Enteropathogenic bacteria

The bacteria most commonly incriminated in canine and feline diarrhoea include Clostridium perfringens, Clostridium difficile, Campylobacter spp., Escherichia coli and Salmonella spp. However, the clinical significance of isolation of enteropathogenic bacteria in dogs and cats as a cause of diarrhoea is clouded by the existence of many of these organisms as normal constituents of the indigenous intestinal flora. Veterinary surgeons are faced with a quandary when attempting to diagnose suspected bacterial-associated diarrhoea in small animals because the isolation rates for putative bacterial enteropathogens are indeed often similar in diarrhoeic and non-diarrhoeic animals, and because the incidence of bacterial-associated diarrhoea is extremely variable. The indications for performing faecal enteric panels are poorly defined, resulting in indiscriminate testing and misinterpretation of results. Faecal cultures and toxin analysis should be reserved for dogs and cats developing diarrhoea after kennelling or show attendance, in animals with an acute onset of bloody diarrhoea in association with evidence of sepsis, and in diarrhoea outbreaks occurring in more than one pet in a household. Screening for Clostridium difficile, Campylobacterspp. or Salmonella spp. is also indicated when zoonotic concerns are present because of an immunocompromised owner.

Clostridium perfringens

Clostridium perfringens is an anaerobic, spore-forming, Gram-positive bacillus that has been associated with outbreaks of acute, often severe diarrhoea in humans, horses, dogsand cats. The elaboration of four major toxins (α, β, and ε) is the basis for typing the organism into five toxigenic phenotypes, A to E. Each type may also express a subset of at least 10 other established toxins, including C. perfringens enterotoxin (CPE), a well characterized virulence factor whose production is supposedly co-regulated with sporulation. Dogs with C. perfringens-associaleti diarrhoea frequently exhibit large bowel diarrhoea characterized by increased frequency of bowel movements with tenesmus, faecal mucus and haematochezia; however, clinical signs of enteritis or enterocolitis are also commonly seen. A strong association has also been detected between the CPE (detected via an enzyme-linked immunosorbent assay, ELISA) and acute haemorrhagic diarrhoeal syndrome (AHDS). CPE was detected in the faeces of 8 of 12 dogs (67%) that had clinical signs consistent with AHDS. Of the four dogs that had peracute symptoms and died as a result of the disease, all had faecal specimens positive for CPE.

Diagnosis

Currently, diagnosis of C. perfringens-associated diarrhoea in the dog is made based on detection of CPE in faecal specimens in conjunction with clinical signs of disease. The value of quantitative faecal culture and faecal spore counts have been shown to be of poor diagnostic value as the organism is isolated from more than 80% of healthy dogs, and there is no correlation between spore counts and detection of enterotoxin. There is only one commercially available ELISA kit (Techlab Inc.) for detection of CPE in faecal specimens; however, the performance characteristics of this assay have not been validated in the dog to date.

Treatment

Antibiotics that have been recommended for the treatment of canine C. perfringens-associated diarrhoea include oral ampicillin (22 mg/kg p.o. q8h), metronidazole (10 mg/kg p.o. q12h) and tylosin (15 mg/kg p.o. q12h). Tetracycline use should be avoided due to the high incidence of tetracycline resistance.

Clostridium difficile

Clostridium difficile is a Gram-positive, anaerobic spore-forming bacillus and is the major cause of antibiotic-associated pseudomembranous colitis in human patients. C. difficile has also been associated with diarrhoea and enterocolitis in foals and adult horses, as well as diarrhoea in dogs. C. difficile-associated diarrhoea is less common in cats and a recent study by the author documented an incidence of 5% in diarrhoeic cats. Two toxins, A and B, are thought to be primarily responsible for disease associated with the organism, although other toxins may also play a role.

