During the present study intestinal scrapings were collected from sheep suspected for enterotoxaemia. Samples were subjected for the isolation by repeated culturing in Reinforced Clostridium medium. Growth on blood agar revealed characteristic colonies of Clostridium perfringens after 18-24 hours. Biochemical and mice inoculation tests were performed. Isolated organism was identified by indirect ELISA. The pure growth was subcultured in RCM in bulk quantity and calculated the dose for experimental infection.
In the second part of the study, sheep and goats were procured, dewormed and kept in the experimental house of UVAS, Lahore. The experimental infection comprised of the whole culture of C. perfringens type D was inoculated intraduodenally via para-mid line between animals of group A and B while in animals of group C and D inoculated only starch solution to achieve the objectives. Accumulative clinical score in sheep was found to be 9 to 16, 13 to 22 and 15 to 23 at 10, 20 and 30 hours PI respectively while in goats the accumulative scores varied from 5 to 15, 9 to 16 and 14 to 21 at above mentioned time intervals. The highest mean score for clinical findings in sheep was anorexia, frothing followed by dehydration while in goats, the highest mean score was recorded for diarrhea dehydration, and anorexia. No significant clinical findings were noted in control groups.
There was a significant increase in blood glucose, urea and serum creatinine in infected group of sheep and mean values reached up to 141 mg/dl, 92 mg/dl and 7.5 mg/dl respectively at 30 hours PI while in goats a similar pattern was observed with the mean values raised to 142 mg/dl, 111 mg/dl and 10.2 mg/dl for blood glucose, urea and serum creatinine respectively. There was no significant change found in RBC and platelet count of both species but there was an early increase in the mean WBC count of sheep 19.7x103/µl at 10 hours but then decreased to 14.7x103/µl at 30 hours PI while in goats it was 23.6x103/µl and then decreased to 15.3x103/µl. The mean PCV % age increased in animals of both infected groups but more in goats and reached to 52% in 30 hours.
During the third part of the present study, the animals were slaughtered and PME performed. The accumulative score for gross lesions were recorded and it was found between 14 to 24 in animals of group A with the highest score for congestion and edema of different organs whereas in goats it was between 12 to 22 with congestion and hemorrhages of intestine having highest scores. The samples were kept in formalin for histopathological examination and accumulative lesions score was noted in different organs. The highest mean score in sheep was recorded in kidneys and lungs and in goat intestine, lung and kidneys were the major organs affected.
A polymerase chain reaction was optimized under our own laboratory condition for the detection of alpha and epsilon toxins of Clostridium perfringens type D from different tissues. Alpha gene was amplified at annealing temperature 52.2oC with amplicon size 247bp and ETX gene at the annealing temperature.
50.2oC with amplicon size 665bp.
The erythrocytes of different species were used to know the sensitivity against culture supernatants of C. perfringens type D. It was observed maximum hemolysis occurred in human erythrocytes (68%) followed by mice (57%) at 37oC. It was also recorded that a significant increase was found at 37oC as compared to25oC except for dog and rabbits where no significant difference was observed.