Clinical and immunological assessment of therapeutic immunization with a subunit vaccine for recurrent ocular canine herpesvirus-1 infection in dogs.

Latent canine herpesvirus-1 (CHV-1) infections are common in domestic dogs and reactivation of latent virus may be associated with recurrent ocular disease. The objectives of the present study were to evaluate the ability of a subunit CHV-1 vaccine to stimulate peripheral CHV-1 specific immunity and prevent recurrent CHV-1 ocular disease and viral shedding. Mature dogs with experimentally-induced latent CHV-1 infection received a 2-dose CHV-1 vaccine series. Recurrent ocular CHV-1 infection was induced by corticosteroid administration in the prevaccinal, short-term postvaccinal (2 weeks post-vaccination), and long-term postvacccinal (34 weeks post-vaccination) periods. Immunological, virological, and clinical parameters were evaluated during each study period. Quantitative assessment of peripheral immunity included lymphocyte immunophenotyping, proliferation response, and interferon-γ production; and CHV-1 virus neutralizing antibody production. In the present study, vaccination did not prevent development of ocular disease and viral shedding; however, there was a significant decrease in clinical ocular disease scores in the short-term postvaccinal period. Significant alterations in peripheral immunity detected in the dogs during the short-term and long-term postvaccinal periods included increased T and B lymphocyte subpopulation percentage distributions, increased lymphocyte expression of major histocompatibility complex class I and II, increased CHV-1 virus neutralizing antibody titers, decreased lymphocyte proliferation, and decreased interferon-γ production. Vaccination of latently infected mature dogs with the selected subunit CHV-1 vaccine was not effective in preventing recurrent ocular CHV-1 infection and viral shedding induced by corticosteroid administration. The vaccine did induce long-term CHV-1 specific immunity and may decrease the severity of clinical ocular disease in the immediate postvaccinal period.

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.

OBJECTIVE: To determine the frequency of spontaneous canine herpesvirus-1 (CHV-1) reactivation and ocular viral shedding in latently infected dogs and the effect of topical ocular administration of cyclosporine. ANIMALS: 8 mature Beagles with experimentally induced latent CHV-1 infection. PROCEDURES: Following induction of primary ocular CHV-1 infection, the presence of reactivatable CHV-1 latency was confirmed by systemically administering prednisolone to the dogs. Dogs were then monitored for 36 weeks via clinical examination and conjunctival sample CHV-1 PCR assay performed at 4-day intervals and CHV-1 virus neutralization antibody assay performed at 2-week intervals. During weeks 16 to 32, dogs were administered 0.2% cyclosporine ointment in both eyes twice daily and blood cyclosporine concentrations were monitored. During weeks 33 to 36, the presence of reactivatable CHV-1 latency was reconfirmed via systemic administration of prednisolone. RESULTS: Reactivation of latent CHV-1 was not detected via clinical examination or viral shedding during the initial 32 weeks, including before and during topical ocular administration of cyclosporine, and there were no significant differences in CHV-1 virus neutralization titer increases between the study periods. Blood cyclosporine concentrations were less than assay detection limits in all dogs on the sampling days. Systemic administration of corticosteroids repeatedly resulted in ocular disease and viral shedding. CONCLUSIONS AND CLINICAL RELEVANCE: Spontaneous CHV-1 reactivation did not occur frequently in latently infected mature dogs, and this was not altered by topical ocular administration of cyclosporine. This characteristic may be a factor contributing to the lower frequency of recurrent herpetic ocular disease in dogs relative to other host species and their associated alphaherpesviruses.

A serologic study of canine herpes virus-1 infection in the Norwegian adult dog population.

