The Veterinary Journal 200 (2014) 257–260
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Serological and virological detection of canine herpesvirus-1 in adult dogs with and without reproductive disorders A. Pratelli *, V. Colao, M. Losurdo Department of Veterinary Medicine, University of Bari, Strada per Casamassima km 3, 70010 Valenzano, Bari, Italy
A R T I C L E
I N F O
Article history: Accepted 3 March 2014 Keywords: Canine herpesvirus 1 Immunofluorescence PCR Serum neutralization Viral shedding
A B S T R A C T
Canine herpesvirus 1 (CaHV-1) is known to cause reproductive disorders in adult dogs and neonatal mortality in puppies. The seroprevalence of CaHV-1 has not been documented in Italy. Sera from 865 dogs were screened for CaHV-1 using a serum neutralization assay (SN). All CaHV-1 positive sera and 100 CaHV-1 negative sera were also tested using an in-house immunofluorescence (IF) test. Thirteen bitches with reproductive disorders and three bitches with no history of reproductive diseases were also examined clinically so that lesions associated with CaHV-1 and CaHV-1 DNA could be identified using PCR analysis of vaginal swabs. An overall seroprevalence of 14.6% was observed using SN, and 18.6% using IF. The correlation between SN and IF was moderate. The SN assay demonstrated a greater sensitivity than IF, with a few exceptions. None of the vaginal swabs tested positive for CaHV-1 DNA. The differences in the seropositivity rates between SN and IF were not statistically significant (P = 0.16). Using the SN test as the reference standard, the sensitivity and specificity of IF were 29% and 95%, respectively. These results suggest that CaHV-1 is common in canine populations and could pose a threat to neonatal survival and canine fertility in breeding kennels in Italy. Vaccination of breeding bitches should be recommended if there is a history of reproductive disorders. © 2014 Elsevier Ltd. All rights reserved.
Introduction Canine herpesvirus 1 (CaHV-1) is classified in the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. The virus was first isolated and identified almost simultaneously by three different research groups in 1965 from cases of fatal disease in puppies and fetuses and from dog kidney cell cultures (Carmichael et al., 1965; Spertzel et al., 1965; Stewart et al., 1965). In susceptible puppies, the virus can produce a fatal systemic necrotizing and haemorrhagic disease, but the infection is usually subclinical in puppies >2 weeks old (Carmichael et al., 1965; Evermann et al., 2011). In adult dogs, CaHV-1 is associated with clinical disorders of the reproductive system, including embryonic death and/ or resorption, abortion and stillbirth. Infection can cause vesicular lesions in the vestibule and vaginal mucosa and/or severe vaginitis in bitches, as well as vesicles on the penis and the preputial mucosa of dogs. CaHV-1 is sporadically associated with upper respiratory and ocular disease in older dogs (Ledbetter, 2013). Natural infection of susceptible puppies primarily occurs via the oronasal route from the birth canal during parturition (Appel, 1987), but ve-
* Corresponding author. Tel.: +39 080 4679835. E-mail address: [email protected] (A. Pratelli). http://dx.doi.org/10.1016/j.tvjl.2014.03.001 1090-0233/© 2014 Elsevier Ltd. All rights reserved.
nereal and transplacental transmission is also possible (Evermann et al., 2011). After either acute disease or subclinical infection, dogs become latent viral carriers and CaHV-1 can be isolated from the trigeminal and lumbo-sacral ganglia, the salivary glands, tonsils and liver, even in the absence of clinical signs (Burr et al., 1996). Reactivation of latent virus might be associated with stress or, experimentally, if immunosuppressive drugs or anti-lymphocyte sera are administered (Ledbetter et al., 2012). CaHV-1 is considered poorly immunogenic and neutralizing antibodies can fluctuate for 4–8 weeks following exposure, rarely lasting more than few months after infection (Carmichael and Greene, 1998; Nöthling et al., 2008). Numerous epidemiological factors could affect canine seroprevalence. Larger kennels and breeding kennels with histories of neonatal infection are more likely to have positive dogs, and one study reported significantly higher anti-CaHV-1 titres with increasing age, in male dogs that were mated and older bitches (Ronsse et al., 2004). It is presumed that CaHV-1 is widespread worldwide, but there is a paucity of prevalence studies reporting viral shedding and antibody titres. A long-term survey of dogs in a rehoming centre documented CaHV-1 in 9.6% of lung and 12.8% of tracheal specimens (Buonavoglia and Martella, 2007). Along with canine minute virus (CnMV), CaHV-1 remains a major and severe health problem in breeding dogs and therapeutic measures to counteract the infection are not available (Pratelli and
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A. Pratelli et al./The Veterinary Journal 200 (2014) 257–260
Table 1 Sequence and position of the oligonucleotides used in PCR testing for canine herpesvirus (CaHV). Primer
Sequence 5’ to 3’
Polarity
Target gene
Positiona
Amplicon size
CaHV-1 CaHV-2
TGCCGCTTTTATATAGATG AAGCGTTGTAAAAGTTCGT
+ −
TK
283–301 758–776
493 bp
a
Oligonucleotide positions are referred to the sequence of CaHV-1 thymidine kinase (TK) gene (GenBank accession no. X75765) for gel-based PCR amplifications.
