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Kerala Veterinary and Animal Sciences University, Wayanad

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2016 - 20193
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Subject: Veterinary Parasitology × End date: 2019 × Start date: 2010 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    DETECTION AND MOLECULAR CHARACTERIZATION OF BENZIMIDAZOLE RESISTANCE IN GASTROINTESTINAL NEMATODES OF GOATS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-06-23) ASHA RAJAGOPAL; Lucy Sabu
    The study was conducted with the objectives of assessing the status of benzimidazole resistance in gastrointestinal (GI) nematodes of goats in Kerala, detection of single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance in the β-tubulin gene of predominant GI nematode species and to evaluate the efficacy of the egg hatch assay, larval development assay and PCR-RFLP in detection of benzimidazole resistance. Microscopical examination of 520 faecal samples collected from goats from 10 organized farms and 16 small holder farmers’ flocks in eight agro-ecological zones of Kerala revealed an overall prevalence of 81.5 + 5.54 per cent strongyles in goats. There was significant difference between the prevalence of strongylosis in organized farms (91.19 + 4.33 %) and small holder farmers’ flocks (65.34 + 10.27 %). The mean faecal egg counts (FECs) also differed significantly between organized farms and small holder farmers’ flocks. The prevalent genera of strongyles identified on coproculture were Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp. Faecal egg count reduction test (FECRT) done for screening the benzimidazole resistance status in 10 organized farms and 16 small holder farmers’ flocks identified benzimidazole resistance in all the organized farms. Among the small holder farmers’ flocks, 43.75 per cent were found to be resistant to benzimidazoles. Susceptibility was identified in 37.5 per cent of the small holder farmers’ flocks while resistance was suspected in 18.75 per cent of the flocks. Statistical analysis revealed significant association between the resistance status and farm type. Haemonchus spp. was found to be the most predominant GI nematode in post-treatment faecal cultures indicating that it is the major species responsible for benzimidazole resistance. Resistance status was found to be significantly correlated with the frequency of deworming in flocks. Molecular genotyping by PCR-RFLP revealed E198A polymorphism in isotype 1 β-tubulin gene in Haemonchus spp. with an overall frequency of 0.516 for the resistant allele (r). The overall prevalence of homozygous resistant genotype (rr) at codon 198 in Haemonchus spp. was 25.6 per cent. No polymorphism was identified at codons 167 and 200 in Haemonchus spp. in this study. In Trichostrongylus spp., F200Y polymorphism was identified in isotype 1 β-tubulin gene with an overall gene frequency of 0.337 for the resistant allele and with 28 per cent of the larvae genotyped being homozygous resistant (rr). Susceptible genotype was identified at codons 167 and 198. All the Oesophagostomum spp. larvae genotyped were found to be of the susceptible genotype at codons 198 and 200. In vitro detection of benzimidazole resistance was done by egg hatch assay (EHA) and larval development assay (LDA). Correlation of the results of FECRT, EHA, LDA and PCR-RFLP revealed significant correlation (p < 0.05) between FECR per cent, ED50 in EHA, Pdd (proportion of larvae hatching at the discriminating dose of 0.02 µg/ml) in LDA and the percentage of homozygous resistant (rr) genotype in PCR-RFLP. There was significant correlation between ED50 and Hdd (hatching ratio of strongyle eggs at the discriminating dose of 0.1 µg/ml) values in egg hatch assay. Pdd values were found to be significantly correlated with other resistance parameters indicating that it is a better criterion for resistance detection than LD50 in LDA. To predict the genotypic resistance using phenotypic resistance indicators, regression equations were derived with rr genotype per cent as dependent variable and FECR per cent, ED50 and Pdd as the independent variables.
