Toxin genotypes of Clostridium perfringens isolates from common quail (Coturnix coturnix) with or without acute necrotic enteritis.

Clostridial diseases are one of the foremost causes of mortality in quails which occur by Clostridium colinum, Clostridium perfringens (C. perfringens), and Clostridium sordellii. C. perfringens genotypes responsible for quail enteritis are not well understood. In this study, the prevalence of C. perfringens genotypes was investigated in common quail (Coturnix coturnix) farms that suffered from acute necrotic enteritis (diarrhoeic) and compared with healthy (non-diarrhoeic) quails. Toward this end, C. perfringens isolates were collected and genotyped for 16s rRNA, cpa, cpb, cpb2, etx, iap, cpe, netB, and tpeL genes, using PCR. It was revealed that 42, 23, and 19 isolates belonged to toxinotypes A, F, and G, respectively, and the other toxinotypes were not obtained. The recovery ratio of C. perfringens from diarrhoeic farms roughly doubled in non-diarrhoeic farms (40.0% versus 21.5%, p = 0.03). Also, we observed a high isolation ratio of the cpb2 genotype (90.48%), which was significantly eminent in the diarrhoeic group (94.2%, p < 0.05). Although the prevalence of tpeL genes was low (15.48%), there was an interesting relationship between this gene and cpb2, so we did not obtain cpb2-tpeL-. This study showed the prevalence of types A, F, and G of C. perfringens in quail enteritis. Also, we reported tpeL+C. perfringens strains in quail for the first time and its frequent co-occurrence with the cpb2 gene. These results highlight the necessity of more accurate investigations of the C. perfringens genotype in different hosts to verify the exact role of these toxins in quail enteritis.

Prevalence, characteristics and antimicrobial susceptibility patterns of Clostridioides difficile isolated from hospitals in Iran.

OBJECTIVES: Clostridioides (previously Clostridium) difficile is a major growing cause of nosocomial diarrhoea known as C. difficile infection (CDI). This study investigated the prevalence and antimicrobial resistance patterns of C. difficile isolated from patients suffering from diarrhoea in Iran between 2016-2018. METHODS: A total of 151 stool specimens were collected and were screened for the presence of C. difficile. Specimens were examined for toxins by culture, enzyme immunoassay (EIA) and PCR. Antimicrobial susceptibility testing was performed for 12 antibiotics (metronidazole, vancomycin, clindamycin, tetracycline, erythromycin, ciprofloxacin, levofloxacin, moxifloxacin, fusidic acid, piperacillin, piperacillin/tazobactam and rifampicin) by the disk diffusion method according to the guidelines of the CLSI, EUCAST and CA-SFM. RESULTS: Of 151 stool specimens, 66 (43.7%) were positive for C. difficile by PCR, whereas 2 (1.3%) were only positive for C. difficile toxins based on EIA. A total of 292 clostridial isolates were obtained from specimens by culture, of which 133 (45.5%) were finally confirmed as C. difficile by PCR. Of 121 isolates resistant to at least one antibiotic, 107 (88.4%) were resistant to three or more antimicrobials and thus were defined as multidrug-resistant (MDR). Different and diverse resistance patterns to the antimicrobial drugs were seen among the isolates. CONCLUSION: This is the first report of the isolation of C. difficile from different governmental hospitals of Iran and indicates that CDI might be an important nosocomial infection in different hospital wards. Moreover, this study provides a comprehensive picture of the MDR phenotype characteristics of C. difficile isolates in Iran.

A Recombinant Probiotic, Lactobacillus casei, Expressing the Clostridium perfringens α-toxoid, as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

Orally administered recombinant Lactobacillus casei vector vaccine expressing ß-toxoid of Clostridium perfringens that induced protective immunity responses.

Clostridium perfringens types B and C cause enteritis and enterotoxemia in animals. The conventional vaccine production systems need time-consuming detoxification and difficult quality control steps. In this study, a modified ß-toxoid gene was synthesized, cloned into the pT1NX vector, and electroporated into Lactobacillus casei competent cells to yield L. casei-ß recombinant strain. Surface expression of the recombinant ß-toxoid was evaluated by ELISA and confirmed by immunofluorescence microscopy. Vaccinated BALB/c mice with L. casei-ß induced potent humoral and cell-mediated immune responses that were protective against lethal challenges with 100 MLD/mL of the ß-toxin. Safety and efficacy of the recombinant clone was evaluated and the presumptive toxicity of L. casei-ß was studied by toxicity test and histopathological findings, which were the same as negative controls. Our results support the use of L. casei as a live oral vector vaccine, and that the recombinant L. casei-ß is a potential candidate for being used in the control of enterotoxemia diseases caused by C. perfringens types B and C.

Seroepidemiological study of Q fever in small ruminants from Southeast Iran.

The aim of the present study was to determine the prevalence of Coxiella burnetii antibodies in small ruminants in Southeast Iran. A total of 368 small ruminant blood samples (241 caprine blood samples and 127 ovine blood samples) were collected from January to May of 2011 in Southeast Iran. A commercial ELISA test kit was employed to identify specific antibodies against C. burnetii in the sheep and goats. Seropositivity in the examined counties ranged from 17.1% to 39.2%. Of the animals tested, 97 animals (26.4%), including 43 sheep (33.9%) and 54 goats (22.4%), had antibodies to C. burnetii. The results of the current study reveal the high prevalence of antibody positivity in small ruminants in Southeast Iran. Thus, sheep and goats are important reservoirs in this area. Additionally, we performed a logistic regression to the identify risk factors for positivity and concluded that age was an important risk factor (P<0.001).