Canine herpes virus-1 (CHV1) causes a fatal hemorrhagic disease in neonatal puppies and is associated with reproductive problems in female dogs. This serologic study was conducted to assess the seroprevalence of CHV1 infection in Norway. Blood samples were collected from clinically healthy dogs (n = 436) one yr of age and older of both genders, supplied by four small animal clinics (A, B, C and D) in different parts of the country. The immunoperoxidase monolayer assay was used for testing of CHV1 antibodies. Serum titers were recorded as the reciprocal value of the highest dilution producing specific cell staining. Titers equal to or above 80 were considered positive for exposure to CHV1. In total, 80.0% of the dogs had titers ≥80 and were classified as positive. Mean age for seronegative dogs was 4.7 yrs (95% CI 4.1-5.4) and for seropositive dogs 5.0 yrs (95% CI 4.7-5.4). Of the dogs, 32.8% displayed a weakly positive titer of 80, whereas 41.5 and 5.7% fell into the moderately (titer 160 and 320) and strongly (titer ≥640) positive categories, respectively. No association was demonstrated when comparing CHV1 antibody titers to gender or reproductive parameters like previous matings, pregnancies, births or number of puppies born. Age, visit in foreign countries and clinic explained together 78% of the variation in antibody titer categories. The percentage of positive samples differed significantly between the four clinics (A 98%, B 58.5%, C 74.6%, D 89.5%). A reasonable explanation for this finding has not been established. No information about an ongoing outbreak of CHV1 infection was available. In conclusion, this study strongly indicates that CHV1 infection is endemic in the dog population of Norway. There are significant differences in seroprevalence between geographic regions in the country.

Seroprevalence of canine herpesvirus-1 in Turkish dog population.

Canine herpesvirus-1 (CHV-1) is the agent of reproductive and respiratory disorders in adult dogs, and the infection generally results in haemorrhagic disease conditions and neonatal death. In this study, virus neutralisation test that used complement (VNT) as well as in-house ELISA were utilised to investigate the CHV-1 seroprevalence in the Turkish dog population. Among the 560 serum samples, 39.3% of the samples tested by ELISA were CHV-1 positive while 29.4% of the samples tested by VNT were CHV-1 positive. Compared to the individual dogs (39.0%), there was a higher CHV-1 seroprevalence (62.1%) found in the colony dogs (62.1%) (p=0.0002). However, there was an insignificant difference between male and female dogs. Although the highest antibody prevalence (56.7%) was found in Golden Retrievers, there were no significant variations detected among the dog breeds used in this study. Neutralizing antibody titres were very low (⩽1:16) in a high portion of the tested animals, confirming the rapid decrease of CHV-1 antibodies after the course of infection. The results of this study show that CHV-1 seroprevalence is moderately high in the Turkish dog population.

Serological evidence of canine herpesvirus-1 in dogs of Kerman city, south-east of Iran.

Canine herpes virus-1 (CHV-1) is an alphaherpesvirus, which causes foetal and neonatal death as well as fertility problems in dogs. The virus is presumed to be enzootic in dogs all over the world, but no information was found about the seroprevalence of CHV-1 from middle-east countries. Therefore, this study was aimed to determine the seroprevalence of CHV-1 among dogs in Kerman (south-east of Iran). Blood samples were taken from 47 privately owned and 35 kennelled dogs, respectively. The entire sampled dogs were apparently healthy. Indirect immunofluorescence antibody (IFA) assay was used to detect antibodies against CHV-1 in all sera. The overall CHV-1 seroprevalence was estimated 20.7%, which was 22.9% and 19.1% for kennelled and owned dogs, respectively. Sex, parity and raising status (owned or kennels) did not differ significantly between seropositive and seronegative dogs. However, the infection rate was significantly higher in dogs older than 3 in comparison with younger groups (15.9% vs. 4.8%, P ≤ 0.05). In conclusion, this study revealed that CHV-1 could be considered endemic in Iran, and more epidemiological researches are needed to identify the geographical distribution of diseases in Iran.

A serological survey of infectious disease in Yellowstone National Park's canid community.

