Screening of the equine intestinal microflora for potential probiotic organisms

REASONS FOR PERFORMING STUDY: Probiotics have not been demonstrated to provide any beneficial health effects in horses, possibly because of improper selection of probiotic organisms. This study was designed to identify lactic acid bacteria of equine origin with predetermined beneficial properties which might make them useful as therapeutic probiotics. HYPOTHESIS: A small percentage of lactic acid bacteria that are native to the intestinal tract of horses possess properties that may be useful in the treatment and/or prevention of gastrointestinal disease in horses. METHODS: Faecal samples were collected from healthy mature horses and foals. Lactic acid bacteria were isolated and tested for the ability to grow in acid and bile environments, aerotolerance and in vitro inhibition of enteropathogens. One isolate that possessed these properties was administered orally to healthy mature horses and foals and gastrointestinal survival was assessed. RESULTS: Of the 47 tested organisms, 18 were deemed to be adequately acid- and bile-tolerant. All were aerotolerant. Four organisms markedly inhibited Salmonella spp. One isolate, Lactobacillus pentosus WE7, was subjectively superior and chosen for further study. It was also inhibitory against E. coli, moderately inhibitory against S. zooepidemicus and C. difficile and mildly inhibitory against C. perfringens. After oral administration, this isolate was recovered from the faeces of 8/9 (89%) foals and 7/8 (87.5%) mature horses. CONCLUSIONS: Lactobacillus pentosus WE7 possesses in vitro and in vivo properties that may be useful for the prevention and treatment of enteric disease in horses. POTENTIAL RELEVANCE: The beneficial in vitro and in vivo properties that L. pentosus WE7 possesses indicate that randomised, blinded, placebo-controlled efficacy studies are warranted.     

Determination of the incidence of Salmonella spp., Campylobacter jejuni, and Clostridium perfringens in wild birds near broiler chicken houses by sampling intestinal droppings

Several methods were evaluated for collecting fecal and intestinal samples from wild birds found near broiler chicken houses. A few intestinal samples and cloacal swabs were obtained from European starlings and house sparrows. Most of the samples collected consisted of wild bird droppings found on or near the houses. Samples were collected from each of four farms of a broiler integrator during a grow-out cycle: a cycle in the summer for farm A, fall for farm B, and spring, summer, fall, and winter for farms C and D. Of the 25 wild bird intestinal and fecal samples collected from a broiler house on farm A during a grow-out cycle in July-August 1997, 24% were positive for Salmonella spp., 4% for Campylobacter jejuni, and 28% for Clostridium perfringens. Of the nine fecal samples collected from broiler house B in a grow-out cycle in September-November 1997, 33% were positive for Salmonella spp., 11% for C. jejuni, and 22% for C. perfringens. For farms C and D, of the 23 samples collected in March-April 1998, 0 were positive for Salmonella spp., 11% for C. jejuni, and 52% for C. perfringens; of 27 samples collected in June-July 1998, 4% were positive for Salmonella spp., 0 for C. jejuni, and 13% for C. perfringens; of 24 samples collected in August-October 1998, 14% were positive for Salmonella spp., 5% for C. jejuni, and 4% for C. perfringens; of 14 samples collected December 1998-January 1999, 0 were positive for Salmonella, 50% for C. jejuni, and 14% for C. perfringens. The incidence of these bacterial enteropathogens in wild birds near the broiler chicken houses suggests that wild birds that gain entry to poultry grow-out houses have the potential to transmit these pathogens to poultry.     

