This study shows that the majority of clinically healthy bitches presented to the Canine Reproductive Clinical Unit at the Norwegian School of Veterinary Science, have been infected by CHV1.
In a previous Norwegian study including both genders, we demonstrated significant geographical differences ranging between 58.5% in eastern Norway and 98% in mid-Norway . When comparing the seroprevalence in the two dog populations originating from eastern Norway and collected in the same period, there is a difference of 85.5% (breeding bitches in this study) vs 58.5% (dogs of both genders in the previous study). This finding might indicate that reproductively active bitches admitted to the reproduction clinic have a greater risk of CHV1 exposure and subsequent infection compared to the general dog population in this part of the country.
All bitches in the present study were in proestrus or estrus. Although clear evidence of an association between circulating reproductive hormone concentrations and viral activation have not been demonstrated, Ronsse et al. reported fluctuations in CHV1 antibody titers related to cycle stage. They found slightly higher titers in estrus and significantly lower titers in early di-estrus . Both 17-β estradiol and medroxyprogesterone acetate have been shown to promote herpes simplex virus type 1 reactivation in mice [26, 27]. Evermann et al. listed risk factors for reproductive disease in the bitch, and cycle stage was identified as one of 8 factors having a positive correlation with disease. Whether estrus itself is a potential stressor which can reactivate a latent infection or increase susceptibility for a new infection remains to be seen. However, clinical signs of reactivation have been described to occur more frequently during heat and around parturition . Psychological stress has also been suggested to contribute to reactivation of latent herpes simplex virus in humans [28, 29].
In Norway, few breeding dogs are kept in kennels, so our data are not directly comparable to kenneled dogs in studies from other countries. Babaei et al. and Ronsse et al. could not demonstrate differences in CHV1 seroprevalence between privately owned in- house pets and kennelled dogs. Nöthling et al. observed that seroprevalence was independent of kennel size, whereas Ronsse et al. found higher titers in kennels with 6–30 dogs than in those with fewer dogs.
There was no difference in age between the seronegative and the seropositive bitches, and the seroprevalence did not increase with age. This is in contrast to our previous study  and can be explained by less variation in age in the breeding bitches in this study compared to dogs in the general population.
In the current study, there were no significant correlations between titer category and breeding status, such as previous matings, previous whelping, failing to get the bitch pregnant or conditions of puppies. This is in accordance with Ronsse et al.. In the present study, the time between previous mating and sampling is variable, and the titer at previous mating is unknown. But, interestingly, owners reported increasing problems of getting the bitches pregnant after previous mating in the highest titer category compared to the others. Recent CHV1 infection coinciding with early stage of pregnancy may result in fetal loss and this could be a plausible explanation. Several authors have demonstrated an association between CHV1 serological status and reproductive problems. Dahlbom et al. found that dogs from kennels with reproductive problems had significantly higher CHV1 titers than dogs from kennels without reproduction problems. Ronsse et al. demonstrated an association between serological status and a history of abortion in bitches. Furthermore, Van Gucht et al. found a relation between the presence of positive breeding bitches and neonatal death and/or infertility in the kennel.
Travel abroad with breeding dogs is quite common in Norway and is confirmed by dog-owners in this investigation for both antibody positive and –negative dogs. However, travel abroad had no influence on the titer values. This was also the fact when this was tested as a separate risk factor in our previous study of dogs in different parts of Norway . By multivariable analysis, it was shown that the variable travel abroad contributed to a better classification of seropositive dogs in our previous study, which was not the case in this study with fewer dogs included. Surprisingly, significantly fewer dogs within all positive titer categories had participated in dog shows, competitions or hunting trials compared to dogs with negative titers. This might be related to the fact that the owners of the bitches in this study are committed, well-informed breeders and even though they travel or participate in different competitions, they keep their dogs under more controlled conditions than dog owners in general. The attitude and behavior of the owners in addition to the very low number of dogs not participating in competitions, might therefore be possible confounders in this study. We can conclude that the endemic status of CHV1 infection in the Norwegian dog population provides ample opportunities for spread of the virus. Staying abroad or participation in competitions/shows does not seem to add an extra risk of contracting infection in this study.
