The emergence of Canine parvovirus type 2 (CPV-2) as a widespread disease in dogs around mid 1978 has since then continued to invite scientific inputs in the areas of diagnostic and preventive measures for enteritis caused by CPV-2. The CPV-2 infection rates have been found increasing during recent past in India. The clinicians are interested in effective control prophylactic and therapeutic measures. Particularly in an early and accurate diagnosis of the infection as shedding of the virus in faeces of incubative, clinical and convalescent carrier for a longer period helps the agent to find newer host. Therefore, early detection of CPV-2 during such periods is very important to initiate suitable therapeutic and control measures.

A variety of serological tests having desirable qualities have been documented for diagnosis of CPV-2 infections, but with conflicting reports about their sensitivities. It is in this context, that the comparative study designed to study their efficacies and hence the present study was taken up.

A total of 65 faecal swabs were collected from dogs showing clinical sings of gastroenteritis and diarrhoea and brought to Zaveri Clinics, Veterinary College, Anand and two other private/government clinics. The faecal samples were transported to laboratory on ice and preserved at −20°C till processing. 10 percent (w/v) suspension of each faecal sample in PBS was prepared and centrifuged at 3000 rpm for 20 min at 4°C. The supernatants were tested for CPV-2 by Agar gel immunodiffusion (AGID), Counter immunoelectrophoresis (CIE) and Dot-ELISA, while crude faecal samples by Sandwich ELISA (INGENASA, Hnos Garcia Noblejas, 4128037 Madrid, Spain).

Commercially available live CPV-2 vaccine (Mega Vac-6) was used to immunize rabbits to produce hyperimmune serum against CPV as per method of Ramdass and Khader (1982) with some modifications. The obtained antiserum was tested by AGID to confirm the presence of CPV antibodies using the vaccine virus.

AGID was carried out as per the method of Williams and Chase (1971) with some modifications. The faecal sample from a healthy dog was kept as a negative control. CIE was carried out as per the method of Hudson and Hay (1980) with some modifications using agarose (1.2%) in 0.5X barbitone acetate buffer and a constant current of 12 mA per slide for on hour.

CPV ELISA Kit based on double antibody sandwich enzyme immunoassay method was procured and used. Briefly, one gram of each faecal sample was homogenized with two ml of sample diluent and centrifuged at 1000 rpm at 4°C. The supernatant was tested by Sandwich-ELISA as per the protocol supplied with the kit. The absorbance of each well (OD values) was measured in an ELISA reader (Bio-Rad, Japan) at 405 nm wavelength. Cut off OD values (double than that of the negative control) were determined and the test samples showing higher OD values than the cut off values were recorded as positive for CPV antigen.

Dot immunobinding assay was applied as per the method described by Chauhan and Singh (1992) with slight modification. Two μl of a faecal supernatant was charged on Nitrocellulose membrane (NICM) and incubated at 37°C for 5 min. The unsaturated sites of NCM were then blocked by blocking buffer (2% Bovine serum albumin) and incubated at 37°C for 1h. The NCM was washed in washing solution (PBS with Tween 20) thrice and incubated in 1:100 diluted anti-CPV hyperimmune serum for 30 min. at 37°C. Known CPV negative antigen control, positive antigen control and CPV negative control serum were also included. The NCM was washed thrice in washing solution and incubated in enzyme conjugate (Goat anti-rabbit HRPO, Bangalore Genie) for 30 min. at 37°C. The NCM was washed thrice in PBST, immersed in freshly prepared substrate solution and incubated at room temperature in dark for 10 min. The reaction was stopped by washing the strip in distilled water followed by air drying. A positive reaction as indicated by development of brown colour was recorded.

AGID detected three (4.61%) of the total 65 faecal samples as positive for CPV antigen, which is in agreement with reports on detection of many CPV positive samples by AGID (Saseendranath et al., 1992; Joshi, 1996) Despite the lesser sensitivity of AGID over other assays economical on site and useful preliminary assay for CPV detection, especially when other facilities or sophisticated tests are not available.

CIE for CPV antigen detection has been reported to be a useful rapid method for CPV detection which corroborotes with the results of work published earlier (Joshi, 1996; Deepa and Saseendranath, 2000; Singh, 2002). In the present study, CIE detected six (9.23%) samples as positive for CPV antigen

In Sandwich ELISA, four samples (6.15%) including one that turned out negative in AGID were detected as positive for CPV antigen. The sensitivity of Sandwich ELISA as compared to other as says has been reported to be higher by some workers (Ruz and Banex, 1996) lower by others (Mortinello and Porspenis, 1996). In the present study, the sensivity of sandwich ELISA was found to be lesser than AGID.

Of late, many workers have reported use of Dot-ELISA for detection of CPV antigen or antibody (Joshi et al., 2001; Singh 2002). Although a qualitative test, the usefulness of Dot-ELISA lies in its simplicity and applicability at field level. In the present study, seven samples showed positive reaction in Dot-ELISA, which indicates its improved sensitivity over Plate-ELISA. The assay was found to be simple, rapid and convenient to perform. The results could be read by naked eye without any difficulty and the NC strips could be stored in the dark for long periods without significant loss in colour reaction.

In the present study three and four samples that were negative in AGID could be detected as positive by CIE and Dot-ELISA, respectively. Dot-ELISA also detected three more samples positive than Sandwich-ELISA. Thus, Dot-ELISA and CIE proved better tests than the others for CPV antigen detection, the CIE gave almost comparable result with Dot-ELISA, while AGID was found least sensitive.

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