titre exceeded 5 log2 by 10 weeks of age. The HI antibody titre remained at very similar level of age. The HI antibody titre remained at very similar level throughout the experiment up to 72 weeks of age with throughout the experiment up to 72 weeks of age with minor fluctuation. minor fluctuation.

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109 109

Co-application of conventional live ND vaccine with Co-application of conventional live ND vaccine with rHVT-ND at day-old resulted in higher HI titres at 3, 4 and 6 rHVT-ND at day-old resulted in higher HI titres at 3, 4 and 6 weeks of age compared to the group vaccinated with rHVTweeks of age compared to the group vaccinated with rHVTND only. This effect was transient, at 10 and 15 weeks of ND only. This effect was transient, at 10 and 15 weeks of age no positive effect of the conventional live vaccine coage no positive effect of the conventional live vaccine coadministration at day-old was detected on the humoral administration at day-old was detected on the humoral immune response. immune response. Booster vaccination with LaSota strain vaccine at 6 Booster vaccination with LaSota strain vaccine at 6 weeks of age increased significantly the antibody level, weeks of age increased significantly the antibody level, however this effect was transient; no difference among the however this effect was transient; no difference among the vaccinated groups could be detected at 15 weeks of age (9 vaccinated groups could be detected at 15 weeks of age (9 weeks p.v.). weeks p.v.). Vaccination of groups 2 and 3 with killed ND vaccine Vaccination of groups 2 and 3 with killed ND vaccine at 15 weeks of age resulted in significant booster effect in at 15 weeks of age resulted in significant booster effect in both groups. Group 3 which received 2 booster vaccinations both groups. Group 3 which received 2 booster vaccinations (live and killed) showed significantly higher titre increase (live and killed) showed significantly higher titre increase compared to group 2. All booster vaccinated groups had compared to group 2. All booster vaccinated groups had significantly higher level of HI titres than the group vaccisignificantly higher level of HI titres than the group vaccinated with rHVT-ND alone throughout the laying period. HI nated with rHVT-ND alone throughout the laying period. HI titres remained on the same level with some fluctuations, titres remained on the same level with some fluctuations, but no decreasing tendency up to the latest sampling date but no decreasing tendency up to the latest sampling date (55 weeks of age in group 3 and 72 weeks of age in groups (55 weeks of age in group 3 and 72 weeks of age in groups 1 and 2) could be detected. 1 and 2) could be detected. 3.2.2. ELISA results 3.2.2. ELISA results Non-vaccinated controls proved to be negative from 3 Non-vaccinated controls proved to be negative from 3 weeks of age onwards. weeks of age onwards. Humoral immune response induced by rHVT-ND alone Humoral immune response induced by rHVT-ND alone could be first detected at 4 weeks of age: although the titres could be first detected at 4 weeks of age: although the titres were far below the positivity limit of the test kit, they were were far below the positivity limit of the test kit, they were significantly higher than in the non-vaccinated controls. At significantly higher than in the non-vaccinated controls. At 6 weeks of age mean titre approached the positivity limit of 6 weeks of age mean titre approached the positivity limit of ELISA, but a high percentage of birds was still sero-negative ELISA, but a high percentage of birds was still sero-negative based on the positivity threshold set by the ELISA kit manbased on the positivity threshold set by the ELISA kit manufacturer. From 10 weeks of age, the vast majority of the ufacturer. From 10 weeks of age, the vast majority of the vaccinated chickens found to be positive. vaccinated chickens found to be positive. Application of live conventional vaccine together with Application of live conventional vaccine together with the rHVT-ND had significant effect on the humoral the rHVT-ND had significant effect on the humoral immune-response during the first 6 weeks of life. The geoimmune-response during the first 6 weeks of life. The geometric mean ELISA titre of group 2 which received both metric mean ELISA titre of group 2 which received both the rHVT-ND and conventional live ND vaccine at day-old the rHVT-ND and conventional live ND vaccine at day-old differed significantly from the one of group 1 that was vacdiffered significantly from the one of group 1 that was vaccinated only with the rHVT-ND vaccine. From 10 weeks to cinated only with the rHVT-ND vaccine. From 10 weeks to 15 weeks of age, difference between the groups immunized 15 weeks of age, difference between the groups immunized with rHVT-ND only or with the combination of rVTM-ND with rHVT-ND only or with the combination of rVTM-ND and live vaccine at day old became negligible, statistically and live vaccine at day old became negligible, statistically not significant. Booster vaccination with the live vaccine not significant. Booster vaccination with the live vaccine at 6 weeks of age, resulted in significantly higher antibody at 6 weeks of age, resulted in significantly higher antibody level in the rHVT-ND&live(1)&live(2) group, compared to level in the rHVT-ND&live(1)&live(2) group, compared to the other ones, however this difference was short-lived and the other ones, however this difference was short-lived and was not significant already at 15 weeks of age., Booster vacwas not significant already at 15 weeks of age., Booster vaccination with the killed vaccine at 15 weeks of age induced cination with the killed vaccine at 15 weeks of age induced significant titre increase. The increase was significantly significant titre increase. The increase was significantly higher in group 3 which received booster vaccination with higher in group 3 which received booster vaccination with conventional live vaccine at 6 weeks of age. conventional live vaccine at 6 weeks of age. Level of ELISA titres reached the plateau by 10–15 weeks Level of ELISA titres reached the plateau by 10–15 weeks of age in the rHVT-ND alone vaccinated group and by 25 of age in the rHVT-ND alone vaccinated group and by 25 weeks of age in the booster vaccinated groups, then it weeks of age in the booster vaccinated groups, then it remained stable throughout the rest of the laying period, remained stable throughout the rest of the laying period, with slight fluctuation. with slight fluctuation.

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Table 2 Detection of HVT vaccine virus by PCR in spleen samples collected at different ages. Group

HVT vaccination

1 2 4 (control)

rHVT-ND rHVT-ND&live(1)&killed No

Ratio of HVT positive spleen samples at different ages (age in weeks) 2

7/10 9/10 NT

6/10 8/10 NT

9/10 10/10 0/5

5/5 NT NT

9/10 8/10 0/5

8/8 7/8 NT

7/10 8/10 NT

8/10 10/10 NT

17/20 11/12 0/5

Number of positive samples/number of tested samples.

Fig. 1. Detection of humoral immune response to different vaccination regimes based on HI test results. Same letters indicate statistically homogeneous groups (comparison of different groups sampled at the same date) above the graph. * indicates statistically signiﬁcant difference between two consecutive sampling dates of the same group. Live boosted group was tested from 10 weeks of age (same results for this group are shown as the ones of rHVT-ND&live group from 3 to 6 weeks of age, since before the live booster these two groups were treated in the same way).

3.3. Efﬁcacy against NDV challenge 3.3.1. Clinical protection Level of clinical protection (i.e., prevention of ND speciﬁc clinical signs and mortality) measured during the consecutive challenges from 3 to 72 weeks of age is shown

in Table 3. SPF control birds were included for validation of the challenge system at early challenge dates only (3 and 4 weeks of age). Effect of maternally derived antibodies on the outcome of challenge was detectable only at 3 weeks of age, resulting in 20% clinical protection in the non-vaccinated control group. All SPF controls died at

Fig. 2. Detection of humoral immune response to different vaccination regimes based on ELISA test results. Same letters indicate statistically homogeneous groups (comparison of different groups sampled at the same date) above the graph. * indicates statistically signiﬁcant difference between two consecutive sampling dates of the same group. Live boosted group was tested from 10 weeks of age (same results for this group are shown as the ones of rHVT-ND&live group from 3 to 6 weeks of age, since before the live booster these two groups were treated in the same way).

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3.3.2. Protection against egg-drop after velogenic NDV challenge Efﬁcacy of vaccination against egg-drop induced by NDV challenge was checked at 33 weeks of age at the peak of egg production. Results obtained during the acclimatization and post-challenge observation period are shown in Table 4 and Fig. 3. Egg production in the different groups during the acclimatization period was different (mean egg production in this period ranged between 81% and 100%) but after challenge this difference among the groups became smaller indicating different level of transportation induced stress for the birds. Since the number of hens was small and the rearing conditions were not the ones used for commercial ﬂocks, such differences are acceptable. Effect of challenge infection was analyzed by comparison of preand post-challenge egg production within the same group to overcome the pre-existing differences.

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10w-D3-G4

6w-D7-G4

10w-D3-G1

10w-D3-G3

b bc c

5 4 3 2 1 10w-D7-G4

10w-D7-G3

10w-D7-G2

10w-D7-G1

72w-D7-G4

72w-D7-G2

72w-D7-G1

72w-D3-G4

10w-D3-G2

72w-D3-G2

72w-D3-G1

55w-D7-G4

55w-D7-G3

oro-nasal swab

6w-D7-G2

6w-D7-G1

6w-D3-G4

55w-D7-G2

6w-D3-G2

6w-D3-G1

4w-D7-G4

4w-D7-G2

4w-D7-G1

4w-D3-G4

55w-D7-G1

55w-D3-G4

a Eight days long acclimatization period was included before the challenge infection. b All groups contained the same number of birds (30). Post-challenge observation period was 13 days long. Negative control group was mockchallenged with PBS.

55w-D3-G3

91% 98% 96%

4w-D3-G2

Post-challengeb

4w-D3-G1

55w-D3-G2

81% 100% 92%

55w-D3-G1

1 (rHVT-ND) 2 (rHVT-ND&live(1)&killed) 4 (negative control)

oro-nasal swab a b b a b b

Egg production Pre-challengea

40w-D7-G4

100% 100%b NT 0%a NT

Table 4 Egg production during the pre-challenge acclimatization and the postchallenge observation periods. Group

3w-D7-G4

NT: not tested. Different superscript letter indicate statistically different groups (p < 0.05).

4–5 days post-challenge validating the challenge system. From 4 weeks of age onwards ND specific mortality in the commercial layer control group was 100% (4–7 days postchallenge). Protective immunity induced by vaccination was detected as soon as the first challenge at 3 weeks of age. Similar level of protection (74–75%) was observed in all vaccinated groups. Difference in the level of protection between the vaccinated groups and the control group proved to be highly significant. After the challenge at 4 weeks of age, only 1 bird died among the vaccinated chickens, in the group receiving rHVT-ND vaccine alone. Except this single bird, all other chickens were clinically protected in the vaccinated groups (95–100%). The bird that died had an HI titre <1:2 and very low ELISA titre (88). There were 3 further chickens in this group with similar antibody level that were completely protected against the challenge. This indicates that complete clinical protection can be achieved without detectable humoral immune response to NDV (with the serological methods used in the study). Following the challenges performed at 6, 10, 15, 25, 33, 40, 55 and 72 weeks of age, all vaccinated birds receiving the different vaccination regimes were clinically protected against NDV challenge (group 3, receiving live booster at 6 weeks of age was challenged only at 10 and 55 weeks of age). No additional positive effect of the live vaccines (application at day-old or at day-old and 6 weeks of age) could be detected regarding clinical protection, since the rHVT-ND vaccine alone provided 100% protection.

40w-D7-G2

100% 100%b 100%b 0%a NT

b c a

72 b

3w-D7-G2

100% 100%b NT 0%a NT

40w-D7-G1

100% 100%b 100%b 0%a NT

40w-D3-G4

100% 100%b NT 0%a NT

15, 25, 33, 40 b

3w-D3-G2

95% 100%b NT 0%a 0%

10 b

40w-D3-G2

74% 75%b NT 20%a 0%

6 b

3w-D3-G1

rHVT-ND rHVT-ND&live(1)&killed rHVT-ND&live(1)&live(2)&killed No No

4 b

7 6 5 4

No tendency of decrease in egg production as an effect of challenge virus replication could be detected in any of the challenged groups. Comparison of egg production before and after challenge in the vaccinated groups revealed no significant reduction Result of Mann–Whitney test for group 1 was p = 0.001, indicating significant increase of egg production, while for group 2 was p = 0.241 indicating no significant difference between the production levels before and after challenge. By comparing the trend of production levels of the vaccinated challenged groups with the one of the mock-challenged negative control group no significant difference could be shown (ANOVA for the slope of regression lines, p = 0.577). 100% of the non-vaccinated controls succumbed to challenge.