A prospective study of the roles of clostridium difficile and enterotoxigenic Clostridium perfringens in equine diarrhoea

Faecal samples from adult horses and from foals with diarrhoea or with normal faeces were evaluated for the presence of Clostridium difficile, C. difficile toxins, C. perfringens enterotoxin (CPE) and C. perfringens spore counts. Clostridium difficile was isolated from 7/55 horses (12.7%) and 11/31 foals (35.5%) with colitis, but from 1/255 normal adults (0.4%) and 0/47 normal foals (P<0.001). Clostridium difficile toxins A and/or B were detected in 12/55 diarrhoeic adults (21.8%) and 5/30 diarrhoeic foals (16.7%) but in only 1/83 adults (1.2%) and 0/21 foals with normal faeces (P<0.001 and P<0.05, respectively). Clostridium perfringens enterotoxin was detected in 9/47 diarrhoeic adults (19%) and 8/28 diarrhoeic foals (28.6%), but was not detected in 47 adult horses (P<0.002) or 4 foals (P = 0.22) with normal faeces. The positive predictive value of isolation of C. perfringens with respect to the presence of CPE was only 60% in adult horses and 64% in foals. There was no association between total C. perfringens spore count and CPE in the faeces. The overall mortality rate from colitis was 22% for adult horses and 18% for foals. Clostridium difficile toxin-positive adult horses with colitis were less likely to survive than C. difficile-negative horses with colitis (P = 0.03). This study provides further evidence that C. difficile and enterotoxigenic C. perfringens are associated with equine enterocolitis.     

Fecal shedding of Salmonella spp by horses in the United States during 1998 and 1999 and detection of Salmonella spp in grain and concentrate sources on equine operations

OBJECTIVE: To estimate prevalence of fecal shedding of Salmonella spp among horses in the US horse population and prevalence of Salmonella spp in grain or other concentrate used as horse feed on equine operations in the United States. DESIGN: Cross-sectional survey. SAMPLE POPULATION: Horses on 972 operations in 28 states. PROCEDURE: Fecal samples were collected from horses resident at each operation. Only a single sample was collected from any individual horse; number of horses from which samples were collected on each operation was determined on the basis of number of horses on the operation. A single sample of grain or concentrate was also collected from each operation. All samples were tested for Salmonella spp by means of bacterial culture. RESULTS: Overall, 0.8% (SE, 0.5) of resident horses shed Salmonella spp in their feces. The overall prevalence of operations positive for fecal shedding of Salmonella spp (i.e., operations with > or = 1 horse shedding Salmonella spp in its feces) was 1.8% (SE, 0.7). Prevalence of grain or other concentrate samples positive for Salmonella spp was 0.4%. Serotypes of Salmonella spp that were identified in grain or other concentrate were not those typically associated with clinical disease in horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the national prevalence of fecal shedding of Salmonella spp by horses in the United States was 0.8%, and that prevalence of Salmonella spp in grain or other concentrate used for horse feed was 0.4%.     

Adsorptive effects of di-tri-octahedral smectite on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies

OBJECTIVE: To determine the adsorptive capability of di-tri-octahedral smectite (DTOS) on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies. SAMPLE POPULATION: 3 C perfringens exotoxins and 9 colostral samples. PROCEDURES: Alpha, beta, and beta-2 exotoxins were individually co-incubated with serial dilutions of DTOS or bismuth subsalicylate, and the amount of toxin remaining after incubation was determined via toxin-specific ELISAs. Colostral samples from healthy mares were individually co-incubated with serial dilutions of DTOS, and colostral IgG concentrations were determined via single radial immunodiffusion assay. RESULTS: Di-tri-octahedral smectite decreased the amount of each C perfringens exotoxin in co-incubated samples in a dose-dependent manner and was more effective than bismuth subsalicylate at reducing exotoxins in vitro. Decreases in the concentration of IgG were detected in samples of colostrum that were combined with DTOS at 1:4 through 1:16 dilutions, whereas no significant decrease was evident with DTOS at the 1:32 dilution. CONCLUSIONS AND CLINICAL RELEVANCE: Di-tri-octahedral smectite effectively adsorbed C perfringens exotoxins in vitro and had a dose-dependent effect on the availability of equine colostral antibodies. Results suggested that DTOS may be an appropriate adjunctive treatment in the management of neonatal clostridiosis in horses. In vivo studies are necessary to fully assess the clinical efficacy of DTOS treatment.     

