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Año VIII / enero 2011 Órgano Oficial de la Asociación de Porcinocultura Científica
A vision of what we really know about an old “acquaintance”: Porcine parvovirus Juan Antonio Mesonero Escuredo (Veterinarian, HIPRA GPM Swine Business Unit) Jaime Maldonado García (Veterinarian, HIPRA Diagnos Manager) Pere Riera Pujadas (Veterinarian, HIPRA Swine Technical Services) Maximiliano Cesio Acuña (Biologist, HIPRA R&D) Carlos Martínez Dávila (Clinical Veterinary OPP)
The Reference in Prevention for Animal Health www.hipra.com
A vision of what we really know about an old “acquaintance”:
Porcine parvovirus Juan Antonio Mesonero Escuredo (Veterinarian, HIPRA GPM Swine Business Unit) Jaime Maldonado García (Veterinarian, HIPRA Diagnos Manager) Pere Riera Pujadas (Veterinarian, HIPRA Swine Technical Services) Maximiliano Cesio Acuña (Biologist, HIPRA R&D) Carlos Martínez Dávila (Clinical Veterinary OPP)
Porcine Parvovirus (PPV) is responsible for producing reproductive failure characterized by infection and embryonic and/or foetal death; sows normally show no external clinical signs. The disease mainly develops when animals are seronegative (and therefore susceptible). They are exposed to PPV by oronasal route or through venereal disease during the first half of pregnancy (Mengelin, 2006). Subsequently, the offspring of these animals will be infected before becoming immunocompetent (at approximately day 67 of gestation). PPV causes infertility, foetal mummification, and litters with piglets born weak and/or small litters. The infection is also associated with poor growth of piglets in the lactation period (Taylor 2006). PPV virus particles have been detected and isolated from diarrhoeal stools and skin lesions, although its involvement in infections of this type remains under debate. PPV is ubiquitous worldwide and infection is endemic on most farms on which its presence has been investigated. Diagnostic evidence indicates that PPV is a major cause of death in pig embryos and foetuses. Recently, it has been suggested experi-
mentally that PPV may enhance the negative effects during the post-weaning period caused by porcine circovirus type II (PCV2) and the clinical symptoms known as “wasting” (PMWS).
AETHIOLOGY PPV belongs to the Parvovirus genus (Latin parvus, parva, parvum which means small) of the family Parvoviridae. PPV isolates commonly isolated are antigenically similar, but not identical. This is how “variant” isolates that differ genetically or antigenically in relation to reference strains or vaccinal strains present in widespread use in Europe have recently been described. (De Zeeuw et al. 2007). PPV is also close antigenically to other viruses of the same genus, but their identity can be confirmed by serological tests and virus neutralization (VN), haemagglutination inhibition (HI) or ELISA. PPV (the genome of which is DNA) is a virus highly resistant to temperature and a wide range of disinfectants and enzymes.
Image 1. Infection routes and processes of the PPV.
