艰难梭菌毒素及致病基因的研究进展

艰难梭菌是一种革兰阳性厌氧芽胞梭菌,是人类肠道感染的主要致病菌,以肠道病理损伤及菌群失调为主的感染性疾病.使用抗菌药物后可导致艰难梭菌过度生长,分泌大量毒素A(肠毒素)和毒素B(细胞毒素),通过毒素介导致病.毒素A启动细胞损伤后,毒素B即可侵入肠黏膜,引起细胞病变,导致一系列与感染相关的临床表现.少数强毒株艰难梭菌还可以产生二元毒素,该毒素可以导致细胞骨架破坏,增强毒素A和毒素B的作用导致严重病变.研究发现,人和动物艰难梭菌之间具有同源性,成为人类感染艰难梭菌的传染源.对艰难梭菌产生毒素的致病机理进行综述,为治疗艰难梭菌感染提供依据.     

魏氏梭菌α毒素分子生物学研究进展

魏氏梭菌是引起各种坏死性肠炎、肠毒血症,同时也是引起创伤性气性坏疽以及人类食物中毒的主要病原菌之一,其致病因子是该菌产生的外毒素。根据主要致病的毒素α、β、ε和ι,将该菌分为A、B、C、D、E 5个毒素型。各型魏氏梭菌均产生α毒素,它是魏氏梭菌最主要的毒力因子。α毒素是依赖锌离子的磷脂酶C,具有细胞毒性、溶血活性、致死性、皮肤坏死性、血小板聚集和增加血管渗透性等特性。     

A型产气荚膜梭菌α毒素的研究进展

α毒素（CPA）是产气荚膜梭菌最重要的毒素之一,而且也是A型产气荚膜梭菌最主要的毒力因子。Macfarlane首先发现α毒素的本质为酶,它具有细胞毒性、溶血活性、致死性、皮肤坏死性、血小板聚集和增加血管渗透性等特性。产气荚膜梭菌是引起各种动物坏死性肠炎、肠毒血症、人类的食物中毒和创伤性气性坏疽的主要病原菌之一,该菌分为A、B、C、D、E5型,其致病因子是菌体产生的外毒素。本文主要综述了A型产气荚膜梭菌α毒素的基本特征、结构、功能、检测技术等方面的研究进展。     

貉肉毒梭菌中毒症的救治

1 发病原因 本病是由于貉采食被肉毒梭菌毒素污染过的饲料、饮水后而引发的一种中毒性疾病,肉毒梭菌毒素可分为A、B、C_α、C_β、D、E、F、G8种类型,引起貉中毒的毒素是C型毒素。毒素主要作用于神经、肌肉结合点,引起机体由下而上的麻痹,肉毒梭菌毒素有很强的毒力,能使多种动物致病,人也可以感染发病。     

产气荚膜梭菌ε毒素研究进展

产气荚膜梭菌是一种重要的人畜共患细菌性传染病病原,在自然界中分布极广,一定条件下可引起多种疾病。该菌常通过其外毒素危害人和动物机体,所产毒素主要有α、β、ε和ι4种外毒素。根据毒素产生的情况,将产气荚膜梭菌分为A、B、C、D、E、F和G 7个型。ε毒素毒力最强,主要由B型和D型菌分泌。论文通过致病机理、病理变化、流行特点及相关疫苗研究方面对产气荚膜梭菌ε毒素研究进展进行综述,以期为产气荚膜梭菌病的防控提供参考。     

卵黄抗体对艰难梭菌感染的治疗潜力

<正>常规抗生素会改变正常肠道菌群的数量和复杂性,进而会降低这些具有保护作用的微生物在胃肠道内的竞争性定植的能力。因此,容易导致艰难梭菌感染(Clostridium Difficile Infection,CDI)。一旦在结肠中定植,艰难梭菌会产生毒素A(Clostridium difficile Toxin A,Tcd A)和毒素B(Clostridium difficile Toxin B,Tcd B),引起包括腹泻、伪膜性结肠炎、中毒性巨结肠及可能致命的多系统器官衰竭等临床症状。     

