Diagnosis of Clostridium perfringens intestinal infections in sheep and goats.

Clostridium perfringens produces enteric diseases, generically called enterotoxemias, in sheep, goats, and other animals. This microorganism can be a normal inhabitant of the intestine of most animal species, including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates and produces potent toxins that act locally or are absorbed into the general circulation with usually devastating effects on the host. History, clinical signs, and gross postmortem findings are useful tools for establishing a presumptive diagnosis of clostridial enterotoxemia in sheep and goats. Definitive diagnosis requires laboratory confirmation. Isolation of some types of C. perfringens (e.g., B and C) can be of diagnostic value, but other types (e.g., A) are so commonly found in the intestine of normal animals that isolation is meaningless from a diagnostic point of view. The most accepted criterion in establishing a definitive diagnosis of enterotoxemia is detection of C. perfringens toxins in intestinal contents. Also, histopathological examination of brain is very useful for diagnosis of type D disease, as lesions produced by epsilon toxin in the brains of sheep and goats are pathognomonic for type D enterotoxemia. Ancillary tests, such as measuring urine glucose or observing Gram-stained smears of intestinal mucosa, can be used. However, although such tests have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxemia when negative.     

Clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: 13 cases (1979-1982).

Enterotoxemia attributable to Clostridium perfringens type D in goats is difficult to diagnose because of a lack of specific clinical signs or postmortem lesions, on which to base the diagnosis. This report describes the clinical signs, postmortem lesions, and clinical responses to treatment and vaccination in 4 goat herds, in which a diagnosis of enterotoxemia was confirmed. Four clinical cases had the diagnosis confirmed on the basis of signs of diarrhea or sudden death and the isolation of C perfringens and epsilon toxin from the feces at the time of admission. The 10 necropsy cases were diagnosed on the basis of the isolation of C perfringens (not typed) or epsilon toxin from the intestinal contents of goats that died with clinical signs compatible with enterotoxemia and without lesions associated with a second serious disease. Enterocolitis was the most consistent lesion reported at necropsy in the 10 goats with enterotoxemia. Ovine enterotoxemia vaccines were of limited value in preventing enterotoxemia. These observations imply that naturally induced enterotoxemia in goats involves a different pathophysiologic mechanism than that associated with enterotoxemia in sheep.     

Differences in signs and lesions in sheep and goats with enterotoxemia induced by intraduodenal infusion of Clostridium perfringens type D

Enterotoxemia was induced in 4 lambs and 4 goat kids by continuous intraduodenal infusion of a whole culture of Clostridium perfringens type D. Clinical signs, hematologic values, biochemical alterations, and postmortem lesions in the lambs and goat kids were compared. The 4 lambs and 4 goat kids died within 25 hours of beginning the infusions. Lesions were not observed in the gastrointestinal tract of the 4 lambs; however, severe hemorrhagic enterocolitis was found in the 4 goat kids. This difference between the lambs and goat kids in the lesions caused by experimentally induced enterotoxemia may explain the discrepancies reported between sheep and goats in clinical signs, response to treatment, and efficacy of vaccination observed in naturally induced enterotoxemia in the 2 species.     

Ulcerative enterocolitis in two goats associated with enterotoxin- and beta2 toxin-positive Clostridium perfringens type D

Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa.     

产气荚膜梭菌ε毒素及其疫苗研究进展

产气荚膜梭菌病是由产气荚膜梭菌引起的一种重要的人兽共患传染病,可导致山羊、绵羊等动物的肠毒血症或坏死性肠炎,并且可引起动物脑、心、肺和肾组织的水肿.B型和D 型产气荚膜梭菌所产生的ε毒素是引起动物上述病理变化和死亡的重要因素之一.虽然甲醛灭活的毒素疫苗能对动物产生保护性抗体,但是,灭活苗潜在的安全性原因使其在应用上受到限制.因此,基于ε毒素基因的重组疫苗和弱毒疫苗就成为人们研究的目标.     

