Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 55 issue 2 (february 2021) : 217-221

Occurrence of Canine parvovirus-2 and Canine adenovirus-1 Infections in Dogs: A Hospital Based Study

G.E. Chethan1,2, Mithilesh Singh3,*, Vishal Chander4, Akhilesh1, Degpal Singh3, J.B. Rajesh1,2, Hridayesh Prasad2, Ujjwal Kumar De1
1Division of Medicine, ICAR-Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India.
2Department of Veterinary Medicine, College of Veterinary Sciences and Animal Husbandry, Selesih, Aizawl-796 014, Mizoram, India.
3Immunology Section, ICAR-Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India.
4Centre for Animal Disease Research and Diagnosis, ICAR-Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India.
Cite article:- Chethan G.E., Singh Mithilesh, Chander Vishal, Akhilesh, Singh Degpal, Rajesh J.B., Prasad Hridayesh, De Kumar Ujjwal (2020). Occurrence of Canine parvovirus-2 and Canine adenovirus-1 Infections in Dogs: A Hospital Based Study . Indian Journal of Animal Research. 55(2): 217-221. doi: 10.18805/ijar.B-3936.
Canine parvovirus-2 (CPV-2) and canine adenovirus-1 (CAV-1) are the major viral agents causing enteric diseases in dogs worldwide despite several control measures. The objective of the present study was to investigate the hospital based occurrence of CPV-2 and CAV-1 in dogs in teaching veterinary clinical complex of northern India from August, 2017 to March, 2019. Faecal samples were collected from the dogs with gastroenteritis and screened for CPV-2 and CAV-1 infections by polymerase chain reaction (PCR). Simultaneously, data regarding age, breed, sex and vaccination status were also recorded from the affected dogs. A total of 216 faecal samples were collected during the study period out of which 90 (41.67%), 12 (5.56%) and 26 (12.04%) samples were found to be positive for CPV-2, CAV-1 and co-infection, respectively. The analysis of positive samples revealed that the occurrence of CPV-2 and CAV-1 infection was highest in young dogs of the age group 0-3 months (CPV-2=44.44% and CAV-1=33.33%), Labrador Retriever breed (CPV-2=35.55% and CAV-1=50.0%) and unvaccinated dogs (CPV-2=68.88% and CAV-1=83.33%). Sex wise analysis revealed that the positivity of CPV-2 infection was higher in female (51.11%) than male dogs (48.88%) but, no such difference was noticed with respect to CAV-1 infection. It is concluded that the occurrence of CPV-2 and CAV-1 infections was very high in the study region and proper control measures are recommended. However, analysis of large number of samples including those from the field are required for further validation of the study as well as  sequential analysis of CPV-2 to ensure the prevalent field antigenic variant of CPV-2 in the region. 
Gastroenteritis is one of the major causes of morbidity and mortality in canine population throughout the world. Among the viral diseases, canine parvovirus (CPV) is considered as the major cause of severe gastroenteritis in dogs (Sakulwira et al., 2003). Canine parvovirus gastroenteritis, an acute, fatal and contagious disease is mainly seen in puppies belonging to the age group of 6 to 20 weeks (Carmichael, 2005). Two distinct parvoviruses are now known to infect dogs, mild or non-pathogenic CPV-1 or the minute virus of canine (MVC) and the pathogenic, CPV-2. CPV-2 is a single-stranded DNA virus with an icosahedral symmetry. CPV-2 is one of the most important pathogenic viruses with high morbidity (100%) and frequent mortality up to 10% in adult dogs and 91% in pups (Nandi and Kumar, 2010).
       
Canine adenovirus-1 (CAV-1) infection is an important viral disease of canines and causes a specific condition known as infectious canine hepatitis (ICH) (Hechinger et al., 2017). CAV-1 infection causes severe clinical disease mainly in members of the Canidae and characterised by acute necrohaemorragic hepatitis, corneal edema (“blue eye”), uveitis and interstitial nephritis that may occur as a consequence of circulating immune complex deposition (Greene, 2012). Clinical signs in dogs that survive from the acute viremic period include vomiting, abdominal pain and diarrhea with or without evidence of haemorrhage. The mortality rate in ICH normally ranges from 10% to 30% but can be increased in the presence of co-infections with other viruses (Decaro et al., 2007).
       
