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Purpose: Crohn's disease is a chronic debilitating intestinal syndrome of unknown aetiology that is thought to result in part from an imbalance (dysbiosis) of the intestinal microbial populations, known as the microbiota. In this study we sought to compare the microbiota at the mucosal and submucosal levels at the resection margin in Crohn's disease to those in other intestinal dysbiotic disease controls to determine the level of bacterial translocation., Methodology: 16S microbiota sequencing was performed on DNA extracted from mucosal and submucosal samples from resected intestinal tissues from Crohn's disease and controls., Results: Grossly normal appearing tissue at the resection margin showed early signs, suggesting bacterial translocation, with two bacterial families having penetrated the mucosal surfaces. In contrast, 4 and 13 bacterial families were present within submucosal tissues at the disease centre and disease margin, respectively. Although there was no significant difference in biodiversity, there was increased bacterial richness in the Crohn's disease group as compared to non-IBD controls., Conclusion: The presence and/or absence of certain bacteria suggested disease-specific ecological or micro-environmental pressures driving or excluding certain organisms in Crohn's disease. The data suggest that several of the dysbiotic conditions previously reported for Crohn's disease are not unique but common to general dysbiosis. The examination of multiple intestinal sites in advanced disease may provide a spectrum of disease from early onset at the resection margin to active disease at the disease margin and late-stage fibrostenotic disease at the centre of the lesion, and a unique etiopathogenic view of Crohn's disease.

Crohn's disease is characterized by increased permeability of the intestinal mucosal barriers and an abnormal or dysregulated immune response to specific and/or commensal bacteria arising from the intestinal lumen. To determine the types of bacteria that are transgressing the mucosal barrier and colonizing the intestinal submucosal tissues, we performed 16S rRNA gene microbiota sequencing of the submucosal and mucosal tissues at the advancing disease margin in ileal Crohn's disease. Microbial populations were compared between mucosa and submucosa and non-inflammatory bowel disease (non-IBD) controls, as well as to microbial populations previously found at the centre of the disease lesion. There was no significant increase in bacteria within the submucosa of non-IBD controls at any taxonomic level when compared to the corresponding superjacent mucosa, indicating an effective mucosal barrier within the non-IBD population. In contrast, there was a statistically significant increase in 13 bacterial families and 16 bacterial genera within the submucosa at the advancing disease margin in Crohn's disease when compared to the superjacent mucosa. Major increases within the submucosa included bacteria of the Families Sphingomonadaceae, Alicyclobacillaceae, Methylobacteriaceae, Pseudomonadaceae and Prevotellaceae. Data suggest that the primary site of bacterial translocation across the mucosal barrier occurs at the margin between diseased and normal tissue, the advancing disease margin. The heterogeneity of the bacterial populations penetrating the mucosal barrier and colonizing the submucosal intestinal tissues and, therefore, contributing to the inflammatory processes, suggests that bacterial translocation is secondary to a primary event leading to a breakdown of the mucosal barrier.

Background: The advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications., Results: We herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample., Conclusions: Laboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings.

Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Since Crohn's disease is a transmural disease, we hypothesized that examination of deep submucosal tissues directly involved in the inflammatory disease process may provide unique insights into bacterial populations transgressing intestinal barriers and bacterial populations more representative of the causes and agents of the disease. We performed deep 16s microbiota sequencing on isolated ilea mucosal and submucosal tissues on 20 patients with Crohn's disease and 15 non-inflammatory bowel disease controls with a depth of coverage averaging 81,500 sequences in each of the 70 DNA samples yielding an overall resolution down to 0.0001% of the bacterial population. Of the 4,802,328 total sequences generated, 98.9% or 4,749,183 sequences aligned with the Kingdom Bacteria that clustered into 8545 unique sequences with <3% divergence or operational taxonomic units enabling the identification of 401 genera and 698 tentative bacterial species. There were significant differences in all taxonomic levels between the submucosal microbiota in Crohn's disease compared to controls, including organisms of the Order Desulfovibrionales that were present within the submucosal tissues of most Crohn's disease patients but absent in the control group. A variety of organisms of the Phylum Firmicutes were increased in the subjacent submucosa as compared to the parallel mucosal tissue including Ruminococcus spp., Oscillospira spp., Pseudobutyrivibrio spp., and Tumebacillus spp. In addition, Propionibacterium spp. and Cloacibacterium spp. were increased as well as large increases in Proteobacteria including Parasutterella spp. and Methylobacterium spp. This is the first study to examine the microbial populations within submucosal tissues of patients with Crohn's disease and to compare microbial communities found deep within the submucosal tissues with those present on mucosal surfaces. Our data demonstrate the existence of a distinct submucosal microbiome and ecosystem that is not well reflected in the mucosa and/or downstream fecal material.

