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.     

Survival of Clostridium difficile and its toxins in equine feces: implications for diagnostic test selection and interpretation.

Although Clostridium difficile is recognized as a cause of enterocolitis in horses and humans, there has been little work published regarding the lability of C. difficile and its toxins in feces. A significant decrease in recovery of C. difficile from inoculated equine fecal samples occurred during storage. Recovery after storage in air at 4 degrees C decreased from 76% (37/49) after 24 hours to 67% (33/49) at 48 hours and 29% (14/ 49) after 72 hours. In contrast to aerobic storage, 25 of 26 samples stored anaerobically at 4 degrees C yielded growth of C. difficile for 30 days, whereas the organism was only detected for 2.5 +/- 2.52 days (x +/- SD) in paired samples stored aerobically. The use of an anaerobic transport medium was effective in maintaining viability of C. difficile. These findings indicate that poor aerotolerance is the reason for the rapid decrease in culture yield. In contrast to C. difficile organisms stored aerobically at 4 degrees C, C. difficile toxins were considerably more stable and could be detected by enzyme-linked immunosorbent assay in both broth and inoculated fecal samples for at least 30 days. The poor survival of C. difficile but the stability of its toxins when feces are stored aerobically must be considered when submitting samples for diagnosis of C. difficile-associated enterocolitis in horses and when interpreting laboratory results.     

Isolation of environmental Clostridium difficile from a veterinary teaching hospital.

An environmental survey of a veterinary teaching hospital for the presence of Clostridium difficile was performed using contact plates and cycloserine-cefoxitin-fructose with 0.1% sodium taurocholate agar. Clostridium difficile was isolated from 24 of 381 sites (6.3%). Growth was obtained from 4.5% (9/202) of sites sampled in the Large Animal Clinic, from 8.1% (13/160) of sites within the Small Animal Clinic, and from 20% (2/10) of sites sampled elsewhere. Fourteen of 21 strains tested produced toxins in vitro. A geographic association was found with areas in the large animal clinic where nosocomial C. difficile diarrhea in horses had previously been diagnosed. Several other sites with a potential for nosocomial transmission of the organism were identified. Areas from which C. difficile was isolated tended to be areas with high animal traffic, with increased chance of fecal contamination, and with rough, difficult to clean surfaces. This study documents the prevalence of this organism in the environment and its potential role in nosocomial disease.     

Clostridium difficile associated with acute colitis in mature horses treated with antibiotics

Clostridium (C.) difficile, or its cytoxin, was demonstrated in faecal samples from 10 of 25 (40%) mature horses investigated with acute colitis treated primarily with antibiotics for disorders other than diarrhoea. C. difficile was not found in faecal samples from 140 horses without signs of enteric disorders, from 21 nondiarrhoeic horses treated with antibiotics, nor from 22 horses with colitis untreated with antibiotics. Except for C. difficile neither Salmonella nor any other investigated intestinal pathogen was isolated in any of the diarrhoeic horses. The findings strongly support some earlier reports that C. difficile is associated with acute colitis in mature horses treated with antibiotics. Of the 10 horses, 4 proved positive for C. difficile both in culture and in the cytotoxin test, 4 in culture only and 2 only in the cytotoxin test. Eight of 10 horses positive for C. difficile were or had recently been hospitalised, indicating that C. difficile may be a nosocomial infection in horses. All horses positive for C. difficile were treated with beta-lactam antibiotics.     

Clostridium difficile: prevalence in horses and environment,and antimicrobial susceptibility

