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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↗
PubMed1993

Antibodies to Mycobacterium paratuberculosis and nine species of environmental mycobacteria in Crohn's disease and control subjects.

Cultural and serological studies have provided limited, often conflicting, evidence of a role for mycobacteria in the pathogenesis of Crohn's disease. Interest has focussed on Mycobacterium paratuberculosis, previously considered to be common in the environment with no major role as a human pathogen. Whether a specific serum antibody response to mycobacteria occurs in Crohn's disease or ulcerative colitis was investigated. Sera from patients with Crohn's disease (n = 38), ulcerative colitis (n = 15), and a healthy control population (n = 30) were assayed in an enzyme linked immunosorbent assay (ELISA) using eight filtered sonicate mycobacterial preparations and a purified protein derivative made from the bovine tubercle bacillus. In addition, IgG, IgM, and IgA levels to M paratuberculosis were determined in sera from patients with active (n = 24) or inactive (n = 29) Crohn's disease and the control populations. There was strong evidence of contact with environmental mycobacteria in all patients and control populations, with the greatest responses to preparations of M avium, M tuberculosis, and M kansasii. A large proportion of patients with Crohn's disease had antibodies that bound most antigens tested but there were no statistical differences between these values and those of the control population. Similarly, there were no differences in antibody levels to M paratuberculosis in patient and control groups. Although a subset of patients with active Crohn's disease (25%) had IgG concentrations that exceeded the control mean by more than 2 SD, this phenomenon may not be specific to Crohn's disease: 20% of a small group of patients with coeliac disease had similarly raised IgG levels to M paratuberculosis. These findings do not provide serological evidence of a role for this organism in the pathogenesis of Crohn's disease.

Antibodies, Bacterial↗
PubMed1997

Search for Mycobacterium paratuberculosis DNA in orofacial granulomatosis and oral Crohn's disease tissue by polymerase chain reaction.

BACKGROUND: Although intestinal Crohn's disease has long been suspected to have a mycobacterial cause, possible mycobacterial involvement in orofacial granulomatosis (OFG) and oral lesions of Crohn's disease has not yet been investigated. AIMS: As the slow growing Mycobacterium paratuberculosis has been implicated in the aetiology of intestinal Crohn's disease, the potential involvement of this mycobacterial species in OFG and oral lesions of Crohn's disease was investigated. PATIENTS: To attempt detection of the organism in OFG and oral Crohn's disease tissue samples, a polymerase chain reaction (PCR) assay was used on archival formalin fixed, paraffin wax embedded oral tissue sections from 30 patients with OFG, seven with Crohn's disease, and 12 normal controls. METHODS: The PCR assay used was based on primers targeting the 5' region of the multicopy IS900 DNA insertion element of the M paratuberculosis genome. In order to achieve maximum sensitivity, two rounds of PCR were carried out and amplicons confirmed by Southern blot hybridisation to a digoxigenin labelled IS900 DNA probe. RESULTS: None of the OFG and oral lesions of Crohn's disease samples were positive for M paratuberculosis and all normal controls were also negative. CONCLUSIONS: These results suggest that M paratuberculosis may not be a major aetiological agent in OFG or oral Crohn's disease lesions, although the use of paraffin wax embedded tissue as opposed to fresh tissue as a sample source could underestimate the true prevalence of the organism.

Blotting, Southern↗
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↗
PubMed1998

Human exposure to Mycobacterium paratuberculosis via pasteurised milk: a modelling approach.

Paratuberculosis is a disease of cattle caused by infection with Mycobacterium paratuberculosis, and it has been suggested that this bacterium may also play a role in the aetiology of Crohn's disease in humans. M paratuberculosis is shed in the milk and may be able to survive pasteurisation. Therefore, people may be exposed to it by the consumption of pasteurised milk. The risk of such exposure has been analysed using a modelling approach and the model has been used to evaluate the effects of intervention measures at different points in the potential route of transmission. On the basis of data from the literature and expert opinion, an initial point estimate of the exposure level of about 0-5 cfu/litre pasteurised milk was derived, mainly due to milk from clinically affected animals. The model indicates the need for quantitative data on variations in the shedding rates of M paratuberculosis in faeces and milk, and the levels of faecal contamination of milk. Such data are essential for a proper analysis of potential exposure, and may result in a 100-fold increase in the estimated median level of exposure.