反刍动物梭菌性肠胃炎的临床症状及病理变化

由梭菌引起的真胃炎和肠炎是在所有家养反刍动物中参见的重要消化系统疾病.这些疾病最常见的病原是A、B、C、D和E毒素型的产气荚膜梭菌过度生长引起的,病伴随着细菌外毒素的释放导致皱胃和肠道黏膜坏死.此外,艰难梭菌也可能与犊牛的肠炎有关,但比产气荚膜梭菌引起的临床病例相比数量较少.本文就反刍动物胃肠道梭菌病的病因、临床症状、...     

G型产气荚膜梭菌NetB毒素研究进展

G型产气荚膜梭菌可引起鸡坏死性肠炎和肠毒血症,致病力强,在自然界中广泛存在,严重危害养鸡业的发展。坏死性肠炎B样毒素(necrotic enteritis B-like toxin, NetB)是近年来发现的是一种新的β-成孔毒素(β-pore-forming toxin,β-PFT),是G型产气荚膜梭菌的主要致病性毒素和保护性抗原,深入研究该毒素的结构功能及致病机理对于鸡坏死性肠炎的防控具有重要意义,但至今尚无相关系统性综述报道。鉴于此,论文主要对国内外关于G型产气荚膜梭菌的流行概况、NetB毒素的分子结构特征、生物学特性、致病机理及其相关防治制剂等方面的研究进行综述,为进一步基于NetB毒素研制鸡坏死性肠炎的防治制剂提供参考。     

羊恶性水肿病的病因、诊断与防控

羊恶性水肿病主要是由腐败梭菌引起的急性、创伤性传染病,经口感染腐败梭菌时称快疫,是常见病,经创伤感染腐败梭菌时称恶性水肿,较少见。1病因分析引起本病的主要病原为腐败梭菌,其次A型魏氏梭菌也可感染致病。病菌由伤口进入受伤组织中,在厌氧条件下进行繁殖产生毒素,损害血管引起局     

鹅肉毒梭菌中毒的防治

<正>本病是由于食人了肉毒梭菌产生的外毒素而引起的急性中毒性疾病。本病特征是全身性麻痹,头下垂,软弱无力,故又称软颈病。1病原病原为肉毒梭菌,但细菌本身不致病,而是其产生的肉毒梭菌毒素,有极强的毒力,对人、畜、禽均有高度致死性。该毒素有较强的耐热性,100℃60min才被破坏。肉毒梭菌毒素分7个型,引起家禽中毒的主要是A型和C型,以C型毒力     

Clostridium perfringens type C and Clostridium difficile co-infection in foals

Clostridium perfringens type C is one of the most important agents of enteric disease in newborn foals. Clostridium difficile is now recognized as an important cause of enterocolitis in horses of all ages. While infections by C. perfringens type C or C. difficile are frequently seen, we are not aware of any report describing combined infection by these two microorganisms in foals. We present here five cases of foal enterocolitis associated with C. difficile and C. perfringens type C infection. Five foals between one and seven days of age were submitted for necropsy examination to the California Animal Health and Food Safety Laboratory. The five animals had a clinical history of acute hemorrhagic diarrhea followed by death and none had received antimicrobials or been hospitalized. Postmortem examination revealed hemorrhagic and necrotizing entero-typhlo-colitis. Histologically, the mucosa of the small intestine and colon presented diffuse necrosis and hemorrhage and it was often covered by a pseudomembrane. Thrombosis was observed in submucosal and/or mucosal vessels. Immunohistochemistry of intestinal sections of all foals showed that many large bacilli in the sections were C. perfringens. C. perfringens beta toxin was detected by ELISA in intestinal content of all animals and C. difficile toxin A/B was detected in intestinal content of three animals. C. perfringens (identified as type C by PCR) was isolated from the intestinal content of three foals. C. difficile (typed as A(+)/B(+) by PCR) was isolated from the intestinal content in 3 out of the 5 cases. This report suggests a possible synergism of C. perfringens type C and C. difficile in foal enterocolitis. Because none of the foals had received antibiotic therapy, the predisposing factor, if any, for the C. difficile infection remains undetermined; it is possible that the C. perfringens infection acted as a predisposing factor for C. difficile and/or vice versa. This report also stresses the need to perform a complete diagnostic workup in all cases of foal digestive disease.     

