Formaldehyde, ethanol, xylol, paraffin, hematoxylin, and eosin were from M/S Merck KGaA, Darmstadt, Germany. Equipment, such as a light microscope, thermocycler, and AlphaImager Mini Imaging System, was from Nikon (Japan), Bio-Rad (UAE), and ProteinSimple (UK), respectively. Tryptose sulfite cycloserine agar and agar agar were from Oxoid, UK, API 20A kits were from bioMérieux, Marcy l'Étoile, France, Column Extraction Kit was from Bio-Basic, Canada, and other chemicals for PCR were from Wuhan Zokeyo Co. Ltd., China. The Minitab software for Windows was from Minitab Ltd., Coventry, UK.

The current study was conducted at desert conditions (Cholistan) of district Bahawalpur during January 2020. Cholistan is known as a tropical desert. This desert is in South Punjab between 27°42′ and 29°45′N latitude and 69°52′ to 75°24′E longitude. The desert is stretched on an area of about 26,330 km² and includes three districts Bahawalpur, Bahawalnagar, and Rahim Yar Khan. Tobas (artificial ponds filled by rainwater) are famous for the compensation of permanent water sources and are well-known common sites for drinking purposes even including nomads. About more than 50% of the agroecological area of South Punjab is inhabited with 32% of the total human population of the province, which is directly or indirectly related to dairy animals.

The socioeconomic and livelihood of the people of this area is based and centered on agriculture and dairy animals. This area has tropical climatic conditions at 48°C in summer. December to February are the colder months, where the average temperature remains below 15°C. The rearing of small ruminants in semiarid areas in this region is famous. Most of the people reared different breeds of goat for sacrificial use and to earn their livelihood. Moreover, the nomadic system in this region is often diversified with small ruminants (sheep and goats), sharing of dairy animals, the seasonal movement of dairy animals for fodder, and water purposes. Despite the importance of these animals, there is a scarcity of proper husbandry measures in this region, and mixed livestock-rearing practices are employed for different dairy animals, which result in outbreaks of various diseases.

Private herds of two breeds of goat (Makhi Cheeni and Beetal) and one non-descriptive native (Teddy) herd were reared in the desert area of district Bahawalpur. There were three herds (n = 153), i.e., Makhi Cheeni (n = 84), Beetal (n = 39), and non-descriptive Teddy goats (n = 30). Out of these, there were 63 male and 90 female goats. Of the total goats, 43, 61, and 49 were <1 year, 1–2 years, and 3 and above 3 years, respectively. These animals were mainly kept in barrens under similar husbandry practices/management on the sandy floor. All the animals were grazed daily for about 2–3 h (9 a.m. to 12 noon). All the animals were offered lush green fodder (lucerne/alfalfa). On the day of mortality, these animals were grazed on oats, and there was a sudden change in weather; there was heavy rain in cold weather. The goats were vaccinated against various diseases including enterotoxemia, peste des petits ruminants, and pleuropneumonia. Deworming was regularly carried out thrice a year. All the animals were carefully monitored for any obvious/visible clinical and behavioral signs daily. Morbid animals were separated from the other animals and treated according to the nature of the condition/disease.

During the cold, rainy, and foggy week of January 2020, history from the owner/attendants indicated that the animals were grazed early in the morning and, in the evening, offered green fodder ad libitum. However, on the day of mortality, there was heavy rain, and animals were grazed on oats. Whole oat (Avena sativa L.) is a rich source of carbohydrate (66%), though protein (17%) and some fats are also present (34). After 2–3 h, the animals started showing clinical signs (watery diarrhea) and became sick. In spite of treatment therapy, there were high morbidity and peracute deaths. Percentage of morbidity, mortality, and case fatality was calculated as given below:

As the farms where outbreaks of enterotoxemia occurred were near the university premises, we were able to conduct necropsy within the shortest possible time (30–60 min). All the visceral organs were examined thoroughly for lesions. Based on severity, the lesions were scored as mild, moderate, and severe. At necropsy, all the goats (carcasses) exhibited similar gross lesions. Tissues from different visceral organs including the lungs, spleen, heart, intestines, liver, and kidneys were fixed in 10% formaldehyde solution (35) and shifted to the Laboratory, Department of Pathology, Faculty of Veterinary and Animal Sciences, IUB. After a few days of preservation/fixation, all the tissues were washed, dehydrated in ascending grades of ethanol, cleared in xylol, and embedded in melted paraffin. For the study of microscopic changes, 4-μm-thick slices of all the visceral organs were cut, processed, and stained with hematoxylin and eosin technique (36). Prepared slides were examined under a light microscope. The scoring of microscopic lesions was made based on severity (mild, moderate, and severe).

