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).

Acetazolamide attenuates transvascular fluid flux in equine lungs during intense exercise.

During intense exercise in horses the transvascular fluid flux in the pulmonary circulation (Jv-a) represents 4% of cardiac output (Q). This fluid flux has been attributed to an increase in pulmonary transmural hydrostatic forces, increases in perfused microvascular surface area, and reversible alterations in capillary permeability under conditions of high flow and pressure. Erythrocyte fluid efflux, however, accounts for a significant fraction of Jv-a. In the lung the Jacobs-Stewart cycle occurs with diffusion of CO2 into alveolar space with possible accompanying chloride (Cl-) and water movement from the erythrocyte directly into the pulmonary interstitium. We hypothesised that inhibition of carbonic anhydrase in erythrocytes inhibits the Jacobs-Stewart cycle and attenuates Jv-a. Five horses were exercised on a treadmill until fatigue without (control) and with acetazolamide treatment (30 mg kg(-1) 30 min before exercise). Erythrocyte fluid efflux, plasma fluid flux across the lung and Jv-a were calculated using haemoglobin, haematocrit, plasma protein and Q. Fluid fluxes were used to calculate erythrocyte, plasma and whole blood Cl- fluxes across the lung. Cardiac output was not different between control and acetazolamide treatment. During exercise erythrocyte fluid efflux and Jv-a increased in control (9.3±3.3 and 11.0±4.4 l min(-1), respectively) and was higher than after acetazolamide treatment (3.8±1.6 and 1.2±1.2 l min(-1), respectively) (P<0.05). Plasma fluid flux did not change from rest in control and decreased after acetazolamide treatment (-4.5±1.5 l min(-1)) (P<0.05). Erythrocyte Cl- flux increased during exercise in control and after acetazolamide treatment (P<0.05). During exercise plasma Cl- flux across the lung did not change in control; however, it increased with acetazolamide treatment (P=0.0001). During exercise whole blood Cl- flux increased across the lung in control (P<0.05) but not after acetazolamide treatment. The results indicate that Jv-a in the lung is dependent on the Jacobs-Stewart cycle and mostly independent of transmural hydrostatic forces. It also appears that Jv-a is mediated by Cl- and water egress from erythrocytes directly into the interstitium without transit through plasma.

Comparison of microbial populations in the small intestine, large intestine and feces of healthy horses using terminal restriction fragment length polymorphism.

BACKGROUND: The composition of the microbiota of the equine intestinal tract is complex. Determining whether the microbial composition of fecal samples is representative of proximal compartments of the digestive tract could greatly simplify future studies. The objectives of this study were to compare the microbial populations of the duodenum, ileum, cecum, colon and rectum (feces) within and between healthy horses, and to determine whether rectal (fecal) samples are representative of proximal segments of the gastrointestinal tract. Intestinal samples were collected from ten euthanized horses. 16S rRNA gene PCR-based TRFLP was used to investigate microbiota richness in various segments of the gastrointestinal tract, and dice similarity indices were calculated to compare the samples. RESULTS: Within horses large variations of microbial populations along the gastrointestinal tract were seen. The microbiota in rectal samples was only partially representative of other intestinal compartments. The highest similarity was obtained when feces were compared to the cecum. Large compartmental variations were also seen when microbial populations were compared between six horses with similar dietary and housing management. CONCLUSION: Rectal samples were not entirely representative of intestinal compartments in the small or large intestine. This should be taken into account when designing studies using fecal sampling to assess other intestinal compartments. Similarity between horses with similar dietary and husbandry management was also limited, suggesting that parts of the intestinal microbiota were unique to each animal in this study.

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses.

BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, ß2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.

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 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).

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.

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.

Transvascular fluid flux from the pulmonary vasculature at rest and during exercise in horses.

Exercise causes changes in pulmonary haemodynamics through redistribution of blood flow, increase in the pulmonary surface area, and increase in pulmonary vascular pressures. These changes contribute to the increase in fluid exchange across the alveolar-capillary barrier. To determine the extent of the fluid exchange across the alveolar-capillary barrier at rest and during exercise, six horses were exercised on a high-speed treadmill until fatigue. Arterial and mixed venous blood were sampled at rest and during exercise and recovery. Blood volume changes across the lung (DeltaBV; measured in percentage) were calculated from changes in plasma protein and haemoglobin concentration, and haematocrit. Cardiac output (Q) was calculated using the Fick equation. Fluid flux (J(V-A); measured in l min(-1)) across the alveolar-capillary barrier was then quantified based on Q and DeltaBV. At rest, no fluid movement occurred across the pulmonary vasculature (0.6 +/- 0.6 l min(-1)). During exercise, the amount of fluid moved from the pulmonary circulation was 8.3 +/- 1.3 l min(-1) at 1 min, 6.4 +/- 2.9 l min(-)(1) at 2 min, 10.1 +/- 1.0 l min(-1) at 3 min, 12.9 +/- 2.5 l min(-1) at 4 and 9.6 +/- 1.5 l min(-1) at fatigue (all P < 0.0001). Erythrocyte volume decreased by 6% (P < 0.01) across the lungs, which decreased the colloid osmotic gradient in the pulmonary vasculature. Decrease colloid osmotic gradient along with increased hydrostatic forces in the pulmonary vasculature would enhance displacement of fluid into the pulmonary interstitium. In conclusion, exercise caused large increases in transpulmonary fluid fluxes in horses. Here, we present a simple method to calculate transpulmonary fluid fluxes in different species, which can be used to elucidate mechanisms of lung fluid balance in vivo.

Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions.

The recovery of Clostridium difficile from the stools of patients with C. difficile-associated diarrhea was evaluated by use of an enrichment broth (cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate [TCCFB]) and was compared to that from selective agar (cycloserine-cefoxitin fructose agar [CCFA]) and alcohol shock followed by inoculation onto blood agar (AS-BA). TCCFB was superior to CCFA and AS-BA, and neither the storage time nor the storage temperature affected the recovery rate.

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 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.

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.

Rhodococcus equi pleuropneumonia in an adult horse.

A 10-year-old warmblood gelding was evaluated for intermittent pyrexia, dullness, weight loss, and progressive respiratory disease. Multifocal necrotic pneumonia and pleuritis due to Rhodococcus equi infection was diagnosed. Case management is discussed, as well as factors that may have led to this rare cause of pleuropneumonia in an adult horse.