Fig. 4. (a and b) Challenge virus shedding measurement result obtained during the growing period (up to 10 weeks of age) and during the egg-production period (from 40 to 72 weeks of age) from oro-nasal swabs by real-time PCR. Identiﬁcation of samples is given by the abbreviation of the “age at challenge”“day of sampling post-challenge”-“group number” (e.g. 3w-D3-G1). Different sampling dates within the same challenge are separated with dotted lines, different challenge dates are separated by solid lines. D7-G4 samplings mean the results from dead controls at 5–7 days post-challenge (maximum shedding). Vaccination of different groups: group 1: vHVT-ND alone, group 2: vHVT-ND&live(1)&killed, group 3: vHVT-ND&live(1)&live(2)&killed, group 4: non-vaccinated control. Statistically different groups are shown with different letters above the graph. All sampling dates had been analyzed separately.

Fig. 3. Daily egg production during the pre-challenge acclimatization and the post-challenge observation period. First day of acclimatization was excluded due to the strong effect of previous transportation. Negative control group was mock infected at the date of challenge of vaccinated groups (on the 8th day of acclimatization).

3.3.3. Reduction of challenge virus replication, protection against shedding Challenge virus replication was measured after the challenges at different ages, excluding the ones at 15, 25 and 33 weeks of age. Swab samples were taken from the choanal slit (“oro-nasal swab”) and from the cloaca at 3 and 7 dpch of the chickens which were alive, and on the day of mortality from the dead birds. Effect of vaccination was analyzed in comparison to the challenged controls at 3 dpch. Since only a few control birds survived until 7 dpch, reduction of challenge virus replication at 7 dpch is shown in comparison with the results obtained from the dead birds at 5–6 dpch in the challenged control groups (usually all control birds died from 4 to 7 dpch). There was no signiﬁcant difference between the results obtained at 5 or 6 dpch from dead control birds, therefore results obtained from them at 5–6 dpch were combined for the evaluation. Statistical analysis was performed on the basis of medians to reduce the effect of great individual heterogeneity at

3 2 1 0 40w-D3-G1

3 1 2 3 4 (control) SPF control

Clinical protection at different ages (age in weeks)

lg ELD50/0.1 ml

ND vaccination

lg ELD50/0.1 ml

Group

V. Palya et al. / Veterinary Immunology and Immunopathology 158 (2014) 105–115

3w-D7-G1

Table 3 Clinical protection against NDV challenges performed at different ages.

112

3w-D3-G4

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certain samplings and the presence of outliers. Results are shown in Figs. 4a, 4b, 5a and 5b. Significant reduction of challenge virus replication was achieved by all vaccination programmes as early as 3 weeks of age, even when the clinical protection was still not complete. The only exception was the cloacal swabs taken 3 days after the 1st challenge, when the difference between the vaccinated groups and the control group did not prove to be statistically significant, due to the high heterogeneity of individual results. After the subsequent challenges up to 72 weeks of age the effect of vaccination(s) was more evident. Oro-nasal replication of challenge virus was suppressed less efficiently compared to the cloacal shedding by all vaccination programmes tested. Effect of vaccination with rHVT-ND alone (group 1) or in combination with live conventional vaccine at day old and boosted with killed vaccine before the start of the laying period (group 2) on the level of challenge virus excretion proved to be similar after the challenges

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b b

cloacal swab a b b a b b

b a

b b b

lg ELD50/0.1 ml

7 6 5 4 3 2 1

10w-D7-G4

10w-D7-G3

10w-D7-G2

10w-D7-G1

10w-D3-G4

10w-D3-G3

10w-D3-G2

6w-D7-G4

72w-D7-G4

10w-D3-G1

6w-D7-G2

6w-D7-G1

6w-D3-G4

6w-D3-G2

6w-D3-G1

4w-D7-G2

4w-D7-G4

4w-D7-G1

72w-D7-G2

cloacal swab b a b b

72w-D7-G1

4w-D3-G4

4w-D3-G2

4w-D3-G1

40w-D7-G4

3w-D7-G4

40w-D7-G2

3w-D7-G2

3w-D7-G1

40w-D7-G1

3w-D3-G4

40w-D3-G4

3w-D3-G2

40w-D3-G2

40w-D3-G1

3w-D3-G1

lg ELD50/0.1 ml

7 6 5 4 3 2 1 72w-D3-G4

72w-D3-G2

72w-D3-G1

55w-D7-G4

55w-D7-G3

55w-D7-G2

55w-D7-G1

55w-D3-G4

55w-D3-G3

55w-D3-G2

55w-D3-G1

Fig. 5. (a and b) Challenge virus shedding measurement result obtained during the growing period (up to 10 weeks of age) and during the egg-production period (from 40 to 72 weeks of age) from cloacal swabs by real-time PCR. Identiﬁcation of samples is given by the abbreviation of the “age at challenge”“day of sampling post-challenge”-“group number” (e.g. 3w-D3-G1). Different sampling dates within the same challenge are separated with dotted lines, different challenge dates are separated by solid lines. D7-G4 samplings mean the results from dead controls at 5–7 days post-challenge (maximum shedding). Vaccination of different groups: group 1: vHVT-ND alone, group 2: vHVT-ND&live(1)&killed, group 3: vHVT-ND&live(1)&live(2)&killed, group 4: non-vaccinated control. Statistically different groups are shown with different letters above the graph. All sampling dates had been analyzed separately.

performed between3 and 25 weeks of age. However after the challenges carried out at 40 or 55 weeks of age the oro-nasal replication of the challenge virus was significantly higher in group 1 compared to group 2. Vaccination programme comprising rHVT-ND&live(1)&live (2)&killed vaccines (group 3) resulted in significantly reduced challenge virus replication at the oro-nasal site compared to the single vaccination with rHVT-ND. Difference between group 2 and 3 was smaller than the difference existing between groups 1 and 3, being significant only at 7 dpch. Comparison of results obtained during the consecutive challenges revealed stronger reduction in challenge virus excretion following the vaccination programmes that included the use of live and killed vaccines along with rHVT-ND. Cloacal shedding was not detectable or was negligible from 6 weeks of age in all vaccinated groups, which indicates efficient immunity against viraemia and generalized replication of the challenge virus. There was no significant

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difference among the vaccinated groups at any sampling date regarding cloacal shedding. 4. Discussion Commercial light brown layers with moderate level of MDA were vaccinated at day old s.c. with rHVT-ND vaccine alone, or rHVT-ND s.c. and conventional live ND vaccine by eye drop. A third group of chickens received the same vaccination at day old, but was boosted with a lentogenic live ND vaccine at 6 weeks of age. Both groups that received the conventional live ND vaccine(s) were boosted with a killed ND vaccine at 15 weeks of age. Single application of the rHVT-ND vaccine resulted in excellent clinical protection against challenge with a recent Asian NDV isolate belonging to genotype VII from 4 weeks of age up to 72 weeks of age (95% at 4 weeks of age and 100% at all later challenges). The immunity induced by this rHVT-ND vaccine was so strong, that from 6 weeks of

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age the clinical protection against NDV challenge was not further improved by the inclusion of conventional live or inactivated ND vaccines in the vaccination programme. Vaccination with rHVT-ND alone efficiently reduced the cloacal shedding to very limited or sometimes non-detectable level, which means 6–7 log10 reduction compared to the non-vaccinated controls. This result indicates very good systemic immunity which could very efficiently reduce or prevent viraemic infection by the challenge virus. Oro-nasal replication of the challenge virus was also significantly reduced, however based on this parameter the efficacy of vaccination with rHVT-ND alone was weaker, resulting in detectable virus replication in the majority of vaccinated birds and 3–5 log10 reduction (although there were some birds at each challenge occasion in which no challenge virus replication could be detected). Local replication of the challenge virus at the oro nasal mucosa was further reduced by the booster vaccination(s): best efficacy against oro-nasal shedding was found in the group that received vaccination with both conventional live and killed ND vaccines in addition to the day-old rHVTND vaccination. The added value of conventional live ND vaccine administration at the time of rHVT-ND vaccination compared to the rHVT-ND-only vaccination was moderate. Antibodies induced by the rHVT-ND vaccination alone could be detected by HI test from 4 weeks and by ELISA kit from 6 weeks of age. The antibody level elicited by the single application of rHVT-ND vaccine reached its maximum by 10 weeks of age and remained stable for 72 weeks of age covering a usual laying period. Application of live conventional vaccine at day old together with the rHVTND significantly increased the humoral immune-response during the first 6 weeks of life, indicating a synergic effect on the stimulation of immune system by the two vaccines, which may contribute to an earlier onset of immunity to ND. Similar effect could be seen after the application of the inactivated ND vaccine at 15 weeks of age. The rHVT-ND vaccine virus could be detected by PCR throughout the tested period (from 2 to 74 weeks of age) verifying life-long persistence of the virus in vaccinated birds and thereby ensuring strong and long lasting immunity to ND through the continuous antigen stimulus. Efficacy results obtained in this study in layer-type chickens together with the ones already published in broilers (Palya et al., 2012) indicate, that the strong immunity elicited by the rHVT-ND vaccine expressing the F gene from a genotype I NDV strain is able to provide full clinical protection along with efficient reduction of shedding even after a single application at day-old against different recent genotypes of NDV strains. The level of immunity induced by the rHVT-ND vaccine – considering antibody response, resistance to challenge as well as reduction of challenge virus shedding – was further increased by the concomitant application of a live ND vaccine at day-old, as well as by the application of a booster vaccination with live lentogenic vaccine (given by spray at 6 weeks of age) and by a killed ND vaccine administered at 15 weeks of age. The possibility of optimally controlled mass application of the rHVT-ND vaccine in ovo or s.c. to day-old chicks at the hatchery in combination with the live apathogenic ND vaccine and its characteristic to induce efficient immunity

even when applied in face of maternally derived antibodies makes this type of ND vaccination programme a good choice and a promising tool for the prevention of ND. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgements We thank Edit Fodor, Magdolna Lénárd and Gabriella Somfai for the excellent technical assistance and Zalán Homonnay for his contribution to shedding measurement and result analysis. References Alexander, D.J., 2008. Newcastle disease. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 6th ed. OIE, pp. 576–589. Bwala, D.G., Abolnik, C., van Wyk, A., Cornelius, E., Bisschop, S.P.R., 2009. Efficacy of a genotype 2 Newcastle disease vaccine (Avinew® ) against challenge with highly virulent genotypes 5d and 3d. J. S. Afr. Vet. Assoc. 80 (3), 174–178. Cattoli, G., Susta, L., Terregino, C., Brown, C., 2011. Newcastle disease: a review of field recognition and current methods of laboratory detection. J. Vet. Diagn. Invest. 23 (4), 637–657. Czeglédy, A., Újvári, D., Somogyi, E., Wehmann, E., Werner, O., Lomniczi, B., 2006. Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications. Virus Res. 120, 36–48. Dortmans, J.C.F.M., Koch, G., Rottier, P.J.M., Peeters, B.P.H., 2011. Virulence of Newcastle disease virus: what is known so far? Vet. Res. 42, 122. Dortmans, J.C.F.M., Peeters, B.P.H., Koch, G., 2012. Newcastle disease virus outbreaks: vaccine mismatch or inadequate application? Vet. Microbiol. 160, 17–22. Hu, S., Ma, H., Wu, Y., Liu, W., Wang, X., Liu, Y., Liu, X., 2009. A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics. Vaccine 27, 904–910. Kapczynski, D.R., King, D.J., 2005. Protection of chickens against overt clinical disease and determination of viral shedding following vaccination with commercially available Newcastle disease virus vaccines upon challenge with highly virulent virus from California 2002 exotic Newcastle disease outbreak. Vaccine 23, 3424–3433. Kumar, S., Nayak, B., Collins, P.L., Samal, S.K., 2011. Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus Type 3 vector in chickens. J. Virol. 85 (13), 6521–6534. Lomniczi, B., Wehmann, E., Herczeg, J., Ballagi-Pordany, A., Kaleta, E.F., Werner, O., Meulemans, G., Jorgensen, P.H., Mante, A.P., Gielkens, A.L.J., Capua, I., Damoser, J., 1998. Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype (VII). Arch. Virol. 143, 49–64. Miller, P.J., King, D.J., Alfonso, C.L., Suarez, D.L., 2007. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 25, 7238–7246. Miller, P.J., Estevez, C., Yu, Q., Suarez, D.L., King, D.J., 2009. Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses. Avian Dis. 53, 39–49. Miller, P.J., Decanini, E.L., Afonso, C.L., 2010. Newcastle disease: evolution of genotypes and the related diagnostic challenges. Inf. Gen. Evol. 10, 26–35. Morgan, R.W., Gelb, J., Schreurs Jr., C.S., Lutticken, D., Rosenberger, J.K., Sondermeijer, P.J., 1992. Protection of chickens from Newcastle and Marek’s diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein. Avian Dis. 36, 858–870. Morgan, R.W., Gelb Jr., J., Pope, C.R., Sondermeijer, P.J., 1993. Efficacy in chickens of a herpesvirus of turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: onset of protection and effect of maternal antibodies. Avian Dis. 37, 1032–1040. Palya, V., Kiss, I., Tatár-Kis, T., Mató, T., Felföldi, B., Gardin, Y., 2012. Advancement in vaccination against Newcastle disease: recombinant

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EXTRACT FROM WESTERN POULTRY DISEASE CONFERENCE 2013 (PP. 17-19).