REASONS FOR PERFORMING STUDY: Clostridium difficile has been associated with acute colitis in mature horses. OBJECTIVES: To survey C. difficile colonisation of the alimentary tract with age, occurrence of diarrhoea and history of antibiotic therapy; and to study the occurrence and survival of C. difficile in the environment and antimicrobial susceptibility of isolated strains. METHODS: A total of 777 horses of different breeds, age and sex were studied. Further, 598 soil samples and 434 indoor surface samples were examined. Antimicrobial susceptibility of 52 strains was investigated by Etest for 10 antibiotics. RESULTS: In horses that developed acute colitis during antibiotic treatment, 18 of 43 (42%) were positive to C. difficile culture and 12 of these (28%) were positive in the cytotoxin B test. Furthermore, C. difficile was isolated from a small number of diarrhoeic mature horses (4 of 72 [6%]) with no history of antibiotic treatment, but not from 273 healthy mature horses examined or 65 horses with colic. An interesting new finding was that, in normal healthy foals age < 14 days, C. difficile was isolated from 1/3 of foals (16 of 56 [29%]). All older foals (170) except one were negative. Seven of 16 (44%) nondiarrhoeic foals treated with erythromycin or gentamicin in combination with rifampicin were also excretors of C. difficile. On studfarms, 14 of 132 (11%) outdoor soil samples were positive for C. difficile in culture, whereas only 2 of 220 (1%) soil samples from farms with mature horses were positive for C. difficile (P = < 0.001). By PCR, it was demonstrated that strains from the environment and healthy foals can serve as a potential reservoir of toxigenic C. difficile. The experimental study conducted here found that C. difficile survived in nature and indoors for at least 4 years in inoculated equine faeces. The susceptibility of 52 strains was investigated for 10 antibiotics and all were susceptible to metronidazole (MIC < or = 4 mg/l) and vancomycin (MIC < or = 2 mg/l). CONCLUSIONS: C. difficile is associated with acute colitis in mature horses, following antibiotic treatment. Furthermore, C. difficile was isolated from 1 in 3 normal healthy foals age < 14 days. POTENTIAL RELEVANCE: Strains from healthy foals and the environment can serve as a potential reservoir of toxigenic C. difficile.     

Molecular characterization of Clostridium difficile isolates from horses in an intensive care unit and association of disease severity with strain type

OBJECTIVE: To determine molecular characteristics, antimicrobial susceptibility, and toxigenicity of Clostridium difficile isolates from horses in an intensive care unit and evaluate associations among severity of clinical disease with specific strains of C difficile. DESIGN: Prospective study. ANIMALS: 130 horses. PROCEDURES: Feces were collected from horses admitted for acute gastrointestinal tract disease with loose feces and submitted for microbial culture and immunoassay for toxin production. Polymerase chain reaction assays were performed on isolates for toxins A and B genes and strain identification. RESULTS: Isolates were grouped into 3 strains (A, B, and C) on the basis of molecular banding patterns. Toxins A and B gene sequences were detected in 93%, 95%, and 73% of isolates of strains A, B, and C, respectively. Results of fecal immunoassays for toxin A were positive in 40%, 63%, and 16% of horses with strains A, B, and C, respectively. Isolates in strain B were resistant to metronidazole. Horses infected with strain B were 10 times as likely to have been treated with metronidazole prior to the onset of diarrhea as horses infected with other strains. Duration from onset of diarrhea to discharge (among survivors) was longer, systemic inflammatory response syndromes were more pronounced, and mortality rate was higher in horses infected with strain B than those infected with strains A and C combined. CONCLUSIONS AND CLINICAL RELEVANCE: Horses may be infected with a number of heterogeneous isolates of C difficile. Results indicated that toxigenicity and antimicrobial susceptibility of isolates vary and that metronidazole-resistant strains may be associated with severe disease.     

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 in Horses Admitted to a Veterinary Teaching Hospital

Fecal samples were collected from 724 horses admitted to, and 232 horses hospitalized at, a veterinary teaching hospital between October 2000 and June 2001, and cultured for Salmonella. Salmonella was isolated on 24 occasions from 12 horses. S. Newport was the most commonly isolated serotype. The estimated prevalence (95% confidence interval [CI]) of Salmonella shedding on admission was 0.5% (0.1, 2.0). The estimated incidence rate of Salmonella shedding during hospitalization was 4.3% (95% CI, 2.2-8.1). The highest incidence of Salmonella shedding during hospitalization occurred during June 2001, but no significant (P > .5) temporal clusters of horses shedding Salmonella were detected.Horses shed Salmonella in their feces at a low rate. Horses shedding Salmonella occurred only sporadically and no obvious serotype pattern was observed. The occurrence of fecal shedding probably reflected the prevalence of inapparent Salmonella infection in the hospital source population, providing a baseline against which future potential outbreaks can be identified.

