EuroPRRS2011 - НАУЧНИ ИНСТИТУТ ЗА ...EuroPRRS2011 intends to gather PRRS...

2 EUROPRRS2011, Novi Sad, Serbia

EuroPRRS2011

“Understanding and combating PRRS in Europe”

12th – 14th October 2011

Hotel Park, Novi Sad, Serbia

COST Action FA902


Scientific Committee Dr Tahar Ait –Ali, University of Edinburgh, UK; Dr Ann Brigitte Cay, CODA-CERVA-VAR Groeselenberg 99, B-1180 Brussels, Belgium Professor Hans Nauwyck, Ghent University, Faculty of Veterinary Medicine Salisburylaan, Belgium Dr Lars Erik Larsen, Technical University of Denmark, Denmark Professor Tomasz Stadejek, National Veterinary Research Institute, Poland Dr Tamas Petrovic, Scientific Veterinary Institute Novi Sad, Serbia Dr Sava Lazic, Scientific Veterinary Institute Novi Sad, Serbia Dr Enric Mateu De Antonio, CReSA-UAB Edifici V, Spain Dr Cinta Prieto Suarez, University Madrid, Veterinary faculty, Spain Dr Ivan Toplak, Veterinary faculty, National Veterinary Institute, Slovenia Dr Dinko Novosel, Croatian Veterinary Institute, Zagreb, Croatia Prof Elisabeth Grosse-Beilager, University of Veterinary Medicine Hannover, Field Station for Epidemiology Hannover, Germany Dr Massimo Amadori, Izsler, Brescia, Italy Organizing Committee Dr Tahar Ait –Ali, University of Edinburgh, UK; Dr Tamas Petrovic, Scientific Veterinary Institute Novi Sad, Serbia; Professor Tomasz Stadejek, National Veterinary Research Institute, Poland; Vesna Milicevic, Scientific Veterinary Institute Serbia, Belgrade, Serbia; Dr Sava Lazic, Scientific Veterinary Institute Novi Sad, Serbia; Samuel Thevasagayam, Pfizer Organizing Committee support team MSc Radoslav Dosen, Scientific Veterinary Institute Novi Sad, Serbia; Dr Vojin Ivetic, Scientific Veterinary Institute Serbia, Belgrade, Serbia; MSc Jasna Prodanov, Scientific Veterinary Institute Novi Sad, Serbia; Jelena Maksimovic-Zoric, Scientific Veterinary Institute Serbia, Belgrade, Serbia; MSc Diana Lupulovic, Scientific Veterinary Institute Novi Sad, Serbia; Dr Vladimir Radosavljević, Scientific Veterinary Institute Serbia, Belgrade, Serbia

Content


Preface 5 Programme 6-9 Keynote address 10-11 Diagnostics of PRRS infection 12-38 Epidemiology of PRRS infection 39-60 Latest development in PRRS research 61-81 PRRS control and eradication and economical impact 82-105 Role of vaccination and/or biosecurity in PRRS control 108-118 List of participants 119

PREFACE


In recent decades there have been great improvements in pig production systems. Genetics, nutrition, flow management, and building design have helped to improve production efficiency, profitability and animal welfare. However, infectious disease is still a major stumbling block on the road to sustainability of the pig industry. Thus, animal breeders and producers, veterinarians and researchers need to establish a coordinated approach to develop more effective means of controlling infectious diseases of farmed animal species. This requirement is driven not only by economic losses, but also by public pressure to reduce the prophylactic use of antimicrobials, to increase animal welfare and to improve food safety. Thus, control and eradication of endemic and infectious diseases are crucial to the development and sustainability of the pig industry and to satisfy consumer demand. More than 20 years after its emergence, PRRS is still having major impact on pig health and welfare worldwide. Strategies based on husbandry, biosecurity and vaccination have been devised to eradicate the disease. However PRRS frequently re-emerges in farms after eradication, indicating that these control strategies are not effective. The difficulties of controlling PRRS are increased by the differences in, inadequacy or even absence of control strategies across many countries. Further research and greater coordination of research activities would inform the development of more effective control strategies. EuroPRRSnet is the new European network COST FA902 dedicated to understand and combat PRRS disease. EuroPRRSnet is funded by COST. More than 20 European countries have joined this network. The aim of this network is to develop more effective multidisciplinary collaborative PRRS research centred on PRRSV epidemiology, immunopathology, vaccine development and harmonization of diagnostics tools. EuroPRRS2011 intends to gather PRRS researchers, veterinarians, pharmaceutical companies, pig breeding companies from Asia, USA and EU to debate on Diagnostic and control of PRRS and to review the latest achievements in PRRS research. In particular, it will address the key goals of:

• Dissemination of the recent developments in PRRS research and development of new strategies

• Increase the global interaction between Europe, Asia and North America aiming at better understaning methods of detection and control of PRRS

• Development of adequate control methods to combat PRRSV in Eastern Europe and Asia. • To train the next generation of young researchers in PRRS research, diagnosis and control

We are grateful to all of you for joining the debates and making this event a reality. In particular we would like to present our sincere appreciation to the speakers who have accepted to present the latest of their research. Finally, this conference would have never been possible without the exceptional sponsorship from Pfizer Animal Health, the Scientific Veterinary Institute „Novi Sad“, Novi Sad, Serbia, the COST office and the university of Edinburgh. We are also grateful for participation of US and Chinese colleagues. We wish the participants fruitful EuroPRRS2011 discussions and a warm welcome to Serbia. Tamas Petrovic and Tahar Ait-Ali


PROGRAMME

Wednesday, 12 October, 2011

12:00 – 13:30 Lunch for the Management committee members ( 30 people)

1 3 : 3 0 – 1 5 : 0 0 Management committee meeting (30 people)

15:00 – 15:15 Coffee break

15:15 – 16:30 Management committee meeting (30 people)

16:30 – 18:00 Registration of participants

Opening ceremony

Welcome Address: Tamas Petrovic (Scientific Veterinary Institute „Novi Sad“, Novi Sad, Serbia) Tahar Ait-Ali Coordinator of the COST FA0902 EuroPRRS.net (The Roslin Institute, University of Edinburgh, Roslin, Scotland) Dragica Stojanovic (Director of Scientific Veterinary Institute „Novi Sad“, Novi Sad, Serbia)

18:00 – 19:00

Keynote Address: Molecular evolution of Porcine Reproductive and Respiratory Syndrome virus – what can it really tells us; A trees and forest metaphor! Frederick Leung (School of Biological Sciences, The University of Hong Kong)

20:00 – 22:00 Dinner cocktail (80-120 people)

Thursday, 13 October, 2011

08:30 – 10:30 Session 1 Diagnostics of PRRS infection

Chairs: Ann Brigitte Cay, Ivan Toplak

08 :30 – 09 :00 Keynote speech: Diagnosis of PRRSV Lars Erik Larsen (National Veterinary Institute, Technical University of Denmark, Denmark)

09 :00 – 09 :30 Keynote speech: PRRS diagnostics, a veterinary diagnostic manufacturer’s point of view Eric van Esch (BioChek BV, Reeuwijk, NL)


9 : 3 0 – 1 0 : 3 0 Four 15 min oral presentations:

1. Analysis of circulation of porcine reproductive and respiratory syndrome virus in 22 Polish pig farms: implications for diagnosis and control (Tomasz Stadejek, NVRI, Pulawy, Poland)

2. Internal evaluation of new applied molecular methods for routine diagnosis of PRRSV RNA in different pig clinical samples (Sandra Revilla-Fernandez, Institute for Veterinary Disease Control, Modling, Austria)

3. The challenges of detecting the PRRS virus by Real-Time PCR (Rolf Rauh, Tetracore Inc, Rockville, USA)

4. Recombinant non-structural Nsp7 protein of PRRS virus – diagnostic use (Vladimir Celer, Faculty of Veterinary Medicine, Brno, Cz)

Posters: 1. Expression and serological reactivity of Nucleocapsid N and NSP7 proteins of PRRS genotype I virus (Jitka Jankovna, Brno, Cz)

2. Presence of antibodies against porcine reproductive and respiratory syndrome virus in farm swine in eastern part of Croatia from 2009 to 2010 (Andreja Jungic, Croatian veterinary institute, Zagreb, Croatia)

10:30 – 11:00 Coffee break with posters

11:00 – 13:00 Session 2 Epidemiology of PRRS infection

Chairs: Tomasz Stadajek, Balka Gyula

11 :00 – 11 :30 Keynote speech: Tracing and tracking the molecular epidemiology and evolution of PRRSV Manreet Brar (School of Biological Sciences, The University of Hong Kong)

11 :30 – 12 :30 Four 15 min oral presentations:

1. Genetic analysis of PRRSV isolates from pig farms in Slovakia (Štefan Vilcek, University of Veterinary Medicine and Pharmacy, Kosice, Slovakia)

2. Molecular detection and genetic analysis of Serbian PRRSV isolates (Tamas Petrovic, Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia)

3. Porcine reproductive and respiratory syndrome virus (PRRSV) infection in Lithuanian wild bors (Sus Scrofa) population (Arunas Stankevicius, Faculty of Veterinary Medicine, Kaunas, Lithuania)

4. PRRSV outbreak with high mortality in Northern part of Denmark (Lise Kvisgaard, National Veterinary Institute, Copenhagen, Denmark)

12 :30 – 13 :30 Lunch

13 :30 – 15 :45 Group discussion (joint epidemiology and diagnostics topics): - Epidemiology topics discussion chaired by Frederick Leung and Tomasz

Stadejak - Diagnostics topics discussion chaired by Lars Larsen and Ivan Toplak


16:15 – 18:00 Session 3 Latest developments in PRRS research: virus – host interaction and host genetics

Chairs: Enric Mateu, Tahar Ait-Ali


16 :15 – 16 :45 Keynote speech: Recent advances in PRRS immunology: innate responses and development of regulatory T cells Enric Mateu de Antonio (CReSA-UAB Edifici V, Spain)

16 :45 – 17 :15 Keynote speech: PRRSV entry into the porcine macrophage: completing the picture Wander Van Bredam (Ghent University, Belgium)

17 :15 – 18 :00 Three 15 min oral presentations: 1. Immunohistochemical characterization of type II pneumocyte

proliferation after PRRSV (Type I) challenge (Balka Gyula, Faculty of Veterinary Science, Budapest, Hungary)

2. Identification of T cell epitopes of porcine reproductiove and respiratory syndrome virus – 1 using a proteome-wide synthetic peptide library (Helen Mokhtar, AHVLA, UK)

3. Comparative analysis of the pathogenesis of Porcine Reproductive and Respiratory Syndrome virus strains (Annemarie Rebel, CVI Lelystad, University Wageningen, NL)

Posters: 1. Estimation of time-dependent infectiousness of pigs infected by the Porcine Reproductive and Respiratory Syndrome virus (PRRSV): correlation with the viral genome load in blood, nasal swabs and the serological response (Marie-Frederique Le Potier, ANSES, France)


18:15 – 19:30 Group discussion on immunity and virus – host interaction, chaired by Enric Mateu and Simon Graham

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20:30 –24:00 Gala Dinner (120 people)

Friday, 14 October 2011

08:00 – 10:00 Session 4 PRRS control and eradication and economic impact of PRRS infection

Chairs: Bob Morrison, Marina Stukelj

08 :00 – 08 :30 Keynote speech: How to control and eliminate PRRS from swine herds on farm and regional level Robert Morrison (University of Minnesota, St Paul, Minnesota, USA)

08 :30 – 09 :00 Keynote speech: Swine herds classification regarding PRRS status Derald Holtkamp (Iowa State University, Lloyd Veterinary Medical Center, Ames, Iowa, USA)


09:00 – 10:00 Four 15 min oral presentations:

1. Economical impact of PRRSV-outbreaks in sow herds (Tom Duinhof, Animal Health Service, Deventer, The Netherlands)

2. Eradication of PRRS in one site farrow-to-finish Greek farms: is it feasible? (Spyridon Kritas, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece)

3. Evaluation of efectivnes of an acclimatization programme to control porcine reproductive and respiratory syndrome (PRRS) in a farm of an endemically infected area of North-Eastern Italy (Michele Drigo, Veterinary College, University of Padua, Italy)

4. Risk assessment and risk management – use of applied epidemiology for PRRSV control in large swine production of Croatia (Dejvid Sabolek, College of Medicine and Veterinary Medicine, University of Edinburgh, UK)

Posters: 1. Coordinated programs on PRRSV in The Netherlands (Tom Duinhof, Animal Health Service, Deventer, The Netherlands)


10:30 – 12:00 Session 5 Role of vaccination and/or biosecurity in PRRS control

Chairs: Spyros Kritas

10:30 – 11:00 Keynote speech: Biosecurity in PRRSV control/eradication: Research update and field applications Satoshi Otake (Swine Disease Eradication Center, University of Minnesota, College of Veterinary Medicine, USA)

11:00 – 12:00 Three 15 min oral presentations:

1. Evaluating regional PRRSV eradication perspectives based on a herd-level risk index for virus persistence and reintroduction (Anna Fahron, Veterinary Health Public Institute, Bern, Switzerland)

2. Elimination of porcine reproductive and respiratory syndrome (PRRS) with serumization (Marina Stukelj, Veterinary Faculty, Ljubljana, Slovenia)

3. Determination of the correlation between cross reactivity profiles of neutralizing antibodies and cross-protection in PRRSV infections (Francisco Javier Martinez-Lobo, Veterinary Faculty, Madrid, Spain)

12:00 –13:00 Lunch

13:00 –15:00 Group discussion on PRRS control and biosecurity and vaccination chaired by Robert Morrison

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1 5 : 0 0 End of meeting


Keynote Address

Molecular Evolution of Porcine Reproductive and Respiratory Syndrome virus – What Can It Really Tells Us; A Trees and

Forest metaphor!

Leung FCC. School of Biological Sciences, The University of Hong Kong

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an

economically important virus which infects domestic swine populations. The virus is highly dynamic in nature: on one hand it is continuously and rapidly evolving, generating “new variants” and expanding its diversity; on the other hand, modern food production systems act as a super-spreader that distributes viruses nationwide and even worldwide. To better understand the dynamic character of this virus, we applied a selection of avant-garde phylogeny-based methods which incorporate temporal, geographic, and genetic information to reveal viral evolutionary history and molecular epidemiological insights.

PRRSV can be divided into two genotypes: Type 1 & 2, whose circulation

mainly centered in Europe and North America respectively. For Type I PRRSV, the molecular dating analyses revealed that it had been endemic in Europe for a long time (around 30~50 yr) before the “original” epidemic, although the earliest record of Type 1 outbreak was in the early 1990s. A potential geographical origin of Type 1 PRRSV is the very Eastern part of Europe. Compared to Type 1, Type 2 PRRSV has slightly smaller diversity, with a tMRCA dated back to the late 1970s. Type 2 PRRSV is most likely originated in North America, but the exact origin remains uncertain.

With the help of phylogenetic methods, we resolved the mystery of several

PRRSV outbreaks in the field. We have also put specific focus on characterizing PRRSV in China owing to the immensely large susceptible swine population and economic consequence. The Bayesian Skyline Plot indicated an exponential expansion in the effective viral population shortly before the recognized re-emergence of type 2 PRRSV (designated HP-PRRSV) in 2006 – a variant evolved from existing Chinese PRRS viruses but with heightened pathogenicity. In this case, the role of human intervention, vis-a vis vaccination or possible change in swine management practices, on top of virus evolution could have played a key role to help the virus overcome geographic and/or biological barrier(s), which later resulted in epidemic-type spread of virulent variants in the field.


The intensive sampling of Type 2 PRRSV also allows us to reveal viral spatial dynamics, which is beyond the predictions arising from standard epidemiological surveillance. International dissemination of PRRSV is sporadically occurring (mainly due to introduction of breeding animals, semen, and live attenuated vaccines), but more frequent dissemination has been observed among places where pig movement is prominent (e.g. Western Europe, North America). Based on North American sampling, we are able to illustrate that the transmission frequency of PRRSV highly reflected the asymmetrical pig flow among various pig-production countries and regions involved in the multiple site production system, indicating the huge influence of the modern food production system on virus dissemination.

In summary, PRRSV is a rapidly evolving organism whose transmission

has been greatly amplified by the modern swine production system. Therefore, to control and prevent PRRS, it is important to keep pace with changing viral dynamics using phylogenetic tools. In my presentation, I shall provide a “century vision” on combating this viral disease based on our current understanding of the molecular evolution of PRRSV.


Session 1 Diagnostics of PRRS infection

Chairs: Ann Brigitte Cay, Ivan Toplak


Diagnosis of PRRSV

Lars Erik Larsen

National Veterinary Institute; Technical University of Denmark ([email protected]) Abstract Since its discovery in the late 80ties, PRRSV has emerged to be the most prevalent infectious disease of pigs worldwide (Lunney et al., 2010). Alone in the US, the annual costs of PRRSV has been estimated at $560 million (Neumann et al., 2005). The impact of PRRSV on the production of swine in Europe has only been assessed in a few countries (Brouwer et al., 1994), but is probably comparable to the figures in the US. Until 2010 there has been no joint European initiatives regarding PRRSV and therefore the procedures for the diagnosis, control and prevention of this important infection in the different European countries have been developed on the national level and as a result these procedures and diagnostic programs may differ considerably throughout Europe. The present review is an attempt to present state of the art in respect to PRRSV diagnostic tests for virus and antibody detection. Detection of PRRS virus Traditional methods Isolation of PRRSV in continuous cell lines derived from green monkey kidney epithelial cells (MA104, CL2621, and MARC-145) or in primary cells (i.e. pulmonary alveolar macrophages – PAMs) have previously been the golden standard for detection of PRRSV in diagnostic samples (Bautista et al., 1993;Mengeling et al., 1995;Mengeling et al., 1996a;Mengeling et al., 1996b). This procedure is laborious and time demanding and has a reduced sensitivity since it requires living virus which may be a problem for samples collected in the field due to the time lap and suboptimal storing conditions until test (Benson et al., 2002). On the other hand, isolation of the virus allows downstream biological characterisation of the virus and this procedure is also less prone for false negative outcome caused by genetic drift. Protocols for detection of PRRSV by in situ hybridization and immuno-histochemistry have been published (Cheon & Chae, 2000;Larochelle & Magar, 1997;Shin & Molitor, 2002;Sur et al., 1996). Similarly, antigen ELISA for detection of PRRSV antigen in serum and tissue has been described (Cai et al.,


2009). None of these methods are presently widely used for routine diagnosis of PRRSV. RT-PCR assays Molecular based methods have been the preferred principle for detection of PRRS virus for the last decade or so. A vast number of conventional RT-PCR assays have been published using different chemistry and employing visualization by either gel electrography (Chen et al., 2010a;Chen et al., 2008;Chen et al., 2010b;Guarino et al., 1999;Li & Ren, 2011;Mardassi et al., 1994;Oleksiewicz et al., 1998;Qin et al., 2009;Spagnuolo-Weaver et al., 1998) or by post amplification ELISA (Guarino, Goyal, Murtaugh, Morrison, & Kapur, 1999;Spagnuolo-Weaver, Walker, McNeilly, Calvert, Graham, Burns, Adair, & Allan, 1998). Primers for PRRSV detection has also been included in a number of conventional multiplex RT-PCR assays (Liu et al., 2011;Ogawa et al., 2009). In recent years, real time RT-PCR has replaced conventional RT-PCR as the preferred tool for the detection of PRRS virus in serum and tissues. Several different technical platforms and assay chemistry have been described, but most published assays rely on either unspecific detection of amplicoms by CyBr Green (Lurchachaiwong et al., 2008;Martinez et al., 2008;Tian et al., 2010;Yang et al., 2006), by Taqman probes (Egli et al., 2001;Kleiboeker et al., 2005;Lurchachaiwong, Payungporn, Srisatidnarakul, Mungkundar, Theamboonlers, & Poovorawan, 2008), or, more recent, by the PrimerProbe energy transfer (PriProET) system (Balka et al., 2009;Balka et al., 2010). In addition to the above mentioned in-house developed assays, there are several commercial conventional and real time RT-PCR assays available. There are conflicting reports on the performance of these assays and the major drawback is that the primer sequences are not publicly exposed. On the other hand, several of the commercial assays have been toughly evaluated in order to obtain market authorization and the performance is continuously monitored by certification bodies. The use of RT-PCR are superior to other methods in that these assays are very sensitive, faster than virus isolation and do not require living virus. In general, real time RT-PCR are preferred for conventional RT-PCR since these assays are more sensitive than conventional assays and there is less risk of laboratory cross-contamination because post amplification handlings are avoided. Furthermore, real time assays are semi quantitative and allows real-time detection of products. The diagnostic performances of a given RT-PCR assay relay on several factors and in general it is difficult to make a sound bench marking of the different assays, however, some important general factors can be highlighted. Most of the available diagnostic RT-PCR assays are targeting the gene coding for the N protein (ORF7). Sequence comparison between and among European (type 1) and North American


(type 2) types of PRRSV have revealed significant sequence variability in ORF7 (Drew et al., 1997;Indik et al., 2005;Meng et al., 1995;Stadejek et al., 2006;Thanawongnuwech et al., 2004). This may have profound impact on the sensitivity of the assays. Indeed, several differences are revealed between the primer and probe sequences of widely used RT-PCR assays and the binding sites on the most diverse ORF7 sequences (Stadejek et al., 2008) (Figure 1).

Figure 1. Differences between primer and probe sequences of a widely used real time PCR assay (Egli, Thur, Liu, & Hofmann, 2001) and the most diverse Type 1 sequences (Stadejek, Oleksiewicz, Scherbakov, Timina, Krabbe, Chabros, & Potapchuk, 2008). With permission from Charlotte K. Hjulsager

Thus, even though assays targeting ORF7 is useful for combining detection and typing of PRRSV, assays targeting more conserved PRRSV genes would be more robust. Other factors than sequence diversity may affect the performance of RT-PCR assays. Especially the choice of RNA extraction protocols have been shown to have a major impact on assay performance and it necessary to make optimization for each kind of sample material – i.e. a protocol than works great on serum may not be applicable on semen (Christopher-Hennings et al., 2006;Guarino et al., 1997). To avoid false negative results it is also recommended to include extraction controls in the set-up (Revilla-Fernandez et al., 2005). RT-PCR are routinely used for the diagnosis of PRRSV infection based on tissue (often lungs) from diseased animals, fetal material in case of reproductive problems or on serum in the case of PRRSV monitoring, certification in connection to export or as a part of herd profiling. Furthermore, PRRSV specific RT-PCRs are used for screening of semen. Recently, studies have shown that paper disks sucked in blood (Inoue et al., 2007) or oral fluids collected by robes (Kittawornrat et al., 2010;Prickett et al., 2008) may serve as convenient alternative sampling materials. Further validations under field conditions are, however, needed before these new approaches can replace serum as sample material. To reduce cost related or/and to increase the sampling size, pooling of samples from 5-10 animals are often used (Rovira et al., 2007). Serology Serology is used for surveillance for PRRSV and may also be suitable for the diagnosis of acute PRRSV outbreaks in herds (Botner, 1997). Two different tests


are routinely used in diagnostic laboratories – ELISA and immune-peroxidase assays (i.e. IPMA). A vast number of in-house ELISA has been published, but most of these assays have been developed for use in research projects and only a few of these are routinely used for diagnosis and monitoring of PRRSV. Both indirect and blocking ELISAs employing whole virus suspensions as antigens seem to be particular popular (Bogdanova et al., 2007;Cho et al., 1996;Ferrin et al., 2004;Mortensen et al., 2001;Sorensen et al., 1997;Sorensen et al., 1998;Takikawa et al., 1996), but recombinant expressed PRRSV proteins have also been used in a number of assays (Chen et al., 2011;Chu et al., 2009;Dea et al., 2000;Denac et al., 1997;Jiang et al., 2005;Kwang et al., 1999;Lopez et al., 2009;Pyo et al., 2010;Seuberlich et al., 2002;Witte et al., 2000). A few studies employed synthetic peptides as antigen (Oleksiewicz et al., 2005;Plagemann, 2006). Only a few of these tests are able to discriminate between PRRSV type 1 and type 2. In general, the performances of ELISA was found to be comparable to or better than the performance of IPMA on the herd level (Cho et al., 1997;Houben et al., 1995;Nodelijk et al., 1996;Yahara et al., 2002). Similarly, in a comparative study performed in the scope of an EU concerted action, the performance between laboratories and different tests were found to be acceptable on the herd level (Drew, 1995). A variety of commercial kits are available and albeit some problems with false positive results have been described (Okinaga et al., 2009) the most popular of those seem to perform as good as the most reliable in house assays. Serum is the sample material of choice for serology, but alternatives such as meat juice (Molina et al., 2008) have been used for monitoring of PRRSV in i.e. Danish pigs (Andreasen et al., 2000;Mortensen, Strandbygaard, Botner, Feld, & Willeberg, 2001). More recently, the use of oral fluids collected by robes have been used for detection of PRRSV specific antibodies (Prickett, Simer, Christopher-Hennings, Yoon, Evans, & Zimmerman, 2008). Furthermore, a few studies have validated the use of pooling of samples for herd screenings (Rovira et al., 2008). Discussion and perspectives A huge variety of different RT-PCR assays and protocols are used in public and private diagnostic laboratories throughout Europe. Only some of the assays have been published and validation data are in general not available. It is therefore of outermost importance that research activities are launched with the focus to improve, validate, implement and standardizes the diagnostic procedures used in Europe and globally. A first step would be to perform ring trials with participation of laboratories throughout Europe. Furthermore, PRRSV is a single stranded RNA virus, which is prone for antigenic drift. As described, studies have shown that some European countries such as Lithuania, Latvia, Belarus and Russia harbor exceptionally diverse EU-genotype PRRSV strains (Stadejek, Oleksiewicz, Scherbakov, Timina, Krabbe, Chabros, & Potapchuk, 2008). Alignments of these strains with the primer and probe sequences of published PRRSV PCR assays indicated that the majority of the


assays would not be able to recognize these recent strains. Updated PRRSV sequence data from the rest of Europe are very sparse; therefore, it is not clear if the various assays used gain a substantial number of false negatives when field samples are tested. Thus, it is obvious that there is a need for surveillance programs with the aim to continuously monitor the drift of PRRSV by sequencing subsets of circulating strains and by building joint PRRSV databases with public accessibility. The sequencing should focus on – but not be limited to - ORF5 and ORF7. Thus, if sequence data for the full genome of a diversity of isolates, it might be possible to identify a more conserved region of the genome which could be target for more sensitive and robust real time RT PCR assays. References:

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PRRSV DIAGNOSTICS, A VETERINARY DIAGNOSTIC MANUFACTURER’S POINT OF VIEW

Eric van Esch

BioChek BV, Reeuwijk, Netherlands, [email protected] Developing diagnostics, basic questions There is great need for diagnostic tools in the field for a better understanding of PRRSV infections and control of PRRSV. For that reason many different assays are developed and are used successfully. In development of these diagnostics tools a lot has to be taken care off. Any development will start with a lot of questions which have to be answered before designing and developing any assay. One basic question will be what has to be detected, antigen or antibodies. Where antigen detection is more reflecting the present, antibodies are more reflecting the past. Another basic question will be in what kind of situation the assay will be used. Roughly four different important situation can be described:

I. Clinical diagnostics. Samples are taken to make a good diagnosis, the antigen is present on a farm and leads from time to time to clinical cases

II. Screening herds. Samples are taken to establish a herd status, the antigen is or was present on a farm and is or was leading to clinical cases from time to time.

III. Eradication. The herds status is known and all efforts are directed to eliminate the antigen from a herd. Samples are taken to control this process.

IV. Herd monitoring. Samples are taken to monitor the freedom of disease in a herd.

Situation I and II can be described as detection of disease, where situation IV is described as detection of freedom of disease. Situation III is in between. It is essential to make these differences for the reason that the performance of an assay will be determined by the situations. To get answers on these questions it will be obvious that feedback from out of the field will be necessary. Development, the next step When the basic questions are answered the next step will be the set up of the assay. To fulfill the user’s need the most suitable setup (e.g. ELISA, PCR) will be chosen. For ELISA the desired antigen has to be found and for PCR the right primer set has to be chosen. Principal research is essential in this step and is needed to select the right things. Additional question will come up in this phase


such as must the assay be able to discriminate between vaccinated animals and field infections and is there a need that the assay is able to discriminate between different types or strains. Other questions as detecting limits and specificity will rise here as well. Development is working together When the desired setup is chosen and an experimental kit is available a technical and diagnostic evaluation is needed to check the performance of the assay. Where for technical evaluation samples from animal experiments are desirable, for diagnostic evaluation samples from out of the field are needed. This means that the development is a joined effort of the developer, scientists and field workers (veterinary laboratories and veterinarians). This co-work will not only be limited to set up the assay at the developers lab, but also sending out the first kits to laboratories to work with this kit and give feedback on the kit, both in performance and user friendliness. In this phase of the development the parameters sensitivity, specificity, predictive value, repeatability and reproducibility will be established and the assay is checked on the fitness for purpose. During these trials the first baselines and interpretation of the results will be established. Development stops when kit is on the market? No, development will never stop on assays on the market. It is essential to receive feedback from users to have a constant monitoring on the kit performance. During the first development of an assay not everything can be checked because the field is so broad and various. Exceptional situations will only be found when a assay is used more often. During many years of experience baselines will be established better and interpretation of results will be more clear (or not?). A continuous feedback on unexpected results, both positive and negative, is essential to monitor if the assay is still fit for purpose. Diagnostics, check-do-act-react It may be clear, veterinary diagnostics developments are not a one man show and feedback from the field and science is essential. The well-known circle Check-Do-Act-React is applicable in diagnostics as well and this is a joined effort from all parties involved. We cannot act without each other!


Analysis of circulation of porcine reproductive and respiratory syndrome virus in 22 Polish pig farms: implications for diagnosis

and control

T. Stadejek*, A. Jablonski, K. Chabros, E. Skrzypiec, Z. Pejsak

National Veterinary Research Institute, OIE Reference Laboratory for PRRS, Pulawy, Poland

Porcine reproductive and respiratory syndrome (PRRSV) is currently one the most important viruses of swine worldwide. Its control is difficult but even complete elimination is possible. Any control approach must involve careful evaluation of PRRSV circulation in the herd. PRRSV circulation depends on many factors of which herd management is likely the most important. In the present paper we communicate results of detailed analysis of PRRSV viremia and seroconversion performed in 22 Polish farrow to finish pig farms. The analyzed herds were infected with PRRSV between 1996 and 2010. The farms represented enterprises with sow herds from 70 to 1400 heads. Different pig flow systems and PRRS control measures were applied. Serum was obtained from pigs of different age (between 2 and 23 weeks of age, depending on the farm, differing by 2-3 weeks). In most herds also sows were sampled. To assess homogeneity of the virological and serological status of a given age group pigs of the same age from several pens were sampled. In total, 3610 serum samples were obtained, (70 to 574 samples per farm). The sera were tested for the presence of PRRSV specific antibodies by in house indirect ELISA and for the presence of viral RNA by Real Time RT-PCR (PRRS NextGen, Tetracore). Serum samples were tested individually by ELISA and pooled (by 5) by PCR. Results obtained in 9 farms (with sow herds of 70 to 850 heads) indicated no virus circulation in sows (PCR negative) nor in weaners (PCR negative and no, or waning colostral antibodies). In 5 of these farms PRRSV circulation was maintained in fattening units (PCR positive, increasing with age proportion of seropositive fatteners), while in remaining 4 farms no viremia nor seroconversion profile suggestive of infection was detected in any of the analyzed groups. In those, only seroconversion in sows and in young piglets was noted. In 6 farms of this group Porcilis PRRS (Merck Animal Health) and/or Progressis (Merial) vaccination was applied in gilts (3 herds) and/or sows (3 herds). In the remaining 13 farms viremia was detected by PCR in sows (4 herds) and piglets, before, or soon after weaning. Only 4 of these farms vaccinated sows and/or gilts against PRRS.


The PCR results showed differences in the PCR profiles between the farms. In some farms PRRSV was detectable from 4 up to 18 weeks of age. In others, positive PCR results were obtained only in few samplings. In one farm PCR result was negative despite serological evidence infection. On the other hand ELISA provided consistent results in all farms. In summary, in farms where vaccination was used, in general better results of PRRSV control were obtained. However, herd management practices might contribute to the observed picture as well. Complete picture of PRRSV in a herd can be obtained only by testing large number of animals by serological and PCR methods. Oral fluid testing by ELISA and PCR can be a cost effective alternative to serum analysis.


Internal evaluation of new applied molecular methods for routine diagnosis of PRRSV RNA in different pig clinical samples

Sandra Revilla-Fernández1*, Adolf Steinrigl1, Tatjana Sattler2, Friedrich Schmoll1

1 AGES Institute for Veterinary Disease Control, Robert Koch Gasse 17, A-2340 Mödling 2 Large Animal Clinic for Internal Medicine, University Leipzig, An den Tierkliniken 11,

D-04103 Leipzig * corresponding author, E-mail: [email protected]

Tel. +43 50555 38320, Fax. +43 50555 38309 Abstract: The first purpose of this study was to perform an in-house evaluation of recently established PRRSV molecular diagnostic methods. Five different assays, including 4 real-time RT-PCRs (RT-qPCR), from which 3 of them are commercially available, and one conventional ORF7 duplex RT-nested PCR were tested with a series of PRRSV EU and NA field strains, and the Epizone ring trial samples 2011. Diagnostic and analytical sensitivity and specificity were extensively studied. In a second approach, the pattern of PRRSV shedding in pigs inoculated with an attenuated PRRSV EU vaccine was compared by RT-qPCR between serum and oral fluid samples (3, 6, 7, 8). This was done to determine whether oral fluid samples could be used for routine PRRS diagnosis instead of serum samples. Keywords: Diagnosis, real-time RT-PCR, sensitive, specific, oral fluid Introduction: PPRSV is a small, enveloped, positive single-stranded RNA virus belonging to the Arteriviridae family, recognised world-wide as an important cause of reproductive failure and pneumonia in pigs. Since PRRSV was first described at the beginning of the 1990´s, wide genomic, antigenic and clinical differences have been described in isolates belonging to the European (EU) and North American (NA) genotypes. Several molecular-based methods like conventional RT-PCR and RT-qPCR have been demonstrated to be fit for detection of PRRSV in clinical samples. However, the high mutation rate of PRRSV poses a diagnostic challenge (5, 11, 12), necessitating continuous evaluation of routinely applied methods with the most recent strains. This is essential to control the disease and to guarantee the negative status of the herds (2). Also, specimens like oral fluids represent advantages in terms of costs, labor, time and animal welfare and therefore have been recently proposed for disease monitoring (3). Methods:


Five different one-step RT-PCR-based assays (Table 1), including 4 RT-qPCRs (3 of them commercially available kits) and one conventional ORF7 duplex RT-nested PCR (9), were tested for assay sensitivity and specificity with a series of PRRSV EU and NA field strains from Central Europe and Russia, samples from the Epizone Ring trial 2011 and PRRSV in vitro transcribed RNA standards encompassing the complete ORF5 to ORF7 regions. The target sequences of the commercial RT-qPCR kits were unknown. All but one test distinguish between EU and NA genotypes (Table 1). Runs were performed in two different real-time PCR cyclers. Plasmids encompassing the ORF5 to ORF7 coding regions of PRRSV-EU and PRRSV-NA were constructed based on strains DV (Porcilis® PRRSV-Intervet) and P120 (Ingelvac® PRRSV-Böhringer-Ingelheim), respectively. The ORF5 region of field samples was sequenced and analyzed phylogenetically with Bionumerics Software (Applied Maths), using reference strains of all PRRSV genotypes and subgroups (11, 12, 13). Quantitative RT-qPCR data (Cq values) was analysed statistically by 2-sided paired T-test. Table 1: Summary of methods applied for validation, references and system descriptions

Assay Reference Nr. Cycles

Differentiation NA/EU

RNA Input (µl)

1 ORF6 RT-qPCR 10 45 yes, two-tube 5 2 ORF7 RT-nested PCR 9 30+30 yes, one-tube 5 3 Kit A RT-qPCR commercial 40 yes, one-tube 8 4 Kit B RT-qPCR commercial 40 yes, one-tube 5 5 Kit C RT-qPCR commercial 40 no 5

In a second experiment, ten PRRSV-negative pigs (8 to 20 weeks of age) were injected with modified live Porcilis® PRRSV EU vaccine. Blood and oral fluid samples were taken from each pig before, and at 4, 7, 14 and 21 days after vaccination (p.v.). RNA extraction from all samples was performed with a commercially available kit on an automated RNA extraction platform. Oral fluid samples were taken without previous centrifugation or dilution. An exogenous RNA control was spiked prior to extraction in order to control for extraction efficiency and to identify false negative results due to PCR inhibition. After RNA extraction, samples from all animals and time-points were analysed with RT-qPCR assay 1. Moreover, seroconversion was monitored by IDEXX PRRS X3 ELISA. Results: In general, the commercially available RT-qPCR kits A and B showed higher sensitivity than the other methods tested (Fig. 1). However, specificity problems were seen with methods 1, 3 (kit A) and 5 (kit C) with some non EU-1 strains, principally those from EU-2 and EU-unassigned genotypes. Also, kit C showed massive specificity problems with high-pathogen NA strains from the ring trial panel. In contrast, EU-1 field strains from Austria, Germany, Slovenia and Russia were equally well detected by all assays tested.


Fig.

1: Cq

values of PRR

SV RT-qPC

R assa

ys obtai

ned with samples

from the

Epizone Ring trial 2011 After taking into account different RNA sample inputs, statistical analysis of Cq values confirmed relevant differences between assays: lowest Cq values, indicating highest analytical sensitivity, were consistently observed with kits A and B. However, kit A did not efficiently detect EU-2 and EU-unassigned strains, whereas kit B showed problems resulting from crosstalk between fluorescence channels in one of the real-time PCR cyclers used for validation. This indicates that various laboratory technical specifications should be considered to avoid false results. Regarding PRRSV shedding after vaccination with an attenuated vaccine, PRRSV RNA was detected more often in serum samples than and in oral fluids. PRRSV RNA detection in serum peaked at day 7 days p.v. (Table 2). Table 2: No. of pigs with PRRSV RNA (PCR) and antibody (Ab) detection in serum and

oral fluid following vaccination with PRRSV modified live vaccine.

day 0 (no vaccination)

day 4 after vaccination day 7 day 14 day 21

PCR Ab PCR Ab PCR Ab PCR Ab PCR Ab Serum 0 0 3 0 7 0 5 7 3 8 Oral fluids 0 0 0 0 2 0 2 2 0 3

Whereas one animal was positive for PRRSV RNA in all serum samples taken p.v., PRRSV RNA was not at all detected in two of the vaccinated pigs. All animals remained Ab-negative until day 7 p.v. The two pigs that tested PRRSV RNA negative also remained serologically negative throughout the trial.


Recommendations: In conclusion, this validation shows that there is currently no assay or commercial kit with optimal analytical and diagnostic sensitivity. To overcome this key aspect in PRRSV diagnosis, continuous re-validation of applied methods is necessary. As shown, in-house validation may be helpful to identify the best-suited methods for PRRSV diagnosis. In contrast to other studies (3) our results indicate that further assay optimization is needed before oral fluid samples can replace serum samples for PRRSV molecular detection. Therefore, present challenges like salivary specific PCR inhibition (1) and differences between strains with regard to PRRSV shedding into oral fluid still remain open. References: 1. Chittick WA, Stensland WR, Prickett JR, Strait EL, Harmon K, Yoon KJ, Wang C,

Zimmerman JJ. (2011): Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens. J Vet Diagn Invest. 23(2):248-53.

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3. Kittawornrat A, Prickett J, Chittick W, Wang C, Engle M, Johnson J, Patnayak D, Schwartz T, Whitney D, Olsen C, Schwartz K, Zimmerman J. (2010): Porcine reproductive and respiratory syndrome virus (PRRSV) in serum and oral fluid samples from individual boars: will oral fluid replace serum for PRRSV surveillance? Virus Res. 154(1-2):170-6.

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6. Prickett J, Simer R, Christopher-Hennings J, Yoon, KJ, Evans RB, Zimmermann JJ. (2008): Detection of Porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: a longitudinal study under experimental conditions. J Vet Diagn Invest. 20:156-63.

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RNAs for qualitative and quantitative detection of PRRSV in porcine semen. J Virol Methods. 126:21-30.

11. Shi M, Lam TT, Hon CC, Hui RK, Faaberg KS, Wennblom T, Murtaugh MP, Stadejek T, Leung FC. (2010): Molecular epidemiology of PRRSV: a phylogenetic perspective. Virus Res. 154(1-2):7-17.

12. Stadejek T, Oleksiewicz MB, Scherbakov AV, Timina AM, Krabbe JS, Chabros K, Potapchuk D. (2008): Definition of subtypes in the European genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in Europe. Arch Virol. 153(8):1479-88.

13. Shi M, Lam TT, Hon CC, Murtaugh MP, Davies PR, Hui RK, Li J, Wong LT, Yip CW, Jiang JW, Leung FC. (2010): Phylogeny-based evolutionary, demographical, and geographical dissection of North American type 2 porcine reproductive and respiratory syndrome viruses. J Virol. 84(17):8700-11

The challenges of detecting the PRRS virus by Real-Time PCR

Rauh R., Nelson W., Pillay S.

Tetracore Inc, 9901 Belward Campus Drive, Suite 300, Rockville, MD 20850, USA

Abstract

Due to its potentially high economic impact, Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most significant diseases in the swine industry. PRRS has spread rapidly around the world through pig sales, semen, and airborne transmission. The risk of PRRS spreading to other countries is increasing, possibly due to increased airline traffic and passengers who carry the virus on their clothing, shoes, or equipment while traveling. There are two major genotypes of the PRRS virus, the European genotype (type 1) and the North American genotype (type 2). These two genotypes display profound variation in both their genomic sequence and antigen composition, and viral recombination between distinct PRRS viruses has been reported. These characteristics of the PRRS virus, combined with limited access to high quality viral sequences, make the design of a real-time PCR assay for the simultaneous detection of both PRRS genotypes complicated.

For a diagnostic assay to reliably detect all PRRS strains, the use of multiple amplification targets for each PRRS genotype is necessary. Further, an effective procedure for sample preparation is essential, and requires verification with an internal assay control. Multiplexing both the amplification targets and the internal control is a challenge in itself. The choice of the real-time PCR instrument and associated reagents additionally limits the flexibility for multiplexing the different target regions and internal control.


After successfully developing the assay, quality assurance and quality control procedures are required in order to guarantee the continued performance of the assay. Proficiency panel testing (i.e., ring trials) should be used to verify the quality of the assay and to ensure the laboratory’s ability to perform it. While real-time PCR detection of PRRS remains challenging, recent advances in instrumentation and reagents offer promise for affordable and reliable assays in the future. Keywords: PRRS, real-time PCR, multiplex assays

Recombinant nonstructural Nsp7 protein of PRRS virus – diagnostic use

Jitka Janková1, Vladimír Celer1,2

1) Institute of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine,

University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1-3, 612 42 Brno, Czech Republic

2) CEITEC – Central European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

Abstract

The aim of our study was to express recombinant structural N and nonstructural Nsp7 proteins of PRRS virus and to use these virus antigens for analysis of antibody response of pig herds with different PRRS virus status and vaccination regime.

The expression of N and Nsp7 proteins was tested in Escherichia coli strains. Resulting recombinant proteins were purified by IMAC using a polyhistidine tag under denaturing conditions from the insoluble fraction of bacterial lysate. Purified N and Nsp7 proteins were applied as antigens in indirect ELISA tests. In examined sera, antibodies against N protein appeared early after infection and in higher titers, followed with Nsp7 antibody response with several weeks delay. Nsp7 serological reactivity was a hallmark of virus replication in PRRS positive or modified live vaccine vaccinated animals. Combination of N and nsp7 antigens in ELISA test improved sensitivity and specificity of PRRS serological detection, moreover assessment of antibody response against both virus antigens allowed discrimination of post infection and post vaccination antibodies in herds vaccinated using inactivated vaccine and identification of field virus breakthrough in inactivated vaccine vaccinated herds.

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a widespread disease of swine characterized by late-term abortions, high incidence


of stillborn and mummified piglets and respiratory problems in suckling, weaned and growing pigs (Hopper et al., 1992; White, 1992).

The herd diagnostics of PRRS infection is usually done by serological testing. At present, several ELISA tests are commercially available to diagnose the disease; these tests are mostly based on virus nucleocapsid protein because specific antibodies against N protein are produced during PRRS infection in high titers (Meulenberg et al., 1995).

Antibody response against PRRS virus infection is directed not only against structural virus proteins, but was also recorded against nonstructural virus proteins (Johnson et al., 2007). Nonstructural proteins (Nsp) include proteases (Nsp1α, Nsp1β and Nsp2), the RNA-dependent RNA polymerase (Nsp9), helicase (Nsp10) and endonuclease (Nsp11), which are expressed in virus infected cells only. The remaining nonstructural proteins including Nsp7 protein are not well described and their main role in a virus life cycle still remains to be established. Nsp specific antibodies are produced in high titers as the response to virus replication, especially against Nsp1α, Nsp1β, Nsp2 and Nsp7 (Brown et al., 2009; Den Boon et al., 1995; Fang and Snijder, 2010; Han et al., 2009) and it can be speculated that these antibodies could be used to differentiate post-infectious and post-vaccination antibodies in animals vaccinated by inactivated vaccines. Recombinant Nsp7 protein of PRRS virus was used as an antigen in an indirect ELISA test and showed good sensitivity and specificity for identification and differentiation of type I and type II of PRRS virus. Furthermore, the Nsp7 ELISA test resolved 98 % of samples suspected to be false-positive in IDEXX ELISA test (Brown et al., 2009).

Serological tests never reach 100 % accuracy, and PRRS ELISA tests are no exception. Additionally, some tests can give false-positive results. A combination of tests based on different antigens would therefore be beneficial to improve the accuracy of PRRS diagnostics. The inclusion of Nsp7 protein in serological diagnostic tests could increase the specificity and sensitivity of used diagnostic tests.

The goal of this work is to express recombinant ORF7 and nonstructural Nsp7 proteins in E. coli cells and to compare their diagnostic sensitivity and specificity on a panel of swine sera from pig herds with different PRRS status. Another goal of the work is to analyze the use of Nsp7 antigen for the differentiation of post-infection and post-vaccination antibodies in animals vaccinated with inactivated vaccine (KV). In our experiments serological profile of several pig farms with different health status concerning PRRS infection and with different PRRS vaccination regime was determined using ORF7 and Nsp7 ELISA test.

Results 1) In PRRS negative farm saws vaccinated with inactivated vaccine all pigs demonstrated antibodies against ORF7 protein; none of them was Nsp7 antibodies positive (Fig. 1).


Fig.1. Comparison of number of ORF7 or Nsp7 positive animals (%) in PRRS negative MLV vaccinated farm.

2) In PRRS negative farm, piglets were vaccinated using modified live vaccine (MLV) vaccine in the age of 3 weeks; ORF7 specific antibodies appeared early after vaccination and 100% prevalence was achieved at 12 weeks of age. Nsp7 specific antibodies appeared at 12 weeks in 20% animals and reached 100% positivity at 18 weeks (Fig. 2).

Fig.2. Number of ORF7 or Nsp7 positive animals in MLV vaccinated piglets (weeks of age). 3) PRRS negative farm, all sows were vaccinated using MLV vaccine. Percentage of ORF7 as well as Nsp7 positive piglets reached 100% at week 13 (Fig. 3). Seroconversion in piglets was likely stimulated by the spread of transplacentally transmitted vaccine virus.

Fig. 3. Percentage of ORF7 or Nsp7 positive piglets at different weeks.


4) PRRS negative herd, sows were vaccinated using inactivated vaccine (Fig. 4). Sows as well as all piglets at week 1 had ORF7 specific antibodies. No Nsp7 specific antibodies were detected. Piglets remained seronegative till the week 15.

Fig. 4. Percentage of ORF7 or Nsp7 positive gilts (G), suckling piglets (S1) and piglets post weaning (W).

5) PRRS positive herd, sows were vaccinated using inactivated vaccine. ORF7 specific antibodies were detected at week 10 in 80% piglets. Increase of Nsp7 antibodies positive piglets at week 16 is due to infection with field PRRS strain of the virus (Fig. 5).

Fig. 5. Number of ORF7 or Nsp7 positive piglets (%) at different weeks of age. Conclusion

Nsp7 protein increases the sensitivity and specificity of serological detection based on ORF7 protein as the only antigen. Moreover we have successfully used Nsp7 based ELISA test to differentiate post-infection and post-vaccination antibodies in herds vaccinated using inactivated vaccine. These antibodies indicated replication of the field or vaccine virus in the host organism but surprisingly appeared only 4-6 week following ORF7 specific antibodies.


References

1. Brown, E., Lawson, S., Welbon, C., Gnanandarajah, J., Li, J., Murtaugh, M. P., Nelson, E. A., Molina, R. M., Zimmerman, J. J., Rowland, R. R., Fang, Y., 2009 Antibody response to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural proteins and implications for diagnostic detection and differentiation of PRRSV types I and II. Clin.Vaccine Immunol 16, 628-635.

2. Den Boon, J. A., Faaberg, K. S., Meulenberg, J. J., Wassenaar, A. L., Plagemann, P. G., Gorbalenya, A. E., Snijder, E. J., 1995 Processing and evolution of the N-terminal region of the Arterivirus replicase ORF1a protein: Identification of two papainlike cysteine proteases. J.Virol 69, 4500-4505.

3. Fang, Y., Snijder, E. J., 2010 The PRRSV replicase: Exploring the multifunctionality of an intriguing set of nonstructural proteins. Virus Res 154, 61-76.

4. Han, J., Rutherford, M. S., Faaberg, K. S., 2009 The porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities. J.Virol 83, 9449-9463.

5. Hopper, S. A., White, M. E., Twiddy, N., 1992 An outbreak of blue-eared pig disease (porcine reproductive and respiratory syndrome) in four pig herds in Great Britain. Vet.Rec 131, 140-144.

6. Johnson, C. R., Yu, W., Murtaugh, M. P., 2007 Cross-reactive antibody responses to nsp1 and nsp2 of Porcine reproductive and respiratory syndrome virus. J.Gen.Virol 88, 1184-1195.

7. Meulenberg, J. J., Bende, R. J., Pol, J. M., Wensvoort, G., Moormann, R. J., 1995 Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies. Clin.Diagn.Lab.Immunol 2, 652-656.

