Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples.

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth.

Phenotypic and genetic characterization of resistance against macrolides and lincosamides in Streptococcus gallolyticus strains isolated from pigeons and humans.

The macrolide and lincosamide (ML) resistance phenotype of 65 pigeon and 30 human Streptococcus gallolyticus strains was determined by the disk diffusion method. Constitutive resistance against the tested antibiotics was seen in 13 human and 28 pigeon strains. Simultaneous screening for the presence of erm(B) and mef(A) genes using PCR revealed that the erm(B) gene was present in 40 out of these 41 phenotypically resistant S. gallolyticus strains while the mef(A) gene was detected in only one resistant and one susceptible human-derived strain. The erm(B) genes of 10 human and 10 pigeon S. gallolyticus strains were sequenced and compared. Four human and seven pigeon strains possessed exactly the same sequence for the erm(B) gene. The sequence of the erm(B) gene of the remaining strains differed in one to five nucleotides. These findings could indicate a possible exchange of resistance genes between human and pigeon strains.

Association of Streptococcus gallolyticus strains of high and low virulence with the intestinal tract of pigeons.

We investigated the ability of a high virulence (STR 357) and a low virulence (STR 598) strain of Streptococcus gallolyticus to attach to the intestinal tract of pigeons. For that purpose, first of all, two groups of six pigeons were anesthetized and ligatures were placed at the beginning of duodenum, jejunum, ileum, and colon. The obtained intestinal loops of the birds of the first and second group were injected with S. gallolyticus strains STR 357 and STR 598, respectively. At 15, 30, and 60 min postinoculation, two pigeons of each group were euthanatized and the various intestinal loops were sampled for histologic, immunohistochemical, and electron microscopic examination. Both the high and low virulence strains were able to adhere to the intestinal mucosa. Indeed, all samples dearly showed numerous coccal-shaped bacteria that stained positively with S. gallolyticus antiserum and were lining up against the intestinal epithelium. Likewise, on electron microscopic examination, cocci were seen in the mucus covering the intestinal epithelium. Second, the association of S. gallyticus strains of differing virulence with the intestinal tissue was determined quantitatively. Experiments were performed as described above. The number of S. gallolyticus bacteria that adhered to the intestinal epithelium was determined by plating out 10-fold serial dilutions of the segments. No significant differences in the number of adhered bacteria were found between the strains of high and low virulence.

Prevalence of antimicrobial resistance among pigeon isolates of Streptococcus gallolyticus, Escherichia coli and Salmonella enterica serotype Typhimurium.

Thirty-three Streptococcus gallolyticus, 60 Escherichia coli and 18 Salmonella enterica serotype Typhimurium var. Copenhagen strains isolated from homing pigeons (Columba livia) were tested for susceptibility to the antimicrobials most commonly used to treat pigeons. Minimal inhibitory concentrations were determined using the agar dilution technique. Aminoglycosides (gentamicin and kanamycin), trimethoprim and flumequine were relatively inactive against the streptococci tested. Acquired tetracycline resistance amounted to 85%, and lincomycin and macrolide (erythromycin) resistance to 48 and 45%, respectively. Fluoroquinolone (enrofloxacin) resistance was found in four S. gallolyticus strains. All strains were susceptible to ampicillin. With the E. coli strains, resistance was found to all antibiotics tested. Over one-half of them were resistant to tetracycline and to broad-spectrum penicillins (ampicillin); however, none showed extended spectrum beta-lactamase activity, implying that the cephalosporins (ceftiofur) remained active. Resistance to trimethoprim, aminoglycosides and fluoroquinolone ranked next. In contrast to the S. gallolyticus and E. coli strains, the S. enterica strains were susceptible to all the antimicrobials tested.