2
PRIMARY REPLICATION (regional ganglion)
3
VIRAEMIA
4
UTERUS
PP virus
1
AEROSOL
5
PP virus
0-10 days pi
10-14 days pi
8-21 days pi
FOETUS
Table 1. Effect of PPV infection in seronegative pigs in different stages of pregnancy. Infection of the sow
Infection time of the foetus
Result of the infection
Clinical disease
10-30
Death and reabsorption of embryo
Large number of sows returning to oestrus
30-70
Foetal death and mummification
Smaller litters with mummified foetuses
70-tĂŠrmino
Normally there are no harmful effects. Infected immunocompetent foetuses produce antibodies
Habitually none
Days of gestation <56
>56
PATHOGENY PPV infects pigs via the oronasal or venereal routes and reaches the bloodstream about 10 days after infection causing transient leucopoenia (Taylor, 2006). Viraemia facilitates the passage of the virus to the reproductive tract of the animal 10-14 days after infection. The virus can be found in sperm of boars by days 5-9 post-infection, and in seminal vesicles and testicular tissue by 8-21 dpi (Taylor 2006). PPV adheres to the pellucid area of ovocytes and infects and makes non-immunocompetent embryos and foetuses unviable by about day 67 of gestation. The virus does not affect females infected 1-4 weeks before insemination but crosses the placenta in those that become infected during insemination or in the following 90 days. Embryos and foetuses that die before days 33-35 of pregnancy can be reabsorbed completely, whereas those that die after that time may become mummified, be stillborn or be aborted (abortions from PPV do not occur when the infection is after 70 days of gestation). The infection spreads from pig to pig along the uterus. In pigs infected at the end of gestation, piglets that do not die may develop high levels of serum neutralizing antibodies or become immunotolerant and remain infected for more than 8 months after birth (Johnson and Collings 1971). Infected piglets that recover from infection show a significant reduction in growth. Serum antibodies appear in the mother at 7-10 days post-infection and their titres rapidly increase. The virus is shed in low con-
centrations in urine, faeces, tonsillar peeling and nasal secretions as of the second week post-infection. In persistently infected pigs, this shedding occurs for approximately two weeks. Affected pigs recovering from the infection have strong humoral immunity (antibodies) transmitted via colostrum to offspring that can be detected up until 4-6 months of age (this varies according to the diagnostic techniques). Pigs are susceptible to reproductive failure induced by PPV if they become infected at any time during the first two trimesters of gestation. This range of maternal susceptibility is supported by numerous experimental studies (Joo et al. 1976; Mengelin 1979; Mengelin and Cutlip 1976; Mengelin et al. 1980). If the sow is infected in the first 56 days of pregnancy, embryonic death and resorption (10-30 days gestation) or death and mummification (30-70 days gestation) may occur. After day 56 of gestation, the infection in sows moves to the placenta, when the foetus is at about day 70 of gestation, and there may be an immune response and survival of the future piglet.
EPIDEMIOLOGY The most common routes of infection in pigs in the postnatal and prenatal period are the oronasal and transplacental routes, respectively. Infection is endemic on most farms in areas of high concentration of swine. A large proportion of primiparous pigs are infected with PPV before insemination. Gilts that have not been exposed to infection before their first pregnancy are at high risk of infection and reproductive disease. Piglets
that have ingested colostrum from well immunized mothers receive a very high amount of antibodies against PPV. These passively acquired titres decrease over time. The haemagglutinating serum antibodies acquired by piglets decrease to undetectable titres 3-6 months after weaning. However, neutralizing antibodies persist for a longer period. The first important point about passive immunity (colostrum) is that it interferes with the development of active immunity (postinfection). High levels of colostral antibodies can prevent infection, but low levels only minimize the spread by infected animals (Taylor 2006). Therefore, some gilts are not fully susceptible to PPV until just before insemination or during the early stages of pregnancy. Farm areas contaminated by PPV are probably the largest reservoirs of the disease. The virus is heat stable, resists the action of most disinfectants and can persist for months with infectivity in secretions and excretions of infected animals. It has been experimentally shown that the virus is only shed in the two weeks after exposure; in pens, however, it can persist for up to 4 months. PPVâ&#x20AC;&#x2122;s ubiquity and resistance to environmental conditions facilitate the permanent infection and re-infection of some pigs and periodic shedding of virus to the environment. The shedding of virus beyond the acute period of infection has not been demonstrated, but the possibility of occurrence of immunotolerant PPV carriers, resulting from in utero infection, has been postulated. When gilts are infected with PPV before day 55 of gestation, their piglets can be born infected but without antibodies, since the foetus is not immunocompetent during this period. The virus has been isolated from the kidneys, testes and seminal fluid in pigs sacrificed at different ages up to 8 months of age. Likewise, in studies in which sows were infected during early pregnancy and their piglets born infected - but without antibodies - the development of immuno- tolerance (Cartwright et al. 1971) was also suggested. Johnson et al (1976) reported the case of an immunotolerant boar. Boars may play an important role in the spread of disease caused by PPV, in that, during the acute phase of
the disease, the virus can be shed by different routes, including semen. The isolation of PPV in semen has been demonstrated previously (Cartwright and Huck 1967, Cartwright et al. 1969; MacAdaragh and Anderson 1975). However, it is important to note that semen may become contaminated with from faeces containing PPV or in a sowâ&#x20AC;&#x2122;s own reproductive tract.