Objective: To determine whether bacterial pathogens can be detected within the diseased submucosal tissues of patients with Crohn's disease by molecular techniques independent of cultural methods., Design: We designed a quantitative polymerase chain reaction to detect 32 virulence genes and transposons within submucosal tissues of patients with Crohn's disease and controls and compared the microbiome of the submucosa with mucosal bacterial populations., Results: Within submucosal tissues, the bacterial invasion/adherence genes eaeA and invA were detected in 43% of patients (P=0.01 and 0.008 vs. mucosa and controls, respectively) and the Mycobacterium-specific IS900 and 251F genes detected in 50% of patients (P=0.03 vs. mucosa and controls). These findings were mutually exclusive: invasion/adhesion genes and Mycobacterium-associated transposons were not detected in the same patient. Metagenomic sequencing and quantitative polymerase chain reaction results confirmed effective separation of the submucosal and mucosal microbiome and the existence of a submucosal bacterial population within diseased tissues., Conclusions: This study is the first to examine the microbial populations of submucosal tissues during intestinal disease and provide evidence of a distinct submucosal microbiome and biotypes within Crohn's disease. These data suggests that Crohn's disease may not be a single disease, but a spectrum that can be divided into distinct biotypes based on the presence of invasion/adherence genes or Mycobacterium-associated transposons. If corroborated by larger population studies, these findings could revolutionize the diagnosis, management, and treatment of Crohn's disease by the identification of patient biotypes and the application of targeted chemotherapeutic treatments that go beyond supportive in nature.

It has been more than 25 years since Mycobacterium paratuberculosis was first proposed as an etiologic agent in Crohn's disease based on the isolation of this organism from several patients. Since that time, a great deal of information has been accumulated that clearly establishes an association between M. paratuberculosis and Crohn's disease. However, data are conflicting and difficult to interpret and the field has become divided into committed advocates and confirmed skeptics. This review is an attempt to provide a thorough and objective summary of current knowledge from both basic and clinical research from the views and interpretations of both the antagonists and proponents. The reader is left to draw his or her own conclusions related to the validity of the issues and claims made by the opposing views and data interpretations. Whether M. paratuberculosis is a causative agent in some cases or simply represents an incidental association remains a controversial topic, but current evidence suggests that the notion should not be so readily dismissed. Remaining questions that need to be addressed in defining the role of M. paratuberculosis in Crohn's disease and future implications are discussed.

Disease caused by Mycobacterium paratuberculosis involves a complex interaction of lymphoid and phagocytic cells of the peripheral and mucosal immune responses. For resistance to develop, animals must generate an effective cellular immune response to primary infections as well as multifocal exogenous and endogenous reinfections. If an effective immune response does not develop, infected animals transgress through a complex immunologic spectrum in which the immunologic reactions themselves are responsible for the disease manifestations.

Available literature on the controversial role of Mycobacterium paratuberculosis as an etiologic agent in human Crohn's disease is reviewed. Despite almost 15 years of investigation, the question of causal or consequential association between Johne's disease and Crohn's disease continues to linger.

Balb/C, C57/B10 and C57/B6 mice were examined for their susceptibility to disseminated Mycobacterium paratuberculosis infection after intraperitoneal inoculation with a suspension of organisms containing mineral oil. Animals were examined monthly by histopathology and bacterial tissue counts of liver and spleen over a 6-month period. Only Balb/c mice maintained a steady infection with an average of 4.1 x 10(5) +/- 7.8 x 10(3) and 8.1 x 10(5) +/- 2.6 x 10(4) colony forming units (cfu) per gram of liver and spleen, respectively, during the course of the study. In contrast, C57/B10 mice reduced the bacterial counts in the liver and spleen from 6.8 x 10(4) and 1.3 x 10(5) to 7.1 x 10(2) and 4.3 x 10(3), respectively during the first 120-150 days after infection. The reduction in cfu was associated with the development of caseous necrotic lesions. C57/B10 mice were of intermediate resistance, slowly reducing cfu in the liver, but not the spleen, during the 6-month period. Balb/c was found to be a suitable mouse strain for the study of chronic M. paratuberculosis infection.