BACKGROUND: Gray wolves (Canis lupus) were reintroduced into Yellowstone National Park (YNP) after a >70 year absence, and as part of recovery efforts, the population has been closely monitored. In 1999 and 2005, pup survival was significantly reduced, suggestive of disease outbreaks. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed sympatric wolf, coyote (Canis latrans), and red fox (Vulpes vulpes) serologic data from YNP, spanning 1991-2007, to identify long-term patterns of pathogen exposure, identify associated risk factors, and examine evidence for disease-induced mortality among wolves for which there were survival data. We found high, constant exposure to canine parvovirus (wolf seroprevalence: 100%; coyote: 94%), canine adenovirus-1 (wolf pups [0.5-0.9 yr]: 91%, adults [>or=1 yr]: 96%; coyote juveniles [0.5-1.5 yrs]: 18%, adults [>or=1.6 yrs]: 83%), and canine herpesvirus (wolf: 87%; coyote juveniles: 23%, young adults [1.6-4.9 yrs]: 51%, old adults [>or=5 yrs]: 87%) suggesting that these pathogens were enzootic within YNP wolves and coyotes. An average of 50% of wolves exhibited exposure to the protozoan parasite, Neospora caninum, although individuals' odds of exposure tended to increase with age and was temporally variable. Wolf, coyote, and fox exposure to canine distemper virus (CDV) was temporally variable, with evidence for distinct multi-host outbreaks in 1999 and 2005, and perhaps a smaller, isolated outbreak among wolves in the interior of YNP in 2002. The years of high wolf-pup mortality in 1999 and 2005 in the northern region of the park were correlated with peaks in CDV seroprevalence, suggesting that CDV contributed to the observed mortality. CONCLUSIONS/SIGNIFICANCE: Of the pathogens we examined, none appear to jeopardize the long-term population of canids in YNP. However, CDV appears capable of causing short-term population declines. Additional information on how and where CDV is maintained and the frequency with which future epizootics might be expected might be useful for future management of the Northern Rocky Mountain wolf population.

Seroprevalence of canine herpesvirus-1 and Brucella canis in Finnish breeding kennels with and without reproductive problems.

We compared the serological status of Brucella canis and canine herpesvirus-1 (CHV-1) in Finnish breeding kennels with and without reproductive problems. Dogs from kennels with reproductive problems had significantly higher CHV-1 titres than dogs from kennels having no reproductive problems (p < 0.001). In dogs from kennels with reproductive problems 100% (32/32) had positive titres, whereas in dogs from kennels without reproductive problems 65% (22/34) had positive titres. The median titre for dogs from kennels with reproductive problems was 1 : 160 and for dogs from kennels without reproductive problems 1 : 80. The high prevalence of positive CHV-1 titres in this study indicates that prevention of the disease is difficult and reinforces the need to minimize the reproductive problems caused by CHV-1. All 388 dogs from 94 kennels had negative B. canis titres.

Seroprevalence of canine herpesvirus in breeding kennels in the Gauteng Province of South Africa.

Canine herpesvirus (CHV-1) causes neonatal deaths as well as infertility due to embryonal death, abortion and stillbirths in breeding kennels. The aim of this study was to determine the prevalence of antibodies against canine herpesvirus in the serum of dogs older than 1 year in breeding kennels in the Gauteng Province of South Africa. A serum neutralization test (SNT) and a newly developed enzyme linked immunosorbent assay (ELISA) were used to test the serum samples of 328 dogs in 38 breeding kennels. With SNT as well as ELISA, 22% of sera were positive (P>0.9). Seventeen kennels (45% of total kennels) each had at least one positive dog on SNT compared with twenty kennels (53% of total kennels) that each had at least one positive dog on ELISA (P=0.6). The prevalence of positive dogs in positive kennels was 42+/-26% (n=17 kennels) for SNT and 39+/-26% (n=20 kennels) for ELISA. Pairwise comparison of kennels showed that the prevalence of SNT positive dogs was similar to the prevalence of ELISA positive dogs (P=0.3, n=38 kennels). Seroprevalence was independent of age, gender or colony size. This study suggests that canine herpesvirus is sufficiently common in breeding dogs in the Gauteng Province of South Africa to pose a threat to neonatal survival and fertility.

Compatibility of a bivalent modified-live vaccine against Bordetella bronchiseptica and CPiV, and a trivalent modified-live vaccine against CPV, CDV and CAV-2.

