Enterotoxemia in a 2-day-old lamb produced by a Clostridium perfringens type D lambda toxin–positive strain

Abstract

Clostridium perfringens type D is a gram-positive bacterium that causes enterotoxemia in sheep, goats, and, less frequently, other animals. This microorganism encodes 2 major toxins, alpha (CPA) and epsilon (ETX). Enterotoxemia occurs when epsilon prototoxin (pETX) is produced in the intestine and is activated by one or more proteases before being absorbed into the general circulation. Traditionally, it was believed that neonatal animals were not susceptible to type D enterotoxemia due to the trypsin-inhibitory action of colostrum in the intestinal tract and the lack of protease activation of pETX, although cases of enterotoxemia have been reported in 2 neonatal goat kids. A 2-d-old lamb, with a history of frailty, hunched posture, shallow breathing, and diarrhea followed by death, was submitted for postmortem examination and diagnostic workup. Autopsy revealed hydropericardium, pulmonary edema, and congested intestines. Histologically, there was pulmonary congestion and edema, and intestinal congestion. C. perfringens type D carrying the gene for lambda toxin was isolated from the small intestine and colon. Both CPA and ETX were detected in the small intestine and colon contents. We speculate that lambda toxin (a protease) activated pETX, and that ETX, in turn, produced enterotoxemia in this lamb.

Enterotoxemia is a disease produced by the absorption into the blood of toxins generated in the intestine, affecting distant organs such as the brain, lungs, heart, and others. ⁸ Clostridium perfringens is one of the most important agents of enterotoxemia in animals. This microorganism is classified into 7 types based on the coding of 6 major toxins, namely alpha [CPA], beta, epsilon [ETX], iota, enterotoxin [CPE], and necrotic enteritis–like toxin. ⁶ C. perfringens type D encodes 2 of these major toxins (i.e., CPA and ETX), although some type D isolates can encode CPE and several other, so-called, minor toxins. ⁸ CPA is a lecithinase that acts on cell membranes, producing hemolysis and necrosis of cells. ¹² The role of CPA in intestinal disease of mammals is controversial, although most evidence indicates that this toxin does not produce significant intestinal damage and it is not associated with C. perfringens type D enterotoxemia.^10–12 ETX is produced as a relatively inactive, ~33-kDa polypeptide named epsilon prototoxin (pETX) that is activated by proteases present in the intestinal environment to the active ~29-kDa ETX.^3,4,8 Trypsin and chymotrypsin, which remove 14 C-terminal amino acids, are the main enzymes responsible for the activation of pETX.^3,5 However, lambda toxin produced by some strains of C. perfringens type D can also activate pETX in vitro, and it has been suggested that lambda toxin can also activate this prototoxin in the intestine of neonatal goat kids. ¹ ETX is a pore-forming toxin that is responsible for the neurologic, respiratory, and other clinical signs and lesions observed in animals with type D enterotoxemia.^3,7

Type D enterotoxemia occurs in sheep and goats around the world. ¹¹ The pathogenesis of enteric infection with C. perfringens type D requires the presence of this microorganism in the intestine, usually associated with a change in the enteric microenvironment, which favors massive multiplication of C. perfringens, and the consequent production of toxins. ⁸ Small ruminants suddenly fed large amounts of grains or concentrate are highly susceptible, thus the synonym “overeating disease.” ⁸

The traditional belief was that neonatal animals are not affected by type D enterotoxemia given the low trypsin activity in the intestinal content due to the trypsin-inhibitory action of colostrum.^3,10 However, this dogma was revised when enterotoxemia type D was diagnosed in 2 neonatal goat kids infected with lambda-positive C. perfringens type D. ¹ Specifically, it was speculated that lambda toxin produced in the intestine by C. perfringens type D activated pETX, in the absence of trypsin. ¹ However, a search of Google Scholar, CAB Direct, PubMed, and Web of Science did not reveal any published information on type D enterotoxemia in neonatal lambs or cases of ovine disease associated with lambda-positive C. perfringens type D. Here we describe a case of enterotoxemia in a neonatal lamb, associated with lambda toxin–positive C. perfringens type D, and we propose that lambda toxin activated pETX in the intestine of this lamb.

A 2-d-old Suffolk–Hampshire mixed, male lamb was submitted to the Tulare Laboratory of the California Animal Health and Food Safety System (CAHFS; University of California–Davis [UC Davis], Davis, CA, USA) for postmortem examination. The lamb had been born clinically healthy and initially nursed normally. A few hours later, however, it was found frail, hunched, and with frothy diarrhea. The animal died soon after.

