Detection of Epsilon Toxin in Goats Affected with Acute Enterotoxaemia and the Haemato-biochemical Alterations

Enterotoxaemia is an infectious disease observed in well fed animals, mainly in sheep, less often in goat and rarely in cattle (Jemal et al., 2016).  Peracute form of this disease occurs in animals of 3-10 weeks old, where as acute and chronic forms can occur in both young and old sheep and goats (Tariq Javed et al., 2009).

The disease is caused by a gram positive, anaerobic, spore forming and toxin producing bacilli, Clostridium perfringens type D (Uzal et al., 2014) in goats, whereas C. perfringens type A is most commonly isolated type in ovine enterotoxaemia (Fahimeh et al., 2018). However, type A, C and D isolate were detected respectively in 75.6 per cent, 0.4 per cent and 0.4 per cent of total faecal samples from healthy goats (Santana et al., 2018). It is a normal component of the intestinal flora of healthy warm-blooded animals and human beings and soilin fairly low numbers (Songer, 1996).

C. perfringens can secrete 4 major toxins like alpha, beta, epsilon and iota and accordingly, the pathogen is typed into 5 toxins types namely A, B, C, D and E (Sakurai, 1995). Excess carbohydrate-rich feed or sudden change in diet leads to reduced peristaltic action resulting in rapid and excessive proliferation of organisms and production of lethal toxins in the intestine. Epsilon toxin (ETX) is the most important one which enters the blood stream and cause perivascular edema in tissues such as kidneys, lungs, heart and brain (Popoff, 2011). It causes sudden death in goats of all age groups (Veschi et al., 2008) and the case-fatality rate approximates 100% (Constable et al., 2017).

Though sheep enterotoxaemia is well documented, the situation is less clear in goats on account of limited studies carried in the goats. Availability of literature on haematologic and clinical biochemistry studies on enterotoxaemia is also limited (Pawaiya et al., 2020). This paper aims to report the incidence of classical enterotoxaemia in goats of different age groups with specific signs by qualitative detection of epsilon toxin in faeces, therapeutic management and haemato-biochemical analysis in the affected goats.

Thirty goats of under 1-4 years age group, both male and female, and Kodi, Kanni and Salem Black breeds were presented to Medicine section, Veterinary Clinical Complex, Veterinary College and Research Institute, Tirunelveli in 2021 with the neurological signs like violent convulsions, opisthotonos, nystagmus, bruxism, ataxia, lateral recumbency, paddling, ptyalism and coma (Fig 1a,1b and 1c). Diarrhoea with semisolid, watery or haemorrhgic faeces could be observed in some of the cases.All the goats were reported to have no history of insecticide poisoning in their premises. Faecal samples were collected by using a rectal scoup from all these cases and tested immediately on spot by immunochromatographic test kit (BioX Diagnostics®, France) to identify the Clostridium perfringens Epsilon toxin. The test faecal sample was diluted in the buffer provided in the sample vial and the immunochromatographic device was placed into the liquid sample. The positive reaction was read if pink line appeared on test line indicating weak or strong positivity without development of reaction in control line (Fig 2) Serum samples were collected from the positive cases for haematobiochemical analysis (Table 1). Peripheral blood smears were also collected to rule out haemoprotozoan infections in all the cases.



​

​

In this study, 36.6 per cent of the goats were found to be positive by the immunochromatographic test. The definitive diagnosis of enterotoxaemia is mostly based only on the detection of epsilon toxin in the intestinal contents and other supportive tests could be measurement of urine glucose levels or examination of Gram-stained smears of intestinal mucosa (Smith and Sherman, 2011). Molecular test like, multiplex PCR is generally used for toxin genotyping of C. perfringens (Mohiuddin et al., 2016). Recently, immuno chromatographic test is found to detect ETX with fivefold detection limits in different matrices such as serum and intestinal contents (Feraudet-Tarisse et al., 2017). The peripheral blood smears revealed no blood parasites from all the cases.