Eight puppies (group 1) were vaccinated once with a bivalent modified-live vaccine against infectious tracheobronchitis by the intranasal route and at the same time with an injectable trivalent vaccine against canine parvovirus, canine distemper virus and canine adenovirus; a second group of eight puppies (group 2) was vaccinated only with the intranasal bivalent vaccine, and a further eight puppies (group 3) were vaccinated only with the injectable trivalent vaccine. Three weeks later they were all challenged with wildtype Bordetella bronchiseptica and canine parainfluenza virus by the aerosol route, and their antibody responses to the five vaccine organisms were determined. Oronasal swabs were taken regularly before and after the challenge for the isolation of bacteria and viruses, and the puppies were observed for clinical signs for three weeks after the challenge. There were no significant differences in the puppies' titres against canine parvovirus, canine distemper virus and canine adenovirus type 2 between the groups vaccinated with or without the bivalent intranasal vaccine. After the challenge the mean clinical scores of the two groups vaccinated with the intranasal vaccine were nearly 90 per cent lower (P=0.001) than the mean score of the group vaccinated with only the trivalent injectable vaccine, and the puppies in this group all became culture-positive for B bronchiseptica and canine parainfluenza virus. There were only small differences between the rates of isolation of B bronchiseptica from groups 1, 2 and 3, but significantly lower yields of canine parainfluenza virus were isolated from groups 1 and 2 than from group 3.

A virus vector based on Canine Herpesvirus for vaccine applications in canids.

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.

Suitability of canine herpesvirus as a vector for oral bait vaccination of foxes.

Studies were conducted to evaluate the feasibility of using canine herpesvirus (CHV) as a vaccine vector for bait-delivered oral vaccination of wild foxes. To test the viability of CHV in baits, CHV was freeze-dried, incorporated into different baits, stored, and the remaining viral infectivity tested in cell culture after varying periods of time at different storage temperatures. Experimental baits (mouse carcasses) and commercial baits (FOXOFF and PROBAIT) were prepared with either liquid or freeze-dried CHV and tested in two fox trials for their capacity to induce CHV-specific antibodies following oral baiting. Freeze-drying and storage temperatures below 0 degrees C had a stabilizing effect to virus infectivity. When stored at -20 degrees C, freeze-dried CHV retained its full infectivity for up to 3 months in PROBAIT baits, the remaining infectivity in FOXOFF baits was 100-fold less. Oral baiting with CHV induced antiviral serum antibodies in all vaccinated foxes (20/20). None of the vaccinated foxes became ill or shed infectious virus into the environment although viral DNA was detected in body secretions as evaluated by PCR. The results indicate that CHV can be freeze-dried and stored over extended periods of time without loosing much of its infectivity. This is the first report of CHV being used for oral bait vaccination of foxes. It appears that CHV is well suited for use as a recombinant vector for wild canids.

Development of canine herpesvirus based antifertility vaccines for foxes using bacterial artificial chromosomes.

Using bacterial artificial chromosome (BAC) technology, a canine herpesvirus (CHV)-based recombinant vaccine vector was produced for the development of an antifertility vaccine for foxes. Infectious viruses were recovered following transfection of canid cells with a BAC plasmid carrying the complete CHV genome. In vitro growth characteristics of BAC-derived viruses were similar to that of wildtype (wt)-CHV. Two recombinant antigens, fox zona pellucida protein subunit 3 (fZPC) and enhanced green fluorescent protein (EGFP) as control antigen, were inserted into thymidine kinase (TK) locus of the CHV genome and shown to be efficiently expressed in vitro. Inoculation of foxes with transgenic CHVs induced CHV specific antibodies, but was innocuous and failed to elicit transgene-specific antibody responses. Infectious virus or viral DNA was not detected in mucosal secretions or tissues of vaccinated foxes. The CHV-BAC system proved to be a quick and reliable method to manipulate the CHV genome. It will help to readily apply changes in the vector design in order to improve virus replication in vivo.

Prevalence of serum antibodies to canine adenovirus and canine herpesvirus in the European red fox (Vulpes vulpes) in Australia.

