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1.
Uzal  F.A.  Rolfe  B.E.  Smith  N.J.  Thomas  A.C.  Kelly  W.R. 《Veterinary research communications》1999,23(5):275-284
Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro.  相似文献   

2.
Clostridium perfringens isolated from lambs with dysentery (n=117) were analysed by a DNA amplification technique, the polymerase chain reaction (PCR), in order to determine the prevalence of the alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes. The most prevalent toxin type of C. perfringens found was type B, containing the alpha-, beta-, and epsilon-toxin genes, representing 46% of the cases with clostridial dysentery. C. perfringens type C containing the alpha-, and beta-toxin genes was isolated in 20% and type D, which is characterized by the alpha- and epsilon-toxin genes, was isolated in 28% of all isolates. The recently discovered, not yet assigned beta 2-toxigenic type of C. perfringens was represented in 6% of all isolates. No C. perfringens type A containing the alpha-toxin alone and no type E, which harbours the ADP-ribosylating iota-toxin, were found in the diseased animals. None of the samples contained the enterotoxin gene. Only one type of C. perfringens was found in a given herd, revealing the epidemiological use of PCR toxin gene typing of C. perfringens. The animals originated from 79 different herds with sizes ranging from 30 to 250 animals, bred in the area of northern Greece.  相似文献   

3.
产气荚膜梭菌ε毒素基因的克隆、表达及其抗血清的制备   总被引：2，自引：0，他引：2  
PCR扩增B型产气荚膜梭菌C58-2株ε毒素完整基因,并将其插入到pGEM-TEasy载体中,构建克隆载体pGEM-T-ε。对克隆载体pGEM-T-ε进行双酶切,将得到的918bp片段以正确的阅读框架定向克隆于pET-28a(+)中,然后将重组质粒转化进宿主菌BL21(DE3)中,在37℃1mmol/LIPTG诱导下该基因获得良好表达。经SDS-PAGE分析,其表达的蛋白约为36600,与预期值一致。Western-blotting试验显示,该重组蛋白可被D型产气荚膜梭菌血清识别,表明该重组蛋白具备天然毒素相似的反应原性。重组ε毒素蛋白在菌液上清、超声波裂解上清和包涵体中均有分布,表明重组蛋白可同时以胞外、周质和胞浆的形式表达,且以周质方式为主。重组蛋白在胰酶活化前后均可致死小鼠,活化后毒力可为原来的150倍。以重组ε毒素蛋白作为抗原免疫家兔制备血清,效价测定结果表明,重组ε毒素蛋白抗血清每毫升可中和30000个小鼠MLD。毒素-抗毒素中和试验和琼脂扩散试验均表明,该抗血清具有ε毒素特异性。  相似文献   

4.
Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before oligomerization, pore formation and cell death. Still, there is little information about the cellular and molecular mechanism involved in the initial steps of the cytotoxic action of ε-toxin, including the specific binding to the target sensitive cells. In the present report, the binding step of ε-toxin to the MDCK cell line is characterized by means of an ELISA-based binding assay with recombinant ε-toxin-green fluorescence protein (ε-toxin-GFP) and ε-prototoxin-GFP. In addition, different treatments with Pronase E, detergents, N-glycosidase F and beta-elimination on MDCK cells and renal cryosections have been performed to further characterize the ε-toxin binding. The ELISA assays revealed a single binding site with a similar dissociation constant (K(d)) for ε-toxin-GFP and ε-prototoxin-GFP, but a three-fold increase in B(max) levels in the case of ε-toxin-GFP. Double staining on kidney cryoslices with lectins and ε-prototoxin-GFP revealed specific binding to distal and collecting tubule cells. In addition, experiments on kidney and bladder cryoslices demonstrated the specific binding to distal tubule of a range of mammalian renal systems. Pronase E and beta-elimination treatments on kidney cryoslices and MDCK cells revealed that the binding of ε-toxin in renal system is mediated by a O-glycoprotein. Detergent treatments revealed that the integrity of the plasma membrane is required for the binding of ε-toxin to its receptor.  相似文献   