Naturally acquired antibodies against Clostridium perfringens epsilon toxin in goats

Clostridium perfringens type D-producing epsilon toxin is a common cause of death in sheep and goats worldwide. Although anti-epsilon toxin serum antibodies have been detected in healthy non-vaccinated sheep, the information regarding naturally acquired antibodies in ruminants is scanty. The objective of the present report was to characterize the development of naturally acquired antibodies against C. perfringens epsilon toxin in goats. The levels of anti-epsilon toxin antibodies in blood serum of goat kids from two different herds were examined continuously for 14 months. Goats were not vaccinated against any clostridial disease and received heterologous colostrums from cows that were not vaccinated against any clostridial disease. During the survey one of these flocks suffered an unexpectedly severe C. perfringens type D enterotoxemia outbreak. The results showed that natural acquired antibodies against C. perfringens epsilon toxin can appear as early as 6 weeks in young goats and increase with the age without evidence of clinical disease. The enterotoxemia outbreak was coincident with a significant increase in the level of anti-epsilon toxin antibodies.     

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.     

Photosenitization of sheep on kleingrass pasture

The clinical appearance and serum gamma-glutamyl transpeptidase (GGT) activity were studied in 5 groups of sheep (12 per group) on kleingrass (Panicum coloratum) pasture plots and in 1 group of sheep (10 animals) on native buffalograss (Buchlo? dactyloides) pasture. Eleven sheep (at least 1 from each group on kleingrass pasture plots) had elevated serum GGT activity. Seven of 11 sheep with elevated serum GGT activity developed signs of photosensitization. None of the sheep on buffalograss pasture developed signs of photosensitization or elevated GGT activity. The pathologic findings were similar in the sheep that had signs of photosensitization. Grossly, there was icterus and subcutaneous edema. The livers had tapeworms (Thysanosoma actinioides) in the bile ducts, were slightly swollen, and varied in color from yellow to ochre in severe cases of biliary system derangements. Microscopically, there was cholangitis.     

Experimental production of hemorrhagic enterotoxemia by Clostridium perfringens type C in maturing lambs.

Maturing lambs, eight to nine months old, were dosed by the intraduodenal route with various preparations of Clostridium perfringens type C. Whole cultures of this organism or cells suspended in fresh medium, both supplemented with soybean flour as a protease inhibitor, produced acute fatal hemorrhagic enterotoxemia in these animals. The latter preparation was more effective than the former in causing disease. Without the soybean supplement the inocula did not produce fatal disease. Dosing with toxic cell-free culture supernatant fluid, with or without soybean supplement, had no lethal effect. Animals that died showed severe hemorrhagic enteritis with necrosis and sloughing of the mucosal epithelium, involving jejunum, ileum and part of duodenum. These lesions were similar to those seen in natural cases of hemorrhagic enterotoxemia in neonatal animals. This experiment demonstrated that nonimmune animals are normally protected against C. perfringens type C enterotoxemia by adequate levels of pancreatic proteases in the intestine, and that factors which inhibit or reduce these enzymes predispose animals for the development of this disease.     

Bacterial meningitis and diseases caused by bacterial toxins

Bacterial meningitis most commonly occurs in young calves secondary to septicemia. Clinical signs of hyperirritability are usually seen. Meningitis can be confirmed by cerebrospinal fluid analysis and culture or by necropsy. Intoxications by the exotoxins of Clostridium perfringens types C and D, C. botulinum, and C. tetani are difficult to confirm. The clinical signs of these intoxications vary from flaccid paralysis (botulism) to muscular rigidity (tetanus). Treatment of affected cattle has been unrewarding in botulism and enterotoxemia, whereas early aggressive treatment of tetanus cases can often be successfully resolved. Botulism and enterotoxemia can be proved using mouse inoculation tests, whereas tetanus is diagnosed largely by ruling out other diseases.     