CLINICAL SIGNS Repetitions, reproductive failure, reduced litter size, mummified foetuses, stillbirth and very sporadic abortions are the main clinical signs in the syndrome associated with PPV infection; there is a higher incidence of this disease in primiparous sows. In those animals, infection can produce a decrease of 1.1 piglets per litter, a reduction of up to 36% in farrowing rate and an increase in litters of fewer than 5 piglets, as well as the presence of mummified piglets and stillbirths. Pseudogestations and/or irregular repetitions may also be observed. Clinical symptoms can be observed throughout the entire herd susceptible to the disease when it is introduced on a farm. The disease is asymptomatic in males and appears not to affect semen quality but it may be a route of transmission. Late repetitions after positive sonographic diagnosis are accompanied by mummified pigs.
DIAGNOSIS Before entering into considerations about reproductive failure in swine herds caused by infectious agents, it is important to keep in mind three facts that greatly influence
ment of farms, like parvovirus vaccine and erysipelas. Taking into account the factors mentioned above, it is not surprising that attempts to diagnose cases of abortions in sows in the laboratory produce results that can be frustrating because it is not possible to link them to an infectious aetiology. PPV should be considered within a differential diagnosis in swine reproductive failure whenever there is evidence of death of embryos, foetuses or both. One form of the initial clinical and tentative diagnosis of PPV in reproductive failure may be the fact that primiparous (not multiparous) sows are affected by the problem, Another clue could be if clinical pathology in adult sows is not observed during pregnancy and if there are only a few or no abortions. Moreover, we have to take a look at foetal anomalies and whether that evidence suggests an infectious disease. breeding in this species. The first fact is the almost universal replacement of natural mating by artificial insemination (AI). Over the long-term, AI has limited the incidence of venereally transmitted diseases and has facilitated reproductive management overall by segregation of breeding males that are housed in SPF semen production facilities. Secondly, as has been documented recently (Maldonado, 2005), over 90% of reproductive failure, particularly those that occur with expulsion of foetuses with different gestational ages, cannot be associated with an active infection caused by viruses, bacteria or mycoplasma that are known primary abortifacients in pigs. As for other agents considered as emerging and the presence of which has been detected in aborted material (Martinez-Guinó, 2010), their ubiquitous nature and the inability to reproduce the disease in a controlled animal model, does not allow them to be associated with reproductive failure itself. It is possible that some agents may act synergistically with others to trigger reproductive failure that cannot be explained by other causes. Finally, it is important to note that some agents are controlled by massive and systematic vaccination of breeder sows. In some cases, those vaccinations have become part of eradication programmes, such as the case of Aujeszky’s virus, and in some cases vaccination is part of routine health manage-
The relative lack of disease in the mothers, with abortions and foetal abnormalities occurring are indicative of clinical parvovirus as opposed to other infectious causes of reproductive failure that also affect the sow in varying degrees. However, the definitive diagnosis requires laboratory support, in addition to clinical and production assessments by the field veterinarian. Most appropriate samples for sending to the laboratory. The diagnosis of parvovirus, as in the case of most infectious diseases, can be performed directly or indirectly. In the first case, one looks for the virus or parts thereof (antigen or nucleic acid), to show its presence in the aborted material. Indirect diagnosis seeks â&#x20AC;&#x153;tracesâ&#x20AC;? left by the infection in terms of animal response by producing antibodies, i.e., by serology. Serology can be done in the mothers or in immunocompetent foetuses from day 6770 of gestation. If what is sought in the laboratory is PPV, it is important to take into account that the ecology of the farm can facilitate (or not) the presence of the virus in the environment, which can lead to misleading results (positive virus samples from the environment and not from the foetus), particularly due to
poor hygiene in farrowing crates. In regard to serology in mothers, natural contact with the virus in the environment and routine vaccination at peripartum impede objective assessment of this parameter. As a result of the above, a distinction should be made between reproductive failure diagnoses and monitoring of parvovirosis. The first case requires a medical history suggesting disease in mothers with a consequent failure, whereas the second is intended to assess the immunity of livestock against PPV. As a result, samples that are usually sent to the laboratory are embryos, foetuses and placentas as well as blood of females of dif-
13501 - NEG 13502 - 1/16 13503 - NEG 13504 - NEG 13505 - NEG 13506 - NEG 13513 - NEG 13514 - NEG 13515 - NEG 13516 - 1/16 13517 - NEG 13518 - NEG 13519 - NEG 13520 - NEG 13521 - NEG 13522 - NEG 13523 - NEG 13524 - NEG 13525 - NEG 13526 - NEG 13527 - NEG 13528 - NEG 13529 - NEG 13530 - NEG 13531 - NEG 13532 - NEG 13533 - NEG 13534 - NEG 13535 - 1/16 13536 - NEG
Figure 1. Results of haemagglutination inhibition for porcine parvovirus in a group of 30 serum samples from sows. Any non-zero result is considered positive.
