PORCINE CIRCOVIRUS TYPE 2. The virus, the disease and the vaccine by Grupo Asís

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PORCINE CIRCOVIRUS TYPE 2 THE VIRUS, THE DISEASE AND THE VACCINE Joaquim Segalés Servet (División de Grupo Asís Biomedia S.L.) Centro Empresarial El Trovador, planta 8, oficina I Plaza Antonio Beltrán Martínez, 1 • 50002 Zaragoza (España) Tel.: +34 976 461 480 • Fax: +34 976 423 000 • www.grupoasis.com

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PORCINE CIRCOVIRUS TYPE 2 The virus, the disease and the vaccine

PORCINE CIRCOVIRUS TYPE 2 THE VIRUS, THE DISEASE AND THE VACCINE Joaquim Segalés

AUTHOR: Joaquim Segalés. FORMAT: 11 x 20 cm. NUMBER OF PAGES: 132. NUMBER OF IMAGES: 35. BINDING: hardcover, wire-o.

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This book presents, in a rationale and practical manner, the main facts that veterinary students and professionals should know in order to deal with PCV2 and related diseases. Besides general knowledge on the virus and pathogenesis, major emphasis is focused on the immunity, diagnosis and proper use of vaccines. Noteworthy, epidemiology, clinical signs and lesions are fundamental cornerstones to revisit. The author is one of the most prolific researchers on PCVD. He has contributed greatly in terms of expanding knowledge and has laid the foundation for further progress and knowledge of the virus and the associated diseases, thus creating an original and at the same time consistent book with the scientific state of the art.

PORCINE CIRCOVIRUS TYPE 2: The virus, the disease and the vaccine

Presentation of the book Porcine circovirus type 2 (PCV2) has been one of the most striking surprises in the world of pig health in the last 50 years. We now know that this is an old virus, which was found by means of retrospective studies as early as in 1962, although no disease was attributed to this agent until mid-90s. After devastating episodes of disease and economic losses all around the world, a number of PCV2 vaccines appeared in the global market between 2006 and 2008. These vaccines represented one of the major successes in the last 3 decades in the field of disease prevention. In fact, these vaccines contributed not only to counteract the diseases linked to the viral infection, but also to improve the growth of pigs. Such scenario led to the vaccines being used not only on clinically affected farms, but also on subclinically affected ones. Almost 20 years have lapsed since the beginning of the whole story, and 16 years since the discovery of PCV2. Therefore, it is time to face back the history and revise what has happened with this virus and its associated diseases/conditions during this period, as well as to address all practical tips regarding the virus and its prevention/control possibilities. The objective of this book is to present, in a rational and practical manner, all these facts that veterinary students and professionals should know in order to deal with PCV2 and its associated diseases. Besides general knowledge on the virus and pathogenesis, special emphasis is placed on the immunity, diagnosis and proper use of vaccines. Epidemiology, clinical signs and lesions are fundamental cornerstones that are also addressed. The advent of vaccines prompted us to almost forget these latter issues due to their great efficacy; however, any potential lack of expected efficiency must count with the proper tips to assess whether PCV2 would be potentially related or not with these problems. All that glitters is not PCV2, but we must know how to establish its real importance!

Joaquim SegalĂŠs

PORCINE CIRCOVIRUS TYPE 2: The virus, the disease and the vaccine

The author Joaquim Segalés He obtained his DVM from the Universitat Autònoma de Barcelona (UAB) in 1991. In 1996, he was awarded a PhD from the UAB. He also conducted his PhD research at the University of Minnesota for 15 months. In 2000, he was board certified by the European College of Veterinary Pathologists (ECVP), and is a founding member of the European College of Porcine Health and Management (ECPHM). Currently, he is Director of the Centre de Recerca en Sanitat Animal (CReSA) and Associate Professor at the Veterinary School of the UAB (main subjects: pathology and swine clinics). Besides, he is President of the ECPHM for the period 2013-2016.

He has been involved in swine disease research since 1993, mainly on infectious diseases (including infections caused by the porcine reproductive and respiratory syndrome virus (PRRSv), Aujeszky’s disease virus, porcine circovirus type 2 (PCV2), swine hepatitis E virus, swine Torque teno sus viruses (TTSuV), Haemophilus parasuis and Mycoplasma hyopneumoniae). He has co-authored more than 200 articles in international peerreviewed journals.

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He has been working as a Diagnostician at the Department of Pathology of the School of Veterinary Medicine of the UAB since 1996. He was responsible for the Pathological Diagnostic Service in Swine of the UAB (1996-2012).

