equine assay data sheet

Enteric E. coli panel

Test code: P0016 - Qualitative detection and differentiation by PCR of 5 different clinically important categories of diarrheagenic E. coli -- ETEC, EHEC, EIEC, EPEC and EAEC

Diarrheal diseases are a major cause of death for children under 5 years of age in developing countries; the estimated death toll is 12,600 children per day. Causes of diarrhea include a wide range of viruses, bacteria, and parasites. Among the bacterial pathogens, various strains of Escherichia coli are the major culprits.

Although E. coli is the predominant nonpathogenic facultative anaerobic member of the human intestinal microflora, some E. coli strains can cause diseases of the gastrointestinal, urinary, and central nervous systems in humans. The intestinal tracts of almost all birds and mammals, including nonhuman primates and horses, are colonized by E. coli. Infections by pathogenic strains of E. coli also occur in many domestic and wild species.

Pathogenic symptoms induced by these diarrheagenic E. coli can be due to production of toxins or other virulence traits. E. coli strains that induce diarrhea in their hosts can be divided into five main categories on the basis of distinct epidemiological and clinical features and specific virulence determinants:

• ETEC - Enterotoxigenic E. coli: Produce heat-labile toxin (LT) and heat-stable toxin (ST).
• EHEC - Enterohemorrhagic E. coli: Produce shiga-like toxins (SLT) I and II.
• EIEC - Enteroinvasive E. coli: Typically invade and destroy the bowel mucosa.
• EPEC - Enteropathogenic E. coli: Damage the bowel mucosa with characteristic attaching and effacing lesions mediated by a protein encoded by a gene called the attaching and effacing locus (eal).
• EAEC - Enteroaggregative E. coli: The epidemiology and pathogenicity of these strains have not yet been clearly defined, but the presence of a large 60 kD plasmid encoding several virulence factors and toxins is important for their virulence.

Clinically, ETEC induce a watery diarrhea in infected hosts by action of the two toxins, LT and ST. The LT enterotoxin is very similar to cholera toxin in both structure and mode of action. ST is known to bind to and activate a guanylate cyclase enzyme located on apical membranes of host cells. This leads to secretion of fluid and electrolytes resulting in a watery diarrhea. Incubation period is approximately 1-2 days and illness can last 3 days to several weeks.

EHEC are mostly represented by a single strain, serotype O157:H7, which causes a diarrheal syndrome with copious bloody discharge and no fever. There is a toxic effect on the kidneys, and diarrhea caused by this strain can be fatal, particularly in infants, due to acute kidney failure. Infection in humans is often associated with ingestion of inadequately cooked hamburger meat. Incubation period is approximately 3 to 4 days and duration of illness is about 1 week.

EIEC are similar to Shigella in their pathogenic mechanism and clinical symptoms. EIEC bacteria penetrate and multiply within epithelial cells of the colon causing widespread cell destruction. The clinical syndrome is identical to Shigella dysentery and includes a dysentery-like diarrhea with fever. EIEC do not produce LT or ST toxin and, unlike Shigella, do not produce shiga toxin. The incubation period is less than 24 hours.

EPEC cause a watery diarrhea similar to ETEC, but do not produce ST or LT toxins. These strains are a principal cause of infant diarrhea in developing countries. The illness typically lasts 1 to 3 days.

EAEC adhere to epithelial cells in a characteristic stacked-brick pattern known as the aggregative adherence (AA) pattern. When they adhere to small and large bowel mucosal surfaces they stimulate mucus production, leading to a thick mucus-containing biofilm encrusted with EAEC. They can also secrete toxins, such as heat-stable enterotoxin 1 (EAST1), Pet and Pic, which are associated with damage to the mucosa. EAEC were originally recognized as one of the predominant etiologic agents of persistent diarrhea in developing countries and they remain an important cause of acute as well as protracted diarrhea in many parts of the world, including industrialized countries.

Several assays are available for detection of diarrheagenic E. coli, including biochemical reactions, serotyping and phenotypic assays based on virulence characteristics. However, molecular detection by PCR has become a commonly-used method to detect and identify these bacteria because the method gives rapid and reliable results in addition to its high sensitivity and specificity (Bellin et al., 2001; Stacy-Phipps et al., 1995).

Utilities:

• Help confirm the disease causing agent
• Selection of appropriate treatment regimen
• Shorten the time required to confirm a clinical diagnosis
• Help ensure that herds are free of diarrheagenic E. coli
• Early prevention of spread of diarrheagenic E. coli
• Minimize personnel exposure to diarrheagenic E. coli
• Safety monitoring of biological products that derive from horses

References:
Bellin, T., Pulz, M., Matussek, A., Hempen, H.G. and Gunzer, F. (2001) Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 39:370-374.
Stacy-Phipps, S., Mecca, J.J. and Weiss, J.B. (1995) Multiplex PCR assay and simple preparation method for stool specimens detect enterotoxigenic Escherichia coli DNA during course of infection. J. Clin. Microbiol. 33:1054-1059.

Specimen requirements: Rectal swab, 0.5 ml feces or 0.2 ml bacterial culture.

Contact Zoologix if advice is needed to determine an appropriate specimen type for a specific diagnostic application. For specimen types not listed here, please contact Zoologix to confirm specimen acceptability and shipping instructions.

For all specimen types, if there will be a delay in shipping, or during very warm weather, refrigerate specimens until shipped and ship with a cold pack unless more stringent shipping requirements are specified. Frozen specimens should be shipped so as to remain frozen in transit. See shipping instructions for more information.

Turnaround time: 3 business days

Methodology: Qualitative PCR

Normal range: Nondetected