13976 - NEG 13977 - NEG 13978 - NEG 13979 - NEG 13980 - NEG 13981 - NEG 13982 - NEG 13983 - NEG 13984 - NEG 13985 - NEG 13986 - NEG 13987 - NEG 13988 - NEG 13989 - NEG 13990 - NEG 13991 - NEG 13992 - NEG 13993 - NEG 13994 - NEG 13995 - NEG 13996 - NEG 13997 - NEG 13998 - NEG 13999 - NEG 14000 - NEG
Figure 2.
4P-6183 - 1/2048 6P-6115 - 1/4096 6P-2594 - 1/1024 6P-6578 - 1/2048 3P-6559 - 1/2048 7P-6210 - 1/1024 5P-4325 - 1/2048 7P-6314 - 1/128 6P-6292 - 1/64 6P-969 - 1/2048 6P-2394 - 1/1024 5P-4994 - 1/8192 7P-3709 - 1/2048 6P-4924 - 1/8192 4P-6370 - 1/4096 4P-6566 - 1/2048 4P-6597 - 1/8192 6P-4750 - 1/1024 6P-4820 - 1/512 6P-4292 - 1/2048 5P-6516 - 1/64 7P-6226 - 1/4096 5P-4211 - 1/1024 5P-3605 - 1/2048 8P-3824 - 1/64 5P-6523 - 1/2048 5P-7181 - 1/256 5P-4667 - 1/1024 5P-4686 - 1/2048 5P-6599 - 1/1024 5P-6722 - 1/4096 5P-4225 - 1/512 7P-3884 - 1/128 6P-4820 - 1/256 6P-4650 - 1/4096 8P-6671 - 1/4096 6P-4862 - 1/8192 7P-2089 - 1/2048 6P-6627 - 1/4096 6P-3838 - 1/2048 5P-6843 - 1/8192 6P-3620 - 1/2048 7P-4525 - 1/8192 7P-7117 - 1/8192 6P-6579 - 1/1024 6P-4854 - 1/8192
ferent ages. Occasionally, the blood of boars is analyzed for the same purpose of monitoring immunizations. Aborted material: It is recommended that 3-5 foetuses the size and stage of development of which correspond to the second trimester of pregnancy (approximately 16 cm in length) be sent. Failing that, one can send the viscera of the foetuses, including the lungs, liver and intestine for laboratory screening. It is generally considered that older mummified foetuses and stillborn or dead at birth animals are not the best sam-
Figure 3.
