Canine Parvo virus infection is causing fatal disease to dogs resulting in severe morbidity and mortality. It is becoming disastrous for canids in Pakistan. It is important to study the pathogenesis of this deadly virus. Parvovirus infection has become more pathogenic with passage of time and its pathogenicity may be enhanced due to possible mutation in field virus. The possible mutations may be the cause of vaccine failure and rapid outbreaks.
In present study, sum of 50 fecal samples from different areas were collected and properly labelled. Both serological and molecular confirmation was done.
Haemagglutination test was used as a screening test. HA test was performed to check agglutinating activity of samples as described by (Desario et al. 2005). Haemagllutination was done and only 20 samples were found positive. Positive samples were selected for polymerase chain reaction. DNA of selected samples was extracted through kit method according to protocol provided by the manufacturer. Nano drop was done to check quantity and purity of viral DNA. PCR was performed. Already reported primers were used for detection of CPV (Buonavoglia et al. 2001). The PCR product was confirmed by running the PCR product on 1.2% agarose gel. Electrophoresis was performed at 110 Volts for 30 minutes. The amplified PCR product was further purified using GeneJet Gel Extraction Kit using instructions provided by the manufacturer. Purified samples were sent to commercial lab for Sanger sequencing. The obtained sequences were examined using BLAST (Basic Local Alignment Search Tool). Sequences were compared with other sequences already obtainable from the GenBank database (http://www.ncbi.nlm.nih.gov) and were carefully aligned by BioEdit software (version 7.2.5.0) using the ClustalW method. The MEGA software program, version 6.0 was used to construct a phylogenetic tree using the neighborhood joining method.
CHAPTER 6
SUMMARY
Summary
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Complete blood count was performed on whole blood samples. The samples were divided into two groups (infected & non-infected) based on the results of PCR. Paired T-Test was applied on the results obtained and results showed that all parameters statistically showed significant difference between two groups except WBC’s but still the number of WBC’s decreases in infected animal like other parameters. Serum chemistry analysis was performed on serum samples. These samples were also divided into two groups (infected & non-infected) based on the results of PCR. Paired T-Test was applied on the results obtained and results revealed that all parameters statistically showed significant difference between two groups except ALT and creatinine levels.
Results showed that new CPV-2a was found to be the major circulating strain of Pakistan as out of 7 sequenced samples, 6 were identified as CPV-2a and only 1 of them was CPV-2. PCR was found to be the most sensitive assay for the detection of CPV, as compared to HA and ICT- CPV antigen detection kit. The strain in vaccine was identified as CPV-2, this finding was considered important because it indicates one of the reason for vaccine failure. The phylogenetic analysis of strains showed that all the samples collected from different regions of Pakistan falls in one clade and branching pattern is shared with the neighboring countries especially China. This information reveals that our virus is genomically similar to our neighboring countries India and China. Upon hematology and serum chemistry analysis, it was revealed that the virus does effect the blood parameters and all the red blood cell indices were lowered except platelet count.
The present study enabled us to study pathogenesis, to determine currently prevailing strain of CPV in Pakistan, identify the cause of disease occurrence even after vaccine, to study pathogenesis of CPV, to characterize and construct phylogenetic tree of currently prevailing canine parvovirus field isolates.