5.
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.  相似文献   

6.
Nineteen Clostridium perfringens Type C strains and ten foreign control strains of subtypes C1, C3, and C4 were tested for their toxin formation and spore resistance to heat. The 19 Type C strains had been isolated from unweaned piglets in the context of necrotising enteritis outbreaks in the GDR. The Clostridium perfringens Type C strains formed beta-toxin, but they failed to form epsilon-toxin or gamma-toxin, alpha-toxin was successfully recorded from 15 of the 19 strains tested from unweaned piglets. The minor-lethal toxin fractions were also tested, with delta-toxin being recorded from all strains, non-alpha-delta-theta-toxin from six, theta-toxin from five, and K-toxin from one. Tests for delta-toxin, lambda-toxin, and mu-toxin were negative. The Clostridium perfringens Type C strains isolated in the GDR from unweaned piglets with necrotising enteritis were, basically, identical with those described in Denmark by von Hogh (1967) with regard to toxin formation. Clostridium perfringens strains cultured in broilers with necrotising enteritis were characterised by regular toxin production in the context of alpha, theta, delta as well as non-alpha-delta-theta. They failed to form beta, epsilon, gamma and lambda, while mu-toxin was formed by them quite irregularly. They, consequently, are Type A strains. Resistance to chloramphenicol and/or oxytetracycline was exhibited by 78.5 per cent of 237 tested Clostridium perfringens strains which had been isolated from unweaned piglets and broilers with necrotosing enteritis. Multiple resistance was recorded from 33.9 per cent. All strains were susceptible to penicillin, nitrofurantoin, and erythromycin.  相似文献   

7.
确定C型产气英膜梭菌合成培养基使用参数及建立配套的毒素浓缩工艺。比较不同pH值、灭菌温度、配制用水的合成培养基及其不同培养时间和温度下培养产气荚膜梭菌CVCC60102株的毒力;按毒素的分子量及超滤膜的截留分子量设计并比较2种不同浓缩工艺的毒素收获率及透出液的毒力。结果表明,pH值为8．0～8．4、灭菌方式为116℃30min、配制用水为去离子水时产毒最佳,毒力达到500—1000MLD／mL;合成培养基培养产气英膜梭菌CVCC60102株18h后毒力达500—1000MLD／mL,随着时间的延长,毒力不再增强;培养温度为36℃或37℃时,产毒效果最佳;不同截留分子量的超滤膜浓缩,10ku截留分子量的收获率为68％,其透出液静脉接种0．2mL,小鼠2／2死亡;而8ku收获率达80％,其透出液静脉接种0．2mL,小鼠0／2死亡。因此8ku截留分子量膜包适宜对C型菌培养毒素的浓缩。上述结果为C型产气荚膜梭菌合成培养基的应用提供了数据支撑。  相似文献   

8.
The aim of this work was to investigate the potential interactions between intestinal absorbance and ricin poisoning. The Caco-2 cell monolayer and everted intestinal sac (VEIS) models were used. The distribution of ricin in CD-1 mice intoxicated with 0.1 mg/kg of ricin intragastrically was determined by immunohistochemistry. The results showed that ricin could not transfer across the healthy Caco-2 cell monolayer within three hours after poisoning. However, it could pass through the everted rat intestinal wall after 0.5 h of incubation. The toxin in the liver, spleen, lungs and kidneys of mice could be detected as early as 1 h after intoxication. The pathological results were in accordance with the cytotoxicities of ricin in Caco-2, HepG 2, H1299 and MDCK cells, indicating that though no significant symptom in mice could be observed within 3 h after ricin intoxication, important tissues, especially the kidneys, were being injured by the toxin and that the injuries were progressing.  相似文献   

9.
Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4 degrees C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpbl and etx genes were detected in all isolates from samples stored either at room temperature or at 4 degrees C for 24-44 h. Repeated aerobic treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem.  相似文献   

10.
The absorption, metabolism and excretion of 3-acetyldeoxynivalenol (3-aDON) in pigs were studied. Pigs with a faecal microflora known to be able to de-epoxidate trichothecenes were used in the experiment. The pigs were fed a commercial diet with 3-aDON added in a concentration of 2.5 mg/kg feed for 2.5 days. No traces of 3-aDON or its de-epoxide metabolite were found in plasma, urine or faeces. Deoxynivalenol (DON) was detected in plasma as soon as 20 min after start of the feeding. The maximum concentration of DON in plasma was reached after 3 h and decreased rapidly thereafter. Only low concentrations close to the detection limit were found in plasma 8 h after start of the feeding. A significant part of the DON in plasma was in a glucuronide-conjugated form (42 +/- 7%). No accumulation of DON occurred in plasma during the 60 h of exposure. The excretion of DON was mainly in urine (45 +/- 26% of the toxin ingested by the pigs) and only low amounts of metabolites of 3-aDON (2 +/- 0.4%) were recovered in faeces. De-epoxide DON constituted 52 +/- 15% of the total amount of 3-aDON-metabolites detected in faeces. The remaining part in faeces was DON. DON was still present in the urine and faeces at the end of the sampling period 48 h after the last exposure. The results show that no de-epoxides are found in plasma or urine in pigs after trichothecene exposure, even in pigs having a faecal microflora with a de-epoxidation activity. The acetylated form of the toxin is deacetylated in vivo. Furthermore, the experiment shows that the main part of DON is rapidly excreted and does not accumulate in plasma, but a minor part of the toxin is retained and slowly excreted from the pigs.  相似文献   