Diagnosis

Current diagnosis of C. difficile-associated diarrhoea is primarily made based upon detection of toxin A ortoxin B in faecal specimens via ELISA. Isolation of the organism alone is not sufficient for diagnosis due to the presence of non-toxigenicstrains. In addition, previous studies have reported no significant difference in the isolation of C, difficile from diarrhoeic and non-diarrhoeic dogs, although animals that are toxin-positive are invariably culture-positive. Similar to C. perfringens, a strong association was found between the detection of C. difficile toxin A and the presence of AHDS.

Treatment

Metronidazole (10 mg/kg p.o. q12h for approximately 7 days) administration is the therapy of choice for dogs and cats with suspected C. difficile-associated diarrhoea. Although metronidazole-resistant C. difficile isolates obtained from foals and adult horses have been reported, a recent study evaluating the susceptibilities of 70 canine C. difficile isolates showed that all were susceptible to <1 ng/ml metronidazole. The second drug of choice in humans and occasionally in horses is vancomycin; however, it is used only in cases of non-responsive C. difficile-associated diarrhoea or when metronidazole-resistant strains have been demonstrated.

Campylobacter

Campylobacter species are small (0.2-0.5 x 0.5-5 (im), microaerophilic, Gram-negative curved rod-shaped bacteria. Campylobacter species that have been implicated in canine enteric disease include C. jejuni, C. coli, C. heiveticus and C. upsaliensis. It has recently been shown that some selective media can have an inhibitory effect on a number of Campylobacter spp., resulting in more sensitive species, such as C. upsaliensis, or other catalase-negative or weakly positive species being missed. Faecal shedding of C. jejuniis significantly greater in dogs <6 months old and during the summer and autumn. The higher prevalence of infection in puppies versus adult dogs may reflect increased exposure of young animals to faecal excrement and confinement to a limited space. In addition, the unexposed immune system of puppies may increase susceptibility to intestinal colonization by C. jejuni. Other enteric pathogens, such as parvovirus, Giardiaor Salmonella, may play a synergistic role. The isolation of Campylobacter spp. from a diarrhoeic animal does not necessarily implicate Campylobacter as a cause of the diarrhoea. Indeed a recent study documented a significantly higher incidence of Campylobacter in healthy, non-diarrhoeic cats (20%) than in diarrhoeic cats (11%).

Diagnosis

Campyfobacter-like organisms (CLOs) can be identified by examining stained smears (Gram stain or Romanowsky-type stain) of fresh faeces from the patient. The characteristic morphology of the organism (slender, curved rods with an ‘S’ shape or seagull-shaped appearance) allows it to be identified relatively easily. The major limitation of direct examinations is that the procedure fails to differentiate between species of Campylobacter or between related organisms including Helicobacter spp. and Anaerobiospirillum spp. In addition, identification of CLOs is not sufficient to warrant a diagnosis of Campyfobacfer-associated diarrhoea as many healthy dogs and cats can harbor CLOs in their intestinal tract.

For optimal recovery of Campylobacter spp., faeces or faecal swabs should be fresh or placed immediately into anaerobic transport medium before refrigeration at 4°C. For isolation, the use of a formulated selective medium containing antimicrobial agents (e.g. Campy-CVA containing cefoperazone, vancomycin and amphotericin B) gives better recovery than other direct-plating selective media. Microaerophilic incubation conditions should be maintained and the plates should be incubated at 37°C, or at 42°C, when isolation of C. jejuni and C. coll from faeces is attempted. Suspect colonies should be Gram-stained and sub-cultured to 5% sheep blood agar (SBA). Biochemical tests can then be performed to determine the species of all CLOs isolated. The use of selective medium containing cefoperazone should be used when attempting to isolate C. upsaliensis, as the organism is more resistant to cefoperazone than to cephalothin. Characterization of Campylobacter infections or mixed infections with Helicobacter and Campylobacter spp. is best accomplished utilizing molecular structure-based diagnostics, employing genus- and species-specific polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis and 16S rRNA sequence analysis.