OBJECTIVES: To determine the seroprevalence and aspects of the epidemiology of canine adenovirus (CAdV) and canine herpesvirus (CaHV-1) in European red foxes (Vulpes vulpes) in Australia. DESIGN: Serum samples were collected opportunistically from foxes in 1991-1994 in Western Australia (WA) and South Australia (SA) and in 1980-1984 and 1990-1994 in New South Wales (NSW) and the Australian Capital Territory (ACT). The sera were examined for antibody to CAdV and CaHV-1 using ELISAs. Seroprevalence in the different regions was determined for both viruses and the CAdV data were analysed for interactions between decade of collection, age, season, region and gender using logistic regression. RESULTS: The overall prevalence of antibody to CAdV was 23.2% (308/1326) but was significantly higher in sera collected in the eastern states of Australia (47%: 233/498) than in WA (9%: 75/828). Overall, in NSW and the ACT, there was a significantly lower prevalence in juveniles than in adults and the prevalence in juveniles in the 1990s was significantly lower than in the 1980s. The prevalence was also significantly lower in the autumn than in the winter for juveniles but the reverse held for adults. The NSW and ACT data were subdivided into eastern (including the ACT) and western regions. This revealed a significantly higher prevalence in the winter than in the autumn for the west and the reverse in the east. In WA, the northern rangeland regions of WA had lower prevalence (1.9%) than the southern agriculture regions (10.7%). Seasonally, there was a peak prevalence in the spring dropping through the summer and autumn and rising again in the winter. This seasonal pattern was also found in the combined data for all sites in the 1990s. There was no gender difference in prevalence of CAdV either overall or in different regions. The overall prevalence of antibody to CaHV-1 was 2.2% (28/1300). The small number of positives allowed only limited statistical analysis that did not reveal any differences in decade of collection, age, season or region. CONCLUSIONS: CAdV infection is common in the Australian fox population whereas CaHV-1 infection is rare. For CAdV, the age and seasonal patterns of seroprevalence were generally consistent with the recruitment of young susceptible foxes into the population in the spring and the accumulation of infections with age. The differences in regional prevalences correlated with fox density. The low prevalence of antibody to CaHV-1 suggests that CaHV-1 may be a more suitable vector than CAdV for bait delivery of immunocontraceptive antigens to foxes in Australia.

Protection of dogs for 13 months against Bordetella bronchiseptica and canine parainfluenza virus with a modified live vaccine.

Twelve specific pathogen-free (spf) puppies were vaccinated intranasally with a bivalent, modified live vaccine against infectious tracheobronchitis (group 1) and six puppies of the same age and from the same source served as unvaccinated controls (group 2). Both groups were challenged with wild-type Bordetella bronchiseptica and canine parainfluenza virus by the aerosol route 56 weeks after group 1 had been vaccinated, and at the same time six 10-week-old spf puppies from the same source (group 3) were also challenged. Oronasal swabs were taken regularly before and after the challenge, for the isolation of bacteria and viruses, and the dogs were observed for clinical signs for three weeks after the challenge. The control dogs became culture-positive for B bronchiseptica and canine parainfluenza virus, but the isolation yields from the vaccinated group were significantly lower (P<0.05). The mean clinical scores of the vaccinated group were 61 per cent lower than the scores of group 2 (P=0.009), and 90 per cent lower than the scores of group 3 (P=0.001).

Canine herpesvirus-1 (CHV-1): clinical, serological and virological patterns in breeding colonies.

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with reproductive disorders and neonatal mortality. To advise dog breeders towards an effective management of CHV-1 infected colonies, 27 breeding bitches were studied during one reproductive cycle in field conditions: the effect of cycle stage, kennel size, initial antibody titre, mating and gestation on serologic and viral excretion patterns was evaluated, while the association between reproductive disorders and CHV-1 antibody titres and viral excretion was also analysed. All initially seronegative bitches seroconverted, while 40% of the initially seropositive bitches became seronegative at one or two occasions. No difference in antibody patterns was observed between mated and unmated bitches. Of the mated bitches, 46% experienced infertility, foetal resorption or mummification. No difference in antibody patterns was observed depending on the occurrence of reproductive disorders even if a decrease in antibody titres during early or late-di-oestrus was often present. Significantly higher titres were observed at all cycle stages in large kennels. None of the vaginal and nasal samples or buffy coats tested positive for CHV-1 DNA. The mixed image of clinical and sub-clinical carriage in this study demonstrated CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with vaccination of reproducing bitches in kennels with reproductive disorders should therefore be advised.

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations.

Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.

Corticosteroid treatment does not reactivate canine herpesvirus in red foxes.

