Phylogenetic analysis of canine parvovirus partial VP2 gene in India.

A total of 85 samples (58.0 %) were found to be positive for Canine parvovirus (CPV) by PCR assay (Hfor/Hrev primers) out of 158 suspected faecal samples of dogs collected from various states/union territories of India. Nine CPV isolates could be obtained in A-72 cell line. The sequencing of the partial VP2 gene of CPV identified the predominant CPV strain as CPV-2a (Ser297Ala) with one CPV-2b (Ser297Ala) and another CPV-2a variant strain (Ser297Gly). Several non-synonymous and synonymous mutations were also recorded in this study. The phylogenetic tree revealed that most of the CPV sequences from Tamil Nadu (Southern India) and Maharashtra (Western India) obtained during 2011 and few sequences from Northern India obtained during 2012 were grouped together along with CPV-2a (Ser297Ala) strains from China and India and followed the same evolution; although there was definitive indication of separate lineages too by few other sequences.

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens.

AIMS: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. METHODS AND RESULTS: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID50 ml⁻¹, whereas the detection limit of the PCR was 1000 TCID50 ml⁻¹. CONCLUSIONS: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. SIGNIFICANCE AND IMPACT OF THE STUDY: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.

Isolation, molecular characterization and phylogenetic analysis of canine parvovirus.

Canine parvovirus type 2 (CPV-2) causes acute haemorrhagic enteritis in dogs. Canine parvovirus is prone to genetic evolution and has undergone several mutations that produced different strains like CPV-2a, CPV-2b, New CPV-2a, New CPV-2b and CPV-2c in the past three decades. Mutations affecting the VP2 gene of CPV have been responsible for evolution of different antigenic variants. Sequence analysis of VP2 gene of the virus and subsequent characterization is important for molecular epidemiology. The present study was conducted to isolate and to characterize the virus by amplifying partial VP2 gene and further sequence analysis and also to estimate phylogenetic relationship of field virus with the reference strains. Out of 77 samples, 51 samples were found to be positive by PCR and all the 51 samples were subjected for virus isolation in CRFK cell line. Sixteen viruses could be isolated and 10 randomly selected isolates were subjected to sequence analysis along with four random clinical samples. All the 10 isolates and 4 clinical samples were characterized as New CPV-2a (CPV2a with 297-Ser→Ala). One of the field isolates was found to be phylogenetically closely related to New CPV-2a strains of Japan and India; another field isolates was found to share ancestral origins with New CPV-2a strains of Korea, USA, Italy, Brazil, Germany, Taiwan and Vietnam; rest other sequences had distinct lineage but shared molecular relationship with New CPV-2a reference strains.

Molecular Typing of Canine parvovirus Occurring in Pondicherry by Multiplex PCR and PCR-RFLP.

The present study was aimed at molecular typing of Canine parvovirus (CPV) occurring in Pondicherry using PCR based assays. CPV-2a and CPV-2b types were detected by PCR in 68 (53.12%) out of 128 faecal samples/rectal swabs. All the 68 samples found positive by PCR assay were subjected to multiplex PCR assay with CPV-2ab and CPV-2b primer pairs. Sixty-seven (98.52%) samples were characterized as CPV-2b type and one sample (1.47%) was categorized as CPV-2a type. Sixty clinical samples found negative by CPV-2ab primers were subjected to another PCR assay with CPV-555 primer pair for detecting CPV-2c. Though three samples (5%) responded to this PCR, subsequent RFLP of these PCR products with MboII did not show any cleavage indicating the absence of CPV-2c in Pondicherry. It was inferred that CPV-2b was the most prevalent CPV type in Pondicherry. It was further concluded that the CPV-2 variants (CPV-2a, CPV-2b and CPV-2c) currently circulating in the field worldwide could be diagnosed by employing multiplex PCR and PCR-RFLP assays.

Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples.

Comparative efficacy of faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was investigated in 40 clinically suspected cases of Johne's disease in dairy cattle. The sensitivity of faecal culture and PCR assay in this study was 52.5% (21/40) and 90% (36/40) respectively. All isolates appeared only on the mycobactin J supplemented Herrold's egg yolk medium (HEYM) at 8-16 weeks post-inoculation, were acid-fast and were positive for IS900 PCR yielding a single amplicon of 217 bp. A total of 28 faecal samples out of 40 were positive by IS900 primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product of size 217 bp. Twelve faecal samples, which gave negative results in the primary PCR, were subjected to secondary PCR assay. Of the 12 samples, 8 gave positive results in the IS900 nested PCR (nPCR), which yielded a PCR product of 167 bp, proving better sensitivity of nPCR assay than single amplification PCR. PCR could detect additionally 15 samples as positive which were negative by faecal culture. The chi-square analysis showed a highly significant difference between the tests (P< 0.01). This study suggests that IS900-PCR-based detection of Map could be used as a potential diagnostic tool for rapid and effective Johne's disease surveillance.