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PubMed1992

Activity of clarithromycin compared with those of other drugs against Mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol.

Radiometric MICs of clarithromycin, a new macrolide drug, were determined against five mycobactin-dependent strains of Mycobacterium paratuberculosis (including two Crohn's disease clinical isolates) and compared with those of other drugs which included rifampin, ethambutol, amikacin, ofloxacin, ciprofloxacin, and sparfloxacin. Among the drugs screened, clarithromycin was the drug for which MICs were lowest against the five strains tested. As MICs were significantly below the reported Cmax levels (about 4 micrograms/ml), the intracellular activity of clarithromycin against the type strain of M. paratuberculosis maintained in cultured macrophages was screened. Clarithromycin was able to kill the initial inoculum by more than 1 log within 7 days, and this activity was further potentiated by ethambutol. Extracellular drug combination screened by using sublethal concentrations of the drugs showed that ethambutol was able to enhance clarithromycin activity in three out of four M. paratuberculosis strains instead of only one out of four strains (or none in the case of ofloxacin) when associated with other drugs. These results suggest that clarithromycin may be fruitful to treat human disease in which M. paratuberculosis may be etiologically involved.

Animals↗
PubMed1987

Isolation and analysis of restriction endonuclease digestive patterns of chromosomal DNA from Mycobacterium paratuberculosis and other Mycobacterium species.

A relatively rapid and efficient method for the extraction of chromosomal DNA from Mycobacterium paratuberculosis and other mycobacteria was developed. Approximately 25 to 50 micrograms of DNA could be extracted from 100 mg (wet weight) of cells, which was sufficient to perform several restriction endonuclease analyses from a single preparation. The DNA from five Mycobacterium species, including four strains of M. paratuberculosis and four strains of M. avium, was analyzed by this method. Digestion with the restriction endonucleases BstEII and PstI yielded the most definitive restriction patterns. For some strains, the restriction endonuclease analysis results were in agreement with the current identification of these organisms. The two strains of M. avium serotype 2 had identical fragment patterns. Similarly, the two strains of M. avium complex serotype 6 had identical fragment patterns. The three mycobactin-dependent M. paratuberculosis strains were very similar, whereas the mycobactin-independent M. paratuberculosis strain was more similar to the M. avium serotype 2 strains. Although many more cultures would need to be evaluated to determine correct groupings, the results of this study demonstrated the potential of restriction enzyme analysis for the differentiation of slowly growing mycobacteria.

Animals↗
PubMed1989

DNA polymorphism in Mycobacterium paratuberculosis, "wood pigeon mycobacteria," and related mycobacteria analyzed by field inversion gel electrophoresis.

Mycobacterium paratuberculosis strains, mycobacteria from patients suffering from Crohn's disease, "wood pigeon mycobacteria," and representatives of Mycobacterium avium-Mycobacterium intracellulare were compared by restriction endonuclease DraI digestion and field inversion gel electrophoresis. Characteristic profiles were seen for M. paratuberculosis, including isolates from patients suffering from Crohn's disease, for wood pigeon mycobacteria, and for M. avium-M. intracellulare serotypes 2, 16, 18, and 19. Two M. paratuberculosis strains used for vaccine production (St 18 and 316 F) presented patterns different from those of the other M. paratuberculosis strains. Strains St 18 yielded a pattern identical to that of the M. avium type strain serotype 2, whereas 316 F gave a unique pattern. The method developed in this study represents a useful taxonomic tool for the identification and classification of mycobacteria.

Animals↗
PubMed1990

Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens.

A commercial radiometric medium, BACTEC 12B, was modified by addition of mycobactin, egg yolk suspension, and antibiotics (vancomycin, amphotericin B, and nalidixic acid). Decontaminated bovine fecal specimens were filter concentrated by using 3-microns-pore-size, 13-mm-diameter polycarbonate filters, and the entire filter was placed into the radiometric broth. Comparison of the radiometric technique with conventional methods on 603 cattle from 9 Mycobacterium paratuberculosis-infected herds found that of 75 positive specimens, the radiometric technique detected 92% while conventional methods detected 60% (P less than 0.0005). Only 3.9% of radiometric cultures were contaminated. To measure the effect of filter concentration of specimens on the detection rate, 5 cattle with minimal and 5 with moderate ileum histopathology were sampled weekly for 3 weeks. M. paratuberculosis was detected in 33.3% of nonfiltered specimens and 76.7% of filtered specimens (P less than 0.005). Detection rates were directly correlated with the severity of disease, and the advantage of specimen concentration was greatest on fecal specimens from cattle with low-grade infections. Detection times were also correlated with infection severity: 13.4 +/- 5.9 days with smear-positive specimens, 27.9 +/- 8.7 days with feces from cows with typical subclinical infections, and 38.7 +/- 3.8 days with fecal specimens from cows with low-grade infections. Use of a cocktail of vancomycin, amphotericin B, and nalidixic acid for selective suppression of nonmycobacterial contaminants was better than the commercial product PANTA (Becton Dickinson Microbiologic Systems, Towson, Md.) only when specimens contained very low numbers of M. paratuberculosis. Radiometric culture of filter-concentrated specimens generally doubled the number of positive fecal specimens detected over conventional methods, making it a useful tool for diagnosis and control of bovine paratuberculosis.