Toxin production by and adhesive properties of Clostridium difficile isolated from humans and horses with antibiotic-associated diarrhea

Clostridium difficile is a common nosocomial pathogen in humans and animals that causes diarrhea and colitis following antibiotic therapy. Isolates of C. difficile obtained from faecal material from 20 human patients and 6 equine subjects with antibiotic-associated diarrhea were investigated regarding production of toxins A and B, their capacity to adhere to the human intestinal Caco-2 cell line and equine intestinal cells, and for the presence of fimbriae. The results showed that most (17/20) of the human clinical isolates produced both toxins A and B. One of the human isolates proved toxin A-negative/toxin B-positive. All (6/6) horse isolates were positive for both toxins A and B. Both the human and horse isolates possessed the capacity to adhere, to varying degree, to human and equine intestinal cells. It appeared that human isolates produced greater amounts of toxin B, and that there was a host-species dependency on ability to attach to intestinal epithelial cells. No fimbriae were found in any of the investigated isolates.     

Clostridial enteric infections in pigs.

Clostridium perfringens types A and C and Clostridium difficile are the principal enteric clostridial pathogens of swine. History, clinical signs of disease, and gross and microscopic findings form the basis for a presumptive diagnosis of C. perfringens type-C enteritis. Confirmation is based on isolation of large numbers of type-C C. perfringens and/or detection of beta toxin in intestinal contents. Diagnosis of C. perfringens type-A infection, however, remains controversial, mostly because the condition has not been well defined and because type-A organisms and their most important major (alpha) toxin can be found in intestinal contents of healthy and diseased pigs. Isolation of large numbers of C. perfringens type A from intestinal contents, in the absence of other enteric pathogens, is the most reliable criterion on which to base a diagnosis. Recently, beta2 (CPB2) toxin-producing C. perfringens type A has been linked to disease in piglets and other animals. However, implication of CPB2 in pathogenesis of porcine infections is based principally on isolation of C. perfringens carrying cpb2, the gene encoding CPB2, and the specific role of CPB2 in enteric disease of pigs remains to be fully defined. Clostridium difficile can also be a normal inhabitant of the intestine of healthy pigs, and diagnosis of enteric infection with this microorganism is based on detection of its toxins in feces or intestinal contents.     

Real-time PCR and typing of Clostridium difficile isolates colonizing mare-foal pairs

Clostridium difficile infection can occur in the dams of sick foals, but it is unknown if mares and foals share the same isolates. In this study, C. difficile isolates from fecal samples of 11 mares paired with 11 foals were genotyped by arbitrarily primed PCR; two mares and three foals in five mare-foal pairs had diarrhea. Fecal immunoassays were utilized to detect C. difficile common antigen and toxin A. Quantitative real-time PCR (qPCR) systems were developed to detect genes for toxins A and B, as well as for binary toxin B. Sequences of all toxins were present in all isolates, although only one horse was positive for toxin A on fecal immunoassay. Identical strains of C. difficile were present in 4/11 (36.4%) mare-foal pairs. Mare-foal pairs can harbor C. difficile subclinically and are potential reservoirs for colonization of each other.     

