Screening of Faecal Samples of Pet Dogs for the Presence of Canine Parvoviral Infection by using Haemagglutination Assay and Polymerase Chain Reaction Test

Canine parvovirus-2 (CPV-2) is highly infectious and contagious virus in the genus Parvovirus of the Parvoviridae family which affects dogs that cause gastroenteritis.  Previously, the virus was implicated in causing heavy mortality in pups due to myocarditis worldwide. Monoclonal antibodies were used to detect newer antigenic variants of CPV-2. This variant was known as CPV-2a. New variants of CPV, known as CPV-2b and CPV-2c were also reported (Parrish et al., 1985; Parrish et al., 1991; Nakamura et al., 2001; Chinchkar et al., 2014). In India, CPV was isolated in 1982 for the first time (Ramadass et al., 1982). Thereafter, it was reported from various states of the country (Biswas et al., 2006; Nandi et al., 2006; Sagar et al., 2008; Khare et al., 2019).

The main transmission of CPV-2 is through faecal-oral route. Dogs that develop the disease show symptoms of illness within 3 to 10 days. The symptoms include lethargy, vomiting, fever and diarrhea (usually bloody). Quick diagnosis of CPV-2 infection in dogs is essential for effective treatment. The present study reports results of screening of faecal samples of pet dogs (from Mhow and Indore area) suffering from gastroenteritis for the presence of CPV-2 infection by using haemagglutination assay (HA) and polymerase chain reaction (PCR), a molecular test.

Sources of samples, history and place of investigation
 
A total number of 50 faecal samples (from May 2018 to May 2019, 3 in Monsoon and 47 from Summer season) were collected from dogs suffering from diarrhea, vomiting and gastroenteritis problems brought to the Teaching Veterinary Clinic Complex (TVCC) of the Veterinary College campus, Dr. Ambedkar Nagar-Mhow (47 samples; 34 males, 13 female) and private veterinary clinic, situated in Indore (3 samples; 2 male and 1 female) (Table 1). The faecal samples were collected in phosphate buffered saline solution (pH 7.2) and stored at -20°C. History of pet dogs selected for the sample collection was noted (Table 1). The present investigation was conducted at Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Dr. Ambedkar Nagar-Mhow, Indore, Madhya Pradesh, India. The investigation plan for the present study was approved by the institutional level ethical committee.



Vaccine
 
Canishot-K5 vaccine manufactured by Choong Ang Vaccine Laboratories Company Limited (Korea) used as positive control. The combined freeze-dried vaccine contained Live Canine distemper virus, Canine adenovirus type 2, Canine parvovirus and Canine parainfluenza virus.
 
Pig red blood cells
 
A total of 10 ml of blood from a healthy adult swine (Middle white Yorkshire crossbred reared at Piggery unit, Livestock Farm complex of the College) was collected in Alsever’s solution (1:1, v/v), kept at 4°C and used within one week of collection, whenever required. After removal of plasma and buffy coat, pig red blood cells were washed thrice in phosphate buffered saline solution (PBS), pH 7.2, then stored at 4°C and used within 3 days.
 
Processing of faecal samples and haemagglutination assay
 
On thawing, samples were centrifuged at 6,000 revolutions per minutes (rpm) for 15 minutes in the sterile tube (Eppendorf) and supernatant was transferred to another tube. Tubes with supernatants were stored at -20°C till it’s use in haemagglutination (HA) assay. Before HA, the supernatants were heat inactivated at 56°C in temperature-controlled water bath for 30 minutes and treated with 10% Chloroform by keeping at 4°C for 10 minutes and then centrifuged at 10,000 rpm for 10 minutes at 4°C. Final supernatant resulted after Chloroform treatment was used for HA (Parthiban et al., 2011). 25 µl of virus diluents was dispensed in each well of 96 well ‘V’ bottom microtitre plate (Laxbro) (Dahiya and Kulkarni, 2004). The processed faecal sample was serially diluted two-fold in virus diluent (Phosphate buffered saline solution, pH 6.0) and equal quantity (25 µl) of 1% pig red blood cells in the diluent [Phosphate buffered saline solution, 0.3 M phosphates, pH 6.0 containing 3% foetal calf serum (FCS)] was added to the each well. Controls were also kept and the plate was incubated at 4°C for overnight duration and the results were recorded.
 
Polymerase chain reaction test
 
Primers (Forward: 5'-GAAGAGTGGTTGTAAATAATT-3' and Reverse: 5'-CCTATATCACCAAAGTTAGTAG-3') were used to amplify partial VP1/VP2 gene of 681 base pairs specific for CPV-2 in faecal samples (Nandi et al., 2006; Singh et al., 2014). PCR (25 µl) was performed in 0.2 ml sterile nuclease free tubes (12.5 µl of PCR mastermix (Fermentas), 3 µl of forward and reverse primers, 3 µl of faecal sample and 0.5 µl of enzyme Taq polymerase of GeneiTM and 6.5 µl of nuclease free water). Thermocyclic conditions used for amplification of the target DNA as follows (initial denaturation: 95°C, 5 minutes, followed by 30 cycles of a) denaturation:  94°C, 30 seconds, b) primer annealing: 55°C, 2 minutes, c) extension: 72°C, 30 seconds and final extension: 72°C, 10 minutes). Positive (Vaccine) and negative (No template) controls were also kept during the reaction. PCR products were mixed with 6X bromophenol blue loading dye and loaded into a well along with a DNA marker and electrophoresed (Genei®, Bangalore) in 1.5% agarose gel in Tris borate ethylenediaminetetraacetic acid (TBE) buffer for 90 volts/cm for an hour. Ethidium bromide (10 mg/ml) stain to a final concentration of 0.005 µg/ml was added in the gel. Bands in the gel were visualized, digitally recorded by gel documentation system (Alphalmager, USA) and analysed.

Haemagglutination assay
 
CPV-2 haemagglutinates pig red blood cells. Haemagglutination assay (HA) carried out in a 96-well plate format allows rapid processing of many samples (Desario et al., 2005). So, in the study 50 processed faecal samples from individual dogs suffering from gastroenteritis were screened by using HA (Table 2, Fig 1, Fig 2) for knowing the status of CPV-2 infection. Out of 47 dogs (25 days to 14 years of age from TVCC of the College, 30 were male and 17 were female dogs of various breeds (Non-descript (17; 11 male, 6 female); German Shepherd (11; 8 male, 3 female); Labrador (11; 6 male, 5 female); Pomeranian (4; 2 male, 2 female); (Pug 2; 1 male, 1 female); Poodle (1 male) and Cross breed (1 male). Out of 3 dogs of below 6 months of age from private veterinary clinic, Sudama Nagar, Indore, all were male animals (1 non-descript, 1 Dachshund and 1 Cross breed). HA titres of tested faecal samples ranged in between 32 to 1024. These findings are very similar to a finding which reported titres upto 1280 (Puentes et al., 2012). 5 samples (4 male, 1 female with clear matt formation) showing HA titres of 32 or above were treated as positive for CPV-2 infection (Parthiban et al., 2011). Results of the HA study indicate presence of CPV-2 infection in pet dog population in the region, however at lower magnitude. Two dogs (1 non descript female of 1.5 years of age and 1 Cross breed male of 3 months of age) from private veterinary clinic, Indore tested positive in HA test with HA titres of 1:32 and 1:512, respectively. Other three male dogs between 2 months to 6 months of age (2 German Shepherd and 1 Labrador) tested positive in HA. Labrador recorded highest HA titre 1:1024 than German Shepherds (1:32 and 1:256). Overall, per cent positivity for CPV-2 infection in the dog population was 10% as per HA test results. Varying percentages of CPV-2 positivity in HA test were reported earlier (Mathys et al., 1983; Rai et al., 1994). Five CPV-2 positive dogs in the present study were ranging from 2 months to 1.5 years of age. However, from these five positive dogs for CPV-2 infection in HA, 3 grouped in the age group in between 3-6 months of age. However, more cases of CPV-2 infection were recorded in 0-2 months of age group in dogs by other investigators (Khan et al., 2006). Out of five positive dogs in the study, 2 dogs were of German Shepherd breed. Similar higher level of CPV-2 infection in German Shepherd breed of 84% was also reported (Khan et al., 2006). Four male dogs (HA titres ranging from 32-1024) were positive for CPV-2 infection in our study as compared to only single female dog (HA titre of 32) which was positive in HA test. In HA test, 12.12% male (4 positive out of 33) and 5.88% female (1 positive out of 17) tested positive. HA titres ranging from 1:40 to 1:20,480 were also reported from dogs (Mohan et al., 1992) which corresponds to findings of the present investigation. The remaining 45 faecal samples, that were negative in HA test for CPV-2 could possibly be due to i). absence of CPV-2 infection, ii). sedimentation of virus in the faecal samples having low viral titres during centrifugation and iii). viral neutralization or aggregation by copro-antibodies (Carmicheal et al., 1980). The haemagglutinaton inhibition (HI) activity was noticed in HA positive samples by using anti-serum from CPV-2 vaccinated dog confirming the involvement of CPV-2 infection (results are not shown) in pet dog population suffering from gastroenteritis.







