Loading...
Thesis
Browse
4 results
Search Results
ThesisItem Open Access DETECTION AND MOLECULAR CHARACTERIZATION OF SOIL TRANSMITTED HELMINTHS OF ZOONOTIC SIGNIFICANCE IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-12) GNANI CHARITHA, V; CHENGALVA RAYULU, V (MAJOR); MALA KONDAIAH, P; ASWANI KUMAR, K; JAGADEESH BABU, AThe present study was carried out to determine the prevalence of soil transmitted helminths of zoonotic significance among canine faecal and soil samples of Andhra Pradesh (A.P) along with molecular characterization of Toxocara and Ancylostoma spp. The overall prevalence of gastrointestinal (GI) parasites was 83.5 percent (n=745) out of 892 canine faecal samples screened by conventional microscopy. Significantly (P ≤ 0.01) higher prevalence of GI parasites was recorded in Costal Andhra (87.1%) than Rayalseema (75.2%) region of A.P. Eleven parasitic species including five nematodes, four cestodes and two protozoans were isolated from the faecal samples. The most ubiquitous parasites were Ancylostoma spp. (33.5%) and Toxocara canis (20.7%) followed by Strogyloides stercoralis (5.04%), Cystoisospora spp. (3.69%), Toxascaris leonina (3.2%), Trichuris vulpis (2.9%), Taenia spp. (2.5%), Dipylidium caninum (2.1%), Entamoeba spp. (1.23%), Diphyllobothrium latum (0.45%) and Spirometra spp. (0.34%). Concurrent mixed infection was recorded in 7.7% of dogs. Dog associated risk factors such as sex, age, breed and domestication along with effect of urbanization and seasonal influences on the prevalence of parasites were analyzed. The overall prevalence of soil transmitted parasites was 43.5 percent out of 390 soil samples screened. High contamination index of Toxocara spp. (18.5%) followed by hookworms (12.3%) was recorded. Rural soils (48.7%) were found more contaminated than urban areas (44.0%). More number of soil samples were positive for different parasitic stages collected from the garden soils (75.5%) followed by play/school grounds (65.2%), animal dwelling areas (37.3%), parks (34.0%) and veterinary dispensaries (24.0%). The prevalence of Ancylostoma spp. and Isospora spp. were positively correlated (P <0.05) with rainfall while Toxocara and Toxascaris spp. were independent of temperature influence. Responses retrieved from the questionnaire survey revealed that more than half of the pet owners (63.7%) were unaware of zoonotic soil transmitted helminths. Genomic DNA (gDNA) of 151 faecal samples were screened for Toxocara spp. using PCR-RFLP targeting ITS-2 gene. Upon genotyping of PCR amplicons (n=50) with RsaI enzyme, all the canine faecal isolates were identified as T. Canis (287 bp and 244bp). Amplification of gDNA from soil samples (n=72) yielded different product sizes viz., ~540bp (ITS-2) and ~500bp (ITS-2). Genotyping of randomly selected ~540bp product (n=32) from soil isolates with RsaI enzyme confirmed T. cati (37.5%) with three fragments (286bp, 150bp and 103bp) and T. canis (62.5%) with two fragments (287bp and 244bp). Sequencing and phylogentic analysis of ITS-2 products (~540bp) revealed 100% homozygous within the species and with the other geographical isolates of T. canis and T. cati. However, the other amplified product of ~500bp (n=21) with RsaI enzyme yielded two fragments (263bp and 219bp) and the sequencing result revealed 81.8 percent identity with T. cati. The phylogenetic analysis of ITS-2 sequences of 500bp products of A.P constituted a different sub branch and diverging from T. cati, T. canis, T. malayseinsis and T. vitulorum thus categorizing as a separate group or variant. Randomly selected 169 faecal and 48 soil samples from A.P were screened for detection of Ancylostoma spp. using a two step semi-nested PCR targeting ITS-1, ITS-2 and 5.8S gene. On gel electrophoresis, 132 (78.10%) samples showed primary PCR band at ~450bp size while secondary amplicons were observed at ~410bp size. Twenty