The distribution and density of Clostridium difficile toxin receptors on the intestinal mucosa of neonatal pigs

Clostridium difficile is an enteric pathogen affecting a variety of mammals, but it has only recently been diagnosed as a cause of neonatal typhlocolitis in pigs. The most important virulence factors of C. difficile are 2 large exotoxins, toxin A (TcdA) and toxin B (TcdB). TcdA is a potent enterotoxin with effects on host tissues that are dependent upon receptor-mediated endocytosis of the intact toxin. TcdB is an effective cytotoxin, but it apparently does not bind receptors on intact mucosal epithelium. TcdB is much less toxic in vivo unless there is underlying damage to the mucosa, and it is not essential for the virulence of C. difficile. One hypothesis to explain the resistance of most species as neonates (e.g., humans and hamsters) is that they may lack significant numbers of TcdA receptors. The susceptibility of neonatal pigs suggests cells of the gastrointestinal mucosa express sufficient numbers of toxin receptors for lesion development. Immunohistochemical (IHC) assays documented specific binding of TcdA, but not TcdB, to the epithelium of the small and large intestine. The carbohydrate Galalpha1-3beta1-4GlcNAc-R has been described as an important receptor for TcdA. However, IHC indicated a distribution on cell surfaces much different from that of TcdA binding, suggesting a specific interaction of toxin with an alternative receptor.     

猪源艰难梭菌的分离鉴定及特性分析

为了解河南省规模化猪场猪源艰难梭菌的流行、耐药以及毒素基因携带情况,收集来自豫西地区5个养殖场中疑似艰难梭菌感染(clostridium difficile infection,CDI)猪的粪便标本(n=41)。采用厌氧培养法,用艰难梭菌选择性培养基环丝氨酸-头孢西丁-果糖琼脂(cycloserine-cefoxitin-fructose agar,CCFA)完成分离培养。采用表型培养法进行菌株分离和鉴定,以多重PCR同时鉴定菌株并检测tcdA和tcdB毒素基因及二元毒素编码基因cdtA和cdtB。采用E-test法检测艰难梭菌对常用抗生素的最低抑菌浓度(minimal inhibitory concentration,MIC)值并判定菌株对药物的敏感性。结果从分离的41份样本中,经染色镜检及PCR鉴定,分离出符合艰难梭菌生长特性的菌株1株。药敏结果显示分离菌株对万古霉素和甲硝唑呈现高度敏感,对氟喹诺酮类药物以及四环素等表现出不同程度的耐药。艰难梭菌的研究大多集中在人源方面,本研究首次在河南省规模化养猪场中分离出艰难梭菌,对所分离菌株进行毒素基因检测,毒素A、B呈阳性,而无二元毒素基因。分离的猪源艰难梭菌对红霉素、万古霉素、甲硝唑、利福平均呈现高度敏感,对克林霉素、阿莫西林、左氧氟沙星、利奈唑酮和青霉素呈现不同程度的耐药。     

Molecular analysis of Clostridium difficile isolates recovered from horses with diarrhea

Clostridium difficile is an important cause of diarrhea in horses, causing sporadic and epidemic disease of varying severity. This study evaluated the molecular characteristics of 48 C. difficile isolates recovered from diarrheic horses admitted to a veterinary hospital by using PCR-ribotyping and toxin gene profile. Additionally, feces were tested for the presence of C. difficile toxin A/B via enzyme immunosorbant assay (EIA) in 38 horses. The toxin genes tcdA, tcdB and cdtB were present in 27 (56.25%), 35 (72.91%) and 2 (4.1%) strains, respectively. Eight isolates (16.6%) were A(-)B(+) variants. Thirteen of forty-eight isolates (27.0%) did not posses any toxin genes (A(-)B(-)CDT(-)). A positive EIA result was reported in 17 (44%) of the cases. There was no association between the presence of different ribotypes or strains and toxin gene(s) profiles and the clinical outcome.     