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PORCINE CIRCOVIRUS TYPE 2 THE VIRUS, THE DISEASE AND THE VACCINE Joaquim SegalÃ©s

Table of contents 1. PCV2: Where are we coming from? 2. The virus and the pathogenesis: Simple things causing complicated outcomes The virus Pathogenesis of PCV2 infections PCV2-SD reproductionÂ models

3. Epidemiology Distribution of genotypes Species susceptibility Transmission Persistence Risk factors for PCV2-SD development

4. Recognizing infections caused by PCV2: Clinical and pathological findings Clinical signs Pathological findings

5. Do we know how to diagnose PCV2 infections/diseases? Laboratory techniques to detect PCV2 In situ hybridization (ISH) and immunohistochemistry (IHC) Quantitative polymerase chain reaction (qPCR) Standard polymerase chain reaction (PCR)

Laboratory techniques to detect antibodies against PCV2 Diagnostic criteria for PCVDs Porcine circovirus type 2-systemic disease (PCV2-SD) Porcine circovirus type 2-subclinical infection (PCV2-SI) Porcine circovirus type 2-reproductive disease (PCV2-RD) Porcine dermatitis and nephropathy syndrome (PDNS)

Differential diagnosis

6. PCV2 immunity: A double-edged sword? PCV2 and immunomodulation Adaptive immune responses against PCV2 Humoral immunity Cellular immunity

Immunostimulation and immunosuppression in PCV2-SD: the double-edged sword

7. Disease control without vaccination Management and zootechnical measures Concurrent infections Stimulation of the immune system Nutrition â&#x20AC;&#x153;Serum therapyâ&#x20AC;? Genetics PCV2 status of the sow Can we control PCV2-SD without vaccination?

8. The advent of PCV2 vaccines: From experiments to the market 9. Facts about PCV2 vaccines Commercial PCV2 vaccines Vaccination of gilts/sows and boars Effect on piglets Effect on gilts/sows Effect on boars

Vaccination of piglets Mechanisms of vaccine-induced protection

10.The use of PCV2 vaccines: How, when, whom? Vaccination against PCV2 Convenience of vaccinating piglets or breeding stock or both Timing of piglet vaccination

Is vaccination failure a concern? PCV2 vaccination in the future

11.Conclusions 12.Recommended literature

PORCINE CIRCOVIRUS TYPE 2

The International Committee on Taxonomy of Viruses (ICTV) was born within the Virology Division of the International Union of Microbiological Societies (IUMS) in 1973. One of the main functions of this Committee is to develop an internationally agreed taxonomy for viruses. The ICTV is composed of six subcommittees covering fungal (and algae), plant, invertebrate, prokaryotic (including archae), and vertebrate viruses. The sixth subcommittee is responsible for managing ICTV data and maintaining the ICTV database and web sites (http://www.ictvonline.org). Currently, within viruses, there are 7 orders, 104 families, 23 subfamilies, 505 genera and 3,186 species (EC 46, Montreal, Canada, July 2014). One of these families is the Circoviridae family, so far with two genera: Gyrovirus and Circovirus. The genus Gyrovirus only contains one species, which is chicken anaemia virus (CAV), while the genus Circovirus is composed of 11 species. The prototype viral species for the latter genus is porcine circovirus-1 (PCV1). The current classification of the Circoviridae family is under revision by the ICTV; therefore, significant changes are expected in the near future. The reader can imagine that if a virus is named with a number (PCV “1”), more than one viral species should exist with the same name: porcine circovirus-2 (PCV2). The first PCV was initially thought to be a picornavirus-like agent, and it was discovered as a porcine kidney cell line (PK-15) contaminant in 1974 by Dr. Ilse Tischer at the Robert Koch Institut (Germany). Further studies indicated that it was a DNA virus with a curious circular single stranded (ss) DNA in 1982. The main reason to study a cell line contaminant agent at that time was to ascertain replication viral modes, which included the rolling circle replication and the formation of a double stranded (ds) replicative form. In addition, PCV is considered the smallest autonomously replicating animal virus, encoding two major open reading frames (ORF1 and ORF2). The gene products (Rep, Rep’ and Cap) are involved in viral replication, regulation of transcription and cap2

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sid formation. Due to such limited coding capacity (currently ORF3 and ORF4 are also described as coding genes), circoviruses are supposed to rely mainly on the host’s machinery for synthesis of proteins. The contaminant PCV was minimally studied (18 scientific papers published between 1974 and 1997) and during the 80–90s was experimentally confirmed to be a non-pathogenic virus for swine based on the few experimental infections performed at that time. However, everything changed in 1997.

