Clostridium perfringens epsilon prototoxin mutant rpETXY30A/Y71A/H106P/Y196A as a vaccine candidate against enterotoxemia.

Epsilon toxin (ETX) is secreted by Clostridium perfringens (C. perfringens)as a relatively inactive prototoxin (pETX), which is enzymatically activated to ETX by removing carboxy-terminal and amino-terminal peptides. Genetically engineered ETX mutants have been shown to function as potential vaccine candidates in the prevention of the enterotoxemia caused by C. perfringens. In the present study, two recombinant site-directed mutants of pETX, rpETXY30A/Y71A/H106P/Y196A (rpETXm41) and rpETXY30A/H106P/Y196A/F199E (rpETXm42), were synthesized by mutating four essential amino acid residues (Tyr30, Tyr71, His106, Tyr196 or Phe199). Compared to recombinant pETX (rpETX), both rpETXm41 and rpETXm42 lacked the detectable toxicity in MDCK cells and mice, which suggested that both rpETXm41 and rpETXm42 are sufficiently safe to be vaccine candidates. Despite the fact that rpETXm41 and rpETXm42 were reactogenic with polyclonal antibodies against crude ETX, both single- and double-dose vaccination (Vs and Vd, respectively) of rpETXm41 induced a higher level of IgG titer and protection in mice than that of rpETXm42. Therefore, we selected rpETXm41 for the further study. Sheep received Vs of 150 µg rpETXm41 developed significant levels of toxin-neutralizing antibodies persisting for at least 6 months, which conferred protection against crude ETX challenge without microscopic lesions. These data suggest that genetically detoxified rpETXY30A/Y71A/H106P/Y196A could form the basis of a next-generation enterotoxemia vaccine.

Enterotoxemia produced by lambda toxin-positive Clostridium perfringens type D in 2 neonatal goat kids.

Enterotoxemia caused by Clostridium perfringens type D usually affects sheep and goats ≥ 2-wk-old. The main clinical signs and lesions of the disease are produced by the epsilon toxin (ETX) elaborated by this microorganism. However, ETX is produced in the form of a mostly inactive prototoxin that requires protease cleavage for activation. It has traditionally been believed that younger animals are not affected by type D enterotoxemia given the low trypsin activity in the intestinal content associated with the trypsin-inhibitory action of colostrum. Two Nigerian dwarf goat kids, 2- and 3-d-old, with a history of acute diarrhea followed by death, were submitted for postmortem examination and diagnostic workup. Autopsy and histopathology revealed mesocolonic edema, necrosuppurative colitis, and protein-rich pulmonary edema. Alpha toxin and ETX were detected in intestinal content, and C. perfringens type D was isolated from the colon of both animals. The isolates encoded the gene for lambda toxin, a protease that has been shown previously to activate ETX in vitro. Type D enterotoxemia has not been reported previously in neonatal kids, to our knowledge, and we suggest that lambda toxin activated the ETX.

A non-toxic recombinant bivalent chimeric protein rETXm3CSAm4/TMD as a potential vaccine candidate against enterotoxemia and braxy.

Clostridium perfringens epsilon toxin (ETX) and Clostridium septicum alpha toxin (CSA) are lethal and necrotizing toxins, which play key roles in enterotoxemia and braxy of ruminants, respectively. In the present study, we synthesized a bivalent chimeric protein rETXm3CSAm4/TMD comprising ETXm3 (Y30A/H106P/Y196A) and CSAm4/TMD (C86L/N296A/H301A/W342A and a deletion of residues 212 to 222). Compared with recombinant ETX and recombinant CSA, rETXm3CSAm4/TMD showed no cytotoxicity in Madin-Darby Canine Kidney cells and was not fatal to mice. Moreover, rETXm3CSAm4/TMD could protect immunized mice against 10 × mouse LD100 of crude ETX or 3 × mouse LD100 of crude CSA without obvious histopathologic difference. Most importantly, both rabbits and sheep immunized with rETXm3CSAm4/TMD produced high titers of neutralizing antibody which protected the animals against the challenge with crude ETX or crude CSA. These data suggest that genetically detoxified rETXm3CSAm4/TMD is a potential subunit vaccine candidate against enterotoxemia and braxy.

