Haemagglutination test
Out of 50 fecal samples screened by HA test, 09 (18%) samples agglutinated porcine RBCs with a titer ranging from 32-256 (Fig 1). Our findings agreed with
Nair, (2022) who had screened a total of 50 samples by HA test, of them 05 (10%) samples agglutinated porcine RBCs with a titer ranging from 32-256. In a similar study by
(Kumari et al., 2020) HA test was conducted on 342 samples and found that 71 (20.76%) of the samples agglutinated pig RBC with HA titers ranging from 1 in 32 to 1 in 512.
PCR and NPCR
Out of the total 50 samples subjected to PCR and NPCR, 19 (38%) samples were found positive by PCR and produced an amplicon size of 681 bp (Fig 2), whereas 29 (58%) were found positive by NPCR amplifying a product size of 548 bp (Fig 3). Similar results were obtained in a study conducted by
(Khadse et al., 2023) which revealed the prevalence of CPV to be (18/50, 36%) by conventional PCR assay. Consequently, a study by
(Navarro et al., 2017) screened 104 diarrheic fecal samples collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, in which, a total of (25/104, 24%) of dogs were found positive for CPV by PCR.
The present study revealed that NPCR has a substantially higher sensitivity for CPV detection than conventional PCR. These results were similar to the earlier findings of
(Mochizuki et al., 1993), who stated that nested PCR is more sensitive than conventional PCR. Similarly, the results ascertained by
(Khadse et al., 2023) revealed the prevalence of CPV in dogs to be (30/50, 60%). A study by
(Kushwaha et al., 2018) using PCR and NPCR for the detection of CPV-2 reported (18%) and (63%) positive reactions, respectively. These above-stated findings indicated that nested PCR assay was a more sensitive molecular technique than conventional PCR for the detection of CPV-2.
Sequencing analysis
Two representative PCR products each from conventional and Nested PCR (Sample no. C13 from Ayodhya Nagar, Nagpur; OQ594804.1) and (Sample no. C23 from Bhavani mandir, Pardi, Nagpur; OQ594805.1) showed 99.5% to 100% sequence identity with the sequences obtained from China, Australia, Egypt, Myanmar, Italy, Ethiopia,
etc. BLAST analysis of OQ594804.1 showed 99.85% identity with two
Canine parvovirus strains
viz CPV 2b and CPV 2c obtained from different regions of the world whereas the sequences of strain 2b obtained from Australia showed 99.85% identity with the sequence under study (Fig 4). Phylogenetic analysis of sample C-13 (accession no. OQ594804.1) further revealed that it was closely related to
Canine parvovirus VP2 gene sequence obtained from China in 2021 (Acc. No. MZ836323, strain 2c) with 99.85% homology. Our sequence also showed 99.85% homology with that obtained from Pangolin from China (Acc. No. OP208805), though it was distinctly located and placed in a separate node in the phylogenetic tree. Moreover, many other sequences of strain 2c obtained from China, Myanmar,
etc. also showed 99.85% identity with our sequence. This pattern of homology suggests that sample C13 might possess
Canine parvovirus belonging to both strains 2b and 2c. Earlier research by various workers involving the application of multiplex PCR for the detection of CPV strains supported this hypothesis. A study by
(Deng et al., 2018) established multiplex PCR to target
Canine parvovirus-2 and revealed that a single sample suspected for CPV showed the presence of all 3 antigenic strains such as, CPV-2a, CPV-2b and CPV-2c. Another study by
Agnihotri, (2017) revealed that the Hisar CPV isolates showed 99.6% identity with KU244254 (
Canine parvovirus 2c capsid protein VP2),99.7% identity with KR869657 (
Canine parvovirus 2a strain CPV /BJ24/VP and KR002793
Canine parvovirus 2b strain CPV /CN/HB1/2013).
BLAST analysis of OQ594805.1 showed (99.79-100 %) homology with only one strain of
Canine parvovirus viz. strain 2c obtained from different regions. This suggested that the CPV of sample 23 belonged to strain 2c. Similar results were obtained by
Perez et al., (2012) which revealed that CPV-2c was the only strain detected showing high homogeneity in both nucleotide and amino acid sequences. Phylogenetic analysis of sample C-23 (accession no. OQ594805.1) revealed that it was closely related to and placed in the same node with
Canine parvovirus VP2 gene sequence obtained from China in 2021 (Acc. No. ON322829, strain 2c) with 100 % homology (Fig 5). Our sequence showed 99.79% identity with other sequences obtained from the Mizoram state of India and was distantly related to all sequences from India. Similar findings by
Nair, (2022) previously revealed that the sequences of CPV isolates obtained from dog samples in Nagpur, Maharashtra showed homology to sequences obtained from China and Mizoram (India) (strain 2c).