Association of Atypical Enteropathogenic Escherichia coli with Diarrhea and Related Mortality in Kittens

Abstract

Diarrhea is responsible for the death of approximately 900,000 children per year worldwide. In children, typical enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea and is associated with a higher hazard of death. Typical EPEC infection is rare in animals and poorly reproduced in experimental animal models. In contrast, atypical EPEC (aEPEC) infection is common in both children and animals, but its role in diarrhea is uncertain. Mortality in kittens is often attributed to diarrhea, and we previously identified enteroadherent EPEC in the intestines of deceased kittens. The purpose of this study was to determine the prevalence and type of EPEC in kittens and whether infection was associated with diarrhea, diarrhea-related mortality, gastrointestinal pathology, or other risk factors. Kittens with and without diarrhea were obtained from two shelter facilities and determined to shed atypical EPEC at a culture-based prevalence of 18%. In contrast, quantitative PCR detected the presence of the gene for intimin (eae) in feces from 42% of kittens. aEPEC was isolated from kittens with and without diarrhea. However, kittens with diarrhea harbored significantly larger quantities of aEPEC than kittens without diarrhea. Kittens with aEPEC had a significantly greater severity of small intestinal and colonic lesions and were significantly more likely to have required subcutaneous fluid administration. These findings identify aEPEC to be prevalent in kittens and a significant primary or contributing cause of intestinal inflammation, diarrhea, dehydration, and associated mortality in kittens.

Keywords: EPEC; Escherichia coli enterocyte attaching and effacing (eae) gene; animal models; cat; histopathology.

FIG 1

Estimation of aEPEC quantity determined by quantitative PCR (qPCR) amplification of the E. coli enterocyte attaching and effacing (eae) gene from fecal DNA from 61 live kittens with and without diarrhea. (A) Standard curve obtained by inoculating serial dilutions of known numbers of CFU of aEPEC into 100-mg aliquots of cat feces, followed by DNA extraction and qPCR amplification of eae. The graph represents the results of 3 independent experiments each performed in triplicate at each dilution. The linear equation relates y = cycle threshold (C_T) value to x = log₁₀ number of CFU of bacteria. The insert shows an electrophoresis gel of the eae qPCR product from aEPEC log dilutions 3 to 8 (10³ to 10⁸) and a molecular weight ladder demonstrating the size of the PCR product (425 bp). (B) Estimated number of aEPEC CFU per 100 mg of feces determined from the standard curve and the cycle threshold (C_T) values for qPCR amplification of eae from the fecal DNA of live kittens with and without diarrhea. The lines represent the medians and interquartile ranges of the data.

FIG 2

Estimated number of aEPEC CFU per 100 mg feces from apparently healthy kittens euthanized by animal control and kittens that died or were euthanized because of severe diarrhea while in foster care. The lines represent the medians and the interquartile ranges of the data.

FIG 3

Estimated number of aEPEC CFU per 100 mg feces from all kittens with or without diarrhea. Lines represent the medians and the interquartile ranges of the data. Kittens with diarrhea had a significantly larger quantity of aEPEC, as determined by the eae qPCR and standard curve. White symbols, live kittens; black symbols, kittens that died or were euthanized. *, P = 0.013, Mann-Whitney rank sum test.

FIG 4

Pulsed-field gel electrophoresis (PFGE) of 13 aEPEC isolates obtained from 3 apparently healthy kittens euthanized by animal control and 6 kittens that died or were euthanized because of severe diarrhea while in foster care. The results for a typical EPEC strain (E2348/69) and a nonpathogenic E. coli strain (ATCC 25822) are included for comparison. The isolates were clustered using the unweighted pair group method with arithmetic means (UPGMA), and the scale on the left represents the percent similarity between each pulsotype. The serotype, isolate identifier, and clinical outcome (H, healthy; D, diarrhea) for each pulsotype are shown on the right.

FIG 5

Small intestinal and colonic histopathology lesion scores based on results of fecal culture and/or qPCR for eae in apparently healthy kittens and those dying or euthanized due to severe diarrhea. Kittens with aEPEC that died with diarrhea had significantly higher small intestinal (A) and colonic (B) histopathology lesion scores than kittens with other causes of diarrhea and apparently healthy kittens with and without aEPEC. P values were determined by the Mann-Whitney rank sum test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Representative photomicrographs of lesions significantly associated with the presence of aEPEC (determined by culture and/or eae qPCR) in kittens. (A) Photomicrograph of small intestine from a kitten with aEPEC demonstrating (i) epithelial cell loss and (ii) inflammatory infiltrate composed of lymphocytes and plasma cells in the lamina propria. (B) Increased lesion severity. Also noted in this panel are crypt dilation and villus blunting in the small intestine. (C) Inflammatory infiltrate in the lamina propria of the colon along with crypt dilation in a kitten with aEPEC. (D) Severe inflammation composed predominantly of neutrophils obscuring the crypts and architecture of the colon. Hematoxylin and eosin stain. Bars = 500 μm (A to C) and 200 μm (D).

FIG 7

Representative fluorescence microscopy images of focally adherent E. coli in the small intestine of one kitten (A to D) and the colon of another kitten (E to H) that were euthanized due to severe diarrhea. (A and E) Appearance of enteroadherent bacteria with Giemsa stain. Fluorescence in situ hybridization was performed using oligonucleotide probes specific for eubacterial 16S (probe Eub338; 3′ 6-FAM green) (B and F) and specific for E. coli and Shigella (5′ Cy3; red) (C and G). (D and H) Overlay of fluorescence signals. The nuclei in the specimens were counterstained with DAPI (4′,6-diamidino-2-phenylindole; blue).