one out of 48 soil PCR products were found positive in first round PCR and as well as in semi-nested PCR. Genotyping of semi-nested PCR products with BfaI and AhdI restriction enzymes revealed A. caninum (43/50) and A. ceylanicum (7/50) from faecal and only A. caninum (21/21) from soil isolates. The phylogeny of A.P isolates of A. caninum and A. ceylanicum revealed 100% homology within the species and with the other geographical isolates. Taken together, present data suggest the potential role of pet/stary dogs as being the main sources of contamination and signifies the need of integrated approaches to minimize the risk at different settings.ThesisItem Open Access GASTROINTESTINAL PARASITISM IN SHEEP AND GOATS IN CERTAIN DISTRICTS OF HYDERABAD-KARNATAK REGION KARNATAKA STATE WITH SPECIAL REFERENCE TO ANTHELMINTIC RESISTANCE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-10) ADEPPA, J; SREEDEVI, C(MAJOR); Placid E., D’Souza; Ravi Kumar, P; SrinivasaRao, TThe present study was carried out to determine the prevalence of GI parasites and efficacy of commonly used anthelmintics in sheep and goats in Hyderabad-Karnataka region, Karnataka. Faecal samples were collected from sheep (1876) and goats (1745) in H-K region (Bidar, Kalaburgi and Raichur districts), Karnataka for a period of one year to determine the prevalence of gastrointestinal parasitism. Processing of faecal samples by standard faecal floatation and sedimentation techniques revealed an overall prevalence of 65.78 per cent. The rate of prevalence was significantly higher (P<0.05) in goats (67.9%) when compared to that in sheep (63.8%). The rate of prevalence in Bidar, Kalaburgi and Raichur districts was 68.76, 61.8 and 62.28 per cent respectively, without significant (P>0.05) differences in prevalence of GI parasitic infection between districts. The infected small ruminants were parasitized with one or two of eight different species and genera of parasites, each. Out of 3621 faecal samples examined, infection with single parasite (43.94%) was more commonly observed than multiple species infection (21.84%). The most prevalent species were strongyles (27.64%) followed by Eimeria spp. (7.91%), Strongyloides papillosus (3.87%), Trichuris ovis (2.54%), Moniezia spp. (1.02%), amphistomes (0.8%), Buxtonella sulcata (0.11%) and Schistosoma spindale (0.06%). Coproculture studies indicated predominance of Haemonchus contortus larvae. Statistically there was no significant (P>0.05) difference between prevalence of GI parasitic infection and age of animals. The overall prevalence was high in post-weaning lambs and kids (69.61%) than in pre-weaning lambs and kids (68.60%) and adults (60.12%). With regard to the gender of animals, the overall prevalence of GI parasitic infection was significantly (P<0.01) high in female animals (67.9%) than in males (62.85%). The overall prevalence was significantly (P<0.001) high in rainy season (69.04) followed by winter (65.63 %) and summer (62.52%). To determine the efficacy of thiabendazole and ivermectin against H. contortus of sheep and goats in H-K region, egg hatch assay (EHA), larval development assay (LDA) and PCR-RFLP were employed. The results of the EHA revealed significant (P<0.05) inhibitory effect on egg hatching rates in sheep (LD50 = 0.12 μg/mL) and goats (LD50 = 0.115 μg/mL) with thiabendazole. LDA studies also showed significant (P<0.05) inhibitory effect on larval development in sheep (LD50 = 0.133 μg/mL) and goats (LD50 = 0.131 μg/mL) with thiabendazole. Similarly the results of the LDA exhibited inhibitory effect on larval development with ivermectin (LD50 = 0.0428 μg/mL; LD50 = 0.0431 μg/mL). Analyzing the overall situation the study highlights that the thiabendazole and ivermectin had extremely high LD50 than discriminating dose in EHA and LDA, reflecting benzimidazoles and ivermectin resistance. A total of 450 (250 from sheep