Clostridium difficile survives minimal temperature recommended for cooking ground meats.

We quantified the thermal inhibitory effect of 71°C (recommended for cooking ground meats), and re-heating at 85°C, on food- and food-animal-derived Clostridium difficile spores. All C. difficile strains tested (n=20) survived 71°C for 2 h, but 90% died within 10 min when re-heated at 85°C. Current cooking recommendations would need revision to include C. difficile.

Possible seasonality of Clostridium difficile in retail meat, Canada.

We previously reported Clostridium difficile in 20% of retail meat in Canada, which raised concerns about potential foodborne transmissibility. Here, we studied the genetic diversity of C. difficile in retail meats, using a broad Canadian sampling infrastructure and 3 culture methods. We found 6.1% prevalence and indications of possible seasonality (highest prevalence in winter).

High Doses of Halotolerant Gut-Indigenous Lactobacillus plantarum Reduce Cultivable Lactobacilli in Newborn Calves without Increasing Its Species Abundance.

To elucidate the ecological effect of high oral doses of halotolerant (resistant to table salt) indigenous-gut bacteria on other commensals early in life, we conducted a culture-based study to quantify the effect of intestinal Lactobacillus plantarum strain of bovine origin (with remarkable aerobic growth capabilities and inhibitory activity against Escherichia coli O157:H7 and F5) on clinical health and gut lactobacilli/coliforms in newborn calves. In a double-blind placebo-randomized trial twelve colostrum-fed calves, consecutively born at a farm, were fed L. plantarum within 12 hours from birth at low (107-8 CFU/day) or high concentrations (1010-11) or placebo (q24 h, 5 d; 10 d follow-up). We developed a 2.5% NaCl-selective culture strategy to facilitate the enumeration of L. plantarum-strain-B80, and tested 384 samples (>1,152 cultures). L. plantarum-B80-like colonies were detected in a large proportion of calves (58%) even before their first 24 hours of life indicating endemic presence of the strain in the farm. In contrast to studies where human-derived Lactobacillus LGG or rhamnosus had notoriously high, but short-lived, colonization, we found that L. plantarum colonized stably with fecal shedding of 6 ± 1 log10·g-1 (irrespective of dose, P > 0.2). High doses significantly reduced other fecal lactic acid bacteria (e.g., lactobacilli, P < 0.01) and slightly reduced body weight gain in calves after treatment. For the first time, a halotolerant strain of L. plantarum with inhibitory activity against a human pathogen has the ability to inhibit other lactobacilli in vivo without changing its species abundance, causing transintestinal translocation, or inducing clinical disease. The future selection of probiotics based on halotolerance may expand therapeutic product applicability.

Prevalence of netF-positive Clostridium perfringens in foals in southwestern Ontario.

NetF-producing Clostridium perfringens have recently been identified as a cause of necrotizing enteritis in neonatal foals, but little is known about its prevalence in clinically normal foals. Foals (n = 88) ranging in age from < 1 wk to 2 to 4 mo (median age 2 to 4 wk) on 8 horse-breeding farms in Ontario were examined on 1 or 2 occasions for the presence of C. perfringens. Of the foals that tested positive, 5 isolates (n = 675) were examined for the netF and enterotoxin (cpe) genes. Colonization by C. perfringens was most marked in foals < 1 wk of age [4.85 ± 2.70 log10 colony-forming units (CFU)] and declined markedly over time (1.23 ± 1.06 log10 CFU at 1 to 2 mo of age). Only 2 isolates possessed the cpe gene and none possessed netF. We concluded that netF-positive C. perfringens does not colonize young foals with any detectable frequency in Ontario and this organism is not likely to be adapted to the intestine of the horse.

Les isolats de Clostridium perfringens producteurs de NetF ont récemment été identifiés comme une cause d'entérite nécrotique chez les poulains nouveau-nés, mais peu de choses sont connues sur leur prévalence chez des poulains cliniquement normaux. Des poulains (n = 88) variant en âge entre < 1 semaine jusqu'à 2 à 4 mois (âge médian 2 à 4 semaines) provenant de 8 fermes d'élevage en Ontario ont été examinés à 1 ou 2 occasions pour la présence de C. perfringens. Des poulains qui se sont avérés positifs, 5 isolats (n = 675) ont été examinés pour la présence des gènes netF et de l'entérotoxine (cpe). La colonisation par C. perfringens était la plus marquée chez les poulains âgés de < 1 semaine [4,85 ± 2,70 log10 unités formatrices de colonies (UFC)] et diminuait de façon marquée en fonction du temps (1,23 ± 1,06 log10 UFC à 1 à 2 mois d'âge). Uniquement deux isolats possédaient le gène cpe et aucun ne possédait le gène netF. Nous avons conclu que les isolats de C. perfringens net-positif ne colonisent pas les jeunes poulains avec une fréquence détectable en Ontario et que ce microorganisme est peu susceptible de s'adapter à l'intestin du cheval.(Traduit par Docteur Serge Messier).

PCR ribotyping of Clostridium difficile isolates originating from human and animal sources.