ples for detecting the virus. This is because at older ages the foetus is able to establish an immune response and generated antibodies can interfere with laboratory tests. It is also possible to send uteri of sows sent to slaughter because of reproductive problems (anoestrus, etc.) to find the remains of foetuses affected by PPV. Analytical techniques available in the laboratory Direct diagnosis of porcine parvovirus can be performed on aborted material by means
of PCR, which shows the presence of the nucleic acid (DNA) of PPV or by antigen detection systems that detect the virusâ&#x20AC;&#x2122; proteins. It is important to stress that, although positive results may confirm clinical suspicion of infection, one must consider the possibility of positive results being from environmental virus. Serology can be performed on foetuses or pigs. Positive serological results in foetuses indicate in utero infection after 65-70 days of gestation and have a high predictive value (indicating possible infection and disease). Serology in adult animals can be used to assess the different groups present on the farm. For example, batches of replacement sows (future breeders in quarantine before entering the farm) that are seronegative could be affected later in pregnancy. In the case of sows that have already entered the premises of the farm, follow-up serology is customary, although its value is limited to detecting sub-populations of animals with lower than expected titres after revaccination or seronegative due to failures in managing vaccinations. Serology is, therefore, another tool, and its results are subject to interpretation and depend on the clinical study of the farm operation. The following illustrates some of the possible scenarios through real serological analyses performed using the haemagglutination inhibition technique (HI). Figure 1 shows the HI titres in the serum of a set of replacement sows. Most of the samples are negative and only three of them have low titres. One possible interpretation without more information than the age of the animals is that it is a group of unvaccinated, negative gilts, or less likely a vaccine failure due to the vaccine not being applied, since all market vaccines induce a greater or lesser degree of seroconversion after the revaccination, but due to non-vaccination due to neglect or misapplication. Figure 1 Results of haemagglutination inhibition for porcine parvovirus in a group of 30 serum samples from sows. Any non-zero result is considered positive. In case 2, we found a group of negative replacement sows with a high risk of infection during gestation and development of reproductive failure due to PPV.
In case 3, the high titres of 1/1024 could be interpreted as contact with the virus field; these titres are hard to find in animals that have only been vaccinated. The problematic of PPV appears when negative populations at risk of exposure become infected and in those where we find animals with infection titres living together with negative animals. Yet serology is only one tool in the differential diagnosis of reproductive failure and always accompanied by a good anamnesis too. PCR is a useful tool and has the advantage that if we know it is positive, we know that there are virus present, but what could also happen is that we could find negative animals, as in the case of semen, but which are infected by the variability in the excretion (this also occurs in persistently infected or PI animals).
PPV serology in Spain
Multiparous Nulliparous Replacement
Source: Diagnos HIPRA (N=1647).
European PPV serology
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d l lan tuga r Po Po
Source: Diagnos HIPRA (N=2980).
PREVENTION AND CONTROL There is no specific treatment for reproductive failure induced by PPV. As a precaution, gilts should be vaccinated before being inseminated. The possibility of immunization by natural infection has been postulated (even though this is a very inconsistent method) because it is not possible to ensure that negative sows will be in contact with positive pigs that are shedding virus. Nor is it possible to ensure infection following the movement of non-infected animals to areas supposedly contaminated
with PPV. In both scenarios described, there is no guarantee of a homogeneous infection in the entire group of animals, in that natural infection with PPV is a phenomenon in equilibrium with vaccination that, as a whole, maintains high and consistent levels of immunity in a group of breeders. Once the infection begins, the virus spreads quickly. Infection is common and in endemic areas more than half of the gilts are infected before mating (Mengelin 1972). The use of vaccines in nulliparous animals is a practice that enables active immunity against PPV before the first pregnancy. Inactivated vaccines (KV) and modified live vaccines (MLV) have been successfully used for this purpose. (Fujisaki 1978; Mengelin et al. 1979).
Vaccines should be administered several weeks before mating of gilts, with the aim of providing immunity before and during the susceptible gestation period. However, vaccination has to be performed after the disappearance of colostral immunity in these nulliparous animals (Paul and Mengelin 1986). These limits define a short space of time for effective vaccination of gilts to be inseminated at an early age (before 7 months of age). Although inactivated vaccines are safe by definition, there is evidence that sufficiently attenuated live vaccines are unlikely to cause reproductive failure, although administered during pregnancy. The duration of immunity after vaccination is difficult to predict, although different studies show efficacy up to 4 months after administration of an inactivated vaccine ( Joo and Johnson 1977b). Vaccination is recommended for sows and boars. If a farm were negative to PPV, vaccination with an inactivated vaccine would be the choice, although it is unusual to find farms free of the virus when they are infected and the result can be disastrous (Donaldson-Wood et al. 1977). For its part, the vaccination of boars reduces their role in the spread of the disease. Vaccination is the most used preventive and control measure in the swine industry worldwide. The most common vaccination procedures in sows include a primary vaccination with two doses 3-4 weeks apart and revaccination at each lactation, 7-10 days post-partum. In boars the recommendation is a two-dose primary vaccination as with females, and revaccination every 4-6 months.
