CHV-1 is an important and universal cause of neonatal mortalities and reproductive disorders [2, 17, 18], and canine serve as the only hosts for this virus. The fact that this pathogen can cause infertility shows the importance of this infection. So, veterinarians should warn breeders regarding the economic importance of this organism, especially in newborn puppies and pregnant bitches. This is the first study for molecular detection of CHV-1 in reproductive samples of dogs in Iran. According to the results of this study, it is determined that reproductive organs of bitches admitted to the Veterinary Teaching Hospital of Shahid Bahonar University of Kerman have been infected by CHV-1.
After first description of virus by Carmichael et al. (1965), numerous studies have demonstrated CHV-1 to be enzootic in different parts of the world [2, 15]. Serologic investigations in domestic dogs have been reported from 0 to as high as 100% in some areas [6]. Ronsse et al. (2002) reported an overall CHV-1 seroprevalence of 45.75% in the Belgian dog population. In another study reported in 2008, the prevalence of antibodies against CHV-1 in the serum of dogs was determined as 22% using SNT and ELISA in the Gauteng Province of South Africa [19]. Dahlbom et al. (2009) also reported 81.5% seroprevalence of CHV-1 in Finland [11]. Furthermore, the seroprevalence of CHV-1 infection was determined as 85.5% by immunoperoxidase monolayer assay (IPMA) in Norway [20]. Similarly, in a study performed in turkey in which virus neutralization test (VNT) and ELISA were used for investigation of CHV-1 seroprevalence, antibody against CHV-1 was detected in 39.3% of samples by ELISA in comparison to 29.4% of the specimens using VNT [21]. In this area, only one study conducted by Babaei et al. in 2010 and the overall CHV-1 seroprevalence was reported to be 20.7% by using indirect immunofluorescence antibody (IFA) assay. In comparison, CHV-1 DNA was detected in 21 out of 140 (15%) referred dogs using real-time PCR in the current study.
Compared to our results, other researchers have reported various prevalence of CHV-1 using molecular assays. Using real-time PCR technique, Losurdo reported that not only CHV-1 developed a fatal systemic illness clinically and histologically, but also it could result in long-term shedding of the virus through the nasal and ocular secretions and the faeces [3]. So, infected puppies pose a threat for other animals in the colony. Cargnelutti et al. (2015) also described an outbreak of systemic fatal illness causing neonatal mortalities using histopathology, VNT and PCR [22]. In study of Larsen et al. (2015) performed in Denmark, 13 out of 57 (22.8%) dead puppies were positive for CHV-1 infections by real-time PCR (qPCR) while histopathological and in situ hybridization results were inconsistent [23]. Moreover, CHV-1 was defined as a respiratory virus and detected in nasal swabs of dogs via PCR [24, 25]. Adult dogs also can be implicated in spread of infections. Reactivation of lifelong latent infection of adult dogs can be recognized after environmental stressors and CHV-1 reshedding occurs. Ledbetter et al. in 2009 proved that viral reactivation of latently infected adult dogs with CHV-1 can be induced following administration of an immunosuppressive dosage of systemic prednisolone. Similarly, Malone et al. (2010) described a case of disseminated CHV-1 infection in an immunocompromised adult dog [26]. In the mentioned case, blood sample and vaginal swab were positive for CHV-1 by PCR indicating a viremia. Moreover, Gadsden et al. (2012) reported a case of fatal CHV-1 infection in a 9-year-old spayed female Bichon Frise dog which infection was diagnosed by PCR, immunohistochemistry, and in situ hybridization [27]. Surprisingly, the case had no history of immunosuppression. So, the author stated "CHV-1 should be included in the list of differential diagnoses of hepatic necrosis in adult dogs". In a study accomplished in Mexico, CHV-1 infection of dead puppies was confirmed using histopathology, direct immunofluorescence, electron microscopy and PCR [2].
