Sharma Shalini, Dhar Prasenjit, Thakur Aneesh, Sharma Vivek, Sharma Mandeep
Department of Veterinary Microbiology, Dr. G. C. Negi College of Veterinary and Animal Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur - 176 062, Himachal Pradesh, India.
Department of Microbiology, College of Basic Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur - 176 062, Himachal Pradesh, India.
Vet World. 2016 Sep;9(9):964-969. doi: 10.14202/vetworld.2016.964-969. Epub 2016 Sep 12.
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.
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.
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 5 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.
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.
本研究旨在检测喜马偕尔邦腹泻犬中犬细小病毒（CPV）的存在情况，并基于VP2基因的分子分型和序列分析确定CPV最流行的抗原变异株。
从接种或未接种CPV的腹泻或出血性胃肠炎临床病例犬中总共采集了102份粪便样本。使用针对VP2基因的CPV特异性聚合酶链反应（PCR）、用于检测CPV-2a和CPV-2b抗原变异株的多重PCR以及用于检测CPV-2c的PCR对样本进行检测。CPV-2b分离株在Madin-Darby犬肾（MDCK）细胞系上培养，并使用VP2结构蛋白基因进行测序。使用ClustalW和MEGA6进行多序列比对和系统发育分析，并采用邻接法进行推断。
未发现样本对疫苗中通常存在的原始CPV毒株呈阳性。然而，约50%（102份样本中的52份）的样本通过检测野外流行的CPV新变异株的CPV-2ab PCR检测呈阳性。此外，同时鉴定CPV-2ab和CPV-2b的多重PCR检测显示，CPV-2b是受影响犬中存在的主要抗原变异株。一株CPV-2b的PCR阳性分离株适应在MDCK细胞中生长，并在传代5次后产生特征性细胞病变效应。研究中使用的CPV-2b分离株（登录号HG004610）的VP2结构基因的多序列比对与NCBI序列数据库中的其他测序分离株相似，显示出98-99%的同源性。
本研究首次报道了在喜马偕尔邦出血性胃肠炎犬中检测到CPV-2b，且未发现其他抗原类型的CPV。此外，CPV特异性PCR检测可用于野外条件下循环病毒株的快速确认。
