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Research Article
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Detection of Canine Parvo Virus by Polymerase Chain Reaction Assay and its Prevalence in Dogs in and Around Mathura, Uttar Pradesh, India |
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Deepti Singh,
Amit Kumar Verma,
Amit Kumar,
Mukesh Srivastava,
Shanker Kumar Singh,
Arvind Kumar Tripathi,
Ashish Srivastava
and
Iftekhar Ahmed
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ABSTRACT
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The early detection of the Canine Parvo Virus (CPV) is of paramount importance.
The present study was aimed to know the molecular epidemiology of Canine parvo
virus. Canine faecal samples from 100 dogs showing the clinical signs of gastroenteritis
in and around Mathura, Uttar Pradesh, India were collected and DNA was extracted
by phenol-chloroform method. CPV vaccine strain was used as a positive control.
Polymerase Chain Reaction (PCR) was carried out to amplify VPI/VP2 gene using
a set of 20-mer primers [pCPV-RT (Forward): 5-CAT
TGG GCT TAC CAC CAT TT-3; (Reverse):
5-CCA ACC TCA GCT GGT CTC AT-3)]
from position 3136-3155 to 3276-3295 of VP1/VP2 gene. A PCR product of approximately
160 bp was generated with positive faecal samples and CPV vaccine strain. After
screening, 63 dogs were found positive for CPV but no sex variation was noticed
amongst the CPV positive cases. Dogs, of the age group of ≤6 months were
more susceptible in comparison to of >6 months and highest occurrence was
noted in unvaccinated dogs and dogs in co-habitation with other dogs. Breed
wise distribution of CPV in dogs revealed that the prevalence of CPV was the
highest in Doberman (77.78%), followed by Spitz (78.57%), German shepherd (70.00%),
Labrador (68.75%), Pomeranian (45.45%). It is concluded that CPV is prevalent
in the Mathura and nearby area and it is more common in pups of age less than
6 months old and more prevalent in German shepherd, Labrador and Pomeranian
breeds of dog.
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How
to cite this article:
Deepti Singh, Amit Kumar Verma, Amit Kumar, Mukesh Srivastava, Shanker Kumar Singh, Arvind Kumar Tripathi, Ashish Srivastava and Iftekhar Ahmed, 2013. Detection of Canine Parvo Virus by Polymerase Chain Reaction Assay and its Prevalence in Dogs in and Around Mathura, Uttar Pradesh, India. American Journal of Biochemistry and Molecular Biology, 3: 264-270.
DOI: 10.3923/ajbmb.2013.264.270
URL: https://scialert.net/abstract/?doi=ajbmb.2013.264.270
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Received: August 05, 2012;
Accepted: September 20, 2012;
Published: April 18, 2013
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INTRODUCTION
Dogs have been domesticated by human since a long time and are supposed to
be the companion of human since civilization. They have very close association
with human beings and most prominent example of the same is in Mahabharata where
dog followed the eldest Pandav Yudhistir till its life lasted. In present time
tremendous change in socio-economic structure in India has increased the demand
for pet dogs and it has started showing an increasing trend as dogs are now
a day status symbol. This is reflected by the 3.93% growth rate in population
from 1987-1992. In 1992 pet dog population in India has been recorded 21.76
million in 1992 (Anonymous, 1997). Canine parvo virus
is a highly contagious and fatal disease characterized by vomition and haemorrhagic
gastroenteritis in dogs of all age (Appel et al.,
1978; Verma et al., 2009; Kumar
et al., 2010) and myocarditis (Han et al.,
2011; Ying et al., 2012) and subsequently
heart failure in puppies of less than 6 months of age (Zhou
et al., 2009). The disease was first recognized in CPV-2 form in
U.S. in 1978 as a cause of new disease in dogs and in one or two years spread
worldwide in domestic dog populations as well as in wild dogs with high morbidity
(100%) and frequent mortality (up to 10%) which may reach to 91%, if untreated
(Appel et al., 1978). In India, CPV-2 was first
reported in by Ramadass and Khader (1982) and then had
drawn major concerns of animal health professionals. The disease is transmitted
from dog to dog by direct or indirect contact with their infected feces. Therefore,
early and rapid diagnosis is necessary so that infected dogs can be isolated
and supportive treatment can be administered to reduce morbidity and mortality
(Cho et al., 2006). Clinical diagnosis of CPV
infection is difficult, because the main clinical signs of the disease (vomiting
and diarrhea) are common to other enteric diseases as well (De
Castro et al., 2007). However, Polymerase Chain Reaction (PCR) technique
has been widely applied for early and confirmatory laboratory diagnosis of the
disease, due to high sensitivity and specificity (Schunck
et al., 1995; Nandi et al., 2009,
2010). Hence, the present study was conducted to detect
canine parvo virus by polymerase chain reaction assay and its prevalence in
dogs in and around Mathura, Uttar Pradesh, India.
MATERIALS AND METHODS
Animals and collection of samples: During the present
study (February, 2012 to July, 2012), dogs suffering from diarrhea and vomiting
and suspected of CPV infection were selected for the study. The faecal samples
were collected with the help of sterilized swabs from the dogs presented to
the Teaching Veterinary Clinical Complex, DUVASU and places near to Mathura
and a total of 100 samples were collected, out of which 31 were nondescript,
16 Labrador, 14 Spitz, 11 Pomeranian, 10 German Shepard, 9 Doberman, 4 Boxer,
3 Rottweiller and 2 were Bull Mastiff. The information of infected dogs according
to risk factors viz., sex age, vaccination status and co-habitation with other
dogs was collected in a pre designed performa (Annexure 1). The faecal samples
were collected in the form of a rectal swab using pre-sterilized swabs and immediately
transferred to the labeled sterile vials containing Hanks Balanced Salt
Solution (HBBS) in a ratio of 1:9, containing streptomycin (100 mg L-1)
and penicillin (1 lakh IU-1). Collected samples were then stored
in -20°C refrigerator till further processing.
Preparation of faecal sample: All the faecal samples stored in -20°C
refrigerator were brought at room temperature. Then swabs were properly rinsed
with HBSS of vials and taken out. Remaining faecal contents in HBSS were centrifuged
at 10,000 rpm at 4°C for 3 min in a refrigerated centrifuge (Remi centrifuge).
The supernatants were pipette out and filtered through a disposable syringe
filter (0.45 μm) (Millex, Millipore) syringe filter.
Genomic DNA extraction: The genomic DNA from the faecal samples was
extracted by phenol chloroform method (Sambrook and Russell,
2001) with slight modification like to remove inhibitory substances, 200
μL of sample was treated with Sodium Dodecyl Sulphate (SDS) and proteinase
K with a final concentration of 1% and 250 μg mL-1 and kept
at 56°C for 30 min in water bath GFL, Germany).
Polymerase chain reaction (PCR) assay: The primer set pCPV-RT (F) 5-CAT
TGG GCT TAC CAC CAT TT-3 (20-mer) and pCPV-RT (R) 5-CCA ACC TCA
GCT GGT CTC AT-3 (20-mer) from position 3136-3155 to 3276-3295 of VP1/VP2
gene of CPV-2 was used to yield an amplicon of 160 bp in PCR (Nandi
et al., 2009). The PCR was conducted in a thermocycler (Techne, USA)
using a reaction volume of 50 μL containing 5 μL of Taq DNA
polymerase buffer with 15 mM MgCl2 (10x), 200 μM dNTPs, 10 pmol
of each primer, 5 μL of processed sample as source of template DNA and
1 μL of Taq DNA polymerase (1 IU μL-1). The thermal
conditions consisted of initial denaturation at 94°C for 3 min, 30 cycles
consisting of denaturation at 94°C for 30 sec, annealing at 52°C for
1 min and extension at 72°C for 30 sec and a final extension at 72°C
for 5 min. The PCR products were electrophoresed along with 100 bp DNA ladder
in 1% agarose gel containing 0.5 μg mL-1 ethidium bromide and
progress of the mobility was monitored by migration of dye.