References •
Pig Diseases, 8 th edition 2006 D.J. Taylor.
•
Diseases of Swine 9 th edition 2006 Straw, Zimmerman, D’Allaire, Taylor.
•
Maldonado J, Segalés J, Martínez-Puig D, Calsamiglia M, Riera P, Domingo M, Artigas C. 2005. Identification of viral pathogens in aborted fetuses and stillborn piglets from cases of swine reproductive failure in Spain. Vet J.169:454456.
•
Martínez-Guinó L, Kekarainen T, Maldonado J, Aramouni M, Llorens A, Segalés J. 2010. Torque teno sus virus (TTV) detection in aborted and slaughterhouse collected foetuses. Theriogenology 74:277281.
THE VISION ON THE FARM As we know, PPV is a ubiquitous virus and sometimes - because a farm’s routine or work does not permit - it is not given the importance we give to other more current diseases. We can see by observing field samples that sometimes replacement sows are not prepared to face a future pregnancy with guarantees. Therefore, good adaptation to the farm where replacements are sent is of great importance by means of effective vaccination programmes to prevent maternal immunity and generate an appropriate response.
HIPRASUIS PE ®
Inactivated vaccine against Porcine parvovirus (PPV) and Swine erysipelas (SE) in suspension for injection
HIPRASUIS®PE: COMPOSITION PER DOSE (2 ml): Inactivated Porcine Parvovirus, strain NADL-2: HIA ≥ 1/128. Inactivated Erysipelothrix rhusiopathiae, strain R32E11: IRPC ≥ 49.2. Adjuvant based on Aluminium hydroxide gel (expressed as mg of Aluminium): 0.6 mg. INDICATIONS: Swine: Active immunization of gilts, sows and boars from 6 months of age onwards, to reduce reproductive disorders caused by Porcine Parvovirosis (PPV) and to reduce reproductive problems and acute clinical signs from Erysipelas. Onset of immunity was shown 21 days following the second vaccination by challenge with Erysipelothrix rhusiopathiae and 21 days after finished vaccination by serology against PPV. Duration of immunity has been demonstrated for 5 months for Erysipelothrix rhusiopathiae and 6 months for PPV, respectively. Administration route Intramuscular injection in the neck muscles and dosage: Gilts Basic Vaccination (Nuliparous) and sows (Multiparous) Revaccination: 2 ml/animal. It is recommended that the vaccine should be allowed to warm to a temperature of between +15 ºC and +25 ºC before administration. Shake before use. In absence of maternally derived antibodies (from 6 months of age onwards) administer 2 doses 3 weeks apart. The second dose should be administered at least 2 weeks before mating/artificial insemination. Usually the vaccination against Porcine Parvovirus is not effective when administered to animals younger than 6 months of age. Monovalent vaccines against Swine Erysipelas shall be administered in these cases. Revaccination should be done during the lactation period, at least 2 weeks before mating/artificial insemination, or every 6 months (mass vaccination). Boars: basic immunization with 2 doses 3 weeks apart and revaccination every 6 months. WITHDRAWAL PERIOD: Zero days. SPECIAL PRECAUTIONS: Store and transport refrigerated (2 °C – 8 °C). Do not freeze. PACKAGING: Box of 10 vials of 10 doses (10 x 20 ml). Box of 10 vials of 25 doses (10 x 50 ml). Box of 1 vial of 50 doses (1 x 100 ml). Not all pack sizes may be marketed.
Laboratorios Hipra, S.A. Avda. la Selva, 135 17170 Amer (Girona) Spain Tel. (34) 972 43 06 60 Fax (34) 972 43 06 61 hipra@hipra.com www.hipra.com