Evidence that Clostridium perfringens Enterotoxin-Induced Intestinal Damage and Enterotoxemic Death in Mice Can Occur Independently of Intestinal Caspase-3 Activation.

Clostridium perfringens enterotoxin (CPE) is responsible for the gastrointestinal symptoms of C. perfringens type A food poisoning and some cases of nonfoodborne gastrointestinal diseases, such as antibiotic-associated diarrhea. In the presence of certain predisposing medical conditions, this toxin can also be absorbed from the intestines to cause enterotoxemic death. CPE action in vivo involves intestinal damage, which begins at the villus tips. The cause of this CPE-induced intestinal damage is unknown, but CPE can induce caspase-3-mediated apoptosis in cultured enterocyte-like Caco-2 cells. Therefore, the current study evaluated whether CPE activates caspase-3 in the intestines and, if so, whether this effect is required for the development of intestinal tissue damage or enterotoxemic lethality. Using a mouse ligated small intestinal loop model, CPE was shown to cause intestinal caspase-3 activation in a dose- and time-dependent manner. Most of this caspase-3 activation occurred in epithelial cells shed from villus tips. However, CPE-induced caspase-3 activation occurred after the onset of tissue damage. Furthermore, inhibition of intestinal caspase-3 activity did not affect the onset of intestinal tissue damage. Similarly, inhibition of intestinal caspase-3 activity did not reduce CPE-induced enterotoxemic lethality in these mice. Collectively, these results demonstrate that caspase-3 activation occurs in the CPE-treated intestine but that this effect is not necessary for the development of CPE-induced intestinal tissue damage or enterotoxemic lethality.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Haemorhagic enterotoxemia by Clostridium perfringens type C and type A in silver foxes.

Type C and type A of C. perfringens were detected in the seat of natural infections in silver foxes characterized by symptoms of haemorrhagic enterotoxemia. In all of the dead foxes characteristic changes were noted in the small intestine and parenchymatous organs. The production of alpha and beta toxins by isolated bacteria was confirmed by the bioassay using white mice and by PCR. The results of the drug sensitivity testing showed that isolated strains were highly susceptible to amoxicillin with clavulanic acid, metronidazole, doxycycline and penicillin with streptomycin.

Proportional mortality: A study of 152 goats submitted for necropsy from 13 goat herds in Quebec, with a special focus on caseous lymphadenitis.

The objectives of this study were to determine the main causes of mortality, with a special focus on caseous lymphadenits as a cause of death or wasting in caprine herds from Quebec. Goats (n = 152) from 13 herds were submitted for necropsy; the cause of mortality, and the presence, location, and cause of abscesses (if present) were recorded. Proportional mortalities were distributed as: Clostridium perfringens type D enterotoxemia (17.1%), pneumonia (13.8%), paratuberculosis (10.5%), listeriosis (6.6%), pregnancy toxemia (5.3%), caprine arthritis-encephalitis (4.6%), and caseous lymphadenitis (3.9%). Caseous lymphadenitis was diagnosed in 24.3% of the submitted goats, but was not a major cause of wasting or mortality. Abscesses were localized internally in 54.1% of the cases. Paratuberculosis was diagnosed in 29 goats (16 as cause of death) and was considered a major cause of wasting and/or mortality.

Mortalité proportionnelle: Une étude de 152 chèvres soumises pour nécropsie provenant de 13 élevages caprins du Québec, avec une attention particulière à la lymphadénite caséeuse. Les objectifs de cette étude furent de déterminer les principales causes de mortalité avec une attention particulière à la lymphadénite caséeuse comme cause de mortalité ou de dépérissement chez les chèvres du Québec. Cent-cinquante-deux chèvres provenant de 13 élevages différents ont été soumises pour nécropsie; la cause de mortalité, la présence d'abcès, leur localisation et leur cause (s'il y a lieu) furent compilées. Les mortalités proportionnelles furent distribuées ainsi : entérotoxémie de type D (17,1 %), pneumonie (13,8 %), paratuberculose (10,5 %), listériose (6,6 %), toxémie de gestation (5,3 %), arthrite-encéphalite caprine (4,6 %) et lymphadénite caséeuse (3,9 %). La lymphadénite caséeuse a été diagnostiquée chez 24,3 % des chèvres soumises, mais sans être une cause majeure de dépérissement et de mortalité. Les abcès étaient localisés de façon interne dans 54,1 % des cas. Au total, la paratuberculose a été diagnostiquée chez 29 chèvres (16 en étant décédées) et fut considérée comme une cause majeure de dépérissement et/ou de mortalité.(Traduit par les auteurs).

Pathology of Clostridium perfringens type C enterotoxemia in horses.

Clostridium perfringens type C is an important cause of enteritis and enterocolitis in foals and occasionally in adult horses. The disease is a classic enterotoxemia, and the enteric lesions and systemic effects are caused primarily by beta toxin, 1 of 2 major toxins produced by C. perfringens type C. Until now, only sporadic cases of C. perfringens type C equine enterotoxemia have been reported. We present a comprehensive description of the lesions in 8 confirmed cases of type C enterotoxemia in foals and adult horses. Grossly, multifocal to segmental hemorrhage and thickening of the intestinal wall were most common in the small intestine, although the colon and cecum were also frequently affected. All horses had variable amounts of fluid, often hemorrhagic intestinal contents. The most characteristic microscopic lesion was necrotizing or necrohemorrhagic enteritis, with mucosal and/or submucosal thrombosis. Numerous gram-positive rods were occasionally seen in affected mucosa. A definitive diagnosis of C. perfringens type C enterotoxemia in all 8 cases was based on the clinical history, gross and histologic lesions, and detection of the beta toxin in intestinal contents.

The VirS/VirR two-component system regulates the anaerobic cytotoxicity, intestinal pathogenicity, and enterotoxemic lethality of Clostridium perfringens type C isolate CN3685.

Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells.

Development and application of an oral challenge mouse model for studying Clostridium perfringens type D infection.

Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.

Effect of avilamycin, tylosin and ionophore anticoccidials on Clostridium perfringens enterotoxaemia in chickens.

In order to study the prophylactic and metaphylactic effect of antomicrobial growth promoters and ionophorous anticoccidials on the incidence of Cl. perfringens enterotoxaemia in chickens, experimental attempts were performed with 675 chickens in 27 trials. The birds were intraduodenally infected with Cl. perfringens type A (ATCC 3624). The following antimicrobial growth promoters and ionophore anticoccidials were used either on their own or in combination: avilamycin, narasin, monensin and tylosin. While infected and non-medicated trials showed an average incubation period of 1 week, clinical symptoms occurred 2-4 days later in infected and medicated birds. Avilamycin medicated birds had the longest incubation period. In the infected and non-medicated trials, a mortality rate of 16%-36% was noted within 3 weeks post infection. The avilamycin trials showed a mortality rate of 0-8% (0-2 birds died) and the narasin and monensin a mortality rate of 0-8%, respectively. In the combination groups (monensin + avilamycin or narasin + avilamycin), the mortality rate ranged from 0 to 4%. Tylosin showed a very good metaphylactic/therapeutic effect against Cl. perfringens enterotoxaemia. Following infection, medicated birds showed a significantly better bodyweight gain than the chickens, whose feeds had not been supplemented. From epidemiological point of view, the systematic prevention of coccidiosis is a key in the control of Cl. perfringens enterotoxaemia in chickens.

[Study of the role of Clostridium perfringens in bovine enterotoxemia]. / Etude du rôle de Clostridium perfringens dans l'entérotoxémie bovine.

Bovine enterotoxaemia is an acute to peracute syndrome occurring mainly in calves and characterized by the sudden or very rapid death of the calf, with colics, convulsions and nervous disorders as clinical signs, if any. The most pronounced lesion is a necrohaemorrhagic enteritis of the jejunum, the ileum, and sometimes the colon. Suckling beef calves are the most frequently affected ones. In 67% of the 78 field cases investigated, some kind of stress was observed 24 to 36 hours prior to the death: change in diet or pasture, vaccination... The most frequently isolated bacteria, and the one isolated in highest numbers, was non-sporulated non-enterotoxigenic toxinotype A Clostridium perfringens. Reproduction of the lesions was successful in a ligated intestinal loop assay in one calf with a few of these strains, more especially with one of them, which was shown later to produce another recently described toxin, the beta 2 toxin. A role for this beta 2 toxin in bovine enterotoxaemia is thus speculated for future research.

Sudden death of feedlot cattle.

