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ThesisItem Open Access Epidemiology and molecular identification of trematode parasites of duck with special reference to echinostome(College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-03) Borah, Nanswita; Phukan, Sulekha ChoudhuryA survey was conducted in five different districts of Assam for a period of one year from September‟ 2020 to August‟2021 to investigate on the epidemiology and molecular identification of trematode parasites of duck along with the pathological changes caused by the latter and furthermore, identification of the snail harboring the echinostome cercariae and molecular identification of echinostome cercariae. Out of 1411 fresh faecal samples examined, 411 samples were found positive for helminths with 29.13 per cent prevalence. Infectivity percentage was highest in Kamrup (rural) district and lowest in Udalguri district. Trematodes evinced highest prevalence of 18.00 per cent followed by nematodes with prevalence of 17.51 per cent and finally cestodes with lowest prevalence of 2.62 per cent. Study revealed eggs of Notocotylus sp., Prosthogonimus sp., Echinostoma sp., segments of cestodes, larvae of Strongyloides avium, porrocaecum sp., Tetrameres sp., Heterakis sp. in faecal samples of infected ducks. 303 faecal samples were positive for single infection with prevalence of 21.47 per cent and 108 samples were positive for mixed infection with prevalence of 7.65 per cent. The seasonal prevalence of trematodes on faecal examination of ducks from different districts of Assam depicted highest in monsoon with 28.93 per cent and lowest in winter with 7.82 per cent. The prevalence of trematodes on faecal examination of duck raised under free-range was higher (19.91%) than semi-intensive system of rearing (16.10%). Out of 2786 ducks examined on post-mortem, 2092 ducks were found positive for helminths with 75.09 per cent prevalence. Infectivity percentage was highest in Kamrup (rural) district and lowest in Udalguri district, respectively. Cestodes showed overall highest prevalence of 50.90 per cent followed by trematodes with prevalence of 44.29 per cent and finally nematodes with lowest prevalence of 17.26 per cent. 1146 ducks with an overall prevalence of 41.13 per cent were positive for single infection and 946 ducks with prevalence of 33.96 per cent for mixed infection. On the basis of morphological studies, 9 different species of trematodes, 7 different species of cestodes and 4 different species of nematodes were identified and recorded. Trematodes were identified as Echinostoma revolutum (7.11%), Echinostoma paraulum (18.23%), Echinostoma friedi (0.29%), Hypoderaeum conoideum (18.52%), Psilochasmus oxyurus (0.04%), Tracheophilus cymbius (0.04%), Prosthogonimus ovatus (0.04%), Notocotylus attenuates (0.04%). Cestodes were identified as Hymenolepis collaris (14.28%), Hymenolepis lanceolata (11.59%), Fimbriaria fasciolaris (7.93%), Raillietina tetragona (4.74%), Raillietina echinobothridia (5.03%), Raillietina cesticillus (3.30%), and Amoebotaenia cuneata (4.02%). Nematodes were identified as Porrocaecum sp. (0.68%), Heterakis gallinarum (5.53%), Heterakis dispar (4.02%), and Tetrameres sp (7.04%). Notocotylus attenuates and Amoebotaenia cuneata was first time reported from Assam in duck. The prevalence of trematodes on post-mortem examination of ducks on the basis of sex depicted higher prevalence in females (46.00%) than males (42.62%). The prevalence of trematodes in post-mortem examination of ducks on the basis of age showed higher in adults (48.93%) than young ducks (38.95%). The prevalence of trematodes on post-mortem examination of ducks on the basis of breed showed highest in Pati ducks (49.24%), followed by Khaki Campbell ducks (45.76%), Chara-chambelli ducks (42.14%) and lowest in White pekin ducks (39.16%). Seasonal prevalence of trematodes on post-mortem examination of ducks depicted highest in monsoon with 49.62 per cent and lowest in winter with 38.25 per cent. Prevalence of trematodes on ii post-mortem examination