An autopsy was performed ~12 h after death. The carcass was moderately fresh, and in fair nutritional condition, with small amounts of fat reserves, but still well fleshed, and moderately dehydrated. Yellow fluid (~20 mL) with fibrin strands distended the pericardial sac. The lungs were diffusely congested and edematous, and there was a large amount of stable froth in the trachea and lower airways. The small intestine was diffusely dark red, distended with gas, and contained scant amounts of watery yellow fluid. The cecum and spiral colon had scant, creamy, yellow contents. No other significant gross abnormalities were seen in the rest of the carcass.

Samples of lung, heart, liver, spleen, thymus, adrenal gland, lymph node, rumen, abomasum, small intestine, colon, and brain were collected and fixed in 10% neutral-buffered formalin for ~24 h. The brain was then sliced at ~0.5-cm intervals and fixed in fresh formalin for another 7 d, after which samples of parietal cortex, corpus striatum, thalamus, brainstem at the level of the anterior colliculus, cerebellum, anterior cerebellar peduncles, pons, and medulla at the level of the obex, were collected. All of the tissues were processed routinely for the production of 4-µm thick H&E-stained sections. Microscopically, there was diffuse pulmonary congestion and proteinaceous alveolar and interstitial edema. The small intestine and the liver had diffuse congestion.

Additional ancillary tests were performed according to CAHFS standard operating procedures. Samples of lung, liver, and pericardial fluid were inoculated onto Columbia 5% sheep blood agar (Hardy) and MacConkey agar (Hardy) plates, and incubated aerobically or microaerobically, respectively, at 37°C for 48 h. All isolates were identified by conventional biochemical techniques and/or MALDI-TOF mass spectrometry (Bruker). A PCR for Salmonella spp. was performed on colon content. ² Small and large intestinal content were inoculated onto pre-reduced, anaerobically sterilized Brucella blood agar (Anaerobic Systems), pre-reduced, anaerobically sterilized phenylethyl alcohol sheep blood agar (Anaerobic Systems), and egg yolk agar (Anaerobic Systems), and incubated anaerobically at 37°C for 48 h. Additional samples of small intestinal content were inoculated onto cycloserine–cefoxitin–fructose agar (Veterinary Media Services, UC Davis) and incubated anaerobically at 37°C for 48 h. An ELISA for C. perfringens CPA, CPB, and ETX toxins was performed on small intestinal and colon contents using a commercial kit (Bio-X Diagnostics) following the manufacturer’s instructions. A heavy metal screen including arsenic, cadmium, copper, iron, lead, manganese, mercury, molybdenum, zinc, and selenium was performed on the liver.

A small amount of mixed aerobic flora was isolated from the lung. PCR for Salmonella spp. was negative. Large numbers of C. perfringens were isolated from the small and large intestinal content. Five isolates were tested by PCR as described previously for the genes encoding the typing toxins of this microorganism, plus the gene encoding lambda toxin. ⁶ The 5 isolates were positive for the cpa, etx, and lambda toxin genes, identifying these isolates as C. perfringens type D, lambda toxin–positive.

CPA and ETX were detected in small intestinal and colonic content by ELISA. The copper concentration was 24 ppm (RI: 25–100 ppm), and the iron concentration was 680 ppm (RI: 30–300 ppm). All other heavy metals were within RIs or not detected.

Our presumptive diagnosis of enterotoxemia was based on the clinical signs, and gross and microscopic findings, coupled with the isolation of C. perfringens type D from the intestinal content. Confirmation was based on detection of ETX in small and large intestinal content. ¹¹ The latter is considered diagnostic for this condition.^3,8 No gross or microscopic lesions were seen in the brain; although gross lesions are not frequently seen in cases of type D enterotoxemia, microscopic lesions of perivascular and intramural edema are seen in ~90% of cases. ¹¹ There are, however, a few cases in which no gross or microscopic changes are seen in the brain, ¹¹ and the absence of these lesions does not, therefore, preclude a diagnosis of C. perfringens type D enterotoxemia.

Type D enterotoxemia was reported in 2023 in 2 neonatal goat kids associated with lambda toxin–positive C. perfringens type D isolates. ¹ Type D enterotoxemia has also been described in 2 neonatal calves, ¹³ although the C. perfringens isolates in that case were not typed.

The absence of intestinal protease activity in the intestine of newborn animals is usually associated with lack of pETX activation. In our case, we speculate that lambda toxin produced by C. perfringens was responsible for the activation of pETX. Because there are no routine methods available to date, to detect lambda toxin in the intestinal content of animals, ⁹ we could not determine if the toxin was present in the intestinal contents. However, all 5 C. perfringens isolates obtained from the intestinal content had the gene encoding this toxin, and the toxin was likely produced in vivo.

Type D enterotoxemia should be considered as a differential diagnosis in neonatal lamb death. In sheep, C. perfringens type D enterotoxemia is not usually associated with diarrhea, as it occurs in goats. However, in our case, the diarrhea could have been related to an individual imbalance in the intestinal microbiota.