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Subject: Veterinary Parasitology × Author: PATHAK, PALLABI × End date: 2016 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    EPIDEMIOLOGY OF LEECH INFESTATION AND EVALUATION OF ANTI LEECH EFFECTS OF SOME LOCALLY AVAILABLE PLANTS
    (Assam Agricultural University, Khanapara, Guwahati, 2016-07) PATHAK, PALLABI; Islam, Saidul
    An epidemiological study was conducted to record the prevalence of aquatic and terrestrial leech species in fifteen districts of Assam. Out of 1991 number leeches collected five aquatic e.g., Hirudinaria manillensis (57.86%), H. granulosa (11.85%), H. javanica (2.71%), Whitmania laevis (2.61%) and Hemiclepsis marginata (0.80%) and one terrestrial leech e.g., Haemadipsa sylvestris (24.16%) was found prevalent in the study area. Molecular identification of H. manillensis based on coI sequence was done. Phylogenetic analysis showed 95.5% sequence similarity with P. manillensis (Accession No: KT693108.1) and lowest with H. manillensis (GQ368747.1). The sequence was submitted into the GenBank, and assigned the Accession No. GenBank KX579976. Bionomics of H. sylvestris was studied in laboratory conditions. The incidence of leech infestation in man and animal in different leech infested areas was conducted using interview method by means of questionnaire. Total protein concentration of crude antigen of H. granulosa, H. sylvestris and H. manillensis was found to be 4.14g/dl, 4.05g/dl and 3.6g/dl. Total protein concentration of E/S antigen of H. manillensis, H. granulosa and H. sylvestris was 3.39g/dl, 3.44g/dl and 3.4g/dl, respectively. Discontinuous gel electrophoresis revealed protein profile of crude antigen of H. manillensis, H. granulosa and H. sylvestris being almost same. The number and size of peptides varied from 9-10 and 12.5 to 96 kDa, respectively. The number of peptides of E/S antigen for H. manillensis, H. granulosa and H. sylvestris were 5 and size varied from 14-96 kDa. PAS staining of glycoprotein antigen could not be detected for E/S product for all three species. Two glycoprotein antigens could be detected in crude H. manillensis and H. granulosa and three in H. sylvestris which ranged from12.5-26 kDa. A total of two immune-reactive peptides were detected for H. manillensis and H. granulosa crude antigens and one in E/S product of H. sylvestris. Hirudinicidal effect of aqueous and methanolic extracts of leaves of Nicotiana sp., seeds of Camellia sinensis and stems of Carica papaya was evaluated against H. manillensis and compared with ivermectin and nicotine. The methanolic extract of Nicotiana sp. and C. sinensis; aqueous extract of Nicotiana sp. and C. sinensis possessed effective hirudinicidal properties. While the concentration and extraction procedure was considered, the aqueous extract of Nicotiana sp. at 5000 mg/ml and 2500 mg/ml showed faster killing effect. While the method of extractions and the level of concentrations were considered against time of repellency, it was found that barring the methanolic extract of C. papaya at the concentrations of 50 mg/ml and 100 mg/ml, all the other extracts at different concentrations showed highly significant (p<0.001) leech repellent effects. Expression of HSP was evaluated by Dot-ELISA in stressed organism using a panel of monoclonal antibodies which comprised of HSP-60, HSP-70 and HSP-90. After application of pure compounds (nicotine and ivermectin) and extracted products (methanolic and aqueous) of Nicotiana sp., C. sinensis and C. papaya, HSP-60, HSP-70 and HSP-90 were detected in excretory and secretory (E/S) product of leeches. HSP-60, HSP-70 and HSP-90 were also detected both in crude and excretory and secretory (E/S) products of the cold stressed leeches. Extracts of Nicotiana sp. and C. sinensis were subjected to in vitro cytotoxicity studies on Vero cell lines using MTT assay. Two fold dilution of the extracts were used from 10.00 µg/µl up to 0.0096 µg/µl. Both the aqueous and methanolic extracts of Nicotiana sp. and metahnolic extract of C. sinensis had more per cent viability of Vero cells than the aqueous extract of C. sinensis. In no case the per cent viability was below 70. The efficacy of extracts against H. sylvestris and aquatic leech was evaluated on human volunteers. The methanolic extract of C. sinenesis failed to exhibit minimum 50% leech repellent effect. Aqueous extract of C. sinenesis exhibited inconsistent effect with 45.83% repellent effect at 20% concentration, but 26.19% effect at 30% concentration. Both the methanolic and aqueous extract of Nicotiana sp. exhibited 100% repellent effect at all the three levels of concentrations which was comparable with DEET (12% concentration).