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20162
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Subject: Veterinary Parasitology × End date: 2017 × Start date: 2015 × 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).
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    ThesisItemOpen Access
    STUDIES ON Indoplanorbis exustus AND ITS ASSOCIATED SCHISTOSOMES
    (Assam Agricultural University, Khanapara, Guwahati, 2016-07) Bulbul, Kamal Hashan; Das, Manoranjan
    A study on ecology, biology and bionomics of Indoplanorbis exustus along with its associated schistosomes was undertaken for a period of two years i.e. from March 2014 to February 2016 in Barpeta, Nalbari and Kamrup district of Assam. A total of 12 aquatic macrophyte species were recorded from the study area. The highest frequency percent (f %) relative frequency percent (RF %) of different macrophytes was found to be 86.67% and 19.70% respectively. Population density of I. exustus in terms of f% and RF% and man hour collection per meter square area (MHC/m2) showed an increasing trend from July to September and then gradually declining towards November. While the snails were reared in biologically balanced aquarium at constant temperature of 15, 20, 25, 30 and 35C and room temperature (20.02-31.75C), all the snails died before attainment of sexual maturity at 15oC. The growth rate in terms of shell diameter and body weight attained maximum size 10.63+0.162 mm and 435.83+23.367 mg, respectively at room temperature at 6th week of age. Egg to egg cycle ranged from 50 days at 30C and 125 days at 20C. Room temperature was found to be more conducive for fecundity wherein as many as 22833 numbers of eggs were hatched out from 1033numbers of egg capsules. The temperature, pH, DO, free CO2 and total alkalinity of ambient water had direct bearing on population density of snails. The ANOVA of physicochemical properties of water was highly significant (p<0.01) between months and areas of water bodies. Out of 161500 numbers of snails collected in two years showed incidence rate of cercariae of Schistosoma spindale (0.72%), S. indicum (0.50%) and S. nasale (0.17%). The cercariae were identified on the basis of morphological and molecular method. The RS data indicated that the distribution of cercariae of S. indicum group was more in the northern bank of Brahmaputra compared to the Southern bank. By means of molecular dissection, brevifurcate cercaria was identified as Trichobilharzia sp. and the longifurcate as Alaria alata with 87-88% clonal relationship with Denmark, Lithuania and Germany isolates. A total of 420 cattle, 179 buffaloes and 171 goats slaughtered at local abattoirs revealed visceral schistosomosis in 37.38, 35.20 and 12.28%, respectively. In regards to coprological examination it was 14.52, 12.85 and 4.68% in order of same sequence as above. The incidence rate in terms of adult parasites present and faecal examination was the highest in monsoon season and the lowest in pre-monsoon season. Concentration of eggs was more in liver (45.45%) followed by ileocaecal junction (37.50%), caecum (25.97%) and small-intestine (18.18%). Based on worm count methods, mild types of intensity was found to be highest followed by moderate and heavy intensity in slaughtered animals. The incidence of nasal schistosomosis was highest in July (18.92%) and lowest in December (2.70%) when nasal swab was examined for the presence of eggs in 363 cattle irrespective of sex and age. Like visceral schistosomosis, nasal schistosomosis also had a higher infection rate in monsoon season and in animals above 8 years of age. The second internal transcribed spacer (ITS2) sequence of S. spindale, S. indicum and S. nasale were found to be amplified showing different repeatitive band patterns. While the mitochondrial cytochrome c oxidase subunit 1 (CO1) genes and the ribosomal gene repeat, part of the 28S RNA gene (28S) were amplified on 372 bp and 1225 bp when subjected to PCR. Phylogenetic analysis of Schistosoma indicum group based on nucleotide sequences of COI and 28S genes revealed Assam isolates of S. indicum showed a clonal relationship with Bangladesh isolates and S. spindale and S. nasale with Nepal isolates. RAPD-fingerprinting using different random primers showed specific polymorphic markers for susceptible and non-susceptible I. exustus to Schistosoma infection due to genetic variability.