Fatal enterocolitis in Asian elephants (Elephas maximus) caused by Clostridium difficile

Two cases of fatal enteritis caused by Clostridium difficile in captive Asian elephants are reported from an outbreak affecting five females in the same zoo. Post mortem examination including histopathology demonstrated fibrinonecrotic enterocolitis. C. difficile was isolated by selective cultivation from two dead and a third severely affected elephant. Four isolates were obtained and found positive for toxin A and B by PCR. All isolates were positive in a toxigenic culture assay and toxin was demonstrated in the intestinal content from one of the fatal cases and in a surviving but severely affected elephant. PCR ribotyping demonstrated that the C. difficile isolates shared an identical profile, which was different from an epidemiologically unrelated strain, indicating that the outbreak was caused by the same C. difficile clone. It is speculated that the feeding of large quantities of broccoli, a rich source of sulforaphane, which has been shown to inhibit the growth of many intestinal microorganisms may have triggered a subsequent overgrowth by C. difficile. This is the first report of C. difficile as the main cause of fatal enterocolitis in elephants. The findings emphasize the need to regard this organism as potentially dangerous for elephants and caution is recommended concerning antibiotic treatment and feeding with diets containing antimicrobials, which may trigger an expansion of a C. difficile population in the gut.     

Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs.

Clostridium difficile-associated-diarrhea (CDAD) is a nosocomial infection in dogs. Diagnosis of this infection is dependent on clinical signs of disease supported by laboratory detection of C. difficile toxins A or B, or both, in fecal specimens via enzyme-linked immunosorbent assay (ELISA). Unfortunately, to the authors' knowledge, commercially available ELISAs have not been validated in dogs to date. We evaluated 5 ELISAs done on 143 canine fecal specimens (100 diarrheic and 43 nondiarrheic dogs) and on 29 C. difficile isolates. The results of each ELISA were compared with the cytotoxin B tissue culture assay (CTA). Clostridium difficile was isolated from 23% of the fecal specimens. Eighteen of the 143 fecal specimens were toxin positive (15 diarrheic and 3 nondiarrheic dogs). On the basis of multiplex polymerase chain reaction (PCR) analysis for toxin-A and -B genes, 72% of the isolates were toxigenic. The carriage rate of toxigenic isolates in diarrheic dogs was higher than that in the nondiarrheic dogs; however, these differences were not statistically significant. A good correlation was found between CTA, PCR, and culture results. The ELISAs done on fecal specimens collected from diarrheic dogs had low sensitivity (7-33%). In contrast, ELISA for toxin A or B, or both, performed on toxigenic isolates had high sensitivity (93%). These results suggest that commercially available human ELISAs are inadequate for the diagnosis of canine C. difficile-associated diarrhea when tested on fecal specimens. In contrast, the Premier ToxinA/B and Techlab ToxinA/B ELISAs may be useful for the diagnosis of canine CDAD when used on toxigenic isolates.     

Natural and experimental infection of neonatal calves with Clostridium difficile

Clostridium difficile toxins were associated with calf diarrhea in a recent retrospective study; however, no causal relationship has been prospectively investigated. This infection study tested whether the oral inoculation of neonatal calves with a toxigenic strain of C. difficile (PCR-ribotype 077) results in enteric disease. Fourteen 6-24 h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4 x 10(8) +/- 3.5 x 10(8) cfu) (n = 8) or sterile culture broth (n = 6). Calves were euthanized on day 6 or after the onset of diarrhea, whichever came first. Fecal and intestinal samples were blindly cultured for C. difficile, and tested for its toxin A/B (C. difficile TOX A/B II ELISA, Techlab). PCR-ribotyping was used to compare inoculated and recovered isolates. Diarrhea was observed in all control calves and 3/8 of inoculated calves (p = 0.03), but it did not occur in calves that tested positive for C. difficile toxins. Fecal toxins were identified only from two controls. PCR-ribotyping confirmed the presence of C. difficile PCR-ribotype 077 in samples of all inoculated calves, but not from controls. The identification of five other PCR-ribotypes in 3/8 (37.5%) and 2/6 (33.3%) of inoculated and control calves, respectively, indicated early natural infection (< or = 24h of age). Five of 14 cecal samples had C. difficile (p = 0.01). In conclusion, the oral administration of C. difficile PCR-ribotype 077 to neonatal calves resulted in fecal/intestinal colonization but not in detection of toxins, or signs of enteric disease. Further studies are required to investigate the clinical relevance of C. difficile in calves.