Statistical analysis: chi-square analysis: The data obtained
was tested for significant (p<0.05) difference between age, sex, breed, vaccination
status and co-habitation with another dog collected with respect to prevalence
using chi-square test as described by Snedecor and Cochran
(1967).
RESULT
In this study, out of 100 faecal samples from dogs showing the clinical signs
of gastroenteritis tested, an amplicon of 160 bp size was obtained using primer
set (pCPV-2RT) in 63 samples (63.00%) indicating the presence of CPV in all
of these samples. In the positive control, there was amplification of template
DNA whereas, in the negative control, no amplification of template DNA was visualized
on an agarose gel (Fig. 1).
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Fig. 1: |
Agar gel electrophoreses showing the amplified product of
160 bp of CPV (F and R) Primers, Lane M, N: 100 bp DNA ladder, Lane 1: Positive
control (vaccine), Lane 2: Negative control (faecal sample of healthy dog),
Lane 4, 5, 6, 8, 9, 10, 12, 13: CPV suspected faecal sample, Lane 7, 11,
14: CPV negative faecal samples |
The epidemiological factors which are also known as predisposing factors for
the CPV infection were also analyzed from the information collected on performa.
Various informations regarding the association of risk factors like breed, sex,
age, vaccination status and cohabitation with other dogs and canine parvo virus
infection status is shown in Table 1-5,
respectively. In breed wise prevalence analysis we did not include all the breeds
because in some breeds numbers of dogs are very low. Breed wise prevalence analysis
revealed that distribution of canine parvo virus infection varied significantly
(p<0.05) among different breeds (Table 1). It was the highest
in Doberman (77.78%) and the lowest in Pomeranian (45.45%).
Table 1: |
Breed-wise distribution of canine parvo virus infection in
dogs |
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*Significant at p<0.05 |
Table 2: |
Sex-wise distribution of canine parvo virus infection in
dogs |
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Table 3: |
Age-wise distribution of canine parvo virus infection in
dogs |
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*Significant at p<0.05 |
Table 4: |
Vaccination status and canine parvo virus infection in dogs |
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*Significant at p<0.05 |
Table 5: |
Co-habitation with other dog and canine parvo virus infection
in dogs |
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*Significant at p<0.05 |
Sex wise prevalence of the canine parvo virus infection was equally common
in males and females dogs and was 62.82 and 63.64%, respectively (Table
2). Dogs were grouped into two age groups viz., 0-<6 months and ≥6
months. Age wise prevalence results (Table 3) revealed that
the prevalence of the canine parvo virus infection was higher in pups (0-<6
months age group) in comparison to dogs in the age group of ≥6 months and
it was 65.51 and 57.14%, respectively. Association of canine parvo virus infection
and vaccination status of dogs was compared (Table 4). The
results revealed that prevalence of canine parvo virus infection was higher
in unvaccinated dogs (64.00%) in comparison to that of vaccinated dogs (50.00%).
Dogs were grouped into two groups on the basis of co-habitation with other dogs
(Table 5). The results showed that the dogs in cohabitation
with other dogs which were positive for canine parvo virus infection, were significantly
associated with canine parvo virus infection. The prevalence of canine parvo
virus infection was higher in dogs which shared their habitat with other dogs
(70.58%) in comparison to those of which do not share their habitat with other
dogs (61.44%).
DISCUSSION
In the present study, the prevalence of CPV in diarrheic dogs was found as
63.00% indicating the higher presence of CPV in diarrheic dogs. The high prevalence
of CPV in diarrheic dogs is in concurrence to the findings of other workers
(Nandi et al., 2006, 2009;
Biswas et al., 2006; Panda
et al., 2009) in India. The main source of the infection seems to
be the feces of infected dogs because more than 109 virus particles
per gram of feces can be shed during the acute phase of the enteric disease.
