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20191
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Subject: Veterinary Parasitology × Author: GNANI CHARITHA, V × Thesis Type: Ph.D ×
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    ThesisItemOpen 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, A
    The 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.