Introduction

Infection of horses by Salmonella organisms is a serious health issue. It is particularly troubling when outbreaks occur in hospitalized patients because these outbreaks can result in substantial economic losses and have a major impact on the welfare of patients.[1] Establishments with a high-density of horses, including veterinary teaching hospitals (VTHs) and private veterinary clinics, [1 and 2] are most vulnerable to outbreaks of disease attributable to Salmonella infection. Host susceptibility and environmental persistence of Salmonella are also factors contributing to outbreaks.Estimates of the prevalence of Salmonella-shedding horses admitted to veterinary hospitals have generally been made under outbreak conditions.[1] For example, between 1971 and 1982, 245 hospitalized horses (1.7%) at the University of California were found to shed Salmonella.[3 and 4] Three major outbreaks occurred during the study period, with no apparent periodicity. Between 1996 and 1999, 35 hospitalized horses (5.5%) at the Michigan State University were found to shed Salmonella.[5] One major outbreak occurred during the study period. Only one national survey of nonhospitalized horses in the United States for Salmonella infection has been undertaken: the prevalence of fecal shedding of Salmonella was estimated to be 0.8%, and the farm prevalence of shedding was 1.8%.[6]Many factors have been associated with the risk of Salmonella isolation from hospitalized horses, including diarrhea, fever, change in diet, large colon impaction, colic, withholding feed, feeding bran mash, antibiotic treatment, intubation with nasogastric tubes, and average daily ambient temperature.[7, 8, 9 and 10] Many of theses factors are thought to operate primarily through the effect of stress, increasing the susceptibility of horses to infection. Also, if a horse is infected by Salmonella but not shedding the organisms in its feces, the presence of stressors may reactivate fecal shedding. Most studies that have been conducted on risk factors for Salmonella shedding in horses have included horses with clinical salmonellosis, with or without inclusion of horses inapparently infected by Salmonella.[7, 8 and 10] Risk factors for Salmonella fecal shedding versus clinical salmonellosis have not been clearly delineated.An outbreak (epidemic) of disease can be defined as “an occurrence of disease in excess of its anticipated frequency.”[11] To more effectively identify future outbreaks of Salmonella infection in hospitalized horses, it is necessary to have accurate estimates of the prevalence of endemic fecal shedding of Salmonella in horses admitted to VTHs, and the incidence of fecal shedding during hospitalization. The aims of this study were to estimate the prevalence of fecal shedding in horses admitted to a VTH, to estimate the incidence of fecal shedding during hospitalization, and to describe the seasonal distribution of fecal Salmonella-shedding prevalence and incidence.

Materials and Methods

Study Design

Fecal samples were collected from horses admitted to the Purdue University VTH between October 12, 2000 and June 30, 2001. Horses admitted as inpatients were sampled at least on the day of admission, the day after admission, the day of discharge, and once or more in between. All horses admitted to the VTH during the study period were eligible to be sampled. In the case of mares accompanying sick foals, samples were also collected from the mare. Fecal samples were collected generally from freshly voided fecal material in stalls. Samples were stored at 4°C for up to 24 hours before processing.

Data Collection

For all horses included in the study, date of examination (outpatients) or date of hospitalization (inpatients) was recorded. For inpatients, date of discharge or date of death was also recorded. Horse characteristics were recorded as part of each horse's medical record, and included date of birth, sex (mare, stallion, gelding), and specific breed. The outcome of each admission (discharged alive, died, euthanized) and whether a necropsy was performed were also recorded. The number of samples collected per horse was recorded in a laboratory-reporting system, but specific date of collection of each sample (except for the first and last samples collected) was not routinely recorded.

Bacteriologic Cultures

All fecal samples were cultured for Salmonella species using standard techniques. Specimens were streaked onto brilliant green (BG) and xylose-lysine-tergitol (XLT-4) plates, and approximately 10 g of fecal material was put into 100 mL of tetrathionate Hajna broth. BG plates were incubated at 35° to 37°C for 18 to 24 hours and XLT-4 plates were incubated for 24 to 48 hours. Tetrathionate broth was incubated at 35° to 37°C for 24 to 48 hours, and then streaked to BG and XLT-4 plates. These plates were incubated as previously described. Suspect colonies on plates were subcultured and further identified by the Vitek GNI system. All Salmonella isolates were speciated and serotyped (National Veterinary Services Laboratory, Ames, Ia).