8. White, M. E. C., 1992 The clinical signs and symptoms of blue-eared pig disease (PRRS). Pig.Vet.J 28, 62-68.

This work was supported by the Internal Grant Agency of VFU Brno no.: 235/2009/FVL, Grant agency of the Czech Republic, project no. 524/09/0673 and by CEITEC (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.


Poster presentation

EXPRESSION AND SEROLOGICAL REACTIVITY OF NUCLEOCAPSID N AND NSP7 PROTEINS OF PRRS

GENOTYPE I VIRUS

Jitka Janková1, Dana Lobová1, Vladimír Celer1,2

1) Institute of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1/3, 612 42 Brno,

Czech Republic; [email protected] 2) CEITEC – Central European Institute of Technology, University of Veterinary and

Pharmaceutical Sciences Brno Abstract

The aim of this work was to express recombinant structural N and nonstructural Nsp7 proteins of Czech field strain (genotype I) of PRRS virus and to compare their diagnostic sensitivity and specificity. Another goal was to analyze the use of Nsp7 antigen for the differentiation of post-infection and post-vaccination antibodies in pigs vaccinated with an inactivated vaccine.

ORF 7 and Nsp7 genes were cloned into pDest17 vector. The expression of N and Nsp7 proteins was tested in different Escherichia coli strains. Resulting recombinant proteins were purified by IMAC using a polyhistidine tag under denaturing conditions from the insoluble fraction of bacterial lysate. Purified N and Nsp7 proteins were applied as antigens in indirect ELISA tests. Serological reactivity of both proteins was assessed on a panel of 274 swine sera separated to three groups: 1) 44 sera from non-vaccinated herds free of PRRS infection for calculation the cut-off value, 2) 44 PRRS-negative pigs vaccinated by the inactivated vaccine and 3) 186 serum samples from PRRS positive farms for a comparison of both ELISA tests.

Both antigens proved to be suitable for serological detection of PRRS specific antibodies, showing diagnostic specificity of 95.6 %, and sensitivity of 90.5 % for N protein ELISA test and 85.7 % specificity and 97.2 % sensitivity for Nsp7 ELISA test. Nsp7 antigen proved to be suitable for differentiation of post-infection and post-vaccination antibodies. Sera from PRRS free herds vaccinated by inactivated vaccine were compared with N and Nsp7 based ELISA tests. Although 100 % of sera gave positive results in N protein based ELISA test, these sera were tested negative with Nsp7 antigen. Keywords: pig, serology, vaccination, infection, non-structural protein This work was supported by the Internal Grant Agency of VFU Brno no.: 235/2009/FVL, Grant agency of the Czech Republic, project no. 524/09/0673 and by CEITEC (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.


Poster presentation

PRESENCE OF ANTIBODIES AGAINST PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IN FARM SWINES IN EASTERN PART OF CROATIA FROM

2009 TO 2010

Andreja Jungic, Besi Roic

Croatian veterinary institute, Laboratory for serological diagnostics of viral diseases,

Savska cesta 143, 1000 Zagreb, Croatia. Introduction: Porcine reproductive and respiratory syndrome (PRRS) is one of the economically most important swine diseases worldwide. In Croatia PRRS has been reported in 1996 [1,2] and during the last decade has caused great economic losses to Croatia’s domestic pig production. The aim of this study was to investigate the distribution of PRRS in domestic pig population in three Croatian counties with highest pig production from 2009 to 2010 Material and methods: In the period from 2009 to 2010 a total of 1664 blood samples were collected from animals of different age. They were raised on farms with high biosecurity practices, located in a density swine populated area in eastern part of Croatia. All sera were tested using commercial ELISA test (HerdCheck PRRSV X3, IDEXX Laboratories, Westbrook, ME, USA) according to manufacturer’s instructions. Results: The results of our investigation showed that all samples tested in 2009 (n=709) were negative, while in 2010 (n=955) only 1.04% of the tested samples reacted positive for PRRSV antibodies. This low prevalence was detected among the group of sows and fatteners. Conclusion: The obtained results demonstrated that PRRSV infection in pig population in eastern part of Croatia is more sporadic and a low seroprevalence suggests no significant influence of PRRS in pig production units of that area. However, PRRS still remains a problem in domestic pig production. Key words: PRRS, Croatia, ELISA References: 1. Lipej et al (1997): Veterinarski arhiv 67, 19-21. 2. Roic et al. (1997): 1st Croatian Vet. Congress, 197-202.


Session 2

Epidemiology of PRRS infection Chairs: Tomasz Stadajek, Balka Gyula


Tracing and Tracking the Molecular Epidemiology and Evolution

of PRRSV

1Manreetpal Singh Brar*, 1Mang Shi, 1Charles Lai-Yin Wong, 2Susy Carman, 3Michael P. Murtaugh, 1Frederick Chi-Ching Leung

1School of Biological Sciences, The University of Hong Kong, Hong Kong S.A.R. 2 University of Guelph, Laboratory Services Division, Animal Health Laboratory, Box

3612, Guelph, ON, N1H 6R8, Canada. 3Department of Veterinary and Biomedical Sciences, St. Paul, University of Minnesota,

MN, USA Abstract Since the recognized advent of the novel swine pathogen termed porcine reproductive and respiratory syndrome virus, towards the end of the 1980’s, both the global distribution of the virus and economic consequence has been widespread. Characterizing the known genetic diversity and keeping abreast of evolving diversity is vital in framing robust surveillance and control measures. To this end, significant progress has been made in developing a well-organized genotyping system based on a representative phylogenetic framework to classify all current and prospective type 2 viruses. Periodic refinement of this representative framework incorporating new diversity provides users an up-to-date backbone for examining their isolates in a global context. Such a methodology provides more accurate reflections of genetic relatedness than other typing means such as RFLP, serological typing, or protein size polymorphisms. Additionally, the use of phylogenetics provides empirical indications on the origins of epidemiologically significant events e.g. outbreaks, especially when traditional surveillance is uninformative as in the case of the MN184 outbreak. Important insights into the transmission dynamics and viral demographic histories can also be garnered. For example, an examination of viral gene flow in North America revealed the transmittance of viruses occurred on a highly biased scale from Canada to the United States. Reverse flow, however, occurred on a much reduced level. Validation for such inferences could be gauged from swine flow between the two countries which took place in a similar asymmetric manner. All the analyses performed on type 2 viruses in North America were made possible due to the intensive effort placed on sampling and sequencing of isolates together with the public availability of such sequence data. Similar in-depth analysis of type 1 virus transmission in Europe has been hampered by the availability of a much smaller volume of sequence data and one that is frequently not serially sampled. Despite this shortcoming, based on existing knowledge, type 1 virus diversity is still estimated to exceed that of type 2 which is in agreement with individual coalescence estimates for all viruses of each genotype. Current


understanding also indicates type 1 viruses to be established much longer in Eastern Europe than in countries west of the Polish border. And, it was from the circulation of viruses in Western Europe that a single introduction of type 1 genotype occurred into the United States followed by expansion in diversity and geographic distribution. Initiative has also been taken to develop a web-based software tool that would enable analyses of the sort mentioned above to be performed in a manner of ease. This would aid in overcoming the unfamiliarity veterinary or diagnostic researchers would have in performing such type of analyses. Furthermore, such a tool could also develop, upon the consensus of the research community, into a central clearing-house of PRRV related sequences. This would reduce inconvenience of searching not so well organized databases like GenBank. Lastly, the coming into existence of the proposed web-tool would help bring a degree of commonality and consistency in the methodology used when constructing phylogenies by different research groups. This will greatly improve the ability to compare various phylogenies from different researchers and / or time points rather than practices of using biased or arbitrary selection of sequences to accompany analysis of isolates of interest. Keywords: Phylogenetics, molecular epidemiology, genotyping, evolution, virus flow Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a relatively recent addition to the family Arteriviridae and order Nidovirales (Cavanagh, 1997). The virus is notorious for causing abortions in pregnant sows and respiratory-related symptoms in young piglets since its initial recognition back in the late 1980’s (Hill, 1990). The single-stranded, positive sense RNA genome stretches approximately 15 kb with overlapping open reading frames encoding both non-structural (ORF1a and ORF1ab) and structural proteins (ORF2 – 7) (Meulenberg et al., 1995; Ziebuhr et al., 2000). As is typical of RNA viruses, the lack of proofreading capability during genome replication and a relatively high substitution rate contribute to the rapid evolution of the virus. The high diversity at both inter-genotype and intra-genotype levels bear testimony to this statement (Shi et al., 2010a; Shi et al., 2010b). Genotyping PRRSV A host of typing methods for PRRSV have been both proposed and utilized in assessing genetic similarity or relatedness. Some of which include restriction fragment length polymorphism (RFLP) (Cheon and Chae, 2000; Itou et al., 2001), ORF size polymorphisms (Stadejek et al., 2008), and serotyping (Nelson et al.,


1993). The strength of these typing methods rely on simplicity in concept, familiarity for practice, and prevalence in use. However, a major shortcoming of these methods is that they cannot provide a high resolution indication of the evolutionary relationship between strains of high genetic similarity. The general availability and relatively inexpensive cost of DNA sequencing has enabled the widespread use of this technology in obtaining the sequence information of various ORFs of PRRSV isolates, especially ORF5. As a result, a large accumulation of such data has occurred particularly with respect to the type 2 genotype. However, an effort to establish an efficient and informative classification system was lacking for quite some time. In this respect, our initial work entailed organizing and setting up a phylogenetic classification method for type 2 viruses based on existing and publically available sequence data at the time (Shi et al., 2010b). The use of this framework and its revision for updating occurred in our subsequent work pertaining to a large number of serially sampled isolates from Canada (Brar et al., 2011). The utilization of the typing system devised provides a more high resolution indication of genetic relationships of a researcher’s isolate(s) of interest in a context of global diversity compared to other typing methods. Furthermore, phylogeny-based analyses can offer insights into the evolutionary origins of epidemiologically important incidents as in the case of outbreaks, fluctuations in population dynamics, and inter-regional virus flow. A pertinent example of this is the high virus flow estimated to be disseminated into the different swine production regions of the USA from Canada whereas virus flow in the opposite direction occurred on a reduced scale. This phenomenon mirrors the pattern of quantitative swine flow between the North American neighbours. Type 1 PRRSV Although our efforts have also entailed performing similar type of work for type 1 PRRSV as well (Shi et al., 2010a), the improvement of the phylogenetic framework to one that is accurately aligned with actual field diversity, together with characterization of inter-country virus transmission in Europe, has been hampered by the limited sequence data available openly. Nonetheless, from what is available, it is apparent that type 1 diversity is no less diverse than its counterpart – genotype 2. Though sampled later on, isolates from Eastern Europe were diverged much earlier than those from the Lelystad Virus-like cluster. This implies the circulation of PRRSV to the East of the Polish border to pre-date the originally recognized outbreak in Europe. The impact of type 1 PRRSV on the United States swine industry was the result of a single introduction event that occurred from Western Europe. The multisite pig production system and the frequent transportation witin this network has helped disperse type 1 viruses geographically within the United States as was typical in the case of type 2 strains. Conclusions


The rapidly evolving nature of PRRSV requires an equally dynamic system which can keep abreast of changing diversity and present researchers with accurate and informative insights. The knowledge of which can serve purposes of revising or strengthening surveillance measures or generating vaccines efficacious against emerging viral diversity. While the development of software tools that enable user-friendly performance of phylogenetic analyses are in the pipeline, the actual success or value of such tools hinges on individual initiatives to share sequence data. Only then can progress be made in advancing towards a holistic perspective of PRRSV molecular epidemiology and evolution. References Brar, M.S., Shi, M., Ge, L., Carman, S., Murtaugh, M.P., Leung, F.C.C., 2011. Porcine reproductive and respiratory syndrome virus in Ontario, Canada 1999 to 2010: genetic diversity and restriction fragment length polymorphisms. Journal of General Virology 92, 1391. Cavanagh, D., 1997. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Archives of virology 142, 629. Cheon, D.S., Chae, C., 2000. Restriction fragment length polymorphism analysis of open reading frame 5 gene of porcine reproductive and respiratory syndrome virus isolates in Korea. Archives of virology 145, 1481-1488. Hill, H., 1990. Overview and history of mystery swine disease (swine infertility/respiratory syndrome). PrOf Mystery Swine Dis Committee, 29-31. Itou, T., Tazoe, M., Nakane, T., Miura, Y., Sakai, T., 2001. Analysis of open reading frame 5 in Japanese porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism. Journal of veterinary medical science 63, 1203-1207. Meulenberg, J.J.M., Petersen-Den Besten, A., De Kluyver, E.P., Moormann, R.J.M., Schaaper, W.M.M., Wensvoort, G., 1995. Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology 206, 155-163. Nelson, E., Christopher-Hennings, J., Drew, T., Wensvoort, G., Collins, J., Benfield, D., 1993. Differentiation of US and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. Journal of clinical microbiology 31, 3184. Shi, M., Lam, T.T.Y., Hon, C.C., Hui, R.K.H., Faaberg, K.S., Wennblom, T., Murtaugh, M.P., Stadejek, T., Leung, F.C.C., 2010a. Molecular epidemiology of PRRSV: a phylogenetic perspective. Virus Research. Shi, M., Lam, T.T.Y., Hon, C.C., Murtaugh, M.P., Davies, P.R., Hui, R.K.H., Li, J., Wong, L.T.W., Yip, C.W., Jiang, J.W., 2010b. Phylogeny-based evolutionary, demographical, and geographical dissection of North American type 2 porcine reproductive and respiratory syndrome viruses. J Virol 84, 8700. Stadejek, T., Oleksiewicz, M., Scherbakov, A., Timina, A., Krabbe, J., Chabros, K., Potapchuk, D., 2008. Definition of subtypes in the European genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in Europe. Archives of virology 153, 1479-1488. Ziebuhr, J., Snijder, E.J., Gorbalenya, A.E., 2000. Virus-encoded proteinases and proteolytic processing in the Nidovirales. Journal of general virology 81, 853.


GENETIC ANALYSIS OF PRRSV ISOLATES FROM PIG

FARMS IN SLOVAKIA

Stefan Vilcek, Anna Jackova, Valeria Leskova, Michaela Vlasakova University of Veterinary Medicine and Pharmacy, Kosice, Komensky 73, Slovakia ([email protected]) Abstract

Genetic typing of PRRSV isolates in ORF5 and ORF7 originating from pig farms in Slovakia revealed that most viruses (n=11) belong to EU genotype, subtype 1. One isolate was typed as NA genotype. While typical length of ORF7 (128 aa) of EU-1 isolates was determined in 8 isolates, two isolates originating from the same farm showed unusual length polymorphism - 132 amino acids, which was not observed so far.

Keywords: PRRSV, typing, ORF7 length polymorphism Introduction At present, two genotypes of PRRSV, European (EU, Type 1) and North American (NA, type 2) genotype were recognised (Suarez et al., 1996; Le Gall et al., 1998; Allende et al., 1999). Both genotypes are genetically and antigenically highly diverse (Kapur et al., 1996, Forsberg et al., 2002). New nucleotide sequences from PRRSV isolates originating from Poland, Lithuania, Belarus and Russia enlarged knowledge on genetic diversity of PRRSV (Stadejek et al., 2002; 2006; 2008). As result of this analysis, it has been proposed division of EU genotype into three subtypes: pan-European subtype 1 and East European subtypes 2 and 3 (Stadejek et al., 2008). The nucleotide sequences of PRRSV isolates circulating in Slovakia have not been reported yet. This study was focused on the detection and analysis of PRRSV isolates originating from Slovak pig farm at the genetic level.

Material and Methods Twelve viral samples (lymph nodes) originating from PRRS suspected pigs housed in five pig farms in south-western Slovakia collected in period 2007-2009 were analyzed at the genetic level. A 432 bp fragment of ORF5 (Oleksiewicz et al., 1998) and the complete ORF7 regions (Oleksiewicz et al., 1998; Drew et al., 1997) were amplified using RT-PCR. PCR products were sequenced in both directions and aligned with PRRSV nucleotide sequences deposited in GenBank.


The sequences were analyzed and viral isolates were typed by phylogenetic analysis (NJ method from Dnastar and Phylip). Results

Typing of viral isolates in ORF5 and ORF7 The obtained nucleotide sequences from ORF5 (432 nt) and entire ORF7 were aligned and compared to each other as well as to the reference strain Lelystad virus and to another isolates of EU genotype, subtype 1, 2, 3 (EU-1, EU-2, EU-3) and NA genotype selected from the GenBank. The phylogenetic analysis based on nucleotide sequences of ORF5 revealed that PRRSV isolates were typed as EU genotype, subtype 1 (EU-1) according to the classification by Stadejek et al., 2006. The isolates originating from the same farm were clustered in the same phylogenetic cluster and had the same or very similar (around 99%) nucleotide sequence. However one isolate, namely36M, grouped into separated branch. Isolate 36M created cluster together with isolates IAF-EXP 91, IAF 93-653, VR 2332 etc., representing NA genotype. This isolate was very similar to Quebec reference strain IAF-EXP91 (91 % nucleotide identity in ORF5). The phylogenetic analysis carried out in entire ORF7 region confirmed phylogenetic analysis in ORF5, e.g. viral isolates belonged mostly to EU genotype, subtype 1. Unusual length polymorphism of ORF7 for EU-1 isolates The nucleotide sequences of most viral isolates showed similar length of ORF7 - 387 nt (128 aa and stop codon). This size of nucleocapsid protein is typical for all Western European strains of EU genotype, subtype 1. Surprisingly, the exceptional size (132 aa) of nucleocapsid protein was revealed in two identical Slovak isolates – 28M and 29M originating from the same farm. This size of ORF7 was not described in scientific literature for any PRRSV isolate so far.

Conclusions Results of this study provide new information on PRRSV isolates from Slovakia. The isolates were typed as classical EU genotype, subtype 1 except one isolate belonging to NA genotype. The finding of new length polymorphism of ORF7 (132 aa) extend our knowledge on diversity of PRRSV isolates in Europe.

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MOLECULAR DETECTION AND GENETIC ANALYSIS OF SERBIAN PRRSV ISOLATES

Petrovic T.1, Milicevic V.2, Radulovic-Prodanov J.1, Maksimovic-Zoric J.2,

Lupulovic D.1, Dosen R.1, Lazic S.1

1. Scientific Veterinary Institute „Novi Sad“, Novi Sad, Serbia

2. Scientific Veterinary Institute of Serbia, Belgrade, Serbia Abstract The PRRS is highly economically important disease in pig production worldwide. The first introduction of PRRS in Serbia was suspected to be in 2001 and was manifested by massive respiratory disorders with high mortality in pigs on 2 big industrial farms located on the north part of the country. The presence of disease in Serbia is detected and monitor by serological commercially available ELISA tests and by virus detection with RT-PCR method. From 2008-2010, tissue samples (lungs, limphonodes) from the total of 42 PRRS suspected animals were collected for virus detection. Total RNA was extracted from 10% tissue homogenates by TRIreagent (Adiagene) and the amplification 304 bp long nucleotide sequence of PRRSV ORF7 was conducted using Access RT-PCR System and with primers described by Shin et al, 1997. The amplified RT-PCR fragments were commercially sequenced by Macrogen Inc., Seoul, Korea and the phylogenetic analyses of the PRRS isolates were conducted using MEGA version 4. From 42 analysed samples 21 were found as RT-PCR positive. The positive samples were found in all examined regions and in 14 of 20 examined farms. From 21 detected, only 18 RT-PCR positive samples were sequenced. In addition to the ORF7 248 nucleotides long sequences of PRRS isolates obtained from Serbia, a corresponding nucleotide sequence of 27 PRRS viruses published in GenBank, representing both virus genotypes and all recently proposed genotype 1 subtypes, were included in phylogenetic comparison. Phylogenetic analysis revealed that all 18 sequenced Serbian PRRSV isolates belongs to type 1 (former EU genotype) and that are clustered in a few different clusters in pan-European subtype 1. US strains as well as new eastern European strains were not found. The obtained results suggest that more study has to be done regarding the introduction of more and different RT-PCR procedures for obtaining high specificity and sensitivity of detection procedures as well as more PRRS isolates has to be sequenced and characterized to get the real picture on PRRS viruses that circulating in Serbia. Keywords: PRRSV isolates, typing ORF7, Serbia


Introduction: Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide (Lunney et al., 2010). The aetiological agent of PRRS is an RNA virus of the order Nidovirales, family Arteriviridae, and genus Arterivirus. There are two related but antigenically and genetically distinguishable strains: genotype 1, with the prototype Lelystad virus representing the viruses predominating in Europe and genotype 2, represented by VR 2332, the prototype of strains originally mostly found in North America (Snijder et al., 2004). A variant of genotype 2 is the cause of severe disease in Asia. Nucleotide sequences of PRRSV isolates from Spain and Italy as well as recently characterized PRRS isolates from eastern European countries as Lithuania, Belarus and Russia reveal high genetic diversity among European PRRSV isolates (Forsberg et al., 2002; Stadejek et al., 2006; 2008). PRRS was first recognized in North America during 1980s and spread rapidly throughout the world. In Europe, a similar disease caused by a distinct genotype of the virus also spread rapidly in that region during 1990s. The disease is now present throughout the world and in most of swine producing countries it has endemic character. The first data about the PRRS presence in Serbia are from 2001. The presence of the disease was for the first time suspected on the bases of clinical signs - massive respiratory disorders with high mortality in pigs on 2 big industrial farms located on the north part of the Serbia (Vojvodina province), 10 – 30 km from Croatia and Hungarian border. Later on, the presence of the disease on mentioned farms was confirmed by serology test (ELISA). PRRS virus was not isolated or confirmed at that time (Lazić et al., 2003). It was suspected that the virus was introduced by boar’s semen that was illegally obtained from neighbouring countries. During that and following year the respiratory syndrome with high morbidity and moderate mortality, that was clinically diagnosed as PRRS, was observed on many big industrial farms in Vojvodina province and latter on in central Serbia (Došen et al., 2003). The laboratory diagnosis of PRRSV in Serbia is mainly performed by serology testing with commercial ELISA tests. The virus detection is done by molecular testing. Usually, just a limited number of samples from clinical cases have been sent for virus confirmation (Lazić and Petrović, 2007). In this paper, the first study on molecular detection and genetic diversity of Serbian PRRS isolates is presented. Materials and methods Samples Tissue samples (lungs, limphonodes) from the total of 42 moribund and succumbed animals were collected in clinically and/or pathologically suspected


cases from 18 farms during 2008, 2009 and 2010. Most of those farms are big pig production systems located on northern and four of them in central part of Serbia. The autopsy, samples preparation and molecular detection and characterization were performed in the Scientific Veterinary Institute ”Novi Sad” and in Scientific Veterinary Institute of Serbia, Belgrade. RNA extraction and RT-PCR Total RNA was extracted directly from 250 µl of 10% suspension of tissues samples (lungs and limphonodes) in PBS from dead and euthanized moribund animals. RNA extraction was performed using TRIreagent (Adiagen) according to the manufacturer recommendations. A highly conserved sequence of 304 bp within ORF7 of the PRRSV RNA was amplified by forward and reverse primers ORF7-1 (5ʹ′-ATG GCC AGC CAG TCA ATC A-3') and ORF7-2 (5ʹ′-CGG ATC AGG CGC ACA GTA TG-3ʹ′) described by Shin et al, 1997. Amplification was performed by one-step RT-PCR using Access RT-PCR system (Promega, USA) according to the manufacturer recommendations. Sequencing of partial ORF7 and phylogenetic analysis PCR products of 304 nucleotides of PRRS isolates ORF7 genome part were used as templates in cycle sequencing reactions primed with the same ORF7-1 and ORF7-2 primers used in detection. The amplified RT-PCR fragments were commercially sequenced by Macrogen Inc., Seoul, Korea. Phylogenetic analyses of the PRRS isolates were conducted using MEGA version 4 (Tamura, Dudley, Nei and Kumar, 2007). Individual sequence homology search was made via the National Centre for Biotechnology Information (NCBI) using BLAST network service (http://www.ncbi.nlm.nih.gov). The 248 bp long ORF7 nucleotide sequences of PRRS isolates were aligned by Clustal W program together with published sequences in NCBI GeneBank. The phylogenetic tree was constructed using neighbor – joining method based on bootstrap of 1000 replicates. Results and Discussion From 42 analysed samples 21 were found as RT-PCR positive. The positive samples were found in all examined regions and in 14 of 20 examined farms. From 21 detected, only 18 RT-PCR positive samples were conducted for sequencing. The geographical distribution of sequenced PRRS isolates is shown in Figure 1. In addition to the ORF7 248 nucleotides long sequences of PRRS isolates obtained from Serbia, a corresponding nucleotide sequence of 27 PRRS viruses published in GenBank were included in phylogenetic comparison. Among 27 included PRRS virus sequences from GeneBank are the representatives of both virus genotypes and all recently proposed genotype 1 subtypes (regarding to Stadejek et al., 2006 and Stadejek et al., 2008) that were found until now.