Animals↗
PubMed1990

Identification of restriction fragment length polymorphisms in DNA from Mycobacterium paratuberculosis.

DNAs from 34 mycobactin-dependent isolates of Mycobacterium paratuberculosis and 1 isolate of M. paratuberculosis 18 were digested with four restriction endonucleases. Southern hybridization experiments were performed with a 32P-labeled oligonucleotide DNA probe derived from the sequence of IS900, an insertion sequence present in 15 to 20 copies per M. paratuberculosis chromosome. The probe hybridized with DNA from each of the mycobactin-dependent isolates, and restriction fragment length polymorphisms were detected among the isolates with each restriction endonuclease used. Restriction fragment length polymorphism analysis may permit identification of various strains of M. paratuberculosis, which has not been possible with other techniques.

Animals↗
PubMed1990

Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease.

DNA probes that hybridize to a mycobacterial insertion sequence, IS900, present in multiple copies in the genome of Mycobacterium paratuberculosis were found to be highly specific for M. paratuberculosis. DNA sequences derived from IS900 were used to prepare DNA primers for detection and identification of M. paratuberculosis by the polymerase chain reaction. Highly specific direct detection of M. paratuberculosis DNA in feces from cattle with Johne's disease was obtained. The polymerase chain reaction test had a sensitivity equal to or greater than that obtained by standard culture techniques and was much more rapid, taking only hours compared with 6 to 12 weeks for culture.

Animals↗
PubMed1991

Mycobacterium paratuberculosis and Crohn's disease.

The possible aetiological role of Mycobacterium paratuberculosis in Crohn's disease was investigated. The immunological response was studied using an enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunocytochemistry. The antibody response to two protoplasmic antigen preparations of M paratuberculosis in the sera of patients with inflammatory bowel disease was measured by ELISA. IgG and IgM antibodies to these antigens were measured in serum samples from 52 patients with Crohn's disease, 15 patients with ulcerative colitis, and 41 control patients without inflammatory bowel disease. Although there was wide variation in the concentrations of antibody detected, patients with Crohn's disease had concentrations that were not significantly different from those of the other two groups. In addition, mycobacterial antigens were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and the immune response to each antigen was then examined separately and assayed for IgG and IgM in 10 patients from each of the three groups. An indirect peroxidase test was also used to detect M paratuberculosis in sections of tissue from 18 patients with Crohn's disease and 10 with ulcerative colitis. The results were negative in all cases. This study does not support a role for M paratuberculosis in Crohn's disease.

Adolescent↗
PubMed1992

Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues.

Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohn's disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohn's disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohn's disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.

Base Sequence↗
PubMed1985

Control of paratuberculosis (Johne's disease) in goats by vaccination.

After several years of unsuccessful efforts to eradicate paratuberculosis in goats in Norway by conventional methods such as general hygienic precautions and the isolation and slaughtering of clinically affected and serologically positive animals, a vaccination programme was initiated in 1967. The vaccine used consists of two live attenuated strains of Mycobacterium paratuberculosis suspended in a mixture of liquid paraffin, olive oil and pumice powder. The vaccine may be stored at 4 degrees C for two weeks, the dose is 1 ml and the goat kids are vaccinated at the age of two to four weeks. The efficacy of the vaccine has been judged mainly by post mortem examination of vaccinated and unvaccinated goats in the period 1967-82. During this period about 131,000 goats were vaccinated and, based on the post mortem examination of 15,219 goats, the infection rate was reduced from 53 to 1 per cent. Moreover, infection occurred almost exclusively in goats which for some reason or other had not been vaccinated or which had been too old when vaccinated. The results of these examinations showed that the adjuvanted vaccine with live M paratuberculosis bacteria offers a high degree of protection against paratuberculosis in goats.

Animals↗
PubMed1990

Some immune responses in cattle exposed to Mycobacterium paratuberculosis after injection with modified-live bovine viral diarrhea virus vaccine.