Treatment

Although diarrhoea produced by Campylobacter organisms is usually self-limiting, the zoonotic potential of the organism often necessitates medical therapy. It is now recognized that Campylobacter are a leading cause of enteric disease in people and that diarrhoeic and non-diarrhoeic dogs can serve as sources of infection for humans. The drugs of choice are the macrolides (erythromycin at 10-15 mg/kg p.o. q8h) or quinolones (enrofloxacin at 5 mg/kg p.o. q12h). However, due to the high rate of mutational resistance Campylobacter organisms have to the quinolones, a resistance that sometimes occurs while animals are being treated, they are not the drug of choice. Erythromycin is the drug of choice, despite the associated gastrointestinal (Gl) side-effects. The duration of excretion in infected dogs and cats can be as long as 4 months and infected animals should be quarantined away from children during this period.

Salmonella

Salmonella species are primarily motile, non-spore-forming, Gram-negative aerobic bacilli. There are currently over 2000 described serotypes of Salmonella that have been associated with both human and animal disease. Salmonella spp. are one of the most common causes of human food-borne disease. Clinical salmoneliosis in dogs and cats is rare, although prevalences are higher in puppies and kennel populations. Isolation of Salmonella spp. from adult dogs ranges from 0 to 2% in non-diarrhoeic animals, and from 0 to 1 % in diarrhoeic dogs. The isolation rates are similar in non-diarrhoeic and diarrhoeic cats. A recently published study evaluating the prevalence of Salmonella among dogs fed raw food diets, isolated Salmonella spp. from 80% of the diet samples and 30% of the stool samples.

Clinical signs

Most Salmonella-infected dogs and cats are asymptomatic, although some animals may manifest clinical signs of systemic sepsis. Signs of clinical salmoneliosis in dogs include fever, anorexia, diarrhoea (which may be bloody), vomiting, weight loss, nasal discharge, pelvic limb paresis and abortion.

Diagnosis

The traditional diagnosis of salmoneliosis is made based on isolation of the organism in conjunction with clinical signs and assessment of potential risk factors, such as hospitalization, age, environmental exposure and antibiotic administration. However, isolation of Salmonella is not necessarily indicative of involvement in disease as similar isolation rates can be detected in healthy non-diarrhoeic animals. Haematological abnormalities are variable, and include a non-regenerative anaemia, lymphopenia, thrombocytopenia and neutropenia with a left shift. Toxic neutrophils are found in animals with systemic disease and endotoxaemia, findings similar to those documented with canine parvovirus. Fresh faecal specimens should be placed on toone or more select ivemedia, including MacConkey agar, XLD agar, and brilliant-green agar. For enrichment, selenite F, tetrathionate, or Gram-negative broth are recommended.

Treatment

Intravenous fluid therapy may be required depending on the severity of the diarrhoea. Antibiotic therapy is typically indicated for animals with concurrent signs of systemic infection or history of immunosuppression. Antibiotics reported to be effective against Salmonella include fluoroquinolones, chloramphenicol, trimetho-prim-sulphonamide and amoxicillin. Fresh-frozen plasma may be beneficial for dogs with more severe signs of endotoxaemia.

Pathogenic Escherichia coli

Escherichia coliis a pleomorphic, Gram-negative, non-spore-forming rod that is a member of the family Enterobacteriaceae. Several distinct pathogenic categories (pathotypes) of diarrhoeogenic E. coll are now recognized. Although the virulence determinants of each E. coli pathotype are distinct, they can generally be categorized as either colonization factors (adhesins), which enable the bacteria to bind closely to the intestinal mucosa and resist removal by peristalsis, or secreted toxins, which interfere with the normal physiological process of host cells. Despite the occurrence of E. coli as a normal commensal in the canine intestine, there is increasing evidence that certain E. coli pathotypes cause intestinal disease in dogs. The three pathotypes that have been studied in the dog are enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC). Very little is known about pathogenic E. coli in cats, although EPEC was isolated from approximately 5% of cats with diarrhoea, enteritisor septicaemia.