of ducks on the basis of rearing duck showed higher in free-range (47.44%) than semi-intensive system of rearing (40.91%). For molecular identification of trematodes namely, Echinostoma revolutum, Echinostoma paraulum, Hypoderaeum conoideum and Echinostoma friedi (morpholocigally identified) were subjected to PCR (COX -1 gene of Echinostoma revolutum, Echinostoma paraulum, Hypoderaeum conoideum and ITS2 gene of Echinostoma friedi). The trematode, Echinostoma revolutum collected from different districts on the basis of COX-1 sequence identity showed 97.80-99.63% identity with Echinostoma revolutum, Echinostoma paraulum collected from different districts on the basis of COX-1 sequence identity showed 99.07-99.54% identity with Echinostoma robustum, Hypoderaeum conoideum collected from different districts on the basis of COX-1 sequence identity showed 99.08%-99.74% identity with Hypoderaeum conoideum. Echinostoma friedi on the basis of ITS2 sequence identity from Kamrup (rural) and Kamrup metropolitan districts showed 100% identity with Echinostoma friedi. DNA extracted from adult trematode parasites yielded positive results for COX-I genes of E. revolutum, E. paraulum and Hypoderaeum conoideum by PCR analysis, yielded the amplicons size of 818 bp, 216 bp, and 765 bp, respectively and 1000bp for E. friedi for ITS2 gene. On post-mortem examination, intestine of ducks infected with the species under the genus Echinostoma and Hypoderaeum showed slight gross lesions except catarrhal enteritis and congestion in the mucosal surface, mainly in the jejunum, ileum and caecum. Psilochasmus oxyurus showed catarrhal enteritis and congestion in the mucosal surface of the small intestine. Trachea infected with Tracheophilus cymbius exhibited bloody mucosal exudates in the site of attachment. No observable gross pathological lesions could be seen in oviduct and caecum affected with Prosthogonimus ovatus and Notocotylus attenuates, respectively. This may be due to low level of infection encountered in the present investigation. Microscopic changes associated with the species under the genus Echinostoma, Hypoderaeum, and Psilochasmus showed marked thickening with infiltration of inflammatory cells including lymphocytes, eosinophil, plasma cells and macrophages in the intestine. Some of the villi were degenerated and necrosed. Desquamated epithelial lining of intestinal villi were found to be deposited in the lumen of the intestine. Haemorrhagic patches could be seen in the tip of some villi. Hypertrophy of goblet cells was seen in the villi. The glandular epithelium showed vasculation and distortion which have further undergone necrosis. Most of the lymphoid follicles in ileum were found to be enlarged and degeneration of lymphoid cells was also observed. Muscle cells showed necrosis and hyalinization. Blood vessels showed congestion and dilation along with haemorrhages in subserosal layer of intestine. Histopathology, of trachea infected with Tracheophilus cymbius showed heavy infiltration of leucocytes in the mucosa and degeneration with necrosis in submucosal layer. Snail, Indoplanorbis exustus (0.55%) and Lymnaea luteola f. ovalis (0.37%) were recorded as the intermediate host of echinostome cercariae. No cercarial infection was recorded from Lymnaea acuminata f. rufescens, and Bellamya bengalensis f. typica. District-wise Monthly prevalence of echinostome cercaria in Indoplanorbis exustus depicted maximum shedding from the month of June to September from the different districts of Assam. District-wise Monthly prevalence of echinostome cercaria in iii Lymnaea luteola f. ovalis depicted maximum shedding from the month of June to October from the different districts of Assam. Morphological identified echinostome cercariae (4 representative samples) collected from different districts of Assam were subjected for molecular identification where ITS2 and COX -1 gene of the parasite was subjected to PCR. Cercariae of Hypoderaeum conoideum and Echinostoma revolutum (molecularly identified) are commonly prevalent in the snails of Assam.