Delayed-type hypersensitivity responses to Mycobacterium paratuberculosis purified protein derivative (PPD) were decreased in cows experimentally exposed to M. paratuberculosis 7 days after exposure to a modified-live bovine viral diarrhea virus (ML-BVDV) vaccine. In vitro lymphocyte blastogenic responses to phytohemagglutinin were decreased in each of 3 cows 7 days after exposure to ML-BVDV vaccine. Also, decreased lymphocyte blastogenic responses to M. paratuberculosis PPD were observed in cultures of 2 of 3 cows 7 days after exposure to ML-BVDV vaccine. No significant differences in enzyme-linked immunosorbent assay reactions were detected in sera of M. paratuberculosis-infected cattle collected before and at 4 and 12 weeks after exposure to ML-BVDV vaccine.

Animals↗
PubMed2025

The Results After One Year of an Experimental Protocol Aimed at Reducing Paratuberculosis in an Intensive Dairy Herd.

Paratuberculosis or Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease is characterized by a chronic and incurable enteritis in ruminants and it is responsible for significant economic losses, also raising concerns about food safety and animal welfare. Effective control is hindered by diagnostic limitations, long incubation periods, and the environmental resistance of the pathogen. This study aimed to reduce the apparent prevalence of paratuberculosis in a single intensive dairy herd through an integrated approach that combines diagnostics and management strategies. All cows over 24 months of age were tested using both fecal PCR and ELISA serology. Digital PCR (dPCR) was used to quantify MAP shedding in fecal-positive animals, enabling prioritization for removal based on environmental contamination risk. Integrating diagnostic tools allowed the precise identification and quantification of high-risk animals. Meanwhile, structural improvements and biosecurity measures were implemented on the farm. Preliminary outcomes suggest a marked reduction in herd-level MAP prevalence, lowering the seroprevalence from 7.6% to 4.5% and the fecal PCR prevalence from 6.5% to 2.8%. This case highlights the effectiveness of combining laboratory testing (serology and molecular diagnostics) and targeted changes in farm management to control paratuberculosis in high-density dairy systems.

MAP↗
PubMed1979

Paratuberculosis (Johne's disease) in bighorn sheep and a Rocky Mountain goat in Colorado.

Between May, 1972 and February, 1978, six cases of paratuberculosis (Johne's Disease) caused by Mycobacterium paratuberculosis were diagnosed in free-ranging Rocky Mountain bighorn sheep (Ovis canadensis) and one Rocky Mountain goat (Oreamnos americanus) on or near Mt. Evans in Colorado. Diagnosis of paratuberculosis was based on gross and histopathologic examination of the animals and by isolation of M. paratuberculosis from three sheep and the goat. The clinical signs and pathologic changes seen in the bighorn sheep resembled those described in cattle, while the lesions in the goat were similar to those described for domestic sheep and goats.

Animals↗
PubMed1975

Effect of aflatoxin on susceptibility of hamsters to Mycobacterium paratuberculosis.

After oral administration of Mycobacterium paratuberculosis to hamsters, the organism passed the epithelial barrier of the intestine, and infection was established in the small intestine and mesenteric lymph nodes. The addition of aflatoxin to the ration of hamsters did not increase their susceptibility to M paratuberculosis but, rather, seemed to decrease susceptibility to the bacillus. Hamsters not treated with aflatoxin and infected with M paratuberculosis had higher bacterial counts in intestinal tract and mesenteric lymph node on necropsy than did infected hamsters that had been treated with aflatoxin. Aflatoxin-treated hamsters grew slowly, had an unthrifty appearance, and developed lesions of megalocytosis regardless of whether they were infected with M paratuberculosis.

Administration, Oral↗
PubMed1991

Serodiagnosis of bovine paratuberculosis by use of a dot enzyme-linked immunosorbent assay.

A dot ELISA was developed for detection of antibodies to Mycobacterium paratuberculosis. The assay was evaluated by testing sera from cattle that were determined, by bacteriologic culturing of feces, to be infected with M paratuberculosis and were suspected of having clinical disease. Further evaluation involved testing sera from cattle in which M paratuberculosis had not been isolated from feces on several attempts. Results of the dot ELISA were positive for sera from 86 of 101 infected cattle, and results were negative for sera from 64 of 64 noninfected cattle. Results of conventional ELISA and agar gel immunodiffusion (AGID) tests were positive for 79 of 99 and for 51 of 101 infected cattle, respectively. The dot ELISA also was evaluated by comparing results of testing 708 sera with results of bacteriologic culturing of matched fecal samples from 262 cattle in 3 central Ohio dairy herds known to include cattle infected with M paratuberculosis. Results of the dot ELISA were positive for 25 of 39 sera from cattle with positive results on culturing of concurrently obtained fecal specimens. The dot ELISA results were negative for 661 of 669 sera from cattle with negative results to culturing of concurrently obtained fecal specimens. The 39 sera from cattle with positive results on bacteriologic culturing of matched fecal specimens had positive results for ELISA and the AGID test 25 and 14 times, respectively. The 669 sera from cattle with concurrently negative results on bacteriologic culturing of feces had negative results to ELISA and the AGID test 559 and 668 times, respectively.

