PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF CLOSTRIDIUM PERFRINGENS TYPE D INFECTION IN SHEEP
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Date
2018-06
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA
Abstract
Enterotoxemia in sheep is a fatal disease and is attributable to a toxigenic type of Clostridium perfringens type D associated with epsilon and alpha toxins. Hence, the present study was undertaken to know the incidence, to elucidate lesions in various organs, to carry out bacterial isolation and identification and molecular confirmation of Clostridium perfringens type D by multiplex PCR. In the present study, a total of 927 indigenous sheep of either sex and of different age groups were screened for clinical signs of enterotoxemia and of these, 157 (16.93%) were found to be clinically affected. A detailed necropsy was carried out in 27 dead animals out of the affected animals and the representative samples were collected for further studies. Urine samples were also collected from dead animals to test for glycosuria. Clinically, the affected sheep showed peracute signs with sudden death mostly in young animals. In few acute cases, there was diarrhoea and convulsions. At necropsy, bloated carcass and presence of fluid in the abdominal cavity were noticed on external examination. All the animals revealed predominant gross lesions in intestine, kidney, lung, heart and brain with occasional involvement of other organs. Gas filled, distended intestines were seen grossly that revealed haemorrhages and necrosis of the mucosal epithelium microscopically. Fibrinous pseudomembrane formation and blunting of the villi were the other salient features. Kidneys were swollen and congested that appeared soft, pulpy and had jam like consistency grossly. Microscopic lesions included cloudy swelling and necrosis of the tubular epithelium and haemorrhages. Haemorrhages and necrosis were seen in glomerulus and the interstitium showed infiltration of inflammatory cells and severe vascular congestion. The lungs were edematous and congested grossly and the microscopic lesions were characterized by severe congestion, haemorrhages, alveolar edema, emphysema and thickened alveolar septa. In the heart, straw colored fluid in the pericardial sac and haemorrhages on the epicardium and endocardium were noticed. Microscopic lesions comprised of mild congestion and inflammatory cell infiltration in between the cardiac muscle fibres. In the brain, congestion of meninges, softening and bilaterally symmetric focal areas of hemorrhages were seen grossly. Perivascular edema, neuronophagia, vacuolation were the microscopic lesions seen in cerebrum whereas degeneration of purkinjee cell layer and axonal swelling were noticed in cerebellum. Grossly, liver appeared congested. Microscopically, congestion, cloudy swelling, fatty change and multi focal necrosis were observed. In some cases, hepatic sinusoids were dilated and contained fibrin threads and many bacterial rods. In the present study, intestinal contents collected from necropsied sheep was inoculated in Robertson’s cooked meat medium prepared with BHI broth and thioglycollate broth for bacterial isolation and the culture smears were stained by Gram’s method that revealed Gram positive, short and stumpy rods with blunt ends. Further identification of the bacteria was done by litmus milk test that resulted in the stormy clot fermentation indicating the presence of Clostridium perfringens. The urine samples collected showed glycosuria by Benedict’s test. In the present study, the Clostridium perfringens culture obtained from intestinal contents was used for genomic DNA extraction and the obtained DNA samples were subjected to multiplex PCR for diagnosis of Clostridium perfringens type D that produces alpha and epsilon toxins. Multiplex PCR was carried out in 27 samples by using specific primers for toxin genes viz. alpha toxin gene (cpa) and epsilon toxin gene (etx) for Clostridium perfringens type D and also beta toxin gene (cpb) to differentiate Clostridium perfringens type D from Clostridium perfringens type B. On electrophoretic analysis, an amplicon of 324bp for alpha (cpa) and 655bp for epsilon (etx) were obtained in all the samples confirming the presence of Clostridium perfringens type D in the study area.
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