VECTORMUNE® ND

V. Palya et al. / Veterinary Immunology and Immunopathology 158 (2014) 105–115 HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions. Avian Dis. 56, 282–287. Perozo, F., Marcano, R., Afonso, C.L., 2012. Biological and phylogenetic characterization of a genotype VII Newcastle disease virus from Venezuela: efficacy of field vaccination. J. Clin. Microbiol. 50 (4), 1204–1208. Purchase, H.G., Okazaki, W., Burmester, B.R., 1971. Field trials with the herpes virus of turkeys (HVT) strain FC126 as a vaccine against Marek’s disease. Poult. Sci. 50, 775–783. Rauw, F., Gardin, Y., Palya, V., Anbari, S., Lemaire, S., Boschmans, M., van den Berg, T., Lambrecht, B., 2010. Improved vaccination against Newcastle disease by an in ovo recombinant HVT ND combined with an adjuvanted live vaccine at day-old. Vaccine 28, 823–833. Reddy, S.K., Sharma, J.M., Ahmad, J., Reddy, D.N., McMillen, J.K., Cook, S.M., Wild, M.A., Schwartz, R.D., 1996. Protective efficacy of a recombinant

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herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek’s diseases in speciﬁc-pathogen-free chickens. Vaccine 14, 469–477. Saitoh, S., Okuda, T., Kubomura, M., Dorsey, K.M., 2003. Recombinant herpesvirus of turkeys and use thereof. United States Patent application number 20030157703. Sato, H., Oh-hira, M., Ishida, N., Imamura, Y., Hattori, S., Kawakita, M., 1987. Molecular cloning and nucleotide sequence of P, M and F genes of Newcastle disease virus avirulent strain D26. Virus Res. 7, 241–255. van Boven, M., Bouma, A., Fabri, T.H.F., Katsma, E., Hartog, L., Koch, G., 2008. Herd immunity to Newcastle disease virus in poultry by vaccination. Avian Pathol. 37 (1), 1–5. Wise, M.G., Suarez, D.L., Seal, B.S., Pedersen, J.C., Senne, D.A., King, D.J., Kapczynski, D.R., Spackman, E., 2004. Development of a real-time reverse-transcription PCR for detection of newcastle disease virus RNA in clinical samples. J. Clin. Microbiol. 42, 329–338.

EFFECT OF EXPERIMENTAL NEWCASTLE DISEASE CHALLENGE OF LAYING HENS RECEIVING VACCINATION PROGRAMS THAT INCLUDES RECOMBINANT HVT NDV R. Merino Departamento de Medicina y Zootecnia de Aves, FMVZ, UNAM. Mexico, DF, 04510, Mexico ABSTRACT

pullets including recombinant HVT ND vaccines commercially available and the protection against the challenge with a Mexican Chimalhuacan ND strain at 28 wk of age.

Newcastle disease effects on poultry can be reduced by vaccination. We measured the performance of vaccinated White Leghorn type laying hens challenged with the Chimalhuacan ND strain at 28 wk old and observed for five wk post challenge (wpc). Four groups of laying hens were vaccinated at one d old as follows:

MATERIALS AND METHODS Four groups of 27 wk old laying hens were used, coming from commercial farms, identified as Yellow, White, Red and Green, with 24 hens/group. At the farm they received the Newcastle disease vaccination programs shown in Table 1. Hens were housed in cages and received water ad libitum, feed according to standard procedures, and 15 hours of light per day. Ten birds per group were bled prior to challenge and every wk after that. Serum samples were tested by ELISA test (AffiniTech LTD, Arkansas, USA). All hens were challenged at 28 wk old with the Mexican standard challenge strain Chimalhuacan by the ocular route with 106 EID50/0.2mL, then observed by five wk post challenge (wpc) for clinical signs, mortality, egg production, and egg quality (weight, broken, and shell-less). Birds showing clinical signs and bad quality eggs were compared among groups by test of proportions. Percent of laid eggs per wk were compared by ANOVA, transforming the percent to proportion and then transforming to the arcsine-root. The mean egg weight (per week) and ELISA antibody titers were compared by ANOVA; alpha value was established in 0.05.

White-Marek’s disease Red- Marek’s disease + Newcastle (killed virus) Green- recombinant HVT-ND Yellow- recombinant HVT-NDV Vectormune® Vaccination programs included five live NDV-IBV vaccines. Red group also received twice a NDV-AIV killed vaccine. All vaccination programs protected against mortality. The mean egg production in the five wpc was lower in White group than in all others but shell-less eggs were higher. Three doses of killed and five of live ND virus avoided the drop in egg production and quality. Recombinant HVT-NDV Vectormune plus live ND virus fully protected against clinical symptoms. INTRODUCTION Newcastle disease (ND) threatens the poultry industry in several areas around the world, since it causes high morbidity and mortality in susceptible flocks. In order to control and prevent the occurrence of ND, good biosecurity practices need to be established and rigorously accomplished. One of these biosecurity practices is the preventive vaccination of flocks. Nowadays most vaccination programs include the use of lentogenic strains both, live or inactivated (killed) in order to provide high protective immunity and minimal adverse effects on the flock performance; however, the need for safer and more efficacious ND vaccines has lead to the development of new products, including the recombinant ones, as HVT-ND and NDAIV. The aim of this study was to evaluate ND vaccination programs commonly used in commercial

RESULTS When the laying hens were received, all groups were around 90% egg production; however, three d later a mite infestation was detected; apparently the Red group was the source, since it was the most affected. All hens were treated prior to the challenge. The transport and housing stress, plus the infestation severity and treatment, caused a reduction in egg production, with variable intensity. Egg production is shown in Figure 1. Yellow group had 54.17% production at challenge and three wpc reached 95.83%, remaining above 90% until the

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(The full-length article will be published in International Journal of Poultry Science.)

Table 1. Vaccination program against ND in commercial pullet farms. Age (days)

Egg production prior challenge was variable in all groups because of the above mentioned factors; however, recovering of lay was seen soon after challenge but was largely dependent of the antibody titer at challenge. Red group had the lowest production at challenge, but the highest ELISA titer. This high titer

ND+IB live virus, ocular

ND+AI KV, SQ

101

ND+AI KV, SQ

108

ELISA antibody titer and egg production (%) 40

100

90 80

40 30

0 28

DISCUSSION

Figure 1. Egg production and ELISA antibody titer during five weeks after challenge in laying hens inoculated with the Chimalhuacan strain of Newcastle disease virus at 28 wk of age.

REFERENCES 1. Bwala, D.G., Fasina, F.O., Van Wyk, A., Duncan, N.M. Effects of vaccination with lentogenic vaccine and challenge with virulent Newcastle disease virus (NDV) on egg production in commercial and SPF chickens. Int. J. Poultry Sc. 10(2):98-105. 2011. 2. Rauw, F., Gardin, Y., Palya, V., Anbari, S., Lemaire, S., Boschmans, M., van den Berg, T., Lambrecht, B. Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old. Vaccine 28:823–833. 2010.

ND+IB live virus, ocular

HVT-ND

ND+IB live virus, drinking water

Green

ND+IB live virus, ocular

Vectormune HVT NDV Marek’s disease Marek’s disease + ND KV

1 ®

ND+IB live virus, ocular

Group Yellow White Red

ELISA antibody titer, x 1000

was expected, since the hens received three doses of killed virus oil emulsion plus five live virus doses. White group had a live-ND virus only vaccination program, antibody titer at challenge was lower than Red group, but similar to Green and Yellow groups; however, egg production decayed from 75% to 60% two wpc and couldn´t reach 80% in the following weeks, so, the challenge virus didn´t kill the hens, but affected both the production and egg quality, as previously reported (1). Immunity in Yellow group that received the recombinant Vectormune HVT-NDV vaccine was not as high as the Red group, but allowed the egg production increase from 54.17% at challenge to around 95% three to five wpc, which was a little higher than Green group (25%) but couldn´t reach 87% after challenge, even when the antibody titer at challenge was almost the same. As seen in this study, vaccination with recombinant HVT-ND and boosting with live virus vaccines induces a long lasting immunity which protects against mortality and affords clinical protection against strong challenge with velogenic NDV, as previously reported (2), and also can reduce egg-production drop caused by the infection. Each vaccination program must be established according to the immunity level needed based on the challenge in the field, as well as the costs involved.

Egg production (%)

end of the trial. White group had 75% production at challenge but didn’t reach 80% as mean per wk trough the whole experiment. The Red group showed the lowest egg production at challenge (8.7%) but increased to 95.65% as soon as two wpc, remaining above 90% the following weeks. The Green group had 25% production at challenge, increased to 85.12% at three wpc but didn´t reached 87% the remaining weeks. Egg production during the five wpc was higher (P< 0.01) in Yellow and Red groups (85 and 81.49%, respectively) than White group (71.25%); egg production in Green group (76.19%) was not different from all other groups. There was no mortality after challenge. Feed consumption was not affected. Only one hen (4.16%) from Green group started with nervous signs (torticollis and incoordination) at 18 dpc, which increased and last for two wk, then started recovering at 32 dpc. There was no statistical difference (P>0.05) in hens showing clinical symptoms. There was no statistical difference among groups in the amount of broken eggs in the five wk period. The mean egg weight in Green group (54.91 g) was lower (P< 0.05) than in all other groups (Yellow= 56 g, White= 55.86 g, and Red= 56.15 g). The White group produced more shell-less eggs, 4.3% (P< 0.05), than the others (Yellow 0.56%; Red 0% and Green 0.31%). Humoral immune response to vaccination programs and after challenge is shown in Figure 1. ELISA antibody titer prior to challenge was higher (P< 0.01) in Red group than in the others; sero-conversion was seen in all groups except Red group. Antibodies titer remained almost the same until the fourth wpc without difference among groups (P>0.05). A significant reduction (P< 0.05) was seen in White group at five wpc.

30 31 Age (weeks)

Yellow ELISA

White ELISA

Red ELISA

Green ELISA

Yellow

White

Red

Green

FIELD SAFETY AND EFFICACY OF A VECTOR MAREK’S/NEWCASTLE DISEASE VACCINE (RHVT – NDV) AS ASSESSED BY CLINICAL AND PRODUCTIVE PERFORMANCE IN A LARGE POPULATION OF COMMERCIAL BROILERS L. Sesti A, C. Kneipp A, R. Paranhos A, P. Paulet B, and C. Cazaban B Ceva Animal Health A Brazil, B France Therefore, it’s imperative for the industry to have available effective, and predominantly safer, immunological tools to prevent and/or control ND, even in countries whose production systems are free of velogenic ND viruses (2,4). The objective of this work was to investigate the safety and efficacy of a vector rHVT-NDV vaccine in

INTRODUCTION Newcastle disease (ND), along with avian influenza, are the most serious health threat to the modern poultry industry around the world due to their induced severe clinical (mortality and morbidity) and political consequences (export of poultry products).

