DETECTION OF ANTIGENIC VARIANTS OF CANINE PARVOVIRUS BY POLYMERASE CHAIN REACTION AND SEQUENCING
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Date
2011
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
A study was conducted to detect the antigenic variants of canine
parvovirus from faecal samples of clinically suspected dogs by PCR and
sequencing. The samples were screened for the presence of CPV by HA test and
the results were confirmed by HI test. Antigenic characterisation of field strains
and vaccine strains was performed by employing PCR with differential pairs of
primers and PCR-RFLP analysis. The partial sequences obtained from the
amplified CPV VP2 gene fragment of nine samples were analysed to identify the
nucleotide variations.
A total of 78 faecal samples were collected from dogs suspected for CPV
infection that were brought to veterinary hospitals attached to KAU and District
Veterinary Centre, Thiruvananthapuram. Twenty eight (35.90 per cent) out of 78
samples were found to be positive to CPV infection by HA test and the results
were further confirmed by HI test.
Among 78 faecal samples tested by PCR, none of the samples were found
positive for CPV-2, while 57 (73.08 per cent) were found positive with CPV-2ab
primers. Out of 57 CPV-2ab positive samples, 15 were identified as CPV-2a
types while remaining 42 were CPV-2b types. The specificity of PCR was further
confirmed by RE digestion using Hha I enzyme. Thus, CPV-2b was identified as
the predominant CPV type in this study.
Among the four vaccines tested, Megavac-6, Nobivac®
Puppy DP and
Vanguard®
5 vaccines were found to be classical CPV-2 based vaccines while
Duramune® max vaccine was found to contain the CPV-2b variant.
All the 57 CPV-2ab positive samples, when subjected to PCR with CPV555 primer set, yielded a specific amplicon of 583 bp. On PCR-RFLP analysis
with Mbo II enzyme, none of these products showed specific CPV-2c cleavage
pattern of 500 and 83 bp. This indicated the absence of CPV-2c in this area.
Sequence analysis of the CPV VP2 gene from the PCR products of nine
samples revealed that seven were CPV-2b types and the remaining two were
“new CPV-2a” types. No CPV-2c types were detected among the sequenced
samples. Additional mutations at nucleotide 4104 of residue 440 and at
nucleotide 4121 of residue 445 were also observed in the CPV sequences under
study.
Sequence similarity searches of the CPV sequences using BLAST
analysis revealed that the sequences were highly specific to CPV, as indicated by
the maximum identity (99-100 per cent) obtained with VP2 gene sequences of
other CPV strains available in the GenBank. Phylogenetic analysis revealed that
most of the CPV samples under study were related to variants typical to the
country. Hence the Indian isolates were found to be genetically close to Asian
isolates suggesting the migration of variants crossing the borders of different
countries.
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