Clostridium perfringens in Animal Disease: A Review of Current Knowledge

The diseases caused by various types of Clostridium perfringens are critically reviewed in the light of current knowledge. Particular emphasis is placed on information concerning these diseases in Canadian livestock.There are two etiologically clearly-defined acute C. perfringens diseases recognized in Canada: hemorrhagic enteritis of the new born calf, caused by C. perfringens type C, and enterotoxemia of sheep, caused by type D. Clostridium perfringens type A may play a role as a secondary pathological agent in various disease conditions, such as necrotic enteritis of chickens. It may also cause wound infections and may provide a source for human food poisoning outbreaks.There appears to be a considerable lack of knowledge regarding the distribution of C. perfringens types, their pathogenesis, diagnosis and the incidence of diseases caused by this organism.     

A survey of more than 11 years of neurologic diseases of ruminants with special reference to transmissible spongiform encephalopathies (TSEs) in Greece

The first cases of scrapie were detected in Greece in a flock of sheep in October 1986. All the animals of the affected flock and all sheep in two flocks that were in contact were killed and buried. A systematic investigation of all available cases with signs indicating a neurological disease started in sheep and goats in late 1986, as well as in cattle in 1989. The investigation was based on clinical examination, necropsy or macroscopical examination of the brain and viscera, and histological examination of the brain in all animals except those with coenurosis. Histological examinations of specimens from the spinal cord and other tissues, and if considered necessary bacteriological, toxicological and serological examinations were also carried out. In October 1997, scrapie was diagnosed in sheep of a second flock (a mixed flock of sheep and goats), grazing in a pasture close to the place where scrapie was initially detected. All animals of the second flock were also killed and buried. Diagnosis in the first flock was based on clinical signs and histological lesions, and in the second immunoblotting was also used. Distinctive lesions of scrapie were found in the brain and/or the spinal cord of eight sheep with clinical signs from the two flocks. The lesions were revealed in the brain stem and/or in the cervical spinal cord, and tended to be symmetrical. In one sheep, severe lesions in the cortex of cerebral hemispheres and of the cerebellum were also found. In the brain of two sheep from the second flock the pathological isoform of PrP protein was detected. Despite the eradication scheme applied, scrapie in sheep reappeared after 11 years in a place close to where it occurred initially. This may indicate that the effectiveness of the eradication scheme implemented was not adequate and additional approaches may be needed.     

Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model

Epsilon toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice were injected intravenously or intragastrically with CPA or PFO with or without ETX. Sublethal doses of CPA or PFO did not affect the lethality of ETX when either was injected together with the latter intravenously. However, sublethal or lethal doses of CPA or PFO resulted in reduction of the survival time of mice injected simultaneously with ETX when compared with the intravenous effect of ETX injected alone. When PFO was inoculated intragastrically with ETX, a reduction of the survival time was observed. CPA did not alter the survival time when inoculated intragastrically with ETX. The results of the present study suggest that both CPA and PFO have the potential to enhance the ETX lethal effects during enterotoxemia in natural hosts such as sheep and goats.     

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep

Objective To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.
Design A clinical and pathological study.
Procedure Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies.
Results All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection.
Conclusions The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.     

Genotyping of Clostridium perfringens isolated from domestic and exotic ruminants and swine

Clostridium perfringens types A, B, C, D and E are known to cause severe enteritis/enterotoxaemia and diseases (especially caused by type A) belonging to the gas oedema complex in many species. Samples from the small intestine as well as faeces of domestic and exotic animals suffering from enterotoxaemic signs or having died within days after first occurance of toxaemia were submitted for typing C. perfringens toxovars by multiplex PCR. The following species have been investigated: domestic sheep (Ovis ammon; n = 10), domestic goat (Capra aegagrus hircus; n = 26), Japanese serow (Capricornis sumatraensis; n = 4), lechwe waterbuck (Hydrotragus leche; n = 1), blackbuck (Antilope cervicapra; n = 1), European reindeer (Rangifer tarandus tarandus; n = 4), domestic swine (Sus scrofa; n = 52), and collared peccary (Tayassu albirostris; n = 1). Interestingly, the predominant C. perfringens toxovar in domestic sheep was type A. This toxovar could also be diagnosed in all reindeer, in three Japanese serows, one lechwe waterbuck and most pigs (n = 47), the majority of those being at suckling age. Type D was the most prevalent toxovar (n = 18) in domestic goats, but also types A and E could be identified as pathogens in this species. Type C could only be found in domestic swine (n = 5) and in one case of clostridiosis in a Japanese serow. Two cases of enterotoxaemia in goats, one case in reindeer, and a single case in blackbuck and collared peccary were caused by C. perfringens type E. Genotyping of C. perfringens is recommended before starting vaccination programmes as it could be shown, that the importance of specific toxovars has been underestimated in specific species and/or age groups.     

