The Veterinary Journal 200 (2014) 200–203
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Prevalence of asinine herpesvirus type 5 (AsHV-5) infection in clinically normal Lipizzaner horses James Oliver Rushton a,b, Jolanta Kolodziejek a, Barbara Nell b, Norbert Nowotny a,c,⇑ a Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria b Clinic for Surgery, Dentistry and Ophthalmology, Department of Companion Animals and Horses, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria c Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
a r t i c l e
i n f o
Article history: Accepted 31 October 2013
Keywords: Horse Asinine herpesvirus (AsHV) type 5 Equid gammaherpesviruses Lipizzaner
a b s t r a c t The aim of this study was to assess the extent of asinine herpesvirus (AsHV) type 5 infection in ‘closed’ populations of clinically normal Lipizzaner horses. Peripheral blood mononuclear cells plus nasal and conjunctival swabs were obtained on four occasions over an 18 month period from 266 animals as part of a health surveillance programme. Sequence analysis of samples that were positive by nested consensus herpesvirus PCR but negative using quantified equid herpesvirus (EHV) type 2 and 5 PCR, revealed a total of 51 samples from 39 horses positive for AsHV-5. No statistically significant association between animal age, gender or geographical location and infection status was identified. The findings suggest sub-clinical AsHV-5 infection may be encountered more frequently than previously reported. Ó 2013 Elsevier Ltd. All rights reserved.
Asinine herpesvirus (AsHV) infection has been sporadically reported in horses in recent years (Back et al., 2012; De Witte et al., 2012) and associated with pyogranulomatous pneumonia and equine multinodular pulmonary fibrosis. The detection of AsHV-5 in the respiratory fluids of horses not exhibiting signs of respiratory disease has also been documented (Fortier et al., 2009). In donkeys, AsHV-4 and -5 have both been identified in cases of interstitial pneumonia (Kleiboeker et al., 2002), and in one case of herpesvirus-associated neurological disease (Vengust et al., 2008). This study is the first to report the repeated detection of AsHV-5 in peripheral blood mononuclear cells (PBMCs) and in nasal and conjunctival swabs from closed herds of clinically normal horses over an extended period of time. Samples from 266 clinically normal Lipizzaner horses at three geographical locations in Austria (Piber, Vienna, and Heldenberg), were obtained as part of a herpesvirus surveillance programme over a period of 18 months. All survey procedures were approved by the Ethics Committee of the University of Veterinary Medicine, Vienna, and by officials of the Spanish Riding School and Federal stud at Piber. A total of 36 horses were lost to ‘follow-up’ over the study period. Peripheral blood mononuclear cells, nasal (NS) and ⇑ Corresponding author at: Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Tel.: +43 125077 2704. E-mail addresses: [email protected], [email protected] (N. Nowotny). http://dx.doi.org/10.1016/j.tvjl.2013.10.035 1090-0233/Ó 2013 Elsevier Ltd. All rights reserved.
conjunctival (CS) swabs from each horse were taken by a single examiner (JOR) at 6 month intervals over the 18 month survey period. DNA was extracted and samples were stored at 80 °C as described by Rushton et al. (2013). The results of quantified equid herpesvirus (EHV) type 2 and 5 PCRs on PBMCs, NS and CS from all horses have been published elsewhere (Rushton et al., 2013). Samples which tested negative by type-specific qPCRs were subjected to nested consensus herpesvirus (HV) PCR as described by VanDevanter et al. (1996) with some modifications and the target gene was the DNA polymerase. We adapted this protocol for a commercial ‘fast-cycling’ PCR Kit (Qiagen) by optimising the annealing temperatures and Mg2+ ion concentrations for both PCR rounds, and we also assessed other combinations of previously described primers (i.e. as a semi-nested PCR). Contamination was avoided by applying strict precautions, such as the use of specified separate rooms for each step, and by including a negative control after every 12th sample. Nucleotide sequencing on amplification products of the consensus HV PCR was performed using a genetic analyser (ABI PRISM 310 Genetic Analyser, Applied Biosystems). All sequences were compared with each other using an alignment software programme (Align Plus Program, Scientific and Educational Software, Version 3.0, Serial number 43071) and compared to the corresponding HV sequences in GenBank using BLAST.1
1
See: http://blast.ncbi.nlm.nih.gov/Blast.cgi.