Daily variability of equine fecal strongyle egg counts.

Fecal egg counts often are used for diagnosing equine strongyle infections and estimating the number of eggs shed in the feces. An individual egg count should be interpreted in view of the normal fluctuation of egg numbers in an individual horse. In this study, the daily variability of strongyle fecal egg counts from horses was investigated. The Cornell-McMaster egg-counting technique was used to estimate the eggs per gram of feces in repeated daily fecal samples from 39 horses. The variation of the daily egg counts across 4 days was greater than would be expected if a consistent number of eggs were produced and dispersed randomly in the feces. The means and variances of the daily counts from each horse had a logarithmic relationship. For practical purposes, however, the fluctuation of egg counts is low enough for the fecal egg count to be used to identify horses for treatment, to estimate pasture contamination, or to assess response to therapy.     

Evaluation of a PCR to detect Salmonella in fecal samples of horses admitted to a veterinary teaching hospital.

The diagnostic accuracy of a PCR used to identify horses shedding Salmonella spp. in their feces during hospitalization was estimated, relative to bacterial culture of serially collected fecal samples, using longitudinal data. Five or more fecal samples were collected from each of 116 horses admitted as inpatients, for reasons other than gastrointestinal disease, between July 26, 2001 and October 25, 2002. All 873 fecal samples collected were tested with a PCR based on oligonucleotide primers defining a highly conserved segment of the histidine transport operon gene of Salmonella typhimurium, and each sample was cultured for Salmonella spp. One or more samples from 87 (75%) horses were PCR positive, and Salmonella was cultured from 1 or more samples from 11 (9.5%) horses. All culture-positive horses had at least 1 PCR-positive result, whereas only 29 (28%) culture-negative horses were PCR negative on all fecal samples tested. The PCR was most specific, relative to bacterial culture of serially collected fecal samples, when used to test samples from Quarterhorse or breeds other than Thoroughbred or Standardbred, or from clinical (vs. healthy, accompanying horses) cases. Overall, the PCR had the greatest agreement (70%), compared with bacterial culture of serially collected fecal samples, using a cutoff of 2 or more positive PCR test results to define a Salmonella-positive horse. The reasons why some fecal samples, from which Salmonella organisms cannot be isolated, are PCR positive need to be determined before the PCR can be incorporated into Salmonella surveillance programs for hospitalized equine populations.     

Evaluation of the opsonic capacity of core lipopolysaccharide antiserum of equine origin against smooth Escherichia coli 0111:B4, using macrophage chemiluminescence

A study was performed to determine whether equine antiserum to core lipopolysaccharide (LPS) would enhance phagocytosis of smooth gram-negative (GN) organisms by equine macrophages. Five healthy adult horses (group A) were immunized with a bacterin prepared from the J-5 mutant of Escherichia coli 0111:B4 and Salmonella minnesota R595 to produce antibodies to core LPS. Five horses (group B) served as nonimmunized controls and were given physiologic saline solution instead of the rough mutant bacterin. Serum antibody titers to core LPS and to smooth E coli 0111:B4 were determined by indirect ELISA. Four serum pools were prepared: pool 1 = sera from horses in group B prior to immunization; pool 2 = sera from horses in group A prior to immunization (preimmune serum); pool 3 = sera from horses in group B, 7 days after the last saline injection; pool 4 = sera from horses in group A, 7 days after the last immunization (core LPS antiserum). The serum pools, either unheated or heated 30 minutes at 56 C, in 3 dilutions (1/50, 1/100, 1/500) were used to opsonize smooth E coli 0111:B4 in an assay of equine peritoneal macrophage chemiluminescence (CL). Peritoneal fluid was collected from clinically normal horses and the macrophages were purified by adherence to borosilicate glass scintillation vials. Each serum type and dilution was added to triplicate vials containing 10(7) colony-forming units of E coli 0111:B4. Luminol-dependent CL was measured with a liquid scintillation counter in the out-of-coincidence mode. Each serum dilution was tested in duplicate vials without bacteria to asses serum-induced nonspecific CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of equine hemograms using the ADVIA 120 as compared with an impedance counter and manual differential count

