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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 MOLECULAR DETECTION OF HAEMOPROTOZOAN AND HAEMORICKETTSIAL ORGANISMS IN DOGS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-08-19) BORA C., ANGELINE FELICIA; Varghese, AnjuThe objective of the present study was to detect haemoprotozoan (Babesia gibsoni, Babesia vogeli, Trypanosoma evansi and Hepatozoon canis) and haemorickettsial organisms (Anaplasma platys, Ehrlichia canis) affecting dogs in different zones of Kerala. A total of 272 peripheral blood smears and whole blood samples in ethylene diamine tetra acetic acid (EDTA) were collectd from dogs from different zones of Kerala. Peripheral blood smears were examined microscopically after Giemsa staining and whole blood samples in EDTA were used for PCR. The peripheral blood smears examination revealed a prevalence of 15.81(43/272) per cent for B. gibsoni infection. None of the other parasites (B. vogeli, T. evansi and H.canis, A. Platys and E. canis) could be detected in the blood smear examination. Genomic DNA was extracted from all the 272 blood samples and used as a template for all haemoprotozoan and hameorickettsial organisms used in the study. The B. gibsoni genus specific primer targeting 18S rRNA showed an amplicon size of ~1655 bp length by primary PCR. A total of 65 samples were positive for B. gibsoni by primary PCR. The PCR product of primary PCR was utilized as a template for nested PCR using a set of internal primers amplified a ~330 bp fragment of 18S rRNA gene. A total of 110 samples were positive for B. gibsoni by nested PCR. The polymerase chain reaction targeting B. gibsoni TRAP gene for B. gibsoni showed amplification at ~855 bp size, 115 samples showed PCR amplification for TRAP gene for B. gibsoni. A total of 16 samples showed PCR amplification for B. vogeli by 18S rRNA gene with amplicon size of ~546 bp length. Only two samples showed PCR amplification for H.canis by 18S rRNA gene with amplicon size of ~666 bp length. Only a single sample showed PCR amplification for E. canis virB9 gene with amplicon size of ~380 bp. None of the samples in the present study showed PCR amplification for A. platys and T. evansi. In the stastical analysis, pair wise comparison revealed that occurrence of B. gibsoni was significantly higher than B. vogeli followed by all other parasites.A total of 19 amplicons of PCR targeting 18S rRNA of B. gibsoni from different zones of Kerala and nine amplicons of TRAP gene of B. gibsoni were selected for sequencing. A total of 13 amplicons of B. vogeli 18S rRNA, two amplicons of H. canis 18S rRNA and one amplicon of E. canis virB9 gene were also selected for sequencing. Each amplicon was considered as an isolate. All the partial sequences of 18S rRNA of B. gibsoni, B. vogeli and H. canis and TRAP gene of B. gibsoni, virB9 gene of Ehrlichia canis in the present study were subjected to homology search using NCBI-BLAST. A total of 19 isolates of B. gibsoni from Kerala were clustered in clade1 with high bootstrap value of 100 per cent. This is the first report of the phylogenetic analysis of B. gibsoni using the 18S rRNA from Kerala. The phylogenetic analysis of TRAP gene of B. gibsoni sequences indicated that B. gibsoni field isolates of Kerala in the present study formed a single clade1 with Indian and Bangladesh isolates with bootstrap of 89 per cent.The phylogenetic analysis based on 18S rRNA gene revealed Kerala isolates formed a separate clade with high bootstrap value of 100 per cent and was away from the clade formed by Punjab isolate and Philipine isolates. This was the first attempt in Kerala to study the phylogenetic analysis of B. vogeli based on 18S rRNA.The phylogenetic tree of 18S rRNA of H. canis and two Kerala isolates of H. canis indicated that field isolates of Kerala in the present study formed a single clade along with previously published Indian isolates (Thrissur, Wayanad, Gujarat and Ludhiana) and foreign isolates with bootstrap of 75 per cent.The phylogenetic analysis of virB9 gene of E. canis revealed that Kerala isolate formed a clade with Uttar Pradesh and Chennai isolates with boot strap value of 68 per