J.O. Rushton et al. / The Veterinary Journal 200 (2014) 200–203
201
Fig. 1. (a) Nucleic acid alignment of the 689 bp sample obtained in this study: reference asinine herpesvirus (AsHV)-5 (Accession number FJ798319); equine herpes virus (EHV)-7 (Accession number EU165547); àEHV-5 (Accession number JX125459); and EHV-2 (Accession number HQ247790) strains. (b) Nucleic acid alignment of the 51 AsHV5 sequences obtained by nested PCR with a reference AsHV-5 strain (GenBank Accession number FJ798319). Note minor genetic differences in a few of the samples.
Of a total of 255 samples subjected to sequence analysis over the study period, 51 samples from 39 horses were positive for AsHV-5: seven animals were repeatedly positive over this time, in one case in more than one sample (horse 136; Table 1). Twenty-four positive results were identified in PBMCs, 22 in NS and five in CS. Twentyone mares, 16 stallions, and two geldings with a mean age of 9 years (SD ± 5.9) which were found to be positive were distributed over the Styria, Vienna and Lower Austria regions (29, 7 and 3 cases, respectively; Table 1). In one case, the semi-nested PCR resulted in the detection of a 689 bp sequence (deposited in GenBank under accession number KC825357) with 97% coverage and 98% identity to AsHV-5 (FJ798319). In all other samples the bands of the seminested PCRs were too weak for subsequent sequence analysis. Furthermore, the HV sequence which had 91% identity with AsHV-2, is also referred to as EHV-7 (GenBank accession number EU165547; Bell et al., 2008). The maximal nucleotide identity to EHV-5 and -2 was 89% and 87%, respectively (Fig. 1a). The 169 bp sequences of the remaining samples obtained by nested PCR exhibited identities of between 98% and 100% to each other (Fig. 1b).
The results of this surveillance support the previous findings of Fortier et al. (2009) that AsHV-5 may be sporadically encountered in clinically normal horses. However, our findings also indicate that the virus can be detected in ‘non-respiratory’ sample material, suggesting that AsHV-5 follows a similar pattern of infection and latency as EHV-2 and -5. In our study population, there was no evidence of previous contact with donkeys, and no correlation was found between infection status and geographical location, or animal age or gender. Nucleotide sequencing of a ‘DNA-packaging protein’ gene of equine gammaherpesviruses has shown that AsHV-4, -5, and -6 are similar (but distinct) viruses, with the greatest similarity to EHV-5 and -2 (Kleiboeker et al., 2004). However the longest nucleotide sequence of AsHV-5 available in GenBank consists of only 881 nucleotides. It therefore remains possible that AsHV-5 is a strain of EHV-5, -2, or possibly another equid herpesvirus, and has been given its distinct nomenclature based on differences in this short genetic sequence (Bell et al., 2008; Fortier et al., 2009). To date, the isolation of AsHV-4, -5, or -6 has been unsuccessful
202
J.O. Rushton et al. / The Veterinary Journal 200 (2014) 200–203
Fig. 1 (continued)
Table 1 Details of the detection of asinine herpesvirus (AsHV) type 5 by nested consensus herpesvirus PCR in peripheral blood mononuclear cells (PBMCs), nasal (NS) and conjunctival (CS) samples from clinically normal Lipizzaner horses over an 18 month period: The numbers of horses sampled were 266, 261, 249, and 230 at the first, second, third, and fourth sampling points, respectively. Pos, positive result for AsHV-5. M, mare; S, stallion; G, gelding. Geographical location: P, Piber; V, Vienna; H, Heldenberg. April 2011 NR
Gender
Age
Location
10 26 37 39 41 44 48 49 62 80 82 91 97 107 110 114 119 127 129 130 132 136 141 143
S S S M M M M M M M S S M M S M M G S S S M M M
5 15 24 8 15 11 10 5 12 10 3 3 12 11 3 13 5 15 3 5 3 21 6 7
V V H P P P P P P P P P P P P P P P P P P P P P
October 2011 PBMCs
NS Pos Pos
Pos
Pos
Pos
Pos
CS
Location V V H P P P P P P P P P P P P P P P P P P P P P
PBMCs
April 2012 NS
Pos
Pos Pos Pos
Pos Pos Pos Pos
Pos Pos
Pos
CS