Enterotoxigenic E. coli

The true incidence of this pathotype in canine diarrhoea is still unclear, with reported prevalences among diarrhoeic dogs ranging from 0 to 31%. The bacteria colonize the proximal small intestine, where they produce heat-stable (ST) and occasionally heat-labile (LT) enterotoxins. These enterotoxins result in overproduction of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) with consequent development of a secretory diarrhoea.

Enteropathogenlc E. coli

EPEC strains are negative for Shiga-toxin and entero-toxin (ST and LT) genes, but carry the chromosomally located gene, eaeA (E. coli attaching effacing). £ coli isolates from 44 of 122 dogs (36%) dying with diarrhoea were found to have the eaeA gene and E. coli was the sole pathogen identified in 15 of 44 (34%) dogs.

Enterohaemorrhagic E. coli

EHEC strains bind tightly to epithelial cells and produce the same type of attachment-effacement lesions as seen with EPEC. EHECs are minimally invasive but do incite an inflammatory response, predominantly in the large intestine. The prototype EHEC, a strain of E. coli of the serotype O157:H7, is a significant food-borne pathogen of human beings. Haemolytic-uraemic syndrome is the most important complication of E. coli O157 infection, and is characterized by microangio-pathic haemolytic anaemia, thrombocytopenia and acute renal failure in approximately 7% of human cases. To date, only a single report has documented the isolation of this serotype from a dog’s faeces. The isolated strain was found to be identical to a strain isolated from an affected child who had contact with the dog. This finding suggests that similar to cattle, dogs may serve as potential vectors for transmission of EHEC O157.

Clinical signs

Pathogenic E. coli strains are commonly isolated from faeces of apparently healthy dogs, and clinical signs can range from asymptomatic carriage to haemorrhagic diarrhoea. In addition, clinical signs can be variable because of the relatively high incidence of concurrent enteric infections with parvovirus, Clostridium perfringens and intestinal parasites. The predominant clinical sign of enterotoxigenie £ coli infection is profuse watery diarrhoea.

Diagnosis

Because E. coli is a significant component of the commensal canine intestinal flora, isolation of the organism is not diagnostic, nor does it allow differentiation between pathogenic and non-pathogenic strains. However, culture enables the application of molecular techniques for detection of specific toxin genes among isolated organisms. Culture of E. coli involves spreading fresh faecal specimens on to selective media, such as MacConkey agar, which will only support the growth of Gram-negative organisms. Single lactose-positive colonies are then sub-cultured and speciated through biochemical testing. PCR has become one of the most common methods for detecting and differentiating pathogenic strains of E. coli. A recent study showed that it is possible to detect 11 of the major virulence genes of E. coli in faecal specimens from dogs with four multiplex PCRs. Although EPEC and EHEC are associated with characteristic attaching and effacing lesions, molecular techniques for the detection of genes specific for each pathotype is more reliable than histological examination.

Treatment

The use of antimicrobials is controversial for several reasons. These bacteria have a relatively high incidence of inherent resistance to antibiotics because of the presence of a Gram-negative cell wall, and because of the high incidence of conjugative transfer of resistance determinants. In addition, antibiotic therapy may enhance toxin synthesis or promote its release from the bacteria with a consequent increased rate of haemorrhagic colitis. Canine patients with only mild clinical signs probably do not warrant antibiotic treatment, whereas parenteral antibiotics and fluid therapy are indicated in severe cases, particularly if the patient is septicaemic. The Enterobacteriaceae are usually resistant to chloramphenicol, tetracyclines, ampicillin and sulphonamides. Clinically stable animals can be treated with amoxicillin-clavulanate and first- or second-generation cephalosporins until susceptibility results are known. Dogs with life-threatening bacteraemia should be treated with amikacin, a third-generation cephalosporin, orenrofloxacin.

Orally administered autogenous or recombinant vaccines have been studied extensively in farm animals in an effort to help prevent or treat ETEC-mediated diarrhoea. An orally administered autogenous vaccine containing heat inactivated E. coli was administered to diarrhoeic puppies and adult dogs once daily for 14 days, and led to a significant decrease in morbidity and mortality. Additional studies are warranted to ensure the safety of this therapeutic regimen.