Data Analysis

The total number of horses examined (admissions), the total number of examinations (including admissions), the total number of horses hospitalized during the study period, and the number of samples collected per horse were calculated (Excel 2000, Microsoft Corp, Redmond, Wash) from recorded information. The frequency distributions of admissions (1-6) per horse, sex, breed, and patient outcome were calculated based on owner/horse identity and hospital record number, and the frequency distribution of number of samples collected per horse (nil to 8) was calculated from laboratory records and owner/horse identification and laboratory submission number. Length of hospitalization (days) was calculated from recorded date of hospitalization and date of discharge information, and was summarized by median and mean lengths of hospitalization and 95% confidence intervals (CIs), based on the Wilcoxon test (Minitab for Windows, Minitab Inc, State College, Penn) and the normal distribution (Statistix for Windows, Analytical Software, Tallahassee, Fla), respectively. Normality of the distributions of lengths of hospitalization and age were tested using the normal probability plot and Wilks-Shapiro statistic (Statistix).To estimate the incidence of Salmonella shedding, only hospitalized horses that were sampled on at least three occasions were included, because the sensitivity of culture is suboptimal and repeated attempts to culture Salmonella are necessary to increase the sensitivity of this technique.[12] The total number of days at-risk of shedding Salmonella was calculated as the sum of lengths of hospitalization. The incidence density rate (true incidence) of Salmonella shedding was calculated as

(No. horses detected shedding Salmonella)/(total No. horse-days at-risk)

and expressed as the number of horses shedding Salmonella per 1000 horse-days at-risk. For example, if 10, 10, and 30 horses were hospitalized for periods of 3, 6, and 9 days each, horse-days at-risk would be (10.3)+(10.6)+(30.9) = 360. If one case of Salmonella-shedding was detected in this group, the estimated incidence density rate would be one per (1000/360) 1000 horse-days AT-RISK = 2.8 per 1000 horse-days at-risk. Horses that were culture-positive on the first sampling were not considered incident cases of Salmonella-shedding, and therefore were not included in the numerator of the incidence density rate. Horses shedding Salmonella after hospitalization were assumed to have commenced shedding at the midpoint of their hospitalization, and therefore contributed half their length of hospitalization to the rate denominator. A 95% CI for the estimated incidence density rate was calculated assuming shedding Salmonella to be Poisson distributed.[13] Incidence-density rates were also calculated for each month of the study, the contribution of each horse to each month's horse-days at-risk being calculated on the basis of date of hospitalization and date of discharge information. Cumulative incidence (risk) rate (%) was calculated as described for the incidence density rate, except that the denominator used was the number of horses at-risk of Salmonella-shedding when hospitalized. For cumulative incidence rate, a 95% CI was calculated based on the binomial distribution.[14]The prevalence of Salmonella-shedding in horses admitted to the VTH was calculated as

(No. horses detected shedding Salmonella by culturewhen admitted)/(total No. horses admitted and sampled)

Ninety-five percent CIs for prevalence estimates were calculations based on the binomial distribution.[14]The temporal clustering of horses shedding Salmonella was investigated using the scan statistic.[15] For horses shedding Salmonella after hospitalization, the midpoint of their length of hospitalization was used as the date of first occurrence of Salmonella-shedding. The population at-risk used in these analyses was the monthly total number of horse-days at-risk. The occurrence of horses shedding Salmonella was assumed to be Poisson distributed, so the expected number of horses shedding Salmonella in any given time period was proportional to the incidence of Salmonella-shedding during the entire study period. The study period was scanned for clusters of horses shedding Salmonella using a scanning window of as much as 50% (130 days) of the time period (SatScan, Bethesda, Md).

