Diagnosis of Infectious Diarrhea in Dogs and Cats: A Critical Appraisal

Stanley Marks � South Africa United States

The clinical documentation of enteropathogenic bacteria causing diarrhea in dogs and cats is clouded by the presence of these organisms occurring as a normal component of the indigenous intestinal flora. Bacterial culture is the method currently utilized for isolation of Campylobacter spp., Salmonella spp., Clostridium perfringens, and Clostridium difficile. However, culture of single fecal samples is relatively unreliable for incriminating these potentially pathogenic enteric organisms as a cause of diarrhea. Attempts at culturing these bacterial pathogens are frequently made in veterinary diagnostic laboratories. Many animals receive inappropriate antibiotic therapy following isolation of a suspected pathogen; the resolution of clinical signs of diarrhea is often wrongly equated with eradication of the �incriminating� pathogen. Routine fecal cultures often yield disappointing results due to the rarity in recovering enteric pathogens, the self-limiting nature of many bacterial infections, and the difficulties involved with the interpretation of results. In vitro methods applying molecular approaches for identification of toxin genes from bacterial isolates and immunoassays for detection of toxins in feces can be utilized to provide a rapid, more accurate diagnosis of bacterial-associated diarrhea. Fecal cultures may be helpful in animals with a history of exposure to bacterial pathogens, outbreaks of diarrhea among more than one pet in a household, onset of diarrhea after kenneling or show attendance, or concern about a public health hazard. Acute onset of bloody diarrhea in association with evidence of sepsis, or presence of large numbers of fecal neutrophils also warrants fecal culture.

Clostridium perfringens

Clostridium perfringens is an anaerobic, oxygen tolerant, gram-positive bacillus that does not sporulate readily in vitro. The organism is an important agent of enteritis and enterotoxemias in domestic animals and humans and has been identified as a cause of both nosocomial and acquired acute and chronic diarrhea in dogs. Dogs with C. perfringens enterotoxicosis generally exhibit large-bowel diarrhea characterized by increased frequency of bowel movements with tenesmus and fecal mucus and hematochezia. The elaboration of four major toxins (alpha-, ß, €, and i) is the basis for typing the organism into five toxigenic phenotypes; however, C. perfringens enterotoxin (CPE), is the principle toxin involved in C. perfringens food-borne illness and is associated with non-food-borne diarrheal disease. Sporulation and the production of CPE are co-regulated, and the toxin, a single polypeptide of 35 kDa, is released upon lysis of the vegetative cell. The diagnosis of C. perfringens-induced enteropathy in dogs has been contingent upon the simultaneous occurrence of typical clinical signs of colitis, the detection of large numbers of heat resistant C. perfringens endospores, and the immunodetection of enterotoxin in abnormal feces. Because C. perfringens is commonly observed in a vegetative form in normal dog feces, isolation of C. perfringens following fecal culture in diarrheic dogs does not necessarily correlate with clinical disease.

Two commercially available serologic tests can be used to detect C. perfringens enterotoxin in the feces. The reverse passive latex agglutination (RPLA) assay (Unipath Co., Ogdensburg, NY) detects a minimum of 4 ng/mL of purified CPE; however, the assay requires overnight incubation and interpretation of the results is somewhat subjective. An enzyme immunoassay (EIA) (Techlab, Inc., Blacksburg, VA) incorporates a monoclonal antibody to the epitope on CPE that binds to enterocytes, providing specificity. Its diluent is formulated to neutralize fecal proteases and other nonspecific reactions. The test takes three hours to perform (most of this is incubation time), and results are easier to interpret than the RPLA assay.

The prevalence of C. perfringens enterotoxin in feces of dogs with and without diarrhea was recently assessed in 144 dogs, representing hospitalized dogs with (n=41) or without (n=50) diarrhea, and clinically normal dogs treated as outpatients (n=53). The RPLA assay was positive in approximately 25% of dogs, regardless of their clinical status, whereas only 9/144 dogs (6.3%) were EIA positive. The cpe gene was reliably detected by PCR, and most of these strains produced CPE. These findings underscore the high incidence of false positive results obtained with the RPLA assay. Although cytologic examination of fecal smears is a quick and simple screening test to identify endospores of C. perfringens, the results are inconclusive because similar numbers of endospores are observed in smears obtained from healthy dogs and dogs with diarrhea. The number of endospores of C. perfringens in human fecal specimens is within reference range in 69% of enterotoxin positive patients, which indicates that a high number of endospores is not useful in determining cause of diarrhea. The RPLA assay was evaluated in a separate group of 142 dogs with diarrhea in which 51 dogs (36%) tested positive for CPE. The EIA has replaced the RPLA assay at UC Davis, and a recent evaluation of this assay in a separate group of 131 dogs with diarrhea detected CPE in only 11 of the dogs (8%). Five of the EIA positive dogs had no endospores detected on fecal smear. The use of gene probes and PCR assays for detection of toxigenic C. perfringens is currently being evaluated. These methods do not require maintenance of cell cultures for toxin assays, and isolates that have cpe, but do not sporulate in vitro, can be readily detected, reducing false negatives. Molecular approaches will also facilitate the identification of virulence factors, such as the beta2 toxin, which has been associated with necrotic enteritis in piglets and enterocolitis in horses.

Clostridium difficile

Clostridium difficile is a large, gram-positive, anaerobic spore-forming motile rod and is the major cause of antibiotic-associated pseudomembranous colitis in human patients. Specific tests for C. difficile toxins used in the diagnostic laboratory include cell culture, which relies on the presence of biologically active toxin, and an enzyme-linked immunosorbent assay, which detects immunologically active toxin that may or may not be biologically active. PCR techniques can discriminate between toxigenic and nontoxigenic strains of the organism, whereas anaerobic culture methods cannot.