Moschidou, 2012). CaHV-1 has been reported in many countries and the prevalence of antibodies in dogs tested worldwide varies markedly. The use of different serological methods and sampling frames within the canine population also influences test results and CaHV-1 seroprevalences in different geographical locations can vary enormously. In The Netherlands, the proportion of seropositive dogs at the end of the 1990s was particularly high (39.3%; Rijsewijk et al., 1999). The seroprevalence of CaHV-1 among dogs in the SouthEast of Iran was estimated to be 20.7%, specifically 22.9% and 19.1% in kennelled and owned dogs, respectively (Babaei et al., 2010). In the Gauteng Province of South Africa, among 328 canine sera tested with both serum neutralization (SN) and ELISA, 22% of the sera were positive (Nöthling et al., 2008). In a Turkish dog population, the seroprevalence by ELISA was 39.3%, but by SN was 29.4% (Yeşilbağ et al., 2012). An overall CaHV-1 seroprevalence of 45.75% was observed in the Belgian dog population (Ronsse et al., 2002), while in Norway and Finland, the seroprevalence was about 80%, suggesting that CaHV-1 is endemic in those countries (Dahlbom et al., 2009; Krogenæs et al., 2012). Epidemiological studies have not previously been documented in Italy. In this study, sera from 865 dogs from Southern Italy were screened for CaHV-1 antibodies. Furthermore, 16 bitches with and without a history of reproductive disorders were examined clinically to identify CaHV-1 associated lesions; vaginal swabs were tested using PCR to detect viral DNA. Finally, the relationship between reproductive disorders, viral excretion and serology was evaluated to determine the potential importance of subclinical carriers of CaHV-1. Materials and methods Dogs and sampling Dogs of at least 1 year of age and without a history of vaccination against herpesvirus were included in the present study. The study group consisted of 849 serum specimens collected during routine examinations at a Clinical Diagnostic Laboratory in Southern Italy. Venous blood was collected into glass tubes without heparin and serum was withdrawn and stored at −80 °C until analysis was performed. Sixteen bitches from different breeding kennels were included in the study. The vulva and vaginal vestibule were examined for CaHV-1 associated lesions. Three bitches had histories of abortion, seven bitches had histories of infertility, three bitches gave birth to puppies that were either stillborn or died in the neonatal period, and three bitches had no history of reproductive disorders. Vaginal swabs were collected in sample medium (Dulbecco Minimal Essential Medium, DMEM), and serum specimens from each bitch were stored at −80 °C until tested. The 865 serum specimens were grouped into two categories for statistical analysis: 286 sera were collected from kennelled/breeding dogs and 579 from pet dogs. Serum neutralization test The CaHV-1 DK13 strain (supplied by Professor L.E. Carmichael, James A. Baker Institute for Animal Health, Cornell University) was used. The virus was propagated on A72 cell lines grown in DMEM supplemented with 10% fetal calf serum (FCS). The stock virus used in the study was at the ninth passage and had an infectivity titre of 104.5 50% tissue culture infective dose (TCID50)/50 μL. All serum specimens were heat treated at 56 °C for 30 min to inactivate endogenous complement. Two-fold dilutions, starting from 1:2 for each specimen, were performed in duplicate and mixed with 100 TCID50 CaHV-1 strain in 96-well tissue culture microplates (Falcon-Becton, Dickinson Labware). The plates were kept at room temperature for 90 min before 20,000 A72 cells were added to each well. Cell cultures were examined daily for 4 days. The SN titre was defined as the reciprocal of the highest serum dilution that inhibited cytopathic effect (cpe) in 50% of the cell cultures.
