In situ molecular diagnosis and histopathological characterization of enteroadherent Enterococcus hirae infection in pre-weaning-age kittens

Abstract

The bacterial causes of diarrhea can be frustrating to identify, and it is likely that many remain undiagnosed. The pathogenic potential of certain bacteria becomes less ambiguous when they are observed to intimately associate with intestinal epithelial cells. In the present study we sought to retrospectively characterize the clinical, in situ molecular, and histopathological features of enteroadherent bacteria in seven unrelated kittens that were presumptively diagnosed with enteropathogenic Escherichia coli (EPEC) on the basis of postmortem light microscopic and, in some cases, microbiological examination. Characterization of the enteroadherent bacteria in each case was performed by Gram staining, in situ hybridization using fluorescence-labeled oligonucleotide probes, PCR amplification of species-specific gene sequences, and ultrastructural imaging applied to formalin-fixed paraffin-embedded sections of intestinal tissue. In only two kittens was EPEC infection confirmed. In the remaining five kittens, enteroadherent bacteria were identified as Enterococcus spp. The enterococci were further identified as Enterococcus hirae on the basis of PCR amplification of DNA extracted from the formalin-fixed, paraffin-embedded tissue and amplified by using species-specific primers. Transmission electron microscopy of representative lesions from E. coli- and Enterococcus spp.-infected kittens revealed coccobacilli adherent to intestinal epithelial cells without effacement of microvilli or cup-and-pedestal formation. Enterococci were not observed, nor were DNA sequences amplified from intestinal tissue obtained from age-matched kittens euthanized for reasons unrelated to intestinal disease. These studies suggest that E. hirae may be a common cause of enteroadherent bacterial infection in pre-weaning-age kittens and should be considered in the differential diagnosis of bacterial disease in this population.

FIG. 1.

Light microscopic appearance of the small intestinal epithelium from a kitten with enteroadherent E. coli (A) and enteroadherent Enterococcus (B) infection after staining with hematoxylin and eosin. Note the similarity in the histological appearance of the bacteria. After Gram staining of the tissue sections, bacteria from a cat with E. coli stain negative (C; arrow), whereas those from the cat with Enterococcus stain positive (D).

FIG. 2.

Fluorescence in situ hybridization (FISH) of the small intestinal epithelium from a kitten with enteroadherent E. coli infection. (A) The nuclear counterstain, DAPI (4′,6′-diamidino-2-phenylindole), enables visualization of intestinal epithelial and lamina propria nuclei. Nucleic acid within the enteroadherent bacteria (arrow) can be seen in the lumen along the junction of two intestinal villi. (B) FISH using the eubacterial probe Eub338 labeled with FAM (green fluorescence) identifies the bacteria as eubacteria. (C) FISH using an E. coli/Shigella-specific probe labeled with Cy3 (red fluorescence) identifies the eubacteria as E. coli/Shigella. (D) Merged image demonstrating the simultaneous hybridization (light yellow) of enteroadherent bacteria with eubacterial and E. coli/Shigella-specific oligonucleotide probes.

FIG. 3.

FISH of small intestinal villous epithelium from kittens with enteroadherent Enterococcus infection. Merged images demonstrate the simultaneous hybridization of enteroadherent bacteria with DAPI (blue) eubacterial (green) and Enterococcus-specific oligonucleotide probes (red), which generate an orange fluorescence.

FIG. 4.

Transmission electron micrographs of feline small intestinal epithelium from kittens naturally infected with enteropathogenic E. coli (A and C) or enteroadherent E. hirae (B and D). Interactions between the bacteria and the microvillus brush border of the epithelial cells are similar in both infections.