BALB/c mice were infected intraperitoneally with Mycobacterium paratuberculosis and, after allowing the infection to progress for 30 days, were treated with rifabutin at 0, 12.5, 25, and 50 mg/kg of body weight. Rifabutin was administered in drinking water under conditions of water deprivation, whereby the entire daily dose was delivered within a 1-h period. Animals were killed at biweekly intervals from time zero of treatment to 180 days. Spleens and livers from each animal were examined by quantitative bacteriologic culture and histopathology. Restricted water availability was found to be a viable alternative to daily gavage for single-dose bolus administration. Infection, as assessed by bacterial counts, was reduced only in animals that received 50 mg of rifabutin per kg. In these animals, bacterial counts in the liver and spleen were reduced from 7.2 x 10(5) +/- 4.1 x 10(4) and 6.5 x 10(5) +/- 4.1 x 10(4) to 3.0 x 10(3) +/- 1.8 x 10(2) and 3.1 x 10(3) +/- 2.2 x 10(2), respectively, over the 6-month treatment period. Rifabutin may be an appropriate chemotherapeutic drug for long-term treatment of M. paratuberculosis infection and should be considered in any multidrug regimen.

In a previous investigation, we obtained evidence that the major histocompatibility complex (MHC)-restricted proliferative response of CD4+ lymphocytes to Mycobacterium paratuberculosis antigens was depressed in naturally infected and immunized animals. Findings suggested that depression of the response was attributable to an abrogation in the ability of CD4+ cells to respond to specific antigens and/or the actual loss of antigen-reactive cells. In vitro cell experiments indicated that the depression was associated with the presence of gamma/delta+ T cells that modulated CD4+ cell function. Examination of additional animals confirmed and extended these observations and showed that the ability of gamma/delta+ T cells to regulate CD4+ responses were blocked by the presence of CD8+ cells. CD4+ T cells from some exposed animals incorporated [3H]-thymidine in the presence of CD8+, gamma/delta+ cells and/or antigen and antigen-presenting cells, but CD4+ cell proliferation was abrogated when CD8+ were excluded from the assays. Likewise, gamma/delta+ T-cell proliferation was abrogated when CD8+ cells were present. The mechanism by which CD8+ cells blocked gamma/delta+ T-cell responses could not be determined, however, the observed effect resembled the veto cell phenomenon. The data suggest that the development of protective immunity against M. paratuberculosis may be dependent on the capacity of CD8+ cells to modulate the regulatory activity of gamma/delta+ T populations.

Although sarcoidosis has clinical and histopathologic similarities to some forms of tuberculosis and other mycobacterial infections, attempts to establish a mycobacterial etiology have not been successful. Using cytoplasmic antigens derived from a wild strain of Mycobacterium paratuberculosis in an enzyme-linked immunosorbent assay, patients with sarcoidosis were found to have immunoglobulin levels significantly higher than those found in a control population in the IgG, but not in IgA or IgM antibody classes. Results were comparable to those reported from patients with Crohn's disease, where M. paratuberculosis has been intensively studied as a possible etiologic agent. To elucidate these relationships, examination of DNA from sarcoid tissues for possible homology with DNA from M. paratuberculosis and closely related organisms, as well as cultural attempts with techniques and media appropriate for M. paratuberculosis may be warranted.

Peripheral blood T-cell subsets were obtained from an experimentally sensitized bovine and from nine bovines naturally infected with Mycobacterium paratuberculosis and tested for their ability to respond to antigen. It was determined that following antigen challenge, proliferative responses followed a biphasic pattern: an initial CD4+ proliferative response, a period of anergy, and a final response governed by gamma/delta T lymphocytes. The anergic phase was characterized by a dramatic drop in peripheral blood CD4+ cells; the nature of the non-responsiveness could not be determined. The anergic phase was followed by increased proliferative responses of non-MHC restricted gamma/delta T lymphocytes. Although CD4+ cells had the ability to proliferate in response to M. paratuberculosis antigens in the absence of gamma/delta T cells, antigen-primed CD4+ lymphocytes failed to incorporate [3H]-thymidine in the presence of gamma/delta T cells and M. paratuberculosis antigen. It was concluded that M. paratuberculosis-specific gamma/delta T lymphocytes have immunoregulatory function and exhibit cytotoxic activity against antigen-primed CD4+ helper cells. The data suggest that the inability of effector cell populations to prevent intracellular proliferation of M. paratuberculosis may be a result of cytotoxic killing of the T helper lymphocyte population required for macrophage activation.