Vaccination is the most efficient strategy for the control of the CPV-2 and CAV-1 infections (Thomas et al., 2014). In spite of vaccination, the incidence of CPV-2 infection has been increased since last few decades because of emergence of a number of variants like CPV-2a, CPV-2b and CPV-2c that is present throughout the world (Thomas et al., 2017a; Gaykwad et al., 2018). In spite of  significant impact of these infections on canine health, there is a paucity of information regarding epidemiological aspects of their occurrence because a large proportion of canine gastroenteritis remains undiagnosed either because the disease is self-limiting or due to financial constraints on diagnostic procedures. Therefore, the purpose of the present study was to investigate hospital based occurrence of CPV-2 and CAV-1 infections in dogs in a teaching veterinary clinical complex of northern region of India.
Selection of animals and collection of faecal samples
 
The study was conducted at Referral Veterinary Polyclinic and Teaching Veterinary Clinical Complex (RVP-TVCC), ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, India. Dogs with history of pyrexia, weakness, low appetite; diarrhea with or without blood, vomiting, with or without icterus and dehydration were included in the present study. The faecal samples from diarrheic dogs were collected in a sterile swab and transported on ice to laboratory. A total of 216 faecal samples were collected from August, 2017 to March, 2019. The epidemiological parameters like age, breed, sex and vaccination status were recorded from the affected dogs.
 
Screening of CPV-2 and CAV-1 infections
 
The screening of CPV-2 and CAV-1 in faecal samples was carried out by direct detection of viral genome by polymerase chain reaction (PCR) based on VP2 gene and E3 gene of CPV-2 and CAV-1, respectively.
 
Extraction of DNA from the faecal samples
 
The faecal samples collected in a sterile swab were immersed immediately in a tube containing Hanks balance salt solution (HBSS) and stored at -20°C until further analysis. The faecal samples were centrifuged at 8,000 rpm for 5 min at 4°C and 200 μl supernatant was taken and processed for DNA extraction. Total genomic DNA from all the collected faecal samples was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions. The extracted DNA was stored at -20°C following quality check in NanoDrop Spectrophotometer (ND-1000, Thermo Fisher Scientific, Wilmington, USA).
 
Polymerase chain reaction
 
The polymerase chain reaction (PCR) for the identification of CPV-2 was carried out by using primers (FP-TGATTGTAAACCATGTAGACTA and RP-TAAGTCAGTATCAAATTCTTTATC) for targeting VP2 gene (Chander et al., 2016) and the identification of CAV-1 was done by using primers (FP-CGCGCTGAACATTACTACCTTGTC and RP-CCTAGAGCACTTCGTGTCCGCTT) specific to E3 gene (Hu et al., 2001). The PCR reaction was set up in a standard 25 μl reaction containing the reagents as mentioned in Table 1. The PCR-amplified products were resolved on 1.5% agarose gel in Tris-acetate-EDTA (TAE) buffer. The agarose gel was visualized under ultraviolet (UV) light in a gel documentation system (Molecular Imager® Gel Doc™ XR+ System, Bio-Rad, USA).
 

Table 1: Details of reagents used in polymerase chain reaction.


 
Statistical analysis
 
The data were structured in the Microsoft Excel spreadsheet. Descriptive statistics were performed and reported as percent positivity of infections.
Occurrence of CPV-2, CAV-1 and co-infection in dogs with enteritis
 
During the study period, a total of 216 faecal samples were collected from the dogs with gastroenteritis. The diagnosis of CPV-2 and CAV-1 infections was carried out by PCR as evident by the presence of expected 631 bp and 508 bp product in agarose gel under UV transillumination, respectively (Fig 1 and Fig 2). Out of 216 samples, 90 (41.67%), 12 (5.56%) and 26 (12.04%) samples were found positive for CPV-2, CAV-1 and co-infection (both CPV-2 and CAV-1), respectively. It is very difficult to precisely diagnose the CPV-2 and CAV-1 infections solely based on their clinical signs as most of the infectious and non-infectious causes of gastroenteritis produce clinical signs that are overlapping in nature. The present study findings are in accordance with earlier studies as reported from different parts of India (Phukan et al., 2010; Srinivas et al., 2013; Thomas et al., 2014; Behera et al., 2015). The high occurrence of CPV-2 infection might be due to easy transmission of infection by faeco-oral route, resistant nature of the virus to commonly available disinfectants and persistence of virus in the environment for longer times in subtropical humid climatic condition. Relatively lower occurrence of CAV-1 in the present study might be either due to less incidence and low endemicity of the diseases or low rate of faecal secretion as the CAV-1 virus prefers to reside in the kidneys and is excreted in the urine (Greene, 2012).
 

Fig 1: 1.5% agarose gel electrophoresis image of the PCR amplified products for identification of CPV-2


 

Fig 2: 1.5% agarose gel electrophoresis image of the PCR amplified products for identification of CAV-1. Lane M -100 bp ladder