Molecular typing of Clostridium difficile isolates from animals and humans may be useful for evaluation of the possibility for interspecies transmission. The objective of this study was to evaluate C. difficile isolates from domestic animals and humans using PCR ribotyping. Isolates were also tested using PCR for the presence of genes encoding toxins A and B. One hundred and thirty-three isolates of C. difficile from dogs (n = 92), horses (n = 21) and humans (n = 20), plus one each from a cat and a calf, were evaluated. Overall, 23 ribotypes were identified. Of these, nine were identified from dogs, 12 from horses, seven from humans and one each from the cat and calf. In dogs, humans and horses, one or two different ribotypes predominated. Overall, 25 % of isolates from humans were indistinguishable from isolates from one or more animal species. Genes encoding C. difficile toxins A and B were detected in all human, equine and bovine isolates, and in 69 % of canine isolates. While different ribotypes appear to predominate in different mammalian species, several indistinguishable strains may be found in multiple species. This suggests that there is a potential for interspecies transmission of C. difficile and epidemiological studies are warranted.

Suspected Clostridium difficile-associated hemorrhagic diarrhea in a 1-week-old elk calf.

Clostridium difficile-associated diarrhea was suspected in a 1-week-old elk (Cervus elaphus) calf. The isolation of a toxigenic strain of C. difficile from a diarrheic fecal sample, along with exclusion of other enteropathogens, formed the basis of this presumptive diagnosis. Further study is indicated to evaluate the role of C. difficile in neonatal diarrhea in elk.

Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in human plasma.

The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma weak acids (Ka). The aim of this study was to experimentally determine Atot and Ka values for human plasma by using in vitro CO2 tonometry. Plasma Pco2 was systematically varied from 25 to 145 Torr at 37 degrees C, thereby altering plasma pH over the physiological range of 6.90-7.55, and plasma pH, Pco2, and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate Atot and Ka from the measured pH and Pco2 and estimated strong ion difference; the Atot and Ka values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453-467, 1991). The values (mean +/- SD) were as follows: Atot = 17.2 +/- 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); Ka = 0.80 +/- 0.60 x 10-7; negative log of Ka = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for Atot, Ka, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.

Experimental Clostridium difficile enterocolitis in foals.

Despite empirical clinical association of infection with Clostridium difficile with colitis in horses, a causal link has not been confirmed. The objective of this study was to develop a model of C. difficile-associated diarrhea in foals with normal transfer of passive immunity. Nine 1-day-old pony foals were inoculated intragastrically with spores or vegetative cells of C. difficile. Five foals were challenged with spores, with 2 receiving 10(5) colony-forming units (CFUs) and concurrently 3 receiving 10(7) CFUs once daily for 3 days. Clindamycin was administered orally to disrupt gastrointestinal flora. A further 4 foals were challenged by orogastric administration of 10(10) CFUs of vegetative cells once daily for 3 days or until diarrhea developed. This group did not receive clindamycin. Spore and vegetative cell preparations were negative for toxins of C. difficile and common enteropathogens. Clinical signs varied from mild abdominal discomfort and pasty feces to colic and watery diarrhea in 8 of 9 foals. Four of 5 foals challenged with spores developed mild diarrhea, whereas all foals challenged with vegetative cells developed moderate to severe diarrhea. C. difficile was isolated from feces of all foals between 24 and 72 hours after inoculation and toxins A or B or both were detected in the feces of all foals by an enzyme-linked immunosorbent assay. We concluded that spores and vegetative cells of C. difficile are capable of colonizing the gastrointestinal tract, producing toxins, and inducing clinical signs similar to those encountered in naturally occurring cases. This study fulfilled Koch's postulates for C. difficile-associated diarrhea in foals and provides a model for consistent reproduction of the disease for future studies.

West Nile virus encephalomyelitis in horses in Ontario: 28 cases.

West Nile virus encephalomyelitis was diagnosed in 28 horses presented to the Ontario Veterinary College Veterinary Teaching Hospital between August 20 and October 15, 2002. The age range of affected horses was 5 months to 20 years (mean 6.9 years, median 6 years). Clinical signs were highly variable. Duration of hospitalization ranged from < 1 to 12 days (mean 5 days, median 5.4 days). Overall, 16 of the 28 (57%) horses were discharged and, of the 14 from which follow-up information was available, 13 (93%) were reported to be clinically normal 4 to 6 weeks following discharge, while the other horse had markedly improved. This pathogen is emerging as an important cause of neurological disease in Canada.

Prevalence of Clostridium difficile in horses.

Fecal samples were collected to establish the apparent prevalence of Clostridium difficile shedding in Standardbred and Thoroughbred racehorses housed at 4 racetracks and 2 breeding facilities, and in horses admitted to a referral large animal clinic. Forty-one (7.59%) of 540 racetrack horses, seven (5.83%) of 120 breeding farm horses, and four (4.88%) out of 82 horses admitted to the referral clinic were culture-positive for C. difficile. An overall fecal culture prevalence of 7.01% for C. difficile was identified in 742 fecal samples. PCR-ribotyping and toxin gene identification was performed and seventeen 17 PCR-ribotypes were identified among the 52 C. difficile isolates.

Clostridium difficile in retail ground meat, Canada.

Clostridium difficile was isolated from 12 (20%) of 60 retail ground meat samples purchased over a 10-month period in 2005 in Canada. Eleven isolates were toxigenic, and 8 (67%) were classified as toxinotype III. The human health implications of this finding are unclear, but with the virulence of toxinotype III strains further studies are required.