Therefore, feces are accepted as a suitable material to detect the virus in
the enteric form of the disease (Carmichael and Binn, 1981).
The epidemiological factors which are also known as predisposing factors for
the CPV infection were also analyzed from the information collected on proforma.
Various informations regarding the association of risk factors like breed, sex,
age, vaccination status and cohabitation with other dogs and canine parvo virus
infection status is shown in Table 1-5,
respectively. In breed wise prevalence analysis all the breeds were not included
because in some breeds numbers of dogs were very low (Table 1).
Breed wise distribution of CPV in dogs revealed that the prevalence of CPV was
the highest in Doberman (77.78%), followed by Spitz (78.57%), German shepherd
(70.00%), Labrador (68.75%), Pomeranian (45.45%) (Table 1).
The effect of breed has also been noticed earlier in dogs by Kumar
et al. (2011). They reported that the percent positivity was highest
in German shepherd (69.2%), followed by nondescript breed (63.1%), Pomeranian
(61.5%) and Doberman (28.5%) with overall percent positivity of 59.6%. These
findings are almost in concurrence with the findings of present study where
overall positivity is 63%.
Sex wise prevalence of the canine parvo virus infection revealed no discrimination
with percentage positivity in males (62.82%) and females (63.64%) dogs (Table
2) indicating that sex had no influence on the prevalence of CPV in diarrhoeic
dogs. Biswas et al. (2006) also found no sex
variation amongst the CPV positive cases, thus CPV infect/invade both the sexes
uniformly and physiological differences viz., pregnancy, lactation, etc. have
no influences in infectivity of virus or the susceptibility of dogs.
Dogs were grouped into two age groups viz., 0-<6 months and ≥6 months.
Age wise prevalence results (Table 3) revealed that the prevalence
of the canine parvo virus infection was higher in pups (0-<6 months age group)
in comparison to dogs in the age group of ≥6 months and it was 65.51 and
57.14%, respectively. Similarly, Biswas et al. (2006)
also reported that dogs of 0-6 months were more susceptible with highest mortality
rate. The probable reason for low prevalence of CPV in adult dogs might be the
repeated subclinical infection or the exposure of virus in less number and the
prevalent CPV might be boosting the antibody titre high enough to protect the
animal.
Association of canine parvo virus infection with vaccination status of dogs
revealed that prevalence of canine parvo virus infection was higher in unvaccinated
dogs (64.00%) in comparison to vaccinated dogs (50.00%) (Table
4). Although the 50.00% prevalence of CPV in vaccinated dogs was quite high
and it might be because of irregular vaccination or the use of improperly maintained
vaccines. However, vaccination helped in controlling the disease in dogs and
reduced the incidence. The prevalence of canine parvo virus infection was higher
in dogs which shared their habitat with other dogs (70.58%) in comparison to
those which were reared only (61.44%) (Table 5). It indicated
that Canine parvo virus appeared to be more prevalent in areas of intensive
animal husbandry practices such as kennels and dog training centres, thus unhygienic
conditions and improper management also seems to be key factors.
CONCLUSION
From the present study, it can be concluded that CPV is prevalent in the dogs
of Mathura and nearby area and more common in pups of age less than 6 months
old and more prevalent in German shepherd, Labrador and Pomeranian breeds of
dog and more common in the dogs that share their habitat with another dog. This
will be helpful in formulating the control strategies for controlling this highly
contagious disease.
ACKNOWLEDGMENTS
The authors of this study are highly thankful to Dean, College of Biotechnology
and Honble Vice Chancellor, DUVASU, Mathura; for providing funds and necessary
facilities to conduct the study; Smt. Mamta, laboratory staff of Central disease
diagnostic laboratory; for providing technical help in the laboratory; the dog
owners who gave their permission for their dogs to take part in the study.
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