 
Age wise occurrence of CPV-2, CAV-1 and co-infection
 
The occurrence of CPV-2 infection was found highest in the dogs belonging to the age group 0-3 months (44.45%) and samples were found negative from the faecal samples collected from dogs of the age group >1 year. Similarly, the occurrence of CAV-1 infection was highest in the pups of 0-3 months of age and faecal samples collected from 6 months to one year old dogs were found negative. In contrary to the above, the occurrence of co-infection with CPV-2 and CAV-1 was found to be highest in the age group 3-6 months and found nil in the age group 6 months-1 year (Table 2). The CPV-2 and CAV-1 infections are most frequently seen in young animals less than one year old, but, unvaccinated dogs of all ages can be affected (Greene, 2012; Thomas et al., 2014; Duque-Garcia et al., 2017). The CPV-2 replicate  in cells (myocardiocytes, small intestinal crypt epithelial cells, lymphopoietic tissue and bone marrow) which are under rapidly dividing stage and young animals will be having more number of such type of cells in comparison to adult animals (Mylonakis et al., 2016). The CAV-1 replicates in hepatic parenchymal cells and vascular endothelial cells (Decaro et al., 2007). The presence of maternal antibodies plays a significant role to protect dogs from infection. The young puppies are generally protected from CPV-2 and CAV-1 infection by maternally-derived antibodies but susceptibility increases as maternally acquired antibodies starts waning (Greene, 2012; Decaro et al., 2004; Thomas et al., 2017b). It has been reported that dogs under six months of age have higher risk of CPV-2 and CAV-1 infections and the adult dogs are more resistant to their infection due to age related reduction in susceptibility and the presence of specific immunity derived from vaccination or previous infections (Decaro and Buonavoglia, 2012; Greene, 2012; Duque-Garcia et al., 2017).
 

Table 2: Age wise distribution of CPV-2, CAV-1 and Co-infection positive cases.


    
Sex wise occurrence of CPV-2, CAV-1 and co-infection
 
The occurrence of CPV-2 infection was found to be slightly more in female dogs (51.11%) as compared to male dogs (48.89%) but no such difference was noticed with respect to CAV-1 infection. The occurrence of co-infection with CPV-2 and CAV-1 was found more in male (69.23%) than female dogs (30.77%). There is no sex predilection for CPV-2 and CAV-1 infection in dogs (Greene, 2012; Thomas et al., 2014). It is speculated that male animals usually travel more than female and thus they are more susceptible to field infections (Khan et al., 2006).  
 
Breed wise occurrence of CPV-2, CAV-1 and co-infection
 
The occurrence of CPV-2 infection was found highest in Labrador Retriever breed (35.56%) and no faecal samples were found positive in Cocker Spaniel breed. The occurrence of CAV-1 infection was also found highest in Labrador Retriever breed (50.0%) whereas, no faecal samples were found positive in Spitz, German Shepherd (GSD), Dobermann, American Bully, crossbred and Cocker Spaniel breeds. The occurrence of co-infection with CPV-2 and CAV-1 was found to be highest in non-descript breed (30.77%) (Table 3). Labrador is the most reared breed in this region and this may be the reason for relatively high percentage of positive cases observed in Labrador dogs. Similar findings have been reported by previous studies on epidemiological aspects of CPV-2 infection in dogs (Thomas et al., 2014; Behera et al., 2015; Roy et al., 2018). Earlier workers observed no breed or sex susceptibility to CPV-2 and CAV-1 infections. CPV-2 and CAV-1 infections can be seen in dogs of any breed, age, or sex, but puppies below 6 months of age are highly susceptible (Prittie, 2004; Greene, 2012). The maximum occurrence of co-infection with CPV-2 and CAV-1 in non-descript dogs may be because of lack of vaccination awareness among the pet owners of the region.
 

Table 3: Breed wise distribution of CPV-2, CAV-1 and Co-infection positive cases.


 
Occurrence of CPV-2, CAV-1 and co-infection based on vaccination status
 
The occurrence of CPV-2, CAV-1 and co-infection with both the viruses was found to be highest in unvaccinated dogs (68.88%, 83.33% and 53.84%, respectively) and was least in the dogs that received three doses of vaccine (Table 4). Occurrence of infection in immunized dogs is probably due to generation of inadequate antibody response in presence of high level of resident maternal antibody or differences in genetic make-up of viruses between vaccine strain and field strain from natural disease (Maya et al., 2013; Mittal et al., 2014; Miranda and Thompson, 2016). Such differences need to be investigated through molecular epidemiology and extensive characterization of both field and vaccine strains of viruses in order to reveal finer details.
 

Table 4: Analysis of CPV-2, CAV-1 and Co-infection positive cases based on their vaccination status.

In the present study, a total of 216 faecal samples were screened and out of which, 90 (41.66%), 12 (5.55%) and 26 (12.03%) samples were found positive for CPV-2, CAV-1 and co-infection, respectively by PCR. The occurrence of CPV-2 and CAV-1 infections was found to be maximum in young puppies belonging to the age group 0-3 months and Labrador Retriever breed with no or incomplete vaccination status. It is concluded that the occurrence of CPV-2, CAV-1 and co-infection with both the viruses is very high in the present study region and it warrants awareness among pet owners about vaccination for dogs against viral diseases. Screening of large number of samples covering wider geographic regions is required for further validation of the study.
The authors are grateful to the Director of the institute for providing necessary facilities to carry out the present study. CGE thank ICAR-IVRI for granting research fellowship for his research program.
The authors declare that there is no conflict of interest.

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