Immunofluorescence test The SN-positive serum specimens and 100 SN-negative specimens were also tested for antibodies against CaHV-1 using an in-house immunofluorescence (IF) test. The 100 SN-negative sera were selected from owned dogs, from kennelled dogs and from breeding bitches. A72 cells infected with CaHV-1 strain DK13 were placed in multi-chamber culture slides (LAB-TEK Chamber Slide System) and fixed with 80% acetone. Serial twofold dilutions of each serum, starting from 1:50 to 1:800, were performed in phosphate buffered saline (PBS). Diluted specimens (20 μL) were placed in duplicate into wells and incubated for 30 min at 37 °C in a moist incubator. Slides were washed three times in PBS and blotted dry. Aliquots of pre-titrated goat anti-dog IgG conjugated to fluorescein isothiocyanate (FITC; Sigma Chemicals) diluted 1:50 (20 μL) were added to each well. The slides were then incubated for 30 min at 37 °C, washed again in PBS, counterstained with Evans blue (BioMérieux) and examined under a fluorescent microscope. Approximately 75% of the cells in each well were infected, providing an internal negative control for the non-specific binding of antibodies to the cell monolayer. The titre was read as the highest serum dilution that produced detectable levels of fluorescence in foci of virus-infected cells. Antibody titres 2 weeks of age, has been demonstrated (Evermann et al., 2011). Nevertheless, the relationship between maternal serological status and in utero clinical manifestations appears controversial, suggesting that there is no association with CaHV-1 infection. Other studies have not reported associations between CaHV-1 antibody titres and fertility parameters, but seropositive bitches more commonly have history of abortions, and reproductive disorders were also observed in bitches with high antibody titres (Ronsse et al., 2004, 2005). None of the vaginal swabs in this study tested positive on PCR analysis. This could be due to the limited number of bitches tested and the fact that the virological data did not produce statistically significant results. Nevertheless, it is known that the duration of viral shedding can be very short, so it is advisable to sample dogs for
260
A. Pratelli et al./The Veterinary Journal 200 (2014) 257–260
several consecutive days. Furthermore, since none of the breeding bitches had vaginal lesions related to CaHV-1 infection, the probability of detecting the virus was low. After human herpesvirus 2 (HSV2) infection, asymptomatic patients sometimes shed virus only for only 1 day (Wald et al., 1995). Accordingly, the negative results observed in our study might be a consequence of the brief viral shedding documented for CaHV-1 under field conditions, despite the endemic prevalence of the virus and the specific immunity demonstrated in the dogs tested. Moreover, CaHV-1 is mostly associated with neonatal mortality and although the majority of the bitches in this study had histories of reproductive disorders, no clinical signs were observed at the time of sampling. Considering all of these observations, our results suggest that CaHV-1 is common in Italian dogs and could pose a threat to neonatal survival and dog fertility in Italian dog breeding kennels. Since risk factors for seropositivity have not been established, vaccination of breeding bitches in kennels with histories of reproductive disorders is recommended.
Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgements The authors would like to thank their colleagues at the Department of Veterinary Medicine, Section Infectious Diseases at the University of Bari, Italy, whose collaboration was essential for the accomplishment of the present study.
References Appel, M., 1987. Canine herpesvirus. In: Appel, M.J. (Ed.), Virus Infections of Carnivores, Elsevier Science Publishers BV, Amsterdam, pp. 5–15. Babaei, H., Akhtardanesh, B., Ghanbarpour, R., Namjoo, A., 2010. Serological evidence of canine herpesvirus-1 in dogs of Kerman city, South-East of Iran. Transboundary and Emerging Diseases 57, 348–351. Buonavoglia, C., Martella, V., 2007. Canine respiratory viruses. Veterinary Research 38, 355–373. Burr, P.D., Campbell, M.E., Nicolson, L., Onions, D.E., 1996. Detection of canine herpesvirus 1 in a wide range of tissues using the polymerase chain reaction. Veterinary Microbiology 53, 227–237. Carmichael, L.E., Squire, R.A., Krook, L., 1965. Clinical and pathologic features of a fatal viral disease of newborn pups. American Journal of Veterinary Research 26, 803–814. Carmichael, L.E., 1970. Herpesvirus canis: Aspects of pathogenesis and immune response. Journal of the American Veterinary Medical Association 156, 1714–1721. Carmichael, L.E., Greene, C.E., 1998. Canine herpesvirus infection. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat, WB Saunders, Philadelphia, pp. 28–32. Dahlbom, M., Johnsson, M., Myllys, V., Taponen, J., Andersson, M., 2009. Seroprevalence of canine herpesvirus-1 and Brucella canis in Finnish breeding kennels with and without reproductive problems. Reproduction in Domestic Animals 44, 128–131.