Location V V H P P P P P P P P P P P P P P P P P P P P P
PBMCs
October 2012 NS
Pos
Pos Pos Pos
Pos
CS
Location V V H P P P P P P P P P P P P P P P P P P P P P
PBMCs
NS
CS
Pos
Pos Pos Pos
Pos
Pos
Pos Pos Pos Pos Pos
203
J.O. Rushton et al. / The Veterinary Journal 200 (2014) 200–203 Table 1 (continued) April 2011 NR
Gender
Age
Location
165 167 170 177 178 181 187 193 199 207 210 222 236 238 253
G M M M M M M M S S S S S S S
6 5 7 8 14 15 7 7 8 13 7 6 20 22 11
P P P P P P P P V V V H V V H
October 2011 PBMCs
NS
Pos
Pos
Pos Pos Pos
Pos
CS
Location
PBMCs
P P P P P P P P V V V H V V H
Pos Pos
Pos
(Kleiboeker et al., 2004), and our attempts to isolate AsHV-5 in equine dermis cell culture was also unsuccessful. A limitation of this study was the fact that 36 horses were lost to follow-up. Although none of these animals were positive for AsHV-5 when they were available for sampling, their infection status at subsequent time-points remains unknown. Furthermore, given that samples positive for EHV-2 and/or -5 on type-specific qPCR were not subjected to sequence analysis, horses infected with both AsHV-5 and EHV-2/-5 may have been missed. As a result, we cannot definitively determine the exact prevalence of AsHV-5 infection for this population. Our findings highlight that AsHV-5 is present in clinically normal horses, and further research is required to determine whether this virus is a distinct herpesvirus in its own right or is merely a strain of EHV-2 or -5.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements The authors would like to thank the staff of the Federal state stud at Piber, of the Spanish Riding School Vienna, and especially Dr. Maximilian Dobretsberger, for their assistance with the survey. We also would like to express our thanks to the Spanish Riding
April 2012 NS
CS
Location P P P P P P P P V V V H V V H
PBMCs
October 2012 NS
Pos
Pos
Pos
Pos
CS
Location P P P P P P P P V V V H V V H
PBMCs
NS
CS
Pos Pos Pos Pos
Pos
School, Federal stud at Piber, and the University of Veterinary Medicine, Vienna for funding this study. References Back, H., Kendall, A., Grandón, R., Ullman, K., Treiberg-Berndtsson, L., Ståhl, K., Pringle, J., 2012. Equine multinodular pulmonary fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: A case report. Acta Veterinaria Scandinavica 54, 57. Bell, S.A., Pusterla, N., Balasuriya, U.B.R., Mapes, S.M., Nyberg, N.L., MacLachlan, N.J., 2008. Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by realtime PCR. Veterinary Microbiology 130, 176–183. De Witte, F.G., Frank, N., Wilkes, R.P., Novak, J.M., 2012. Association of asinine herpesvirus-5 with pyogranulomatous pneumonia in a mare. Journal of Veterinary Internal Medicine 26, 1064–1068. Fortier, G., Van Erck, E., Fortier, C., Richard, E., Pottier, D., Pronost, S., Miszczak, F., Thiry, E., Lekeux, P., 2009. Herpesviruses in respiratory liquids of horses: Putative implication in airway inflammation and association with cytological features. Veterinary Microbiology 139, 34–41. Kleiboeker, S.B., Schommer, S.K., Johnson, P.J., Ehlers, B., Turnquist, S.E., Boucher, M., Kreeger, J.M., 2002. Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus). Journal of Veterinary Diagnostic Investigation 14, 273–280. Kleiboeker, S.B., Turnquist, S.E., Johnson, P.J., Kreeger, J.M., 2004. Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses. Journal of Veterinary Diagnostic Investigation 16, 67–74. Rushton, J.O., Kolodziejek, J., Tichy, A., Nell, N., Nowotny, N., 2013. Detection of equid herpesviruses 2 and 5 in a herd of 266 Lipizzaners in association with ocular findings. Veterinary Microbiology 164, 139–144. VanDevanter, D.R., Warrener, P., Bennett, L., Schultz, E.R., Coulter, S., Garber, R.L., Rose, T.M., 1996. Detection and analysis of diverse herpesviral species by consensus primer PCR. Journal of Clinical Microbiology 34, 1666–1671. Vengust, M., Wen, X., Bienzle, D., 2008. Herpesvirus-associated neurological disease in a donkey. Journal of Veterinary Diagnostic Investigation 20, 820–823.