The inhibitory and bactericidal, synergistic, and antagonistic activities of rifabutin combined with ciprofloxacin, ethambutol, clofazimine, cefazolin, and amikacin in dual and triple combinations against various human and animal isolates of Mycobacterium paratuberculosis were determined. Synergism was observed when rifabutin was combined with either cefazolin or clofazimine in double combinations. The greatest amount of synergy occurred with the rifabutin-cefazolin combination in which bactericidal synergism was present with all strains. Of the triple combinations examined, only rifabutin in combination with ethambutol and cefazolin or streptomycin and cefazolin showed bactericidal synergism against most of the strains. Although antagonism was not observed in any double combination with rifabutin, antagonism was shown with several of the triple combinations. The rifabutin-cefazolin and rifabutin-streptomycin-cefazolin combinations were found to have the greatest bactericidal synergism at concentrations well within achievable serum and tissue levels and may be appropriate choices for chemotherapeutic use.

Nineteen Mycobacterium paratuberculosis strains, including strains of bovine, caprine, ovine, cervid, subhuman primate, and human origins, were compared with organisms of the M. avium complex by restriction fragment length polymorphism with a 5S rRNA gene probe as the reference DNA. Mycobacterial DNA was extracted, digested with several restriction enzymes, subjected to electrophoresis and Southern blotting, and then hybridized with a 5S rRNA gene probe from Escherichia coli. Hybridizing bands were visualized by autoradiography, and the sizes of the resulting rRNA fragments in kilobases were determined. Base substitutions were calculated on the basis of the number of shared fragments between species and strains. It was determined that M. paratuberculosis and the M. avium complex possess a single copy of the rRNA genes within their genomes and that the M. avium complex and M. paratuberculosis are a group of closely related organisms, likely with a common ancestral link. In proximity to the 5S rRNA gene exists a region or regions which display polymorphisms that are capable of species and subspecies differentiation. M. paratuberculosis strains isolated from humans, subhuman primates, and animals were found to be genetically identical to each other. M. paratuberculosis strains lacked the genetic heterogeneity (restriction fragment length polymorphisms) characteristic of most species, suggesting that this organism has unidirectional genetic selection. It is therefore assumed to be biologically isolated, occupying a unique and specific biological niche. This homogeneity was present in all strains, including those of animal and primate (subhuman and human) origin and strains isolated from different parts of the world.

The MICs and MBCs of various antimicrobial agents for strains of Mycobacterium paratuberculosis isolated from animal and human sources were evaluated. The MICs and MBCs of rifabutin, ciprofloxacin, ethambutol, clofazimine, streptomycin, cefazolin, and amikacin were found to be well below therapeutic levels in serum and tissue.

Transovarian passage of salmonella was evaluated in snakes by cesarean delivery and subsequent bacteriological examination of fetuses. In all cases, the same Salmonella serotype was isolated from the feces of gravid females and their fetuses. The visceral distribution of salmonella in normal snakes was found to involve almost all visceral organs. Of nonenteric organs examined, salmonella was recovered most often from the livers and ureters. Experimental infections with Salmonella typhimurium and Salmonella arizonae were established by oral, intracardial, and intracoelomic routes. Animals infected orally shed the organism in feces, but did not develop humoral antibodies or any detectable adverse effect. Animals injected by the intracardiac and intracoelomic routes developed antibody titers of 1:256 to the respective salmonella serotypes, but remained normal throughout the experiment. On the basis of results, salmonella was regarded as an opportunistic organism in reptiles.