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EXTRACT FROM WVPAC 2013, NANTES, FRANCE.

VECTORMUNE® ND

EXTRACT FROM WVPAC 2013, NANTES, FRANCE.

VECTORMUNE® ND WVPAC2013 - 19-23 august 2013 - Nantes FRANCE

SKP14

The use of rHVT-F vector vaccine improves chicken flocks immunity against the Newcastle Disease under field conditions. GARDIN Yannick1, GACAD Emilio2, UMANDAL Mel2, PANIAGO Marcelo3, LOZANO Fernando3, EL-ATTRACHE John4 1

Ceva Animal Health, Le Clos du Bois, 49460 Cantenay-Epinard, France Ceva Animal Health Philippines Inc., 1605E PSEC Bldg., Exchange Road - Ortigas Center, 1605 Pasig City, The Philippines 3 Ceva Animal Health, 10 avenue de la Ballastiere, 33501 Libourne, France 4 Ceva Animal Health, 8906 Rosehill Road, 66215 Lenexa, KS, USA

Abstract

Newcastle Disease was first described almost a century ago, but since then, despite huge improvements regarding biosecurity, vaccines, vaccination techniques and programs, it has remained in many poultry producing countries, one of the most devastating diseases. Together with the emergence of new genotypes of virus, an increase in prevalence has even been recorded during the past decade in Central America, Africa, South East Asia, China, The Middle East and Eastern Europe. Many non vaccine-related factors can explain this continuous incidence: (i) increase in poultry production, (ii) increase in commercial exchanges, (iii) existence of a permanent reservoir of virus in other avian species, domestic and wild, (iv) persistence of backyard farming and fighting cocks activities, (v) poor biosecurity, etc. But when it comes to vaccines and vaccinations, the two well identified factors explaining protection failures are: (i) the partial / total neutralization of live and inactivated vaccines by maternally derived antibodies, and (ii) the poor quality of vaccine administration when performed at the farm. Many studies conducted with rHVT-F vector vaccines have demonstrated their capacity to replicate and induce reliable, high and long lasting protection in the presence of even high levels of passive immunity (1, 2). Because of this, vaccination can be done at the hatchery and protection always develops, which are key advantages when compared to classical vaccination programs. The objective of this study was to compare, under field conditions, a vaccination program including a rHVTF vaccine (Vectormune ND – Ceva) with a vaccination program combining live and inactivated vaccines applied together at the hatchery, usually considered as the “golden standard” for broilers (3, 4). A total of 6 farms were included in the trials. In each farm, comparable houses were used with flocks of chickens vaccinated according to one of the two vaccination programs described in table 1. At harvest time, 20 blood samples were collected from each house and tested for antibody titer using the HI test. Results indicated absence or much lower percentage of sero-negative chickens and better uniformity in flocks that received the rHVT-F program compared to the reference program.

References 1. 2. 3. 4.

Rauw F. et al. (2010) Vaccine 28 (3) :823-33. Palya V. et al. (2012) Avian Diseases 56 : 282-287. Bennejean G. et al. (1978) Avian Pathology 7(1) :15-27 Kapczynski D. et al. (2005). Vaccine 23 (26): 3424-33.

Keywords: Newcastle Disease; rHVT-F ND vector vaccine; Vaccine prevention

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Scientific File • N°18

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EXTRACT FROM Figure 1. Egg production and ELISA antibody titer during five weeks after challenge in laying hens inoculated L. SESTI, C. KNEIPP, R. PARANHOS, with the Chimalhuacan strain of Newcastle disease virus at 28 wk of age. P. PAULET, AND C. CAZABAN. WVPAC 2013 - NANTES FRANCE. ELISA antibody titer and egg production (%) VECTORMUNE® 40 ND 100

Scientific File • N°19

40 30

Egg production (%)

ELISA antibody titer, x 1000

VECTORMUNE® ND

PROCEEDINGS OF THE AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS ANNUAL MEETING, 2012, SAN DIEGO, CALIFORNIA.

HVT-ND

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0 28

30 31 Age (weeks)

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N°19 • Scientific File

comparison with different ND vaccination programs with conventional live vaccines in a large field trial involving six companies and over nine million broilers in the northeast region of Brazil (states of Pernambuco and Ceará).

feed conversion, vaccination program, sales price per kg of live slaughter age broiler). Statistical analysis. All productivity, clinical and laboratory results were statistically analyzed by completely random analysis of variance and means compared by Tukey HSD All-Pairwise test at p<0.05 level. (Statistix 9.0 software, www.statistix.com)

MATERIALS AND METHODS Testing vaccine. The new ND commercial vaccine tested was a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of the ND virus (Vectormune® ND, from Ceva Animal Health, Lenexa, Kansas, USA). Experimental groups. The data to be presented are from three companies (integrated production systems) which had different conventional ND vaccination programs. The respective ND vaccination programs compared in each company and volumes of broilers tested in each were as follow:

RESULTS Serology. HI seroconversion from rHVT-NDV vaccinated broilers was almost 100% negative in all companies with only a few individuals presenting titers slight above the positive threshold limit (3 log 2) after 42 d of age while the on ELISA serology, a low but clear seroconversion could be observed in all flocks at 42 and 49 d. Most interesting was the significant uniformity (very low CVs) of titers in rHVT-NDV vaccinated broilers after 35 d of age. Histopathology of tracheas. At almost all ages tested (except for Company 1 at 14 d and Company 3 at 28 d), all broilers rHVT-NDV vaccinated presented significantly lower lesion scores (congestion, deciliation, mononuclear inflammatory infiltrate and epithelial hyperplasia) on sections from upper, mid, and lower regions of the trachea when compared to conventional live vaccines vaccinated broilers (Table 1). rHVT PCR. All ten flocks sampled at three wk of age in the rHVT-NDV vaccine group in each company were found to be positive for the molecular detection of the rHVT indicating an excellent vaccination take. Productivity and financial data. The main productivity and financial data sets from both experimental groups are depicted on Table 2. It is evident the very strong trend, and sometimes significant differences, for better results for the rHVTNDV vaccinated broiler group when compared with the conventional live vaccines group. This is clearly demonstrated by the numerically and/or significantly higher values of the European Productivity Index which takes into consideration data of viability, weight, feed conversion, and age at slaughter. In addition, all rHVT-NDV vaccinated groups generate higher values of financial income at slaughter age.

Company 1- rHVT-NDV d one SQ (437,000 broilers) vs. apathogenic Phy.LMV. 42 strain d one spray + lentogenic La Sota 18 d DW (555,000 broilers) Company 2- rHVT-NDV d one SQ (761,000 broilers) vs. apathogenic Phy.LMV.42 strain d one spray (754,000 broilers) Company 3 -rHVT-NDV d one SQ (1,080,000 broilers) vs. lentogenic C2 strain d one spray (1,210,000 broilers). In all companies the populations of broilers used in each group were always contemporaneous (one wk vaccination with each treatment group) and had absolutely the same vaccination program, management procedures, nutrition levels, type of houses and disease challenges in general. Sampling for laboratory analysis. The sampling schedule in ten flocks of each group in each company was as follows: 20 blood samples for HI (8 HU) and IDEXX ELISA serology were taken at 1, 14, 21, 35, 42 and 49 d of age; five tracheas for histopathology at 14, 21, and 28 d of age; and pool of five wing feathers from each of five birds for rHVT polymerase chain reaction (PCR) at 21 d of age. Productivity data. Key productivity parameters (daily weight gain, slaughter weight, feed conversion, final mortality and European productivity index) were collected and statistically analyzed for the entire broiler population used in each group in each company. In addition, a detailed financial output from each experimental group of broilers was calculated taking into account all main broiler production costs and income involved (unit cost of DOC, feed, mortality,

DISCUSSION AND CONCLUSIONS Recently published research results (1,3) have demonstrated that the rHVT-NDV vaccine is quite able to induce some rapid and significant protection against a strong ND challenge in field and laboratory vaccinated commercial broilers.

62nd Western Poultry Disease Conference 2013

The present results strongly indicate that such rHVT-NDV vaccine is also highly beneficial in regions free of velogenic ND where preventive vaccination with conventional live ND vaccines is practiced. Such beneficial effect most probably occurs for two main reasons: a) elimination of the normal post vaccination inflammatory reaction that occurs after the use conventional live ND vaccines and therefore eliminating the metabolic cost associated with it; and, b) elimination and/or prevention of “rolling” post ND vaccination reactions in the field, which not only are naturally growth detrimental by themselves but also many times, interact with reactions from the other respiratory vaccines (IBV and/or aMPV) causing more severe secondary bacterial infections and mortality in four to seven wk broilers. These two main reasons can be confirmed by the significantly lower histopathological lesion scores (Table 1) and higher productivity and economical results generated by the rHVT-NDV vaccinated broilers (Table 2). In conclusion, the rHVT-NDV Newcastle Disease vaccine tested is effective and quite safe for vaccination of broilers in modern poultry production systems at both velogenic ND free and endemic countries.

REFERENCES 1. Lechuga, M., D. Dueñas, A. Soto, F. Lozano, P. Paulet, V. Palya and Y. Gardin. New approaches in the prevention of velogenic Newcastle Disease in Mexico. In: Proceedings of the 61st Western Poultry Disease Conference, Sacramento, California, USA. p 55, 2012 2. Orsi, M. Biological, molecular, immunological and thermal stability characterization of vaccine and field isolates of Newcastle Disease virus from modern poultry and migratory birds in Brazil. DSc thesis School of Medical Sciences, University of Campinas. Campinas, SP – Brazil. 179 pages, 2010. (in Portuguese) 3, Palya, V., I. Kiss, T. Tatár-Kis, T. Mató, B. Felföldi and Y. Gardin. Advancement in vaccination against newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions. Avian Diseases 56:282–287. 2012 4. Thomazelli, L.M., J. de Oliveira, C. de S. Ferreira, R. Hurtado, D.B. Oliveira, T. Ometto, M. Golono, L. Sanfilippo, C. Demetrio, M.L. Figueiredo and E.L. Durigon. Molecular surveillance of the Newcastle disease virus in domestic and wild birds on the North Eastern Coast and Amazon biome of Brazil. Brazilian J of Poultry Science 14(1): 1-7. 2012

Table 1. Histopathology lesion scores at 14, 21, and 28 days of age from tracheas of broilers vaccinated with either an rHVT-NDV vaccine or live conventional vaccines agains ND. rHVT-NDV Company 1 Company 2 Company 3

14 Days 0.38a 0.45a 0.20a

Conventional live vaccine 0.43a 0.52b 0.33b

rHVT-NDV 21 Days 0.53a 0.66a 0.35a

Conventional live vaccine 0.60b 0.86b 0.45b

rHVT-NDV 28 Days 0.69a 0.69a 0.56a

Conventional live vaccine 0.79b 0.99b 0.60a

Table 2. Productivity parameters and economical results from broilers vaccinated with either an rHVT-NDV vaccine or live conventional vaccines against ND. Productivity and Economic Parameters Slaughter Weight Mortality Feed Conversion Extra income Productivity Groups (%) (g/g) (g) Index (US$) * Company 1 (mixed sex; slaughter age = 53.3 days) rHVT-NDV 2.16a 255a 3010a 5.0a 85.2 Phy.LMV.42 a a b b 6.0 2.26 3050 237 + La Sota Company 2 (mixed sex; slaughter age = 50.5 days) 6.3a rHVT-NDV 3100a 2.02a 285a 49.9 a a a Phy.LMV.42 3100 7.8 2.05 276a Company 3 (females; slaughter age = 36.7 days) rHVT-NDV 1830a 3.5a 1.80a 272a 32 a a a C2 1840 3.9 1.83 264a Company 3 (males; slaughter age = 50.7 days) 3190a 6.4a 16.8 rHVT-NDV 2.03a 294a a a a 7.2 C2 3180 2.04 nd 286b 21 62 Western Poultry Disease Conference 2013 * extra income per each group of 1000 broilers from the rHVT-NDV vaccinated group when compared with the income generated by the control group (conventional live vaccines).