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    ThesisItemOpen Access
    DEVELOPMENT AND EVALUATION OF RECOMBINANT PROTEIN BASED ELISA FOR THE DIAGNOSIS OF INTESTINAL SCHISTOSOMOSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) PRIYA M. N.; Bindu Lakshmanan
    Animal schistosomosis caused by Schistosoma spindale is an economically considerable blood fluke infection that adversely affects the livestock sector in India. The aim of the present study was to clone, express, purify and analyse 22.6 kDa tegument protein of S. spindale (rSs22.6 kDa) in prokaryotic system and to assess its usefulness as a diagnostic candidate for seroprevalence studies. An abattoir survey of intestinal schistosomosis in bovines conducted in Thrissur corporation slaughter house, Kuriachira from August, 2017 to July, 2018 revealed an overall prevalence of 25 per cent of intestinal schistosomosis in cattle with 24.34 per cent of S. spindale and 0.66 per cent of S. indicum infection. The highest prevalence of infection (26.32 per cent) was observed during monsoon season. Majority of the samples showed low intensity infections (81.57 per cent). Occurrence of the disease or the intensity of the infection related with seasons did not show any statistical significance. For the production of recombinant 22.6 kDa protein, RNA was isolated from adult live schistosome worms, cDNA synthesized and the specified 22.6 kDa tegument protein coding gene was amplified and cloned in pJET cloning vector and transformed in E. coli Top 10 cells. The confirmed product (573bp) was amplified with primers having restriction site for BbS1 and digested with BbS1 restriction enzymes. Then pET28b expression vector was digested with Xho1 and NCo1 restriction enzymes and transformed to BL21 E. coli cells. Induction of protein was done 0.6mM IPTG for four hours. Purification of the soluble fraction of newly expressed polyhistidine (6X-His) tagged fusion protein was carried out by Nickel chelating affinity chromatography using Ni-NTA agarose column. The SDS PAGE analysis of rSs22.6 protein and the following Coomassie Brilliant Blue staining of the gel revealed a bright single band of size approximately 22.6 kDa. Moreover, immunoblotting of rSs22.6 protein with schistosome positive bovine serum revealed a single immunodominant protein corresponding to the approximate molecular weight of 22.6 kDa without any cross reaction with amphistome positive bovine serum. Standardisation of IgM and IgG based Dot and indirect ELISA was carried with rSs22.6 protein. Excretory secretory antigens (ESA) were also prepared using the mesentery recovered adult schistosomes to conduct ESA based indirect IgG ELISA. Copro PCR was carried out with mitochondrial gene specific primers of Schistosoma spp. and an amplicon of approximately 454 bp size was amplified indicating the presence of S. spindale. Comparison of results of rSs22.6 IgM ELISA with that of copro PCR revealed a sensitivity of 16.67 per cent while that of copro PCR was 30 per cent with a cent percent specificity. The results showed that the sensitivity of rSs22.6 IgM ELISA was lower than that of copro PCR, suggesting unsuitability of rSs22.6 IgM indirect ELISA for seroprevalence studies. The diagnostic performance of rSs22.6 IgG ELISA was compared with ESA IgG ELISA and copro PCR using 38 known positive samples and 13 known negative samples. A sensitivity of 92.11 per cent for rSs22.6 IgG ELISA, 89.47 per cent for ESA IgG ELISA and 31.58 per cent for copro PCR was observed whereas specificity was cent per cent for all the three assays. Sensitivity of rSs22.6 IgG ELISA higher than ESA IgG ELISA. Sensitivity of copro PCR was considerably low. However, there was no statistically significant difference between the sensitivities of rSs22.6 IgG ELISA and ESA IgG ELISA while both these assays showed statistically significant difference from copro PCR. Seroprevalence study with rSs22.6 IgG ELISA of 506 sera samples of cattle from southern, central and northern zones of Kerala revealed a prevalence status of 26.88 per cent of intestinal schistosomosis. Highest prevalence of the infection was observed in Alappuzha district (42.86 per cent) and coastal sandy zone (41.18 per cent) whereas the lowest prevalence was in Ernakulam (17.19 per cent) district and Red loam (15.63 per cent) zone of Kerala without any statistically significant difference between these findings. In silico analysis of the expressed protein revealed that it is a protein with 190 amino acids. Predicted secondary structure of protein revealed the presence of alpha helices (47.89 per cent), extended strands (17.37 per cent), beta turns (8.95 per cent) and random coils (25.79 per cent). The tertiary structure of the protein revealed that it contained α helices and β sheets with EF-hand as a helix-loop- helix domain. Analysis of rSs22.6 protein showed that signal peptides were absent. Presence of transmembrane helices in the predicted protein sequence indicated the absence of transmembrane helices. The NCBI conserved domain prediction revealed the presence of two conserved domains, one EF-hand domain located in its N-terminus (residues 12–71) and a dynein light-chain domain located in its C-terminus (residues 99–186). The possible number and composition of epitopes were predicted by linear epitope prediction in Immune Epitope Database and Analysis Resource tool revealed the presence of seven epitopes with aminoacid sequences ranging from 3 to 20. Phylogenetic analysis using the Maximum Likelihood method in MEGA 5.2. revealed that it was a sister clade of S. haematobium and S. bovis while it is distinct from the clade containing S. japonicum.
