The first molecular detection of Canine herpesvirus-1 in reproductive specimens of adult dogs in Iran

Background Canine herpesvirus-1 (CHV-1) is recognized to be enzootic in the dog population with a widespread distribution. This pathogen leads to a lethal generalized illness in newborn puppies and is associated with reproductive disorders. CHV-1 should be considered as an important pathogen of neonatal death and infertility; so, it appears to pose a threat for breeding kennels. Although serologic data point to the circulation of CHV-1 among dogs of Iran, not definitive diagnosis has been conducted based on the molecular assay. So, this research was done to detect the prevalence of CHV-1 in dogs of Kerman. In this study, the presence of CHV-1 in vaginal specimens and biopsies of the uterus of dogs referring to the Veterinary Hospital of Shahid Bahonar University of Kerman was determined. Samples were collected and evaluated using real-time PCR. Results Viral DNA was detected in 21 samples from a total of 140 (15%) collected samples which were related to 14 uterine samples (20%) and 7 (10%) vaginal specimens. The association of this virus with age, breed, housing, pregnancy and reproductive disorders was not significant. Conclusions This study is the first molecular detection of CHV-1 in reproductive samples of dogs in Iran. Considering the significant prevalence of this virus, it is necessary to carry out management measures in controlling and preventing this disease. Tracing CHV-1 requires further research on this virus in dogs of this region.


Background
Canine herpesvirus-1 (CHV-1), an alpha herpesvirus, is considered as a significant cause of neonatal deaths and reproductive problems in bitches with a widespread distribution [1][2][3]. In 1965, Carmichael et al. described this pathogen for the first time [4]. It has been well documented that the cell tissues of domestic and wild Canidae can only be infected by CHV-1 [5][6][7]. Virus is transmitted through exposure with secretions from respiratory or genital tracts of infected animals. Newborn puppies can be infected in utero, through the birth canal or contact with oronasal and genital mucosal secretions [4,8,9]. CHV-1 infection causes various clinical presentations based on the age of infected animals [5]. Newborn puppies are susceptible to manifest an acute fatal generalized systemic illness which is characterized by progressive multifocal hemorrhagic necrosis in multiple organs [3,9,10] Mild 3 subclinical infection or inapparent clinical illness is observed in puppies older than 3 weeks [11]. Adult domestic dogs can be clinically asymptomatic or display canine infectious respiratory disease complex and genital papulovesicular lesions [12]. Reproductive disorders including abortion, fetal death, mummification, stillbirth, premature or weak newborn puppies are observed in pregnant bitches [9,10,13]. Recently, CHV-1 was correlated to the ocular lesions including conjunctivitis and ulcerative and non-ulcerative keratitis [12]. Lifelong latent infection can be established after recovery [6,12]. In stressful conditions such as overcrowding, transportation, pregnancy and immunosuppression, CHV-1 reactivates and replicates, and therefore reshedding occurs [12,14,15].
CHV-1 infection is diagnosed serologically by serum neutralization test (SNT) and enzyme linked immunosorbent assay (ELISA), virus isolation, fluorescent antibody techniques and electron microscopy. Polymerase chain reaction (PCR) is recognized as the most reliable method of identifying latent infection in adult dogs. A real-time PCR assay has also been established to detect CHV-1 nucleic acid [5].
Although no vaccine is commercially available in Iran and serologic data point to the circulation of CHV-1 among dogs, no definitive diagnosis has been conducted based on the molecular assay. In a recent study from south-east of Iran, the seroprevalence of CHV-1 was reported to be 20.7% in dogs [16]. Various predisposing factors have been reported to influence the incidence of CHV-1, but the conclusions have been inconsistent. Considering the global spread of this virus and its economic importance due to reproductive disorders, this research was conducted to determine the prevalence of CHV-1 in dogs of Kerman by real-time PCR assay and to investigate the possible risk factors.