Polymerase chain reaction test
 
Initially, PCR conditions were tested successfully by amplification of 681 base pairs deoxyribonucleic acid (DNA) product of partial VP1/VP2 gene specific for CPV-2 by using the Canine parvovirus-2 contained in the vaccine (Canishot-K5), directly. No nucleic acid extraction protocols were followed because of positive PCR results on using Vaccine virus without any prior treatments. Positive HA results indicate involvement of CPV-2 infection, however, few workers reported on CPV-2 strains that lacked HA activity (Parrish et al., 1988; Cavalli et al., 2001). So, 12 pet dogs tested positive (4) and negative (8) in HA were randomly selected and processed individual faecal samples from these dogs were directly used as template in PCR as per Nandi et al., (2006). These samples tested negative in PCR.  Because of the above facts and to save the PCR reagents, rest of the faecal samples were pooled (5 faecal samples at the most) and tested negative in PCR. Majority of faecal samples tested negative in PCR in the study can be due to excretion of viruses in faecal material to the undetectable level, presence of PCR inhibitors (Oikarinen and Hyoty 2009) in faeces or unexplained reasons/errors. However, these faecal samples will be used in future studies to extract parvoviral DNA and that will be used in PCR/nested set PCR. A German Shepherd dog (Sample no 23) whose faecal material was collected twice on 2^nd (tested negative in PCR) and 3rd day (tested positive in PCR, may be due to excretion of virus in huge amounts in the faecal material) after the notice of first clinical signs of gastroenteritis. Besides Canine pavovirus-2 molecular detection of other viral infections that cause gastroenteritis in dog population were also reported (Agnihotri et al., 2017). The findings of the study will be helpful in understanding the epidemiology of infectious diseases that cause gastroenteritis in pet dogs of the area and its management, prevention and control.

The results of the present study indicated presence of Canine parvovirus-2 infection at lower magnitude in pet dogs suffering from gastroenteritis in the region. However, it is essential to test these cases for involvement of other parasitic infestations, viral and bacterial infectious agents causing gastroenteritis in pet dogs in the region.

Authors are thankful to Dr. A. Soni, Assistant Professor, Department of Veterinary Medicine and Post Graduate students for their help in handling and restraining pigs for blood collection and also to the staff of the laboratories for providing timely help. We are also thankful to Dr. S. Nanavati/In-charge, Livestock Farm complex of the college for his permission to use pigs for blood collection.