Results

Between October 12, 2000 and June 30, 2001, 724 horses were admitted to the VTH. Six hundred and thirty-two horses (87.3%) were admitted only once during the study period; 69 (9.5%), 15 (2.1%), 2 (0.3%), 5 (0.7%), and 1 (0.1%) horses were admitted on 2, 3, 4, 5, and 6 separate occasions, respectively. Length of hospitalization was not recorded for 2 horses. Three-hundred and sixty (42.3%) of the 854 admissions performed did not result in hospitalization of the horse. The distribution of length of hospitalization of all horses hospitalized during the study is shown in Figure 1. The median and mean lengths of hospitalization (95% CI) were 3.0 (2.5, 3.5) and 4.3 (3.8, 4.7) days, respectively. The minimum and maximum lengths of hospitalization were 1 and 57 days. The distribution of lengths of stay was nonnormally distributed (Wilks-Shapiro statistic, 0.7317).     

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%.     

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.     

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.     

Prevalence of Clostridium perfringens enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic.

OBJECTIVE: To determine prevalence of clostridial enterotoxins in feces of horses with diarrhea and colic, and to determine whether an association exists between detection of clostridial enterotoxins in feces and development of diarrhea as a complication of colic. DESIGN: Prospective case series and case-control study. ANIMALS: 174 horses with diarrhea, colic, or problems not related to the gastrointestinal tract. PROCEDURE: Horses were assigned to 1 of 4 groups: colic with diarrhea (group 1; n = 30); colic without diarrhea (group 2; 30); diarrhea without colic (group 3; 57); and control (group 4; 57). Feces were evaluated by use of ELISA to detect Clostridium perfringens enterotoxin (CPE) and C difficile toxin A (TOXA). Frequency of detection of CPE or TOXA in groups 1 and 3 was compared with that in groups 2 and 4, respectively. RESULTS: Prevalence of enteric clostridiosis in horses in group 3 was 25%. Clostridium perfringens enterotoxin was detected in 9 of 57 (16%), TOXA in 8 of 57 (14%), and both toxins in 3 of 57 (5%) fecal samples collected from these horses. Neither toxin was detected in feces of the age-matched horses in group 4. Clostridial enterotoxins were detected in feces of 7 of 60 (12%) horses with colic (groups 1 and 2), however, a significant association was not found between detection of enterotoxins in feces and development of diarrhea as a complication of colic. CONCLUSIONS AND CLINICAL RELEVANCE: Clostridia are important etiologic agents of diarrhea in horses. Additionally, changes in intestinal flora of horses with colic may allow for proliferation of clostridia and elaboration of enterotoxins regardless of whether diarrhea develops.     

Clostridium difficile diarrhea: infection control in horses.

C difficile has emerged as an important cause of diarrheic disease in horses. C difficile diarrhea is usually diagnosed in mature horses, mostly when they are treated with antimicrobials and hospitalized. It is important for clinicians at veterinary hospitals to have knowledge about the organism and the infection. To prevent C difficile diarrhea, judicious use of antimicrobials is important, as is minimizing different stress factors at the animal hospital or clinic. Infected horses must be isolated. Routine examination for C difficile and toxin A or B is recommended in horses with antibiotic-associated diarrhea. When treating foals for R equi pneumonia, it is important to avoid accidental ingestion of erythromycin by the dams. To reduce the number of environmental spores, thorough cleaning and surface disinfection of the animal hospital and clinic are important. Routine handwashing should be performed by all staff.     

Real-time PCR and typing of Clostridium difficile isolates colonizing mare-foal pairs

Clostridium difficile infection can occur in the dams of sick foals, but it is unknown if mares and foals share the same isolates. In this study, C. difficile isolates from fecal samples of 11 mares paired with 11 foals were genotyped by arbitrarily primed PCR; two mares and three foals in five mare-foal pairs had diarrhea. Fecal immunoassays were utilized to detect C. difficile common antigen and toxin A. Quantitative real-time PCR (qPCR) systems were developed to detect genes for toxins A and B, as well as for binary toxin B. Sequences of all toxins were present in all isolates, although only one horse was positive for toxin A on fecal immunoassay. Identical strains of C. difficile were present in 4/11 (36.4%) mare-foal pairs. Mare-foal pairs can harbor C. difficile subclinically and are potential reservoirs for colonization of each other.     