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Prevalence of asinine herpesvirus type 5 (AsHV-5) infection in clinically normal Lipizzaner horses James Oliver Rushton a,b, Jolanta Kolodziejek a, Barbara Nell b, Norbert Nowotny a,c,⇑ a Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria b Clinic for Surgery, Dentistry and Ophthalmology, Department of Companion Animals and Horses, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria c Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
a r t i c l e
i n f o
Article history: Accepted 31 October 2013
Keywords: Horse Asinine herpesvirus (AsHV) type 5 Equid gammaherpesviruses Lipizzaner
a b s t r a c t The aim of this study was to assess the extent of asinine herpesvirus (AsHV) type 5 infection in ‘closed’ populations of clinically normal Lipizzaner horses. Peripheral blood mononuclear cells plus nasal and conjunctival swabs were obtained on four occasions over an 18 month period from 266 animals as part of a health surveillance programme. Sequence analysis of samples that were positive by nested consensus herpesvirus PCR but negative using quantified equid herpesvirus (EHV) type 2 and 5 PCR, revealed a total of 51 samples from 39 horses positive for AsHV-5. No statistically significant association between animal age, gender or geographical location and infection status was identified. The findings suggest sub-clinical AsHV-5 infection may be encountered more frequently than previously reported. Ó 2013 Elsevier Ltd. All rights reserved.
Asinine herpesvirus (AsHV) infection has been sporadically reported in horses in recent years (Back et al., 2012; De Witte et al., 2012) and associated with pyogranulomatous pneumonia and equine multinodular pulmonary fibrosis. The detection of AsHV-5 in the respiratory fluids of horses not exhibiting signs of respiratory disease has also been documented (Fortier et al., 2009). In donkeys, AsHV-4 and -5 have both been identified in cases of interstitial pneumonia (Kleiboeker et al., 2002), and in one case of herpesvirus-associated neurological disease (Vengust et al., 2008). This study is the first to report the repeated detection of AsHV-5 in peripheral blood mononuclear cells (PBMCs) and in nasal and conjunctival swabs from closed herds of clinically normal horses over an extended period of time. Samples from 266 clinically normal Lipizzaner horses at three geographical locations in Austria (Piber, Vienna, and Heldenberg), were obtained as part of a herpesvirus surveillance programme over a period of 18 months. All survey procedures were approved by the Ethics Committee of the University of Veterinary Medicine, Vienna, and by officials of the Spanish Riding School and Federal stud at Piber. A total of 36 horses were lost to ‘follow-up’ over the study period. Peripheral blood mononuclear cells, nasal (NS) and ⇑ Corresponding author at: Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Tel.: +43 125077 2704. E-mail addresses: [email protected], [email protected] (N. Nowotny). http://dx.doi.org/10.1016/j.tvjl.2013.10.035 1090-0233/Ó 2013 Elsevier Ltd. All rights reserved.
conjunctival (CS) swabs from each horse were taken by a single examiner (JOR) at 6 month intervals over the 18 month survey period. DNA was extracted and samples were stored at 80 °C as described by Rushton et al. (2013). The results of quantified equid herpesvirus (EHV) type 2 and 5 PCRs on PBMCs, NS and CS from all horses have been published elsewhere (Rushton et al., 2013). Samples which tested negative by type-specific qPCRs were subjected to nested consensus herpesvirus (HV) PCR as described by VanDevanter et al. (1996) with some modifications and the target gene was the DNA polymerase. We adapted this protocol for a commercial ‘fast-cycling’ PCR Kit (Qiagen) by optimising the annealing temperatures and Mg2+ ion concentrations for both PCR rounds, and we also assessed other combinations of previously described primers (i.e. as a semi-nested PCR). Contamination was avoided by applying strict precautions, such as the use of specified separate rooms for each step, and by including a negative control after every 12th sample. Nucleotide sequencing on amplification products of the consensus HV PCR was performed using a genetic analyser (ABI PRISM 310 Genetic Analyser, Applied Biosystems). All sequences were compared with each other using an alignment software programme (Align Plus Program, Scientific and Educational Software, Version 3.0, Serial number 43071) and compared to the corresponding HV sequences in GenBank using BLAST.1
1
See: http://blast.ncbi.nlm.nih.gov/Blast.cgi.