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Scientific File • N°20

EXTRACT FROM WVPAC 2013, NANTES, FRANCE

VECTORMUNE® ND

Scientific File • N°21

PROCEEDINGS OF THE 61ST WESTERN POULTRY DISEASE CONFERENCE, 2012, SCOTTSDALE, ARIZONA.

VECTORMUNE® ND

Assessment of a vector Marek’s/Newcastle vaccine through clinical and produc;ve performance of commercial broilers in two diﬀerent epidemiological situa;ons of Newcastle Disease challenge Luiz Ses; 1, Yesenia Vega 2, Jorge Cortegana 2, Pascal Paulet 3, Marcelo Paniago 3, Fernando Lozano 3 Ceva Animal Health / 1 Paulínia, Brazil / 2 Lima, Peru / 3 Libourne, France

luiz.ses;@gmail.com

INTRODUCTION Newcastle Disease (ND), has long been one of the most serious health threat to the modern poultry industry around the world due to their induced severe clinical (mortality and morbidity) and poli<cal/economic consequences (export of poultry products). Therefore, it’s impera<ve for the industry to have available effec<ve, and predominantly safer, immunological tools to prevent and/or control ND in countries like Brazil where produc<on systems are free of velogenic ND viruses (2,4) and in velogenic ND endemic countries (Peru). The objec<ve of this work was to inves<gate the safety and efficacy of a vector rHVT-‐NDV vaccine in comparison with different ND vaccina<on programs with conven<onal live vaccines in a large field trials in a country with no velogenic ND challenge (Brazil) and in another (Peru) where there’s a medium challenge by velogenic ND. MATERIAL AND METHODS Tes;ng vaccine The new ND commercial vaccine tested was a turkey herpesvirus-‐based recombinant vaccine (rHVT) expressing a key protec<ve an<gen (F glycoprotein) of the ND virus (Vectormune® ND -‐ from Ceva Animal Health -‐ Lenexa, Kansas, USA). Experimental groups The data where originated from three companies in Brazil and one company in Peru which had different conven<onal ND vaccina<on programs. The respec<ve ND vaccina<on programs compared in each company and volumes of broilers tested in each were as follow: BRAZIL à Company 1 = rHVT-‐NDV day one SQ (437,000 broilers) vs apathogenic Phy.LMV.42 strain day one spray + lentogenic La Sota 18 days DW (555,000 broilers); Company 2 = rHVT-‐NDV day one SQ (761,000 broilers) vs apathogenic Phy.LMV.42 strain day one spray (754,000 broilers); Company 3 = rHVT-‐NDV day one SQ (1,080,000 broilers) vs lentogenic C2 strain day one spray (1,210,000 broilers). PERU à Company 1 = rHVT-‐NDV in ovo + HB1 strain day one spray (995,250 broilers) vs HB1 strain day one spray + La Sota strain 10 days DW + apathogenic Phy.LMV.42 strain 18 days spray (950,275 broilers). In all companies the popula<ons of broilers used in each group were always contemporaneous (one week vaccina<on with each treatment group) and had absolutely the same vaccina<on program, management procedures, nutri<on levels, type of houses and disease challenges in general. Sampling for laboratory analysis The sampling schedule in 10 flocks of each group in each company was as follows: 20 blood samples for HI (8 HU) and idexx Elisa serology were taken at 1, 14, 21, 35, 42 and 49 days of age; 5 tracheas for histopathology at 14, 21 and 28 days; and pool of 5 wing feathers from each of 5 birds for rHVT PCR (Polymerase Chain Reac<on) at 21 days of age. Produc;vity data Key produc<vity parameters for each broiler flock (daily weight gain, slaughter weight, feed conversion, final mortality and European produc<vity index) were collected and sta<s<cally analyzed for the en<re broiler popula<on used in each group in each company. In addi<on, a detailed financial output from each experimental group of broilers was calculated taking into account all main broiler produc<on costs and income involved (unit cost of DOC, feed, mortality, feed conversion, vaccina<on program, sales price per kg of live slaughter age broiler). Sta;s;cal analysis All produc<vity, clinical and laboratory results were sta<s<cally analyzed by completely random analysis of variance and means compared by Tukey HSD All-‐Pairwise test at p<0.05 level. (Sta<s<x 9.0 somware – www.sta<s<x.com) RESULTS Produc;vity and financial data The main produc<vity and financial data sets from both experimental groups in both countries are depicted on Table 1. It is evident the very strong trend, and some<mes significant differences, for beoer produc<on and financial results for the rHVT-‐NDV vaccinated broiler group when compared with the conven<onal live vaccines group in both countries. Serology HI seroconversion from rHVT-‐NDV vaccinated broilers was almost 100% nega<ve in all companies with only a few individuals presen<ng <ters slight above the posi<ve threshold limit (3 log 2) amer 42 days of age while the on Elisa serology a low but clear seroconversion could be observed in all flocks at 42 and 49 days (Figures 1, 2). Histopathology of tracheas At almost all ages tested in the Brazilian trials (except for company1 at 14 days and company 3 at 28 days), all broilers rHVT-‐NDV vaccinated presented significantly lower lesion scores (conges<on, decilia<on, mononuclear inflammatory infiltrate and epithelial hyperplasia) on sec<ons from upper, mid and lower regions of the trachea when compared to conven<onal live vaccines vaccinated broilers. (Table 2). rHVT PCR All 10 flocks sampled at 3 wk of age in the rHVT-‐NDV vaccine group in each company in both countries were found to be posi<ve for the molecular detec<on of the rHVT. DISCUSSION AND CONCLUSION Recently published research results (1,3) have demonstrated that the rHVT-‐NDV vaccine is quite able to induce some rapid and significant protec<on against a strong ND challenge in field and laboratory vaccinated broilers. The present results strongly indicate that such rHVT-‐NDV vaccine is also highly beneficial in regions free and endemic for velogenic ND where preven<ve vaccina<on with conven<onal live ND vaccines is prac<ced. Such beneficial effect most probably occurs for two main reasons: a) elimina<on of the normal post vaccina<on inflammatory reac<on that occurs amer the use conven<onal live ND vaccines and therefore elimina<ng the metabolic cost associated with it and, b) elimina<on and/or preven<on of “rolling” post ND vaccina<on reac<ons in the field which not only are naturally growth detrimental by themselves but also many <mes interact with reac<ons from the other respiratory vaccines (IBV and/or aMPV) causing more severe secondary bacterial infec<ons and mortality in 4 to 7 weeks broilers. These two main reasons can be confirmed by the significantly lower histopathological lesions scores seen in rHVT-‐NDV vaccinated flocks in Brazil (Table 2) and higher produc<vity and economical results presented by the rHVT-‐NDV vaccinated broilers (Table 1). In conclusion, the rHVT-‐NDV Newcastle Disease vaccine tested is very effec<ve and quite safe for vaccina<on of broilers in modern poultry produc<on systems at both velogenic ND free and endemic countries. In addi<on, with such immunological tool, many countries around the world will have a quite beoer opportunity to work on ND eradica<on programs. BIBLIOGRAPHY 1. Lechuga, M., D. Dueñas, A. Soto, F. Lozano, P. Paulet, V. Palya and Y. Gardin. Proceedings of the 61st Western Poultry Disease Conference, Sacramento, California, USA. p 55, 2012 2. Orsi, M. DSc thesis -‐ School of Medical Sciences, University of Campinas. Campinas, SP – Brazil. 179 pages, 2010. (in Portuguese) 3. Palya, V., I. Kiss, T. Tatár-‐Kis, T. Mató, B. Felföldi and Y. Gardin. Avian Diseases 56:282–287. 2012 4. Thomazelli, L.M., J. de Oliveira, C. de S. Ferreira, R. Hurtado, D.B. Oliveira, T. Omeoo, M. Golono, L. Sanfilippo, C. Demetrio, M.L. Figueiredo and E.L. Durigon. Brazilian J of Poultry Science 14(1): 1-‐7. 2012

Table 1. Productivity parameters and economical results from broilers vaccinated with either an rHVT-NDV vaccine or live conventional vaccines against ND. Slaughter Weight (g)

Productivity and Economic Parameters Mortality Feed Conversion Productivity (%) (g/g) Index

Extra income (US$) *

rHVT-NDV Phy.LMV.42 + La Sota

3010a

Company 1 (mixed sex; slaughter age = 53.3 days) 5.0a 2.16a 255a

85.2

rHVT-NDV Phy.LMV.42

3100a 3100a

rHVT-NDV C2

1830a 1840a

rHVT-NDV C2

3190a 3180a

Groups

3050a

BRAZIL

6.0a

2.26b

237b

Company 2 (mixed sex; slaughter age = 50.5 days) 6.3a 2.02a 285a 7.8a 2.05a 276a Company 3 (females; slaughter age = 36.7 days) a a 3.5 1.80 272a 3.9a 1.83a 264a Company 3 (males; slaughter age = 50.7 days) a a 6.4 2.03 294a 7.2a 2.04a 286b

49.9 32 16.8

PERU

Company 1 (mixed sex; slaughter age = 41.2 days) rHVT-NDV + 2490a 3.7a 1.68a 345a 217.1 HB1 HB1 + La Sota + b b b b 2280 7.1 1.76 291 Phy.LMV.42 * extra income per each group of 1000 broilers from the rHVT-NDV vaccinated group when compared with the income generated by the control group (conventional live vaccines).

Figure 1. Seroconversion proﬁle in broilers vaccinated with rHVT-‐NDV vaccine in ovo + HB1 strain at day one in the Peruvian trial.

Figure 2. Seroconversion proﬁle in broilers vaccinated with rHVT-‐ NDV vaccine at day one and broilers vaccinated with conven<onal live vaccines at day one and in the ﬁeld (control group) in the Brazilian trials(V = rHVT-‐NDV trt and C = control group).

Table 2. tracheas’ histopathology scores in broilers vaccinated with rHVT-‐NDV vaccine at day one and broilers vaccinated with conven<onal live vaccines at day one and in the ﬁeld (control group) in the Brazilian trials.

REFERENCE Ses;, L; Vega, Y; Cortegana, J; Paulet, P; Paniago, M & Lozano, F (2013) Assessment of a vector Marek’s/Newcastle vaccine through clinical and produc;ve performance of commercial broilers in two diﬀerent epidemiological situa;ons of Newcastle Disease challenge. Poster presented at the XVIIIth Congress of the World Veterinary Poultry Associa;on -‐ WVPA, Nantes, France -‐ 19-‐23 August 2013.

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Scientific File • N°22 VECTORMUNE® ND

SUMMARY OF EVALUACIÓN DE UNA VACUNA VECTORIZADA CONTRA LA ENFERMEDAD DE NEWCASTLE COMO PARTE DE UN PROGRAMA DE PREVENCIÓN. DUENAS D. ET AL., 2012. PROCEEDINGS OF THE XXXVII ANECA CONFERENCE, MAY 2-6, PUERTO VALLARTA, MEXICO, PP.105-110.

SUMMARY OF NUEVOS ENFOQUES EN LA PREVENCIÓN DE LA ENFERMEDAD DE NEWCASTLE VELOGENICA. HIGUERA S. ET AL., 2012. PROCEEDINGS OF THE XXXVII ANECA CONFERENCE, MAY 2-6, PUERTO VALLARTA, MEXICO, PP.169-172.

MATERIALS AND METHODS Eight farms of commercial broilers belonging to six different poultry companies were included in this trial. They were located in 4 different high Newcastle disease challenge areas of Mexico. In each farm, one control broiler flock was given the routine ND vaccination program; whereas in the test broiler flocks Vectormune® ND vaccine replaced the killed vaccine (given once or twice, according to the company) or was tested in front of a rHVT-NDV competitor vaccine. In each test group, Vectormune® ND was administered by subcutaneous route, at one day of age, in the hatchery, together with Cevac® Vitapest L given by spray. Challenge: At the age of 17 to 35 days, depending on the trial, ten broilers per group were brought to the laboratory of the University of Mexico for challenge using the local Chimalhuacan NDV strain. Challenge dose was 105.5 EID50 per bird, administered by eye drop. Five SPF chickens of the same age were challenged as well. The birds were monitored for 14 days post-challenge. A total of 12 challenge trials were performed. Tracheal and cloacal swabs were collected 6 days after challenge and submitted to RT-PCR for challenge virus detection. In some trials, no virus shedding was evidenced in any group, so no conclusion could be drawn. Serology: antibody response was assayed by HI test.