Fig. 1: Geographical location of 18 sequenced PRRS virus isolates from Serbia Phylogenetic analysis based on nucleotide sequences of ORF7 revealed that all 18 sequenced Serbian PRRSV isolates belongs to type 1 (former EU genotype) and that are clustered in a few different clusters in subtype 1 (EU-1). US strain was not found between 18 sequenced isolates (Figure 2). Also, neither of the characterized Serbian PRRS virus isolates was similar to the recently characterized eastern European type 1 subtypes. The nucleotide homologies that are found for Serbian isolates compared with the sequences published in Genbank was most similar to Danish, French, Hungarian and Dutch strains and ranged depending of the isolate from 92-95%. The obtained results suggest that PRRS virus infection is widely distributed in Serbia. Many of the big pig production farms were found to be positive. Phylogenetic analysis reviled that all 18 genetically typed isolates belongs to the EU subtype 1 or Lelystad type viruses that are distributed globally in Europe as well as in the other parts of the world. This result was expected regarding the results published from surrounding and other EU countries (Andreyev et al., 2000; Schmoll et al., 2002; Stadejek det al., 2006; Stadejek et al., 2008).


Fig. 2: Phylogenetic tree based on partial ORF7 region nucleotide sequences (248bp) of 18 Serbian PRRSV isolates and strains reported in Genebank, generated by neighbor-joining method (using MEGA version 4) Also, these results are in line with live pig trade between Serbia and other mostly western European and some surrounding countries. Trade with eastern European countries was not done, especially in the recent past or even if it is done it is just


few cases. It could be a reason that eastern European subtypes of genotype 1 PRRS virus was not detected in Serbia. Another reason is that only 18 PRRS isolates was genetically characterized, so if the eastern European strains are present and if they are present in low prevalence, it is possible that they are not detected regarding the small number of analysed isolates in Serbia. The same conclusion could be directed to the American genotype 2 PRRS virus strains that were also not find between analysed PRRSV isolates in Serbia. In some cases the PRRSV was not detected by RT-PCR in clinically and pathologically highly suspected cases possibly due to specificity and sensitivity of the used laboratory test. As just one RT-PCR protocol was used in detection, it is possible that some divergent PRRSV strains were not detected during the study. This could be the third reason why more divergent virus subtypes strains, like eastern European, were not detected in our study. For that reason, in our labs from 2011 many other RT-PCR and real time RT-PCR procedures are introduced in PRRSV detection. The obtained results suggest that more study has to be done regarding the introduction of more and different RT-PCR procedures for obtaining adequate specificity and sensitivity of detection procedures as well as more PRRS isolates has to be sequenced and characterized to get the real picture on PRRS viruses that circulating in Serbia. Acknowledgement This work is part of the research done in the project TR31084 funded by the Ministry of education and science of Republic of Serbia. References

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9. Snijder E.J., Brinton M.A., Faaberg K.S., Godeny E.K., Gorbalenya A.E., MacLachlan N.J., Mengeling W.L. Plagemann P.G.W. (2004): Family Arteriviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomyof Viruses. Edited by: C. M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger and L.A. Ball. London: Elsevier/Academic Press.

10. Stadejek T., Oleksiewicz M.B., Potapchuk D., Podgorska K. (2006): Porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern Europe support the definition of new genetic subtypes. Journal of General Virology 87, 1835 – 1841.

11. Stadejek T., Oleksiewicz M.B., Scherbakov A.V, Timina A.M., Krabbe J.S., Chabros K., Potapchuk D. (2008): Definition of subtypes in the European genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in Europe. Archiv of Virology 153, 1479-1488.

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PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

(PRRSV) INFECTION IN LITHUANIAN WILD BORS (SUS SCROFA) POPULATION

Arunas Stankevicius1, Jurate Buitkuviene1, Jurgita Valanciute1, Rytis Cepulis1,

Tomasz Stadejek2

1Faculty of Veterinary Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania; e-mail:[email protected] 2National Veterinary Research Institute, Pulawy, Poland Abstract Since domestic pigs and wild boars have the same susceptibility to various infections there was major concern to monitor the epidemiological PRRSV situation in feral pigs and to characterize genetically the detected strains. From 659 examined wild boar sera, 43 (6.5 %) were positive to PRRSV antibodies. Antibodies to PRRSV were detected in all age groups, however seroprevalence was significant higher in adults. Wild boars serum samples from 31 locations out of 42 investigated were seropositive for PRRSV. Samples of 13 (8.2%) wild boars tested PRRSV-positive in genotype 1 specific nPCR. No positive results were obtained in genotype 2 specific nPCR. The sequencing and genetic comparison of the selected ORF5 amplicons revealed that these wild boar sequences belonged to two genetic subtypes 3 and 4. The new sequences formed well defined clusters within these subtypes. Interestingly such strains were never found in domestic pigs in Lithuania. Subtype 3 viruses are common in Belarus and subtype 4 was found in two Belarusian and two Latvian farms. This study also has for the first time demonstrated presence of PRRSV in Eastern European wild boars. It has shown that wild boar population can harbour different genetic lineages of PRRSV strains than those found in domestic pigs in Lithuania. The most striking finding is detection of subtype 4 strains in 5 wild boars. Previously this subtype was detected only in Belarus and Latvia. Altogether, these findings are strongly supporting the role of wild boars as a natural reservoir for PRRSV. Keywords: PRRSV, wild boar, seroprevalence, phylogenetic analyses Introduction PRRSV is endemic in most swine-producing countries, and today it is associated with major economic losses. PRRSV strains are divided into two genotypes based on genetic and antigenic characteristics: genotype 1 (formerly European) and genotype 2 (formerly North American) (10). Within the genotype 1, several Eastern European genetic subtypes were defined (11, 12). Antibodies to PRRSV have been found in pigs almost worldwide. The majority pig farms in Lithuania


are also positive for antibodies to PRRSV. Although PRRSV is widespread in domestic swine, very little is known about PRRSV infection in European wild boar. However, comprehensive information on PRRSV infection in wild boars was published only from Germany (7). Wild boars have been found also seropositive to PRRSV in France (1), USA (9), Italy (5), and Croatia (8). Recently PRRSV was identified in hybrid wild boars, also known as special wild pigs in China (14). Thus, most likely, wild boars become infected by domestic swine as a result of seldom direct or indirect contacts. On the other hand, wild boars can act as a reservoir for infectious diseases of domestic pigs and interactions between these two populations can potentially result in the dissemination of these diseases (15). Since domestic pigs and wild boars have the same susceptibility to various infections there was major concern to monitor the epidemiological PRRSV situation in feral pigs and to characterize genetically the detected strains. Materials and methods A total of 659 serum samples from wild boars from 42 locations throughout Lithuania were collected during autumn–winter hunting seasons 2008/2009 and 2009/20010. The wild boars sera were analyzed via different producers ELISA test systems: IDEXX PRRS 2XR Ab, IDEXX HERDCHEK* PRRS X3 antibody test kits (Corporate Headquarters IDEXX Laboratories, Inc., USA), Ingezim PRRS Europa (Ingenasa, Madrid, Spain) and others, according to manufacturer’s instructions and in the ISO/IEC 17025:2005 standard accredited laboratory. For genetic characterisation samples of blood sera and lungs of 159 wild boars were collected from 15 hunting grounds situated in 5 regions of Lithuania during autumn-winter hunting seasons from 2007 to 2011. Total RNA was extracted from homogenate of tissue or serum samples using the GeneJET RNA Purification kit (Fermentas). It was used as template in reverse transcription nested PCR specific for ORF5 of genotypes 1 and 2 PRRSV as described previously (11, 12). Gel-purified 606 bp ORF5 PCR products were cycle sequenced using the BigDye Terminator Cycle Sequencing kit (v2.0, Applied Biosystems) and ABI310 genetic analyzer. Sequence alignment was performed using the Clustal W software. A Phylogenetic tree was constructed with MegAligh program from Lasergene program package. Results From 659 examined wild boar sera, 43 (6.5 %) were positive to PRRSV antibodies. Investigation of PRRSV antibodies with different ELISA kits did not show difference in detection positive serum samples (p>0.05). The results of serological analysis are summarized in Table 1.


Table 1. The results of detection PRRSV antibodies in wild boars samples

Antibodies to PRRSV were detected in all age groups, however seroprevalence was significant higher in adults (Table 2). Wild boars serum samples from 31 locations out of 42 investigated were seropositive for PRRSV.

Table 2. Prevalence of PRRSV antibodies in wild boars serum by age groups

Samples of 13 (8.2%) wild boars tested PRRSV-positive in genotype 1 specific nPCR. No positive results were obtained in genotype 2 specific nPCR. The sequencing and genetic comparison of the selected amplicons revealed that these wild boar sequences belonged to two genetic subtypes 3 and 4. The new sequences formed well defined clusters within these subtypes (Fig.1). Interestingly such strains were never found in domestic pigs in Lithuania. Subtype 3 viruses are common in Belarus and subtype 4 was found in two Belarusian and two Latvian farms.


Figure 1. A Phylogenetic tree based on ORF5 nucleotide sequences. A set of sequences (11) representing the full genetic diversity of genotype 1 PRRSV was used as a reference. Conclusions In spite of the fact that PRRSV is actively circulating in domestic swine of Lithuania, the seroprevalence in wild boars was only 6.5 %. This result indicated very low possibility to contact wild boars and domestic swine, which could present opportunity for PRRSV transmission. Similar results of PRRSV seroprevalence (8.92%) in feral pigs were reported in Croatia (8). However in reports from Italy (5) the prevalence (37.7%) of PRRSV antibodies was quite high and it could be due to PRRSV transmission from domestic pigs to wild boars.


Interestingly, in neighboring countries such as Russia or Poland PRRSV antibodies in feral pigs were not detected (4). This study has for the first time demonstrated presence of PRRSV in Eastern European wild boars. It has shown that wild boar population can harbour different genetic lineages of PRRSV strains than those found in domestic pigs in Lithuania (13). This poses a serious threat for Lithuanian farms where only subtype 2 strains are circulating. Recent studies showed that subtype 3 strains may be highly virulent (3). The most striking finding is detection of subtype 4 strains in 5 wild boars. Previously this subtype was detected only in Belarus and Latvia (11, 13). Altogether, these findings are strongly supporting the role of wild boars as a natural reservoir for PRRSV. Acknowledgement This work was funded by Project No. MIP-48/2010 of Research Council of Lithuania. References

1. Albina et al. (2000). A serological survey on classical swine fever (CSF), Aujeszky’s disease (AD) and porcine reproductive and respiratory síndrome (PRRS) virus infections in French wild boars from 1991 to 1998. Vet Microbiol 77, 43-57.

2. Bonilauri et al. (2006). Presence of PRRSV in wild boar in Italy. Vet Rec 158, 107-108.

3. Karniychuk et al. (2010). Pathogenesis and antigenic characterization of a new East European subtype 3 porcine reproductive and respiratory syndrome virus isolate. BMC Vet Res 6:30.

4. Kukushkin et al. (2008). Investigation of wild boar (Sus scrofa) for porcine reproductive and respiratory síndrome in some territories of Russia. Eur J Wildl Res 54, 515-518.

5. Montagnaro et al. (2010). Prevalence of antibodies to selected viral and bacterial pathogens in wild boar (Sus scrofa) in Compania region, Italy. J Wildl Dis 46, 316-319.

6. Oslage et al. (1994). Prevalence of antibodies against the viruses of European swine fever, Aujeszky’s disease and porcine resiratory reproductive síndrome in wild boars in the federal states Sachsen-Anhalt and Branderburg. Dtsch Tierarztl Wochenschr, 101, 33-38.

7. Reiner et al. (2009). Porcine reproductive and respiratory syndrome virus (PRRSV) infection in wild boars. Vet Microbiol 136, 250-258.

8. Roic et al. (2008). Serological evidence of reproductive and respiratory síndrome virus (PRRSV) in wild boars (Sus scrofa) in Croatia. In IPVS Congr, Durban, 29.

9. Saliki et al. (1998). Serosurvey of selected viral and bacterial diseases in wild swine from Oklahoma, J Wildl Dis 34, 834-838.

10. Shi et al. (2010). Molecular epidemiology of PRRSV : A phylogenetic perspective. Vir Res 154, 7-17.

11. Stadejek et al. (2006). Porcine reproductive and respiratory syndrome virus strains of exceptional diversity in Eastern Europe support the definition of new genetic subtypes. J Gen Virol 87,1835-1841.


12. Stadejek et al. (2008). Definition of subtypes in the European genotype of porcine reproductive and respiratory syndrome virus:nucleocapsid characteristics and geographical distribution in Europe. Arch Virolo 153, 1479-1488.

13. Stankevicius et al. (2007). Exceptional diversity of PRRSV strains in Latvia and Lithuania. In 5 th Int Symp on Emerg and Re-emerg Pig Dis, Krakow, Poland, 178.

14. Wu et al. (2011). Porcine reproductive and resiratory síndrome in hybrid wild boars, China. Emer Infec Dis 17, No 6, 1071-1073.

15. Wyckoff et al. (2009). Feral swine contact with domestic swine: a serologic survey and assessment of potential for disease transmission. J Wildl Dis 45, 422-429.


PRRSV OUTBREAK WITH HIGH MORTALITY IN

NORTHERN PART OF DENMARK

Lise Kvisgaard1, Charlotte Hjulsager1, PH Rathkjen2, Solvej Breum1, Ramona Trebbien1, Lars E Larsen1.

1National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.

2Boehringer Ingelheim Danmark A/S, Copenhagen, Denmark. Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the Arteriviridae family and is the cause of significant respiratory and reproductive disease in swine worldwide. Strains of PRRSV are divided into two genotypes: type 1 and type 2, also referred to as EU and US type, respectively, due to their geographical origin. In Denmark the type 1 virus was first recognized in 1992, and since 1996 both types of PRRSV are widely spread. Approximately 50 % of the herds are seropositive for PRRSV antibodies against either or both types of PRRSV. In November 2010, a severe case of PRRSV with high mortality rate in piglets occurred in Northern Jutland. PRRSV type 2 was detected by real-time RT-PCR in lung tissue from 10 days old piglets. The outbreak was treated by extensive vaccination with Ingelvac® PRRS MLV and strict management procedures. 6 weeks later, the mortality of live born piglets had dropped to normal levels. From week 6 until week 14 after the initial outbreak, up to 75 % of fetuses were born as mummified. PCV2 and PPV have not been detected in the fetuses. 15 weeks after the initial outbreak, the number of liveborn piglets and the mortality until weaning was back to normal. Total losses of piglets until weaning for the 15 week period were about 50 %. Losses in the nursery and finisher barn are still substantial 15 weeks after the initial outbreak. Sequencing of ORF5 and ORF7 confirmed the type of PRRSV to be type 2, and revealed distinct nucleotide differences compared to other Danish PRRSV type 2 sequences in the ORF5 region. Additional sequencing of partial nsp2 further suggests the high diversity of this type 2 strain. We speculate that the virus causing this outbreak is more pathogenic than previously recognized Danish PRRSV type 2 strains. Keywords: PRRSV; NSP2; virulence; epidemiology


Session 3

Latest developments in PRRS research: virus – host interaction and host genetics

Chairs: Enric Mateu, Tahar Ait-Ali


Recent advances in PRRS immunology: innate responses and development of regulatory T cells

E. Mateu1,2, L. Darwich1,2, I. Diaz2, M. Gimeno1,2, L. Kutzemseva2, J. Hernández3

1. Departament de Sanitat I Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 2. Centre de Recerca en Sanitat Animal (CRESA), IRTA-UAB, edicifi CReSA, campus UAB, 08193 Bellaterra, Spain. 3. Centro de Investigación en Alimentación y Desarrollo (CIAD) A.C. Carretera a la Victoria, Km 0,6. Hermosillo, Sonora, Mexico.

Abstract The immunobiology of porcine reproductive and respiratory syndrome

virus is unique based on the abnormal adaptive immune response that develops in infected pigs. The ultimate causes of this abnormal response are not known but it is thought that the origin could be traced back to the events of the innate response, particularly to the interaction of the virus with macrophages and dendritic cells. Also, the development of regulatory T-cells can be an important element in the immunopathogenesis of this infection. The present will review some of these aspects.

Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is one of

the major pathogens for pig industry worldwide. Twenty years after the emergence of this virus, the understanding of the mechanisms by which PRRSV causes disease and alter the functionality of the immune system is limited. Moreover, the role of neutralizing antibodies and cell-mediated immunity in protection are not well understood. In any case, the evidences point to the fact that the effects of PRRSV upon the immune system are profound and lead to an abnormal immune response. In the present paper some aspects of the immune response to PRRSV will be reviewed.

Target cells for PRRSV

Early studies (Pol et al., 1991; Wensvoort et al., 1991) showed that macrophages, particularly alveolar macrophages (AM), were one of the main targets for viral replication. The entrance of the virus in the macrophage is thought to be mediated by an initial attachment to heparin sulphate molecules in the surface of the macrophage and a subsequent interaction of viral GP5/M heterodimers with the porcine sialoadhesin (PoSn or CD169) through the sialic acid residues present in GP5 (van Breedam et al., 2010). The need for this interaction with PoSn to occur for an effective entry of the virion into the


macrophage explains why monocytes are not susceptible to infection with PRRSV. In the process of decapsidation a third co-receptor is need, CD163 (Calvert et al., 2007; van Gorp et al., 2008). Interestingly, CD163 is a scavenger receptor involved in the clearance of haptoglobin-hemoglobin complexes that is being increasingly involved in the innate responses to pathogens. Dendritic cells seem to be susceptible to PRRSV as well although this depends on the precise of DC examined. No other cell targets of relevance have been reported so far in the natural host, the pig.

The interplay between PRRSV and dendritic cells Although AM have been extensively used as a cell system for propagation

of PRRSV and as a representative target for in vivo or ex vivo studies; the role of these cells as antigen presenting cells (APC) is somewhat secondary compared to that of DCs. Actually, DCs are devoid of phagocytic abilities and are fully professional APCs considered to be the central element linking the innate and the adaptive immune system. Thus, depending on the type of response of DCs against a given pathogen, the adaptive immunity will be driven in one direction or another. A simple but useful classification categorize DCs in two main types: myeloid (classical) DC (mDC) that become activated after encountering a pathogen, secrete cytokines and prime naïve T-cells and, plasmacytoid DC (pDC) which role is thought to be focused in anti-viral responses by producing interferon-alpha (IFN-α) and pro-inflammatory cytokines. Other types of DCs may exist, particularly involved in mechanisms of immune tolerance. The activation of DCs of either type is mediated by the interaction of the pathogen with a number of receptors that recognize non-self elements such as bacterial oligosaccharides, double stranded RNA or other generic compounds (generically known as pathogen-associated molecular patterns) that are not found in the mammalian organism. These receptors are designated as Toll-like receptors (TLR). When a TLR is activated by the adequate molecule, the result is the secretion of cytokines.

As mentioned before, PRRSV may infect DCs, or at least some types of DCs. Wang et al. (2007) showed that monocyte-derived mDCs may support PRRSV replication. Infected cells suffered a decrease in the expression of MHC-I and MHC-II molecules, suggesting a potential impairment of antigen presentation by infected DCs. Similarly, Chang et al. (2008) showed that bone marrow derived DCs also support viral replication. In that case, CD80/86 expression –a co-stimulatory molecule required for antigen presentation- was up-regulated simultaneously with the down-regulation of MHC-I. These effects were accompanied by a release of IL-10. Results by Flores-Mendoza et al. (2008), Park et al. (2008), Silva-Campa et al. (2009), and Peng et al. (2009) agreed with those previous observations. Recently, Gimeno et al. (2011) showed that different genotype I strains may have different potential for regulating the expression of MHC-I, MHC-II, CD80/86, CD14 and CD163 although in general the picture was that PRRSV affects the expression of several molecules involved in antigen presentation and delays maturation of DCs. The precise nature of the mechanisms


by which the virus produces such effect is not known but it has been speculated that it could be related to ability to induce cytokines as well as to the replication levels of the virus.

Regarding pDC, Calzada-Nova et al. (2011) showed that both viable and inactivated PRRSV are able to supress IFN-α release in PBMC stimulated with a synthetic oligonucleotide (ODN19, an agonist of TLR-9) or with gastroenteritis transmissible virus. This inhibition of IFN- α took place with six different PRRSV strains, indicating that this was an inherent property of the virus. Interestingly, this inhibition of IFN-α response was not mediated by the infection of pDC. Actually, pDC did not support PRRSV replication and the inhibition of IFN-α response in those cells was probably mediated by some type of interaction with some (unknown) cell surface receptor resulting in the inhibition of the cascade of IFN-α production. This paper leads to interesting and relevant questions about how IFN-α is inhibited by PRRSV and about the biological significance of the results obtained in different cell systems. As discussed later, most studies dealing with the inhibition of type I interferons have been done using AM or MARC-145 cells and pointed to non structural proteins of PRRSV as responsible for this inhibitory effect. TLR responses against PRRS

As commented above, TLRs are the means by which the innate immune system recognizes, in a very broad sense, PAMPs. Activation of these TLRs produces a cascade of events resulting in the secretion of a number of cytokines including interferons and pro-inflammatory cytokines. These cytokine will be involved in driving the adaptive immune response towards a Th1 or a Th2 polarization, in inflammatory processes and also in apoptosis of infected or bystander cells. Each TLR is activated by a different type of molecule. For example, TLR-3 is activated by double stranded RNA (dsRNA) and TLR-7 is activated by single stranded RNA. Since PRRSV is a positive sense RNA virus producing dsRNA, both TLRs could be potentially activated by replication of the virus within the cell. Chaung et al. (2010) showed that PRRSV) inhibited TLR3 and TLR7 expressions in both AM and immature mDCs at early times with an increased response later on. Liu et al., studying mRNA expression in tissues of infected animals indicated an increase in the expression of TLR2, 3, 4, 7 and 8 but with no clear-cut pattern in all tissues and cells examined. Our preliminary results indicate that the expression of TLR-3 and TLR-7 in infected AM depends, at least from a kinetic point of view, on the replication rate of a given viral strain. Thus, infecting AM with strain DV (an attenuated strain developed to replicate very scarcely, if any, in AM) results in a TLR3 or TLR7 expression similar to that of uninfected macrophages; in contrast, use of wild-type PRRSV strains produced in AM resulted on enhanced TLR-3 expression similar to the one induced by a known TLR-agonist. Differences between strains were also noticed. These results point to the participation of TLRs in the innate response against PRRSV.


Interleukin 10 (IL-10) and PRRSV The role of IL-10 in the immunity against PRRSV has been a matter of

great interest in PRRSV research. IL-10 is a pleiotropic cytokine having the potential for suppressing cell-mediated responses. IL-10 can be produced either in the course of the innate immune response –for example by macrophages or mDCs- or in the course of the adaptive responses by T-cells committed to the regulation of the immune response, particularly by controlling or limiting inflammatory responses that could be harmful to the organism.

Silva-Campa et al. (2009) showed that DCs can produce IL-10 upon stimulation with PRRSV and similar results were obtained by Peng et al. (2009). It is worth to note that this ability to induce IL-10 responses in DC seems to be dependent on the strain used for infecting DCs and even, on the type of DCs used (Gimeno et al., 2011). Suradhat et al., (2003) indicated that IL-10 was up-regulated in PBMC stimulated with PRRSV. Feng et al., (2003) also indicated an imbalance of IL-10 and IL-12 mRNA in an in utero model of infection. Diaz et al. (2005, 2006) showed that IL-10 was elevated in PBMC-culture supernatants of infected animals during the initial phases of viraemia and that IL-10 levels correlated inversely with IFN-γ responses.

When the examination of IL-10 responses was examined in an infection model using a high virulence PRRSV isolate (Subramanian et al., 2011) it was shown that IL-10 was not up-regulated by the virus and the authors pointed towards a regulating role of IL-10. However, these results were in contradiction with the observations of Wang et al. (2011). Those authors examined the IL-10 responses in pigs infected with a highly pathogenic Asian PRRSV and observed that both IL-10 and IFN-α were significantly elevated in infected animals compared to uninfected counterparts. Thus, at present the role of IL-10 in PRRSV infection is still in debate.

Regulatory T-cells and PRRSV infection

Regulatory T-cells (Tregs) is a designation that includes T-lymphocytes with the capability to regulate or suppress the response of other T-cells. Regulatory mechanisms are thought to be evolved as a protection against immune-mediated self-inflicted damage because of the recognition of self-antigens or as a mean to limit damage to tissues because of a too strong response. Nevertheless, some pathogens may persist by inducing Tregs that would thus suppress the response against the pathogen. At present, a distinction is made between “natural” Tregs, namely those controlling autoimmunity, developing in the thymus and presenting the “typical” CD4+/CD25+/Foxp3+ phenotype and “inducible” Tregs that develop in the periphery after stimulation of T-cells in the presence of IL-2 and TGF-β. These inducible Tregs can be found among CD4+ T and CD8+ T-cells (see Käser et al., 2011 for details on Tregs in swine). In the case of PRRSV, Silva-


Campa et al. (2009) showed that using PRRSV-infected DCs, CD25+/Foxp3+ with regulatory capabilities could be generated. Wongyanin et al. (2010) indicated that PRRSV infection increased the proportion of inducible CD4+/CD25+/Foxp3+ T-cells. Silva-Campa et al. (2010) suggested that genotype I PRRSV isolates were unable to induce Tregs in contrast to the induction obtained with genotype II isolates. In a recent work, we found that pigs infected with a genotype II strain developed CD4+CD8+CD25+Foxp3+ cells with IL-10 producer capabilities (Tr1) that could explain, at least in part, the delayed onset of the cellular immune response in PRRS.

