Enterocyte Proliferation and Intracellular Bacteria in Animals

Gut 1994; 35: 1483-1486 1483

PROGRESS REPORT Gut: first published as 10.1136/gut.35.10.1483 on 1 October 1994. Downloaded from Enterocyte proliferation and intracellular bacteria in animals

S McOrist, C J Gebhart, G H K Lawson

Considerable proliferation of enterocytes, candidates for the bacteria involved.2 Recent forming adenomatous growths within the work shows that the intracellular bacteria are a intestinal mucosa, is a consistent feature of a new genus of obligate intracellular bacteria, number of enteric conditions in animals, now living within enterocytes. This report describes referred to under the general heading of pro- recent advances in the taxonomic status of the liferative enteropathy. These were originally bacteria and its association with disease patho- described in pigs as adenomas ofthe ileum and genesis in animals, focusing on how this can colon1 and this condition was found to have a aid an understanding of enterocyte prolifera- worldwide occurrence.2 Similar conditions tion. were subsequently described in a number of other animal species, again under the general heading of proliferative enteropathy or Intracellular bacteria enteritis. The degree, however, of mucosal The intracellular bacteria in proliferative proliferation, corresponding to a description of enteropathy in animals have been closely hyperplasia, through adenoma to a more studied in the pig and hamster species, in carcinomatous form, varies between species. which the disease is common and widespread. Occasional reports of the condition in the fox,3 Clinical diagnosis of infection and disease is, horse,4 and guinea pig5 describe a hyperplastic however, difficult2 and the disease might be condition of the mucosa whereas hamsters6 7 recognised more widely if diagnostic tools develop adenomatous lesions similar to the became available. Morphologically, the pig, and the rat8 and ferret9 can develop an bacteria have a wavy trilaminar cell wall and http://gut.bmj.com/ apparently carcinomatous condition with true granular protoplasm typical of Gram negative metastases to local lymph nodes. Study of the bacteria.10 They tend to be curved bacilli, disease was revolutionised in 1974 by the sometimes sigmoid, with ends tapering to a discovery that these lesions in pigs had the con- rounded extremity.14 They divide by trans- sistent presence of intracellular bacteria lying verse septation. Healthy and dividing bacteria free in the cytoplasm of proliferating entero- have only been seen within mammalian enterocytes.'0 These bacteria were not detected in cytes. Some degenerate forms have been on September 23, 2021 by guest. Protected copyright. normal animals or in unaffected portions ofthe seen in mucosal macrophages.2 The healthy intestine. The disease is infectious and is trans- bacterial forms seem similar morphologically mitted by oral exposure of animals to diseased in all variations of host species and pathology. mucosa. Antigenic studies of these bacteria in several The wide variety and occurrence of these host species including pigs, hamsters, ferrets, lesions in animals prompted comparisons with rats, guinea pigs, fox, horse, and deer some ileocolic conditions in humans, such as show that they also share outer membrane Department of studies of the antigens.15 16 These antigens are distinct from Veterinary Pathology, Crohn's disease. Comparative University of lesions, however, and serological testing,'1 12 those of other curved bacteria found in the Edinburgh, Veterinary including a study by Crohn himself,'3 showed gut.'7 Preliminary and more definitive studies Field Station, Easter that proliferative enteropathy in animals had of the DNA of these bacteria show that they Bush, Midlothian delta S McOrist no clear counterpart in humans. There may be form a distinct new genus within the G H K Lawson some mechanistic comparisons with the food group of Proteobacteria.'8 19 Porcine strains of sensitive enteropathies, including coeliac the bacteria have been designated an ileal Division of Their relation to Comparative disease, in which primary crypt cell hyperplasia symbiont intracellularis.'9 Medicine, Medical is thought to play a part in pathogenesis. "l This strains from other hosts is not yet known, but School, University of still leaves open the question of how an enteric results of preliminary antigenic and DNA Minnesota, show that they are almost identical. Minneapolis, USA bacterium, albeit one living inside enterocytes, analyses C J Gebhart can change host cell metabolism to induce a These DNA studies showed a 91% hyperplastic or even carcinomatous state. homology of the 16S rDNA portion of the Correspondence to: Dr S McOrist, Department Previous exploration of this phenomenon was genome to Desulfovibrio desulfuricans.'9 These of Veterinary Pathology, by a lack of knowledge of the are one of the predominant sulphate reducing University of Edinburgh, hampered Veterinary Field Station, bacterium involved. Many early studies bacteria in the colon and can use molecular H2 Easter Bush, Midlothian considered the bacteria morphologically as an electron donor.20 This may point to some EH25 9RG. similar to vibrio or campylobacters and several distant evolutionary or functional relation, or Accepted for publication 12 January 1994 campylobacter species were proposed as both, to the intracellular bacteria. 1484 McOrist, Gebhart, Lawson

