The occurrence of Clostridium perfringens in the intestine of chicks

Commercial broiler chicks brooded either on wire or on used or new litter demonstrated a 75% (62/75) incidence of recovery of "perfringens-like" colonies from the intestine during a 5-week period. Eleven Clostridium spp. were identified from among these "perfringens-like" organisms, which were cultured on SPS selective agar medium. Clostridium perfringens was positively identified only infrequently (five isolates) from among the "perfringens-like" colonies. In contrast, "perfringens-like" colonies were not recovered from the intestinal contents of specific-pathogen-free chicks reared in an isolation unit. However, C. perfringens was isolated from the yolk sac of one embryonated egg and from the intestine of a single 7-day-old chick, indicating the possibility of vertical transmission of this potential pathogen.     

Antimicrobial resistance in Clostridium perfringens isolates from broilers in Belgium

The antimicrobial susceptibility of Clostridium perfringens strains isolated from Belgian broilers between May and September 2007 was investigated. All 39 tested isolates were sensitive to enrofloxacin, erythromycin, tylosin, florfenicol and bacitracin. Twenty-six (66%) and 24 (61%) out of the 39 tested isolates showed acquired resistance to tetracycline and lincomycin, respectively. The C. perfringens isolates were also screened by PCR for the presence of the resistance genes tet(K), tet(L), tet(M), tetB(P), tet(O), tet(W), lnu(A) and lnu(B). In 22/26 tetracycline resistant strains and 7/24 lincomycin resistant strains, resistance could be attributed to one or more of these genes. An extended frequency distribution range of MICs was seen for ampicillin. These data are consistent with data derived from studies carried out in 1980 and in 2004, indicating that no changes in antimicrobial resistance patterns have taken place during time in C. perfringens isolates from broilers in Belgium.     

The occurrence of enterotoxigenic Clostridium perfringens strains in the feces of dogs and cats

A total of 147 faecal specimen of dogs and cats was examined with cultural method for the occurrence of Cl. perfringens and its enterotoxin by using a reversed passive latex agglutination test (Pet-RPLA). Cl. perfringens could be detected in 77.9% of the samples of dogs (n = 68) and 65.6% of cats (n = 32) with diarrhoea in germ counts of 10(4)-10(10) cfu/g faeces. In the group of non diarrhoeic dogs (n = 39) and cats (n = 8) Cl. perfringens was found in 53.9% and 50% of the samples, the germ counts revealed 10(4)-10(8) cfu/g faeces. The enterotoxin of Cl. perfringens was detected in 48.5% of the samples of dogs and 28.1% of cats with diarrhoea. On the other hand this toxin could not be detected in any faecal specimen of non diarrhoeic animals. The in vitro detection of Cl. perfringens enterotoxin was successful at 65% of 20 strains, that had been isolated from faecal samples with enterotoxin. Also 2 Cl. perfringens strains isolated from faeces of non diarrhoeic dogs proved to be enterotoxigenic. The present test results given reason to believe that enterotoxigenic Cl. perfringens strains are involved in the complex of enteric disease of dogs and cats.     

The occurrence and antibiotic resistance of motile Aeromonas in livestock

The present study was carried out to assess the prevalence of motile Aeromonas spp. in the faeces of clinically healthy sheep, cattle and horses and evaluate their susceptibility to some anti-microbial agents. Rectal swabs from 120 sheep, 85 cattle and 20 horses were examined for Aeromonas species using alkaline peptone water (pH 8.4) as the enrichment medium and Aeromonas Selective Agar containing 5 mg/l ampicillin as the isolation medium. Identification and antibiotic resistance of motile Aeromonas strains was performed using Gram Negative Enteric ID panel. Motile aeromonads were isolated from 12 (10%) sheep, 7 (8.2%) cattle and 1 (5%) horse. Of these 20 aeromonad isolates, 13 were A. caviae, 6 were A.sobria and 1 was A. hydrophila. Aeromonas species in the faeces of livestock might pose a public health problem for humans who are in direct contact with contaminated animals. However, further studies should be performed on aeromonads relating to their transmission between animals and humans.     

不同地区鸡源产气荚膜梭菌的毒素型鉴定和药敏试验

为探明我国鸡群中产气荚膜梭菌(Clostridium perfringens,Cp)不同毒素型分布特征及携带主要致病毒素基因和对饲用抗菌素的抗药谱,于2013年7月至2014年5月间从四川等7省17个肉鸡生产较为集中的地区采样692份,分离鉴定获得Cp菌株214株,分离率31%。经多重PCR方法鉴定,其中有206株为A型Cp,8株为C型。利用特异性单一PCR方法检测发现:有87株Cp携带β2毒素基因,阳性率为41%;5株携带Net B毒素基因,阳性率为2.3%。以最小抑菌浓度法(MIC)检测的常见饲用抗菌素敏感性结果显示:那西肽、弗吉尼亚霉素和效霉素有广泛的抑菌作用,四川德阳(2013)、四川眉山(2013)和广东鹤山(2013)的分离菌株对所测试的11种药物均表现敏感,而广西桂林(2014)的菌株仅对那西肽敏感。结果表明我国鸡源Cp具有明显的毒素型、主要致病毒素基因携带状况和抗药谱的地区差异。     

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.     