Conclusions

The ultimate causes explaining the abnormal adaptive immune response PRRSV against are far from being known although considerable advances have been produced in the last years. At present, the interplay of the virus with the innate immune system and the potential mechanism of escape, such as the development of Tregs, are some of the critical targets in the research of the immunopathogenesis of PRRS.

References

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3. Van Breedam W, Van Gorp H, Zhang JQ, Crocker PR, Delputte PL, Nauwynck HJ. 2010. The M/GP(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner. PLoS Pathog, 15: e1000730.

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dendritic cells and up-regulates interleukin-10 production. Clin Vaccine Immunol, 15: 720-725.

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12. Gimeno M, Darwich L, Diaz I, de la Torre E, Pujols J, Martín M, Inumaru S, Cano E, Domingo M, Montoya M, Mateu E. 2011. Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates. Vet Res, 42:9.

13. Calzada-Nova G, Schnitzlein WM, Husmann RJ, Zuckermann FA. 2011. North American porcine reproductive and respiratory syndrome viruses inhibit type I interferon production by plasmacytoid dendritic cells. J Virol, 85: 2703-2713.

14. Chaung HC, Chen CW, Hsieh BL, Chung WB. 2010. Toll-Like Receptor expressions in porcine alveolar macrophages and Dendritic Cells in responding to poly IC stimulation and porcine reproductive and respiratory syndrome virus (PRRSV) infection. Comp Immunol Microbiol Infect Dis, 33: 197-213.

15. Suradhat S, Thanawongnuwech R. 2003. Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus. J Gen Virol, 84: 2755-2760.

16. Feng WH, Tompkins MB, Xu JS, Zhang HX, McCaw MB. 2003. Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol, 94: 35-45.

17. Díaz I, Darwich L, Pappaterra G, Pujols J, Mateu E. 2005. Immune responses of pigs after experimental infection with a European strain of Porcine reproductive and respiratory syndrome virus. J Gen Virol, 86: 1943-1951.

18. Díaz I, Darwich L, Pappaterra G, Pujols J, Mateu E. 2006. Different European-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs. Virology, 351: 249-259.

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22. Wongyanin P, Buranapraditkun S, Chokeshai-Usaha K, Thanawonguwech R, Suradhat S. 2010. Induction of inducible CD4+CD25+Foxp3+ regulatory T lymphocytes by porcine reproductive and respiratory syndrome virus (PRRSV). Vet Immunol Immunopathol, 133: 170-182.

23. Silva-Campa E, Cordoba L, Fraile L, Flores-Mendoza L, Montoya M, Hernández J. 2010. European genotype of porcine reproductive and respiratory syndrome (PRRSV) infects monocyte-derived dendritic cells but does not induce Treg cells. Virology, 396: 264-271.


PRRSV ENTRY INTO THE PORCINE MACROPHAGE: COMPLETING THE PICTURE

Wander Van Breedam & Hans J. Nauwynck

Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; e-mail: [email protected] Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the late 1980s and rapidly became one of the most significant viral pathogens in the swine industry. The virus shows a very narrow in vivo cell tropism and targets specific subsets of porcine macrophages.

The entry of PRRSV into its host cell is the first crucial step in the infection process. Different studies that have focused on virus attachment, internalization and genome release have shed light on these early events in infection and have allowed to sketch a model of PRRSV entry into the porcine macrophage. The current model suggests that initial contact of the virus with the macrophage occurs via heparan sulphate glycosaminoglycans on the cell surface. Subsequently, the virus interacts with the macrophage-specific lectin sialoadhesin via sialic acids on the viral M/GP5 complex. Binding of the virus with sialoadhesin is followed by the uptake of the virus-receptor complex via clathrin-mediated endocytosis. Upon internalization, the viral genome is released into the cytoplasm of the macrophage. This last stage of the entry requires acidification of the endosome. Scavenger receptor CD163 appears to be critical for PRRSV genome release. The role of CD163 as an entry mediator may require interaction with the viral GP2 and GP4 glycoproteins, but further research is necessary to corroborate this. In addition, also cellular aspartic and serine proteases have been implicated in the process of genome release. However, their exact functioning is currently unknown.

Although substantial progress has been made over the last years, several questions regarding PRRSV entry remain unanswered. Further fundamental research is necessary to obtain a complete picture of the PRRSV entry process. Keywords: PRRSV, macrophage, entry


Immunohistochemical characterization of type II pneumocyte proliferation after PRRSV (Type I) challenge

G. Balka1*, A. Ladinig2, M. Ritzmann2, A. Saalmüller3, W. Gerner3, T. Käser3, M.

Rusvai1, H. Weißenböck4

1Department of Pathology and Forensic Veterinary Medicine, Faculty of Veterinary Science, Szent István University 2Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna 3Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna 4Institute of Pathology and Forensic Veterinary Medicine, University of Veterinary Medicine Vienna Abstract

The aim of the study was to characterize histologically and immunohistochemically the lung lesions after a challenge with a recently isolated PRRSV field strain in growing pigs 10 and 21 days post infection (DPI)

In the first phase of the study lung lesions were evaluated on routine HE stained slides. The microscopic evaluation of the lung lesions was performed as a blinded analysis and the lesions were scored based on the following criteria: (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intraalveolar necrotic debris, (4) intraalveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation.

For further characterization of the lung lesions, immunohistochemical stainings were performed using anti-cytokeratin, anti-Ki67, anti-TTF-1 (Thyroid Transcription Factor-1) and anti-myeloid receptor (MAC387) antibodies to identify alveolar epithelial cells, proliferating cells, type II pneumocytes, and macrophages, respectively. The evaluation of the immunohistochemical stainings revealed that humanized anti TTF-1 antibodies can succesfully identify type II pneumocytes in porcine lung tissues. Marked proliferation of these cells was confirmed by a significant (p<0.05) increase of TTF-1 positive cells in acute cases compared to the lungs of control pigs. Cytokeratin labeling marked the type I, and type II pneumocytes as well as bronchial epithelial cells, however this staining was not suitable for cell counting purposes. When the routine histological scores were compared to the number of immunohistochemically positive cells, Ki67 cell counts were found to show positive correlation (p<0.05) with the overall severity of the lesions. Introduction

Type II pneumocytes are cuboidal cells tipically located at the insertion of the alveolar septa. Their most important role is the production of pulmonary surfactant to reduce surface tension in the alveoli to prevent alveolar collapse


during exspiration. The other important function of the type II pneumocytes is based on their ability to proliferate. After injury of type I pneumocytes, that are much more sensitive to harmful effects, type II pneumocytes will serve as progenitor cells to replace damaged and desquamated type I pneumocytes, and finally differentiate into the latter cell type. Clara cells are non-ciliated and non mucus secreting, progenitor cells that can proliferate, and replace ciliated and other non-ciliated cells in the terminal part of the bronchi (Caswell and Williams, 2007).

Thyroid transcription factor (TTF-1) is a 38 kDa homeodomain-containing nuclear protein, member of the Nkx2 transcription factor family. The protein was originally described as a regulator of the thyroid-specific transcription of thyreoglobulin, thyreoperoxidase and thyrotropin receptor. In the lung tissues TTF-1 regulates surfactant gene and Clara cell secretory protein gene transcription (Bohinski et al. 1994; Zhou et al. 1996; Ray et al 1996). As TTF-1 is exclusively expressed in the nuclei of type II pneumocytes and Clara cells in the lungs, it is widely used as a marker for the diagnostics of primary and metastatic lung cancer (Tan et al. 2003).

Porcine reproductive and respiratory syndrome virus is reported to cause interstitial pneumonia characterized by hyperplastic and hypertrophied type 2 pneumocytes, septal infiltration by mononuclear cells, and accumulation of necrotic alveolar exsudate (Halbur et al., 1996, Rossow, 1998) Methods

Nine week-old PRRSV negative pigs were challenged with 2.2 × 105 TCID50 of a Type 1, subtype 1 virulent PRRSV field isolate. Negative control pigs were inoculated with virus free cell culture supernatant. Animals were euthanized on 10 DPI (n=7) and 21 DPI (n=5). Lung lesions were compared to age matched pigs of the non-infected control group. All seven lung lobes were sampled, but only left middle lobes were included in this study to exclude the possible effect of different lesion distribution in the pig’s lung lobes (cranial and middle lobes had always more severe lesions compared to the caudal ones).

Severity (0-3) and distribution (0-3) of macroscopic and microscopic lung lesions were scored, and summarized based on the following criteria: (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intraalveolar necrotic debris, (4) intraalveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation. The microscopical evaluation was performed as a blinded analysis.

For the immunohistochemical analyses anti-cytokeratin, anti-Ki67, anti-TTF-1 (Thyroid Transcription Factor-1) and anti-myeloid receptor (MAC387) antibodies were applied to identify alveolar epithelial cells, proliferating cells, type II pneumocytes, and macrophages, respectively. In case of Ki67, TTF-1 and MAC387 the labeled cells were counted in 50 non-overlapping and consecutively selected high magnification fields of 0.20 mm2. The results of immunohistochemical staining were compared to the overall HE histological score of the lung lobe to find out which antibody score correlates the most with the


histological severity. SPSS software was applied to carry out the statistical analyses: Student’s T-test was used for significance calculations, and Pearson's chi-square test for correlation analyses.

Results and discussion

As expected, the analysis of the HE stained slides revealed significant differences (microscopic lesions: p ≤ 0.0081) between challenged animals and the negative controls.

The analysis of the immunohistochemically stained slides revealed that humanized anti-TTF-1 antibodies successfully identify porcine type II pneumocytes, and Clara cells in the terminal bronchioli. Marked proliferation of these cells was confirmed by a significant (p<0.05) increase of TTF-1 positive cells in acute cases. Upregulation of Ki67 and MAC387 positive cells was also observed, however due to the relative low number of the sample animals and high values of standard deviation, the increase of these values were found not to be statistically significant (Figure 1.)

Cytokeratin labeling clearly identified the different epithelial cell types: type I, and type II pneumocytes as well as bronchial epithelial cells, however this staining was not suitable for cell counting purposes. Type I pneumocytes were flattened cells lining the alveolar spaces providing surface for gas exchange. In contrast type II pneumocytes were rounded cells usually located at the insertion of the alveolar spaces in control lungs. In infected animals, as verified by TTF-1 staining as well, these rounded cells were found in markedly higher numbers.

Figure 1. Mean values and standard deviation of TTF-1, Ki67, and Mac387 labeled cells is shown in the figure. Results were calculated from the positive cell numbers counted in 50 non overlapping microscopic fields. Dashed lines separate the three different (control, acute and chronic) groups.

When scores obtained from the routine HE stained histological slides were compared to the number of immunohistochemically positive cells by Pearson's


chi-square test, from the three different antibodies Ki67 cell counts were found to show positive correlation (p<0.05) with the overall severity of the lesions.

The relatively high variation of the immunohistochemical mean values among the different animals of the PRRSV infected groups indicates great individual differences in the response to the infection both in terms of macrophage infiltration and pneumocyte proliferation. The high standard deviation values of the individual cases point the differences of the positive cell numbers in the different microscopic fields. This feature is in harmony with the routine histological findings, different areas within the same slide show great differences in the severity of the lesions.

Acknowledgements This paper was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Scienes and by the TÁMOP-4.2.2.B-10/1 „Development of a complex educational assistance/support system for talented students and prospective researchers at the Szent István University” project. References

1. Bohinski, R.J., DiLauro, R., and Whitsett, J.A.: Lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3 indicating common mechanisms for organ-specific gene expression along the foregut axis. Mol Cell Biol 14, 5671-5681. 1994.

2. Caswell, C. L. and Williams, K. J.: Respiratory system. In Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Edited by Maxie, M. G. saunders, Elsevier. 2007.

3. Halbur, P.G., Paul, P.S., Meng, X-J., Lum, M.A, Andrews, J.J. and Rathje J.A.: Comparative pathogenicity of nine US porcine reproductive and respiratory syndrome virus (PRRSV) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model. J Vet Diagn Invest 8, 11-20. 1996.

4. Ray, M.K., Chen, C.Y., Schwarts, R.J., and DeMayo, F. J.: Transcription regulation of a mouse Clara cell-specific protein (mCC10) gene by the Nkx transcription factor family members thyroid transcription factor 1 and cardiac muscle–specific homeobox protein (CSX). Mol Cell Biol 16, 2056-2064. 1996.

5. Rossow, K.D.: Porcine reproductive and respiratory syndrome. Vet Pathol 35, 1-20. 1998.

6. Tan, D., Li, Q., Deeb, G., Ramnath, N., Slocum, H.K., Brooks J., Cheney R., Wiseman S., Anderson T., and Loewen G.. Thyroid transcription factor-1 expression prevalence and its clinical implications in non-small cell lung cancer: a high-throughput tissue microarray and immunohistochemistry study. Hum Pathol 34, 597-604. 2003.

7. Zhou, L., Lim, L., Costa, R.H., and Whitsett, J.A. Thyroid transcription factor-1 hepatocyte nuclear factor-3beta, surfactant protein B, C, and Clara cell secretory protein in developing mouse lung. J Histochem Cytochem 44, 1183-1193. 1996.


IDENTIFICATION OF T CELL EPITOPES OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS-1

USING A PROTEOME-WIDE SYNTHETIC PEPTIDE LIBRARY

Helen Mokhtar1, Melanie Eck2, Sophie Morgan1, Nicolas Ruggli2, Artur

Summerfield2, Jean-Pierre Frossard1, Simon P. Graham1*

1Virology Department, Animal Health and Veterinary Laboratories Agency, Addlestone, UK 2The Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland *[email protected] Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive problems in sows and plays a major role in the porcine respiratory disease complex. PRRSV has a huge economic impact but nonetheless an effective universal vaccine is yet to be developed. The immunology of PRRSV is not well understood, but IFN-γ secreting T cells have been linked to protective immunity. There is little knowledge about which PRRSV antigens contain the major T cell epitopes and therefore it is difficult to construct an effective vaccine that stimulates both arms of the immune system. A synthetic peptide library (overlapping 15mer peptides offset by 4 residues) spanning the proteome of PRRSV-1 was designed based on sequence data from open reading frames encoding structural proteins of the Olot91 isolate and the non-structural proteins of the closely related Lelystad isolate. T cells from pigs rendered immune to PRRSV-1 Olot91 through repeated experimental inoculation were screened for reactivity against peptides using an IFN-γ ELISpot assay. We were able to identify 18 antigenic peptides on five of the viral proteins that were recognised by either CD4 or CD8 T cells. In a second experiment, we aimed to address the identification of highly conserved T cell epitopes by screening the library with T cells from animals experimentally infected with Lelystad virus or a PRRSV-1 genotype 3 strain (SU1-bel). While T cells from both groups responded to the same proteins identified in the first experiment, SU1-bel animals mounted the greatest response against peptides representing NSP5 suggesting that this protein contains highly conserved T cell epitopes. Ongoing work is being conducted to define and characterise these conserved antigenic regions.

Keywords: PRRSV-1, T cell, peptides, IFN-γ


Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive problems in sows and plays a major role in the porcine respiratory disease complex (PRDC). Infection with PRRSV causes clinical respiratory signs, but also greatly enhances the susceptibility to secondary viral and bacterial infections which define the PRDC. This has a huge economic impact due to the increased mortality and reduced growth rate in growers and finishing pigs. Despite the effect that PRRSV has globally, a successful vaccine is yet to be developed, which is in part due to the high genetic diversity of the virus (Labarque et al., 2004).

There is limited information about how PRRSV interacts with the host immune system. It is known that the target cells are differentiated macrophages, and that the virus is not cleared for a number of weeks, indicating that the virus manipulates the cells of the immune system. Data has shown that PRRSV infection delays both neutralising antibody (Meier et al., 2000, 2003) and T cell mediated responses (Bautista and Molitor, 1997; Lopez-Fuertez et al., 1999; Meier et al., 2003). While the exact mechanisms underlying protective immunity against PRRSV are not well understood, vaccine induced IFN-γ secreting T cell responses have been associated with protection (Labarque et al., 2000). However, little is known about their specificity, the exception being the identification of epitopes on the surface glycoprotein GP5 (Bautista et al, 1999) which is the best-studied of PRRSV antigens for its vaccine potential. The lack of knowledge about which other PRRSV antigens contain the major T cell epitopes makes it difficult to construct effective vaccines that stimulate both arms of the immune system.

We here describe the application of a PRRSV-1 proteome-wide synthetic peptide library to identify the antigenic regions recognised by T cells from immune pigs. We also utilised experimental infections with a divergent PRRSV-1 strain to identify T cell antigenic regions that are highly conserved.

Materials and Methods

A synthetic peptide library was designed which comprised 15mer peptides off-set by four residues, which is considered an optimal length and overlap for identification of both MHC class-I and –II restricted epitopes. The peptide sequences were designed using the predicted amino acid sequences of the structural proteins of PRRSV-1 Olot91 strain (GenBank Accession No. AY588319.1) and the non-structural proteins of the closely related PRRSV-1 Lelystad strain (GenBank Accession No. X92942.1), since the non-structural protein encoding open-reading frame sequences are not available for Olot91. The library consisted of 1275 peptides which were additionally combined into pools representing the 19 proteins of PRRSV-1.


In a first experiment, peripheral blood mononuclear cells (PBMC) were isolated from three specific-pathogen free pigs rendered immune to PRRSV-1 Olot91 by repeated experimental infection. The pigs received three intranasal inoculations of MARC145-adapted Olot91 virus, at nine weeks, 18 weeks and 14 months of age. The third inoculation was administered two weeks before the beginning of the T cell assays. PBMC were first stimulated with Olot91 PRRSV and peptide pools representing each PRRSV protein. Reactivity was assessed by measuring IFN-γ release by ELISpot assay. Positive pools were identified and peptides were screened individually, again using an IFN-γ ELISpot assay. IFN-γ inducing peptides were confirmed by titration of the respective peptides. CD8β+ and CD4+ T cell depletions were then carried out on PBMC using magnetic sorting and depleted cell subsets were stimulated with the IFN-γ inducing peptides to determine the phenotype of the responding cells.

In a second experiment, groups of 10 pigs were experimentally infected with various genotype 1 PRRSV strains, either Lelystad virus (subtype 1), a recent British subtype 1 isolate (215-06) or with a subtype 3 isolate from Belarus (SU1-bel). PBMC were stimulated with homologous and heterologous viruses over the course of infection to assess the induction of T cell responses by IFN-γ ELISpot assay. Peptides constituted in ‘protein’ pools were screened on PBMC collected from animals 35 days post infection. Antigenic peptides contained within the pools were then determined as described above.

Data was analysed using analysis of variance (ANOVA) with Bonferroni post-test to identify peptides inducing significant IFN- γ responses.

Results and Discussion

Screening of the peptide library with T cells from pigs immune to Olot 91 PRRSV

PBMC from all animals showed significant IFN-γ responses to Olot91 PRRSV and peptides pooled to represent PRRSV proteins. Significant responses were observed against peptide pools representing NSP1b, NSP2, RdRp, GP3, GP4, GP5, and M proteins. Individual peptides making up GP3, GP4, GP5 and M were then screened individually and peptides making up NSP1b, NSP2 and RdRp were screened in pools of 10, due to the length of the viral proteins. From these screens, 5 putative antigenic peptides were identified in NSP1, 9 in NSP2, 7 in RdRp, 4 in GP3, 8 in GP4, 8 in GP5 and 14 in the M protein. Antigenic peptides were confirmed by titration using a 10-fold dilution series. Finally, PBMCs were depleted of either CD4+ or CD8β+ T cells and then stimulated with antigenic or control peptides to determine the phenotype of the responding T cells. We were able to identify 13 antigenic peptides on five of the viral proteins that were recognised by either CD4 or CD8 T cells (Table 1).


Table 1. Summary of the antigenic peptides identified using T cells from Olot91 PRRSV immune pigs

Protein Antigenic Peptide ID Phenotype of responder cells 4 CD4

18 CD8 24 CD4 33 CD8 38 CD8 52 CD8

Non-structural protein1(NSP1)

88 CD4 164 Inconclusive Non-structural protein 2 (NSP2) 175 Inconclusive 94 Inconclusive 54 CD8 RNA Polymerase (RdRp)

55 CD8 14 Inconclusive Glycoprotein 5 (GP5) 16 CD4 4 CD4 5 Inconclusive

40 CD8 Matrix protein (M)

41 CD8

Screening of the Olot91 PRRSV peptide library with T cells from pigs experimentally infected with similar or divergent viral strains

We next aimed to address the identification of conserved T cell epitopes by screening the Olot91 library with T cells from animals experimentally infected with closely related viruses (Lelystad virus and 215-06), or a divergent PRRSV-1 subtype 3 strain (SU1-bel). T cell IFN-γ responses to homologous and heterologous viruses showed that there was a degree of conservation in the specificity of responses. Screening of T cells from all groups with the ‘protein’ pools showed that responses were in general directed against the same proteins identified in the first experiment. Interestingly, all the SU1-bel inoculated animals mounted the greatest response against peptides representing NSP5, suggesting that this protein contained numerous highly conserved T cell epitopes.

Data from the two experiments were collated, analysed and one structural protein, M, and one non-structural protein, NSP2, were chosen for further investigation. Individual peptides from the M protein, pools of 10 consecutive peptides from NSP2 and the antigenic peptides identified in the first experiment were tested on PBMC from these pigs. We were able to confirm that 6 of the 18 peptides which induced a response from PRRSV-1 Olot91 immune pigs, were also able to induce a response in individual pigs that were infected with either of the three PRRSV-1 isolates used. Moreover, we were able to identify antigenic


peptides from M protein and antigenic pools of peptides from NSP2 that were responded to by 20% and 24% of animals, respectively. To investigate how well these antigenic peptides might be conserved between other strains of PRRSV, we performed sequence alignment on the M protein from 64 different European genotype PRRSV isolates and confirmed that this antigenic region lay in a well conserved area of the protein. Genetic material from all pigs is currently being processed to determine the SLA class-I and -II haplotypes of each animal which will allow further analysis of the antigenic regions identified.

In summary, we have been able to successfully identify well conserved antigenic regions in the PRRSV proteome using T cells from pigs inoculated with four different PRRSV strains. This information should be valuable in the efforts to develop effective, broadly cross-reactive vaccines against PRRS. References

1. Bautista, E. M., and T. W. Molitor. 1997. Cell-Mediated Immunity to Porcine Reproductive and Respiratory Syndrome Virus in Swine. Viral Immunology 10:83-94

2. Bautista, E. M., P. Suárez, and T. W. Molitor. 1999. T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus. Archives of Virology 144:117-134

3. Labarque, G. G., H. J. Nauwynck, K. Van Reeth, and M. B. Pensaert. 2000. Effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs. J Gen Virol 81:1327-1334

4. Labarque, G., Van Reeth, K., Nauwynck, H., Drexler, C., Van Gucht, S. & Pensaert, M. 2004. Impact of genetic diversity of European-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy. Vaccine 22:4183–4190

5. López Fuertes, L., N. Doménech, B. Alvarez, A. Ezquerra, J. Domínguez, J. M. Castro, and F. Alonso. 1999. Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection. Virus Research 64:33-42

6. Meier, W., J. Galeota, F. Osorio, R. Husmann, W. Schnitzlein, and F. Zuckermann. 2003. Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination. Virology 309:18 - 31

7. Meier, W., J. Wheeler, R. Husmann, J., F. Osorio, and F. Zuckermann, A. 2000. Characteristics of the immune response of pigs to PRRS virus. Vet. Res. 31:41


Comparative analysis of the pathogenesis of Porcine Reproductive

and Respiratory Syndrome virus strains Johanna Rebel1, Eefke Weesendorp1, Sophie Morgan2, Norbert Stockhofe1 1Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands 2 Veterinary Laboratories Agency, New Haw, Addlestone, Surrey UK Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease and reproductive losses in pigs. It is extremely difficult to control worldwide by the appearance of new virus variants, and the insufficient protection by vaccination. In addition, highly virulent strains have emerged that led to high losses.

The objective of this study, part of the EU PoRRSCon Project, is to compare the pathogenesis of PRRSV strains in order to understand differences in virulence. More specifically, to define if there are differences in target cells that become infected and to dissect the immunological host responses of pigs infected with different strains. Materials/ Methods

An animal trial was performed with 4 groups of sixteen pigs. In group 1, pigs were infected with EU type PRRSV strain Lena, known to induce clinical signs. In group 2, pigs were infected with the recently isolated EU type strain Belgium 07V063, that causes subclinical infections. In group 3, pigs were infected with the EU reference strain Lelystad (LV), that also causes subclinical infections. Group 4 were control pigs. At days 7 and 21 post inoculation (p.i.), 4 pigs per group were vaccinated with a pseudorabies virus (PRV) vaccine to study the immune competence of pigs after PRRSV infection.

Weekly, serum was collected for antigen detection and antibody responses, and peripheral blood mononuclear cells were isolated for IFN-y ELISPOT assay and FACS analysis. With FACS analysis, kinetcs of the haematological changes were studied, focusing on the identification of the lymphocyte sub-populations.

At days 3 and 7 p.i., 4 pigs per group were euthanized for post-mortem examination. At day 35, 8 pigs per group (4 vaccinated and 4 non-vaccinated) were euthanized for post-mortem examination. Several tissues were collected for immunopathological analysis, to reveal PRRSV load and changes in leukocyte number and composition in tissues. Results

Immunopathological analysis, PR serology, virus isolations and PCRs are currently performed and results will be presented during the conference.