The method of entry of the bacteria into affected crypts and normal mucosal crypts host enterocytes is probably by parasite regenerate. Despite the detection by others of directed endocytosis, with attachment of the enteric viruses and Chlamydia sp in some bacteria to the brush border surface, formation experimental inocula or animals,27 29 it is of an entry vacuole, and early release free into probable that ileal symbiont intracellularis Gut: first published as 10.1136/gut.35.10.1483 on 1 October 1994. Downloaded from the cytoplasm, where multiplication and can act as a sole initiating agent of prolifera- division occurs.21 Some of these biological tive enteropathy.28 30 There seems to be some features are similar to that of some chlamydia host specificity in the ability to produce and rickettsia species. For example, R lesions. Although organisms derived from tsutsugamushi lie freely in the cytoplasm of lesions in the pig can produce lesions in orally infected fibroblasts.22 Intracellular bacteria are dosed hamsters,31 these tend not to be as characterised by a wide variety of entry and florid as lesions produced by hamster derived survival mechanisms.23 These mechanisms are isolates. Immunological and DNA analyses, not, however, indicative of any particular including 16S rDNA gene sequence data, evolutionary relation between bacteria that have shown that the organisms within pig and have similar modes of entry and survival.23 hamster enterocytes are almost identical. 16 There does seem to be some common ground Therefore the apparent differences in in the general scheme of regulation of attach- abnormal reactions upon oral transmission of ment, entry, and survival genes and their inocula to a separate host species may only products. Bacteria in the gut lumen must reflect a reduced ability of inoculated bacteria possess the ability to recognise features of their to compete with the differing gut flora of external environment, such as temperature, another host species and effect colonisation. osmolarity or anaerobiosis, to regulate the Dosing ofprimates has not been reported. The expression of genes active in attachment and exact relation of intracellular bacteria to the entry into enterocytes. Recent reviews24 25 carcinomatous lesions in rats has not been provide many examples, such as the detection clearly established, but further experiments of anaerobiosis postulated for the expression may clarify this. of the invasion phenotype of Salmonella Experiments and clinical data show that typhimurium for entry into mammalian cells. animals are most susceptible to infection just After entry into cells, intracellular bacteria after weaning from their mother.32 This may may detect environmental calcium or other reflect a greater proportion of permissive crypt signals24 25 regulating the action of genes epithelial cells within the intestine or a necessary for intracellular survival. more protective intraluminal environment for In some instances, we have found that bacterial colonisation, or both. Recent experi- the bacteria within enterocytes lie closely ments in germ free pigs, recently weaned pigs associated with mitochondria and rough with a normal gut flora, and pigs with a defined endoplasmic reticulum. Similar findings in gut flora of four non-pathogenic bacteria http://gut.bmj.com/ cells infected with other intracellular bacteria supported the concept that the degree of gut have led to the suggestion that these intra- maturity and luminal conditions attained at cellular bacteria may derive some benefit from weaning greatly enhances colonisation and triphosphates produced by the host cells,26 disease due to ileal symbiont intracellularis.28 30 possibly by incorporation of these into The evidence suggests that enterocyte bacterial respiration. proliferation in animals can be caused directly by a transmissible agent, identified as a delta on September 23, 2021 by guest. Protected copyright. group Proteobacteria closely related to the Enterocyte proliferation sulphate reducing bacteria. Current knowledge The remarkable presence of intracellular of proliferative enteropathy is insufficient to bacteria within enterocytes is made even more suggest how a bacterium can influence entero- remarkable by the clear temporal and cytes to start comparatively uncontrolled causative relation between the appearance of growth. Any proposed mechanism would have these bacteria and the development of pro- to take into account the dynamics of crypt liferation of the enterocytes leading to gross epithelial cell production by monoclonal stem hyperplasia, adenoma or carcinoma. Many cells, with subsequent proliferation and early experiments in which pigs were inocu- vertical elevation into adjacent villi, as outlined lated with bacteria derived directly from elsewhere.32 Some possible mechanisms, how- homogenates of affected mucosa7 1427 and ever, can be postulated. Firstly, the bacterium later experiments using ileal symbiont could possess an intrinsic mitogenic factor, intracellularis grown in cultured intestinal which is either expressed constitutively or epithelial cells28 as the inoculum clearly upon intracellular entry. Such a mitogen has showed that the bacteria had entered host been postulated to be a virulence factor of enterocytes by five to seven days after dosing. Bartonella bacilliformis,33 the Gram negative Hyperplasia of infected mucosal epithelium intracellular bacterium that is the cause of was then evident 10 to 14 days after dosing, endothelial cell proliferation in the dermal with gross lesions visible in some cases 21 verruga of Bartonellosis.34 Secondly, the days after dosing. Histological examination of bacteria could share receptor binding capacity lesions points to mitosis and piling up of with natural ligands such as tumour growth immature enterocytes in defined, but long, factor ot or other growth factors, although this branching crypt structures. Some of those would not explain why the organism requires cells may die, appearing as apoptotic bodies. an intracellular phase. A transmissible colonic In recovering lesions, goblet cells reappear in hyperplasia of mice caused by a Citrobacter Enterocyte proliferation and intracellular bacteria in animals 1485

freundii like organism3536 apparently occurs accumulation of IgA in affected enterocytes without any need for intracellular entry, occur- possibly results from the disruption of their ring after attachment of the bacteria to the normal activity by the presence of intracellular brush border. This organism has recently been bacteria, or from the immaturity ofthe affected

shown to contain an attaching and effacing cells. Gut: first published as 10.1136/gut.35.10.1483 on 1 October 1994. Downloaded from locus homologous to the eae gene of In conclusion, the occurrence of a common enteropathogenic Escherichia coli.37 This disease in animals, in which the presence of raises the possibility that a group of gut intracellular bacteria of the delta group of bacteria possess at least two mechanisms to Proteobacteria clearly triggers massive prolifer- change epithelial cell growth, one which acts ation of enterocytes, offers food for thought for extracellularly and one which acts intra- gastroenterologists. Can bacteria in the gut cellularly. Patients with colon adenomas can interact with host cell membrane receptors that have a greater predominance of anaerobes in affect growth? Can some bacteria produce their colonic flora than control subjects.38 39 mitogenic factors acting locally on the bowel? Whether this is merely a by product of colonic The elucidation of the mechanisms underlying stasis or is indicative of a functional relation host cell proliferation induced by these between colonic bacteria and enterocyte pro- bacteria will be the next goal. liferation is not clear. Further investigation of 1 Beister HE, Schwarte LH. Intestinal adenoma in swine. Am the type and taxonomic status of colonic flora J Pathol 1931; 7: 175-85. in adenoma patients may prove of interest. 2 Rowland AC, Lawson GHK. Porcine proliferative enteropathies. In: Leman AD, Straw BE, Mengeling WL, This is not to imply that there is an increased D'Allaire S, Taylor DJ, eds. Diseases of swine. 7th ed. risk of colon cancer from contact with the gut Ames: Iowa State University Press, 1992: 560-9. 3 Eriksen K, Landsverk J. Interstinal adenomatosis in the blue flora of pigs or other animals; no such risk has fox. Nordic Veterinaria Medica 1985; 37: 254-5. been shown clearly in relevant occupational 4 Duhamel GE, Wheeldon EB. Intestinal adenomatosis in a foal. Vet Pathol 1982; 19: 447-50. categories in epidemiological studies.40 Many 5 Elwell MR, Chapman AL, Frenkel JK. Duodenal hyper- other mechanisms have been suggested plasia in a guinea pig. Vet Pathol 1980; 17: 136-9. 6 Frisk CS, WagnerJE. Experimental hamster enteritis: an elec- whereby gut bacteria can participate in local tron microscopic study. Am J Vet Res 1977; 44: 1861-8. cancer formation. For example, production of 7 Jacoby RO. Transmissible ileal hyperplasia of hamsters. I. Histogenesis and immunocytochemistry. Am Jf Pathol carcinogenic bile acid metabolites by anaerobic 1978; 91: 433-50. bacteria may occur in the formation of some 8 Vandenberghe J, Verheyen A, Lauwers S, et al. Spontaneous adenocarcinoma of the ascending colon in Wistar rats: colon cancers in humans.39 the intracytoplasmic presence of a Campylobacter-like bacterium. Jf Comp Pathol 1985; 95: 45-55. 9 Fox JG, Murphy JC, Otto G, et al. Proliferative colitis in ferrets: epithelial dysplasia and translocation. Vet Pathol Other host responses 1989; 26: 515-7. 10 Rowland AC, Lawson GHK. Intestinal adenomatosis in the The immune response to bacterial infection in pig: immunofluorescent and electron microscopic studies. proliferative enteropathy is characterised by a Res Vet Sci 1974; 17: 323-33. 11 MacDonald TT. Epithelial proliferation in response to weak humoral and cellular response.4' 42 gastro-intestinal inflammation. Ann N Y Acad Sci 1992; http://gut.bmj.com/ Specific IgA and IgM are detected in the serum 664: 202-9. 12 Mayberry JF, Rhodes J, Heatley RV. Infections which cause samples of severe clinical cases in pigs, but ileocolic disease in animals: are they relevant to Crohn's carrier or subclinical states could not be disease? Gastroenterology 1980; 78: 1080-4. 13 Crohn BB, Turner DA. Porcine ileitis. Gastroenterology detected serologically.4' Examination of 1952; 20: 350-1. affected intestines shows appreciable accumu- 14 McOrist S, Lawson GHK, Rowland AC, et al. Early lesions of proliferative enteritis in pigs and hamsters. Vet Pathol lation of IgA in the cytoplasm of affected cells, 1989; 26: 260-4.

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