The development of culture independent techniques for determining the presence of infectious agents has revolutionized the approach to detecting both suspected and unsuspected pathogens. For example: PCR with broad range primers has led to the identification of Bartonella henselae as the agent of cat scratch fever and peliosis hepatis. PCR with pathogen specific primers has enabled the accurate differentiation of a growing number of Ehrlichial associated diseases. The development of labeled DNA probes against bacterial 16- or 23s rRNA not only facilitates specific identification of bacterial pathogens, but also their localization within tissues and cells, and antimicrobial resistance e.g., associated with 23s mutations--clarithromycin. This presentation focuses on the application of culture independent techniques to Histiocytic Ulcerative Colitis.

Canine Histiocytic Ulcerative Colitis

HUC: (also known as Granulomatous Colitis of Boxer Dogs) is a severe disease of unknown etiology that typically affects Boxer dogs. Clinically it is characterized by frequent, bloody, mucoid stools, ulceration and inflammation of the colon, anemia, hypoalbuminemia and weight loss. The dominant histological features are an accumulation of lymphocytes and plasma cells, and large numbers of PAS positive macrophages containing inclusions (similar to Whipple's disease) and the loss of colonic epithelium and goblet cells. Immunopathological studies describe an increase in IgG₃ and IgG₄ plasma cells, and PAS⁺ positive macrophages and CD₃-T cells, L1 and MHCII positive cells, with HUC likened to human ulcerative colitis. Several studies have described bacteria within a proportion of macrophages in the mucosa of HUC affected dogs. Ultrastructural studies suggest active phagocytosis of bacteria that in some instances resemble Chlamydia. Known invasive enteropathogens such as Salmonella, Campylobacter Yersinia, and Shigella have not been isolated from dogs with HUC. Mycoplasma have been isolated from the colon and regional lymph nodes of 4 boxer dogs but an attempt to reproduce granulomatous colitis with Mycoplasma was unsuccessful. This failure to identify or isolate an infectious agent has lead to HUC being considered a chronic immune mediated disease. The outcome of treatment with empirical combinations of diet, antimicrobials and immunosuppression is variable, with perhaps the most favorable outcome described in dogs receiving antibiotics such as chloramphenicol and tylosin. Recent reports indicate resolution of clinical signs and histological lesions, including the disappearance of PAS infiltrates in response to enrofloxacin alone or in combination with amoxicillin and metronidazole, re-awakening the possibility that HUC may indeed have an infectious etiology.

Colonic biopsies from affected dogs (13 Boxers with HUC) and controls (27 non-HUC, 11 normal histology) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16srRNA probe. Culture, 16s rDNA sequencing, and histochemistry were used further to define the invasive flora, and guide subsequent FISH. HUC-associated E.coli were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages, serotype, overall genotype, phylogenetic group, and presence of virulence genes.

Intramucosal bacteria, predominantly gram-negative coccobacilli, were present in 100% of GCB, but none of the control samples. Culture and 16srDNA sequencing yielded mostly Enterobacteriaceae, and invasive bacteria hybridized with FISH probes to E.coli. HUC-associated E. coli isolates adhered to, invaded, and persisted within cultured epithelial cells. Invasion triggered a "splash"-type response, was decreased (P<0.05) by cytochalasin-D, genistein, colchicine and wortmannin, and paralleled the behavior of Crohn's disease-associated E. coli LF-82. HUC-associated E.coli isolates and LF-82 were diverse in serotype and overall genotype, but similar in phylogeny (B2 and D), and virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsM-II, iss). HUC-associated E.coli that displayed an adherent and invasive phenotype in cell culture and LF82 were larger in genome size than commensal E.coli, and belonged to novel MLST clonal groups.

Thus HUC of Boxer dogs is associated with selective intramucosal colonization by Escherichia coli. E. coli strains associated with HUC and Crohn's disease have an adherent and invasive phenotype, and resemble extraintestinal pathogenic E. coli in phylogeny and virulence genes. These findings support the thesis that IBD is a consequence of mucosal colonization by a restricted subset of the luminal microflora in a susceptible individual, and point to the association of E.coli resembling extraintestinal pathogenic strains in genotype with chronic intestinal inflammation. As HUC is largely confined to Boxer dogs, it is tempting to speculate that a defect in host defense, perhaps analogous to those identified in some patients with Crohn's disease e.g., NOD2/CARD 15, TLR4, is present in Boxer dogs and linked to bacterial invasion and inflammation.

Acknowledgements

Dr. Simpson is supported by a grant from the US public health service (DK 002938). The author is grateful to Sean McDonough, Mark Rishniw, Belgin Dogan, Richard Goldstein, Patrick McDonough, Alex German, Robin Yates, David Russell, Yung-Fu Chang, Raghavan Palanappian, Martin Weidman, Kendra Nightingale, Roger Hostutler, Jennifer Chaitman, Susan Johnson, Ynte Schukken, Francis Davies, Pat Fisher and Douglas Berg for their input and collaboration on the studies described above.

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