During early-mid 90s, a very sporadic condition of postweaning pigs characterized by wasting (Fig. 1), respiratory distress, jaundice and mortality was described in Central Canada by Dr. John Harding (clinician at Harding Swine Veterinary Services Inc., Saskatchewan, Canada) and Dr. Edward G. Clark (pathologist at the Department of Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Canada).

Figure 1. The smaller pig showing wasting was diagnosed as PMWS in 1995 in Canada. Image courtesy of Dr. John Harding.

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PORCINE CIRCOVIRUS TYPE 2

Initial thoughts pointed out about the possibility of porcine reproductive and respiratory syndrome virus (PRRSV) being the cause of such disease condition, but it was soon ruled out. The affected pigs displayed very consistent microscopic lesions in lymphoid tissues, characterized by lymphocyte depletion and granulomatous inflammation (Fig. 2). Since the aetiology was not known at that time, the disease was named in a descriptive fashion: postweaning multisystemic wasting syndrome (PMWS).

Figure 2. Spleen from a pig diagnosed as PMWS in 1996 in Canada. Moderate lymphocyte depletion and granulomatous infiltration in the periarteriolar lymphoid sheaths. Haematoxylin and eosin stain. Image courtesy of Dr. Edward G. Clark.

These microscopic lesions were relatively similar to those caused by other viruses of the genus Circovirus in avian species (Fig. 3). For such a reason, Dr. Harding and Dr. Clark contacted with Dr. Gordon Allan (virologist at the Department of Agriculture for Northern Ireland, Veterinary Sciences Division, UK), who did his PhD thesis on PCV1 (PCV at that time), and they used a polyclonal antibody raised against PCV to test the presence of viral antigen on formalin-fixed paraffin embedded tissues of the Canadian sick pigs. Surprisingly, the damaged lymphoid tissues showed a highly positive reaction to the immunohistochemical test (Fig. 4), and it was postulated that PCV or a novel PCV-like agent was the potential cause of PMWS. 4

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Figure 3. Bursa of Fabricius from a psittacine bird diagnosed with beak and feather disease. Marked lymphocyte depletion of the follicular areas with moderate histiocytic infiltration and presence of intracytoplasmic inclusion bodies (dark purple). Haematoxylin and eosin stain.

Figure 4. Spleen from a pig diagnosed as PMWS in 1996 in Canada. Moderate amount of PCV2 antigen located in macrophages of the periarteriolar lymphoid sheath. Immunohistochemistry to detect PCV2 antigen; haematoxylin counterstain. Image courtesy of Dr. Edward G. Clark.

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PORCINE CIRCOVIRUS TYPE 2

Controversy about PMWS and its causal agent started immediately after PCV was postulated as the potential infectious cause of the new syndrome. Although sequencing of the PCV found in clinically affected pigs indicated a nucleotide identity of less than 70 % with the cell contaminant PCV, such controversy did not stop. All the opposite, the novel PCV (named as Porcine circovirus-2, PCV2) did not get more credit than its “brother” PCV1, and a number of pros and cons were considered. In fact, such controversy lasted for almost 10 years in which “circo-believers” and “circo-skepticals” discussed extensively about the causality of PMWS (Table 1). “Circo-skepticals” considered that PCV2 was a ubiquitous virus that up-regulated its replication in those pigs suffering from PMWS, but that the causal agent was a yet unknown agent, which was tentatively designated as “agent X”. What was not in discussion was the economic consequences of the disease, although such impact varied among regions of the world and across time. Some calculations performed by Dr. David Burch in the United Kingdom in 2009 indicated the following: “if all the percentage loss of piglets produced and additional weaner/grower and finisher mortality is totalled during the epidemic it comes to 21.2 %, 11.8 % and 16.5 % for each group respectively. If a value of €30, €45 and €75 are put on each category of pig and based on an annual production of 9 million pigs, it can be costed at €216 million, just on increased mortality alone, or €24 million per 1 million pigs produced. If this is scaled up over the EU-27 with 240 million pigs produced/year, the costs could be as high as €5.76 billion”. The same author continued with the following statement: “one can see why this epidemic has been such a major disaster over the last decade, especially in the UK”.

