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Subject: Veterinary Parasitology × Author: DEEPA C.K. × Start date: 2020 ×
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    ThesisItemOpen Access
    MOLECULAR CHARACTERISATION AND SERO- DETECTION OF BABESIA GIBSONI IN DOGS OF KERALA
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD, 2022-03-25) DEEPA C.K.; Reghu Ravindran
    Canine babesiosis is an important tick-borne disease caused by intraerythrocytic piroplasms of the genus Babesia, which affect the domestic and wild canines. The prevalence of canine babesiosis due to Babesia gibsoni has increased throughout the world including the south Indian state of Kerala. Hence, the present study was undertaken with the following objectives, molecular characterisation of B. gibsoni in dogs of Kerala by polymerase chain reaction (PCR); sequencing, cloning and expression of thrombospondin-related adhesive protein (BgTRAP) gene of B. gibsoni; sero- detection of B. gibsoni antibodies in dogs by indirect enzyme-linked immunosorbent assay (ELISA) using the recombinant antigen. Thin peripheral blood smears (n=297) collected from dogs of three different zones of Kerala (north, south and central), revealed the presence of piroplasms of B. gibsoni in 60 samples by microscopy. The DNA isolated from the whole blood samples of the same number of animals were used for the molecular detection of B. gibsoni using PCR assays targeting amplification of different genes. The genus specific primers targeting 18S rRNA gene of Babesia spp. amplified ~1665 bp fragment in 72 samples during the primary PCR while, a ~308 bp B. gibsoni specific product was amplified in 120 samples in nested PCR using another set of primers amplifying its internal region. The polymerase chain reaction targeting the thrombospondin related adhesive protein gene of B. gibsoni revealed the amplification of ~855 bp product in 125 samples. The amplification of apical membrane antigen (AMA1) gene specific for B. gibsoni showed ~1483 bp in 65 samples. Amplicons of ~1500 bp, ~825 bp and ~1938 bp were observed in 50, 85 and 60 samples when they were targeted against B. gibsoni specific genes like secreted antigen 1 (SA1), 50 kDa surface antigen (P50), heat shock protein (HSP70) respectively. Hence, the PCR assays targeting the 18 S rRNA (nested) and TRAP were identified as highly useful assays for the diagnosis of canine babesiosis due to B. gibsoni. There was no significant difference in the relative effectiveness between BgTRAP PCR and nested Bg18S rRNA PCR for the detection of B. gibsoni in canines based on McNemar’s test (P>0.05). The single step BgTRAP PCR was less time consuming, compared to the nested time consuming Bg18S rRNA PCR. Few amplicons of these PCR assays were randomly selected for sequencing. The phylogenetic analysis of Bg18S rRNA and BgHSP70 sequences, revealed the clustering of all isolates of B. gibsoni in a single clade revealing the close genetic relatedness among the isolates of Kerala and those from other countries. The phylogeny of BgTRAP and BgP50 genes revealed the clustering of the isolates from Indian subcontinent, differentiating them from similar isolates from East Asian countries. Similarly, based on phylogeny of BgAMA1 sequences, it was observed that the Japanese isolates were clustered separately from all other available isolates including those from India. Hence, it was concluded that the genetic diversity is very minimum among B. gibsoni isolates. In order to develop a serological screening assay for the detection of B. gibsoni, the prokaryotic expression of the thrombospondin-related adhesive protein (BgTRAP) was also performed in the present study. The N-terminal BgTRAP gene fragment was cloned into prokaryotic expression vector (pET32a) and transformed into BL21 Escherichia coli cells. The recombinant protein was purified and used for the indirect ELISA. The recombinant protein detected sero-reactivity in 125 (46.12 per cent) out of 271 samples. There were no cross-reactivity for this ELISA with the known positive sera of the dogs infected with the helminths like, Ancylostoma caninum, Dirofilaria immitis, D. repens, Spirometra spp., Toxocara canis and haemoprotozoans like Trypanosoma evansi, B. vogeli, Hepatozoon canis and Ehrlichia canis. The newly standardised rBgTRAP ELISA when analysed for the relative effectiveness using McNemar's test against both BgTRAP PCR and nested Bg18S rRNA PCR, no significant difference was observed. The sensitivity and specificity of rBgTRAP ELISA in comparison with BgTRAP PCR as the reference test, was 84.00 and 73.33 per cent respectively. When the Kappa statistics was used for the evaluation of agreement of the newly standardised rBgTRAP- ELISA with reference test BgTRAP PCR, the newly developed ELISA showed fair to good agreement for the detection of B. gibsoni organism in dogs (Kappa value=0.566). Hence, the indirect ELISA using the recombinant BgTRAP antigen could be considered as an adjunct tool for the surveillance of B. gibsoni infection in dogs.