Decaro, N., Amorisco, F., Desario, C., Lorusso, E., Camero, M., Bellacicco, A.L., Sciarretta, R., Lucente, M.S., Martella, V., Buonavoglia, C., 2010. Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1. Journal of Virological Methods 169, 76–180. Engels, M., Mayr-Bibrack, B., Ruckstuhl, B., Metzler, A., Wyler, R., 1980. Seroepizootiology of canine herpesvirus in Switzerland and preliminary trials of a vaccine. Zentralblatt für Veterinärmedizin. Reihe B 27, 257–267. Evermann, J.F., Ledbetter, E.C., Maes, R.K., 2011. Canine reproductive, respiratory, and ocular diseases due to canine herpesvirus. The Veterinary Clinics of North America. Small Animal Practice 41, 1097–1120. König, M., Neiseke, J., Thiel, H.-J., 2004. Prevalence of canine herpesvirus 1 (CHV-1) in German kennels. Tierärztliche Umschau 59, 559–565. Krogenæs, A., Rootwelt, V., Larsen, S., Sjøberg, E.K., Akselsen, B., Skår, T.M., Myhre, S.S., Renström, L.H.M., Klingeborn, B., Lund, A., 2012. A serologic study of canine herpes virus-1 infection in the Norwegian adult dog population. Theriogenology 78, 153–158. Ledbetter, E.C., da Silva, E.C., Kim, S.G., Dubovi, E.J., Schwark, W.S., 2012. Frequency of spontaneous canine herpesvirus-1 reactivation and ocular viral shedding in latently infected dogs and canine herpesvirus-1 reactivation and ocular viral shedding induced by topical administration of cyclosporine and systemic administration of corticosteroids. American Journal of Veterinary Research 73, 1079–1084. Ledbetter, E.C., 2013. Canine herpesvirus-1 ocular diseases of mature dogs. New Zealand Veterinary Journal 61, 193–201. Nöthling, J.O., Hüssy, D., Steckler, D., Ackermann, M., 2008. Seroprevalence of canine herpesvirus in breeding kennels in the Gauteng Province of South Africa. Theriogenology 69, 276–282. Pratelli, A., Moschidou, L., 2012. Host range of Canine minute virus in cell culture. Journal of Veterinary Diagnostic Investigation 24, 981–985. Reading, M.J., Field, H.J., 1999. Detection of high levels of canine herpesvirus-1 neutralizing antibody in kennel dogs using a novel serum neutralization test. Research in Veterinary Science 66, 273–275. Reading, S.A., Dimmock, N.J., 2007. Neutralization of animal virus infectivity by antibody. Archives of Virology 152, 1047–1059. Rijsewijk, F.A.M., Luiten, E.J., Daus, F.J., van der Heijden, R.W., van Oirschot, J.T., 1999. Prevalence of antibodies against canine herpesvirus 1 in dogs in The Netherlands in 1997–1998. Veterinary Microbiology 65, 1–7. Ronsse, V., Verstegen, J., Onclin, K., Guiot, A.L., Aeberlé, C., Nauwynck, H.J., Poulet, H., 2002. Seroprevalence of canine herpesvirus-1 in the Belgian dog population in 2000. Reproduction in Domestic Animals 37, 299–304. Ronsse, V., Verstegen, J., Onclin, K., Farnir, F., Poulet, H., 2004. Risk factors and reproductive disorders associated with canine herpesvirus-1 (CHV-1). Theriogenology 61, 619–636. Ronsse, V., Verstegen, J., Thiry, E., Onclin, K., Aeberlé, C., Brunet, S., Poulet, H., 2005. Canine herpesvirus-1 (CHV-1): Clinical, serological and virological patterns in breeding colonies. Theriogenology 64, 61–74. Schulze, C., Baumgärtner, W., 1998. Nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies. Veterinary Pathology 35, 209–217. Spertzel, R.O., Huxsoll, D.L., McConnell, S.J., Binn, L.N., Yager, R.H., 1965. Recovery and characterization of a herpes-like virus from dog kidney cell cultures. Proceedings of the Society for Experimental Biology and Medicine 120, 651–655. Stewart, S.E., David-Ferreira, J., Lovelace, E., Landon, J., 1965. Herpes-like virus isolated from neonatal and fetal dogs. Science 148, 1341–1343. Takumi, A., Kusanagi, K., Turchiya, K., Xuan, X., Azetaka, M., Takahashi, E., 1990. Serodiagnosis of canine herpesvirus infection-development of an enzyme immunosorbent assay and its comparison with two improved methods of serum neutralization test. Nihon Juigaku Zasshi 52, 241–250. Van Gucht, S., Nauwynck, H., Pensaert, M., 2001. Prevalence of canine herpesvirus in kennels and the possible association with fertility problems and neonatal death. Vlaams Diergeneeskundid Tijdschrift 70, 825–839. Wald, A., Zeh, J., Selke, S., Ashley, R.L., Corey, L., 1995. Virologic characteristics of subclinical and symptomatic genital herpes infections. The New England Journal of Medicine 333, 770–775. Yeşilbağ, K., Yalçin, E., Tuncer, P., Yilmaz, Z., 2012. Seroprevalence of canine herpesvirus-1 in Turkish dog population. Research in Veterinary Science 92, 36–39.
