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ThesisItem Open 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 RavindranCanine 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.ThesisItem Open Access FORMULATION OF A COMBINED TARGETED SELECTIVE TREATMENT STRATEGY FOR SUSTAINABLE CONTROL OF GASTRO INTESTINAL NEMATODOSIS IN GOATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, MANNUTHY, THRISSUR, 2020-12-18) K. SYAMALA; K. DevadaGastro intestinal parasitism and mounting anthelmintic resistance pose serious impediments to goat production in humid tropical state of Kerala. In order to develop a sustainable parasitic control strategy ideal for goats of the state, a comprehensive research on epidemiology of gastro-intestinal parasitism, parasitological indicators and helminth control modules was carried out with the following objectives; 1) conduct a survey on epidemiology and current treatment strategies of gastro-intestinal parasitism among goats in Kerala; 2) validate the combined targeted selective treatment (C-TST) indicators such as FAMACHA©, body condition score (BCS) and dag score (DS); 3) compare the impact of treatment strategies like routine anthelmintic treatment (RT), strategic prophylactic treatment (SPT) and C-TST on the growth performance and anthelmintic resistance in goats; and 4) develop an anaemia eye colour chart based on the physiological status suitable for goats in humid tropics. Detailed survey on veterinary practitioners in Kerala on their awareness on parasite control practices revealed that updates on advances in veterinary parasitology in relation to dose calculation, development and management of anthelmintic resistance and alternate methods of parasite control with limited chemotherapy need to be intensified. A glance into the parasite management strategies followed by the goat farmers of Kerala indicated that majority of them were clueless of the importance of correct dosage, frequency of anthelmintic treatment and integrated parasite management methods. Epidemiological survey of gastro-intestinal parasitism in goats in the state was carried out in 13 agro-ecological zones for a period of one year in six organised farms and 120 small holder flocks. It revealed an overall prevalence of 67.2 per cent strongylosis with a mean faecal egg count (FEC) of 390.79 ± 19.17, ranging from 0 to 8968 and standard deviation of 716.745. Highly significant influence of season and month (p 3, BCS was ≤ 1.5, dag score was ≥ 2 and/ or FEC >500. In the case of routine treatment bimonthly anthelmintic treatment was carried out whereas in the case of strategic prophylactic treatment (SPT) anthelmintic treatment was given before South West monsoon (June) and after North East monsoon (October). After 13 months of treatment, the impact of the C-TST on parasitological and production parameters and mortality was assessed. More number of doses (256) were required for the RT while for SPT only 152 doses were required for administration. The least number of doses (82) only were given to animals in C TST, where treatment was applied based on the TST indicators. The impact of the C-TST on anthelmintic resistance was assessed by FECRT and molecular methods, and no statistically significant difference was detected before and after treatment and between the treatment groups. There was no significant difference in prolificacy, weight gain and mortality between C-TST and the other two treatment regimes. . Molecular genotyping of Haemonchus contortus by PCR-RFLP revealed the presence of polymorphism at 198 and 200 codons, while at codon 167 no heterozygous genotype were detected. In Trichostrongylus sp. F200Y polymorphism was identified in isotype 1-β tubulin, whereas at codon 167 only resistant genotypes were detected. The gene frequency was 0.16 for resistant allele before treatment and 0.14 after treatment. In this species, no polymorphism was detected at E198A in all the treatment categories. The most frequently identified polymorphism in H. contortus was associated with E198A, while F200Y was found to be the least common. In Trichostrongylus sp., F200Y was found to be the most common. No significant difference could be observed between the genotype frequencies at the three codons in the two species of strongyle parasites before and after treatments.ThesisItem Open 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 SabuThe 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.ThesisItem Open 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 LakshmananAnimal 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.ThesisItem Open 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.DEVADABovine 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.ThesisItem Open Access THE EFFECT OF CERTAIN BIGPESTICIDES AND IRRADIATIGN GN THE DEVELOPMENTAL STAGES OF MYIASIS PRODUCING FLIES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1998) SUBRAMANIAN., H.; Rajamohanan, K.; SUBRAMANIAN., H.A Study was undertaken on the prevalence of cutaneous myiasis in domestic animals and its control using biopesticides and gama ray irradiation. The prevalence of cutaneous myiasis in domestic animals were found to be 205 (2.08 per cent) among the 9861 animals screened. The peak of infestation was noted in the month of January. In-host-wise and parasite-wise the highest incidence was noted in cattle (63.41 per cent) and the majority of infestation was produced by Chrysomyia bezziana larvae (90.73 per cent) . Methoprene at 1 to 50 ppm concentration caused only moderate mortality on larvae but significantly increased the mortality rate on eggs, prolonged the larval phase, increased the formation of larval pupal intermediaries and adult deformities and reduced the adult emergence. Diflubenzuron at 0.5 to 5 ppm caused 55 to 100 per cent larvicidal effect due to lowered chitin content of 18.42 to 52.11 per cent in larval cuticle. Bacillus thuringiensis var israelensis produced only moderate larval mortality at 160 to 800 ppm in myiasis producing flies. Azadirectin at 10.5 to 15 ppm produced 100 per cent mortality in eggs and larvae. Significant antifeedant, ovipositional deterrent and repellant effects were also produced by Azadirectin. Among the extracts of Acorus calamus, studied, petroleum ether extract at 2.5 per cent concentration gave 82.5 to 100 per cent mortality of the larvae. Moderate antifeedant, ovipositional deterrent and repellant effects were also noticed. The petroleum ether extract produced 100 per cent sterility at 0.1 to 1 per cent concentration by preventing the development of ovarian follicles. Three day old pupae of myiasis producing flies exposed to rays gave excellent sterility effect at 2000 to 4000 rads radiation exposure without any other deleterious effect in the flies. Diflubenzuron at 5 ppm concentration showed the highest larvicidal effect. (88.5 per cent) in natural cases of cutaneous myiasis.