Results
In this study, a total of 140 reproductive samples were collected and submitted to real-time PCR targeting the gB gene to detect CHV-1. Viral DNA was detected in 21 out of 140 (15%) reproductive samples collected from the referred dogs. In uterine samples, 14 specimens (20%) were positive for viral DNA whereas CHV-1 was identified in 7 out of 70 (10%) vaginal samples ( Table 2). Out of 14 positive uterine samples, 6 specimens were collected from adult dogs ≤2 years old, 7 samples were 4 belonged to dogs between 2 to 7 years old and one case was above 7 years old age. Although, no statistically significant differences were observed between all groups (P = 1), the rate of positive samples was greater in dogs less than 7 years old age. Regarding vaginal samples, in the group of adult dogs, two dogs which were under 2 years old age were positive for CHV-1 DNA. Furthermore, viral DNA was amplified in three out of 37 cases aged between 2 to 7 years old and two of 13 dogs which were above 7 years old age. No association was identified between age and presence of CHV

Discussion
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 5 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  [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 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 7 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]. 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".

Conclusion
Until now, this study is the first molecular detection of the presence of CHV-1 in Iran. According to the results of this study, reproductive samples of dogs can harbor CHV-1 and being exposed to this population and their excretions may lead to a higher prevalence of infection. These findings are in agreement with previous study conducted by Babaei et al. (2010) in southeast of Iran, which has demonstrated that CHV-1 infection is endemic in the dog population of Iran and could pose a huge threat for breeders regarding neonatal mortalities and reproductive problems. Since risk factors for infections have not been established, vaccination of bitches seems logical particularly in problematic kennels with valuable breeding dogs. To avoid outbreaks, early diagnosis and good management are necessary. Accordingly, this is advisable to reduce environmental stressors for the dam and puppies, to improve sanitary and nutritional conditions and to enhance temperature during 3 weeks after birth for newborn puppies [10]. Further epidemiologic researches are required for better understanding the distribution of this pathogen in Iran. For each animal, a detailed questionnaire was prepared to record predisposing factors including age, breed, type of housing (indoor or outdoor), health status, and history of pregnancy, whelping or reproductive disorders during sampling. Dogs belonging to the study group and risk factors predisposing to CHV-1 infection are shown in Table 1. CHV-1 vaccine was not administered for any of the dogs.

Sample collection and DNA extraction
Vaginal swab samples were collected by sterile rayon tipped applicators (Puritan®, Maine, USA) and transferred into the sterile 1.5 ml microtubes containing 500 µl sterile phosphate buffered saline (PBS). For uterine samples, a full-thickness specimen (a 3×3 mm piece) was acquired from each dog during ovariohysterectomy surgery. The uterine specimens were also preserved in a sterile 1.5 ml microtube comprising 70% ethanol. DNA was extracted from specimens using the DNA extraction Mini Kit (GenAll®Exgene TM, Korea), according to the manufacturer's instruction and stored at -20 °C. A negative extraction control was included with every 3 samples.

Molecular detection of CHV-1 (Real-time PCR assay)
Real-time PCR was performed to screen the glycoprotein B (gB) gene as previously described (Decaro et al., 2010). Total volume of each PCR reaction was 20 µl containing 10 µl of RealQ Plus 2x Master Mix Green (Ampliqon, Denmark), 0.3 µl of each primer (work stock solution was 10 pmol/µl), 2.5 µl of DNA template and 6.9 µl of distilled water. In gB-positive samples, forward (5′-ACAGAGTTGATTGATAGAAGAGGTATG-3′) and reverse (5′-CTGGTGTATTAAACTTTGAAGGCTTTA-3′) primers will result in amplicons of 439-465 bp. The PCR thermal conditions were 1 cycle at 95ºC for 15 min (for activation of hot start Taq DNA polymerase) followed by 40 cycles including denaturation (95ºC for 15 s), annealing (65ºC for 20 s) and elongation (72ºC for 20 s). Also melting step (95ºC for 10 s, 65ºC for 10 s and 97ºC for 1 s) was performed for more analysis. Distilled water and DNA extracted from a slide of CHV-1 indirect immunofluorescence antibody (IFA) test kit (Mega Screen FLUO C.HV; MegaCor, Horbranz, Austria) were considered as negative and positive controls, respectively. After completion of the real-time PCR run, threshold cycle number (CT) and melting temperature (Tm) of each amplicon were automatically determined via a software on LightCycler 96® System (Roche, Germany).

Statistical analysis
Statistical analysis was carried out using SPSS software (version 21; SPSS Inc.) and p <0.05 was considered statistically significant.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.