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RESULTS The following table summarizes the various ND vaccination programs applied in the farms. Red color highlights the differences between groups in each “trial”.

All unvaccinated SPF birds died within less than 6 days post-challenge, hence validating the severity of the challenge model. PROTECTION AFTER LAB CHALLENGE FIELD COMMERCIAL BROILERS

Trial

Test groups

Age at challenge (days)

Control groups

100 90

Vectormune® ND, SQ Live ND spray

D1 D1 + D12

Killed ND, SQ Live ND spray

D1 D1 + D12 + D23

Vectormune® ND, SQ Live ND spray, eye drop, spray

D1 + D12 D1 + D12 + D23

Killed ND, SQ Live ND spray, eye drop, spray

Protection rate (%)

D1 D1 + D12

70 60 50 40 30 20

D1 D1 + D12 + D28

Vectormune® ND, SQ Live ND spray, eye drop, dw

D1 + D12 D1 + D12 + D28

Killed ND, SQ Live ND spray, eye drop, dw

1(35)

2(35)

3(35)

4(23)

5(35)

6(17)

7(21)

8(28)

9(35) 10(28) 11(28)

Trial no. (age in days at challenge in each trial)

D1 D1 + D15 + D24 + D35

Vectormune® ND, SQ Live ND spray, eye drop, spray, spray

D1 D1 + D15 + D24 + D35

Competitor rHVT-ND + SB1, SQ Live ND spray, eye drop, spray, spray

D1 D1 + D15 + D24 + D35

Vectormune® ND, SQ Live ND spray, eye drop, spray, spray

D1 D1 + D15 + D24 + D35

Competitor rHVT-ND + SB1, SQ Live ND spray, eye drop, spray, spray

REDUCTION OF SHEDDING, VECTORMUNE® ND VERSUS CONVENTIONAL PROGRAM

Vectormune® ND, SQ Live ND spray, eye drop, spray, spray

D1 D1 + D12 + D24 + D35

Killed ND, SQ Live ND spray, eye drop, spray, spray

D1 D1 + D12 + D24 + D35

Vectormune® ND, SQ Live ND spray, eye drop, spray, spray

D1 + D12 D1 + D12 + D24 + D35

Killed ND, SQ Live ND spray, eye drop, spray, spray

D1 D1 + D12 + D24 + D35

Vectormune ND, SQ Live ND spray, eye drop, spray, spray

D1 + D12 D1 + D12 + D24 + D35

Killed ND, SQ Live ND spray, eye drop, spray, spray

% drop in challenge virus shedding (tracheal/cloacal)

100

D1 D1 + D12 + D24 + D35

D1 D1 + D8

Vectormune® ND, SQ Live ND spray, eye drop

D8 D1 + D8 + D18

Killed ND, SQ Live ND spray, eye drop, dw

D1 D1 + D8

Vectormune® ND, SQ Live ND spray, eye drop

D1 + D8 D1 + D8

Killed ND, SQ Live ND spray, eye drop

100

97.5

95.5

94.9

90 80 67.4

70 60 50 40 30 20 10

Vectormune®

Controls

Trial no.

HI TITRES AT CHALLENGE AND 2 WEEKS POST-CHALLENGE 12

D1 D1 + D8

Vectormune® ND, SQ Live ND spray, eye drop

D1 + D8 D1 + D8

Killed ND, SQ Live ND spray, eye drop

28 HI titre (Log2)

8 6 4

98 /

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Conventional

Vectormune® ND

Conventional

Vectormune® ND

Final

Conventional

Vectormune® ND

5 Initial

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

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N°22 • Scientific File

EVALUACION DE UNA VACUNA VECTORIZADA CONTRA LA ENFERMEDAD DE NEWCASTLE COMO PARTE DE UN PROGRAMA DE PREVENCION

The following graph depicts the rise in HI titers after challenge (mean HI titer 2 weeks postchallenge minus mean HI titer at challenge). This gives an indication regarding the ability of the challenge virus to invade or not the chicken’s body.

1 1 1 2 3 3 David Dueñas , Andres Soto Mario Lechuga , Fernando Lozano , Pascal Paulet , Yannick Gardin (1) Ceva Animal Health - Mexico; (2) Ceva Animal Health - United States; (3) Ceva Animal Health - France david.duenas@ceva.com Tel. +52 777 3621800, fax. +52 7773145575

DELTA IN HI TITRES AFTER AND AT CHALLENGE 10

Resumen.

La enfermedad de Newcastle (EN) es una de las mayores preocupaciones para la industria avícola en México. Con el fin de evaluar la protección del pollo de engorda vacunado con programas de vacunación convencionales o con vacuna vectorizada (rHVT+EN), se corrieron cuatro desafíos en laboratorio con virus virulento de EN. Grupos de 10 pollos de engorda de 35 días de edad siguiendo diferentes programas de vacunación, incluyendo la vacuna rHVT-EN, fueron llevados a unidades de aislamiento y alojados junto con 5 aves SPF de la misma edad y desafiados con el virus de la EN cepa 6 Chimalhuacan a una dosis de 10 DIEP50 por vía ocular. Los parámetros medidos fueron: Signos clínicos, viabilidad, respuesta sexológica, excreción viral usando RT-PCR y ganancia de peso. Todas las aves mostraron 100% de protección contra signos clínicos y mortalidad, y todas las aves SPF murieron. La excreción viral fue significativamente disminuida en los grupos vacunados con rHVT-EN. La Vacuna HVT-EN en combinación con virus vivos puede ser aplicada en condiciones de producción de pollo de engorda, generando igual protección que un programa convencional pero disminuyendo sustancialmente la excreción viral.

6 4 2

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

Vectormune® ND

Conventional

-2

Vectormune® ND

In most trials, the rise in HI titers was lower in Vectormune®-vaccinated groups than in controls.

CONCLUSIONS: In a Newcastle disease endemic country like Mexico, the introduction of Vectormune® ND in a vaccination program including several live vaccine administrations enabled to: • Remove the use of killed oily ND vaccines, i.e. less handlings, less stress to the chickens, less time spent in vaccination procedure; • Induce similar, if not higher clinical protection, compared to the heavy conventional vaccination program; • Significantly reduce virus shedding after challenge which is a critical parameter when dealing with multiple flocks management and aiming at eradication of the disease.

Keywords: Newcastle, vector, vaccine Introducción

La Enfermedad de Newcastle (EN) es sin duda alguna una de las mayores preocupaciones de la industria avícola en México debido a las

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105

grandes pérdidas económicas que causa cuando se presenta de manera clínica o subclínica (8). El vEN fue reportado por primera vez en México en 1946 (10), aunque es muy probable fuera introducido al país mucho tiempo atrás, sin embargo, como la avicultura en esa época consistía en pequeños gallineros, las pérdidas nunca se consideraron de gran magnitud. A partir de los años 50’s, la avicultura mexicana se desarrolló como una industria de alta producción, y desde entonces se volvió imprescindible proteger a cada parvada contra la EN (4). Además de las buenas prácticas de bioseguridad, el control de la EN consiste en la vacunación preventiva, utilizando la combinación de vacunas vivas e inactivadas (6); los tipos de vacuna y programas de vacunación varían dependiendo de la amenaza potencial, esto es, de la virulencia de la cepa presente, fin zootécnico de las aves y calendario de producción. Aunque la vacunación contra la EN es una práctica normal de manejo, las cepas velogénicas son endémicas y responsables de importantes pérdidas directas e indirectas (8). Con las estrategias actuales de vacunas y vacunación, la mortalidad y morbilidad se han reducido pero no han eliminado la infección ni la excreción viral, lo cual es crítico para el control de la EN (5). Estos brotes en parvadas vacunadas sugieren que el nivel de cobertura de la vacunación es bajo, o que la vacunación no provee inmunidad perfecta permitiendo al virus diseminarse entre poblaciones. También es importante notar que en regiones donde no circula o aparentemente no circula el virus virulento, las empresas prefieren usar

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programas de vacunación que epidemiológicamente son no-óptimos debido a los efectos colaterales negativos de la vacunación (7), aunado al hecho de que la infección, diseminación y transmisión de la EN velogénica se puede presentar sin signos clínicos aparentes (3,7). Finalmente, no se puede ignorar que la presencia de altos niveles de anticuerpos maternos interfiere con el desarrollo de una inmunidad sólida después de la vacunación (1,9) al día de edad. Por lo tanto, los brotes continuos en parvadas comerciales vacunadas contra la EN demuestran que las estrategias actuales no son totalmente eficaces, éstas pueden ser mejoradas por una nueva generación de vacunas como son las vacunas vectorizadas. La vacuna vectorizada de la Enfermedad de Marek HVT que expresa al gen de la proteína F del vEN (rHVT-NDV) han mostrado eficacia al aplicarse dentro de un programa de vacunación contra la EN (2). La protección que estas vacunas ofrecen se basa en la fuerte respuesta de tipo celular que el HVT induce y que adicionalmente, al expresar la proteína F del vEN, genera anticuerpos neutralizantes. Tan solo los anticuerpos contra la proteína F evitan la transmisión de célula a célula. La eficacia de la vacuna rHVTNDV ha sido probada en desafíos con cepas velogénicas en laboratorio (5), y también en condiciones de producción comercial en diferentes partes del mundo (Y. Gardin, comunicación personal) con considerable éxito. El objetivo de éste trabajo es demostrar el nivel de protección generada por la vacuna rHVT_NDV incluida en programas de vacunación de pollos de engorda criados en

condiciones comerciales y desafiadas en laboratorio con una cepa velogénica de la EN (Chimalhuacan) en términos de viabilidad y excreción viral, con la finalidad de mostrar que es factible su utilización en condiciones de producción propias de México.

Tabla 1. Programas de vacunación contra la Enfermedad de Newcastle por prueba No. Prueba 1

Material y método Para el desarrollo de este trabajo se corrieron cuatro experimentos con el mismo diseño, comparando parvadas vacunadas con rHVTNDV y virus vivos con parvadas con programas de vacunación que incluyen virus vivos e inactivos. Aves. Se tomaron 80 pollos de engorda de 30 días de edad provenientes de 8 granjas distintas, ubicadas en cuatro zonas geográficas diferentes. De cada zona se tomaron 10 pollos de una granja vacunados contra la EN con rHVT-NDV más virus vivos y 10 pollos de otra granja diferente que fueron vacunados con virus vivos e inactivados (Tabla 1). Cada grupo de 10 aves se alojó en unidades de aislamiento con aire filtrado tipo Horsfall-Bauer junto con 5 aves SPF de la misma edad.

Programa de vacunación experimental rHVT-NDV, 1 día Virus vivo, 1 y 12 días

Programa de vacunación control Inactivada, 1 días Virus vivo, 1 y 12 días

rHVT-NDV, 1 día Virus vivo, 1 y 12 y 23 días

Inactivada, 1 y 12 días Virus vivo, 1, 12 y 23 días

rHVT-NDV, 1 día Virus vivo, 1 y 12 y 28 días

Inactivada, 1 y 12 días Virus vivo, 1,12 y 28 días

rHVT-NDV, 1 día Virus vivo, 1, 12 y 28 días

rHVT-NDV, 1 día Virus vivo, 1, 12 y 25 días

Desafío. A cada ave, incluyendo las aves SPF, se les aplicó 0.3 ml vía ocular de la cepa velogénica de la EN cepa Chimalhuacán con 6 un título de 10 DIEP50. El virus de desafío fue originalmente aislado en México, y pertenece a la clase II, genotipo V, con un IPIC de 1.89. Parámetros: Las aves fueron observadas por 14 días posdesafío (PD) por signos clínicos y mortalidad. El día que en que las aves fueron inoculadas se tomaron muestras de sangre para correr las pruebas de ELISA (IDEXX) y de HI (diluciones decimales seriadas) con 4 UH. A los siete días PD, se tomaron muestras de hisopos cloaca les para medir la excreción viral por medio de la prueba de Reacción en cadena por la Polimerasa de Tiempo Real (PCR-TR). La técnica de PCR-TR es la que se realiza en el laboratorio de diagnóstico de Investigación Aplicada, S. A. de C.V., Tehuacan Puebla). De cada grupo se tomaron 10 hisopos, mismos que se dividieron en dos pool.