BACKGROUND: The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. OBJECTIVES: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. METHODS: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various breeds, both sexes, and 2-6 years of age. Samples were stored for up to 12 hours at 4 degrees C and then analyzed on the ADVIA 120 and the Hemat 8. A 100-cell to 200-cell differential leukocyte count was performed by 3 independent observers on May-Grünwald-Giemsa-stained smears. Intra-assay precision of the ADVIA 120 was determined by analyzing 5 replicates each of 10 of the blood samples. RESULTS: Results from the ADVIA were significantly higher than those from the impedance counter for RBC count, total WBC count, hemoglobin concentration, red cell distribution width, MCH, and MCHC, and significantly lower for HCT and platelet count. Significantly higher neutrophil and basophil counts and significantly lower lymphocyte counts were obtained with the ADVIA 120 compared with manual counts. Based on Passing-Bablok regression analysis, RBC and platelet counts were in good agreement between the 2 analyzers; a constant and proportional bias was present for other values. Coefficients of variation for erythrocyte parameters on the ADVIA were <1%, but were higher for platelet (6%), total WBC (2%), differential WBC (4%-30%), and reticulocyte (75%) counts. CONCLUSIONS: Results obtained with equine samples on the ADVIA 120 were comparable with those obtained on an impedance counter; reference intervals differed statistically but overlapped. The ADVIA had poor precision for reticulocyte and differential leukocyte counts such that the latter should always be verified on smears.     

Nosocomial infections and bacterial antibiotic resistance in a university equine hospital

A base-line study of bacteria isolated from horses admitted to the Veterinary Medical Teaching Hospital during a 6-month period was performed to determine the extent of multiresistant nosocomial infections caused by gram-negative aerobic bacteria other than Salmonella spp. Results of this study indicated that 21.9% of the 105 horses from which cultures and sensitivities were available had developed nosocomial gram-negative aerobic infections, with high rates of resistance to gentamicin, kanamycin, and trimethoprim sulfadiazine, three of the most often prescribed antibiotics in this hospital. In addition, a prospective study of antibiotic-resistant bacteria of fecal origin was performed to determine whether there was a change in the degree of antibiotic resistance of a horse's intestinal flora while the horse was hospitalized. Bacterial culturing for gram-negative lactose fermenting bacteria was done on fecal specimens collected directly from the rectum on day 1 and day 7 of a horse's hospitalization. Susceptibility testing was done on each isolant. Of the 24 paired fecal specimens obtained, Escherichia coli and Klebsiella sp isolated on day 7 were resistant to a significantly higher number of antibiotics than day 1 isolants (P = 0.003, P = 0.043, respectively).     

Influence of bacteria on Clostridium perfringens infections in young chickens

When monoflora chickens with Lactobacillus acidophilus or Streptococcus faecalis were inoculated with Clostridium perfringens either in broth culture or resuspended in Gifu anaerobic medium broth or supernatant fluid, few or no chickens died. Approximately 50% of germ-free chickens died after inoculation of C. perfringens culture, whereas no conventional birds died after inoculation of broth culture. C. perfringens in the contents of duodenum from germ-free chickens numbered about 10(4) colony-forming units (CFU)/g after inoculation 10(8) CFU broth culture per bird, but in gnotobiotic and conventional chickens this organism decreased or was not detected. When C. perfringens was cultured in intestinal contents collected from germ-free chickens, C. perfringens proliferated but alpha toxin was not detected. These findings indicate that the pathogenicity of C. perfringens was suppressed by L. acidophilus or S. faecalis administered previously or inhibited by normal intestinal flora.     