For bacterial isolation and culturing, intestinal contents (fecal samples) were collected from all the morbid animals and were shifted to the laboratory immediately. All the collected samples were cultured in reinforced clostridial media (Oxoid, CM0149) and incubated at 37°C for 24 h anaerobically. A loopful from the crude broth culture was streaked onto tryptose sulfite cycloserine agar (TSC, Oxoid, CM0587B) having selective supplement. Incubation was carried out under anaerobic conditions at 37°C for 24 h. The isolated colonies were stained for bacterial morphology. Typical blackened colonies on TSC plates, characteristic beta hemolysis on 5% sheep blood agar, and production of opaque halo around colonies on egg yolk agar confirmed the isolated colonies as C. perfringens. The pure bacterial isolates were further biochemically characterized using API 20A kits (bioMérieux, Marcy l'Étoile, France).

For DNA extraction, pure colonies from 18-h culture of C. perfringens were mixed in sterile water and centrifuged at 6,000 × g for 4–5 min. After centrifugation, the pellets were suspended in 200–250 μl of cold Tris EDTA (TE) buffer. DNA extraction was conducted using EZ-10 Spin Column Extraction Kit (Bio-Basic, Canada).

Multiplex PCR was carried out for inquisition of cpα, cpb, etx, iap, cpβ₂, and iota toxin genes (Table 1) using previous specific primers (37–39). The reaction mixture contained 1.2 U of Taq DNA polymerase, 1 × PCR buffer, 4 mM MgCl₂, 250 μM dNTPs, 0.12 μM forward and reverse primers of alpha (α), epsilon (ε), beta (β), iota (i), and β₂ gene, and 1.5 μl of sample DNA (100–200 ng/μl). The PCR conditions included 95°C for 10 min of predenaturation step, followed by 94°C for 45 s, 55°C for 60 s, 72°C for 90 s (35 cycles) followed by 72°C for 7 min (40, 41). Gel electrophoresis was carried out in a 1.5% agarose gel, and bands were observed under UV transillumination, photographed using AlphaImager Mini Imaging System (CA, USA).

The C. perfringens isolates identified from fecal samples of goat that died of enterotoxemia were screened by multiplex PCR, and the toxinotypes were presented in a tabularized form. The prevalence percentage was calculated. Chi-square test was applied using the Minitab software for data analysis, and the significance level was considered at p < 0.05.

No clinical signs were observed in peracute cases. However, there were different clinical abnormalities such as watery diarrhea, acute anorexia, depression, dullness, and dehydration in acute cases. Other than these, the following were observed: pale mucus membranes, icterus, mild-to-moderate color of urine, herding in corner and opisthotonus, bloody diarrhea, stretching their bodies at the time of standing and walking, and reluctant to eat and drink. In case of convulsion, animals were unable to stand, laying on the ground by extending the legs, with their head and neck stretched back on its withers. At this stage, frothy discharge from the mouth and abdominal discomfort was obvious.

Morbidity, mortality, and case fatality in Makhi Cheeni, Beetal, and non-descriptive (Teddy) goats caused by enterotoxemia were 87.58, 75.81, and 76.11%, respectively. In Makhi Cheeni goats, morbidity (χ²-value = 25.419), mortality (χ²-value = 24.879), and case fatality (χ² value = 26.432) were significantly (p < 0.0001) higher compared with these parameters in Beetal and non-descriptive goats (Table 2). Morbidity, mortality, and case fatality based on sex and age showed non-significant differences (Table 3).

