Susceptibility of Balb/c, C57/B6 and C57/B10 mice to infection with Mycobacterium paratuberculosis.

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.

Infectious necrotic hepatitis caused by an unclassified anaerobic bacillus in the water snake.

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.

Biochemical characteristics of various strains of Mycobacterium paratuberculosis.

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.

Characterization of Mycobacterium paratuberculosis of bovine, caprine, and ovine origin by gas-liquid chromatographic analysis of fatty acids in whole-cell extracts.

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.

Transmission of the white-tailed deer cutaneous fibroma.

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.

Eastern white-tailed deer as a reservoir of ruminant paratuberculosis.

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.

Immunology: resistance to paratuberculosis.

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.

Paratuberculosis: a potential zoonosis?

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.

Antimicrobial activity of rifabutin in combination with two and three other antimicrobial agents against strains of Mycobacterium paratuberculosis.

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.

Transovarian passage, visceral distribution, and pathogenicity of salmonella in snakes.

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.

Characterization of Mycobacterium paratuberculosis and organisms of the Mycobacterium avium complex by restriction polymorphism of the rRNA gene region.

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.

Bactericidal activities of various antimicrobial agents against human and animal isolates of Mycobacterium paratuberculosis.

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.

Crohn's disease and the mycobacterioses: a review and comparison of two disease entities.

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.

Serologic reactivity against Mycobacterium paratuberculosis antigens in patients with sarcoidosis.

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.

The cellular immunology of bovine paratuberculosis: immunity may be regulated by CD4+ helper and CD8+ immunoregulatory T lymphocytes which down-regulate gamma/delta+ T-cell cytotoxicity.

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.

The cellular immunology of bovine paratuberculosis: the predominant response is mediated by cytotoxic gamma/delta T lymphocytes which prevent CD4+ activity.

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.