The clinical signs observed are in consistent with that of Smith and Sherman (2011) and Ortega et al., (2019). Diarrhoea was the most consistent finding which is due to enterocolitis in acute cases and initial softening of faeces in sub acute cases (Pawaiya et al., 2020). The nervous signs observed could be associated with ETX that was known to stimulate the release of glutamate, an excitatory neurotransmitter, by targeting the hippocampal glutamatergic system (Lonchamp et al., 2010 and Popoff, 2011) or vasogenic edema and neuronal toxicity  (Morris et al., 2017). Brain is a prime target of epsilon toxin which initially binds to endothelial cells of the blood-brain-barrier (BBB), resulting in swelling, vacuolation and necrosis and thereby leakage of fluid and proteins, hypoxia of the neural parenchyma (Stiles et al., 2013). However, Kumar (2019) and Singh (2017) observed brain changes as inconsistent in caprine enterotoxaemia. The damage to the vascular endothelium leads to the accumulation of protein-rich fluid effusions in heart, brain and lung (Constable et al., 2017).

In this report, neutrophilia and leukocytosis were observed in majority of the cases (90.0%) of enterotoxaemia and statistically, a highly significant increase (P<0.001) in the neutrophilsand leukocytes count and a highly significant decrease (P<0.001) in the lymphocyte count were noticed in the positive cases when compared to the controls. Reduction in total erythrocyte count (TEC) was observed in 40.0 per cent of the cases, however, statistically, no significant difference was observed in positive cases when compared to controls. The findings are in accordance with Kumar (2019) who observed a significant increase in the white blood cells (WBC) in affected goats, with a significant reduction in mean erythrocyte (RBC) count and haemoglobin level. Reduction in RBC levels could be associated with haemorhhagic enteritis caused by the ETX and the leukocytosis with  clostridial proliferation in the GI tract.

In this report, elevated blood urea nitrogen (BUN) creatinine, glucose, sodium, alkaline phosphatise (ALP) and chloride levels were observed in positive cases. Statistically, a significant increase (P<0.05) in the levels of BUN, creatinine, glucose, ALP and chloride levels was observed, and statistically a significant increase (P<0.001) in the sodium level was observed. This finding was in accordance with Khan et al., (2017) who recorded a significant increase in blood glucose, liver enzymes, serum creatinine, total bilirubin caprine enterotoxaemia, pulpy kidney was not consistently reported, but with petechial and ecchymotic subcapsular haemorrhages (Uzal et al., 1994).Pronounced hyperglycem, blood urea and glucose in goats affected with enterotoxaemia.

The elevated BUN and creatinine could be associated with lesions in kidney. However, in ia (150 to 200 mg/dL) due to the mobilization of hepatic glycogen and marked glycosuria are reported to be characteristic in the terminal stage of enterotoxaemia (Constable et al., 2017). Glycosuria is said to be strongly indicative of enterotoxaemia in both sheep and goats (Uzal and Songer, 2008), where as Pawaiya et al., (2020) reported the hyperglycaemia and glycosuria to be inconsistent in goats.

All the animals were treated with parentral administration of sulphadimidine @ 150 mg /kg body weight to prevent further growth of the bacterium and toxin production, flunixin meglumin @2.2 mg//kg body weight to alleviate toxaemia, mannitol @2.0 gms/kg body weight to reduce the cerebral oedema, diazepam @ 0.5/kg body weight as an anticonvulscent, dextrose normal saline and Ringer’s lactate to alleviate dehydration along with recommendation of oral sulphadimidine, activated charcoal as toxin binder, bloat relieving suspension and rumenotorics (Smith and Sherman, 2011). However, all the cases were not presented for follow up therapy which might possibly be due to the fatal outcome of the disease, as the treatment is generally not effective due to acute nature of the disease.

In conclusion, acute enterotoxaemia mostly leads to a fatal outcome due to the encephalitic form not only in young kids but also in adults. Hence, periodical prophylactic vaccination of sheep and goats against enterotoxaemia especially before rainy season in endemic areas will help prevent the onset of disease and the mortality.

There is no conflict of interest with any of the authors either directly or indirectly to the content of this article.

The authors thank the Dean, veterinary College and Research Institute, Tirunelveli and Director of Clinics, Tamil Nadu veterinary and Animal Sciences University for providing necessary facilities to carry out the present work.