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Serological and virological detection of canine herpesvirus-1 in adult dogs with and without reproductive disorders A. Pratelli *, V. Colao, M. Losurdo Department of Veterinary Medicine, University of Bari, Strada per Casamassima km 3, 70010 Valenzano, Bari, Italy
A R T I C L E
I N F O
Article history: Accepted 3 March 2014 Keywords: Canine herpesvirus 1 Immunofluorescence PCR Serum neutralization Viral shedding
A B S T R A C T
Canine herpesvirus 1 (CaHV-1) is known to cause reproductive disorders in adult dogs and neonatal mortality in puppies. The seroprevalence of CaHV-1 has not been documented in Italy. Sera from 865 dogs were screened for CaHV-1 using a serum neutralization assay (SN). All CaHV-1 positive sera and 100 CaHV-1 negative sera were also tested using an in-house immunofluorescence (IF) test. Thirteen bitches with reproductive disorders and three bitches with no history of reproductive diseases were also examined clinically so that lesions associated with CaHV-1 and CaHV-1 DNA could be identified using PCR analysis of vaginal swabs. An overall seroprevalence of 14.6% was observed using SN, and 18.6% using IF. The correlation between SN and IF was moderate. The SN assay demonstrated a greater sensitivity than IF, with a few exceptions. None of the vaginal swabs tested positive for CaHV-1 DNA. The differences in the seropositivity rates between SN and IF were not statistically significant (P = 0.16). Using the SN test as the reference standard, the sensitivity and specificity of IF were 29% and 95%, respectively. These results suggest that CaHV-1 is common in canine populations and could pose a threat to neonatal survival and canine fertility in breeding kennels in Italy. Vaccination of breeding bitches should be recommended if there is a history of reproductive disorders. © 2014 Elsevier Ltd. All rights reserved.
Introduction Canine herpesvirus 1 (CaHV-1) is classified in the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. The virus was first isolated and identified almost simultaneously by three different research groups in 1965 from cases of fatal disease in puppies and fetuses and from dog kidney cell cultures (Carmichael et al., 1965; Spertzel et al., 1965; Stewart et al., 1965). In susceptible puppies, the virus can produce a fatal systemic necrotizing and haemorrhagic disease, but the infection is usually subclinical in puppies >2 weeks old (Carmichael et al., 1965; Evermann et al., 2011). In adult dogs, CaHV-1 is associated with clinical disorders of the reproductive system, including embryonic death and/ or resorption, abortion and stillbirth. Infection can cause vesicular lesions in the vestibule and vaginal mucosa and/or severe vaginitis in bitches, as well as vesicles on the penis and the preputial mucosa of dogs. CaHV-1 is sporadically associated with upper respiratory and ocular disease in older dogs (Ledbetter, 2013). Natural infection of susceptible puppies primarily occurs via the oronasal route from the birth canal during parturition (Appel, 1987), but ve-
* Corresponding author. Tel.: +39 080 4679835. E-mail address: [email protected] (A. Pratelli). http://dx.doi.org/10.1016/j.tvjl.2014.03.001 1090-0233/© 2014 Elsevier Ltd. All rights reserved.
