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Journal of Immunology and Immunopathology
Year : 2006, Volume : 8, Issue : 2
First page : ( 141) Last page : ( 142)
Print ISSN : 0972-0561.

Development and standardization of PCR for the detection of canine parvoviral DNA in the stool samples of canines

Nandi S., Pandey A.B., Sharma K., Audarya S.D., Chauhan R.S.

Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar, U.P. 243 122.

Abstract

Canine parvovirus (CPV-2) infection in dogs has become an alarming problem in India causing high morbidity and low mortality. PCR has been developed and standardized to diagnose the disease in the stool samples which is sensitive, specific and rapid and can be adopted both small as well as large laboratories which in turn will be helpful in managing the disease in an effective and efficient manner.

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Key words

Canine parvovirus, Polymerase chain reaction, DNA, antigenic drift.

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Introduction

Canine parvovirus 2 (CPV-2), the causative agent of acute haemorrhagic enteritis and myocarditis in dogs, is one of the most important pathogenic viruses. It is a highly contagious and often fatal disease (Parrish et al., 1991). CPV-2 was first recognized in 1977 and since then it has been well established as an enteric pathogen of dogs throughout the world with high morbidity (100%) and frequent mortality up to 10%. The disease is characterized by 2 prominent clinical forms (i) enteritis with vomition and diarrhea in dogs of all ages (ii) myocarditis and subsequent heart failure in pups of less than 3 months of age. After the initial appearance of CPV type 2 in 1979, it has been well established as an enteric pathogen of dogs throughout the world including India. In 1984, two new antigenic variants of CPV with altered antigenicity due to antigenic drift became widespread and have been designated as CPV type 2a (CPV-2a) and CPV type 2b (CPV-2b) respectively (Parrish, 1999). The disease is endemic in India with high degree of prevalence (Khadilkar et al., 1994).

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Materials and Methods

The stool samples of dogswere collected in HBSS containing Penicillin (1 lakh I.U./litre) and streptomycin (100 mg/litre) at the ratio of 1:10 from the ailing dogs suspected for CPV infections. The stool samples have been centrifuged at 10,000 rpm for 5 minutes and supernatant used in the PCR. The custom synthesized forward and reverse primers specific for the VP1/VP2 gene of the CPV genomic DNA was used to amplify the DNA of 681 bp in length having the following cyclic conditions. Initial denaturing temperature of 94°C for 5 min one cycle, 94°C for 30 sec, 55°C for 2 min, 72°C for 2 min 30 cycles and final extension of 5 min at 72°C. After the run agarose gel electrophoresis was carried out to visualize bands against the 100 bp DNA ladder.

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Results and Discussion

In this study, PCR has been developed and standardized to diagnose the canine parvovirus infections directly from the stool samples. Out of 21 stool samples tested by PCR, 16 were found to be positive for the canine parvoviral genomic DNA. Although a galaxy of serological tests and isolation of virus in cell culture are available to diagnose the canine parvovirus infections, they suffer from a number of drawbacks such as high cost, low sensitivity and specificity, requirement of cell culture facility, time consuming and requirement of costly equipments. In the PCR, diagnosis can be provided within 6 hours and can be adopted in the small as well as large laboratories. The test is simple, prompt and highly sensitive and can detect the genomic DNA from a minute quantity of processed stool samples. In our studies, PCR has been standardized to detect the genomic DNA in the stool samples suspected for canine parvovirus infections to amplify the 681 bp product of VP1/VP2 gene of CPV. The results have been corroborated well with the Pereira et al., (2000). The quick and accurate diagnose of the CPV infections in canines by PCR may lead to the efficient and effective management of the disease and implementation of the suitable control strategies to bring the disease under control.

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References

KhadilkarM V, BatraP, SinghviN M. (1994). Clinico-pathogenic features of canine parvoviral enteritis. Journal of Remount Veterinary Corps, 33: 81–84.

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ParrishC R. (1999). Host range relationship and the evolution of caine parvovirus. Veterinary Microbiology, 69: 29–40.

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ParrishC R, AquadroC F, StrassheimM L, EvermannJ F, SgroJ Y, MohammedH O. (1991). Rapid antigenic type replacement and DNA sequence evolution of canine parvovirus. Journal of Virology, 65: 6544–6552.

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PereiraC A, MoneziT A, MehnertD U, D'AngeloM, DurigonE L. (2000). Molecular characterization of canine parvovirus in Brazil by PCR. Veterinary Microbiology, 75: 127–133.

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