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Table 1. Controversial elements regarding PMWS causality by PCV2 known between 1997 and 2007. The advent of commercial PCV2 vaccines demonstrated the central importance of PCV2 regarding the aetiology of the syndrome and such controversy disappeared. In favour of PCV2 as the cause of PMWS

Against PCV2 as the cause of PMWS

•

PCV2 was genetically different to PCV1, considered as nonpathogenic for swine.

•

PCV2 was systematically detected in PMWS-affected pigs to a higher load compared to healthy animals.

Since PCV1 was considered nonpathogenic, PCV2 was assumed to be not that different.

•

PCV2 was a ubiquitous virus.

•

PCV2 was present in farms with and without PMWS.

•

PCV2 was present in countries reporting the disease as well as in countries not describing the condition.

•

Retrospective detection of PCV2 at least in 1962, at a time where the disease was not presumably present in the livestock.

•

Genetically, PCV2 isolates were considered to be relatively homogeneous, so, very difficult to explain differences among farms and countries.

•

Although in some (few) experimental studies PMWS was reproduced using PCV2 alone, the participation of other pathogens could not be completely ruled out.

•

PCV2 was always found within the lesions of PMWS-affected animals by means of immunohistochemistry or in situ hybridization.

•

In some (few) experimental studies, PMWS was reproduced using PCV2 alone.

•

PMWS was easily reproduced with PCV2 together with infectious or non-infectious coadjuvants, but PCV2 participation was compulsory.

•

Initial PCV2 vaccine prototypes pointed out as an efficient way to control viraemia load.

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PORCINE CIRCOVIRUS TYPE 2

The first step to scientifically understand a disease is to create a model to reproduce the condition. This was intended by several research groups all over the world with variable results. Major conclusions of these studies were that PMWS was hardly reproduced using only PCV2 as inoculum, although mild to moderate lymphoid lesions typical of the disease were achieved. However, the use of concomitant infections (mainly porcine parvovirus [PPV] and PRRSV) together with PCV2 increased significantly the likelihood of PMWS reproduction. Moreover, an experimental model using gnotobiotic pigs infected with PCV2 and immunostimulated with a non-infectious product (keyhole limpet hemocyanin) reproduced the disease unequivocally. Compiling all performed studies, Henle-Kochâ&#x20AC;&#x2122;s postulates (Table 2) were considered not completely fulfilled, since in most of the cases other elements besides PCV2 were needed to reproduce PMWS. Noteworthy these did not always work equally efficiently, depending on the animal model used (age of animals at inoculation, use of gnotobiotic pigs, snatch-farrowed-colostrum-deprived pigs or conventional pigs, timing of adjuvant elements use, etc.). Table 2. Henle-Kochâ&#x20AC;&#x2122;s postulates (Rivers, 1937). 1

The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease.

It occurs in no other disease as a fortuitous and non-pathogenic parasite.

After being fully isolated from the body and repeatedly grown in pure culture, it can induce the disease anew.

These experimental findings, together with some of the epidemiological facts known at that time (Table 1), suggested considering PMWS as a prototype of multifactorial disease. Although a number of researchers already considered PCV2 as the pivotal factor to develop this multifactorial condition, the subsequent advent of PCV2 commercial vaccines reinforced the central role of PCV2. In fact, one may consider that this virus did not fulfill 8

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completely the Henle-Kochâ&#x20AC;&#x2122;s postulates, but in turn fulfilled completely the so-called Evans postulates. These latter postulates were proposed in 1976 and they represent a set of guidelines that take into account not only the infectious agent, but also the host and the spectrum of host responses to establish a relationship between causation and disease (Table 3). Table 3. PCV2 clearly fulfilled Evans postulates (Evans, 1976). 1

Prevalence of the disease should be significantly higher in those exposed to the risk factor than those not.

Exposure to the risk factor should be more frequent among those with the disease than those without.

In prospective studies, the incidence of the disease should be higher in those exposed to the risk factor than those that have not been exposed to it.

The disease should follow exposure to the risk factor with a normal or log-normal distribution of incubation periods.

A spectrum of host responses along a logical biological gradient from mild to severe should follow exposure to the risk factor.

A measurable host response should follow exposure to the risk factor in those lacking this response before exposure or should increase in those with this response before exposure. This response should be infrequent in those not exposed to the risk factor.

In experiments, the disease should occur more frequently in those exposed to the risk factor than in controls not exposed.

Reduction or elimination of the risk factor should reduce the risk of the disease.

Modifying or preventing the host response should decrease or eliminate the disease.

All findings should make biological and epidemiological sense.