Determination of the prevalence of antimicrobial resistance genes in canine Clostridium perfringens isolates

Clostridium perfringens is a well documented cause of a mild self-limiting diarrhea and a potentially fatal acute hemorrhagic diarrheal syndrome in the dog. A recent study documented that 21% of canine C. perfringens isolates had MIC's indicative of resistance to tetracycline, an antimicrobial commonly recommended for treatment of C. perfringens-associated diarrhea. The objective of the present study was to further evaluate the antimicrobial susceptibility profiles of these isolates by determining the prevalence of specific resistance genes, their expression, and ability for transference between bacteria. One hundred and twenty-four canine C. perfringens isolates from 124 dogs were evaluated. Minimum inhibitory concentrations of tetracycline, erythromycin, tylosin, and metronidazole were determined using the CLSI Reference Agar Dilution Method. All isolates were screened for three tetracycline resistance genes: tetA(P), tetB(P) and tetM, and two macrolide resistance genes: ermB and ermQ, via PCR using primer sequences previously described. Ninety-six percent (119/124) of the isolates were positive for the tetA(P) gene, and 41% (51/124) were positive for both the tetA(P) and tetB(P) genes. No isolates were positive for the tetB(P) gene alone. Highly susceptible isolates (MIC< or = 4 microg/ml) were significantly more likely to lack the tetB(P) gene. One isolate (0.8%) was positive for the ermB gene, and one isolate was positive for the ermQ gene. The tetM gene was not found in any of the isolates tested. Two out of 15 tested isolates (13%) demonstrated transfer of tetracycline resistance via bacterial conjugation. Tetracycline should be avoided for the treatment of C. perfringens-associated diarrhea in dogs because of the relatively high prevalence of in vitro resistance, and the potential for conjugative transfer of antimicrobial resistance.     

Binding of epsilon-toxin from Clostridium perfringens in the nervous system

Epsilon-toxin (varepsilon-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. varepsilon-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for varepsilon-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An varepsilon-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of varepsilon-toxin to myelin structures. varepsilon-Prototoxin-GFP and varepsilon-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by varepsilon-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of varepsilon-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system.     

Antimicrobial susceptibility of canine Clostridium perfringens strains from Switzerland

Fifty Clostridium perfringens strains were isolated from individual dogs with acute diarrhoea that were not given antibiotics. Toxin types and minimal inhibitory concentrations of 15 antibiotics were determined for each of them. All strains harboured the alpha-toxin gene, 12 of them had both the alpha- and entero-toxin gene and 5 had both the alpha- and beta2-toxin gene. Eighteen percent of the isolates showed resistance to tetracycline and 54 % showed decreased susceptibility to metronidazole which is one of the most frequently used antibiotics in the treatment of canine diarrhoea. Apart from that, all isolates were susceptible to the remaining antibiotics tested. These findings lead to the conclusion that despite a general susceptibility to antibiotics in C. perfringens, resistance is developing in isolates from dogs. Therefore, careful identification of the pathogenic agent and antibiotic susceptibility testing should be performed prior to therapy in order to minimise further selection of antibiotic resistance.     

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.     

Foaling-management practices associated with the occurrence of enterocolitis attributed to Clostridium perfringens infection in the equine neonate

Enterocolitis associated with Clostridium perfringens (C. perfringens) infection in neonatal foals is often severe and has been associated with a high case-mortality risk. We designed a premises-based survey to evaluate the associations of regional foaling practices, premises environmental management, periparturient foal and brood-mare management, and periparturient brood-mare ration with the occurrence of neonatal enterocolitis attributed to C. perfringens infection. Potential risk factors individually associated with enterocolitis were breed type, housing type at foaling and in the first three days of life, ground/floor surface type at foaling and in the first three days of life, brood-mare ration before and after foaling, and the presence of livestock other than horses on the premises in the past. From the multivariable-logistic regression models, six variables were significantly associated with an increased risk of the outcome of interest (p<0.05): foals of the stock horse type, housing in a stall or drylot in the first three days of life, other livestock present on the premises in the past, foal born on dirt, sand or gravel surface, and low amounts of grass hay and grain fed post-partum. Low grain amounts fed pre-partum represented a decreased risk of the outcome of interest.     

The partial purification of Clostridium perfringens beta toxin.

An attempt was made to purify Clostridium perfringens Beta toxin. Crude toxin prepared by ammonium sulphate precipitation of culture supernatants was purified by chromatography on Sephadex G50, Sephadex G100 and DEAE cellulose. This material, although highly purified was not homogeneous on polyacrylamide gel electrophoresis. It had a toxicity of 800 000 mouse MLDs/mg N, a typical protein absorption spectrum in the UV region, an iso-electric point of 5, 6 and the main component had a molecular mass of 42 000 +/- 2 000 (estimated by electrophoresis in sodium dodecyl sulphate containing polyacrylamide gels).     

Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema

The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline.