LONGITUDINAL STUDY OF CLOSTRIDIUM DIFFICILE, CLOSTRDIUM PERFRINGENS, SALMONELLA AND ESCHERICHIA COLI IN HEALTHY HORSES.

Clostridium difficile, Clostridium perfringens and Salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. Point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. Additionally, antimicrobial resistance is a pressing concern. Commensal E.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. The objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for E. coli.
Fecal samples were collected from 25 adult horses from five farms on a monthly basis over the course of one year. Selective cultures were performed for C. difficile, C. perfringens, Salmonella and E. coli. C. difficile isolates were characterized via toxin gene PCR and ribotyping. Broth microdilution was performed to assess antimicrobial susceptibility profiles of E. coli.
Clostridium difficile was isolated from 15/275 (5.45%) samples from 11/25 (44%) horses. Four horses were positive on more than one occasion, three were positive in two consecutive months. Different ribotypes were found in two of the latter horses. Most isolates were ribotype 078 (n=6) with ribotype 001 (n=5) and ribotype C (n=4) also identified. Ribotypes 078 and C possessed genes encoding toxins A, B and binary toxin, while ribotype 001 only possessed toxin A and B genes. Despite a detection threshold of 9cfu/g feces, C. perfringens was not detected in any samples, nor was Salmonella. E coli was isolated from 117/225 (52%) samples. Resistance to =1 antimicrobial was present in only 19/117 (16.9%) isolates. Multidrug resistance (= 3 antibiotics) was present in 5/117 (4%). Most commonly, isolates were resistant to sulfisoxazole (17/117) and trimethoprim sulfamethoxazole (16/117).
The overall detection rate for toxigenic C. difficile in fecal samples of healthy horses was 5.4% which is consistent with previous studies. The cumulative prevalence of 44% was striking but only one horse shed the same strain for more than one month, indicating C. difficile shedding is a transient and dynamic state. The predominant isolation of ribotype 078 is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. The low prevalence of C. perfringens and Salmonella is in agreement with some other studies. The low prevalence of antibiotic resistance in commensal E. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria.