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. 2016 Sep;9(9):964-969.
doi: 10.14202/vetworld.2016.964-969. Epub 2016 Sep 12.

First detection of canine parvovirus type 2b from diarrheic dogs in Himachal Pradesh

Shalini Sharma  1 , Prasenjit Dhar  1 , Aneesh Thakur  1 , Vivek Sharma  2 , Mandeep Sharma  1
Affiliations

First detection of canine parvovirus type 2b from diarrheic dogs in Himachal Pradesh

Shalini Sharma et al. Vet World. 2016 Sep.

Abstract

Aim: The present study was conducted to detect the presence of canine parvovirus (CPV) among diarrheic dogs in Himachal Pradesh and to identify the most prevalent antigenic variant of CPV based on molecular typing and sequence analysis of VP2 gene.

Materials and methods: A total of 102 fecal samples were collected from clinical cases of diarrhea or hemorrhagic gastroenteritis from CPV vaccinated or non-vaccinated dogs. Samples were tested using CPV-specific polymerase chain reaction (PCR) targeting VP2 gene, multiplex PCR for detection of CPV-2a and CPV-2b antigenic variants, and a PCR for the detection of CPV-2c. CPV-2b isolate was cultured on Madin-Darby canine kidney (MDCK) cell lines and sequenced using VP2 structural protein gene. Multiple alignment and phylogenetic analysis was done using ClustalW and MEGA6 and inferred using the Neighbor-Joining method.

Results: No sample was found positive for the original CPV strain usually present in the vaccine. However, about 50% (52 out of 102) of the samples were found to be positive with CPV-2ab PCR assay that detects newer variants of CPV circulating in the field. In addition, multiplex PCR assay that identifies both CPV-2ab and CPV-2b revealed that CPV-2b was the major antigenic variant present in the affected dogs. A PCR positive isolate of CPV-2b was adapted to grow in MDCK cells and produced characteristic cytopathic effect after 5th passage. Multiple sequence alignment of VP2 structural gene of CPV-2b isolate (Accession number HG004610) used in the study was found to be similar to other sequenced isolates in NCBI sequence database and showed 98-99% homology.

Conclusion: This study reports the first detection of CPV-2b in dogs with hemorrhagic gastroenteritis in Himachal Pradesh and absence of other antigenic types of CPV. Further, CPV-specific PCR assay can be used for rapid confirmation of circulating virus strains under field conditions.

Keywords: canine parvovirus; molecular typing; phylogenetic analysis; sequencing.

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Figures

Figure 1
Figure 1
Canine parvovirus 2ab polymerase chain reaction (PCR) assay amplifies VP2 gene and generates a 681 bp PCR product. Lane M represents ladder, sample 1 is negative control, sample 2 is positive control, and sample 3 is positive fecal sample.
Figure 2
Figure 2
Canine parvovirus (CPV)-2ab/CPV2b multiplex polymerase chain reaction (PCR) assay simultaneously detects CPV-2a and CPV-2b antigen types. The multiplex assay generates 681 and 427 bp PCR products with CPV-2b antigenic strain. Lane M represents ladder, sample 1 is negative control, sample 2 is positive sample, sample 5 is positive fecal sample, and 3, 4, and 6 are negative samples.
Figure 3
Figure 3
Cytopathic effects produced by canine parvovirus on Madin-Darby canine kidney cell culture at different time points (100×), (a) Cell monolayer after 24 h showing no visible change, (b) cell monolayer after 48 h showing slight rounding of cells, (c) cell monolayer after 72 h showing rounding of cells, (d) cell monolayer after 96 h showing rounding and sloughing of cells, (e) cell monolayer after 120 h showing complete sloughing of cells.
Figure 4
Figure 4
Phylogenetic tree constructed using canine parvovirus sequence under study ([CPV-2b], HG004610) and reference CPV-2b sequences, KM457125.1 (UY317, Uruguay), KC196095.1 (M258, Uruguay), DQ340409.1 (BR-183-85, Brazil), U22896.1 (VP-1 and VP-2, USA), JF414817.1 (Arg5, Argentina), AY742932.1 (USA), DQ340407.1 (BR145-80, Brazil), FJ005261.1 (G162/97, Italy), AB120725.1 (HNI-3-4, Vietnam), JQ335985.1 (PALAM-8, India), JQ335982.1 (PALAM-5, India), JQ743892.1 (CPV-BG [11], China), JQ743890.1 (CPV-4 [10], China), GU380299.1 (02/09, China), JQ335978.1 (PALAM-1, India), JF701924.1 (MB26b, India), and JF900762.1 (KP6204, India).
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