cent. The present investigation provided the genetic basis for designing and testing the efficacy of different diagnostic molecules for detection of haemoprotozoan and haemorickettsial organisms in the field. Serological techniques in conjunction with PCR are reliable tools for their accurate and large scale epidemiology.ThesisItem Open Access SEROLOGICAL DETECTION OF TOXOCARA CANIS INFECTION IN DOGS OF KERALA USING RECOMBINANT CATHEPSIN-L1 AND TOXOCARA EXCRETORY SECRETORY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-08-19) A, NANDINI; Varghese, AnjuPresent study was conducted on the expression of two important proteins of Toxocara canis viz. cathepsin-L1 and TES-26 and to evaluate the diagnostic potential of these two recombinant antigens in canine toxocarosis. Adult T. canis worms were collected from puppies and total RNA was isolated and cDNA was synthesized. The coding sequences of cathepsin-L1 and TES-26 genes, respectively were amplified using specific forward and reverse primers. The PCR products obtained from cDNA got amplified at 1083 and 793 bp, respectively of cathepsin-L1 and TES-26. These proteins were subsequently expressed in Escherichia coli pPROEXHT-b expression vector. Recombinant proteins were purified using Ni-NTA chromatography. Recombinant cathepsin-L1 and TES-26 proteins were evaluated for their potential in the sero-detection of T. canis infection in both owned and stray dogs by IgG ELISA. Sera collected from 157 owned dogs were screened for anti-T. canis IgG antibodies with recombinant cathepsin-L1 and TES-26 antigen. ELISA with recombinant cathepsin-L1 showed sero-reactivity of 39/157 (24.8 per cent) in owned dogs and stray dogs showed 18/40 (45 per cent). Likewise, ELISA with recombinant TES-26 gave seroreactivity with 78/157 (49.7 per cent) owned dogs. When the stray dogs were screened with the same antigen showed a positive percentage of 28/40 (70 per cent) was recorded. As a result of this comparative study of two recombinant antigens, TES-26 gave highest positive ELISA reactivity for stray dogs followed by cathepsin-L1. rTES-26 antigen is highly sensitive in the detection of T. canis infection in owned and stray dogs compared to cathepsin-L1. Cross reactivity of these antigens were checked with dog sera which is positive for other helminths like, Ancylostoma caninum, Dirofilaria immitis, D. repens, Diphyllobothrium latum, Spirometra spp. and Babesia gibsoni. The above results showed that two recombinant antigens are cross-reacting with all the above canine parasites. The two antigens are sensitive in the detection of T. canis infection in dogs; however, the specificity of these antigens in canines needs to be further studied.ThesisItem Open Access DETECTION OF HAEMOPARASITES AND HAEMOPLASMAS IN DOMESTIC CATS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-09-19) MALANGMEI, LANCHALUNG; K. G., AJITH KUMARThe present study was conducted with the objective of detection of haemoparasites and haemoplasmas of domestic cats using microscopy and polymerase chain reaction (PCR). A total of 111 cat blood samples were collected from four different districts of Kerala viz., Wayanad, Kozhikode, Ernakulam and Thiruvananthapuram. Peripheral blood smears were prepared and stained with Giemsa’s stain and were microscopically examined for the presence of any organisms. The examination of blood smears could not detect any parasitemia in any of the smears. The whole blood samples collected in EDTA vials were processed for the isolation of genomic DNA for PCR amplification. The genomic DNA were used as template for all PCR reactions. The piroplasmid primers targeting 18S rRNA gene with a product size of 358 bp showed amplification in 13 samples. Sequence analysis and BLAST results of the PCR products revealed 10 samples infected with Hepatozoon felis (9.01 per cent), and three infected with Cytauxzoon felis (2.70 per cent). The phylogenetic tree constructed for Hepatozoon felis based on 18S rRNA gene revealed the isolates of Kerala being diverged into two clades. Nine of the isolates grouped with H. felis sequences of cats from Hyderabad, India. One isolate of Kerala clustered with sequences of domestic cats from Japan