Transmission of capripoxvirus

The transmission of capripoxvirus to sheep, using an aerosol suspension of a Yemen isolate of the virus, was demonstrated. Capripoxvirus was also transmitted by contact to sheep and goats kept with animals infected with virus isolates from the Yemen, Sudan, India and Nigeria. The incubation period for capripoxvirus infection in sheep and goats was approximately eight to 12 days. Animals that had well developed clinical signs transmitted capripoxvirus more rapidly than animals which died of peracute disease or animals that had only mild clinical signs.     

Aquaporin-4 in acute cerebral edema produced by Clostridium perfringens type D epsilon toxin

Sheep, particularly lambs, with high circulating levels of Clostridium perfringens type D epsilon toxin develop severe neurologic signs and often die suddenly. On microscopic examination, in the brain, there is microvascular endothelial injury and diffuse vasogenic edema. The aquaporin (AQP) family of membrane water-channel proteins, especially AQP-4, is important in the regulation of water balance in the brain and facilitates reabsorption of excess fluid. In rats given epsilon toxin, generalized cerebral edema was demonstrated by marked albumin extravasation and was correlated with widespread upregulation of AQP-4 in astrocytes. These results suggest that AQP-4 has a role in the clearance of edema fluid from brains damaged by this clostridial toxin.     

Clostridium perfringens toxin types in hooded seals in the Greenland Sea, determined by PCR and ELISA

Very little is known about the occurrence of Clostridium perfringens and of diseases caused by this anaerobic bacterium in marine mammals, especially those that are free-living. During a scientific expedition to the Greenland Sea (West Ice) in spring 1999, faeces samples from 70 hooded seals (Cystophora cristata) were taken to isolate C. perfiringens. Subsequently, PCR analysis of the isolates was performed with oligonucleotide primers of the genes encoding the four major lethal toxins (alpha, beta, epsilon and iota) for classification of toxin type and of the genes encoding C. perfringens beta2-toxin and enterotoxin for further subclassification. In addition, a commercial ELISA kit for detection of C. perfringens alpha, beta- and epsilon-toxin was used. C. perfingens was isolated in samples from 38 (54.3%) hooded seals. All isolates were C. perfringens toxin type A (alpha-toxin positive). This is the first report on the occurrence of C. perfringens in this arctic marine mammal species. Myositis and enterotoxemia caused by C. perfrigens were described in other marine mammals and it may be assumed that the pathogenesis of an outbreak of disease is similar to that encountered in terrestrial animals. Although there is some controversy surrounding the enteropathogenicity and virulence of alpha-toxin (concerning enterotoxemia), this study suggests that a possible outbreak of enterotoxemia caused by C. perfringens type A in hooded seals may, however, not be excluded.     

Isolation and characterization of Clostridium perfringens from apparently healthy animals of the Shandong province of China

In a pilot study the presence and frequency of Clostridium (C.) perfringens was investigated among apparently healthy farm animals in the Shandong province of China. 748 faecal samples were collected from 9 pig-, 4 sheep-, 7 cattle- and 5 rabbit farms. C. perfringens was isolated from 124 samples (16.6%). The isolates were classified into major toxin types by using PCR analysis detecting the genes encoding these toxins. All isolates were identified as C perfringens toxin type A. There are also some reports from different regions in China linking C. perfringens toxin type A strains to gastrointestinal diseases. Therefore further investigations about the epidemiologic role of C perfringens toxin type A strains in the Shandong region are necessary. Currently, cases of enterotoxemia from this region are investigated for the presence of C perfringens.