nereal and transplacental transmission is also possible (Evermann et al., 2011). After either acute disease or subclinical infection, dogs become latent viral carriers and CaHV-1 can be isolated from the trigeminal and lumbo-sacral ganglia, the salivary glands, tonsils and liver, even in the absence of clinical signs (Burr et al., 1996). Reactivation of latent virus might be associated with stress or, experimentally, if immunosuppressive drugs or anti-lymphocyte sera are administered (Ledbetter et al., 2012). CaHV-1 is considered poorly immunogenic and neutralizing antibodies can fluctuate for 4–8 weeks following exposure, rarely lasting more than few months after infection (Carmichael and Greene, 1998; Nöthling et al., 2008). Numerous epidemiological factors could affect canine seroprevalence. Larger kennels and breeding kennels with histories of neonatal infection are more likely to have positive dogs, and one study reported significantly higher anti-CaHV-1 titres with increasing age, in male dogs that were mated and older bitches (Ronsse et al., 2004). It is presumed that CaHV-1 is widespread worldwide, but there is a paucity of prevalence studies reporting viral shedding and antibody titres. A long-term survey of dogs in a rehoming centre documented CaHV-1 in 9.6% of lung and 12.8% of tracheal specimens (Buonavoglia and Martella, 2007). Along with canine minute virus (CnMV), CaHV-1 remains a major and severe health problem in breeding dogs and therapeutic measures to counteract the infection are not available (Pratelli and
258
A. Pratelli et al./The Veterinary Journal 200 (2014) 257–260
Table 1 Sequence and position of the oligonucleotides used in PCR testing for canine herpesvirus (CaHV). Primer
Sequence 5’ to 3’
Polarity
Target gene
Positiona
Amplicon size
CaHV-1 CaHV-2
TGCCGCTTTTATATAGATG AAGCGTTGTAAAAGTTCGT
+ −
TK
283–301 758–776
493 bp
a
Oligonucleotide positions are referred to the sequence of CaHV-1 thymidine kinase (TK) gene (GenBank accession no. X75765) for gel-based PCR amplifications.
Moschidou, 2012). CaHV-1 has been reported in many countries and the prevalence of antibodies in dogs tested worldwide varies markedly. The use of different serological methods and sampling frames within the canine population also influences test results and CaHV-1 seroprevalences in different geographical locations can vary enormously. In The Netherlands, the proportion of seropositive dogs at the end of the 1990s was particularly high (39.3%; Rijsewijk et al., 1999). The seroprevalence of CaHV-1 among dogs in the SouthEast of Iran was estimated to be 20.7%, specifically 22.9% and 19.1% in kennelled and owned dogs, respectively (Babaei et al., 2010). In the Gauteng Province of South Africa, among 328 canine sera tested with both serum neutralization (SN) and ELISA, 22% of the sera were positive (Nöthling et al., 2008). In a Turkish dog population, the seroprevalence by ELISA was 39.3%, but by SN was 29.4% (Yeşilbağ et al., 2012). An overall CaHV-1 seroprevalence of 45.75% was observed in the Belgian dog population (Ronsse et al., 2002), while in Norway and Finland, the seroprevalence was about 80%, suggesting that CaHV-1 is endemic in those countries (Dahlbom et al., 2009; Krogenæs et al., 2012). Epidemiological studies have not previously been documented in Italy. In this study, sera from 865 dogs from Southern Italy were screened for CaHV-1 antibodies. Furthermore, 16 bitches with and without a history of reproductive disorders were examined clinically to identify CaHV-1 associated lesions; vaginal swabs were tested using PCR to detect viral DNA. Finally, the relationship between reproductive disorders, viral excretion and serology was evaluated to determine the potential importance of subclinical carriers of CaHV-1. Materials and methods Dogs and sampling Dogs of at least 1 year of age and without a history of vaccination against herpesvirus were included in the present study. The study group consisted of 849 serum specimens collected during routine examinations at a Clinical Diagnostic Laboratory in Southern Italy. Venous blood was collected into glass tubes without heparin and serum was withdrawn and stored at −80 °C until analysis was performed. Sixteen bitches from different breeding kennels were included in the study. The vulva and vaginal vestibule were examined for CaHV-1 associated lesions. Three bitches had histories of abortion, seven bitches had histories of infertility, three bitches gave birth to puppies that were either stillborn or died in the neonatal period, and three bitches had no history of reproductive disorders. Vaginal swabs were collected in sample medium (Dulbecco Minimal Essential Medium, DMEM), and serum specimens from each bitch were stored at −80 °C until tested. The 865 serum specimens were grouped into two categories for statistical analysis: 286 sera were collected from kennelled/breeding dogs and 579 from pet dogs. Serum neutralization test The CaHV-1 DK13 strain (supplied by Professor L.E. Carmichael, James A. Baker Institute for Animal Health, Cornell University) was used. The virus was propagated on A72 cell lines grown in DMEM supplemented with 10% fetal calf serum (FCS). The stock virus used in the study was at the ninth passage and had an infectivity titre of 104.5 50% tissue culture infective dose (TCID50)/50 μL. All serum specimens were heat treated at 56 °C for 30 min to inactivate endogenous complement. Two-fold dilutions, starting from 1:2 for each specimen, were performed in duplicate and mixed with 100 TCID50 CaHV-1 strain in 96-well tissue culture microplates (Falcon-Becton, Dickinson Labware). The plates were kept at room temperature for 90 min before 20,000 A72 cells were added to each well. Cell cultures were examined daily for 4 days. The SN titre was defined as the reciprocal of the highest serum dilution that inhibited cytopathic effect (cpe) in 50% of the cell cultures.