Nosocomial diarrhea associated with enterotoxigenic Clostridium perfringens infection in dogs

A retrospective analysis of the medical records of 30 consecutive cases of diarrhea occurring in dogs that were hospitalized in a teaching hospital was performed. A prospective analysis of culture results for Clostridium perfringens of dogs with diarrhea were compared with those of a control nondiarrheal group. Hospital-acquired diarrhea in dogs was found to be associated with multiple serotypes of enterotoxigenic Clostridium perfringens. Other potential etiologic agents could not be isolated. Clinical signs were variable, and included mild depression, anorexia, and soft to watery diarrhea with or without frank blood, mucus, and tenesmus. Fever was not present. There were no hematologic or serum biochemical abnormalities, nor were there any consistent virologic or parasitologic findings. Salmonella spp or Campylobacter spp were not identified by fecal culture. No risk factors could be identified. A dog that was euthanatized on the day it developed diarrhea had intestinal histologic findings suggestive of clostridial enteritis. Dogs with diarrhea had significantly higher fecal clostridial counts than did dogs without diarrhea (mean log10 counts +/- SD = 6.34 +/- 1.79 vs 4.75 +/- 2.07). Enterotoxin was found in the feces of 41% of diarrheic dogs but in only 7% of dogs without diarrhea.     

Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs

The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.     

A comparative study of the intestinal microbiota of healthy horses and those suffering from equine grass sickness

This study compares quantitatively the microbiota of the gastrointestinal tract of healthy horses with that of horses with equine grass sickness (EGS). Faecal and ileal samples were cultured quantitatively on selective and non-selective media. Confirmed anaerobes were identified to species level.Overall faecal counts gave a ratio of aerobes:anaerobes of approximately 1:1. However, the mean counts in healthy horses of 4.4x10(8) aerobes:3.7x10(8) anaerobes per gram wet weight were different from counts in EGS (means were 10-100-fold higher), with statistically significant differences for the anaerobes (p=0.04). There were 10-100-fold more anaerobic cocci in EGS samples compared to healthy controls. Most of the seven species of anaerobic cocci were found in both healthy horses and EGS. Differences in clostridia isolated between health and disease were notable: fourteen species were isolated from EGS cases, compared to only one (C. bifermentans) in controls. The mean faecal clostridial counts in chronic disease were higher than in controls (10-fold) and in acute EGS (100-fold). In contrast, mean counts for ileal samples from acute cases, showed a 10-fold increase for clostridia compared to 1000-fold reduction in chronic cases (compared to faecal counts). Results indicate an increase in the bacterial numbers in the GI tract of animals with EGS compared to the controls and clostridia are prominent in EGS. Whether the increase in clostridia is the cause of GI stasis or a consequence remains uncertain.     

Factors associated with Salmonella shedding among equine colic patients at a veterinary teaching hospital

OBJECTIVE: To evaluate factors potentially associated with fecal Salmonella shedding among equine patients hospitalized for colic at a veterinary teaching hospital and to determine the effects of probiotic treatment on fecal Salmonella shedding and clinical signs. DESIGN: Longitudinal study and controlled trial. ANIMALS: 246 equine colic patients. PROCEDURE: History and medical information were obtained from patient records. Fecal and environmental samples were submitted for aerobic bacterial culture for Salmonella enterica. Fifty-one patients were treated with a commercially available probiotic; 46 were treated with a placebo. Logistic regression was used to evaluate data. RESULTS: Salmonella organisms were detected in feces from 23 (9%) patients at least once during hospitalization. Patients were more likely to shed Salmonella organisms if diarrhea was evident < or = 6 hours after hospitalization and duration of hospitalization exceeded 8 days (odds ratio [OR], 20.3), laminitis developed during hospitalization (OR, 12.0), results of nasogastric intubation were abnormal (OR, 4.9), leukopenia was evident < or =6 hours after hospitalization (OR, 4.6), or travel time to the teaching hospital exceeded 1 hour (OR, 3.5). Horses treated with the probiotic did not differ from control horses in regard to likelihood of fecal Salmonella shedding (OR, 1.5) or prevalence of clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that certain risk factors are associated with fecal shedding of S enterica among equine patients hospitalized at a veterinary teaching hospital because of colic and that pathogen monitoring in patients and the hospital environment and use of barrier nursing precautions for equine colic patients are beneficial.     

Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever)

Fifty-five horses were inoculated IV and/or SC with materials containing Ehrlichia risticii, ie, infected whole blood, buffy coat cells, or cell culture, to study clinical and hematologic features of equine monocytic ehrlichiosis (Potomac horse fever). Major clinical and hematologic features of induced E risticii infection were biphasic increase in rectal temperature with peak increases of 38.9 C and 39.3 C on postinoculation days (PID) 5 and 12, respectively; depression; anorexia; decreased WBC count (maximal decrease of 47% on PID 12); and diarrhea from PID 14 to PID 18. Increased WBC count was an inconsistent feature, with a maximal increase of 51.5% on PID 20. During times of decreased and increased WBC counts, lymphocyte/neutrophil ratios remained fairly constant. However, not all horses had all clinical and hematologic features, and these features were present in different degrees among horses. Increased rectal temperature, depression, anorexia, and decreased WBC count were more consistent features, whereas diarrhea developed in 73% of the horses. Of 55 horses, 39 (71%) had all clinical and hematologic features of the disease (classic disease), whereas 16 (29%) horses did not have greater than or equal to 1 of these features (nonclassic disease). The E risticii titer in the blood (ehrlichemia) was maximum during the peak increase in rectal temperature. In 55 horses, mortality was 9%. Significant differences (P greater than 0.5) in clinical and hematologic features were not detected between horses that survived and those that died of E risticii infection.     

Equine immune-mediated hemolytic anemia associated with Clostridium perfringens infection

Background: Previous studies have suggested an association between equine immune-mediated hemolytic anemia and clostridial infections or neck abscesses.
Objective: The purpose of this report was to describe and characterize the hematologic abnormalities in a horse with Clostridium -associated immune-mediated hemolytic anemia. We also retrospectively evaluated hematologic abnormalities in 8 horses with clostridial myositis or subcutaneous emphysema.
Methods: A 7-year-old Foreign Warm-Blood gelding was evaluated for anemia and a cervical abscess. CBCs and reticulocyte counts were obtained using an Advia 120 analyzer and evaluation of Wright's-stained smears. All cases of equine Clostridium spp. myositis or subcutaneous emphysema over a 7-year period were identified in a retrospective search of the University of Minnesota Veterinary Teaching Hospital database. Clinical, hematologic, and microbiological findings were recorded.
Results: Clostridium perfringens genotype A was isolated from the neck wound of the gelding. The CBC was characterized by severe regenerative anemia, intravascular hemolysis, and RBC agglutination. A direct Coombs' test was positive. Moderate numbers of spheroechinocytes were observed. The total automated reticulocyte count was 4.5% (56,700/μL), with most reticulocytes having low absorbance (mature reticulocytes). The anemia responded to penicillin and steroidal and nonsteroidal immunosuppressive drugs. Of 8 horses with myositis, all of which involved the cervical region, 5 were anemic, 1 had a positive direct Coombs' test, and 2 had increased numbers of type III echinocytes and spheroechinocytes.
Conclusions: The presence of type III echinocytes or spheroechinocytes may be helpful in diagnosing immune-mediated hemolytic anemia associated with clostridial infections in horses. Automated reticulocyte counts may detect very low levels of reticulocytosis in hemolytic anemia in horses.     