Animals↗
PubMed2006

Lateral gene transfer in Mycobacterium avium subspecies paratuberculosis.

Lateral gene transfer is an integral part of genome evolution in most bacteria. Bacteria can readily change the contents of their genomes to increase adaptability to ever-changing surroundings and to generate evolutionary novelty. Here, we report instances of lateral gene transfer in Mycobacterium avium subsp. paratuberculosis, a pathogenic bacteria that causes Johne's disease in cattle. A set of 275 genes are identified that are likely to have been recently acquired by lateral gene transfer. The analysis indicated that 53 of the 275 genes were acquired after the divergence of M. avium subsp. paratuberculosis from M. avium subsp. avium, whereas the remaining 222 genes were possibly acquired by a common ancestor of M. avium subsp. paratuberculosis and M. avium subsp. avium after its divergence from the ancestor of M. tuberculosis complex. Many of the acquired genes were from proteobacteria or soil dwelling actinobacteria. Prominent among the predicted laterally transferred genes is the gene rsbR, a possible regulator of sigma factor, and the genes designated MAP3614 and MAP3757, which are similar to genes in eukaryotes. The results of this study suggest that like most other bacteria, lateral gene transfers seem to be a common feature in M. avium subsp. paratuberculosis and that the proteobacteria contribute most of these genetic exchanges.

Gene Transfer, Horizontal↗
PubMed1983

Pathology of spontaneous and experimental infection of North American wild ruminants with Mycobacterium paratuberculosis.

Spontaneous paratuberculosis was studied in free-ranging and captive bighorn sheep (Ovis canadensis), and Rocky Mountain goats (Oreamnos americanus). Lesions of paratuberculosis in these species resembled the disease in domestic sheep and goats. Mycobacterium paratuberculosis cultured from bighorn sheep was used to orally infect bighorn x mouflon (Ovis musimon) hybrid sheep, elk (Cervus elaphus nelsoni), mule deer (Odocoileus hemionus), and white-tailed deer (Odocoileus virginianus). Clinical paratuberculosis developed only in mule deer and was characterized by poor growth and diarrhea. Gross lesions were mild in all species. Enlargement of mesenteric lymph nodes was mild to moderate; the wall of the distal small intestine was affected minimally. Focal to diffuse infiltrates of epithelioid macrophages and giant cells occurred in the cortex of mesenteric lymph nodes, around mesenteric lymphatics, and in the intestinal mucosa. Extraintestinal lymph nodes, spleen, liver, and lung were involved in some animals; focal necrosis and mineralization was present in all species but was severe and widespread in the cervids.

Animal Population Groups↗
PubMed2005

Comparison of blood polymerase chain reaction and enzyme-linked immunosorbent assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep.

A study was carried out to compare the performance of enzyme-linked immunosorbent assay (ELISA) and blood polymerase chain reaction (PCR) for diagnosis of paratuberculosis in cattle and sheep. For cattle, a set of 278 samples from 1 paratuberculosis-affected Friesian farm was used; it included 80 ELISA-positive samples and 198 ELISA-negative samples from an age-matched group. Ninety-four samples were from heifers and 184 were from 2-5-year-old cows. The overall analysis showed a clear association (Fisher exact test [FET] P = 0.0049) but a weak negative agreement (45.3%, kappa = -0.1665 +/- 0.0994) between the 2 tests. It reflected a moderate agreement among heifers (87.7%, kappa = 0.4471 +/- 0.2435) and a moderate disagreement among cows (62.7%, kappa = -0.3670 +/- 0.1057). For sheep, 496 blood samples from 53 Latxa dairy flocks were used; 180 of the blood samples were from dam/offspring pairs. The overall association between the 2 tests on ovine samples was strong (FET, P = 0.0005), whereas the agreement was low (kappa = 0.1622 +/- 0.1188). There was slightly better agreement for ewes (kappa = 0.2135 +/- 0.1992) than for lambs (kappa = 0.1193 +/- 0.1301). There was also a highly unlikely proportion of dam/offspring positive results (FET, P < 0.0001, kappa = 0.6269 +/- 0.1854). Four of 6 lambs that were necropsied 1 year after testing had paratuberculosis microscopic lesions in the ileocecal valve (3 lambs) or a PCR-positive result (4 lambs). These results suggest that blood PCR testing might be a potentially useful new approach in paratuberculosis diagnosis, especially in young animals.