Pilot studies were done to assess the pathogenicity of a Mycobacterium which had been recovered from the diseased ileum of a patient with Crohn's disease. In four separate studies, pairs of infant goats served as subjects. One of each pair received an oral inoculum of freshly harvested Mycobacterium species strain Linda suspended in cream. A littermate or stablemate which received only cream served as control. Necropsies were done at three, five, six, and 10 months postinoculation. Each of the four inoculated animals developed segmental granulomatous disease of the ileum or ileum and more proximal segments of small intestine, and regional lymph nodes. The earliest lesion occurred in Peyer's patches of the ileum and consisted of granulomatous clusters of epithelioid cells and giant cells, without caseation, which often occurred in a mantle of lymphocytes between the germinal centers and the muscularis mucosae. Nine of 10 such granulomas were free of acid-fast bacilli. In more advanced lesions, there was confluence of granulomas and ulceration of the mucosal surface. Two of the four inoculated animals also had lymphocytic lymphangitis in affected segments. Although the Mycobacterium Linda was recovered from intestinal segments of all four animals, acid-fast bacteria were not demonstrable in the intestines in two of them. Control animals remained free of lesions and acid-fast bacilli and were negative by bacteriologic culture. The Mycobacterium species strain Linda represents an enteric pathogen capable of inducing granulomas of the distal small intestine of susceptible species. The lesions produced have distinct similarities to those occurring in Crohn's disease.

Crohn's disease is a chronic granulomatous ileocolitis, of unknown etiology, which generally affects the patient during the prime of life. Medical treatment is supportive at best, and patients afflicted with this disorder generally live with chronic pain, in and out of hospitals, throughout their lives. The disease bears the name of the investigator who convincingly distinguished this disease from intestinal tuberculosis in 1932. This distinction was not universally accepted, and the notion of a mycobacterial etiology has never been fully dismissed. Nevertheless, it was 46 years after the distinction of Crohn's disease and intestinal tuberculosis before research attempting to reassociate mycobacteria and Crohn's disease was published. Recently, there has been a surge of interest in the possible association of mycobacteria and Crohn's disease due largely to the isolation of genetically identical pathogenic Mycobacterium paratuberculosis from several patients with Crohn's disease in the United States, the Netherlands, Australia, and France. These pathogenic organisms have been isolated from only a few patients, and direct evidence for their involvement in the disease process is not clear; however, M. paratuberculosis is an obligate intracellular organism and strict pathogen, which strongly suggests some etiologic role. Immunologic evidence of a mycobacterial etiology, as assessed by humoral immune determinations, has been conflicting, but evaluation of the more relevant cellular immunity has not been performed. Data from histochemical searches for mycobacteria in Crohn's disease tissues have been equally conflicting, with acid-fast bacilli detected in 0 to 35% of patients. Animal model studies have demonstrated the pathogenic potential of isolates as well as elucidated the complexity of mycobacterial-intestinal interactions. Treatment of Crohn's disease patients with antimycobacterial agent has not been fully assessed, although case reports suggest efficacy. The similarities in the pathology, epidemiology, and chemotherapy of Crohn's disease and the mycobacterioses are discussed. The issue is fraught with controversy, and the data generated on the association of mycobacteria and Crohn's disease are in their infantile stages so that a general conclusion on the legitimacy of this association cannot be made. While no firm evidence clearly implicates mycobacteria as an etiologic agent of Crohn's disease, the notion is supported by suggestive and circumstantial evidence and a remarkable similarity of Crohn's disease to known mycobacterial diseases.

The in vitro susceptibility of three strains of an unclassified Mycobacterium sp., isolated from three patients with Crohn's disease, to 23 antimicrobial agents was determined by a modified broth dilution method with 7H9 broth containing oleic acid-albumin-dextrose-catalase, Tween 80, and mycobactin J. All three strains were susceptible to streptomycin, viomycin, rifampin, clofazimine, cefazolin, amikacin, and kanamycin and resistant to p-aminosalicylic acid, cycloserine, 2-thiophenecarboxylic acid hydrazide, trimethoprim, diaminodiphenylsulfone, sulfamethoxazole, sulfadimethoxine, polymyxin B, metronidazole, neomycin, and carbenicillin. Variable results between strains were encountered with ethambutol, ethionamide, capreomycin, amoxicillin, and cephalothin.