Emerging outbreaks associated with equine coronavirus in adult horses

The purpose of this study was to describe clinical, hematological and fecal PCR results from 161 horses involved in outbreaks associated with ECoV. The outbreaks happened at four separate boarding facilities between November 2011 and April 2012 in the States of CA, TX, WI and MA. Following the molecular detection of ECoV in the feces from the initial index cases, the remaining herdmates were closely observed for the development of clinical signs. Fecal samples were collected from sick and healthy horses for the PCR detection of ECoV. All four outbreaks involved primarily adult horses. Fifty-nine horses developed clinical signs with 12–16 sick horses per outbreak. The main clinical signs reported were anorexia, lethargy and fever. Four horses from 3 different outbreaks were euthanized or died due to rapid progression of clinical signs. The cause of death could not be determined with necropsy evaluation in 2 horses, while septicemia secondary to gastrointestinal translocation was suspected in 2 horses. Blood work was available from 10 horses with clinical disease and common hematological abnormalities were leucopenia due to neutropenia and/or lymphopenia. Feces were available for ECoV testing by real-time PCR from 44 and 96 sick and healthy horses, respectively. 38/44 (86%) horses with abnormal clinical signs tested PCR positive for ECoV, while 89/96 (93%) healthy horses tested PCR negative for ECoV. The overall agreement between clinical status and PCR detection of ECoV was 91%. The study results suggest that ECoV is associated with self-limiting clinical and hematological abnormalities in adult horses.     

Longitudinal study of Clostridium difficile and antimicrobial susceptibility of Escherichia coli in healthy horses in a community setting

Point prevalence studies have reported carriage rates of enteric pathogens in healthy horses, but longitudinal data are lacking. Commensal E. coli is an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data for horses. The objectives of this study were to investigate and molecularly characterize isolates of Clostridium difficile, Clostridium perfringens and Salmonella, collected sequentially over a one year period, and to determine the antibiotic susceptibility profile for E. coli. Fecal samples were collected monthly from 25 adult horses for one year. Selective cultures were performed for all above bacteria. C. difficile isolates were characterized via PCR toxin gene profiling and ribotyping. Broth microdilution was performed to assess antimicrobial susceptibility profiles of E. coli. Toxigenic Clostridium difficile was isolated from 15/275 (5.45%) samples from 10/25 (40%) horses. Four horses were positive at multiple sampling times but different ribotypes were found in three. Ribotypes included 078 (n=6), 001 (n=6) and C (n=3). C. perfringens was not isolated, nor was Salmonella. E. coli was isolated from 232/300 (77%) fecal samples. Resistance to ≥1 and ≥ 3 antimicrobials was present in 31/232 (13.4%) and 6/232 (2.6%) respectively. Only two horses shed the same strain of toxigenic C. difficile for more than one month, indicating that shedding is transient. The high number of ribotype 078 is consistent with recent emergence of this strain in the local horse population. The low prevalence of antibiotic resistance in commensal E. coli suggests that healthy horses are not likely a major reservoir of resistance for enteric bacteria.     

Immediate postnatal inoculation of a microbial barrier to prevent neonatal diarrhea induced by Clostridium difficile in young conventional and gnotobiotic hares

The cecal content of a healthy young hare (a 10-fold dilution prepared in anaerobiosis) was transferred into gnotobiotic recipient mice. The fecal flora of recipient mice was inoculated into 49 conventional young hares (kept in a closed building) immediately after birth. All the inoculated hares survived until weaning whereas 35% of 48 non-treated conventional hares died of diarrhea. Clostridium difficile and C perfringens were present in all the diarrheal noninoculated hares, whereas these species were present only in 50% of healthy inoculated or noninoculated hares at low concentrations (less than 10(6)/g of feces). A microbial barrier effect against challenge-exposure strains of C difficile and C perfringens was evidenced in the intestinal tract of recipient gnotobiotic mice and young hares. Seemingly, the protective effect of the microbial inoculum was due to antagonisms exerted against C difficile and C perfringens in the intestinal tract of inoculated conventional hares.