Vacunas. La vacuna rHVT-NDV (Vectormune® HVT_NDV) fue producida por Ceva-Biomune (USA). En todas las pruebas, la vacuna que se aplicó al día de edad fue la cepa PHY-LMV 42. La revacunación con virus vivos se realizó con: Prueba 1: PHY-LMV 42; Prueba 2: LaSota rND-AI; Prueba 3: LaSota y; Prueba 4: LaSota. Las vacunas inactivadas que se aplicaron en las pruebas 1,2 y 3 son elaboradas con la cepa LaSota, provenientes de diferentes laboratorios comerciales.

las aves SPF, todas murieron entre 3 y 6 días PD. Serología. En la tabla 2 se presentan los resultados de las pruebas de HI y de ELISA Los títulos de HI obtenidos por los grupos vacunados con rHVT-NDV fueron bajos como se esperaba, dado que es la proteína F del vEN el que genera la respuesta inmune protectiva y no la Hemaglutinina, aunque tampoco ésta prueba fue capaz de detectar los anticuerpos generados por los virus vivos administrados en el mismo programa de vacunación. En los grupos vacunados con rHVT-NDV, el título HI inicial es el mínimo, excepto en la prueba 3 que probablemente se debió a que la aplicación de un virus vivo es la más reciente en comparación con otras pruebas. En todas las pruebas, el grupo control tuvo títulos de HI inicial más alto, y después del desafío mostraron una respuesta más alta con excepción de la prueba cuatro, debido a que en ésta ambos grupos recibieron la vacuna rHVT-NDV.

Resultados Viabilidad.. Ninguno de los cuatro grupos experimentales ni de los grupos control tuvo mortalidad, e incluso tampoco se presentaron signos clínicos que reportar. Con respecto a

106 102 /

En este trabajo que se presenta, la prueba de ELISA-IDEXX no fue sensible para detectar los anticuerpos generados por la proteína F, y tampoco fue muy sensible al estímulo

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N°22 • Scientific File

generado por los virus vivos aplicados en los

mismos programas de vacunación.

reducción en la excreción viral en estas pruebas fue significativamente menor en los grupos que recibieron la vacuna rHVT-NDV. En las mismas pruebas, las aves que recibieron vacuna inactivada, prácticamente no redujo la excreción viral, o la reducción de de menos de un logaritmo.

Tabla 2. Títulos de HI y de ELISA obtenidos antes del desafío y 14 días después

Prueba

Inhibición de la Hemoaglutinación Antes desafío Posdesafío

Grupo rHVT-NDV Control

rHVT-NDV Control

3.32±0.00 4.82±1.27

rHVT-NDV Control

3.32±0.00 4.65±0.71

ELISA Antes desafío c

Posdesafío c

5.72±1.71 8.92±1.43

125±109 552±443

1799±1008 5608±4105

Discusión.

8.02±1.64 10.02±0.67

253±248 467±374

2865±1862 20039±6499

5.82±1.27 6.42±2.08

5.02±1.06 9.72±0.84

697±383 608±983

2588±1757 14293±8764

3.32±0.00 5.72±1.07

9.52±1.69 8.82±2.22

84±63 999±719

24760±8061 18245±8462

La vacuna rHVT-NDV es una vacuna vectorizada que utiliza al virus Herpes de Pavo HVT para expresar al gen de la proteína F del virus de la EN que se aplica en la incubadora al día de edad, o que pude aplicarse también in ovo para generar inmunidad contra las enfermedades de EN y de Marek. Después de correr cuatro pruebas de desafío en unidades de aislamiento con un virus velogénico de la EN cepa Chimalhuacan en pollos de engorda criados en granja, se pudo observar que:

C=Diferencia estadísticamente significativa p≤0.01 por la prueba t-Letus 123. Los resultados de HI se presentan en Log 2.

Excreción viral. Los resultados de prueba de excreción viral medida por PCR-TR se muestran en la gráfica 1. Las barras de la gráfica se agrupan en pool 1 y pool 2 de cada grupo.

Hubo 100% de protección contra signos clínicos y mortalidad en todos los grupos probados, tanto los experimentales vacunados con rHVT-NDV como en los grupos control. Si bien es cierto que no hubo diferencias en este punto de análisis, es importante para el objetivo del estudio hacer notar que la coincidencia en el resultado demuestra que todas las vacunas actuaron en tiempo y forma de acuerdo a su diseño, generando inmunidad suficiente. La norma mexicana para este tipo de desafíos indica que por lo menos el 80% de las aves desafiadas deben estar protegidas, y que por lo menos el 80% de las aves testigo deben morir o mostrar signos de la EN; en este caso la protección de los grupos probados fue de 100%, y así mismo, la mortalidad de todos los grupos de aves SPF murieron mostrando signos y lesiones de la EN, por lo que las cuatro pruebas se consideran satisfactorias.

Gráfica 1. Excreción viral medida por PVR-TR y expresada en copias por ml Log 2.

6 Copias por ml Log (2)

5 4 rHVT-NDV

Control

2 1 0

Prueba 1

Prueba 2

Prueba 3

Prueba 4

La dosis inoculada expresada en copias/ml Log 2 fue de 5.93

Como se puede observar en la gráfica 1, solo se detectó excreción viral en las pruebas 1 y

108

104 /

2, muy probablemente relacionado con el programa de vacunación utilizado. La

El grupo de aves que recibieron la vacuna rHVT-NDV más virus vivos no generaron una

respuesta serológica detectable por la prueba de HI y tampoco por la prueba de ELISAIDEXX. Es conocido que la respuesta inmune la genera la proteína F y no la proteína HN, así que se esperaba este resultado. Aunque también es importante mencionar que las dos pruebas serológicas tampoco fueron sensibles para detectar la respuesta generada contra las vacunas con virus vivos a los 35 días de edad. Así mismo, debido a la ausencia de respuesta serológica a la prueba de ELISA_IDEXX, se asume que es necesario contar con una prueba que detecte anticuerpos contra la proteína F del vEN. Como la respuesta serológica generada por el programa que recibió rHVT-NDV no es detectable por la prueba de HI, ésta prueba puede utilizarse como sistema DIVA (Differenciate Infected from Vaccinated Animals) para detectar infecciones por virus de campo. En estos estudios, la respuesta serológica no estuvo relacionada con la protección como se ha mostrado en otros estudios donde se utilizan vacunas vivas e inactivadas. Este punto será importante para la interpretación de resultados serológicos cuando se utilice la vacuna rHVT-NDV en explotaciones comerciales. En las pruebas de excreción viral con la prueba de PCR-TR, solo se detectaron virus en las pruebas 1 y 2, y aunque no tenemos una explicación o pruebas contundentes, especulamos que el tipo de vacunas con virus vivo que se usaron en estos programas incidieron en el resultado pues en las pruebas 3 y 4 se utilizaron vacunas conteniendo la cepa LaSota mientras que en las pruebas 1 y 2 se utilizaron vacunas apatógenas y LaSota recombinante, sin embargo, es necesario hacer pruebas complementarias para poder afirmarlo. Una observación importante es, que las aves vacunadas con rHVT-NDV en las pruebas 1 y 2 excretaron una cantidad significativa menor que las que recibieron vacunas inactivadas. La excreción viral de los

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N°22 • Scientific File

PROCEEDINGS OF THE 61ST WESTERN POULTRY DISEASE CONFERENCE, 2012, SCOTTSDALE, ARIZONA.

VECTORMUNE® ND

grupos rHVT-NDV en las pruebas 1 y 2 redujeron en un 60-65% y totalmente en las pruebas 3 y 4, por lo que podemos afirmar que ésta vacuna tiene un fuerte potencial para reducir significativamente la transmisión del vEN velogénico de una manera más eficiente que los programas con vacunas inactivadas, ofreciendo un método de control de la EN más eficaz que lo que se utiliza actualmente. Finalmente, el objetivo de este trabajo es conocer el potencial de la vacuna rHVT-NDV para ser utilizada dentro de un programa de vacunación contra la EN, en condiciones de producción comercial de pollo de engorda en un medio ambiente propio de las principales zonas avícola de México, de tal manera que concluimos que ésta vacuna puede ser incluida en programas de vacunación esperando un mejor desempeño que los programas convencionales.

Moreno R. La Enfermedad de Newcastle y algunos avances recientes de diagnóstico. Ciencia Veterinaria (FMVZ-UNAM) 6:49-72. 1994

Rauw F., Gardin Y., Palya V., Anabari S., Lemarie S., Boschmans M. Improved vaccination against Newcastle Disease by in ovo recombinant HVT-NDV combined with an adjuvanted live vaccine at day old. Vaccine 28:823-833.2010

Senne D., King D., Kapczynski D., Control of Newcastle by vaccination. Dev. Biol. 119:165-70. 2004

VAnBoven M., Bouma A. Fabri T., Katsma E., Hartug L. and Koch G. Herd immunity to Newcastle Disease virus in poultry by vaccination. Avian Pathol. 37(1):1-5. 2008

Villegas P. Viral respiratory syatem. 77:1143-45. 1998

Referencias. 1.

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Guittet B., Picault J., Bouquet J., Devaux B., Gaudry D. and Moreau Y. Vaccination of one day old chicks against Newcastle Disease using inactivated oil adjuvant vaccine and/or live vaccine. Avian Pathol. 7:15-27. 1978 Heckert R., Riva S., Cook J., McMillen J. Schwartz J. Onset of protective immunity in chickens after vaccination with a recombinant Herpesvirus of Turkeys vaccine expressing Newcastle Disease virus Fusion and HemaglutininNeuroaminidase antigens. Avian Dis. 40 (4):770-7. 1996 Kapczynski D., King D., Protection of chickens against overt clinical disease

NUEVOS ENFOQUES EN LA PREVENCION DE LA ENFERMEDAD DE NEWCASTLE VELOGENICA

and determination of viral shedding following vaccination with commercially available Newcastle Disease virus vaccines upon challenge with highly virulent virus from the California 2002 exotic Newcastle Disease outbreak. Vaccine 23:3424-33. 2005

Sergio Higuera, Francisco Martínez, Mario Lechuga, David Dueñas, Andrés Soto Ceva Salud Animal Aunque la vacunación contra la Enfermedad de Newcastle (EN) es una práctica normal de manejo, las cepas velogénicas continúan siendo endémicas en muchos países y son responsables de pérdidas directas e indirectas. Con las estrategias actuales de vacunas y vacunación, la mortalidad y morbilidad se han reducido pero no han eliminado la infección ni la excreción viral, lo cual es crítico para el adecuado control de la EN. Estos brotes en parvadas vacunadas sugieren que el nivel de cobertura de la vacunación es bajo, o que la vacunación no provee inmunidad “perfecta” permitiendo al virus diseminarse entre poblaciones. También es importante notar que en regiones donde no circula o aparentemente no circula el virus velogénico de la EN, las empresas prefieren usar programas de vacunación que epidemiológicamente no son óptimos debido principalmente a los efectos colaterales de la vacunación, aunado al hecho de que la infección, diseminación y transmisión de la EN velogénica se puede presentar con pocos signos aparentes. Finalmente, no se debe ignorar que la presencia de altos niveles de anticuerpos interfiere con el desarrollo de una inmunidad sólida después de la vacunación en los primeros días de vida del ave. Por lo tanto, los brotes continuos en parvadas comerciales vacunadas contra la EN demuestran que las estrategias actuales no son del todo eficaces y son susceptibles de ser mejoradas por una nueva generación de vacunas originadas de la ingeniería genética.

Diseases of Poultry Sci.