Disruption of the colonic microflora together with the presence of toxigenic C. difficile, are the prerequisites for disease. C. difficile produces two antigenically distinct toxins which damage colonic epithelium: toxin A (enterotoxin) and toxin B (cytotoxin). Toxin A causes hemorrhagic fluid accumulation and detachment of epithelial cells. Toxin B acts synergistically with toxin A as a cytotoxin only after the epithelium has been injured by toxin A. Extravasation of plasma proteins and alteration of water and electrolyte transport follow. Approximately 10% of asymptomatic dogs shed toxigenic C. difficile in feces. A high percentage of human infants are colonized with toxigenic C. difficile, but do not show signs of disease.

The fecal prevalence of C. difficile in dogs who were patients at the University of California Veterinary Medical Teaching Hospital was 18.4% (28 of 152 dogs) based on direct plating on Cycloserine cefoxitin fructose agar (CCFA) and confirmation by a latex particle agglutination test. Isolates from 14 of these patients (50%) contained the genes for toxins A and B as determined by PCR analysis with confirmation by Southern blot hybridization. Diarrhea was a clinical finding in five (35.7%) of the dogs carrying toxigenic isolates of C. difficile, whereas diarrhea was only noted in two of 14 dogs (14.3%) shedding nontoxigenic isolates. The frequency of shedding of C. difficile from dogs with and without diarrhea was not significantly different; however, the carriage rate of C. difficile was significantly higher for inpatients versus outpatients. The concurrent use of antibiotics or immunosuppressive drug therapy was not found to be significantly different between dogs that were C. difficile positive or negative and between the toxigenic and nontoxigenic isolates.

The prevalence of C. difficile in fecal specimens from cats who were patients at the University of California Veterinary Medical Teaching Hospital was 9.4% (23 of 245 cats) based on culture on selective media and identification by a latex particle agglutination test. Toxin A and B sequences were identified in 8/23 isolates and were confirmed by Southern blot hybridization. All of the cats colonized with toxigenic C. difficile had > of the risk factors (antibiotic use, antineoplastic therapy) associated with C. difficile infection in human patients. Four of the eight cats colonized with toxigenic C. difficile were treated with metronidazole and had resolution of the diarrhea and negative fecal culture on subsequent testing. The significance of isolating nontoxigenic C. difficile in asymptomatic human patients is uncertain because the organism is a normal component of the colonic microflora in some individuals. Multiple strains of C. difficile are often isolated during outbreaks of C. difficile-associated diarrhea; AP-PCR can be utilized to identify the different genotypes of C. difficile within a hospital population. This method has confirmed the potential role of environmental contamination in bacterial transmission by identifying bacteria in the environment with identical genotypes to those isolated from the patient.

Campylobacter jejuni

Diarrhea produced by this small, curved, motile, microaerophilic gram-negative rod is seen primarily in younger animals, although it has been seen in animals of all ages. It can be isolated from the feces of a high percentage (approximately 40%) of healthy animals that have been kenneled, particularly in animal control facilities. Poultry and poultry products, as well as unpasteurized milk, are major sources for human infection. Puppies and kittens are also sources for humans; however, human beings may also be a source of infectious organisms for dogs and cats. Campylobacter can survive for days in surface water and as long as four weeks in feces. The duration of excretion in infected dogs and cats can be as long as four months and infected animals should be quarantined away from children during this period.

C. jejuni can colonize the jejunum, ileum, cecum, and colon; however, histologic changes are largely restricted to the colon. The organism adheres to the intestinal epithelium via an outer surface protein and produces an enterotoxin that results in a secretory diarrhea mediated by cyclic AMP. Campylobacter organisms also elaborate a cytotoxic enterotoxin that is probably responsible for the epithelial damage. Clinical signs range from mild transient diarrhea to mucous laden bloody stools with associated signs of colitis. Infection by a Campylobacter spp. may be suspected when the presence of their characteristic curved or spiral-shaped gram-negative rods is noted on a Gram-stained smear of fresh feces from the patient. Feces can also be cultured using one of several selective media designed to inhibit the growth of most other intestinal flora (e.g., CVA agar, FDA agar). Plates are incubated under microaerophilic conditions at 42 C.

Campylobacter spp. was isolated from 13 fecal specimens obtained from 279 dogs (4.7%) with diarrhea at the UC Davis VMTH, whereas fecal smears demonstrated Campylobacter-like organisms in 4/277 dogs (1.4%). Fecal specimens obtained from cats with diarrhea yielded positive cultures in 4/68 cats (5.9%), whereas Gram-stained fecal smears demonstrated Campylobacter-like organisms in 7/66 cats (10.6%). These findings underscore the limitations of relying on fecal smears for diagnosing Campylobacter jejuni as a cause of diarrhea. Although diarrhea produced by Campylobacter organisms is usually self-limiting, the zoonotic potential and the age of the animal necessitates treatment with antimicrobial agents. Effective drugs are the macrolides (erythromycin) or quinolones (enrofloxacin). Erythromycin is the drug of choice, despite the associated gastrointestinal side effects. Although enrofloxacin lacks gastrointestinal side- effects, the rapid development of resistance to this class of antimicrobials, and the potential for inducing joint abnormalities in young animals makes this drug a second choice for managing Campylobacter infections.

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