Loading...
Theses
Browse
4 results
Search Results
ThesisItem Open Access STUDIES ON Dirofilaria immitis IN DOGS AND ITS ASSOCIATION WITH Wolbachia SPECIES(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781022, 2013-06) Borthakur, Sonjoy Kumar; Deka, Dilip Kr.Dirofilaira immitis is an important canine filarial nematode. An epidemiological study was carried out to record the prevalence of D. immitis in dogs in two different geographical locations viz., Guwahati, Assam and Aizawl, Mizoram of North Eastern Region in India, from February, 2011 to July, 2012. The study also included to evaluate the persistence of Wolbachia endosymbiont with D. immitis. In the present study, dogs were grouped into three categories, i.e., stray, pet and working dogs, their respective numbers being 413, 266 and 103 irrespective of the study regions. Three different methods were used for the study, i.e., microscopy (wet film and KCT), immunological (Ag ELISA by SNAP®4Dx kit) and molecular techniques (PCR). The study revealed overall heartworm prevalence in Guwahati to be higher (18.23%) than in Aizawl (17.68%) irrespective of categories of dogs. Sex-wise, the infection was higher in male (18.12%) than in female (17.90%), though the difference was statistically non-significant. The overall efficacy percentage for detection of heartworm by wet film, KCT, Ag ELISA and PCR test revealed 6.26, 11.38, 18.03 and 13.93 percent, respectively. Ag ELISA test was found to be the best amongst the three types of tests compared. Using molecular tools, prevalence of D. immitis in dogs was 13.52 percent in Guwahati and in Aizawl was 14.62 percent. With PCR, 4 cases of D. repens could be diagnosed in stray dogs from Guwahati. The study revealed overall 22.69 percent occult infection, of which, highest cases were recorded in working dogs (60%). Occult infection was calculated by finding the difference between heartworm prevalence based on Ag ELISA and PCR test. Dot ELISA test using monoclonal antibody of D. immitis for detection of heartworm antigen in dog blood samples was standardized. The test revealed 72% specificity against known positive D. immitis blood samples at SNAP®4Dx commercial kit. Molecular technique using PCR was standardized to detect D. immitis using published primers with slight modification of thermal condition. Two different primers were used viz., specific primers for D. immitis only and another, pan filarial primers for detecting six different canine filariids. Both the primers resulted desired amplification product size against different filarial parasites. Molecular cloning and characterization of D. immitis for ITS-2 region of Guwahati isolates were conducted. The results showed the Guwahati isolates had a close relationship with that of South Asian isolates of D. immitis. Pair-wise homology analysis revealed 98.6 - 98.9% similarity with a few sequences available at NCBI GenBank. Similarly, phylogenetic analysis of D. repens encountered in Guwahati isolate was also done. Endosymbiont Wolbachia association with D. immitis worm as well as in heartworm infected blood was revalidated by PCR method. The findings were substantiated with the presence of the organisms in the worm’s lateral cord by fine structural studies conducted through transmission electron microscopy (TEM). Molecular evidence followed by sequence analysis of Wolbachia revealed 99.4 to 99.8% similarities with other sequences available in NCBI GenBank for Wolbachia endosymbiont of D. immitis. Finding of the present studies establish the endemicity of D. immitis in North East India and validates the association of Wolbachia endosymbiont in D. immitis. Record of D. repens warrants further detail studies owing to its zoonotic significance.ThesisItem Open 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, SaidulAn 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).ThesisItem Open Access STUDIES ON Indoplanorbis exustus AND ITS ASSOCIATED SCHISTOSOMES(Assam Agricultural University, Khanapara, Guwahati, 2016-07) Bulbul, Kamal Hashan; Das, ManoranjanA 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 35C and room temperature (20.02-31.75C), 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 30C and 125 days at 20C. 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.ThesisItem Open Access STUDIES ON HAEMOPARASITIC INFECTIONS OF DOG WITH SPECIAL REFERENCE TO Dirofilaria immitis(Assam Agricultural University, Khanapara, Guwahati, 2011-08) Bhattacharjee, Kanta; Sarmah, P.C.The present research “Studies on haemoparasitic infections of dog with special reference to Dirofilaria immitis” was undertaken with a broad aim to assess by various methods the prevalence of haemoparasites in Assam of North East India, their clinical and clinicopathological significance and antigenic characterization of D. immitis. Microscopic study of Giemsa stained blood smears performed prospectively between January 2009 and December 2010 revealed a climate suited and highly enzootic situation with vector borne haemoparasites viz. Babesia gibsoni (47.16%), Ehrlichia platys (8.49%), Dirofilaria immitis (2.83%), Ehrlichia canis (2.12%), Babesia canis (1.41%), Hepatozoon canis (1.41%) and Ehrlichia ewingii (0.47%) in single or mixed infections. The prevalence was 57.31% in the hospital population comprising pet (58.03%) and working (54.54%) dogs and 63.64% in stray dog population in the prospective study against 23.19% record in the retrospective study. Clinical illness due to haemoparasites in the hospital dogs was recorded throughout the year. Variations in the infection rate due to age, breed and sex of dogs and seasons of the year were statistically non significant. Infection with Ehrlichia and Babesia were detected in dogs of all ages while in the case of D. immitis 80% positive cases were from 2-7 years age and 20% in the 8-10 years age group. Dogs below 2 years and above 10 years age were found microfilaria negative. Microscopic identity of Babesia species was confirmed in polymerase chain reaction (PCR), Indirect fluorescent antibody test (IFAT) using commercial B. canis IFA IgG antibody and B. gibsoni IFA IgG antibody Kits (Fuller Laboratories, USA) supplemented with conventional animal experimentation (sub-inoculation and splenectomy). PCR detection of B. canis and B. gibsoni in 16.36% and 3.63% respectively as single infection and 49.09% as mixed infection in tested samples was a significant finding which claimed 5.45% and 58.18% false negativity in B. gibsoni and B. canis detection respectively by microscopic method. In view of usual mixed infection found in PCR analysis detection of B. gibsoni in blood smear during field diagnosis could be considered as the biomarker of B. canis for the treatment purpose since the drug regimen against the two species are different. ELISA based commercial diagnostic kit, SNAP 3Dx (IDEXX, USA) confirming the microscopic identity of E. canis detected antibody in 32% dogs in a small group within the hospital population which was higher than the microscopic detection (2.12%). Among the hospital group the infection was found higher in working dogs than in the pet dogs. Seventy five percent E. canis seropositive dogs had E. platys inclusions as mixed infection suggesting the latter’s presence as biomarker for the former also. Haemoparasitic infection was confirmed in 57.31% cases through the assessment of parasite specific and non specific clinical symptoms. Regenerative anaemia characterized by biphagic erythrocyte sedimentation rate (ESR), appearance of nucleated erythrocyte, polychromasia, howell jolly body, thrombocytopenia with presence of giant platelets, variable leucogram usually with neutrophilia in blood smears were the prominent haematological findings in clinical babesiosis. In Ehrlichia, Hepatozoon and Dirofilaria infected clinical cases the anaemia was of non-regenerative type. Variable neutropenic leucopenia, thrombocytopenia with appearance of giant platelets, total absence or rare presence of howell jolly body, nucleated RBC and polychromasia in blood smear were the additional features specific to E. canis infection. However in mixed infection with Babesia and Ehrlichia features of regenerative anaemia were predominant. Biochemical evaluation showed wide variation leading to low albumin, elevated levels of total protein, globulin, creatinine and bilirubin. Dogs died of experimental and spontaneous babesiosis with nervine symptoms showed severe congestion, perivascular oedema with microabscess formation, neuronal degeneration in the brain tissue during histopathological examination. Enlarged heart with thickening of myocardium, right ventricular dilatation, thrombus formation in pulmonary artery and extensive areas of mild to moderate fatty changes were the pathological changes observed in heartworm positive carcasses. Antigenic analysis conducted in crude somatic male (SMA) and somatic female antigen (SFA) showed 9 and 17 clear protein bands in 12% SDS-PAGE of which 14, 20, 28 and 59 kDa protein fractions were common in both the antigens. Hyperimmune rabbit sera reacted with 3 protein bands in SMA and 5 protein bands present in SFA. Antibody ELISA performed in sera samples from hospital and stray dogs revealed 9.37% positivity at 1:50 dilution, 6.25% at 1:100 and 5.20% in 1:200 dilution. Necessity of a cost effective sensitive test to detect D. immitis during the amicrofilaraemic prepatent period was discussed.