Immunofluorescence test The SN-positive serum specimens and 100 SN-negative specimens were also tested for antibodies against CaHV-1 using an in-house immunofluorescence (IF) test. The 100 SN-negative sera were selected from owned dogs, from kennelled dogs and from breeding bitches. A72 cells infected with CaHV-1 strain DK13 were placed in multi-chamber culture slides (LAB-TEK Chamber Slide System) and fixed with 80% acetone. Serial twofold dilutions of each serum, starting from 1:50 to 1:800, were performed in phosphate buffered saline (PBS). Diluted specimens (20 μL) were placed in duplicate into wells and incubated for 30 min at 37 °C in a moist incubator. Slides were washed three times in PBS and blotted dry. Aliquots of pre-titrated goat anti-dog IgG conjugated to fluorescein isothiocyanate (FITC; Sigma Chemicals) diluted 1:50 (20 μL) were added to each well. The slides were then incubated for 30 min at 37 °C, washed again in PBS, counterstained with Evans blue (BioMérieux) and examined under a fluorescent microscope. Approximately 75% of the cells in each well were infected, providing an internal negative control for the non-specific binding of antibodies to the cell monolayer. The titre was read as the highest serum dilution that produced detectable levels of fluorescence in foci of virus-infected cells. Antibody titres 2 weeks of age, has been demonstrated (Evermann et al., 2011). Nevertheless, the relationship between maternal serological status and in utero clinical manifestations appears controversial, suggesting that there is no association with CaHV-1 infection. Other studies have not reported associations between CaHV-1 antibody titres and fertility parameters, but seropositive bitches more commonly have history of abortions, and reproductive disorders were also observed in bitches with high antibody titres (Ronsse et al., 2004, 2005). None of the vaginal swabs in this study tested positive on PCR analysis. This could be due to the limited number of bitches tested and the fact that the virological data did not produce statistically significant results. Nevertheless, it is known that the duration of viral shedding can be very short, so it is advisable to sample dogs for
260
A. Pratelli et al./The Veterinary Journal 200 (2014) 257–260
several consecutive days. Furthermore, since none of the breeding bitches had vaginal lesions related to CaHV-1 infection, the probability of detecting the virus was low. After human herpesvirus 2 (HSV2) infection, asymptomatic patients sometimes shed virus only for only 1 day (Wald et al., 1995). Accordingly, the negative results observed in our study might be a consequence of the brief viral shedding documented for CaHV-1 under field conditions, despite the endemic prevalence of the virus and the specific immunity demonstrated in the dogs tested. Moreover, CaHV-1 is mostly associated with neonatal mortality and although the majority of the bitches in this study had histories of reproductive disorders, no clinical signs were observed at the time of sampling. Considering all of these observations, our results suggest that CaHV-1 is common in Italian dogs and could pose a threat to neonatal survival and dog fertility in Italian dog breeding kennels. Since risk factors for seropositivity have not been established, vaccination of breeding bitches in kennels with histories of reproductive disorders is recommended.
Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgements The authors would like to thank their colleagues at the Department of Veterinary Medicine, Section Infectious Diseases at the University of Bari, Italy, whose collaboration was essential for the accomplishment of the present study.
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