Main gross lesions were quantified into severe, moderate, and mild (Table 4). At necropsy, severe serosal petechiation (93.96 %), and straw-colored and light pink fluid were present in the thoracic, peritoneal, and abdominal cavity. Grossly hyperemic mesentery, congestion, ballooning degeneration, multifocal hemorrhagic enteritis, ulcerated mucosa, and necrotizing enteritis were the striking features in the ilium. Severe congestion, hyperemic mucosa, ballooning, and severe hemorrhagic enteritis (85.34%) were observed in the large intestines of goats. Both the small and the large intestines contained mucoid exudate and were severely hemorrhagic. In the majority of cases, the jejunal mucosa was necrosed and had multiple hemorrhage areas. Hemorrhagic enteritis (Table 4), congestion, brown color fluid with smell, and intense mucosal and serosal hyperemia were the most striking lesions observed in the intestine. Grossly, the liver of infected goats was congested, swollen, dark black in color, friable, and had a petechial necrotic lesion.

The heart of goats that died of peracute enterotoxemia showed congestion along with multifocal severe hemorrhage on the epicardium (92.24%) and myocardium. The heart contained clotted blood in the ventricles. A large amount of straw-colored fluid was present in the pericardial sac. The ventricles were enlarged and had necrotic foci.

The lungs of goats were hyperemic, consolidated, severely congested (Figure 1a), and edematous (88.79%). The cut sections of the lungs of infected goats indicated the presence of frothy material in all parts of the airways including the bronchioles. The trachea was severely congested and exhibited extensive frothy fluid (Figure 1b). The spleen of infected goats in the majority of cases was congested and edematous, and in a few cases, the spleen indicated engorgement of the small blood vessels. The kidneys both right and left in the majority of cases (90.51%) were severely hemorrhagic, swollen, and soft in consistency (Table 4). Grossly, the cut sections of the kidneys were severely congested (Figure 1c). The urinary bladder was filled with light pink color urine. In some cases, the urinary bladder contained chocolate color urine.

Lesions recorded through a microscopic study were also quantified into severe, moderate, and mild (Table 5). Lungs of all the infected goats showed extensive mononuclear cell infiltration in the alveoli, emphysema, interstitial pneumonia, congestion, fibrinous exudate, and hemorrhage. The predominant histopathological changes in the lungs were severe eosinophilic proteinaceous edema, broncho-interstitial pneumonia (90.51%), embolic pneumonia, and inflammatory exudate within the bronchioles, bronchi, and alveoli. Histological observation of liver tissues showed extensive necrosis of hepatocytes, fatty change, congestion, and hemorrhage.

Microscopic analyses of the kidneys indicated severe glomerular hemorrhage (87.06%), severe necrosis (Figure 2) and degeneration of the renal tubules, sloughing of the endothelium of the renal tubules, congestion, atrophy of the glomeruli, hemorrhage, and increased urinary spaces. The small and large intestines had severe pathological changes in the infected goats (Figure 3A). In these tissues, the microscopic changes mainly comprised of congestion, hemorrhage, necrosis of the mucosa and submucosa, necrosis of the villi, edema in the submucosa, and mononuclear cell infiltration in the lamina propria. There were severe necrosis and sloughing of the epithelium of the villi and fusion of the villi (88.89%) (Figure 3B). Histologically, spleen sections showed necrosis of the red and white pulp, congestion, hemorrhage, and depletion of the lymphoid tissue in the white pulp (80.17%). Histopathological analyses of heart sections of different goats showed extensive neutrophilic infiltration (76.72%), edema, congestion, hemorrhage, coagulative necrosis, degeneration of cardiac cells, and severe myocarditis.

All goats at the farms (dead and live) were screened for the presence of alpha, beta, epsilon, iota, and cpβ2 genes using specific primers. About 116 samples were positive for C. perfringens indicated by the presence of an alpha gene (cpα) screened using simplex PCR. These 116 positive isolates were further subjected to mPCR to identify the toxinotypes involved in the current outbreak (Figure 4). Deaths due to C. perfringens type D were significantly higher (χ²-value = 10.288; p <0.001) compared with deaths by C. perfringens type A. The multiplex PCR has shown that 47.4% of the isolates were positive for cpα + etx, and 20.7% for cpα, etx, and cpβ2 genes (Table 6). All these isolates positive either for cpα and etx or cpα, etx, and cpβ2 belonged to C. perfringens type D. The remaining 31.9% isolates were type A, whereas 11.2% of these type A isolates were also harboring cpβ2 gene. Occurrence of these toxinotypes showed that cpα + etx was significantly (χ²-value = 25.179; p < 0.0001) high as other toxins. Neither beta nor iota gene was identified from the isolates via mPCR indicating the absence of C. perfringens types B, C, and E among the outbreak animals.