Preliminary data show that the animal trial resulted in the expected clinical pictures, with the Lena strain causing fever and respiratory symptoms, while the Belgium and LV strain caused subclinical infections.

All pigs inoculated with the Lena, Belgium or LV strain were infected and developed antibodies, detectable from 10 days p.i. Virus could be isolated in serum from the Lena and Belgium infected pigs between days 3-33 p.i, and from LV infected pigs between days 3-26. All control pigs remained uninfected.

FACS analysis showed differences in cell populations between the infected and control pigs (NK cells and γδ T) and between strains (B cells and CD4 memory T).

The IFN-y ELISPOT assays showed an PRRSV specific IFN-y response at day 26 p.i., with the highest number of IFN-y secreting cells from Belgium infected pigs, followed by LV infected pigs and the lowest number by Lena infected pigs. Discussion

The significance of the results will be discussed during the conference. The immunolopathological analysis will reveal if there are differences in PRRSV load between tissues and target cells that become infected. It is hypothesized that the more virulent Lena strain will infect more cell types of the monocytic cell lineage than the low virulent Belgium and LV strains. Based on the preliminary results, it can already been concluded that there are differences in pathological and immunological host responses between the Lena strain on one hand, and the Belgium and LV virus strain on the other hand. Poster presentation Estimation of time-dependent infectiousness of pigs infected by the Porcine Reproductive and Respiratory Syndrome virus (PRRSV): correlation with the viral genome load in blood, nasal swabs and

the serological response

C. Charpin (1), S. Mahé (1), R. Cariolet (1), F. Madec (1), C. Belloc (2), M.-F. Le Potier (1), N. Rose (1)

(1) Anses, BP53, 22440 Ploufragan, France

(2) INRA-Oniris, Oniris, Atlanpole la Chantrerie, BP 4070, 44307 Nantes Cedex 03, France

Abstract

The time-dependent transmission rate of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and the correlation between infectiousness


and virological parameters and antibody responses of the infected pigs were studied in experimental conditions. Seven successive transmission trials involving a total of 77 specific pathogen-free piglets were carried out from 7 to 63 days post inoculation (DPI). A semi-quantitative RT-PCR was developed to assess in inoculated and contact pigs, the evolution of the viral genome load in blood and from nasal swabs with time. Virus genome in blood was detectable in inoculated pigs from 7 to 84 DPI, whereas the viral shedding was detectable from nasal swabs from 2 to 48 DPI. The infectiousness of inoculated pigs, assessed from the frequency of occurrence of infected pigs in susceptible groups in each contact trial, increased from 7 to 14 DPI and then decreased slowly until 42 days post infection (3, 7, 2, 1 and 0 pigs infected at 7, 14, 21, 28 and 42 DPI, respectively). Using those data, the time-dependent infectiousness was modeled by a lognormal-like function with a latency period of 1 day and led to an estimated basic reproduction ratio, R0 of 2.6 [1.8, 3.3]. The evolution of infectiousness was mainly correlated to the course of viral genome load in blood whereas the decreasing part of the infectiousness was strongly related to the rise of total antibodies


Session 4 PRRS control and eradication and economic

impact of PRRS infection Chairs: Bob Morrison, Marina Štukelj


How to control and eliminate PRRS from swine herds on farm and regional level

Robert Morrison DVM, MBA, PhD

PRRS CAP Regional Project Coordinator

College of Veterinary Medicine, University of Minnesota Abstract:

Porcine reproductive and respiratory syndrome virus (PRRSv) can have a significant economic impact on swine herds due to reproductive failure, preweaning mortality and reduced performance in growing pigs. Control at the farm level is pursued through different management procedures (e.g. pig flow, gilt acclimation, vaccination, air filtration). PRRSv is commonly eliminated from sow herds by a procedure called herd closure whereby the herd is closed to new introductions for a period of time during which resident virus dies out. However, despite thorough application of biosecurity procedures, many herds become re-infected from virus that is present in the area. Consequently, some producers and veterinarians are considering a voluntary regional program to involve all herds present within an area. Such a program was initiated in Stevens County in west central Minnesota in 2004. PRRSv has been eliminated from most sites within the region and the area involved has expanded to include adjacent counties. The program has been relatively successful and reflects local leadership, a cooperative spirit, and a will to eliminate virus from the region. The concept of voluntary, producer-led, coordinated regional PRRS control has spread and there are now approximately 20 such programs in North America. In pig-dense regions of United States, an increasing number of sow farms are modifying air intake such that all air is filtered. Reports suggest an 85% reduction in the rate of outbreaks of farms after filtration when compared to the 5 years prior to filtration. Analysis of rate of return on filtration suggests that this is an attractive investment option for sow farms situated in pig-dense regions. Taken together, cooperative control in low-medium dense regions, and air filtration in high dense regions, we are well positioned for effective PRRS control and eventual elimination. Introduction: PRRSv is wide spread throughout the US. In 2000, it was estimated that the virus was present in 21.4%, 17.5% and 16.6% of the sow, nursery and finishing herds respectively according to the National Animal Health and Surveillance study. In 2006, a similar survey was conducted and it was estimated that 27.3%, 26.6% and


30.2% of the sow, nursery and finishing herds respectively, were thought to have experienced health problems related to PRRS in the previous 12 months. Although significant progress has been made in understanding the routes for PRRSv transmission and which biosecurity measures are the most effective at preventing PRRSv infection in swine herds, there is a need to further expand control measures at the herd and regional level. In addition, the epidemiological features of PRRSv highlight the need for regional, geographically defined or national collaborative control and elimination programs. The purpose of this article is to describe the current methods for PRRSv control and elimination at the herd level and to report the progress being made in regional elimination programs in the US and thus provide insight into future and larger elimination programs (Corzo et al 2010). The first objective in controlling PRRSv is to produce PRRSv negative (not infected) weaned pigs from sow herds. Several management strategies have been reported to assist in achieving this goal. Semen - Based on the fact that PRRSv can be transmitted to sows via semen, it is imperative to avoid introducing contaminated semen into the sow herd. Presently, most boar studs in the US are PRRSv negative and are routinely testing for PRRSv to detect and eliminate PRRSv from the stud should an infection occur. McRebel - McCaw (2000) introduced this concept to control spread of pathogens in suckling pigs. The concept acronym McRebel stands for “Management Changes to Reduce Exposure to Bacteria to Eliminate Losses”. Measures such as decreasing cross-fostering to a minimum, eliminating poor doing non-responsive pigs, changing needles between litters or pens and taking extra care of the smallest pigs are included. Breeding farms that undergo a PRRSv elimination process that did not include thorough and continued McRebel management practices often have recurrent recirculation of the virus in the piglet population. Gilt Acclimation - Once a breeding herd has become infected with PRRSv, gilt introduction becomes one of the most important factors for PRRSv control. Gilts are susceptible to PRRSv infection and PRRSv recirculation if they have not developed protective immunity prior to introduction into the herd. Additionally, gilt introduction has an important influence on the production of PRRSv negative pigs at weaning. If gilts become viremic during the breeding period, they become a source of virus for the herd which results in transmission to the neonatal pigs. Gilts that are infected with PRRSv in their growing phase will create future breeding animals that once introduced into the breeding herd could be at least partially immune to reinfection. Therefore, the goal in a PRRSv acclimation program is to expose gilts to the same strain of virus that is resident in the herd into which they will be introduced and at a young enough age such that they are


fully recovered before entering the breeding herd. Early in the acclimation program, gilts can be exposed to PRRSv using different programs. Three ways to accomplish this are (1) to expose gilts to viremic nursery pigs which may transmit the virus to the gilts, (2) intentionally expose incoming gilts to the resident PRRSv by live virus injection, and (3) vaccinate gilts during the acclimation period. Vaccination - Both inactivated and modified live virus (MLV) vaccines have been used in gilts, sows and growing pigs for the control of PRRSv. MLV vaccine has shown efficacy in reducing mortality and poor growth. Also, MLV vaccines have been used to aid in the elimination of field virus from infected breeding herds. Inactivated PRRSv vaccines have been reported to improve farrowing rate, return to estrus and piglets weaned per sow in endemically infected populations. However, others have reported that inactivated commercial vaccines have not conferred protection when administered to gilts which were subsequently experimentally challenged. Air Filtration - Experimental evaluation of the potential for air filtration to reduce exposure and infection rates has been reported. In a long-term side-by-side evaluation, Dee et al. (2011) observed a PRRS infection in 28/65 replicates (43%) in non-filtered control groups as compared to 0/101 replicates in filtered treatment groups. The first field experiences with filtering boar studs and then sow farms have been positive (Spronk et al 2010) and this has led to substantial uptake in the industry. Recent data from the field indicate an 85% reduction in the annual rate of outbreaks at farms after filtration when compared to the 5 years prior to filtration [Darwin Reicks, Gordon Spronk and Paul Ruen, personal communication]. Elimination methods Different methods have been described for eliminating PRRSv from sow herds including test and removal, whole herd depopulation/ repopulation and herd closure. Test and removal - Elimination of PRRSV through this method has been documented and although rarely done, could be applicable within a sow herd or boar stud where spread is slow. The main disadvantage is cost of testing and cost of premature removal of positive animals from a productivity point of view. Whole herd depopulation and repopulation - This method comprises the elimination of all breeding and/or growing swine from the farm, disinfecting the facilities and restocking the farm with PRRSv negative pigs. Although this method is highly effective, whole herd repopulation is costly since all the breeding females need to be replaced. An off-site breeding program can be implemented to mitigate the disruption in weaned pig production. One important advantage is that this method can also eliminate other pathogens and improve genetics simultaneously.


Herd Closure and Rollover - Herd closure and rollover has become the most widely used method for eliminating PRRSv from sow herds. This method was first described by Torremorell et al. (2003) and consists of interrupting the introduction of incoming replacement females into the breeding herd for at least 6 months plus the elimination of seropositive animals over time. The objective of stopping the introduction of new animals into the herd is to decrease the number of susceptible animals in which the pathogen can replicate, thereby favoring the elimination of the virus. Herd closure has been reported to be the least expensive method to eliminate PRRSv (Schaefer and Morrison, 2007). Planned exposure of the breeding herd with homologous virus or MLV vaccine as a last step before stopping the introduction of the last infected replacement animals, increases herd immunity when closure is initiated. The objective of exposing all breeding animals at once is to ensure that all sows have been exposed and had an opportunity to mount an immune response. Once all animals have mounted an immune response transmission should decrease. Although persistently infected animals may exist temporarily, if there are no susceptible animals remaining in the herd, the ability of the virus to circulate within the herd will be significantly reduced or eliminated. Future introductions must be with PRRSv negative gilts to maintain the herd free of the virus. PRRSv regional control / elimination A regional control program can be defined as “reduction of disease incidence, prevalence, morbidity or mortality to a locally acceptable level as a result of deliberate efforts. Continued intervention measures are required to maintain the reduction.” A regional control program might just entail testing herds, surveillance, sharing information & acting accordingly. In contrast, a regional elimination program is a: “reduction to zero incidence of a pathogen in a defined geographical area as a result of deliberate efforts. This requires continued measures to prevent re-establishment of virus transmission.” Either approach creates the opportunity to lessen the impact of PRRS. (http://www.cdc.gov/MMWR/preview/mmwrhtml/su48a7.htm) For certain animal diseases, regional elimination of the pathogen has been the only effective way to control the disease. Regional elimination programs have been reported for diseases such as foot and mouth disease (FMD) where mass vaccination and movement restrictions have proven to be effective. Recently, Chile and Sweden reported the successful elimination of PRRSV from the countries. The term “disease eradication” often refers to draconian efforts, such as the historical CSF and PRV programs of the 1960’s and 1990’s. Disease eradication is driven by statutes and enforced by the authorities. In contrast, the implementation of regional PRRS virus elimination projects is motivated by a desire to increase


profitability. The process is driven by communication, education and local leadership. Immediate benefits include improved biosecuity and an increased awareness of the impact of infectious disease. In the longer term, these efforts culminate in the stable elimination of PRRS. In the much longer term, the infrastructure developed to eliminate PRRSV will lead to the elimination of other infectious diseases that impact animal health and profits. In a voluntary control/elimination program there is no assurance of 100% participation. However, 100% success is not dependent on 100% participation. Some predictors of success include: - Local unified leadership from producers and veterinarians, - Good communication among participants, - A long term commitment and willingness to adapt as new challenges arise, - Few pigs entering the region from PRRS-positive or herds with unknown PRRSV status. - The presence of natural borders (mountains, rivers, etc.) or a low density of herds at the perimeter of the region, - Few exhibition pigs in the region or the willingness of hobby farmers to participate, - In general, the higher the herd density in a region, the more difficult the process. Brief history of PRRS projects: The first regional project aimed at controlling PRRS virus spread started in 2002 in eastern Rice Cy, Minnesota. Some progress was made but in retrospect, the region did not have the ideal attributes to determine whether a voluntary, regional, coordinated, PRRS control & elimination program is feasible. Stevens County, Minnesota - A second regional project started in 2004 in Stevens County in west central Minnesota and the project has been a resounding success. Approximately 90% of the producers in the county have participated and the prevalence of PRRS has decreased from approximately 50% of sites to no known sites having PRRS. The project has expanded twice and now includes all of northern Minnesota. Getting started: Having defined a “region”, and leadership, the first step in developing a regional elimination project is to get the word out. Engaging local producers is a never ending task and is ideally done at the local level by producers and veterinarians. The elements of a plan can be as follows.

1. Establish a leadership team, establish meeting frequency & communication methods. Transparency in communication is important.

2. Discuss the risks of disclosing status and how the group wants to mitigate this risk (producer agreement, hold harmless).

3. Map all locations with pigs, preferably with GPS coordinates. Categorize each site in terms of type of production and approximate size.


4. Characterize the presumed PRRS status for every location. PRRS status needs to be openly discussed by regional elimination. As discussed below, there are risks to the disclosure of PRRS virus status.

5. Determine the biosecurity risk of participating farms by conducting a PADRAP evaluation. This is a detailed questionnaire that describes biosecurity management for a site and the risk of the farm for introducing and maintaining PRRS virus infection.

6. Develop a pig flow map for the entire region. (The region size may change based on pig flow.)

7. Develop herd plans for eliminating the virus at the herd level, including sampling. Get exhibition folks on board right away including biosecurity training and innovative programs to incentivize showing PRRS negative pigs.

8. Go for early successes. Producers watch and respect local experiences more than research results from elsewhere. There will be much skepticism at the beginning, however, once positive results appear, a change in mindset follows.

9. Establish a protocol for outbreak investigation & notification. 10. Education program. Bring in guest speakers to discuss various aspects of

PRRS control, sampling, share experiences, etc. 11. Once a PRRS-negative status is achieved maintain a risk-based testing

system to assure that regional status is maintained. Current Projects: With the success of the N212MN project, USDA PRRS CAP allocated 10% of its budget to initiate six other regional projects around United States. Each of these projects has a coordinator and is progressing at various rates towards achieving PRRS control. Each Project’s Progress & Challenges: The projects vary in their progress and challenges. For example, the N212MN project (P.I. Dr. Montse Torremorell) has an extremely large area with 4 clusters of pork production and the majority of the region being very low density. As well, with Stevens having no known sites being PRRS positive, the region employs a risk-based surveillance system to identify sites with higher risk that deserve more frequent testing. The north central IL project (P.I. Dr. Noel Garbes) has the challenge of relatively large farrow to finish single sites. Also, they are using attenuated PRRS vaccines in their region and will need to eliminate this virus as well in the long term. The west central IL project (P.I. Dr. Dyneah Classen) has struggled with participation rate and is in the process of designing a survey of attitudes so we can learn about factors affecting perceptions about adoption rate of regional, voluntary, coordinated PRRS control and elimination. The Iowa County project (P.I. Dr. Derald Holtkamp) is trying to gain understanding of the impact of pigs entering the control region. The Cuming County project (P.I. Dr. Alan Snodgrass) is working with BI to incorporate a novel mapping program (BioPortal) that includes PRRS virus sequence as well as location and PRRS status of farms. And finally, the Pennsylvania project (P.I. Dr


Tom Parsons) has been studying the role of geographic location and topography on apparent lateral transmission of PRRS virus. Each project coordinator submits a quarterly progress report (all available for review at PRRS.Org). The progress report includes a table summarizing herd inventory and PRRS status for the region and a bar chart showing change in PRRS status of sow herds over time. Table 1. Example of regional progress report from Minnesota project.

Figure 1. Change in sow herd status within Minnesota regional project.

A concern is that every project is using different sampling frequency and numbers to assess sow farms and growing pig sites. So while the sow herd classification guidelines exist (Holtkamp et al 2009), they are not generally being applied. Discussing the risks of disclosure: A key part of a voluntary, regional, coordinated disease control program is the sharing of disease status. There will be a range in reactions from “sure” to “no way!” One fear is that a participating


producer might be held liable by a neighbor for alleged spread of virus. And as a region makes progress, anticipate increasing attention and collective pressure being given to new breaks. To help manage the risk of disclosure, most projects have all participants sign a participation agreement (consent letter) &/or a hold harmless contract that discusses the risks of the project and gives the project coordinator access to the farm status. Despite having these agreements in place, there might be an unexpected negative reaction to a new disclosure. Service to a PRRS virus positive farm might be discontinued by one or more commercial truckers, the farm might be turned away from one or more local feed mills or by manure pumping contractors. A source farm may terminate delivery, commercial rodent control service may refuse service, and other externally provided services may be in jeopardy. Also, a producer might feel pressured to instigate a control or elimination program more quickly than h/she would otherwise have done. These are all unlikely possibilities, but need to be discussed before they occur.

-‐ Knowing a neighbor’s PRRS status is a privilege. -‐ Pressure a neighbor or withdraw services and a project runs the risk of

driving PRRS underground. Developing a budget: Testing - The major project cost for a region will be diagnostic testing. This will include veterinary costs and diagnostic lab fees. Many producers in the region may already be testing and might continue to pay those costs. Some producers who are not currently testing may pay for the tests at their farms. A challenge will be paying testing costs for farms that are willing to participate but not willing to cover the costs. Coordination - There may also be costs for a project coordinator, travel, and communications (phone, etc). Volunteer time on the part of producers and veterinarians may cover the coordination, education, communications, etc. Vaccine - In some regions, some producers may include PRRS vaccine as part of their control approach. The challenge for all regions is to evolve into financially sustainable projects that are totally funded by the producers. There is much enthusiasm for regional PRRS projects with at least 20 projects underway in North America. As we might expect, challenges arise as we tackle this complex problem. There are 4 working groups currently addressing the following issues:

-‐ Develop a standard of practice (SOP) that might serve as a template for managing and sampling sow herds as we work to eliminate virus.

-‐ Develop a generally accepted minimum sampling program for sow herds in low dense or PRRS free regions. Secondly, develop sampling guidelines for growing pig sites.


-‐ Incorporate oral fluid sampling into the sampling guidelines. -‐ Develop guidelines for managing the risk of disclosure.

Conclusions / Recommendations:

Dramatic progress has been made in relatively few years going from 1 to over 20 regional projects in just two years. We are already seeing some regional projects coalescing as the interest in regional PRRS control grows. This, coupled with the advent of filtering sow farms in hog dense regions and the negative pig flow resulting from such effort, one can imagine the industry entering a new era of widespread voluntary, regional coordinated PRRS control & elimination. References:

1. Corzo C et al. / Virus Research 154 (2010) 185–192 187. 2. Dee S et al. Journal of Swine Health and Production — September and October

2011. 3. Holtkamp D et al. Terminology for classifying swine herds by porcine

reproductive and respiratory syndrome virus status. Journal of Swine Health and Production January and February 2011 44.

4. McCaw, M., 2000. Effect of reducing cross fostering at birth on piglet mortality and performance during an acute outbreak of porcine reproductive and respiratory syndrome. J Swine Health Prod. 8 (1), 15–21.

5. Schaefer, N., Morrison, R., 2007. Effect on total pigs weaned of herd closure for elimination of porcine reproductive and respiratory syndrome virus. J Swine Health Prod. 15 (3), 152–155.

6. Spronk GD et al. Prevention of PRRSV infection in large breeding herds using air filtration. Veterinary Record (2010) 166, 758-759.

7. Torremorell, M., Henry, S., Christianson, W.T., 2003. Eradication using herd closure. In: Zimmerman, J., Yoon, K.J., Neumann, E. (Eds.), PRRS compendium. National Pork Board, Pork Checkoff, pp. 157–160.

Swine herds classification regarding PRRS status

Dr. Derald Holtkamp

Department of Veterinary Diagnostics and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA

Standardized terminology related to porcine reproductive and respiratory syndrome virus (PRRSV) and the PRRSV status of herds is necessary to facilitate communications between veterinarians, swine producers, genetic companies, and


other industry participants. Likewise, a standardized system for herd classification based upon a set of related definitions is required for implementation of regional and national efforts towards PRRSV control, elimination, or both. The purpose of this paper is to provide a herd classification system for describing the PRRSV status of herds based upon a set of definitions that reflect the biology and ecology of PRRSV as it is understood today. The system described here was developed by a definitions committee formed jointly by the American Association of Swine Veterinarians (AASV) and the United States Department of Agriculture (USDA) PRRS-Coordinated Agricultural Project (PRRS-CAP) and was approved by the AASV Board of Directors on March 9, 20101. The committee included veterinarians from private practice and industry, researchers, and representatives from AASV and the National Pork -Board. Classification of breeding herds Breeding herds, with or without growing pigs on the same premises, are categorized as Positive Unstable (Category I), Positive Stable (Category II), Provisional Negative (Category III), or Negative (Category IV) on the basis of herd shedding and exposure status. Positive Unstable (Category I) breeding herds have positive shedding and exposure statuses. It is also the default category when herd shedding and exposure statuses have not been confirmed and when herds have not been tested. Herds going through a clinical PRRS outbreak and those with chronically recurring shedding of virus will fall into Category I. Positive Stable (Category II) breeding herds have an uncertain shedding status and positive exposure status. Absence of clinical signs of PRRS in the breeding-herd population and confirmation of a sustained lack of detectable viremia in sampled weaning-age pigs (and growing pigs if present) for a minimum of 90 days is required. This classification requires negative PCR herd tests in weaning-age pigs for at least 90 days. A minimum of four consecutive negative PCR herd tests in weaning-age pigs sampled every 30 days or more frequently is required. The exposure status of the breeding herd remains positive. The possibility that animals may still be infected and later shed the virus cannot be ruled out. A distinction is made between Category II breeding herds that are not undergoing PRRSV elimination (Category II-A) and those that are (Category II-B). A breeding herd is undergoing elimination if it has initiated an elimination procedure. An elimination procedure begins when the last seropositive breeding replacements are introduced or when the last intentional exposure to any live PRRSV, wild-type or any vaccine (live or killed or both), occurs in the herd, whichever is later. Confidence that the weaning-age pigs and growing pigs moving from Category II breeding herds are not shedding PRRSV increases over time if the breeding herd is undergoing PRRSV elimination, since steps are being taken to eliminate viral shedding in the herd undergoing elimination. These steps will include closure of the herd to


introductions of breeding replacements and may also include restrictions on cross-fostering and initiation of whole-herd exposure to live virus or vaccination. All herds that are undergoing PRRSV elimination by herd rollover will meet the criteria to be classified as Category II-B at some point during the elimination process. Category II-A is the goal for herds that are trying to control, rather than eliminate, the virus. Provisional Negative (Category III) breeding herds have a negative shedding status. For a herd to be classified as Category III, sustained introduction of negative breeding replacements without their seroconversion to the PRRSV is required. Lack of seroconversion in introduced animals is considered sufficient evidence to confirm that PRRSV is no longer being transmitted in the herd. The negative breeding replacements must have been in contact with previously positive animals and remain seronegative by ELISA for a minimum of 60 days after entry into the breeding herd. Some adult breeding animals in the herd may still have antibodies to the PRRSV due to prior infections. If growing pigs are present at the same premises, confirmation of a negative exposure status in that subpopulation is also required. Category III herds include those intentionally pursuing elimination via herd rollover by entering negative breeding replacements. Negative (Category IV) breeding herds have a negative shedding and exposure status. Confirmation of the negative exposure status of Category IV breeding herds varies depending on how the herd is established negative. For herds established negative by herd rollover, diagnostic information is required to confirm the negative exposure status of adult breeding animals, but information from production records may also be used in conjunction with diagnostic information to confirm the negative status. Production records may be used to identify the breeding animals on the inventory list just before the first negative breeding replacements are introduced: this is the population of animals that must have been removed. When the current inventory contains no adult breeding animals that were on that list, the herd has been completely “rolled over” once and may be classified as Category IV. Alternatively, a negative exposure status may be confirmed with a negative herd test based upon sampling adult breeding animals to confirm that they are seronegative after a minimum of 1 year from the start of Category III status. Individual animal records are not required for the alternative criterion. If growing pigs are present at the same premises, confirmation of a negative exposure status in that subpopulation is also required. Category IV also encompasses new premises populated with negative breeding replacements or premises that were completely depopulated and repopulated with negative breeding replacements that have remained seronegative. For these herds, a negative ELISA herd test in adult breeding animals is required at least 30 days after the premises are populated to define the negative exposure status of the herd. Classification of growing-pig herds


Growing-pig herds are categorized as Positive or Negative. Premises with growing pigs only are classified as either positive or negative. Positive herds have a positive shedding status or a positive exposure status or both. It is also the default category when diagnostic information is inadequate to classify a herd as negative. Negative herds have a negative shedding status and a negative exposure status. References 1. Holtkamp D.J., Polson D.D., Torremorell M. and committee members Morrison B. (chair), Classen D.M., Becton L., Henry S., Rodibaugh M.T., Rowland R.R., Snelson H., Straw B., Yeske P., Zimmerman J. 2011. Terminology for classifying swine herds by PRRS virus status. J. Swine Health Prod. 19:1 44-56.


