Enterotoxemia is a fatal enteric disease that affects animals and humans. Enterotoxemia is an economically important disease of livestock and causes heavy economic loss among feedlot and pasture-reared lambs than every other disease combined, if vaccination is not applied¹¹. Sheep of all ages are affected by enterotoxaemia, but lambs under 10 weeks of age are most susceptible because they are nursed by lactating ewes and the weaned lambs on lush pasture. It is caused by Clostridium perfringens exotoxins. Clostridium perfringens is an anaerobic gram positive bacteria that is categorized into five toxino types (A, B, C, D, and E)⁷. C. perfringens is a normal inhabitant of the intestine of animals and humans. Once the intestinal environment is changed by sudden changes in the diet, the microorganism grows rapidly, proliferates, and secretes powerful exotoxins that act locally or systemically resulting in disturbing effects on animal species¹². The diagnosis of enterotoxemia is made by necropsy, and histopathological examinations. The present study investigated the molecular identification of C. perfringens in sheep as well as pathological changes in lungs.

The affected tissue samples of lungs from carcasses of sheep were collected for proposed investigation irrespective of sex, age groups and breeds from various Veterinary hospitals, rural areas in and around Bikaner district of Rajasthan. The samples received from field veterinarians in the Department of Veterinary Pathology were also included in this study. In the present investigation, detailed necropsy was performed on 362 sheep carcasses. Out of these, lungs samples from 123 sheep suspected for enterotoxemia were further preserved in 10% formalin. The parts of affected tissue measured 2-5 mm thickness and presenting the lesions with normal tissue, were used for fixation and histopathological examination. For histopathological examination, processing of tissue was done by paraffin embedding using acetone and benzene technique³. The tissue sections of 4-6 micron thickness were cut and stained with hematoxylin and eosin staining method as a routine.

Intestinal contents were collected from enterotoxemia suspected necropsied sheep. Samples were collected in sterilized biohazard polybags and transport to laboratory in icebox filled with ice, and stored at 4°C until used.

DNA from the intestinal contents samples of sheep was using QIA amp Fast DNA stool mini kit (Cat. No. 51604). The quality and quantity of isolated genomic DNA was measured by agarose gel electrophoresis and UV-Spectrophotometer method.

16S rRNA gene based species specific identification of C. perfringens type D was done as per the method described^1,5. The sequence of the primer pair used was as follows: Forward primer: 5’ TAA CCT GCC TCA TAG AGT 3’ Reverse Primer: 5’ TTT CAC ATC CCA CTT AAT C 3’ The expected product size was 481bp.

The PCR amplification was performed in a 0.2 ml PCR tube in 25 microlitre reaction. The reaction mixture included 12.5 microlitres of Master mix (Thermo Fisher Scientific), 1 microlitre each of forward and reverse primer (10 pM/μl), 3 microlitres template DNA and 7.5 μl MiliQ water. The PCR conditions used was initial denaturation at 95°C for 15 min followed by 35 cycles of denaturation (94°C) for 30 sec, annealing (49.5°C) for 1 min, extension (72°C) for 1.30 min and final extension at 72°C for 10 min. The PCR was performed in Applied Bio System. In the present investigation, a detailed necropsy was performed on 362 sheep irrespective of age, sex and breeds. Out of these, 123 sheep suspected for enterotoxemia were processed for molecular confirmation of C. perfringens type D from intestinal contents by using PCR. After conducting PCR, 66 sheep found positive for C. perfringens type D. An overall incidence of enterotoxemia infection in sheep was recorded as 18.23%.

On gel electrophoresis analysis, amplicons of 481bp for species specific (Fig. A) gene were seen.

Grossly, the affected lungs were enlarged, congested, edematous and hemorrhagic (Fig. B). Some of cases showed multifocal haemorrhage. On cut section, variable amounts of froth were appeared in the trachea, bronchi and bronchioles. Various histopathological conditions is presented in Table-1.

Microscopically, lungs revealed congestion, edema, emphysema and mononuclear and polymorphonuclear cells infiltration in most cases (Fig. C). Lungs showed proteiceous edematous fluid. Lungs showed haemorrhages and mononuclear cells and polymorphonuclear cells infiltrations. Interstitial pneumonia in which irregular thickening of interalveolar septa due to proliferation of septal cells and infiltration of mononuclear cells and polymorphonuclear cells were observed in some cases. Some sections showed desquamation of epithelial cells in bronchiole with infiltration of mononuclear and polymorphonuclear cells in interstitium.

An overall incidence of enterotoxemia infection in this study was 18.23%. Lower incidence was reported in India which was 1.5% by previous workers⁹. However, comparatively higher incidence was reported by earlier workers⁴ in sheep and goat as 31% and 22%, respectively.

Similar gross and microscopic lesions were recorded by earlier studies^2,8,10. Microscopically, congestion and oedema was observed in 64 cases (96.96%), haemorrhages in 46 cases (69.69%) and interstitial pneumonia in 43 cases (65.15%). Comparatively lower incidence was reported by previous observers⁶ who recorded 65% interstitial pneumonia. Pulmonary emphysema was recorded in 11 cases (16.66 %).

Similar to the present study⁵ identified the species specific gene yielding amplified products at the same regions.

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