Longitudinal study of antimicrobial-resistant commensal Escherichia coli in the faeces of horses in an equine hospital

The increasing prevalence of antimicrobial resistance in bacteria represents a considerable problem for human and veterinary medicine, causing complications in the treatment of infections. Resistance in Escherichia coli from horses has been documented in commensal and pathogenic strains, but little information exists regarding the prevalence of such bacteria in hospitalised horses or associated risk factors. A longitudinal cohort study was conducted of 103 horses admitted to a referral equine hospital for more than 48 h, with faecal samples collected on hospital admission and subsequently every two days until discharge. Horses undergoing radioactive gamma scintigraphic examination, un-weaned foals and mares with un-weaned foals were excluded. Data were collected from enrolled animals, including antimicrobial treatment history and hospitalisation details. Samples were cultured for resistant E. coli; isolates had their antimicrobial resistance profile determined. High sample prevalence for resistant E. coli was identified for all antimicrobials examined except co-amoxiclav. The prevalence of resistance was consistently lower at admission, rising to a peak 4 days post-admission. Risk factors were analysed using multilevel, multivariable modelling, which identified significant clustering of resistance outcomes within horses. For all outcomes except trimethoprim resistance, the day the sample was obtained was significant, with increased risk of resistance for samples taken on day 2 or later. Antimicrobial treatment in the previous seven days and increased total daily dosages of cotrimoxazole prescribed in the hospital in the previous 24-48 h were associated with increased risk. Location within the hospital and admission reason were significant risk factors for some resistance outcomes. High levels of multidrug-resistant E. coli (47.7% of samples) and extended spectrum β-lactamase-producing E. coli (27.3% of samples) were recovered; such bacteria could significantly complicate treatment if they were the cause of infection and may represent a risk to personnel in close contact with hospitalised horses.     

A simple diagnostic technique to better determine the prevalence of tapeworms

A simple diagnostic technique was developed to improve the estimated prevalence of tapeworms on farms, by examining post-deworming fecal samples. All horses (n=162) on one farm were dewormed with a triple dose of pyrantel pamoate^a and a fecal sample was collected before deworming and 24 hours after deworming. The results showed that the Cornell-Wisconsin centrifugal fecal floatation technique on pre-deworming fecal samples had a 32% sensitivity and 98% specificity when compared to the same technique on post-deworming fecal samples. One barn of horses (n=24) had fecals collected at 17, 24, 39 and 48 hours to determine the optimum time to collect fecals after deworming. The results suggested that the optimum time to collect fecals was 24 hours post-deworming.     

THE PREVALENCE AND EPIZOOTIOLOGY OF SALMONELLOSIS AMONG GROUPS OF HORSES IN SOUTH EAST QUEENSLAND

Over a 3-year period, 1178 faecal samples were cultured from 462 horses admitted to the equine clinic of the University of Queensland; 185 samples were positive for salmonella yielding 213 isolations consisting of 21 serotypes. S. anatum was the predominant serotype isolated (54%) followed by S. ohio (11.27%) and S. typhimurium (9.4%). One hundred and ten horses (23.81%) were positive on one or more occasion, and 42 (9.09%) on more than one occasion.
S. anatum was the most common serotype isolated (71.43%) from the main drains in the stable block (33.57% positive samples).
The prevalence of salmonella excretors among a large non-clinic population of horses in south east Queensland was 1.65%.
Acute salmonellosis did not occur in the hospitalised animals. However, salmonellas were incriminated in 6 cases of chronic diarrhoea, which all yielded S. anatum , although the most severe involved both S. anatum and S. typhimurium , and these serotypes were isolated from multiple locations at the subsequent autopsy of 3 cases.     