11.
When monoflora chickens with Lactobacillus acidophilus or Streptococcus faecalis were inoculated with Clostridium perfringens either in broth culture or resuspended in Gifu anaerobic medium broth or supernatant fluid, few or no chickens died. Approximately 50% of germ-free chickens died after inoculation of C. perfringens culture, whereas no conventional birds died after inoculation of broth culture. C. perfringens in the contents of duodenum from germ-free chickens numbered about 10(4) colony-forming units (CFU)/g after inoculation 10(8) CFU broth culture per bird, but in gnotobiotic and conventional chickens this organism decreased or was not detected. When C. perfringens was cultured in intestinal contents collected from germ-free chickens, C. perfringens proliferated but alpha toxin was not detected. These findings indicate that the pathogenicity of C. perfringens was suppressed by L. acidophilus or S. faecalis administered previously or inhibited by normal intestinal flora.  相似文献   

12.
Investigations were performed on shedding of C. perfringens in sows from four different pig farms. In two farms where no outbreaks of necrotizing enteritis had been observed, no strains of C. perfringens producing beta-toxin were detected in the faeces of sows. In contrast, C. perfringens strains producing beta-toxin were detected in sows on both farms suffering outbreaks of acute necrotizing enteritis. Strains of C. perfringens producing beta-toxin were invariably positive for the beta 2-toxin gene. However, strains carrying the beta 2-toxin gene only (i.e. negative for beta-toxin) were present in animals on all farms with roughly similar frequencies (mean 28.2% carriers). Some sows carried C. perfringens strains of both toxin genotypes simultaneously. Whereas these data further support the role of betatoxin as a cause of necrotizing enteritis, the role of beta 2-toxin in intestinal disease of piglets remains unclear. To establish the role of faecal shedding vs. environmental contamination as reservoirs of C. perfringens type C, strains were isolated from teats and feedlot trough swabs (toxin genotype beta/beta 2), as well as from fodder (genotype beta 2). However, sows carried this pathogen intermittently and in small numbers. This renders an individual, reliable diagnosis of carrier sows very difficult. Ribotyping of 34 C. perfringens isolates of different toxin genotypes showed five distinct profiles. Different toxin genotypes can belong to the same ribotype, and the same toxin genotype can be present in different ribotypes. Thus, even if a majority (79.4%) of strains investigated in a limited geographic region belonged to ribotype 1, ribotyping offered discrimination of strains beyond toxin typing.  相似文献   

13.
Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.  相似文献   

14.
The effect of cadmium (Cd) on the number of different faecal indicator bacteria in sewage, and on species composition of different indicator bacteria, was studied. Different amounts of Cd were added to aliquots of a sewage sample, and after 0, 4% and 24 h of Cd exposure at 20°C conforms, faecal coliforms, faecal streptococci and Clostridium perfringens were enumerated by the membrane filter method.The Cd-induced reduction in the number of coliforms and faecal coliforms during exposure was found to be greater than the decrease in the number of faecal streptococci. In the case of C. perfringens the Cd concentrations used produced no observable effect on the cell number. The addition of Cd changed the faecal coliforms and faecal streptococci density relationship. Escherichia coli seems to be more resistant to Cd than other coliforms and Streptococcus faecalis var. liquefaciens and Streptococcus durans more resistant to Cd than other faecal streptococci. No influence of Cd on gas production by faecal coliforms was observed.Faecal streptococci and C. perfringens seem to be better indicator bacteria than coliforms and faecal coliforms in evaluating the hygienic quality of Cd polluted sewage.  相似文献   