Pathology and Pathogenesis of Brain Lesions Produced by Clostridium perfringens Type D Epsilon Toxin.

Clostridium perfringens type D epsilon toxin (ETX) produces severe, and frequently fatal, neurologic disease in ruminant livestock. The disorder is of worldwide distribution and, although vaccination has reduced its prevalence, ETX still causes substantial economic loss in livestock enterprises. The toxin is produced in the intestine as a relatively inactive prototoxin, which is subsequently fully enzymatically activated to ETX. When changed conditions in the intestinal milieu, particularly starch overload, favor rapid proliferation of this clostridial bacterium, large amounts of ETX can be elaborated. When sufficient toxin is absorbed from the intestine into the systemic circulation and reaches the brain, two neurologic syndromes can develop from this enterotoxemia. If the brain is exposed to large amounts of ETX, the lesions are fundamentally vasculocentric. The neurotoxin binds to microvascular endothelial receptors and other brain cells, the resulting damage causing increased vascular permeability and extravasation of plasma protein and abundant fluid into the brain parenchyma. While plasma protein, particularly albumin, pools largely perivascularly, the vasogenic edema becomes widely distributed in the brain, leading to a marked rise in intracranial pressure, coma, sometimes cerebellar herniation, and, eventually, often death. When smaller quantities of ETX are absorbed into the bloodstream, or livestock are partially immune, a more protracted clinical course ensues. The resulting brain injury is characterized by bilaterally symmetrical necrotic foci in certain selectively vulnerable neuroanatomic sites, termed focal symmetrical encephalomalacia. ETX has also been internationally listed as a potential bioterrorism agent. Although there are no confirmed human cases of ETX intoxication, the relatively wide species susceptibility to this toxin and its high toxicity mean it is likely that human populations would also be vulnerable to its neurotoxic actions. While the pathogenesis of ETX toxicity in the brain is incompletely understood, the putative mechanisms involved in neural lesion development are discussed.

Potential Therapeutic Effects of Mepacrine against Clostridium perfringens Enterotoxin in a Mouse Model of Enterotoxemia.

Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin that causes the symptoms of common bacterial food poisoning and several non-foodborne human gastrointestinal diseases, including antibiotic-associated diarrhea and sporadic diarrhea. In some cases, CPE-mediated disease can be very severe or fatal due to the involvement of enterotoxemia. Therefore, the development of potential therapeutics against CPE action during enterotoxemia is warranted. Mepacrine, an acridine derivative drug with broad-spectrum effects on pores and channels in mammalian membranes, has been used to treat protozoal intestinal infections in human patients. A previous study showed that the presence of mepacrine inhibits CPE-induced pore formation and activity in enterocyte-like Caco-2 cells, reducing the cytotoxicity caused by this toxin in vitro Whether mepacrine is similarly protective against CPE action in vivo has not been tested. When the current study evaluated whether mepacrine protects against CPE-induced death and intestinal damage using a murine ligated intestinal loop model, mepacrine protected mice from the enterotoxemic lethality caused by CPE. This protection was accompanied by a reduction in the severity of intestinal lesions induced by the toxin. Mepacrine did not reduce CPE pore formation in the intestine but inhibited absorption of the toxin into the blood of some mice. Protection from enterotoxemic death correlated with the ability of this drug to reduce CPE-induced hyperpotassemia. These in vivo findings, coupled with previous in vitro studies, support mepacrine as a potential therapeutic against CPE-mediated enterotoxemic disease.

Intramural Vascular Edema in the Brain of Goats With Clostridium perfringens Type D Enterotoxemia.