To study canine herpesvirus (CHV) reactivation from red foxes (Vulpes vulpes), 29 foxes with varying CHV antibody and CHV carrier status were treated with methylprednisolone acetate, a glucocorticosteroid drug with prolonged immunosuppressive effect in dogs. In the first experiment, 17 foxes with unknown CHV carrier status were treated once with methylprednisolone: in the second experiment, five foxes were treated twice, 4 mo after being intravenously CHV infected; and in the third experiment, six foxes were treated five times, 11 mo after peroral CHV infection. Infectious CHV was not isolated after treatment from either naturally or experimentally CHV-infected foxes or from untreated, CHV-seronegative in-contact foxes. Canine herpesvirus DNA was not detectable in mucosal secretions or white blood cells of any of the foxes, whereas all trigeminal ganglia of experimentally CHV-infected foxes were polymerase chain reaction-positive. In CHV-seropositive foxes, anti-CHV antibody titers did not change with time after treatment, and CHV-seronegative in-contact controls did not seroconvert. Hematologic parameters remained mostly unchanged. We conclude that CHV is not as easily reactivated in foxes following corticosteroid treatment as in dogs, although there was no obvious sign of immunosuppression. Canine herpesvirus was not spread from virus carriers to naive in-contact foxes, which may be among possible explanations for the reported low CHV prevalence in wild foxes.

Risk factors and reproductive disorders associated with canine herpesvirus-1 (CHV-1).

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with fertility disorders and neonatal mortality. In this study we screened for risk factors affecting CHV-1 antibody titers and investigated the association between antibody titers and reproductive disorders. Therefore, serum from 545 dogs used for reproduction was analysed with an ELISA. Using a forward stepwise procedure and retaining significant risk factors (P<0.05), best fitting multifactorial generalized linear model (glm) procedures were built for males and females. The effect of antibody titers on reproductive disorders was analysed with logistic regression analysis. The association between reproductive disorders and seroprevalence was analysed in chi-square analyses using contingency tables. In both sexes, kennel cough and breeding management were found to have an impact on the CHV-1 antibody titer. Also, the influence of kennel cough on the antibody titer was correlated to the hygienic status of the kennel. In females, age, kennel size and cycle stage had an effect on CHV-1 antibody titers. Furthermore, kennel size and hygiene were found to be correlated. In males, mating experience had an impact on CHV-1 antibody titers. An association was observed between serological status and a history of abortion in bitches. In conclusion, this study suggests CHV-1 antibody titers may be affected by many factors, both on an environmental and host level. Therefore, interpretation of the serological status requires precaution. Furthermore, oronasal and venereal transmission seem to play a role in the spreading of infection.

Seroprevalence of canine herpesvirus-1 in the Belgian dog population in 2000.

Canine herpesvirus-1 (CHV-1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme-linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV-1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV-1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non-breeding dogs.

Experimental infection of European red foxes (Vulpes vulpes) with canine herpesvirus.

We report on the pathogenicity of canine herpesvirus (CHV) for European red foxes. In the first experiment, we inoculated 10 adult foxes intravenously with a canine isolate of CHV. All foxes became infected and shed CHV in saliva and genital secretions for up to 14 days post-inoculation (p.i.) as evaluated by PCR and/or by virus isolation. All foxes developed clinical signs such as fever, lethargy and evidence of respiratory tract disease. Two foxes died on day 6 p.i., one on day 7 p.i., and one fox was euthanased on day 6 p.i. Tissues taken from the four dead foxes were positive for CHV by PCR. The remaining six foxes recovered after approximately 14 days p.i. Virus particles with morphology typical of herpesviruses were found by electron microscopy in the liver of an infected animal. All surviving foxes developed serum anti-CHV antibodies. In a second experiment, six foxes were dosed perorally with CHV and paired with six untreated controls. Neither the perorally dosed nor the in-contact control foxes developed clinical signs of disease. Infectious CHV was not isolated from any of the dosed or the in-contact foxes but all perorally-infected foxes and one of the in-contact foxes tested PCR-positive for CHV on several occasions p.i. All perorally-infected foxes, but none of the in-contact foxes, seroconverted. In summary, intravenous CHV inoculation caused a clinical disease in adult foxes much more severe than observed in experimentally-infected adult dogs. No clinical disease or virus spread was observed after peroral dosing although viral infection occurred as evidenced by seroconversion.