Based on all abovementioned aspects, and before the advent of PCV2 vaccines, a number of researchers already defined PMWS as a multifactorial disease in which PCV2 was a necessary but insufficient cause.

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PORCINE CIRCOVIRUS TYPE 2

In subsequent years, the disease-linked scope of PCV2 expanded significantly. Although PMWS was considered the most significant condition related to this virus, a number of them, collectively named as porcine circovirus diseases (PCVDs – main terminology used in Europe) or porcine circovirus associated diseases (PCVADs – main terminology used in North America), were also described. Table 4 describes the pathological conditions or diseases that have been historically associated with PCV2. Table 4. Conditions associated with PCV2 infection in the last 20 years. The right column provides some considerations about each of these conditions. PCVD name/s

Comments

PMWS (currently denominated PCV2-systemic disease, PCV2-SD)

•

The initially described disease causing devastating losses in the pig industry, and almost eliminated from farms by means of PCV2 vaccination.

PCV2-subclinical infection (PCV2-SI)

•

Probably the most economically significant PCVD since it causes significant loss of growth.

•

Discovered after the use of PCV2 vaccines in non-PCV2-SD-affected farms.

•

PCV2 is a pathogen able to replicate in foetuses and cause organic damage in them.

•

It is believed that its prevalence is low or very low due to the existence of herd immunity.

•

PCV2 is a systemic viral infection so it is possible to find the virus in the respiratory tract.

•

Although initially proposed as a separate entity from PCV2-SD, nowadays it is believed that PCV2 mainly exerts its effect on the respiratory system within the scope of the PCV2-SD or PCV2-SI.

PCV2-reproductive disease (PCV2-RD)

PCV2-lung disease (also PCV2-respiratory disease, including porcine respiratory disease complex and proliferative necrotizing pneumonia)

➔ 10

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It was mainly in 2006 (North America) and 2004-07 (Europe) when the “circo-believer” side took significant advantage over the skeptical group. Several vaccine manufacturing companies had decided a number of years earlier to bet on PCV2 as an agent able to cause or trigger clinical disease. Therefore, the first results of PCV2 vaccine administration on farms severely affected by PMWS got available. These results were simply stunning.

PCVD name/s PCV2-enteric disease

Porcine dermatitis and nephropathy syndrome (PDNS)

Congenital tremors type AII

Comments

•

PCV2 is a systemic viral infection so it is possible to find the virus in the digestive tract.

•

Although initially proposed as a separate entity from PCV2-SD, nowadays it is believed that PCV2 mainly exerts its effect on the digestive system within the scope of the PCV2-SD or PCV2-SI.

•

Known before the first description of PMWS and considered a type III hypersensitivity reaction (immunecomplex disease).

•

Circumstantial evidences pointed out PCV2 as the associated antigen, but it has never been fully demonstrated. The use of PCV2 vaccines caused almost disappearance of this syndrome.

•

Already proposed as being caused by PCV1 during early 90s.

•

PCV2 was suggested as the aetiological agent of this condition during early 2000s, but subsequent studies could not confirm such proposal.

•

It is currently not considered as a PCVD.

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PORCINE CIRCOVIRUS TYPE 2

These biological products were able to counteract almost completely the devastating effect of the disease, with a dramatic decrease in mortality and clinical signs related to wasting (growth retardation, runts, etc). Vaccines against PCV2 are currently the most used worldwide, with a significant number of important pig producing countries vaccinating close to 100 % of the pigs reaching the slaughter. Today, year 2017, the PCV2 story is still being written, but so far it is a great success story:

• Successful from the animal’s point of view: less disease, lower mortality, fewer co-infections and improved growth; at the end, better pig welfare.

• Successful from the producer’s point of view: better average daily weight gain, fewer antibiotic treatments and more productivity; at the end, better profitability.

• Successful from the veterinarian’s point of view: capacity to deal with an immunosuppressive disease, better results of the implemented treatments and increased know-how on fighting against diseases; at the end, better professionalism.

• Successful from the vaccine manufacturers’ point of view: increased technological capabilities, improved product manufacturing innovation and novel ways of product registration; at the end, they delivered an outstanding product with great added value to the swine industry.

• Successful from the scientific point of view: discovery of a novel virus causing disease, improved understanding of multifactorial diseases and paved the path to vaccine development; at the end, an increase in pig disease knowledge (more than 1,500 scientific papers on PCV2 published between 1997 and 2015).

• And successful for the society: better animal production, safer meat and lower use of antibiotics; at the end, an increase in expectations fulfilment on meat production. 12

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