J.O. Rushton et al. / The Veterinary Journal 200 (2014) 200–203
201
Fig. 1. (a) Nucleic acid alignment of the 689 bp sample obtained in this study: reference asinine herpesvirus (AsHV)-5 (Accession number FJ798319); equine herpes virus (EHV)-7 (Accession number EU165547); àEHV-5 (Accession number JX125459); and EHV-2 (Accession number HQ247790) strains. (b) Nucleic acid alignment of the 51 AsHV5 sequences obtained by nested PCR with a reference AsHV-5 strain (GenBank Accession number FJ798319). Note minor genetic differences in a few of the samples.
Of a total of 255 samples subjected to sequence analysis over the study period, 51 samples from 39 horses were positive for AsHV-5: seven animals were repeatedly positive over this time, in one case in more than one sample (horse 136; Table 1). Twenty-four positive results were identified in PBMCs, 22 in NS and five in CS. Twentyone mares, 16 stallions, and two geldings with a mean age of 9 years (SD ± 5.9) which were found to be positive were distributed over the Styria, Vienna and Lower Austria regions (29, 7 and 3 cases, respectively; Table 1). In one case, the semi-nested PCR resulted in the detection of a 689 bp sequence (deposited in GenBank under accession number KC825357) with 97% coverage and 98% identity to AsHV-5 (FJ798319). In all other samples the bands of the seminested PCRs were too weak for subsequent sequence analysis. Furthermore, the HV sequence which had 91% identity with AsHV-2, is also referred to as EHV-7 (GenBank accession number EU165547; Bell et al., 2008). The maximal nucleotide identity to EHV-5 and -2 was 89% and 87%, respectively (Fig. 1a). The 169 bp sequences of the remaining samples obtained by nested PCR exhibited identities of between 98% and 100% to each other (Fig. 1b).
The results of this surveillance support the previous findings of Fortier et al. (2009) that AsHV-5 may be sporadically encountered in clinically normal horses. However, our findings also indicate that the virus can be detected in ‘non-respiratory’ sample material, suggesting that AsHV-5 follows a similar pattern of infection and latency as EHV-2 and -5. In our study population, there was no evidence of previous contact with donkeys, and no correlation was found between infection status and geographical location, or animal age or gender. Nucleotide sequencing of a ‘DNA-packaging protein’ gene of equine gammaherpesviruses has shown that AsHV-4, -5, and -6 are similar (but distinct) viruses, with the greatest similarity to EHV-5 and -2 (Kleiboeker et al., 2004). However the longest nucleotide sequence of AsHV-5 available in GenBank consists of only 881 nucleotides. It therefore remains possible that AsHV-5 is a strain of EHV-5, -2, or possibly another equid herpesvirus, and has been given its distinct nomenclature based on differences in this short genetic sequence (Bell et al., 2008; Fortier et al., 2009). To date, the isolation of AsHV-4, -5, or -6 has been unsuccessful
202
J.O. Rushton et al. / The Veterinary Journal 200 (2014) 200–203
Fig. 1 (continued)
Table 1 Details of the detection of asinine herpesvirus (AsHV) type 5 by nested consensus herpesvirus PCR in peripheral blood mononuclear cells (PBMCs), nasal (NS) and conjunctival (CS) samples from clinically normal Lipizzaner horses over an 18 month period: The numbers of horses sampled were 266, 261, 249, and 230 at the first, second, third, and fourth sampling points, respectively. Pos, positive result for AsHV-5. M, mare; S, stallion; G, gelding. Geographical location: P, Piber; V, Vienna; H, Heldenberg. April 2011 NR
Gender
Age
Location
10 26 37 39 41 44 48 49 62 80 82 91 97 107 110 114 119 127 129 130 132 136 141 143
S S S M M M M M M M S S M M S M M G S S S M M M
5 15 24 8 15 11 10 5 12 10 3 3 12 11 3 13 5 15 3 5 3 21 6 7
V V H P P P P P P P P P P P P P P P P P P P P P
October 2011 PBMCs
NS Pos Pos
Pos
Pos
Pos
Pos
CS
Location V V H P P P P P P P P P P P P P P P P P P P P P
PBMCs
April 2012 NS
Pos
Pos Pos Pos
Pos Pos Pos Pos
Pos Pos
Pos
CS