and Austria and also with wild felids. This indicated the existence of two genetically different populations of H. felis infecting the domestic cats of Kerala. The phylogenetic tree based on 18S rRNA gene plotted for C. felis showed that the three sequences of C. felis from Kerala formed a separate clade and do not cluster with any other isolates of pathogenic C. felis, C. manul and undetermined Cytauxzoon sp. of Europe, proving the existence of a non pathogenic population of C. felis existing in the apparently healthy cats of Kerala. Similarly, PCR amplification of 16S rRNA gene specific for Mycoplasma spp. yielded ~600 bp product in 10 samples. NCBI-BLAST of nucleotide sequences revealed the existence of three different populations of Mycoplasma spp. (9.01 per cent) circulating in the blood of infected cat viz., Mycoplasma haemofelis in two cats, Candidatus M. haemominutum in seven cats and Candidatus M. turicensis in one cat. The phylogenetic analysis of feline 85 Mycoplasma spp. isolates from Kerala using 16S rRNA gene revealed two sequences of Mycoplasma haemofelis clustered with M. haemofelis isolates from South Africa, USA, UK and Australia, seven sequences of Candidatus M. haemominutum belonged to the same clade with Candidatus M. haemominutum isolates of other countries and one sequence of Candidatus M. turicensis grouping in one clade with Candidatus M. turicensis isolates from other countries. Thus, the present study established the prevalence of H. felis, C. felis and three population of Mycoplasma spp. viz., Mycoplasma haemofelis (1.80 per cent), Candidatus M. haemominutum (6.31 per cent) and Candidatus M. turicensis (0.90 per cent) in domestic cats of Kerala.ThesisItem Open Access MORPHOLOGICAL AND MOLECULAR STUDIES ON THE INTRAMOLLUSCAN STAGES OF AMPHISTOMES AND SCHISTOSOMES(College of Veterinary and animal Science,Mannuthy, 2019) ANBARASU K.; Asha RajagopalThe study was undertaken in central Kerala region during the period from June 2018 to May 2019 with the objectives of determining the occurrence of snails in different habitats and seasons and detecting amphistome and schistosome infection in the predominant snail species by morphological and molecular techniques. A total of 1037 snails were collected from three habitats viz., uncultivated paddy fields, permanent water bodies and catchment areas of dams and screened for presence of trematode infection during monsoon, post-monsoon and pre-monsoon seasons. Five species of snails were identified morphologically viz., Indoplanorbis exustus, Lymnaea luteola, Melanoides tuberculatus, Pila globosa and Bellamya spp. Indoplanorbis exustus was found to be the most predominant snail species with a prevalence rate of 45.41 per cent. The prevalence rates of L. luteola, M. tuberculatus, P. globosa and Bellamyia spp. were 22.85, 15.62, 10.70 and 5.40 per cent, respectively. Highest prevalence of snails was recorded during monsoon (37 %), followed by post-monsoon (35.5 %) and pre-monsoon (27.5 %). Statistically significant association was found between the prevalence of different species of snails and seasons with I. exustus occurring more in monsoon and L. luteola more during pre-monsoon. Habitat-wise analysis of prevalence of snails revealed I. exustus and L. luteola to be predominant in permanent water bodies and uncultivated paddy fields where as M. tuberculatus showed significantly high prevalence in catchment areas of dams. Overall prevalence of trematode infection in snails was found to be 13.4 per cent. Five types of trematode cercariae were identified viz., amphistome, mamamalian schistosome, strigeid, avian schistosome and echinostome cercariae. Highest prevalence was recorded for echinostome (36 %) followed by avian schistosome (26.6 %), strigeid and amphistome (15.8 % each) while mammalian schistosome was the least prevalent (5.8%). Amphistome, avian schistosome and strigea showed higher prevalence in monsoon while echinostome infection was more prevalent in pre-monsoon and mammalian schistosome in post-monsoon. Trematode infection in snails was significantly higher in uncultivated paddy (42.4 %) followed by permanent water bodies (36 %) and catchment areas of dams (21.6 %). Highest prevalence of amphistome infection in snails was observed in permanent water bodies. Uncultivated paddy fields and catchment areas of dams showed higher prevalence of infection with trematodes of birds compared with that of animals. Molecular identification of I. exustus was done by PCR targeting 229 bp region of 16S rRNA gene. Molecular detection of amphistome and schistosome infection in snails was done by primers targeting mitochondrial sequences. Multiplex PCR was standardised for simultaneous detection of amphistome and schistosome and I. exustus DNA.ThesisItem Open Access MORPHOLOGICAL STUDIES ON THE TICK FAUNA OF GOATS AND DETECTION OF ACARICIDE RESISTANCE(College of Veterinary and animal Science,Mannuthy, 2019) TAMBE KAJAL ANNASAHEB; Bindu LakshmananA comprehensive study of the morphology of different species of ticks under light and electron microscopy and detection of acaricide resistance status among ticks infesting goats in Kerala was done. A total of 1200 ticks were collected from goats that were reared in 13 organised and unorganised farms of Thrissur and Palakkad districts of Kerala as well as from those presented to University Veterinary Hospital at Mannuthy and Kokkalai. It was observed that ear pinna was the most preferred site for tick attachment followed by tail, head, eyes, neck region. Gross morphological features were observed by light microscopy and four tick species under the genus Haemaphysalis and two species under the genus Rhipicephalus were identified. Out of the 1200 ticks examined, 1190 ticks (99.1 per cent) belonged to Haemaphysalis genus and 10 ticks (0.83 per cent) to Rhipicephalus genus. Among the genus Haemaphysalis, the most prevalent species on goats was H. bispinosa (63.02 per cent), followed by H. intermedia (36.55 per cent), H. megalaimae (0.25 per cent) and H. kutchensis (0.16 per cent). Among the genus Rhipicephalus, R. haemaphysaloides (50 per cent) and R. sanguineus (50 per cent) were identified. The present study reports H. megalaimae and H. kutchensis for the first time in Kerala. Ultrastructural morphology of external surface of Haemaphysalis spp. was studied by scanning electron microscopy. Larval packet test was used to detect acaricide resistance for deltamethrin and amitraz against Haemaphysalis spp. the most prevalent tick infesting goats. In the present study, Ottapilavu, Mundupalam and Poomala isolates were found susceptible, while Vadakkanchery isolate was resistant. All the tested population viz., Mundupalam, Poomala, Vadakkanchery and Choondal isolates of Haemaphysalis spp. were susceptible to amitraz. Log probit analysis was done to derive the LC50 and LC90 of resistant and susceptible isolates. Adult immersion test was performed to assess the status of resistance to deltamethrin and amitraz in Haemaphysalis spp. isolates from Thrissur and Palakkad districts. The per cent mortality and reproductive indices were measured at different concentrations as well as at discriminating dose. In the present study, Ottapilavu isolates of Haemaphysalis survived upto 240 ppm deltamethrin and was considered resistant. The per cent mortality in Amaloor isolates was 90 per cent at 120 ppm and hence was considered resistant. The predicted mortality at 75 ppm for Vennur isolates was 91 per cent and was considered susceptible. Log probit analysis was done to derive the LC50 and LC90 of resistant and susceptible isolates. It was observed that as the concentration of deltamethrin increased, the reproductive index decreased and the oviposition was inhibited. At 240 ppm there was only approximately 55 per cent inhibition of oviposition in resistant Ottapilavu isolates, while in Amaloor isolates 100 per cent inhibition was recorded. All the isolates in the present study were susceptible to amitraz. Haemaphysalis spp. on goats are developing resistance to deltamethrin. Amitraz may be recommended for tick control in goats in areas where deltamethrin resistance has been observed. Future investigations are to be conducted on molecular characterisation of resistance in these species of ticks as well as to detect the status of resistance to different group of acaricides throughout the state.