ECONOMICAL IMPACT OF PRRSV-OUTBREAKS IN SOW HERDS

N. Nieuwenhuis a,b, T. Duinhof a, A.van Nesb

a GD Animal Health Service, PO Box 9, Deventer, The Netherlands, [email protected] b Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Introduction After the first recognition of porcine reproductive and respiratory syndrome virus (PRRSv) in 1987, PRRSv is now endemic in most pig producing countries, including the Netherlands. The epidemic phase of the disease presents itself by massive reproductive failure during the last month of gestation (Collins et al, 1991) and the economic losses of PRRS are considered to be high (Holck, 2003). The aim of this study was to quantify the economical effects of an outbreak of PRRSv in sow herds. Materials and Methods Nine breeding herds were selected based on a confirmed PRRSv outbreak. The economical impact during the 18-week period following the outbreak was calculated, based on the technical production results and costs (medication, diagnostics, labour) compared to 26 weeks preceding the outbreak. We also calculated the costs after the outbreak, e.g. costs to eradicate PRRSv. Results and discussion An outbreak of PRRSv resulted in a reduced number of sold pigs per sow of 1.7. The economical impact varied from €59 to €379 per sow per 18-week outbreak period. The two nucleus production herds had the biggest losses per sow per outbreak, with an average loss of €305 per sow during the outbreak period. The average loss in a regular sow herd was €75 per sow per outbreak. An overall loss per sow per outbreak was €126. The costs after the outbreak, varied greatly from €3 to €160 per sow, due to the variety in decisions of farmers to eradicate PRRSv or just stabilize the herd.


The calculated costs in this study are in line with the costs of the initial outbreak in the Netherlands in 1991 (€98/sow, Brouwer et al, 1994). Table 1. Losses during and after the period of outbreak

Herd Type of herd *

# sows

No. of piglets born alive/litter Absolute change

Pre-weaning mortality rate (%) Absolute change

Post-weaning mortality rate (%) Absolute change

Farro-wing index Absolute change

No. of sold feeder pigs Percentile change

Total loss (€)

Loss/sow during outbreak (€)

Loss/sow after the outbreak (€)

1 M 375 -1.39 +2.1 +0.8 -0.05 -15,5% 31,522 83 5 2 B 250 -1.62 +16.5 +3.5 -0.12 -38.6% 95,725 379 164 3 M 275 -0.20 +6.6 +3.1 -0.01 -12.5% 33,523 85 16 4 B/M 440 -0.20 +3.6 +2.1 +0.07 -4.7% 27,853 59 60 5 M 385 -1.10 +3.0 +2.1 -0.19 -20.2% 35,040 81 5 6 M 360 -1.38 +3.6 +1.7 +0.05 -14.4% 26,084 61 3 7 M 1175 -0.67 +1.9 +1.1 -0.26 -19.2% 105,854 90 3 8 B 585 -1.50 +4.5 +1.5 -0.04 -20.8% 134,292 230 66 9 M 1075 -0.50 +3.7 == -0.19 -15.9% 68,293 64 5 Mean -18,0% 62,021 126

* M=Multiplier, B=Breeder Keywords: PRRS, economical impact, sow herd References:

1. Brouwer, J., Frankena, K., de Jong, M.F., Voets, R., Dijkhuizen, A., Verheijden, J., Komijn, R.E. (1994) PRRS: effect on herd performance after initial infection and risk analysis. Veterinary Quarterly 16. pp. 95-100.

2. Collins, J.E., et al. Swine infertility and respiratory syndrome (mystery swine disease). (1991). In Proceedings of Minnesota Swine Conference for Veterinarians, University of Minnesota, MN.

3. Holck JT, Polson DD. (2003) Financial impact of PRRS. In: Zimmerman JJ, Yoon K-J, eds. The porcine reproductive and respiratory syndrome compendium. 2nd ed. Des Moines: National Pork Board. pp. 51–58.

4. Neumann, E.J., Kliebenstein, J.B., Johnson, C.D., Mabry, J.W., Bush, E.J., Seitzinger, A.H., Green, A.L., Zimmerman, J.J. (2005) Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. Journal of the American Veterinary Medical Association 227. pp. 385-392.

Full article submitted to Veterinary Record


ERADICATION OF PRRS IN ONE SITE FARROW-TO-FINISH

GREEK FARMS: IS IT FEASIBLE?

Spyridon K. Kritas 1, Georgios Filioussis 1, Evanthia Petridou 1, Konstantinos Papageorgiou 1, Georgios Christodoulopoulos 2, Angeliki R. Burriel 3, Tomasz

Stadejek 4

1 Department of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Macedonia, Greece; 2 Clinic of Medicine and 3 Laboratory of Microbiology, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece; 4 National Veterinary Research Institute, Department of Swine Diseases, Pulawy, Poland Contact person: [email protected]. Abstract The objective of the study was to review whether the epidemiological situation of PRRS in Greece favors possible eradication in individual or local basis. For this reason sera from 38 farms of different sizes throughout the entire Greek territory were collected and examined for the presence of antibodies against PRRS virus. Almost 75% of the examined farms were positive for antibodies to PRRS virus at the end of the fattening period. There was statistically significant correlation of this antibody presence and a) the distance to neighboring farms, b) application of biosecurity measures within the farm, c) purchase of gilts from other farms, d) the presence of other health problems in the farm. A strong tendency was found between PRRS seropositivity and a) sow vaccination against PCV2 or b) the application of all-in, all-out system in the farm. During the last years several successful efforts had been made to eradicate PRRS virus from USA farms. However, apart from the virus strains, the type of pig farming differs a lot from that in Europe. Since the vast majority of the farms in Greece are one site farrow-to-finish type, a discussion on factors and methods that can be important for possible disease eradication will be made. Keywords: PRRS, eradication, epidemiology, Greece


Evaluation of the effectiveness of an acclimatization programme to control porcine reproductive and respiratory syndrome (PRRS) in

a farm of an endemically infected area of North-Eastern Italy

Michele Drigo, DVM, PhD

Department of Public Health, Comparative Pathology and Veterinary Hygiene, Veterinary College, University of Padua,Viale dell’Università 16, 35020 Legnaro, Padua,

Italy Introduction In Italy PRRSV infection is widespread, with circulation of several viral strains. In breeding herds the key to break virus circulation is to expose replacement animals to the farm specific PRRSV strain to induce a good immune response prior to their introduction into the herd. Therefore, a rigorous bio-security policy is needed to avoid the incoming of new strains into a positive farm. The aim of this study was to monitor the serological, virological and productive trends of an endemically PRRSV infected breeding herd where the control of the infection was based on the implementation of an effective acclimatization program and of strict bio-security measures. Keywords: PRRSV; control; acclimatization; herd stability; Italy Materials and Methods The study was carried out in a 1500 sows farrow-to-wean farm, infected by PRRSV since 1995. As farmers decided not to vaccinate animals for PRRSV, an acclimatization program was improved over time. 52 3-week-old PRRSV negative gilts were introduced into the farm every month and at once exposed to PRRSV through the contact with weaned pigs, used as donor sources of virus. During the last 3 months of acclimatization viremic pigs were turned over every 2 weeks and at last taken away one month prior to the introduction of gilts into the stimulation unit. The aim of the program was to fertilize 8-month-old gilts after they became immune but no longer viremic. For 24 consecutive months, from April 2007 to March 2009, sera samples were collected from 1185 gilts at the end of acclimatization. In the same period, 5 cross-sectional monitorings were carried out almost seasonally, sampling a total of 607 pigs. Between October and November 2008 a PRRS outbreak occurred in the farm and thoracic fluid was collected from some aborted fetuses. PRRSV RNA was extracted from both sera and thoracic fluid samples, analyzed by RT-PCR, purified and sequenced in ORF7 region. Sera samples from gilts were also checked for the presence of a specific PRRSV immunity by an ELISA commercial kit. Performance Trend Analysis (PTA) was obtained by the software of the farm,


and analyzed statistically using Chi-square test for count data and one sample T-Test for continuous variables. Results After a first period of low efficacy of the acclimatization program, especially in July and November 2007, both prevalence of seropositive gilts and s/p ratio mean increased in the next months. This trend may be the consequence of seasonality and of a better management of the viremic pigs used as virus donors. Furthermore a 100% of positive pools in 3 gilts groups during cold season and a range from 2/16 positive pools in July 2008 to 23/23 in February 2009 in the 5 cross-sectional monitorings confirm that season play a key role in the viral circulation. The outbreak consisted in a reproductive failure with 27 abortions and in a significant fall of the productive performance: farrowings number, farrowing rate, piglets weaned, piglets weaned/sow/year, total born/litter, liveborn/litter, piglets weaned/litter.

Fig. 1 Serological and virological data of the 24 groups of gilts. The comparison of these parameters between the months before and the ones after the outbreak didn’t show significant differences demonstrating that in a well acclimatization system the return to normality can be achieved rapidly. Phylogenetic analysis assessed the presence of a single homologous PRRSV variant on the farm and revealed the incoming of a new strain (9,4-10,8% of nucleotide p-distance) causing the outbreak and no longer detected in the following monitorings. DISCUSSION – This work confirms that an acclimatization program like the one described in this study is able to maintain the circulation of a unique viral strain over time, in not reproductive units and that it is able to restore herd stability after an outbreak. Phylogenetic analysis is more and more often a useful tool to help farmers and vets to understand the causes of the failures and to find successful control measures.


Risk Assessment and Risk Management – Use of Applied epidemiology for PRRSv control in large swine productions of

Croatia

Dejvid Sabolek DVM MSc IAH

College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK, [email protected]

Abstract: Following the devastating impact PRRSv infection has left on Croatian swine productions during the first decade of this century, there is a growing trend among producers towards measures for complete eradication of this pathogen from their production systems. Measures most commonly applied include complete or partial depopulation followed by extensive cleaning and disinfection of premises, in turn followed by restocking (repopulation) with animals from certifiable sources free of the PRRS virus (notably SPF herds). In an effort to maintain the PRRSv free status, productions that have undergone the eradication process are investing enormous energy and resources in maintaining stringent bio-security protocols while on the other hand leaving the PRRSv out of the vaccination programmes, resulting in the creation of a PRRSv naive populations accounting for some 50% of the total Croatian controlled swine production. The aim of this paper is to augment the urgency for use of applied epidemiology tools namely Risk Assessment process (RA), that will in turn provide important data for the Risk Management process (RM), resulting in the implementation of measures which will reduce the risk of PRRSv re-introduction to an acceptable and manageable level, thereby facilitating production at the most economically viable level. Recommended Risk Assessment tools include surveillance and monitoring of uncontrolled swine productions (Backyard pig production) in the immediate vicinity of the farm, on the regional as well as national levels and surveillance of wild boar populations, all in an effort to gain the greater understanding of the natural history of the virus in the field and identification of the most likely sources of infection. Risk management process will use the collected information to create the most efficient bio-security strategies, vaccination protocols and measures that will reduce the consequences should an incursion of PRRS virus occur. Key words: Risk Assessment, Risk Management, PRRSv, eradication, swine production


Introduction: Croatian swine production can best be described by dividing it into two segments, controlled and uncontrolled. The controlled segment, which makes up only 20% of the total national swine numbers, is composed of large integrators (17%) with farms most often averaging 500+ sows and small family owned farms (3%) ranging in size form 50 up to 200 sows. The remaining 80% of the total swine numbers make up the uncontrolled segment, the so called “Backyard production”, where individual producers keep one or two sows and their progeny for personal use, as well as for providing additional family income by sale on the market (Croatian Agricultural Agency, 2011). The terms “controlled” and “uncontrolled” are used to describe the level of veterinary supervision in each of the segments, where as the controlled segment generally has a well organized and constant veterinary health service, the uncontrolled segment is receives little or no attention by the veterinary services, largely owing to the economic situation in the rural areas and the general lack of education by the individual producers. Croatian experience with PRRS virus has been quite painful and expensive. Ever since its first appearance in 1995 (Lipej et al., 2011) it has managed to bring the already struggling industry to its knees and forced the producers to re-evaluate their production methods and bio-security protocols. Concomitant infections with PCV2 virus further augmented the problem resulting in productions with losses reaching up to 50% (from farrowing to slaughter) of the already low live-born rate of piglets, high reproductive failures and unsustainable growth rates. Desperate situation called in for drastic measures and over the past few years the majority of large integrators have opted for either complete or partial de-pop/re-pop method or completely renewed their production systems with modern new farms. Farms that have undergone such measures were restocked form sources certifiably free of PRRS and other economically important swine pathogens (SPF herds). As a result of such a policy over 63% of large swine productions have managed to bring the problem under control and are currently relying on stringent bio-security measures to prevent the reintroduction of the virus. Past experience and general lack of trust in effective vaccine has resulted in PRRSv being left out of the vaccination protocols and the creation of a large pool of immunologically naive animals placing the entire industry in serious jeopardy. Risk Assessment (RA) and Risk Management (RM) are applied epidemiology tools most commonly used to assess the threats of importation of a pathogen into a country, evaluate the consequences of such incursions and consequently implement measures which reduce the risk to an acceptable and manageable level (Thrusfield, 2005). Simple modification of these processes could provide invaluable tools for the swine industry to implement into the decision making processes and general management practices decreasing the chances for potential gaps in the systems that would normally allow pathogens to become established.


Main text: The general guidelines on the methodology of Risk Assessment and Risk Management are provided in the section 2.1 of the Terrestrial Animal Health Code of the World Organisation for Animal Health (OIE) under the heading of Import Risk Analysis (OIE, 2010). Risk Assessment (RA) is a process composed of different components which follow a sequence by which we try to answer a series of questions (qualitative) that will allow us to estimate probabilities (quantitative) of certain events (hazards). This paper is concerned with Risk Assessment and Risk Management of PRRSv, although it can be applied to any economically important pathogen. Risk Management is the decision making process that follows Risk Assessment with the aim of identification, selection and implementation of measures that will reduce the risk of a pathogen’s entry and establishment. Risk analysis:

1. Hazard identification – is the first step in the Risk analysis process and its aim is to identify the pathogenic agent which could produce adverse consequences to the industry; in our case this is PRRS virus.

2. Risk Assessment – for the purposes of this paper, the risk source needs to be identified and named. Given that large swine productions are closed entities, we will assume that new animals will only be introduced from sources certifiably free of PRRSv. The source of risk for these productions is therefore identified as “Backyard production” whose status remains unknown, but given the national PRRS status is most certainly positive for the virus, as well as Wild Boar population which has been proven to be PRRS positive by numerous authors in the past (Roić et al. 2006; Ž. Župančić et al. 2002; J. et al Tončić 2006). Risk assessment process is comprises several steps:

a) Release assessment – Is concerned with describing the biological pathways necessary for the hazard to be released (introduced) into a particular environment and estimating the probability of that event occurring. Some of the inputs that might be required in this section are numbers of swine, structure of productions, breeds, age structure, agent’s predilection sites, vaccination status, evaluation of veterinary services, incidence/prevalence of the agent, ease of contamination, effects of transport and natural migration of wildlife.

b) Exposure assessment – this part is concerned with describing biological pathways necessary for exposure of animals and humans on the swine production unit to the hazard. One important thing to keep in mind is the fact that humans working on the production units most often live in the immediate vicinity of the sources of risk and could therefore be viewed as potential risk source of the hazard and could be discussed under the previous heading. Some of the inputs that may be required under this


segment include the properties of the agent, presence of potential vectors, personnel and animal demographics (animal structure), management practices, environmental characteristics, disposal practices etc.

c) Consequence assessment – deals with the relationships of the specified exposures to a hazard and the consequences of such exposures. A causal process should exist by which exposure produces adverse health consequences. This assessment should be able to describe the potential consequences to a particular exposure and estimate the probability of them occurring. Some examples include Direct consequences (animal infection, production losses, public health consequences), indirect consequences (control costs, compensation costs, trade losses, adverse environmental effects, socio-economic losses)

d) Risk estimation – is a process that integrates all of the results obtained during the risk analysis process to produce overall measures of risk associated with a potential hazard (PRRSv). It encompasses the overall pathway from hazard identification to consequence assessment.

All of the above components of Risk assessment may be either quantitative or qualitative in nature. 3. Risk Management – based upon the input from previous sections of the

analysis, Risk Management is a process that will serve to decide upon and implement the measures to achieve appropriate level of protection of the production in question, ensuring the minimal negative effects on the trade and production. Some of the components of Risk Management include:

a) Risk evaluation - comparing the level of risk measured with the productions own level of protection

b) Option evaluation – identification and selection of the most feasible measures to provide the appropriate protection; focus on technical, operational and economical factors affecting the implementation of risk management options.

c) Implementation – following through with risk management decision and ensuring risk management measures are in place

d) Monitoring and review – ongoing process by which risk management measures are continually audited to ensure intended results.

Conclusion/Recommendations: Risk Assessment and Risk Management (Risk analysis) are useful epidemiological tools that should be incorporated into management practices of large swine productions in Croatia and elsewhere where similar situations occur. Risk analysis can and should be undertaken for every economically important swine pathogen in order to design and implement the best possible bio-security protocols, as well as reduce the negative effects of potential incursions to a minimum should they occur. Given its history with PRRS virus, and the general lack of information on the incidence and prevalence of the virus in “Backyard productions”, Croatian swine industry needs to take immediate steps to urge the policy makers to


implement measures that will correct the problem and provide them with invaluable information for the protection of their productions. References:

1. Cost Action FA0902, 2011. Euro PRRS. net - The Balkan Meeting on PRRS

Diagnostics. In Euro PRRS.net. Split, pp. 1 - 29. 2. Croatian Agricultural Agency, 2011. Annual Report 2010. Pig Breeding,

Križevci. Available at: www.hpa.hr. 3. Michael Thrusfield, 2005. Veterinary Epidemiology Third edit., Oxford:

Blackwell Publishing company. 4. OIE, 2010. Terrestrial Animal Health Code 14th ed., Paris (France): World

Organisation for Animal Health. Available at: www.oie.int. 5. Roić, B. et al., 2006. A serological survey of classical swine fever virus in wild

boar (Sus scrofa) from Croatia. Veterinarski Arhiv, 76, p.S65–S72. Available at: http://www.vef.unizg.hr/vetarhiv/papers/2006-76-7-9.pdf [Accessed September 26, 2011].

6. Tončić, J. et al, 2006. Health and Genetic Status of the European Wild Boar in Croatia. Radovi Šumarskog Instituta Jastrebarsko, (9), pp.223-236.

7. Župančić, Ž. et al., 2002. Prevalence of Antibodies to Classical Swine Fever , Aujeszky ’ s Disease , Porcine Reproductive and Respiratory Syndrome , and Bovine Viral Diarrhoea Viruses in Wild Boars in Croatia. Journal of Veterinary Medicine, B 49, pp.253-256.


Poster presentation

COORDINATED PROGRAM ON PRRSV IN THE

NETHERLANDS

T. Duinhof a, B. van Damb

a GD Animal Health Service, PO Box 9, Deventer, The Netherlands, [email protected] b Product Board for Livestock and Meat, PO Box 460, 2700 AL Zoetermeer, The Netherlands Initiated by the Dutch Product Board for Livestock and Meat, a national coordinated program on controlling PRRSv has been started in the Netherlands. The Dutch swine industry has a sound basis for an economic successful production, based on management- and biosecurity- practises, transport legislation, identification of pigs and registration of farms. Nevertheless, PRRSv should be controlled to further enhance the health status of the Dutch swine population. Controlling PRRSv will also lead to a reduced influence of other swine pathogens as well as a reduced level of antibiotic usage. These are extra arguments to start this program. Based on the successful results of PRRSv-control programs in the US, the goal is to apply these principles for control and eradication on swine farms in the Netherlands. As a first step a deskstudy has been performed to generate an overview of the science based tools that are available to control and eventually eradicate PRRSv. Results of the deskstudy indicate that the control of PRRSv is based on the same principles that will lead to eradication. These principles are based on sound internal and external biosecurity measures, including air filtration. The best chances to achieve success in controlling and eventually eradicating PRRSv will be found in a regional program, starting in an area of low swine density. Training and further education of workers in swine farms and swine transport will be inevitable to ensure the required level of hygiene and biosecurity to maintain the reached level of PRRSv-control. The process is now in the phase of identifying the right region to start the program with motivated swine farmers and veterinarians. Keywords: PRRSv, Control program, The Netherlands


Session 5 Role of vaccination and/or biosecurity in PRRS

control Chairs: Spyros Kritas


Biosecurity in PRRSV control/eradication: Research update and field applications

*Satoshi Otake1,2, Scott Dee1,3, Andrea Pitkin1,5,

Gordon Spronk3, Darwin Reicks4, Paul Ruen5, John Deen1

1Swine Disease Eradication Center University of Minnesota, MN, USA. 2Swine Extension & Consulting, Japan. 3Pipestone Veterinary Clinic, MN, USA. 4Swine Vet Center, MN, USA. 5Fairmont Veterinary Clinic, MN, USA. *Contact information: [email protected] or [email protected] Abstract For “sustainable freedom from PRRSV” (Dee 2011) to be a reality in US swine

industry, we must understand and manage the risk of area spread, especially airborne spread. The objectives of this paper are to update recent research information and field applications of biosecurity in PRRSV control/eradication, including SDEC production region model, and the use of air filtration to reduce the risk of PRRSV introduction in large commercial sow farms in high swine-dense regions in US. The results from these studies suggest that air filtration is an effective means to significantly reduce the risk of external PRRSV introduction to large breeding herds located in high swine-dense regions. Introduction PRRSV can be eliminated from farms1-2. However, re-infection is a frequent

event (Area spread)3. PRRSV elimination is a long term goal in US, committed by AASV (American Association of Swine Veterinarians) 2005, NPPC (National Pork Producer Council) 2010 and NPB (National Pork Board) 2011. For “sustainable freedom from PRRSV” (Dee 2011) to be a reality in US swine industry, we must understand and manage the risk of area spread, especially airborne spread. The objectives of this paper are to update recent research information and field

applications of biosecurity in PRRSV control/eradication. 1. Aerosol spread of PRRSV Recently, certain numbers of significant research findings were obtained

regarding aerosol transmission of PRRSV, such as: ・ Probability of aerosol transmission of PRRSV is virus variant dependent4. ・ Risk factors of aerosol spread of PRRSV can exist at both population base and

meterological base5-6. ・ Aerosol transport of infectious PRRSV can occur over long distances for up to

4.7km7 and 9.1km8 under field conditions.