and 200 from goats) adult male H. contortus from different areas in H-K region were genotyped at codon 200 in β-tubulin isotype 1 gene for the detection of BZ resistance by PCR-RFLP. The genotypic frequencies of three genotypes (homozygous resistant ‘rr’, heterozygous ‘rS’ and homozygous susceptible ‘SS’) for BZ resistance in sheep and goats varied significantly (P<0.01) in the studied area. Out of 250 parasites genotyped in sheep, 43 (17.2%) were ‘rr’, 179 (71.6%) were ‘rS’ and 28 (11.2%) were ‘SS’ type. Among 200 parasites genotyped in goats, 38 (19.0%) were ‘rr’, 129 (64.5%) were ‘rS’ and 33 (16.5%) were ‘SS’ type. Overall, the proportion of BZ resistant (‘r’) allele was significantly (P<0.01) high when compared to per cent prevalence of susceptible allele (‘S’) in worms collected from sheep and goats in H-K region. Among different areas of H-K region the frequency of resistant allele was significantly higher (P<0.01) in Sedam and Lingasugur regions in sheep and goats, respectively. Findings of PCR-RFLP corroborated with the results of EHA and LDA.ThesisItem Open Access Molecular characterization of Fasciola species in sheep and goats of Andhra Pradesh, India(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-11) JYOTHI SREE, Ch; MALAKONDAIAH, P(MAJOR); CHENGALVA RAYULU, V; Rama Devi, V; RAMANI PUSHPA, R.NABSTRACT: The present investigation has been undertaken to study the “Molecular characterization of Fasciola species in sheep and goats of Andhra Pradesh, India”. Slaughter/ necropsy examination of 5192 sheep and 2070 goats livers revealed 124 (2.38%) and 29 (1.40%) positive for Fasciola spp. with an overall prevalence rate of 2.10%. Higher prevalence was recorded in female sheep (3.50%) and goats (1.70%) than males of sheep (2.10%) and goats (1.24%), respectively. Highest prevalence was revealed in youngs (2.61% and 1.62%) than in adults (1.86% and 1.25% in 1-3 yrs and 2.11% and 1.22% in more than 3 years age) in sheep and goats, respectively. Highest infection rate was recorded in summer (2.89% & 1.53%) followed by winter (2.35% & 1.51%) and rainy seasons (1.19% & 0.91%) in sheep and goats, respectively. Present investigation also revealed that the prevalence of Fasciola spp. was the highest in April (6.05%) in sheep and in March (4.13%) for goats. Morphometric analysis was displayed large leaf like appearance of flukes with length and width ranged between 27.1 - Name of the author 36.5mm and 4.8 - 6.3mm in sheep and 27.5 - 38.1mm and 4.5 - 7.4mm in goats, respectively. Allometric values of Ventral Sucker to Posterior end (VS-P) and ratio of body length and width of adult flukes were 24.35-29.8mm and 4.71-6.29mm in sheep whereas 24.6-34.8mm and 4.89-6.73 mm in goats, respectively. Allometric values of the flukes collected in the present study were within the standard range (VS-P=26.8- 50.09mm, BL/BW=3.4-6.78mm) of F. gigantica and confirmed that the flukes from different parts of Andhra Pradesh were morphologically F. gigantica. Amplification of 275 genomic DNA samples (175 sheep and 100 goats) with their respective primers yielded a 433bp (ITS 1) and 550bp (ITS 2) products, respectively and confirmed them as Fasciola spp. Upon digestion with Rsa1 and BspHI restriction enzymes, the PCR-RFLP revealed a fragment of 233bp and 200bp in ITS 1 and 377bp and 173bp products in ITS 2 from all isolates of sheep (50) and goats (50), respectively. Sequencing analysis of randomly selected ITS 2 PCR products (3 sheep, 2 goats and 1 buffalo) revealed 100% homozygous within the species and a very few variations with the other geographical isolates. Pair wise distance analysis revealed 0% divergence in between them and 1-25% with the other geographical isolates of F. gigantica whereas 6% divergence with the F. hepatica. The phylogenetic analysis of ITS 2 sequences (6) revealed a close relationship with F. gigantica isolates of Asia (India, Iran, China, Thailand, South Korea, Vietnam, Indonesia, Pakistan) and Africa (Egypt, Zambia, Kenya, Mauritania). While the F. hepatica from Iran, Egypt, Australia and France were also exhibited relationship with the AP isolates and observed as a separate sub branch. The phylogeny of Andhra Pradesh isolates revealed that they were clustered in a same sub branch with regardless of their host, geographical origin and maternally inherited from F. gigantica.ThesisItem Open Access CLONING, EXPRESSION AND CHARACTERIZATION OF PARAFLAGELLAR ROD GENE OF TRYPANOSOMA EVANSI(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-01) SIVAJOTHI, S; CHENGALVA RAYULU, V(MAJOR); MALA KONDAIAH, P; SREENIVASULU, D; SRILATHA, ChABSTRACT : Present study was undertaken with an objective to isolate, clone, express and characterize the paraflagellar rod gene of Trypanosoma evansi. Local isolate of Trypanosoma evansi collected from naturally infected cow was multiplied in Wistar rats. Total RNA was extracted from DEAE-cellulose column chromatography purified trypanosomes by using Trizol LS reagent. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription using oligo dT primers. RT-PCR was standardized to amplify the cDNA by targeting 1800 bp unique for PFR 2 gene of T. evansi. Amplification of cDNA was confirmed on agarose gel electrophoresis. The concentration of PCR amplicon was found to be 40 ng/μl after extracting from the gel. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into pTZ57R/T vector system. Transformation of competent Escherichia coli DH5α cells with ligated PFR 2 gene T. evansi was successfully carried out in LB agar with X-Gal and IPTG. Developed recombinants were observed as white colonies and non-recombinants as blue colonies. Presence of inserts was confirmed initially by Colony-PCR and then by Plasmid-PCR. Nucleotide sequence of the PFR 2 gene of T. evansi S.V.V.U. isolate (Accession No. KT277497) of the present study revealed 100 % homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. Variation in nucleotide mutations at 4 positions with T. evansi Izatnagar and 3 positions with T. evansi Bikaner isolates were observed. The amino acid mutations in the PFR 2 gene of T. evansi S.V.V.U. isolate displayed regularity at 4 positions when compared to T. evansi China, Izatnagar and Bikaner isolates. Tree topology based on the Neighbor-joining (NJ) method of phylogenetic analysis has showed a close homology with other Trypanosomatidae species sequences with 100% bootstrap values. Restriction digestion of insert DNA of PFR 2 gene as well as pET 32a vector was carried out with EcoR I and Hind III enzymes and subjected for ligation by using T4 DNA ligase. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. A high level of expression of recombinant protein of PFR 2 gene of T. evansi was noted following four hours of induction with 1 mM IPTG. Molecular weight of the Ni-NTA column chromatography purified recombinant protein of PFR 2 gene of T. evansi was found to be approximately 90 kDa after resolving by SDS-PAGE. PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE analysis and western blotting against hyper immune serum. Indirect ELISA was optimized for detection of specific antibody titre against recombinant protein of PFR 2 gene of T. evansi. Based on the ELISA result, it is evident that PFR 2 gene products are eliciting very good immune response. However, further study is required to know the protective effect of the antibodies in laboratory animal models and to explore the PFR 2 gene of T. evansi as potential candidate for diagnostic and vaccine target against surra. Findings of the present study confirmed the existence of PFR 2 gene in Indian cattle isolate of T. evansi. Cloning, expression and characterization of PFR 2 gene of T. evansi of cattle isolate carried out in the present investigation is probably the first report in India.