15.
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.  相似文献   

16.
OBJECTIVE: To evaluate the morphologic and physiologic changes induced by Clostridium perfringens type A (alpha toxin in the ileum and colon of sheep. SAMPLE POPULATION: 16 ligated intestinal loops in 4 Merino lambs and 18 explants of ileum and colon from slaughtered lambs. PROCEDURE: alpha Toxin-induced fluid accumulation was evaluated in ligated ileal and colonic loops of sheep. Tissues were evaluated morphologically by use of gross and histologic examination. Effects of toxin on in vitro intestinal net water transport were tested in modified Ussing chambers. RESULTS: Ovine ileal and colonic loops incubated with C perfringens type A alpha toxin retained more fluid than control loops. Histologically, in the ileum of lambs inoculated with 300 LD50 of alpha toxin/mL, there was a mild to moderate multifocal infiltration of neutrophils in the lamina propria and submucosa. The colonic loops of lambs inoculated with 30 or 300 LD50 of alpha toxin/mL had excessive mucus in the lumen, a moderate amount of neutrophils mixed with mucus in the intestinal lumen, and moderate multifocal infiltration of the lamina propria and submucosa with neutrophils; the blood vessels of these layers were engorged with neutrophils. In vitro measurements of water transport also revealed inhibition of net epithelial water absorption in ileum and colon incubated with alpha toxin on the mucosal side. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that alpha toxin induces alterations in sheep intestine. Clostridium perfringens type A organisms that produce alpha toxin could be responsible for diseases of intestinal origin in some ruminants.  相似文献   

17.
多重PCR检测圈养牛、猪和羊源魏氏梭菌   总被引：4，自引：0，他引：4  
采用多重PCR对圈养源魏氏梭菌的α，β，ε，ι毒素基因进行了检测，结果证实该方法具有很高的特异性。通过对山东德州、枣庄、泰安、蒙阴曾经流行过魏氏梭菌病的猪场、牛场和羊场的162个粪便样品进行检测，检出率为19．1％，均为A型。应用该方法鉴别魏氏梭菌血清型快速、简便，结果对于预防治疗均具有重要的指导作用。  相似文献   

18.
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.  相似文献   

19.
In piglets inoculated with partially purified exfoliative toxin (pp-shET) produced by Staphylococcus hyicus subsp. hyicus, exfoliation was observed at 12 h after injection. Chickens inoculated with the same dose of pp-shET also showed exfoliation within 30 min of injection. However, exfoliation was not demonstrated in mouse, rat, guinea pig, hamster, dog or cat inoculated with pp-shET until 24 h after injection. In cultured cell lines, especially L-929 and Hep-2, the rounding effect occurred after incubation with pp-shET for 1 h. The rounding effect was also seen in five other cultured cells (NCTC 2544, HeLa/S3, HmLu-1, CHO and BHK-21) 6-24 h after exposure to pp-shET. These round cells survived for 72 h after inoculation and formed a monolayer 24 h after changeover to a toxin-free medium. The rounding effect was observed in cells after the formation of the monolayer, but not before. It was suggested that the rounding effect was not caused by the increase in cyclic AMP in cells inoculated with pp-shET but by the cleavage of intracellular contacts.  相似文献   

20.
为验证重组α毒素对携带非典型cpb2基因的A型产气荚膜梭菌的免疫保护性,本研究应用PCR技术,从某牛场牛源A型产气荚膜梭菌G1分离株中扩增出1 194 bp的α毒素编码基因(cpa)和795 bp的β2毒素编码基因(cpb2)。经BLAST分析显示,G1分离株携带的cpb2基因与14个菌株的非典型cpb2基因的氨基酸序列同源性为95.1%～98.9%,与典型cpb2基因(L77695)的氨基酸序列同源性为61.7%。这表明,G1的cpb2基因为非典型cpb2基因。同时分别将cpa和cpb2基因扩增产物克隆于原核表达载体中,构建重组表达质粒pET-a和pET-b2,重组菌经IPTG诱导表达重组蛋白,将其纯化后单独及联合免疫小鼠进行免疫保护试验。结果显示,单独免疫重组α毒素蛋白组以及联合免疫重组β2毒素蛋白组的小鼠,均可以抵抗至少6倍最小致死量(MLD)的G1外毒素(包含α毒素和非典型β2毒素)的攻击,也可以完全抵抗至少6 MLD的G2外毒素(不包含β2毒素)的攻击。表明,重组α毒素蛋白对含有及不含有非典型β2毒素的A型产气荚膜梭菌均具有良好的免疫保护作用。  相似文献   

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