Determination of the in vitro susceptibility of feline tritrichomonas foetus to 5 antimicrobial agents.

BACKGROUND: The nitroimidazole, ronidazole, has been demonstrated to have in vitro and in vivo activity against the protozoan Tritrichomonas foetus in cats. The purpose of this study was to evaluate the in vitro susceptibility of feline T. foetus isolates obtained from naturally infected cats to 5 antimicrobial agents and to compare the in vitro time kill of ronidazole and metronidazole. HYPOTHESIS: We hypothesized that nitroimidazoles have in vitro activity against T. foetus, whereas furazolidone, omeprazole, and paromomycin do not. ANIMALS: Fecal specimens were cultured from 4 naturally infected Bengal cats with a history of T. foetus-associated diarrhea. METHODS: A 24-hour susceptibility assay was performed on all 4 isolates for the 5 antimicrobial agents. A time-kill microdilution method was performed on 2 isolates for metronidazole and ronidazole. RESULTS: Paromomycin and omeprazole showed no in vitro effect at concentrations < or = 80 microg/mL. There was no significant difference in 24-hour susceptibilities among metronidazole, ronidazole, and furazolidone. In addition, only the results of the highest concentration tested (80 microg/mL) and concentrations of 1.25 and 2.5 microg/mL revealed significant differences in the rate of trophozoite killing, with ronidazole having a faster reduction in trophozoite survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Time-kill assays demonstrated ronidazole had a higher lethal activity compared with metronidazole. These findings contrast with a previously published report and may reflect strain variation, different methodologies, or both. The lack of clinical response seen with metronidazole administration to treat feline trichomoniasis may not reflect inherent resistance but rather in vivo events involving drug distribution and pharmacokinetics.

Determination of the prevalence of antimicrobial resistance genes in canine Clostridium perfringens isolates.

Clostridium perfringens is a well documented cause of a mild self-limiting diarrhea and a potentially fatal acute hemorrhagic diarrheal syndrome in the dog. A recent study documented that 21% of canine C. perfringens isolates had MIC's indicative of resistance to tetracycline, an antimicrobial commonly recommended for treatment of C. perfringens-associated diarrhea. The objective of the present study was to further evaluate the antimicrobial susceptibility profiles of these isolates by determining the prevalence of specific resistance genes, their expression, and ability for transference between bacteria. One hundred and twenty-four canine C. perfringens isolates from 124 dogs were evaluated. Minimum inhibitory concentrations of tetracycline, erythromycin, tylosin, and metronidazole were determined using the CLSI Reference Agar Dilution Method. All isolates were screened for three tetracycline resistance genes: tetA(P), tetB(P) and tetM, and two macrolide resistance genes: ermB and ermQ, via PCR using primer sequences previously described. Ninety-six percent (119/124) of the isolates were positive for the tetA(P) gene, and 41% (51/124) were positive for both the tetA(P) and tetB(P) genes. No isolates were positive for the tetB(P) gene alone. Highly susceptible isolates (MIC< or = 4 microg/ml) were significantly more likely to lack the tetB(P) gene. One isolate (0.8%) was positive for the ermB gene, and one isolate was positive for the ermQ gene. The tetM gene was not found in any of the isolates tested. Two out of 15 tested isolates (13%) demonstrated transfer of tetracycline resistance via bacterial conjugation. Tetracycline should be avoided for the treatment of C. perfringens-associated diarrhea in dogs because of the relatively high prevalence of in vitro resistance, and the potential for conjugative transfer of antimicrobial resistance.

Epidemiologic evaluation of diarrhea in dogs in an animal shelter.

OBJECTIVES: To determine associations among infectious pathogens and diarrheal disease in dogs in an animal shelter and demonstrate the use of geographic information systems (GISs) for tracking spatial distributions of diarrheal disease within shelters. SAMPLE POPULATION: Feces from 120 dogs. PROCEDURE: Fresh fecal specimens were screened for bacteria and bacterial toxins via bacteriologic culture and ELISA, parvovirus via ELISA, canine coronavirus via nested polymerase chain reaction assay, protozoal cysts and oocysts via a direct fluorescent antibody technique, and parasite ova and larvae via microscopic examination of direct wet mounts and zinc sulfate centrifugation flotation. RESULTS: Salmonella enterica and Brachyspira spp were not common, whereas other pathogens such as canine coronavirus and Helicobacter spp were common among the dogs that were surveyed. Only intestinal parasites and Campylobacterjejuni infection were significant risk factors for diarrhea by univariate odds ratio analysis. Giardia lamblia was significantly underestimated by fecal flotation, compared with a direct fluorescent antibody technique. Spatial analysis of case specimens by use of GIS indicated that diarrhea was widespread throughout the entire shelter, and spatial statistical analysis revealed no evidence of spatial clustering of case specimens. CONCLUSIONS AND CLINICAL RELEVANCE: This study provided an epidemiologic overview of diarrhea and interacting diarrhea-associated pathogens in a densely housed, highly predisposed shelter population of dogs. Several of the approaches used in this study, such as use of a spatial representation of case specimens and considering multiple etiologies simultaneously, were novel and illustrate an integrated approach to epidemiologic investigations in shelter populations.

Bacterial-associated diarrhea in the dog: a critical appraisal.

The clinical documentation of enteropathogenic bacteria causing diarrhea in dogs is clouded by the presence of many of these organisms existing as normal constituents of the indigenous intestinal flora. The diagnosis of a putative bacterial enteropathogen(s) in dogs should be made based on a combination of parameters, including signalment and predisposing factors, clinical signs, serologic assays for toxins, fecal culture, and PCR. Relying on results of fecal culture alone is problematic, because C perfringens, C difficile, Campylobacter spp, and pathogenic and non-pathogenic E coli are commonly isolated from apparently healthy dogs [10,13,33]. Nevertheless, culture may be useful in procuring isolates for the application of molecular techniques, such as PCR, for detection of specific toxin genes or molecular typing of isolated strains to establish clonality in suspected outbreaks. The oversimplistic attempt to characterize bacterially associated diarrhea by anatomic localization of clinical signs should be discouraged, because most of the previously mentioned bacteria have been associated with small and large intestinal diarrhea. Accurate diagnosis of infections may require diagnostic laboratories to incorporate PCR-based assays using genus- and species-specific primers to facilitate detection of toxin genes and differentiation of species that appear phenotypically and biochemically similar. There has been tremendous interest in the application of microarray technology for the simultaneous detection of thousands of genes or target DNA sequences on one glass slide. This powerful tool could be used for detection of specific pathogenic bacterial strains in fecal specimens obtained from dogs in the future.

Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs.

Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution.

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.