Enterotoxemia caused by Clostridium perfringens type D is an important disease of sheep and goats with a worldwide distribution. Cerebral microangiopathy is considered pathognomonic for ovine enterotoxemia and is seen in most cases of the disorder in sheep. However, these lesions are poorly described in goats. In this article, we describe the vasculocentric brain lesions in 44 cases of caprine spontaneous C. perfringens type D enterotoxemia. Only 1 goat had gross changes in the brain, which consisted of mild cerebellar coning. However, 8 of 44 (18%) cases showed microscopic brain lesions, characterized by intramural vascular proteinaceous edema, a novel and diagnostically significant finding. The precise location of the edema was better observed with periodic acid-Schiff, Gomori's, and albumin stains. Glial fibrillary acidic protein and aquaporin 4 immunostaining revealed strong immunolabeling of astrocyte foot processes surrounding microvessels. The areas of the brain most frequently affected were the cerebral cortex, corpus striatum (basal ganglia), and cerebellar peduncles, and both arterioles and venules were involved. Most of the goats of this study showed lesions in the intestine (enteritis, colitis, and typhlitis), although pulmonary congestion and edema, hydrothorax, hydropericardium, and ascites were also described. Although the intramural edema described, for the first time, in these caprine cases is useful for the diagnosis of enterotoxemia when observed, its absence cannot exclude the disease.

New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia.

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is responsible for diseases that occur mostly in ruminants. ETX is produced in the form of an inactive prototoxin that becomes proteolytically-activated by several proteases. A recent ex vivo study using caprine intestinal contents demonstrated that ETX prototoxin is processed in a step-wise fashion into a stable, active ∼27 kDa band on SDS-PAGE. When characterized further by mass spectrometry, the stable ∼27 kDa band was shown to contain three ETX species with varying C-terminal residues; each of these ETX species is cytotoxic. This study also demonstrated that, in addition to trypsin and chymotrypsin, proteases such as carboxypeptidases are involved in processing ETX prototoxin. Once absorbed, activated ETX species travel to several internal organs, including the brain, where this toxin acts on the vasculature to cross the blood-brain barrier, produces perivascular edema and affects several types of brain cells including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, affected animals show edema within the vascular walls. This edema separates the astrocytic end-feet from affected blood vessels, causing hypoxia of nervous system tissue. Astrocytes of rats and sheep affected by ETX show overexpression of aquaporin-4, a membrane channel protein that is believed to help remove water from affected perivascular spaces in an attempt to resolve the perivascular edema. Amyloid precursor protein, an early astrocyte damage indicator, is also observed in the brains of affected sheep. These results show that ETX activation in vivo seems to be more complex than previously thought and this toxin acts on the brain, affecting vascular permeability, but also damaging neurons and other cells.

Comparative neuropathology of ovine enterotoxemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives.

Clostridium perfringens type D causes enterotoxemia in sheep and goats. The disease is mediated by epsilon toxin (ETX), which affects the cerebrovascular endothelium, increasing vascular permeability and leading to cerebral edema. In the present study, we compared the distribution and severity of the cerebrovascular changes induced in lambs by C. perfringens type D strain CN1020, its isogenic etx null mutant, and the ETX-producing complemented mutant. We also applied histochemical and immunohistochemical markers to further characterize the brain lesions induced by ETX. Both ETX-producing strains induced extensive cerebrovascular damage that did not differ significantly between each other in nature, neuroanatomic distribution, or severity. By contrast, lambs inoculated with the etx mutant or sterile, nontoxic culture medium did not develop detectable brain lesions, confirming that the neuropathologic effects observed in these infections are dependent on ETX production. Lambs treated with the wild-type and complemented strains showed perivascular and mural vascular edema, as well as serum albumin extravasation, particularly severe in the cerebral white matter, midbrain, medulla oblongata, and cerebellum. Brains of animals inoculated with the ETX-producing strains showed decreased expression of glial fibrillary acidic protein and increased expression of aquaporin-4 in the end-feet processes of the astrocytes around blood vessels. Early axonal injury was demonstrated with anti-amyloid precursor protein immunohistochemistry. Perivascular accumulation of macrophages/microglia with intracytoplasmic albumin globules was also observed in these animals. This study demonstrates that ETX is responsible for the major cerebrovascular changes in C. perfringens type D-induced disease.

Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known.

Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains and causes enterotoxemia, a highly lethal disease with major impacts on the farming of domestic ruminants, particularly sheep. ETX belongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to other toxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this high potency, ETX is considered as a potential bioterrorism agent and has been classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States. The protoxin is converted to an active toxin through proteolytic cleavage performed by specific proteases. ETX is absorbed and acts locally in the intestines then subsequently binds to and causes lesions in other organs, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine and its possible use as a biological weapon have drawn the attention of researchers and have led to a large number of studies investigating ETX. The aim of the present work is to review the existing knowledge on ETX from C. perfringens type B and D.

Diagnostic exercise: hemolysis and sudden death in lambs.

Within a 24-hour period, 7 out of 200 three- to four-week-old pastured Katahdin lambs died after showing clinical signs of hemoglobinuria, red-tinged feces, weakness, and recumbency. One of the lambs that was examined clinically before natural death also had abdominal pain, trembling, tachycardia, and severe anemia with a packed cell volume of 4%. Pathologic findings included icterus, hemoglobinuric nephrosis, dark red urine, pulmonary edema, hydrothorax, splenomegaly, and acute centrilobular to midzonal hepatocellular degeneration and necrosis with cholestasis. The differential diagnoses and diagnostic workup to achieve the diagnosis are briefly discussed.

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).

lesion development in a new intestinal loop model indicates the involvement of a shared Clostridium perfringens virulence factor in haemorrhagic enteritis in calves.

Clostridium perfringens-associated enterotoxaemia is a fatal disease in fast growing suckler and veal calves. An intestinal loop model was developed to study the pathogenesis of the disease. Loops were injected with stationary and logarithmic C. perfringens cultures with or without, a milk protein-based commercial milk replacer for calves. Isolates tested were from cases of bovine enterotoxaemia and from calves without signs of enterotoxaemia, in addition to netB-positive and -negative isolates from poultry, a type C isolate from piglets and the human isolate JIR325. All isolates induced necrohaemorrhagic lesions in combination with milk replacer, while all control loops (i.e. medium plus milk replacer) remained histologically normal. In addition, time-course experiments were conducted using an isolate from an outbreak of bovine enterotoxaemia. Histological examination showed that the earliest lesion was congestion of the capillaries, starting within 30 min of inoculation. Haemorrhage and mucosal necrosis began at the tips of the villi 3-4 h after bacterial inoculation. These lesions are similar to those observed in natural cases of bovine enterotoxaemia. Therefore, in this model, necrohaemorrhagic lesions can be induced by C. perfringens isolates from diverse origins, suggesting that the lesions may be caused by one or more virulence factors that are shared by these isolates.

The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats.

Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.

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.

Focal symmetrical encephalomalacia in a goat.

Focal symmetrical encephalomalacia (FSE) is the most prominent lesion seen in the chronic form of enterotoxemia caused by Clostridium perfringens type D in sheep. However, this lesion has not been reported in goats. The current paper reports a case of FSE in a goat from the state of Paraíba in the Brazilian semiarid region. As reported by the farmer, 30, 4-48-month-old animals from a flock of 150 goats died after showing nervous signs, including blindness and recumbence, for periods varying between 1 and 14 days. The flock was grazing native pasture supplemented with wheat and corn bran. Additionally, lactating goats were supplemented with soybeans. A 4-month-old goat with nervous signs was examined clinically and then necropsied 3 days after the onset of clinical signs. Bilateral, focal, and symmetrical areas of brown discoloration were observed in the internal capsule and thalamus. Histologic lesions in these areas consisted of multifocal, bilateral malacia with a few neutrophils; endothelial cell swelling; perivascular edema; and hemorrhages. The etiology of these lesions was not determined. However, FSE is considered pathognomonic for C. perfringens type D enterotoxemia in sheep, and it is speculated that this microorganism was the etiologic agent in the present case. The flock had been vaccinated against type D enterotoxemia only once, approximately 3 months before the beginning of the outbreak. Insufficient immunity due to the incorrect vaccination protocol, low efficacy of the vaccine used, and a diet including large amounts of highly fermentable carbohydrates were suspected to be predisposing factors for this outbreak.