Location V V H P P P P P P P P P P P P P P P P P P P P P
PBMCs
October 2012 NS
Pos
Pos Pos Pos
Pos
CS
Location V V H P P P P P P P P P P P P P P P P P P P P P
PBMCs
NS
CS
Pos
Pos Pos Pos
Pos
Pos
Pos Pos Pos Pos Pos
203
J.O. Rushton et al. / The Veterinary Journal 200 (2014) 200–203 Table 1 (continued) April 2011 NR
Gender
Age
Location
165 167 170 177 178 181 187 193 199 207 210 222 236 238 253
G M M M M M M M S S S S S S S
6 5 7 8 14 15 7 7 8 13 7 6 20 22 11
P P P P P P P P V V V H V V H
October 2011 PBMCs
NS
Pos
Pos
Pos Pos Pos
Pos
CS
Location
PBMCs
P P P P P P P P V V V H V V H
Pos Pos
Pos
(Kleiboeker et al., 2004), and our attempts to isolate AsHV-5 in equine dermis cell culture was also unsuccessful. A limitation of this study was the fact that 36 horses were lost to follow-up. Although none of these animals were positive for AsHV-5 when they were available for sampling, their infection status at subsequent time-points remains unknown. Furthermore, given that samples positive for EHV-2 and/or -5 on type-specific qPCR were not subjected to sequence analysis, horses infected with both AsHV-5 and EHV-2/-5 may have been missed. As a result, we cannot definitively determine the exact prevalence of AsHV-5 infection for this population. Our findings highlight that AsHV-5 is present in clinically normal horses, and further research is required to determine whether this virus is a distinct herpesvirus in its own right or is merely a strain of EHV-2 or -5.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements The authors would like to thank the staff of the Federal state stud at Piber, of the Spanish Riding School Vienna, and especially Dr. Maximilian Dobretsberger, for their assistance with the survey. We also would like to express our thanks to the Spanish Riding
April 2012 NS
CS
Location P P P P P P P P V V V H V V H
PBMCs
October 2012 NS
Pos
Pos
Pos
Pos
CS
Location P P P P P P P P V V V H V V H
PBMCs
NS
CS
Pos Pos Pos Pos
Pos
School, Federal stud at Piber, and the University of Veterinary Medicine, Vienna for funding this study. References Back, H., Kendall, A., Grandón, R., Ullman, K., Treiberg-Berndtsson, L., Ståhl, K., Pringle, J., 2012. Equine multinodular pulmonary fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: A case report. Acta Veterinaria Scandinavica 54, 57. Bell, S.A., Pusterla, N., Balasuriya, U.B.R., Mapes, S.M., Nyberg, N.L., MacLachlan, N.J., 2008. Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by realtime PCR. Veterinary Microbiology 130, 176–183. De Witte, F.G., Frank, N., Wilkes, R.P., Novak, J.M., 2012. Association of asinine herpesvirus-5 with pyogranulomatous pneumonia in a mare. Journal of Veterinary Internal Medicine 26, 1064–1068. Fortier, G., Van Erck, E., Fortier, C., Richard, E., Pottier, D., Pronost, S., Miszczak, F., Thiry, E., Lekeux, P., 2009. Herpesviruses in respiratory liquids of horses: Putative implication in airway inflammation and association with cytological features. Veterinary Microbiology 139, 34–41. Kleiboeker, S.B., Schommer, S.K., Johnson, P.J., Ehlers, B., Turnquist, S.E., Boucher, M., Kreeger, J.M., 2002. Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus). Journal of Veterinary Diagnostic Investigation 14, 273–280. Kleiboeker, S.B., Turnquist, S.E., Johnson, P.J., Kreeger, J.M., 2004. Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses. Journal of Veterinary Diagnostic Investigation 16, 67–74. Rushton, J.O., Kolodziejek, J., Tichy, A., Nell, N., Nowotny, N., 2013. Detection of equid herpesviruses 2 and 5 in a herd of 266 Lipizzaners in association with ocular findings. Veterinary Microbiology 164, 139–144. VanDevanter, D.R., Warrener, P., Bennett, L., Schultz, E.R., Coulter, S., Garber, R.L., Rose, T.M., 1996. Detection and analysis of diverse herpesviral species by consensus primer PCR. Journal of Clinical Microbiology 34, 1666–1671. Vengust, M., Wen, X., Bienzle, D., 2008. Herpesvirus-associated neurological disease in a donkey. Journal of Veterinary Diagnostic Investigation 20, 820–823.