In our data, 57.3% of the dogs had been pregnant prior to sampling. No difference in seroprevalence between this group of bitches and those being mated for the first time was demonstrated, indicating that the oronasal infection route is the most likely way of virus transmission between dogs. This finding is in accordance with other studies [12, 15, 18] of adult dog populations. However, Babaei et al., suggest that oronasal transmission of CHV1 may be epidemiologically less important than venereal transmission. In their study, no seropositive dog was detected in animals younger than 12 months of age. It might be important to emphasize that the overall CHV1 seroprevalence was estimated to 20.7%, which is very low.
In spite of the high seroprevalence in reproductively active dogs, we have the opinion that fertility problems and loss of newborn puppies due to CHV1 infection at present are of minor importance in Norway. There are no epidemiological data available regarding the prevalence of reproduction disorders in bitches. However, two recent studies on postnatal puppy mortality have been published. IndrebØ et al. reported a total puppy loss to be 6.9% during the first three weeks of life in a selected population of four breeds. Etiological diagnosis was not recorded, and the authors concluded that impact of CHV1 infection was unlikely because of rare occurrence and no evidence indicating this disease. TØnnessen et al. reported perinatal mortality in a large-scale observational study including 10.810 litters and 224 breeds. In total, perinatal mortality was observed in 8% of the puppies (4.3% stillborn and 3.7% died before age of 8 days). Autopsy was not performed and causes for perinatal mortality not pursued. Obviously, there is need for follow-up investigations to find etiological causes of these clinical manifestations. At the Norwegian Veterinary Institute in Oslo, CHV1 has been diagnosed sporadically in newborn puppies (Øyvor KolbjØrnsen, personal communication). There is most likely an underdiagnosing of CHV1 infection because few puppies are submitted for autopsy due to high costs. In Norway, dogs are kept in households usually including one family dog, occasionally two, and there are few big breeding kennels . Management and hygienic conditions are generally good and the stress level low probably contributing to less impact of CHV1. The fact that most bitches are seropositive to CHV1 following natural immunization, may protect the puppies against disease. Further, satisfactory vaccine coverage against other diseases, provide fetuses and puppies with good general protection against infection.
Interestingly, moderately positive and highly positive bitches were found only in dogs sampled during summer and fall, whereas no negative or weakly positive dogs were sampled in these seasons. This might indicate that the dogs are more exposed to new infections or re-expression of latent infections during the summer and fall, which could be due to increased outdoor activities in general and contact with other dogs in this period. Our findings may explain observations by TØnnessen et al. that litters born during fall had a higher risk of experiencing early neonatal mortality than litters born during other seasons.
The three parameters of season, previous birth and participation in competitions/shows increase the probability of correct classification between the different titer categories to between 70 to 80%.
Vaccination against CHV1 infection is used in some countries. In Norway, the herpesvirus vaccine is a non-core vaccine. Vaccination is not routinely recommended for breeding bitches. Nevertheless, veterinarians experience an increased demand for vaccination from owners concerned about CHV1 infection and impact of their breeding success. In case of an increased risk of CHV1 infection by recent contact with other bitches that have aborted, borne weak puppies or suffered increased neonatal mortality due to CHV1, vaccination should be considered in young females entering into their first or second pregnancy. In bitches with a positive CHV1 antibody titer from natural infection, protective efficacy is difficult to evaluate. We consider the status of majority of these dogs as being protected. Moreover, if reactivation of virus occurs, this is likely to boost the immune response and increase level of immunity. However, there might be relevant to consider vaccination even though naturally acquired antibodies are present, for example in older breeding bitches from about six years age. Here a reactivation may be more likely to cause problems since aging is associated with decreased immune responsiveness and increased susceptibility to infectious diseases [20, 35, 36].