Abstract Book of EAVLD2012 - eavld congress 2012

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S3 - O - 09 DIFFERENT DIAGNOSTIC TOOLS FOR A BROAD RANGE OF MACAVIRUSES AND THEIR RESERVOIR AND SUSCEPTIBLE HOSTS Christine Foerster 1 , Matthias Koenig 1 , Heinz-Juergen Thiel 1 , Jens-Ove Heckel 2 1 Institute of Virology, diagnostic laboratory, Justus-Liebig-University Giessen, Germany 2 Zoo Landau, Germany Macavirus, malignant catarrhal fever, reservoir and susceptible host Introduction Malignant catarrhal fever (MCF) is a fatal disease of Artiodactyla, which is caused by different macaviruses within the subfamily Gammaherpesvirinae (GHV) of the family Herpesviridae. Until now there are at least 6 distinct macaviruses known to cause MCF: Alcelaphine herpesvirus 1 (AlHV-1), ovine herpesvirus 2 (OvHV-2), caprine herpesvirus 2 (CpHV-2), white tailed deer- MCF-virus (MCF-WTD), an unclassified macavirus from ibex and a virus resembling alcelaphine herpesvirus 2 (AlHV-2). Other closely related viruses are the hippotragine herpesvirus 1 (HiHV- 1 in roan antelope) and GHV in springbok, impala, oryx, muskox and aoudad. MCF usually occurs only sporadically. Within the framework of differential diagnostics the detection of macaviruses was stimulated since the first cases of bluetongue disease were noticed in northern Europe. Typical reservoir hosts which do not develop clinical disease are sheep, goat and wildebeest. Several species of Artiodactyla are susceptible to MCF, for example bison, cattle, deer and moose. In addition pigs, goats and experimentally infected rabbits show typical clinical symptoms like fever, mucosal erosions, nasal and lacrimal discharge, corneal opacity and skin lesions. Undirected virological methods like virus propagation in cell culture and electron microscopy are not promising with regard to macaviruses. PCR is the method of choice for laboratory diagnosis. Until now most laboratories use the OvHV-2 specific PCR (Baxter et al., 1993) for samples of cattle. Objective of this work was to compare and improve the diagnostic tools for the detection of a broad spectrum of potential MCF-viruses. Discussion & conclusions The competitive ELISA was clearly superior to SNA with regard to time, effort and quality of result. ELISA is recommended for routine diagnostic use, especially for reservoir hosts. The importance of broadly reactive PCR methods was demonstrated by 18 positive results of wildlife samples. All 18 cases led to negative results in the OvHV-2-specific PCR, but reacted positive in the PAN-Macavirus-PCR. Cloning, sequencing and phylogenetic analyses of the amplificates led to the detection of CpHV-2, CpHV-2-like and BoHV-6-like macaviruses. For the first time CpHV-2-induced MCF was detected in two Banteng-cattle. Until now, CpHV-2 has been considered as a cause of MCF only in deer and moose. This implies that routine OvHV-2-specific-diagnostic in cattle is insufficient. The establishment of an OvHV-2-CpHV-2-discriminating realtime PCR allowed rapid differentiation between the two most common causative agents of MCF in Europe. This method is suitable for application in routine diagnostics of small ruminants and cattle. For the application in wildlife samples the conventional PCR is more suited, because primer pairs were selected for a broad range of macaviruses and the conventional PCR appeared to be more sensitive. References 1. Baxter, S. I., Pow, I., Bridgen, A. and Reid, H. W. (1993). PCR detection of the sheep-associated agent of malignant catarrhal fever. Arch Virol 132 (1-2), 145-59. 2. Russell, G. C., Stewart, J. P. and Haig, D. M. (2009). Malignant catarrhal fever: A review. Vet J 179 (3), 324-35. Materials & methods Two new conventional PCR-methods and an OvHV-2-CpHV-2- discriminating realtime PCR were established, which allowed to detect a broad spectrum of macaviruses. They were compared to the commonly used OvHV-2-specific PCR. Amplified fragments were cloned, sequenced and used for phylogenetic analyses. Furthermore two antibody-test-systems, ELISA and serum neutralisation assay (SNA), were compared to each other. Samples of 276 wild and zoo animals, comprising 54 different genera of the family Bovidae, Cervidae, Camelidae and Giraffidae, 230 goats and 58 sheep were examined. Results 39 cases out of 276 wild ruminants (14 %) led to a positive result in at least one procedure. In approximately half of the samples (19) OvHV-2 was identified, in 18 CpHV-2 or CpHV-2 like virus. Samples of one waterbuck contained sequences related to BoHV-6. 19 of 39 macavirus-positive animals showed MCF like disease. CpHV-2 was identified in conjunction with two cases of MCF in Banteng cattle. 5 out of 230 goats (2 %) showed MCF like symptoms and 4 of them also pathological and histological signs of MCF. In all samples of these animals OvHV-2 could be demonstrated. Samples of 92 (42 %) goats obtained positive results in at least one PCR method. Interestingly single and also double infection of OvHV-2 and CpHV-2 were found. In contrast to the goats only OvHV-2 was found in 27 sheep samples (47 %). Realtime PCR was able to detect OvHV-2 and CpHV-2 in all cases of MCF-diseased animals. In latently infected animals high cycle treshold values were found. In some reservoir hosts antibodies were found but no viral DNA. 33 of 59 animals (56 %) were found positive via ELISA, but negative by SNA, while only one animal (1,6 %) was estimated positive by SNA and negative via ELISA.