An unclassified Mycobacterium species has been isolated from two patients with Crohn's disease (CD). Antibodies to the unclassified mycobacteria cross-reacted with Mycobacterium paratuberculosis. Because of this cross-reactivity, an enzyme-linked immunosorbent assay (ELISA) was used to examine the sera of inflammatory bowel disease (IBD) patients, both CD (N = 56), and ulcerative colitis (UC) (N = 34), for antibodies to M. paratuberculosis, Mycobacterium kansasii, and Mycobacterium tuberculosis. Controls consisted of healthy, PPD-negative individuals (N = 67), and from PPD-positive patients (N = 41). Eighteen resected CD patients were also examined. CD patients had a statistically significant increase in antibody titer (P = 0.0003) to M. paratuberculosis compared to healthy controls. Although patients with positive PPD had elevated titers to this organism, the positive response of CD patients was not related to PPD responsiveness, area of involvement in the gut, nor to activity of the disease process.

The prevalence of bovine paratuberculosis in New England was determined to be 18% based on a random survey of abattoir cattle. Six tissues from each of 100 animals were examined by histologic and bacteriologic methods. The cecal lymph node and the ileocecal valve yielded positive cultures most frequently, but isolations were also made from liver, tonsil, colon, and ileum. On the basis of the prevalence data, the economic significance of paratuberculosis to the New England dairy industry was estimated in excess of +15.4 million annually. Field studies identified 25 infected herds in New England. One of the infected herds from Connecticut was sold at public auction, an act which resulted in the dissemination of 95 potentially infected cattle to 28 farms in 8 different states.

Mycobacterium paratuberculosis infection was documented in a colony of stumptail macaque monkeys (Macaca arctoides), with 29 (76%) of 38 monkeys infected and shedding organisms in feces. Thirteen deaths have occurred during the past five years. Animals without overt clinical disease were shedding as many as 2 X 10(6) colony-forming units of M. paratuberculosis/g of feces. Intestinal tissues from animals dying of this disease contained up to 10(8) colony-forming units of M. paratuberculosis/g of tissue. The clinical and pathological features of paratuberculosis in this species were comparable to those reported for paratuberculosis in ruminants and Mycobacterium avium infections in primates. By enzyme-linked immunosorbent assay, antibodies to M. paratuberculosis were found in 79%-84% of the animals. Antibodies could not be detected in six animals with clinical disease. These findings extend the natural host range of M. paratuberculosis to include nonhuman primates and add support to current suggestions that M. paratuberculosis may be pathogenic for humans.

The genome size of an unclassified Mycobacterium species, isolated from a patient with Crohn's disease, was determined by measurement of DNA renaturation kinetics as 3.1 X 10(9) Da. The percentage of DNA homology of this organism to DNA of related mycobacteria was evaluated by measurement of DNA renaturation with heterologous DNA. These studies supported the classification of this organism as Mycobacterium paratuberculosis but failed to distinguish between M. paratuberculosis and organisms of the Mycobacterium avium-intracellulare groups.

A previously unrecognized Mycobacterium species was isolated from two patients with Crohn's disease. The organism is an acid-fast, mycobactin-dependent Mycobacterium that has characteristics which do not conform to any of the presently recognized species. It belongs to the Runyon group III mycobacteria and is most closely related to Mycobacterium paratuberculosis. Animal inoculation revealed pathogenicity for mice when injected intravenously or intraperitoneally, but not for rats, guinea pigs, rabbits, or chickens. The mice developed hepatic and splenic granulomas which contained numerous acid-fast mycobacteria. A 7-day-old goat which was inoculated orally with 50 mg of the organism developed both humoral and cell-mediated immunologic responses in two to three weeks and granulomatous disease of the distal small intestine, with noncaseating tuberculoid granulomas in five months. Acid-fast bacilli were not demonstrable in sections of the intestine, but a single organism was seen in each of two microgranulomas of the mesenteric lymph node. The Mycobacterium species was reisolated from the lymph node but not from intestine. Our findings raise the possibility that a Mycobacterium plays an etiologic role in at least some cases of Crohn's disease.

Ten white-tailed deer were shot and killed at a Connecticut farm that had a 6-year history of bovine paratuberculosis, and organs from these animals were examined for evidence of paratuberculosis. Mycobacterium paratuberculosis was isolated from the cecal lymph node, terminal ileum, and ileocecal valve of 1 deer and from the cecal lymph node of another. Characteristic lesions and acid-fast bacilli were not observed. It was concluded that infected deer could serve as sources of infection for domestic stock.