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    ThesisItemOpen Access
    DEVELOPMENT OF ELISA BASED DIAGNOSTICS FOR EARLY DETECTION OF COPROANTIGENS IN BOVINE AMPHISTOMOSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2016-12-30) H. SHAMEEM; K.DEVADA
    Bovine amphistomosis is a relatively neglected snail borne trematode disease causing great economic loss to dairy farmers. Conventional ova detection often underestimates the true prevalence of the disease as many of the amphistomes are seasonally egg producing and the pathogenicity of the disease is due to immature flukes which do not lay eggs. The present study identified ten species of amphistomes viz. Fischoederius cobboldi, Gastrothylax crumenifer, F. elongatus, Carmyerius spatiosus, Paramphistomum epiclitum, P. cervi, Ceylonocotyle scoliocoelium, Cotylophoron cotylophorum, C. indicum and Explanatum explanatum from rumen collected from Municipal Corporation slaughter house, Thrissur. Fischoederius cobboldi was found to be the most predominant species. Protein profile of whole worm antigen of five species of amphistomes namely Fischoederius cobboldi, Gastrothylax crumenifer, F. elongatus, Carmyerius spatiosus, and Paramphistomum spp. and excretory-secretory antigens were analysed in one dimensional SDS-PAGE followed by western blotting with amphistome positive bovine sera. A 34 kDa polypeptide common to both whole worm antigen and excretory-secretory antigen was identified as immunogenic and useful for further diagnostic studies. Somatic antigens of G. crumenifer was partially purified by column chromatography and subjected to SDS-PAGE and immunoblotting. Cross reactivity of excretory-secretory antigen was ruled out by immunoblotting with schistosome and strongyle positive bovine sera. Excretory-secretory antigen was used to raise hyperimmune serum in guinea pigs and rabbits for use as detection antibodies. A coproantigen sandwich ELISA protocol was standardised using guinea pig and rabbit hyperimmune serum which could detect minimum 3 ng/µl of excretory-secretory antigen in dung. Out of 515 faecal samples collected from six agro-ecological zones of central Kerala, 362 (70%) were found to be positive by sandwich ELISA as against 165 (32%) by ova detection. Dot ELISA was also standardised as a rapid test for coproantigen detection in amphistomosis. Sensitivity and specificity of two tests were determined. Sandwich ELISA possessed a sensitivity of 90 per cent, specificity of 100 per cent while dot ELISA had a sensitivity of 76 per cent and specificity of 100 per cent. Statistical tests also depicted the reliability and high discriminating power of sandwich ELISA in early detection of amphistomosis.Statistical analysis showed no significant difference in the prevalence of amphistomes between the six agro-ecological zones of central Kerala but recorded highest infection in Palakkad plains (86%) followed by Central Midlands (80%). Molecular characterisation of four prevalent amphistomes namely F. cobboldi, G. crumenifer, F. elongatus, and Paramphistomum spp. was done by PCR targeting ITS-2+ rDNA sequences which yielded amplicons of 494 bp, 503 bp, 514 bp and 494 bp respectively. Nucelotide sequence analysis revealed eight base pair differences between pouched and unpouched species and also two base pair difference between G. crumenifer and Fischoederius spp. suggesting the utility of ITS-2+ region to be used as a species marker. In silico RE map analysis of four amphistomes identified unique recognition sites which could differentiate G. crumenifer from other three amphistomes. Phylogenetic analysis revealed clustering of Kerala isolates with Chennai, Shillong and Bareilly isolates.