Gastrointestinal Parasites of Stabled and Grazing Horses in Central and Northern Greece

The aim of this investigation was to compare parasites found in feces from stabled and grazing horses in Greece. For this, a total of 223 fecal samples were collected from horses from various parts of Central and Northern Greece. One hundred fifty were stabled and 73 were grazing horses, used for riding, working, or breeding. One hundred came from seven riding clubs, 73 from one breeding farm, and 50 were work horses from five farms. Parasitologic investigation was performed by applying three fecal examination techniques (Teleman's, zinc flotation, and stained smears) to detect ova, larvae, and oocysts of parasites. It was found that 77 the 223 horses (34.5%) in the study were infected with one or more parasite species. In the stabled horses, the most common parasites detected were eggs of strongyles, Strongyloides spp, Anoplocephala spp, Habronema spp, and Parascaris equorum and oocysts of Eimeria spp and Cryptosporidium spp; in the grazing horses, Anoplocephala spp. Strongyles were significantly more prevalent in the group of stabled horses than in the other group (P < .05). The results of the current study demonstrate that parasite infection is highly prevalent in horses in Greece.     

Comparison of fecal samples collected per rectum and off the ground for estimation of environmental contamination attributable to beef cattle

OBJECTIVES: To determine whether sampling feces off the ground replicates prevalence estimates for specific pathogens obtained from fecal samples collected per rectum of adult cows, and to determine characteristics of feces on the ground (fecal pats) that are associated with subsequent identification of Campylobacter spp, Cryptosporidium parvum, and Giardia duodenalis. ANIMALS: A random sample of adult beef cattle from 25 herds located throughout California. PROCEDURE: 1,115 rectal and ground fecal samples were obtained. Samples were submitted for culture of Campylobacter spp and examined, using a direct fluorescent antibody assay, to detect C parvum oocysts and G duodenalis cysts. Characteristics of fecal pats, such as volume and consistency, were recorded. RESULTS: Prevalence of Campylobacter spp was 5.0% (20/401) for rectal fecal samples, which was significantly greater than prevalence determined for ground fecal samples (2/402; 0.5%). Most isolates were C jejuni subsp jejuni. Prevalence of C parvum was higher in rectal fecal samples (6/557; 1.1%) than in ground fecal samples (1/558; 0.2%), but this difference was not significant. Prevalence of G duodenalis did not differ for rectal (36/557; 6.5%) versus ground (26/558; 4.7%) fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Evaluation of ground fecal samples may not accurately indicate the prevalence of Campylobacter spp or C parvum in cattle but may reflect prevalence of G duodenalis. Differences in prevalence estimates between the 2 methods suggest inactivation of pathogens in feces after cattle have defecated. Prevalence estimates generated by evaluation of ground fecal samples, however, may more accurately estimate environmental pathogen burden.     

Caecal intussusceptions in horses: a New Zealand perspective

Objective To establish the prevalence of intussusceptions involving the caecum in a population of horses admitted to a university hospital for colic. Design Retrospective clinical study Methods Medical records of all horses admitted to the Massey University Veterinary Teaching Hospital between 1991 and 2004 were examined for information of those horses diagnosed with an intussusception involving the caecum. Results A total of 135 horses were admitted for colic surgery during the study period and 61 horses had a diagnosis of ileocaecal (37), caecocaecal (5) or caecocolic intussusception (19) made either at surgery or necropsy. Of the horses with ileocaecal intussusception, 32 had an incomplete hand-sewn ileocaecostomy without reduction and 29 survived to discharge. All the horses with caecocaecal intussusceptions were diagnosed preoperatively via rectal examination and/or transabdominal ultrasound: 2 were euthanased at surgery and 3 survived to discharge. In the 19 horses with caecocolic intussusceptions, manually reduction at surgery was performed in 6 and 5 of them survived to discharge. A typhlectomy was performed via a colotomy in 6 horses, 3 of which survived to discharge. Conclusions The high prevalence of intussusceptions involving the caecum seen at this referral centre may indicate a higher prevalence in New Zealand than is reported elsewhere in the world. Clinical relevance Intussusceptions involving the caecum should be considered as a differential diagnosis in horses presenting with chronic low-grade colic. Transabdominal ultrasound is useful for identifying caecocaecal and caecocolic intussusceptions. Hand-sewn side-to-side incomplete ileocaecostomy is a quick, effective and safe method of surgical treatment of ileocaecal intussusceptions.