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.

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.

Use of rifabutin in treatment of systemic Mycobacterium paratuberculosis infection in mice.

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.

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.

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: 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.

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.

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.

Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides).

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.

Determination of genome size and DNA homology between an unclassified Mycobacterium species isolated from patients with Crohn's disease and other mycobacteria.

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.

Experimental disease in infant goats induced by a Mycobacterium isolated from a patient with Crohn's disease. A preliminary report.

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.

Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.

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.