Keywords: PRRSV, wild boar, seroprevalence, phylogenetic analyses

Transcription

1 54 EUROPRRS2011, Novi Sad, Serbia PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) INFECTION IN LITHUANIAN WILD BORS (SUS SCROFA) POPULATION Arunas Stankevicius 1, Jurate Buitkuviene 1, Jurgita Valanciute 1, Rytis Cepulis 1, Tomasz Stadejek 2 1 Faculty of Veterinary Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania; sarunas@lva.lt 2 National 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

2 55 EUROPRRS2011, Novi Sad, Serbia 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/ 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 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). Gelpurified 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.

3 56 EUROPRRS2011, Novi Sad, Serbia 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.

4 57 EUROPRRS2011, Novi Sad, Serbia 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.

5 58 EUROPRRS2011, Novi Sad, Serbia 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 Vet Microbiol 77, Bonilauri et al. (2006). Presence of PRRSV in wild boar in Italy. Vet Rec 158, 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: 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, 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, 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, Reiner et al. (2009). Porcine reproductive and respiratory syndrome virus (PRRSV) infection in wild boars. Vet Microbiol 136, Roic et al. (2008). Serological evidence of reproductive and respiratory síndrome virus (PRRSV) in wild boars (Sus scrofa) in Croatia. In IPVS Congr, Durban, Saliki et al. (1998). Serosurvey of selected viral and bacterial diseases in wild swine from Oklahoma, J Wildl Dis 34, Shi et al. (2010). Molecular epidemiology of PRRSV : A phylogenetic perspective. Vir Res 154, 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,

6 59 EUROPRRS2011, Novi Sad, Serbia 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, 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, Wu et al. (2011). Porcine reproductive and resiratory síndrome in hybrid wild boars, China. Emer Infec Dis 17, No 6, Wyckoff et al. (2009). Feral swine contact with domestic swine: a serologic survey and assessment of potential for disease transmission. J Wildl Dis 45,