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↗
PubMed2000

Mycobacterium paratuberculosis infection in two free-ranging Alpine ibex.

The authors report two cases of Mycobacterium paratuberculosis infection in free-ranging Alpine ibex (Capra ibex) from two different herds in the Western Alps, Italy. One ibex, found dead in October 1998, was in poor condition. The second animal died due to trauma following capture with a dart gun. The only gross lesions observed were the enlargement of the mesenteric and iliac lymph nodes. Samples from both ibex tested positive to polymerase chain reaction for a primer set specific for the M. paratuberculosis insertion sequence IS900 and one ibex also tested positive to the Zielh-Nielsen stain. Isolation by bacterial culture was not successful. The infected ibex originated from herds in which seroreactors to M. paratuberculosis had been found previously. Seroreactors to M. paratuberculosis were also detected in sympatric cattle.

Animals↗
PubMed1994

Mycobacterium paratuberculosis in intestinal tissue from patients with Crohn's disease demonstrated by a nested primer polymerase chain reaction.

BACKGROUND: The etiology of Crohn's disease remains unknown, but current research has concentrated on autoimmunity and/or mycobacterial infection. The polymerase chain reaction (PCR) enables the detection of genetic material even when very few microorganisms are present. METHODS: A nested primer PCR for detection of a multi-copy insertional element (IS900) specific for Mycobacterium paratuberculosis was applied to DNA extracted from fresh and from paraffin-embedded intestinal tissue obtained from patients undergoing surgery. RESULTS: In fresh intestinal tissue from 11 of 24 patients with Crohn's disease, from 2 of 10 patients with ulcerative colitis, and from 3 of 28 patients with other colonic disorders, specific M. paratuberculosis DNA was found. In paraffin-embedded Crohn's disease tissue the presence of specific M. paratuberculosis DNA was also increased. CONCLUSIONS: Whether the presence of M. paratuberculosis is connected to the inflammatory bowel disease or is a mere coincidence cannot be stated. We find this presence interesting and encouraging for further investigations.

Adolescent↗
PubMed2002

Paratuberculosis in dairy cattle: variation of the antibody response in offspring attributable to the dam.

The objective of this study was to examine transmission of paratuberculosis in dairy cattle attributable to the dam. Milk samples were collected from 8131 cows in 110 Danish dairy herds. The level of antibodies to Mycobacterium avium subspecies paratuberculosis was determined by use of an ELISA. Information on dam and sire was obtained from the Danish Cattle database. The following two data sets were analyzed: Data set A contained all cows < or = 400 days in milk (n = 7410); data set B contained 1056 dam-daughter pairs present simultaneously in herds at the day of sampling. Cows > 400 days in milk were excluded. Linear mixed models were used to obtain variance components for the effect of sire in data set A and the effect of sire and dam-daughter pairs in data set B. Models for both data sets A and B included information previously shown to confound antibody level and information of the relative prevalence of paratuberculosis in the herd. In data set A, the effect explained by sire was 1.9%, whereas it was 6.3% in data set B. The effect from dam-daughter pairs was 7.7%. Those effects were all significant. It was concluded that the parental contribution was significant, and both heritability of susceptibility and vertical transmission should be considered in any control programs on paratuberculosis in dairy cattle.

Animals↗