To the knowledge of the authors, the present study is the first molecular detection of CHV-1 in reproductive samples in Iran. Particularly, there was no study regarding detection of viral DNA in uterine specimens in literature. In the present study, 14 out of 70 uterine samples (20%) were found positive for viral DNA while CHV-1 was detected in 7 out of 70 (10%) vaginal samples. In contrast to our findings, Ronsse et al. (2005) indicated that despite seroconversion, none of the vaginal and nasal swabs or buffy coats were confirmed positive for CHV-1 [13]. In the study of Pratelli et al. (2014) in Italy, an overall seroprevalence of 14.6% and 18.6% was determined using SNT and in-house immunofluorescence (IF), respectively; but CHV-1 DNA was not found in none of the vaginal swabs [18]. In another study conducted in Italy, none of the submitted sera were CHV-1 positive by SNT and nested PCR assays [7]. Li et al. in 2016 reported that replication and invasion of CHV-1 in vaginal mucosa is experimentally better than respiratory mucosa according to the latitude and penetration depth of the plaques of viral antigen positive cells [17]. It seems that the reproductive system is considered a better target rather than the respiratory system for virus.
Our finding is similar to other studies in which there was no statistical difference between age and detection of CHV-1 [7, 14, 19, 21]. In contrast to our findings, other researchers indicated that the rate of the infection was significantly higher in older dogs compared with younger ones [16, 25, 28]. We did not found breeds of dogs as a predisposing factor for infection. In accordance with our findings, other researchers also could not find any relation between CHV-1 and the breed of infected dogs [7, 19]. Despite insignificant difference in study of Yeşilbağ et al., prevalence of CHV-1 was higher in Golden Retrievers (56.2%), followed by Terriers (50.0%) in turkey [21]. Moreover, we did not find any significant difference between CHV-1 detection in privately owned and stray dogs in current study. In agreement with our findings, other investigators demonstrated that the CHV-1 prevalence is not necessarily associated with various housing types [8, 16, 18]. However, there have been a number of reports in which higher prevalence was observed in dog colonies rather than client-owned dogs [21].
Relation between CHV-1 infection and reproductive disorders has been established in previous studies. Dahlbom et al. (2009) found an association between the rates of antibody titers and reproductive problems [11]; although, other researchers demonstrated no significant relationship between CHV-1 infections and reproductive abnormalities [14, 18] which is in accordance with our study. In the study conducted by Ronsse et al., 2004, no considerable association was determined between reproductive conditions and CHV-1 antibody titers in spite of a tendency to more abortions in seropositive breeding kennels [13, 28]. There were no statistically significant differences among pregnancy status and CHV-1 infections in this study. Some authors also did not find any association between CHV1 antibody titers to reproductive parameters such as pregnancy [7, 14]. Ström Holst et al., 2012 indicated pregnancy presumably does not result in reactivation of the infection when there is good sanitary and managements [15]. Excretion of CHV-1 in the vaginal mucosa during whelping was considered as a risk factor for puppies by Kapil, (2015). In study of Ström Holst et al. in 2012, viral reactivation of latent CHV infection was evaluated during gestation on pregnancy consequence or in the course of non-pregnant luteal phase. For this purpose, twelve mated and eight control bitches were assessed by detection of antibody against CHV-1 in blood samples and viral DNA in vaginal swab using real-time PCR. Interestingly, any dependable difference in antibody titers was not observed in these stages and vaginal swab was not CHV-1 positive [15].
The prevalence of CHV-1 infection depends on the geographic regions, variation in sampling, and also the largely various study populations. The difference between sensitivity of the various diagnostic methods can also affect the results. CHV-1 is considered as a poorly immunogenic virus which antibodies against of it disappear during a few months after exposure [7, 28]. Moreover, serologic assays have not been standardized and different results can be extracted from various labs. This emphasizes that the seroprevalence of infection can be underestimated using serologic techniques. In the current study, real-time PCR was used to detect CHV-1 in reproductive samples. PCR is well known as one of the most reliable methods for detecting CHV-1 in puppies and latent infections of adult dogs [5, 6]. Kapil in 2015 optimized a PCR test for detection of CHV-1 DNA in formalin-fixed, paraffin embedded (FFPE) sections and stated "The gB gene offered a suitable PCR target for CHV-1 detection in FFPE samples".