2. SDEC production region model The objectives of this study were to evaluate airborne spread of PRRSV and

Mycoplasma hyopneoumoniae (M.hyo) under field conditions, and to test the efficacy of air filtration for the reduction of the risk of the airborne transmission and transport of those agents. Additionally, attempts to identify meteorological data associated with the airborne spread of both agents were made. SDEC UMN (Swine Disease Eradication Center, University of Minnesota) production region model was used for the study. The production region model incorporated 4 different facilities to represent 4 different farms in endemically PRRSV-infected region. The infected population source facility was located in the middle of the region with 3 other facilities of different biosecurity levels; High-1 (MERV 16, 14, or electrostatic mechanical air filtration system, along with insect, fomite, personnel and transport protocols), High-2 (antimicrobial air filtration system), and Medium (matching protocols except for filtration), surrounding it at equal distance of 120 m. The study was planned to run for 3 years and 39 replicates each of 4 weeks in duration. PRRSV MN-184, 1-18-2, 1-26-2, and M.hyo 232 were used to inoculate the pigs in the infected population source. Serum and nasal swabs were collected weekly from all pigs in the 3 recipient facilities in order to monitor the infection status of PRRSV and M.hyo in each population. Air was collected using a cyclonic collector inside of each recipient facility as well as from the exhaust fan of the infected population source facility daily during the study period, and tested for those agents. Finally, on-site weather station was placed on the study site and real-time weather data were collected. The study was completed on December 2010, and the summarized results and

conclusion will be presented during the meeting to support our observations: (1) airborne spread of PRRSV and M.hyo could occur under field conditions. (2) air filtration system successfully protected susceptible swine populations from the airborne spread of both agents. (3) certain weather patterns were documented during the episodes of airborne transmission of those agents. 3. Use of air filtration to reduce the risk of PRRSV introduction in large commercial sow farms in high swine-dense regions The objective of this study was to evaluate the efficacy of air filtration in large

commercial sow herds in swine-dense regions and calculation of its cost: benefit using data from filtered and non-filtered herds. Participant herds had to meet the following criteria. Filtered (treatment) herd: A PRRSV-negative sow herd with an inventory of > 2400 sows to which a validated air filtration system has been installed. Filtered herds have historically received naïve gilt replacements and semen from naïve AI centers and have practiced a scientifically validated program of biosecurity for indirect routes of PRRSV transmission such as personnel/fomites, transports, and insects. Participant herds have experienced > 3 new PRRSV introductions over the past 4 years. The herds were located in areas with > 4 pig sites within a 2-mile radius and neighboring sites had experienced PRRSV infection and clinical disease 3-6 months prior to the initiation of the


study. Unfiltered (control) herd: A sow herd which met the criteria defined for filtered herds, but which has not installed an air filtration system. To assess the impact of air filtration, we measured the followings: (1) differences in the frequency of virus introduction across treatment and control herd, defined as the detection of a PRRSV that differed by 2% in the ORF 5 region from previous viruses found in the herd. (2) cost of implementation of air filtration system on large sow herds. (3) differences in performance and profitability between treatment and control herds following analysis of production and financial data. The study was planned to run for 4 years (2008-2012), and is currently on going. As preliminary results from past 3 years, new PRRSV introduction has been

documented in 2/10 filtered herds; however, transport and personnel breaches were confirmed via diagnostic data and security camera recording. On the other hand, new virus introduction was observed in 28/30 (93%) in non-filtered herds. Of these 28 farms, 17 (62%) were infected one time, 7 (25%) were infected twice and 4 (13%) were infected three times. Chi-square analysis indicated that treatment herds were significantly less likely to become infected when compared to control herds (P = 0.0001). In preliminary conclusion, air filtration appears to be an effective means to reduce the risk of PRRSV-introduction to large commercial sow herds in swine-dense regions. Conclusion The results from these studies suggest that air filtration is an effective means to significantly reduce the risk of external PRRSV introduction to large breeding herds located in high swine-dense regions. With this air filtration technology proven to be efficacious over time, along with strict biosecurity practices for other means including people/fomites, insects and transport, the problems on area spread of PRRSV can be finally solved and “sustainable PRRSV freedom” (Dee 2011) in swine-dense regions will be reality. Acknowledgements The authors would like to thank: ・ PRRS CAP 1 and 2 ・ National Pork Board ・ Minnesota Pork Board ・ University of Minnesota Swine Disease Eradication Center board members ・ Pipestone Veterinary Clinic ・ Swine Vet Center ・ Fairmont Veterinary Clinic ・ Dr. Steve Pohl from South Dakota State University References 1. Dee et al. (1998) Vet Rec. 146, 211-213 2. Torremorell et al. (2000) Proc Leman Swine Conf. 27, 59-62 3. Lager et al. (2002) Swine Health Prod. 10, 167-170 4. Cho et al. (2006) Can J Vet Res. 70, 297-301


5. Dee et al. (2010) Virus Res. 154, 177-184 6. Dee et al. (2010) Vet Rec. 167, 976-977 7. Dee et al. (2009) Vet Res. 40, 1-13 8. Otake et al. (2010) Vet Microbiol. 145, 198-208


EVALUATING REGIONAL PRRSV ERADICATION PERSPECTIVES BASED ON A HERD-LEVEL RISK INDEX

FOR VIRUS PERSISTENCE AND REINTRODUCTION

Anna Fahrion1*, Heiko Nathues2, Elisabeth grosse Beilage2, Marcus Doherr1

1Veterinary Public Health Institute, Vetsuisse Faculty, University of Bern, Switzerland *corresponding author e-mail address: [email protected]

2Field Station for Epidemiology, University of Veterinary Medicine Hannover, Germany Abstract Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is widely distributed in the German pig population with an estimated herd prevalence of 85 to 90%. The perspectives for extensive PRRSV eradication efforts are difficult to predict. On herd level, various strategies have proven effective to achieve freedom from PRRSV, but the cost-effectiveness of such efforts depends on the risk of reintroduction of the virus once it has been eradicated. In this study, we want to substantiate the perspectives for the pig herds in two pig-dense counties in Northwestern Germany of staying endemically infected or getting re-infected when eradication is attempted. The objective is to propose a methodological approach that can support assessing the feasibility of a sustainable PRRSV eradication for herds and regions. It may allow to geographically locate areas with higher or lower chances of maintenance of a PRRSV-free status when undergoing eradication. For this purpose, anonymized data for all pig herds (n = 2416) of two Lower Saxonian counties were obtained from their veterinary administrations. Datasets included exact herd sizes as reported by the farmers to the reimbursement fond for epizootic diseases, as well as geo-reference data providing information on proximity to neighboring herds. Using this information, and based on knowledge from literature and expert opinion, an index was calculated for each herd, relating to its respective risk of endemic virus circulation in the herd and reinfection during and after virus eradication. When analyzing the geographical distribution of the indexed herds, we identified regions with comparably high risk of (re)introduction and endemic circulation of the virus as well as “lower risk” regions with better chances for sustained disease freedom past eradication. On this basis, suggestions on implementation of eradication measures could be developed and possibly get extended to other regions in Germany. Keywords: PRRSV eradication, Geo-spatial analysis, Northwestern Germany


ELIMINATION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME (PRRS) WITH SERUMIZATION

– preliminary results

Marina Štukelj1, Ivan Toplak2, Zdravko Valenčak1

1University of Ljubljana, Veterinary faculty, Institute for the health care of pigs, Gerbičeva 60, 1000 Ljubljana, Slovenia

2University of Ljubljana, Veterinary faculty, Institute for microbiology and parasitology, Gerbičeva 60, 1000 Ljubljana, Slovenia

Contact Email: [email protected],

Abstract One of the methods for elimination of PRRSV from infected farm is serumization. Serumization consists of the intramuscular injection of all breeding pigs with serum derived from acutely infected pigs, which contains a farm-specific isolate of PRRSV. Serumization was performed on two PRRSV infected farms where the decrease production results and reproductive failure were noticed. Farm 1 had 74 breeding pigs and farm 2 134. PRRSV positive animals were identified by RT-PCR method and positive sera from these animals were used as inoculum for serumization. Blood samples were collected from all breeding pigs, before starting with herd closure and serumization, the immune status of herds was controlled with serological testing of all breeding animals 3 and 6 months after serumization. On both farms all breeding pigs were immune and without virus 6 months after serumization. During the study on the farm 1 new boar was introduced into the farm without any testing as well as group of fatteners. Also, own gilts were introduced to the breeding herd. One year after serumization decrease of production results and the new outbreak was confirmed by detecting PRRSV by RT-PCR. The farmer on farm 2 strictly followed the biosecurity protocol and herd closure. Six months after serumization the S/P ratio in breeding herds decreased and breeding animals were without PRRSV and some of the sows were already negative. Only in group of weaners (age of two months) the PRRSV still circulate. The partial depopulation of weaners was suggested for elimination the PRRSV from the herd. Key words: pig, PRRS, elimination, serumization Introduction Porcine reproductive and respiratory syndrome PRRS is endemic in most swine-producing countries and is associated with major economic losses (Wayne et al., 1994). The disease is characterized by severe reproductive failure including late-term abortions, early farrowing, stillbirths, and weak born piglets in sows and increased mortality in neonates, nursery and growing pigs as well as by a respiratory tract illness that can be especially severe in neonatal and nursery-age pigs (Wensvoort, 1993).


Control efforts begin by increasing breeding herd immunity, than work progressively toward control in growing pigs through partial depopulation, all-in /all-out pig flow, vaccination and intentional exposure to field virus or a combination of approaches (Zimmerman, 2008). An attempt for PRRS control in the commercial pig industry was described with many vaccine schemes, commercial and autogenous vaccines, serum inoculations and natural exposure. Increasing evidence indicates that PRRSV strains differ in virulence and are biologically, antigenically, and genetically heterogeneous (Meng, 2000). Therefore, it appears that the vaccines currently available contains a single strain of PRRSV, may not be effective and protecting against infections with genetically different strains of PRRSV (Meng, 2000). Consequently, procedures that expose pigs to the homologous herd strain have represented successful approach being implemented in many countries (Batista et al., 2002). Serumization is the intentional immunization of pigs with homologous strain of PRRS virus originated from the infected farm (Dee, 2009). Herd closure is required to achieve herd stability. This applies to both internal replacements as well as replacements purchased from a breeding stock company (Torremorell et al., 2002). The very important measure is to follow the strict biosecurity protocol. The objective of this study was to eliminate PRRSV from two small pig farms in Slovenia with serumization. Materials and methods Two pig farms Both farms were free of Aujeszky disease and classical swine fever. Farm 1 has two boars and 72 breeding sows. Farm 2 has four boars and 130 breeding sows. On farm 1 the serumization was performed once on day 30, on the farm 2 two times in period of three months, first time on day 30 (one months after confirmation of PRRS) and second time on day 120 that was three months after first serumization with prepared inoculum for serumization in doses 2 ml/pig. The owners accepted strict biosecurity protocols and herd closure for at least 200 days. The herd closure did not allow introduction of new breeding pigs even from their own production. Sampling Farm 1: Sampling for serology: On farm 1all together 292 blood samples were taken (Table 1). Sampling for PRRSV detection (RT-PCR method for PRRSV genome detection): Day 9: 5 weaners 8 weeks of age, 5 weaners 10 weeks of age and 5 weaners 12 weeks of age. Day 357: 4 weaners 6 weeks old, 5 weaners 8 weeks old, 5 growers 10 weeks old, 5 growers 12 weeks old and 5 growers 14 weeks old. Farm 2:


Sampling for serology: On farm 2 all together 519 blood samples were taken (Table 2). Sampling for PRRSV detection (RT-PCR method for PRRSV genome detection): Day 6: 5 weaners 6 weeks of age, 5 weaners 8 weeks of age and 5 growers 10 weeks of age. Day 129: 5 weaners 8 weeks old, 5 growers 10 weeks old and 5 growers 12 weeks old. Day 255: 133 breeding sows. Day 319: 88 breeding sows and 20 growers. ELISA The HerdChek, IDEXX Laboratories, PRRS X3 ELISA test was used for detection of antibodies. The interpretation results of samples were divided in four groups as followed: negative samples, samples with S/P less than 1 (low positive), samples with S/P between 1 and 2 (positive) and samples with S/P more than 2 (high positive). Detection of PRRSV with gel-based RT-PCR and direct sequencing of PRRSV positive samples Serum samples form both farms were tested individually or as pools (5 samples in pool) by One-step RT-PCR (One-Step RT-PCR Kit, Qiagen, Germany) using degenerated primer sequences based on the open reading frame 7 (ORF7), with the forward primer P1 5’-CCA GCC AGT CAA TCA RCT GTG-3’ and the reverse primer P2 5’-GCG AAT CAG GCG CAC WGT ATG-3’ (Donadeu et al., 1999). Fifteen PRRSV positive samples (from farm 1 four samples and from farm 2 eleven samples) were directly sequenced from RT-PCR products in both directions using the Macrogen sequencing service (Macrogen, South Korea) and the RT-PCR amplification primers. Preparation of inoculums for serumization of breeding pigs Inoculums for serumization of breeding pigs were prepared from individual serum samples previously determined by RT-PCR as PRRSV positive. Identified PRRSV positive serum samples on each farm were mixed in one pool. To one part of pool four parts of RPMI-1640 medium (Gibco, Germany) and 1% of Antibiotic-Antimycotic (100x), (Invitrogen) was added. Results Serology In column “pigs” all the categories from the weaning pigs to the fatteners are combined. On the Day 210 (six months after serumization) number of high positive pigs decreased. Four weaners and five fatteners were high positive, one


weaner was positive on the day 348 (eleven months after serumization). The production results on the farm litters at the beginning of the study were fifty percents of returns to oestrus, small with unhealthy piglets, from 12 gilts 6 did not wean any piglet, while other 24 gilts weaned only 4 in average. Three months after serumization the improvement in production results was noticed (9,5 healthy piglets per sow were weaned). Table 1 The results of serology (ELISA for detection of PRRS antibodies) on farm 1.

No. of tested pigs

No. of negative pigs

No. of low positive pigs

No. of positive pigs

No. of high positive pigs

Sampling

Breeding pigs

Pigs Breeding pigs

Pigs Breeding pigs

Pigs Breeding pigs

Pigs Breeding pigs

Pigs

Day 0 10 10 0 0 0 0 2 0 8 10 Day 30 74 0 1 0 4 0 10 0 59 0 Day 90 74 0 1 0 0 0 17 0 56 0 Day 210 75 0 3 0 13 0 24 0 35 0 Day 348 0 10 0 0 0 0 0 1 0 9 Day 375 0 24 0 3 0 2 0 8 0 11

Table 2 The results of serology (ELISA for detection of PRRS antibodies) on farm 2.

No. of tested pigs

No. of negative pigs

No. of low positive pigs

No. of positive pigs

No. of high positive pigs

Sampling

Breeding pigs

Pigs Breeding pigs

Pigs Breeding pigs

Pigs Breeding pigs

Pigs Breeding pigs

Pigs

Day 0 5 5 0 0 0 0 3 0 2 5 Day 22 134 0 13 0 24 0 43 0 54 0 Day 108 134 0 6 0 21 0 51 0 56 0 Day 225 133 0 2 0 18 0 10 0 83 0 Day 319 88 20 2 4 12 3 39 7 35 6

In column “pigs” all the categories from the weaning pigs to the fatteners are combined. Three months after first serumization (Day 108) still six breeding pigs were negative and 21 breeding pigs with low S/P ratios, so we decided to perform the second serumization. Six months after second serumization (Day 319) the stabilization of breeding herd was evident (decrease of S/P ratio were noticed, even some of the breeding sows were negative as well production results improved). PRRSV detection and genetic typing The results of PRRSV detection by RT-PCR method and sequencing of PRRSV on farm 1 From the 15 tested samples, 10 samples were identified as PRRSV positive (detection of RT-PCR products of about 300 bp). Ten PRRSV positive samples in


farm 1 were detected among 8 and 10 weeks old weaners. The sequencing results of the partial nucleocapsid protein gene (ORF7) from farm 1 positive sample with name Stra8t/2009 (HQ213911) revealed the circulation of PRRSV strain which shared 89,1% nucleotide identity with the Lelystad virus. Eleven month after serumization all 10 serum samples collected from 4-14 weeks old weaners and growers were negative for PRRSV by RT-PCR. Six weeks after these negative results, 24 serum samples were collected from weaners in ages between 6 and 10 weeks and tested for the verification of PRRSV negative status of farm 1. Surprisingly out of 24 tested samples, 2 positive pigs (6 and 10 weeks of age) were detected as PRRSV positive. The observed homology in 258 nt of ORF 7 between four sequences of PRRSV strains circulating between 2009 and 2011 was 99,2-100% (Fig. 1). The results of PRRSV detection by RT-PCR method and sequencing of PRRSV on farm 2 From the 15 tested samples, 15 samples were identified as PRRSV positive by gel-based RT-PCR. Four months later PRRSV was detected in 15 serum samples (strain Meol/2011) collected from 8-12 weeks old weaners, with 98,4% nucleotide identity to previous PRRSV (06086t/2010) and second serumization in farm 2 was done by the detected PRRSV strain Meol/2011. Three month later 133 samples from farm 2 (all pigs) were negative for PRRSV by RT-PCR. Two month later another 7 samples (gilts) were negative for PRRSV by RT-PCR. A week later 108 samples were tested again and 100 (breeding sows) were resulted as negative, while 8 serum samples (weaners and growers between 6 and 10 weeks) were PRRSV positive by RT-PCR. The observed sequence homology between eleven sequences of two groups from farm 2 was 96,9-98,4% suggesting that possible second strain was introduced into farm 2 during our study between 2010 and 2011. Discussion Considering heterogenity of PRRS serotypes and importance of homologues immunity, we believe that serumization can be successful measure in combination with herd closure for elimination or even eradication of PRRS from the farm. On both farms, herd closure was implemented. Introduction of new pigs to the farm and introduction of gilts from own production to breeding herd was not allowed. Three months after serumization we confirmed the stability in breeding herd on farm 1 by ELISA testing (Table 1) and great improvement in production results. In that period they weaned 9,5 healthy piglets per sow. Six months after serumization we noticed decrease of S/P ratio in breeding herd. With epidemiological investigation we confirmed that after serumization new boar was introduced to the farm without any testing as well as fatteners for further fattening, and introduced own gilts to the breeding herd. The boar and new fatteners were positive for antibodies against PRRSV. One year after serumization the owner noticed decreased production results. 24 serum samples were collected from weaners and growers. Two pigs (6 and 10 weeks of age) were detected as PRRSV positive. The observed homology between farm strain from 2009 and 2011 was 99,2-100%. The


elimination of PRRSV from the breeding herd can not be achieved without following the rules. At the farm 2, after first serumization the immunity was not sufficient according to results from ELISA. It is possible that the quantity of the virus for the serumization was not sufficient. Three months after the second serumization the breeding herd become stabile and the improvement of production results was noticed. All serum samples tested by RT-PCR from all breeding animals were negative three months after the second serumization. The owner followed the strict biosecurity protocol as well as strict herd closure. Six months after second serumization S/P ratio in breeding herds decreased and some of the sows were negative. All breeding animals were without virus proved by RT-PCR. We achieved stabile breeding herd but in weaners at age of two months the PRRSV still circulate which was proved by RT-PCR. Not implemented the all in all out protocol as well as one site production system is probably the main reasons why we still have the virus in this category of pigs on the farm 2. We believed that the partial depopulation could stop shedding of the PRRSV as was published before (Dee et al. 1993). References

1. Batista, L., Pijoan, C., Torremorell, M., 2002. Experimental injection of gilts with porcine reproductive and respiratory syndrome virus (PRRSV) during acclimatization. J. Swine health Prod. 10(4), 147-150.

2. Dee, S.A., Morrison, R.B., Joo, H.S., 1993. Eradication porcine reproductive and respiratory syndrome (PRRS) virus using two-site production and nursery depopulation. Swine Health band Prod. 5, 20-23.

3. Dee, S.A., 2009. PRRS control. - (accessed on 05/05/2011) http://www.pig333.com/prrs/pig_article/556/prrs-control.

4. Donadeu, M., Arias, M., Gomez-Tejedor, C., Aguero, M., Romero, L.J., Christianson, W.T., Sanchez-Vizcaino, J.M., 1999. Using polymerase chain reaction to obtain PRRSV-free piglets from epidemically infected herds. Swine Health Prod. 7, 225-261.

5. Meng, X.J., 2000. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet. Microbiol. 74, 309-329.

6. Torremorell, M., Christianson, W.T., 2002. PRRS eradication by Herd Closure. Advance in Pork Production. 13, 169-176.

7. Wayne, R.F., Han, S.J., 1994. Cessation of porcine reproductive and resperiratory syndrome (PRRS) virus spread in a commercial swine herd. Swine Health and Production. 2 (1), 13-15.

8. Wensvoort, G., 1993. Lelystad virus and the porcine epidemic abortion and respiratory syndrome. Vet. Res. 24, 117-124.

9. Zimmerman, J., 2008. Porcine reproductive and respiratory syndrome virus (PRRSV): The disease that keeps bugging us. London swine conference – Facing the New Reality, 1-2 April 20.


DETERMINATION OF THE CORRELATION BETWEEN CROSS REACTIVITY PROFILES OF NEUTRALIZING ANTIBODIES AND CROSS-PROTECTION IN PRRSV

INFECTIONS Francisco Javier Martínez-Lobo1, Francisco Díez-Fuertes1, Isabel Simarro1, José

María Castro1, Cinta Prieto1 1Departamento de Sanidad Animal, Facultad de Veterinaria, Madrid, Spain. Abstract Neutralizing antibodies (NAs) are known to play a role in protection against PRRSV infection and consequently cross-reactivity of NA might be relevant in cross-protection. The aim of this study was to determine whether cross-neutralization in vitro is related to cross-protection in vivo. For this purpose, 47 two-month-old PRRSV seronegative pigs were immunized three times with an isolate that induces cross-reactive NAs. 4 weeks after the last immunization pigs were divided into nine groups, to which 3 seronegative age-matched pigs were added as controls. Each group was exposed to one of nine heterologous PRRSV isolates. Blood was taken at selected times post-challenge (p.c.) to study NAs response (4) and viremia by virus isolation. All pigs were sacrificed 21 days p.c. and tonsils were collected to determine PRRSV presence by RT-nPCR. A second study was carried out with the same experimental design but pigs were immunized with an isolate that induces only strain-specific NAs. In both studies pigs developed detectable levels of NAs against the isolate used for immunization. However, and as expected, cross-reacting NAs were frequently found the day of challenge in pigs of most of the groups of Study 1 while were almost absent in most the groups of Study 2. Regarding virological parameters, in study 1 viremia was not observed after challenge with 5 of the heterologous viruses and it was only sporadically recorded in pigs of the remaining 4 groups. On the contrary, viremia was frequently detected in pigs of all groups in Study 2, regardless of the virus used for challenge. Moreover, all tonsils obtained from pigs of Study 2 were positive by RT-nPCR and the nucleotide sequences confirmed that the virus detected was the challenge isolate. On the contrary, in the Study 1, 34 out of 53 tonsil samples were positive by RT-nPCR and only 16 samples were identified as positive for challenge virus. Keywords: Neutralizing antibodies, cross-protection


List of participants

Title First Name Last Name Email

Dr Gor Abgaryan [email protected]

Mrs Zaklin Acinger-Rogic [email protected]

Mrs Sonja Agten [email protected]

Dr Tahar Ait-Ali [email protected]

Dr Massimo Amadori [email protected]

Dr Gyula Balka [email protected]

Dr Andrea Ballagi [email protected]

Dr Stelian Baraitareanu [email protected]

Dr Perica Bicvic

Dr Anette Bøtner [email protected]

Dr Sara Botti [email protected]

Dr Mato Bozic

Dr Vanja Brankov [email protected]

Mr Manreetpal S. Brar [email protected]

Mr Kisin Bratislav [email protected]

Mrs Jurate Buitkuviene [email protected]

Mr Alexel Burgara [email protected]

Mrs Ann Brigitte Cay [email protected]

Prof Vladimir Celer [email protected]

Dr Luminita Costinar [email protected]

Mr M. Kiyaari Dawuda [email protected]

Dr Klaus Depner [email protected]

Dr Vuk Djuric [email protected]

Dr Michele Drigo [email protected]

Mr Tom F. Duinhof [email protected]

Dr Michal Fabisiak [email protected]

Dr Anna Sophie Fahrion [email protected]

Dr Bogdan Faur [email protected]

Dr George Filioussis [email protected]

Dr Jean-Pierre Frossard [email protected]

Prof Mladen Gagrcin [email protected]

Ms Jesús Galán Enguídanos [email protected]

Dr Patrice Gracieux [email protected]

Dr Simon Graham [email protected]

Prof Elisabeth Grosse Beilage [email protected]

Dr Bernd Grosse Liesner [email protected]

Prof Viorel Herman [email protected]

Dr Charlotte K Hjulsager [email protected]

Dr Derald Holtkamp [email protected]

Dr Anna Jackova [email protected]

Dr Jitka Jankova [email protected]

Dr Djana Jelic [email protected]

Dr Andreja Jungic [email protected]

Prof Avo Karus [email protected]

Dr Tamás Kis [email protected]

Dr Stane Kosorok [email protected]


Prof Spyridon Kritas [email protected]

Mr Branislav Kureljusic [email protected]

Miss Lise Kirstine Kvisgaard [email protected]

Prof Lars Larsen [email protected]

Prof Sava Lazic [email protected]

Mrs Marie-Frédérique Le Potier [email protected]

Prof Frederick Leung [email protected]

Dr Zoran Lipej [email protected]

Mrs Jelena Maksimovic-Zoric [email protected]

Dr Beno Marinic [email protected]

Dr Francisco Javier Martinez-Lobo [email protected]

Dr Radoslav Martinov [email protected]

Dr Enric Mateu [email protected]

Dr Velimir Mikalacki [email protected]

Mr Sinisa Milic [email protected]

Dr Vesna Milicevic [email protected]

Mr Jovan Mirceta [email protected]

Mr Vijay R Monger [email protected]

Prof Robert Morrison [email protected]

Dr Dinko Novosel [email protected]

Ms Jelena Obadovic [email protected]

Prof Vassilis Papatsiros [email protected]

Dr Corina Pascu [email protected]

Dr Alojz Petroci [email protected]

Dr Tamas Petrovic [email protected]

Dr Vladimir Polacek [email protected]

Dr Cinta Prieto [email protected]

Mrs Jasna Prodanov-Radulovic [email protected]

Mr Rolf Rauh [email protected]

Dr Annemarie Rebel [email protected]

Dr Sandra Revilla-Fernández [email protected]

Dr Dragan Ristevski [email protected]

Dr Trpe Ristoski [email protected]

Dr Dragan Rogozarski [email protected]

Prof Peter Rottier [email protected]

Dr Drazen Rukovalski [email protected]

Dr Dejvid Sabolek [email protected]

Dr Björn Schröder [email protected]

Mr Radoslav Sevic [email protected]

Ms Eva Slaninková [email protected]

Prof Tomasz Stadejek [email protected]

Dr Arunas Stankevicius [email protected]

Dr Perica Stankov

Dr Ognjen Stevancevic [email protected]

Mr Nenad Stojanac [email protected]

Mrs Marina Stukelj [email protected]

Dr Samuel Thevasagayam [email protected]

Dr Ivan Toplak [email protected]

Dr Zdravko Valencak [email protected]


Dr Wander Van Breedam [email protected]

Dr Eric Van Esch [email protected]

Dr Dejan Vidanovic [email protected]

Prof Stefan Vilcek [email protected]

Dr Jess Waddell [email protected]

Mr Pawel Wróbel [email protected]

Mrs Mihaela Zaulet [email protected]

Dr Spira Zelic [email protected]

Mr Beckei Žolt [email protected]

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