Necrotic hepatitis resembling black disease of ruminants is described in a group of five water snakes (Natrix sipedon pictiventirs). Lesions varied from multifocal granulomas to massive coagulation necrosis. A bacterium recovered from the livers could not be classified, but closely resembled Eubacterium tarantellus. The bacterium was isolated from all snake livers and from snake mites (Ophionyssus natricis) which were probably implicated in the transmission of the disease and it is possible that trematodes were concerned in producing the initial damage to the liver.

The characteristics of an unclassified Mycobacterium sp. isolated from three patients with Crohn's disease are presented. The organism is extremely fastidious and mycobactin dependent and may require up to 18 months of incubation for primary isolation. Colony morphology is rough. Characteristics are unlike those of any presently defined species. The isolates produced postive niacin, catalase, and 2-week arylsulfatase reactions and were susceptible to neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), and rifampin (0.25 micrograms/ml). Chromogenicity, nitrate reduction, quantitative catalase, Tween hydrolysis, urease, tellurite reduction, pyrazinamidase, and 3-day arylsulfatase tests were negative, and the isolates were resistant to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml) and isoniazid (10 micrograms/ml). Optimum growth in broth was determined to be in 7H9 medium with Dubos oleic albumin complex, Tween 80, and mycobactin J at 37 degrees C without CO2 or agitation and in low medium depth. This Mycobacterium sp. may be a subspecies or biovariant of Mycobacterium paratuberculosis, or it may represent a new species of Mycobacterium. It is suggested that this Mycobacterium sp. may play an etiological role in some cases of Crohn's disease.

The cellular fatty acid composition of Mycobacterium paratuberculosis from bovine, ovine, and caprine origin (grown on Herrold's egg yolk medium) was determined by gas-liquid chromatography (GLC) and mass spectrometry and was compared with that of 19 other Mycobacterium spp. A species-specific fatty acid of M paratuberculosis was not demonstrable. Gas-liquid chromatographic analysis of cellular fatty acids was used to develop flow charts for the rapid identification of M paratuberculosis and other Mycobacterium spp. Two charts for distinguishing organisms were developed; one chart was based on GLC alone and the other based on GLC, growth rate, and chromogenicity. Seemingly, the GLC was reliable for detecting Mycobacterium spp rapidly.

Biochemical activities of 20 wild-type strains and of 2 laboratory strains of Mycobacterium paratuberculosis were evaluated. Biochemical activities evaluated were growth at 30 C, 37 C, and 42 C; production of urease, niacin, pyrazinamidase, arylsulfatase, and catalase; hydrolyzation of Tween 80; reduction of nitrate and tellurite; and growth in 5% NaCl. Antimicrobial susceptibility to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml), neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), rifampin (0.25 micrograms/ml), and isoniazid (10 micrograms/ml) also was determined. Generally, M paratuberculosis was biochemically inactive, with only a few strains producing pyrazinamidase and maintaining catalase activity after heating. All strains grew optimally at 37 C, grew slightly at 30 C, and did not grow at 42 C. Wild-type strains did not grow in the presence of neotetrazolium chloride, streptomycin, and rifampin, and grew in the presence of thiophene-2-carboxylic acid hydrazide and isoniazid. Although biochemical evaluation can be used as an aid in the identification of M paratuberculosis, growth rate, and mycobactin dependency remain major criteria for positive identification.

Cutaneous fibromas were successfully transmitted to 7 white-tailed deer (Odocoileus virginianus) inoculated with crude fibroma extracts (2 deer) or with partially purified deer fibroma virus (5 deer). The fibromas were transmitted by intradermal and subcutaneous inoculation and by rubbing the virus preparation into tattoo sites. Inoculation by scarification was not successful. The induced tumors resembled those of naturally occurring fibromas. Tattoo inoculation sites underwent an initial acute inflammatory response followed by mesenchymal proliferation, perivascular lymphocytic infiltration, and finally regression. The deer developed antibody titers against deer fibroma virus as determined by hemagglutination inhibition, using mouse RBC. Viral antigens could not be detected by indirect immunofluorescence in any induced fibroma.

Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.