Westbury H. Comparison of the immunogenicity of Newcastle Disease virus strains V4, Hitchner B1 and LaSota in chickens. Australian Veterinary J. 61:10-3. 1984

Vacunas enfoque

10. World Organization for Animal Health (OIE). International Animal Health Code, Paris In: OIE. 2003

Vectorizadas

nuevo

La vacuna vectorizada de la Enfermedad de Marek que expresa al gen de la proteína F del virus de la EN (rHVT-NDV) ha mostrado eficacia al aplicarse dentro de un programa

de vacunación contra la EN. La protección que estas vacunas ofrecen se basa en la fuerte respuesta de tipo celular que el HVT induce y que adicionalmente, al expresar la proteína F del virus de la EN genera anticuerpos neutralizantes. Tan solo los anticuerpos contra la Proteína F evitan la transmisión del virus de célula a célula. La eficacia de la vacuna rHVT-NDV ha sido probada en desafíos con cepas velogénicas en laboratorio y también en condiciones de producción comercial en diferentes partes del mundo con considerable éxito. El objetivo planteado en un inicio era demostrar el nivel de protección generado por una vacuna rHVT-NDV incluida en diferentes programas de vacunación en pollo de engorda criados en condiciones comerciales y desafiados en laboratorio con una cepa velogénica de la EN (CHimalhuacan) con la finaliad de mostrar la factibilidad para su uso comercial en sitios con alta presión del virus de la EN. De esta manera, en éste artículo se presentan las conclusiones y aprendizajes obtenidos después de 12 pruebas de desafío con las características mencionadas y que nos abren un nuevo enfoque para el control y prevención de la EN a través de la vacunación La experiencia Después de 12 desafíos en aves vacunadas con rHVT-NDV, se obtuvo que la protección en todas las pruebas fue de 100% con excepción de una, que tuvo 90% y se encuentra dentro del rango de muy aceptable. Los grupos controles tuvieron un desempeño muy similar con excepción de tres pruebas con protección alrededor del 80% mostrando algunas diferencias en términos de mortalidad y que cada vacuna y

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N°23 • Scientific File

programa actuó en tiempo y forma de acuerdo a su diseño, generando inmunidad suficiente. La diferencia en el diseño de los programas de vacunación principalmente radicó en que los grupos que recibieron la vacuna rHVT-NDV no fueron vacunadas con ninguna inactivada. Esto último fue el primer beneficio que ofreció la vacuna rHVT-NDV al evitar los manejos que implicaba inyectar a los pollos en dos ocasiones. Manejo de la parvada Es posible reducir manejos por vacunación como se mencionó en el párrafo anterior. En los Estados Unidos, donde la presión por la EN es mínima, se puede utilizar rHVT-NDV como única vacunación. En países donde la presión de la EN va de moderada a alta se debe considerar el uso de rHVT-NDV dentro de un programa de vacunación que incluya la aplicación de por lo menos dos virus activos. En casos extremos de presión de la EN puede considerarse la aplicación de una vacuna hiperconcentrada al día de edad junto con la rHVT-NDV y los virus activos. Excreción viral Un beneficio importante encontrado con el uso de la vacuna rHVT-NDV en los programas de vacunación probados, es la sustancial reducción de la excreción viral medida tanto por PCR cuantitativa como por titulación en

embrión de pollo (ver tabla). Después de la protección contra la mortalidad y los signos clínicos, la excreción viral es el siguiente aspecto de relevancia cuando se habla de control de la EN.

Sistema DIVA

Las aves vacunadas con rHVT-NDV probadas por serología (HI y ELISA) contra la EN mostraron bajos títulos de anticuerpos, o incluso éstos fueron negativos, con un 100% de protección ante el desafío. Esta condición nos mostró que cuando se usa la vacuna rHVT-NDV en un programa, la respuesta detectada por serología es baja aunque lña protección es suficiente, pero además se tiene el potencial para aprovechar las pruebas serológicas como sistema para diferenciar parvadas vacunadas de aquellas infectadas (DIVA por sus siglas en inglés: Differenciate Infected from Vaccinated Animals).

Conclusión Sin duda alguna, las vacunas vectorizadas están modificando o revolucionando la forma en que se controlan las enfermedades en las aves, y en el casod e la EN, rHVT-NDV está cambiando el enfoque de los programas convencionales por programas que de manera integral ayudan a potenciar y optimizar sus resultados redundando en beneficios para el productor.

•

rHVT-NDV

0.091

102

Control

0.094

0.178

Negativo

1.6

0.07

rHVT-NDV

Negativo

Control

Negativo

rHVT-NDV

rHVT-NDV Control

Negativo

34.85

30.43

Control

32.58

32.49

32.04

33.57

rHVT-NDV

36.98

Negativo

33.04

Control

31.72

27.96

34.31

26.35

rHVT-NDV

29.63

37.06

32.64

32.41

Control

29.92

30.93

31.11

29.69

rHVT-NDV

Negativo

Control

Negativo

rHVT-NDV

9.34

4.2

6.28

18.04

Control

32.4

178

312

178

rHVT-NDV

224

109

Control

32.4

178

312

178

Las cantidades están expresadas en millones de copias de ADN por mililitro. Las pruebas 7, 8 y 9 se reportan en valores de Cycle Threshold (Ct) de tala manera que: 25 a 29 son altamente positivos; 30 a 34 son moderadamente positivos; 35 a 36.9 son levemente positivos y >37 son negativos.

Excreción viral* Prueba

170

108 /

Grupo rHVT-NDV Control

RT-PCR Cloacal pool

RT-PCR Traqéal pool

Pool 1 (n=5)

Pool 2 (n=5)

Pool 1 (n=5)

Pool 2 (n=5)

Negativo

0.023

0.86

0.065

rHVT-NDV

Negativo

<10

Control

Negativo

<10

rHVT-NDV

Negativo

1.43x10

<10

Control

2.12x10

8.74x10

<10

3.0

<10

3.0

<10

3.0

<10

3.0

<10

3.0 3.0 3.0

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Scientific File • N°24

N°22 • Scientific File

EXTRACT FROM AAAP, 2012.

VECTORMUNE® ND

Pruebas de potencia % de protección

John K. Rosenberger and Sandra C. Rosenberger AviServe LLC, Delaware Technology Park, 1 Innovation Way, Suite 100, Newark, DE 19711.

SUMMARY A commercially available herpes virus of turkeys (HVT) vector vaccine

Table 1. Experimental design to assess the onset of immunity in commercial turkey poults vaccinated at day of age with VECTORMUNE® HVT NDV

for the control of Newcastle Disease (ND) was subcutaneously

Vaccines

administered to 1-‐day-‐old turkey poults to evaluate the onset of immunity against Newcastle Disease Virus (NDV). The vaccine used is a geneNcally engineered serotype 3 Marek’s disease vaccine (HVT) that expresses the NDV fusion gene. Onset of immunity to NDV in vaccinated poults was determined by challenge with NDV B1 at 1, 2, 3, 4 and 5 weeks of age. Response to

# of Birds Challenged with NDV Number B1 of Week Poults 1 2 3 4 5 50

VECTORMUNE® HVT NDV + SB1

CEVAC® HVT & SB1

Fig. 1. Percent embryo mortality caused by Newcastle disease virus isolated from tracheal swabs collected from poults at 5 days post-‐challenge with NDV B1 strain. 100

Serology: NDV maternal anSbody was present in all birds at 1 day of age. protecSon (44%) at seven days of age in unvaccinated birds and enhanced

Table 3. Onset of Immunity in commercial turkey poults vaccinated at day of age with VECTORMUNE® HVT NDV by the subcutaneous route. Week 3

VECTORMUNE® HVT NDV

9/9 (100%)

6/9 (67%)

3/9 (33%)

8/10 (80%)

10/10 (100%)

VECTORMUNE® HVT NDV + SB1

6/9 (67%)

4/9 (44%)

2/9 (22%)

6/10 (60%)

8/10 (80%)

CEVAC® HVT & SB1

4/9 (44%)

0/9 (0%)

0/8 (0%)

0/10 (0%)

90 80

MATERIALS AND METHODS

70 60

Procedure: Two hundred one-‐day-‐old Nicholas turkey poults were obtained from Sleepy Creek Turkeys, Inc. Goldsboro, North Carolina.

Onset of AcSve Immunity: Protection against NDV challenge resulting from

VECTORMUNE® HVT NDV alone was 80% and 100% at 4 and 5 weeks,

VaccinaNon: One group of 50 poults was vaccinated with VECTORMUNE® HVT

respectively. The addition of SB-1 reduced the efficacy of the recombinant in

NDV in 200 µl per bird. A second group of 50 poults was vaccinated with

turkeys by 20% at 4 and 5 weeks resulting in 60% and 80% protection,

VECTORMUNE® HVT NDV plus SB1 and a third group of 40 poults was vaccinated with convenSonal CEVAC® MD HVT & SB1. All vaccines were administered at day of age via subcutaneous route. At vaccinaSon, the birds were neck banded and placed in glove port isolators by treatment group. At each challenge period, 8 to 10 birds were removed from each treatment group and transferred to a separate isolator according to

respectively (Table 3). With the onset of active immunity the relative amounts of

NDV re-isolated from tracheas of susceptible birds appeared to be reduced

NDV challenge: Each bird to be challenged was inoculated by the intranasal

9-‐11 day-‐old SPF embryos. If NDV was re-‐isolated the bird was considered suscepSble to challenge (unprotected). Serology: At ~1 day of age extra unvaccinated birds were bled and sera assayed for NDV anSbody by hemaggluSnaSon inhibiSon (HI) and commercial anSbody ELISA to determine anSbody status.

VECTORMUNE® HVT NDV CEVAC® HVT & SB1

# Positive/Total # of Turkeys Vaccines

based on lower embryo mortality rates when tracheal swabbings collected from vaccinates were compared to unvaccinated controls (Figure 1) suggesting a

VECTORMUNE® HVT NDV + SB1

Table 2. Newcastle disease anNbody responses at 5 weeks post vaccinaNon.

reduced opportunity for NDV shed in susceptible vaccinates.

CONCLUSIONS 1. The onset of active immunity in turkey poults vaccinated at ~1 day of age with VECTORMUNE® HVT NDV was detected at four weeks providing 80% protection and achieved 100% protection at 5 weeks.

IDEXX ELISA

SYNBIOTICS ELISA

VECTORMUNE® HVT NDV

0/10

4/10

2. Addition of SB-1 to VECTORMUNE® HVT NDV reduced the efficacy of the recombinant in turkey poults as assessed by relative resistance to NDV B1 challenge.

VECTORMUNE® HVT NDV + SB1

0/10

3/10

3. There was an apparent synergism between maternal NDV antibody and VECTORMUNE® HVT NDV as expressed by increased protection against NDV B1 challenge.

CEVAC® HVT & SB1

0/10

route with 103.5 EID of the NDV B1 strain. Five days post-‐challenge, a tracheal swab was taken from each turkey poult and processed for virus isolaSon in

Age (weeks)

the vaccine received and challenged with NDV B1 strain.

110 /

tracheas of protected vaccinates.

Turkeys Protected/Total Challenged

respecNvely.

groups (Figure 1). This was assumed to be associated with less NDV in the

Vaccines

RESULTS

immunity aﬀorded by VECTORMUNE® HVT NDV reached 80% and 100%

birds and markedly diminished with the onset of acSve immunity in vaccinated

vaccine induced protecSon at 1 and 2 weeks (Table 3).

swabbings. Results showed that the vaccine apparently acted unvaccinated controls at 1 and 2 weeks. At 4 and 5 weeks acNve

for embryos inoculated with tracheal swabbing was highest in unvaccinated

Maternal anSbody detected at 1 day provided relaSvely high levels of

VECTORMUNE® HVT NDV

challenge was evaluated by virus re-‐isolaNon aYempts from tracheal synergisNcally with maternal anNbody to provide beYer protecNon than

Mortality in Embryos Inoculated with Tracheal Swabbing: Percent mortality

4. VECTORMUNE® HVT NDV vaccinates susceptible to NDV challenge with or without SB-1 appeared to retain lower concentrations of NDV in tracheas compared to controls thus reducing the potential for NDV shed.

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Notes

112 /

VECTORMUNE速 ND

/ 113

SCIENTIFIC INFORMATION

VECTORMUNE速 ND

114 /