Eimeria macusaniensis associated lesions in neonate alpacas dying from enterotoxemia.

Histopathological analysis of 108 intestine samples (103 grossly affected ileum and 5 jejunum) taken from Clostridium-induced neonatal alpaca (Vicugna pacos) enterotoxemia mortalities collected in the Departments of Arequipa, Puno and Cusco of southern Peru during the 2005-2008 birth seasons (January-March), revealed the presence of large numbers of both asexual and sexual stages of Eimeria macusaniensis in 33/108 (30.55%) of the samples with moderate to severe necrotized and/or hemorrhagic enteritis. It is proposed that damage to the mucosa produced by coccidial infections may facilitate overgrowth of Clostridium perfringens with toxin production leading to fatal enterotoxemia.

Development and application of new mouse models to study the pathogenesis of Clostridium perfringens type C Enterotoxemias.

Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.

Clinicopathologic features of experimental Clostridium perfringens type D enterotoxemia in cattle.

This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.

Lesions in the brains of three cattle resembling the lesions of enterotoxaemia in lambs.

CASE HISTORY: A 3-month-old female Angus calf was found dead, and two adult Friesian dairy cows died soon after developing nervous signs. PATHOLOGICAL FINDINGS: Grossly, bilateral and mostly symmetrical areas of haemorrhage were evident that mainly involved areas of grey matter in the brainstem from the level of the caudal colliculi to the thalamus and, in one, the internal capsule and caudate nucleus. In the occipital and caudal parietal cortex, there was extensive oedema of white matter. Histologically, in addition to haemorrhage, there was protein-rich oedema around arterioles and venules in the cerebrum, hippocampus, internal capsule, thalamus, midbrain, dorsal medulla, and central cerebellar and cerebellar folial white matter. The calf 's brain had bilateral and symmetrical oedema and necrosis affecting several brainstem nuclei and the occipital grey matter overlying areas of oedema of the corona radiata. DIAGNOSIS: Although the cause was not established, the perivascular lesions resembled those produced in calves by the intravenous administration of epsilon toxin. CLINICAL RELEVANCE: It is possible that epsilon toxin-induced enterotoxaemia occurs naturally in cattle, and where bilateral haemorrhage is recognised in the brains of cattle, small intestinal contents should be collected for analysis of epsilon toxin.

The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep.

The pathological findings in sheep with peracute experimental Clostridium perfringens type D enterotoxemia are described. Of 16 animals inoculated intraduodenally with a whole culture of this microorganism and a starch solution in the abomasum, 12 developed clinical signs including increased respiratory efforts, recumbency, paddling, bleating, convulsions, blindness, and opisthotonus. Diarrhea was not observed in any of the animals. The time lapse between the beginning of intraduodenal infusion and onset of clinical signs varied between 30 minutes and 26 hours, and the clinical course varied between 1 and 9 hours. Gross postmortem changes were observed in these 12 animals and included pulmonary edema; excess pericardial, peritoneal, or pleural fluid with or without strands of fibrin; liquid small intestinal contents; leptomeningeal edema; cerebellar coning; and subcapsular petechiae on kidneys. Histological changes consisted of severe edema of pleura and interlobular septa and around blood vessels and airways and acidophilic, homogeneous, proteinaceous perivascular edema in the brain. Five of 12 animals (42%) with clinical signs consistent with enterotoxemia lacked specific histological lesions in the brain. None of the intoxicated or control animals developed nephrosis. Glucose was detected in the urine of 3 of 6 animals that were tested for this analyte. These results stress the importance of the use of histological examination of the brain, coupled with epsilon toxin detection, for a definitive diagnosis of C. perfringens type D enterotoxemia in sheep.