S3 - O - 10 DIAGNOSTIC ASPECTS OF SUID HERPESVIRUS 1 INFECTION IN WILD BOAR Adolf Steinrigl, Sandra Revilla-Fernández, Eveline Wodak, Zoltán Bagó, Friedrich Schmoll AGES Institute for Veterinary Disease Control, Mödling, Austria SuHV-1, wild boar, prevalence, molecular characterization Introduction Many infectious diseases that are strictly controlled in livestock may remain unnoticed in wild animals, posing the risk of entering domestic animal populations with potentially devastating consequences. Suid herpesvirus 1 (SuHV-1), the causative agent of Aujeszky’s disease (AD), was eradicated from domestic pig populations in many European countries, including Austria. At the same time, SuHV-1 infection is common in European wild boar, as shown by a number of studies, reporting detection of SuHV-1 DNA or antibodies in wild boar (1). An additional indicator of SuHV-1 presence in wild boar are occasional cases of AD in hunting dogs, usually in close connection to hunting events (2). Recently, we reported about the first detection of SuHV-1 in Austrian wild boar and the molecular characterization of SuHV-1 field isolates from wild boar and hunting dogs (3). In the course of the Wild Animal Survey 2011, a nationwide study that was partly financed by the Austrian Federal Ministry of Health, additional sera and tissue samples from Austrian wild boar were collected and analysed, with the aim to provide a representative estimate of the prevalence of both, SuHV-1 DNA and antibodies, in Austrian wild boar. This work shows the results of this survey and reflects our experiences with detection and molecular characterization of SuHV-1 in wild boar. Materials & methods Wild boar tissues and sera were sampled according to a survey design that was based on recent wild boar hunting bag statistics. Anti-SuHV-1 gB ELISA, real-time PCR (qPCR), amplification and sequencing of the SuHV-1 glycoprotein C coding region, phylogenetic analysis and virus isolation were performed as described previously (3). Results From April 2011 to January 2012, tissues from 298 wild boars were sampled, corresponding to 78% of the scheduled sample size for the whole country. Wild boar sera of sufficient amount and quality were available from 259 animals. Results of serological and virological screening are shown in Table 1. All qPCR positive wild boars were adult females at 2-4 years of age. One of them was also serologically positive, while the other two animals were seronegative. Table 1: Results of serological (ELISA) and virological (qPCR) screening of Austrian wild boars Samples Samples Prevalence tested positive ELISA 259 59 22.8% qPCR 298 3 1.0% Virus was successfully isolated from one of the qPCR-positive, yet seronegative, wild boars: tissue homogenate from a qPCRpositive tonsil was inoculated onto PK15 cells, resulting in massive cytopathic effect within 24 hours post inoculation. An attempt to isolate virus from the other two qPCR positive animals was unsuccessful. Phylogenetic analysis of SuHV-1 gC sequences amplified from the three qPCR positive wild boars sampled in 2011 showed that all three animals hosted virus that was identical in gC sequence and belonged to one of the previously described lineages of SuHV-1 (3), which are currently present in Austria (Figure 1). Figure 1: Neighbour Joining tree, based on a 639 bp alignment of the SuHV-1 partial gC coding region. Sequences were amplified from wild boars (WB), hunting dogs (HD) and from a domestic pig (DP). Year of isolation is in brackets. Numbers along the branches indicate the percentage of 1000 bootstrap replicates. Discussion & conclusions The presented study confirms the presence of SuHV-1 in Austrian wild boar. SuHV-1 seroprevalence detected during the Wild Animal Survey 2011 was lower than that previously described (3), which is probably due to the more representative sampling strategy applied for the current study. In contrast to the high numbers of wild boars with detectable SuHV-1 antibodies, SuHV-1 DNA was detected by qPCR in only a few animals. Furthermore, Cq-values of qPCR-positive tissue pools or individual organs were relatively high (30 - 38 Cq), indicating a low viral load in these tissues. This is also supported by our previous observation of weak immunohistochemistry signals in qPCR positive tissues (3) and by the fact that virus isolation was positive in a single case only. In summary, our results indicate that most SuHV-1 infected wild boars harbour virus in a latent form, whereas only a small proportion of infected wild boars is actually shedding virus and may infect other susceptible animals. This is also the most likely explanation for the relatively rare cases of AD in hunting dogs, despite rising numbers of wild boars in many European countries and the frequent involvement of hunting dogs in wild boar hunting. Despite the fact that only few potentially virus shedding wild boars were found in this study, direct contact between wild boars and domestic pigs could result in SuHV-1 transmission. Feeding of wild boar intestines to domestic animals, especially pigs, dogs and cats, is another reasonable possibility of transmission and must be strictly discouraged. Acknowledgements This study was financially supported in part by the Austrian Federal Ministry of Health. The excellent technical assistance of the laboratory staff of the Departments for Molecular Biology, Virology and Electron Microscopy, and Pathology is acknowledged. References 1. Müller T, Hahn EC, Tottewitz F, Kramer M, Klupp BG, Mettenleiter TC, Freuling C (2011). Pseudorabies virus in wild swine: a global perspective. Arch. Virol., 156, 1691-705. 2. Cay AB, Letellier C (2009). Isolation of Aujeszky’s disease virus from two hunting dogs in Belgium after hunting wild boar. Vlaams Diergeneeskundig Tijdschrift, 78, 194-195. 3. Steinrigl A, Revilla-Fernández S, Kolodziejek J, Wodak E, Bagó Z, Nowotny N, Schmoll F, Köfer J (2011). Detection and molecular characterization of Suid herpesvirus type 1 in Austrian wild boar and hunting dogs. Vet Microbiol., Dec 30. [Epub ahead of print]
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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