Animals↗
PubMed1990

Probable paratuberculosis in a Sicilian ass.

A presumptive diagnosis of paratuberculosis was made in a Sicilian ass on the basis of a history of chronic diarrhea and weight loss, pasture exposure to a heifer with paratuberculosis confirmed by bacterial culture of feces, postmortem identification of granulomatous inflammation of the intestine containing acid-fast organisms, the absence of acid-fast organisms in extraenteric tissues, and the absence of exposure to tuberculosis. The literature on paratuberculosis in equids is reviewed. The potential for cross-species transmission is emphasized. Justification for consideration of Mycobacterium paratuberculosis infection in the differential diagnoses of equine granulomatous enteritis is discussed.

Animals↗
PubMed1990

Granulomatous enteritis following oral inoculation of newborn rabbits with Mycobacterium paratuberculosis of bovine origin.

To assess the rabbit as a model for the study of paratuberculosis infection, two groups of newborn rabbits were orally inoculated at one to two days of age with Mycobacterium paratuberculosis (ATCC 19698 or field strain 22206) and compared to uninoculated controls. Nine of thirteen rabbits (69%) inoculated with ATCC 19698, and all three rabbits inoculated with 22206, experienced episodes of intermittent diarrhea starting four months after inoculation. Multifocal granulomas containing acid-fast organisms were observed in the sacculus rotundus and vermiform appendix of the cecum in three of nine rabbits (all with diarrhea) that had been inoculated with ATCC 19698. Although M. paratuberculosis was not recovered from inoculated rabbits when fecal cultures were incubated three months in vitro, a slow-growing mycobactin-dependent form of Mycobacterium was recovered from feces and ileal tissue after incubation for 11-15 months. Reduced feed intake, body weight loss and reduced abdominal fat at necropsy, were not observed. Epithelial transport function across the distal ileum in vitro was not altered nine months subsequent to inoculation. Diarrhea and the histological lesions indicate that the rabbit may be a useful model for the study of paratuberculosis infection.

Animals↗
PubMed1989

Comparison of dot immunobinding assay, enzyme-linked immunosorbent assay and immunodiffusion for serodiagnosis of paratuberculosis.

A rapid, simple and inexpensive dot immunobinding assay (DIA) was evaluated for the serodiagnosis of paratuberculosis in cattle. The assay was performed on nitrocellulose strips which were dotted with purified protoplasmic antigen of Mycobacterium paratuberculosis. After incubation with test serum samples, the bound antibodies were detected using an enzyme-amplified immunostaining procedure. The efficacy of DIA as a screening test for paratuberculosis was compared to that of an enzyme-linked immunosorbent assay (ELISA), a modified agar gel immunodiffusion (mAGID) test, and an AGID test using 329 serum samples from cattle which were examined for M. paratuberculosis infection by a sensitive fecal culture technique. The DIA and ELISA had comparable results and both of the enzyme immunoassays had higher sensitivity than tests based on AGID. The sensitivity of all four tests was influenced by the intensity of fecal bacterial shedding. Preabsorption of sera with Mycobacterium phlei increased the sensitivity of both enzyme immunoassays. the specificity but reduced the sensitivity of both enzyme immunoassays.

Animals↗
PubMed1986

Restriction endonuclease analysis of various strains of Mycobacterium paratuberculosis isolated from cattle.

Deoxyribonucleic acid (DNA) preparations from 3 reference strains of Mycobacterium paratuberculosis and from 23 isolates of M paratuberculosis obtained from cattle in New Zealand were characterized by restriction endonuclease analysis, using the enzymes BstE II, Pvu II, and Bcl I. Patterns of DNA fragments for strain 18 (one of the reference strains) differed markedly from patterns of other strains, indicating genetic differences between strain 18 and the other strains of M paratuberculosis evaluated. The other 2 reference strains (TMC 1613 and Weybridge strain 316) and all but 1 of the isolates from cattle had identical patterns with the 3 enzymes. These 2 reference strains differed from each other in their dependence on exogenous mycobactin, but this was not reflected in their restriction patterns. The single variant isolate from cattle had patterns identical to those of the other isolates, using Pvu II and Bcl I, and had only 1 fragment line difference with BstE II. Although close genetic homogeneity of cattle strains of M paratuberculosis prevented development of a typing system on the basis of restriction endonuclease analysis, the results provided a basis for genomic comparison with other closely related organisms.

Animals↗