Prevalence of Mycobacterium paratuberculosis in ileocecal lymph nodes of cattle culled in the United States.

During 1983 and 1984, ileocecal lymph node specimens were obtained from clinically normal cattle at 76 US Department of Agriculture-inspected cull cattle slaughterhouses in 32 states and Puerto Rico. Mycobacterium paratuberculosis was isolated from the lymph nodes of 119 of 7,540 cattle. The prevalence of bovine paratuberculosis was 1.6% overall, with 2.9% in dairy culls and 0.8% in beef culls. The prevalence for females and males did not appear to differ significantly.

Investigation of association of mycobacteria with inflammatory bowel disease by nucleic acid hybridization.

We used DNA-DNA hybridization to characterize a mycobacterial isolate, strain Linda, that was obtained from a patient with Crohn's disease and that has been reported to cause ileitis in experimental animals. We also investigated the association of this mycobacterium with Crohn's disease. Our results identified Mycobacterium strain Linda as a strain of Mycobacterium paratuberculosis, the etiologic agent of Johne's disease, a disease of ruminants that has some superficial resemblance to Crohn's disease. Sequences that hybridized with strain Linda DNA probes were detected in DNA extracted from human intestinal tissues from patients with Crohn's disease, ulcerative colitis, and noninflammatory bowel disease. These hybridizing DNA sequences were more prevalent in the muscle layers than in the intestinal mucosa, making it unlikely that they represented DNA from bacterial contaminants in the intestinal lumen. Measurement of the melting temperatures of the DNA-DNA hybrids formed between strain Linda probes and tissue DNAs indicated that the related sequences detected were of mycobacterial origin but were not identical to each other or to strain Linda DNA. These results do not support the proposed specific relationship between Mycobacterium strain Linda and Crohn's disease. The possible etiologic role of mycobacteria in Crohn's disease is discussed.

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.

In vitro antimicrobial susceptibility of a Mycobacterium sp. isolated from patients with Crohn's disease.

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.

Possible role of mycobacteria in inflammatory bowel disease. II. Mycobacterial antibodies in Crohn's disease.

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.

Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified Mycobacterium species isolated from patients with Crohn's disease.

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.

Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease.

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.

Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.

Intrauterine inoculation of Mycobacterium paratuberculosis into guinea pigs and cattle.

Mycobacterium paratuberculosis was inoculated intrauterinely into guinea pigs and cattle near the time of insemination to assess the effect and subsequent distribution of the organisms. Guinea pigs inoculated intrauterinely with 1 of 3 concentrations of M paratuberculosis or not inoculated were caged with male guinea pigs for 10 days. Guinea pigs given the largest dose had the lowest rate of pregnancy when examined. At necropsy, tissues were cultured to determine extent of infection. Abortion followed hypersensitivity reactions to johnin in some highly infected animals. Mycobacterium paratuberculosis was recovered in culture from the body and horns of the uterus of cows necropsied 1, 2, 3, and 7 days after inoculation and from 1 of 3 cows necropsied 2 weeks after inoculation. The organisms were not detected in extrauterine organs of any cattle or in uterine specimens taken at 3 or 4 weeks after inoculation. The evidence indicates that the small numbers of M paratuberculosis sometimes found in semen from paratuberculous bulls probably would be destroyed, rather than leading to systemic infection of the dam or to persistent hypersensitivity.

Heat inactivation of in vivo- and in vitro-grown mycobacteria in meat products.

Heat inactivation of mycobacteria from lesions and from culture was compared in meat products. In vivo-grown organisms were more easily heat inactivated than were in vitro-grown organisms.

Inactivation of Mycobacterium bovis in meat products.

The time-temperature combinations necessary to destroy Mycobacterium bovis in meat products were determined. In any given time, M. bovis was destroyed at temperatures 6 to 7 degrees C (ca. 12 degrees F) lower than those necessary for destruction of members of the Mycobacterium avium-Mycobacterium intracellulare complex. Hence, any processing heat adequate to kill M. avium-M. intracellulare-complex organisms will provide a very large safety factor with respect to M. bovis. Benzalkonium chloride treatment of wiener specimens for cultural examination effectively destroyed the normal flora of wiener emulsion without reducing the numbers of M. bovis. Treatment with a phenolic disinfectant followed by formaldehyde vapor was effective in disinfecting equipment contaminated with meat emulsion containing M. bovis.

Heat inactivation of Mycobacterium avium-Mycobacterium intracellulare complex organisms in meat products.

Wieners and sausages were prepared which contained the most heat-tolerant representative of the Mycobacterium avium-Mycobacterium intracellulare complex we were able to obtain. They also were prepared with infected tissues obtained from tuberculous swine. Processing conditions were as varied as possible. Neither incorporation of sodium nitrite in the emulsion nor presence of smoke during processing altered the heat susceptibility of the organisms. Substantial killing of the organisms occurred as wieners reached the upper processing temperatures, but hot oil or radiant heating of the "precooked" sausages allowed very short times within the killing range; hence, higher peak internal temperatures were necessary. The lethalities for these organisms of reaching and maintaining various processing temperatures are given.

Heat inactivation of Mycobacterium avium-Mycobacterium intracellulare complex organisms in aqueous suspension.

Isolants from swine and from humans representing serotypes 1, 2, 4, 8, and 10 of the Mycobacterium avium-Mycobacterium intracellulare complex were compared for heat tolerance in aqueous suspension. The most heat-resistant isolant found was a serovar 10 isolated from a human. This isolant was examined further to determine the rate of kill at various temperatures and pH's, the effect of meat protein and fat, and the effect of nitrite. Kill rates were not significant at 60 degrees C or below. Decimal reduction values were 4 min or less at 65 degrees C and 1.5 min or less at 70 degrees C. Kill rates were slightly higher at pH values of 6.5 and 7.0 than at 5.5 or 6.0. the water-soluble fraction of wiener emulsion did not alter kill rates, but the saline-soluble fraction protected the organism somewhat. Fat did not affect the survival of the organisms except to eliminate the protective effect of saline extract when the suspension contained 50% fat. The addition of sodium nitrite to the suspension did not alter the heat sensitivity of the organisms.

Tripeptide hydroxamate from Corynebacterium kutscheri.

A tripeptide hydroxamate was isolated from cultures of Corynebacterium kutscheri grown on an iron-limiting medium. The metabolite was characterized by spectral measurements and by chemical degradation as L-alpha-aspartyl-L-alpha-N-hydroxy-aspartyl-D-cycloserine.

In vitro lymphocyte transformation as a herd survey method for bovine paratuberculosis.

The lymphocyte-transformation (LT) test was evaluated for its potential application as a field test for bovine paratuberculosis. Using a whole blood technique, samples from 3 consecutive collection periods were subjected to 3 mycobacterial antigens and to phytohemagglutinin. The results obtained from LT were compared with conventional serologic and cultural methods. A positive LT response to johnin purified-protein derivative (PPD) or avian PPD (or both) was noted in 40% to 60% of the animals tested. The complement-fixation test yielded 4% to 6.7% positive results, the immunodiffusion test between 1.2% and 1.4%, and the direct fecal culture between 2.4% and 6%. The mean of the stimulation indices of all positively responding animals was highest with johnin PPD. Specific stimulation to mammalian PPD occurred between 2.4% and 6% of the animals. The efficacy of the LT test for determining the incidence of infection with Mycobacterium paratuberculosis is discussed.