Sudden deaths or the sudden death syndrome are perceived as major concerns in cattle feedlots because most of these deaths occur in cattle near market weight. Etiology and preventive measures are poorly defined. The current literature indicates that sudden deaths are associated most commonly with digestive upsets. Death is thought to be the result of interactions between factors including acidosis, bloat, and endotoxemia. Trauma, peracute interstitial pneumonia, and other identifiable events are specifically defined but relatively uncommon. Enterotoxemia is of questionable significance as a cause of sudden deaths.

The characteristics and causes of sheep losses in the Victorian Mallee.

The extent and causes of sheep losses in the semi-arid Mallee region of north-western Victoria were assessed by interviewing the owners of 79 randomly selected farms running 241 flocks in 1987/88 and 245 flocks in 1988/89. Mean annual losses were higher in ram flocks (21%) than in ewe flocks (7%), in flocks of non-Merino sheep (rams 24%, ewes 11%, weaners 5%) than in Merino (rams 11%, ewes 6%, wethers 4%, weaners 4%) and in ewe flocks 3 or more years old (10%) than in young ewe flocks (3.5%). In flocks where losses exceeded 5%, the causes most often reported by farmers were blowfly strike (especially in Merino sheep and weaners), ewe losses in autumn close to lambing, and heliotrope (Heliotropium europaeum) poisoning. Heliotrope poisoning was considered by the authors to be the main reason for the higher losses in old ewes than in young ewes and in non-Merino sheep than in Merino sheep. Losses of ewes associated with pregnancy and lambing were considered by the authors to be often predisposed by liver damage caused by heliotrope poisoning, and high losses in non-Merino ram flocks were attributed to both heliotrope poisoning and their ability to escape through boundary fences. Reasons for continuing high losses due to enterotoxaemia are discussed. Losses due to gastro-intestinal parasites, footrot and foot abscess were low.

Utilization of cholestyramine resin as a preventive treatment for antibiotic (clindamycin) induced enterotoxaemia in the rabbit.

Cholestyramine, an ion exchange resin shown to bind bacterial toxins, was utilized to treat rabbits with antibiotic induced enterotoxaemia. Three groups of 6 rabbits were administered 30 mg/kg clindamycin phosphate intravenously on day 1. One group was untreated; 2 groups were treated daily by gavage with 2 g cholestyramine in 20 ml water until day 21, starting on either day 1 or 3. Daily body weights, faecal output, faecal occult blood, food and water consumption, and body temperatures were determined. Four of 6 rabbits in the untreated group either died or were moribund and euthanased. There were no deaths in either treatment groups. Dramatic decreases in food consumption (86%), water consumption (62%), and faecal output (89%) were noted within 3 days after clindamycin administration in all groups. These parameters remained depressed throughout the study. There was no clear trend in body weight changes, body temperature, or faecal occult blood test results. Cholestyramine was effective in eliminating mortality associated with the intravenous administration of clindamycin and is recommended to prevent the development of enterotoxaemia when pyrogen testing or administering antibiotics known to induce the syndrome in rabbits.

Evaluation of a toxoid for protection of rabbits against enterotoxaemia experimentally induced by trypsin-activated supernatant of Clostridium spiroforme.

Investigations were conducted into an enterotoxaemia caused by Clostridium spiroforme responsible for significant losses in commercial rabbit farms in Western Australia. Two trials using laboratory and farm bred rabbits were performed to evaluate the protective value of a toxoid prepared from the supernatant of C. spiroforme cultures against intraperitoneal challenge with the trypsin-activated toxin of C. spiroforme. The trials showed clearly that a single vaccination at weaning (four weeks) was protective against toxin but more complete and lasting protection was conferred following a second vaccination administered 14 days after the first. Adults likewise showed similar levels of protective antibodies but did not appear to pass on this protection to their kits although ELISA results indicated levels of antibody in kits from unvaccinated mother to be lower than progeny from vaccinated mothers. However antibody levels in kits from vaccinated mothers were very low and did not protect against challenge with toxin.

Pathogenicity of Clostridium perfringens for germ-free guinea pigs after oral ingestion.

Germ-free guinea pigs died with signs and lesions of acute enterotoxemia after oral ingestion of C. perfringens types B, C, D, and E. The signs and lesions observed resembled those seen in acute enterotoxemia of sheep and cattle and the naturally occurring disease seen in "ex-germ-free" guinea pigs. C. perfringens type A was found to be innocuous. Conventional guinea pigs did not become ill after ingestion of any of the five toxigenic types.