Analysis of Antigens in Mycobacterium Paratuberculosis

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good. Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations. Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity. The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

Cultural and Biochemical Characteristics of Mycobacterium Paratuberculosis Isolated from Goats in Norway

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant. The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria. Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Comparative evaluation of the MGIT and BACTEC culture systems for the recovery of Mycobacterium avium subsp. paratuberculosis from milk

AIMS: To compare the detection capabilities of the non-radiometric MGIT (Mycobacteria Growth Indicator Tubes) and radiometric BACTEC 460TB culture systems (Becton Dickinson, Cowley, Oxford, UK) for recovering Mycobacterium avium subsp. paratuberculosis from milk. METHODS AND RESULTS: Ultra heat treated (UHT) milk samples spiked with different levels of M. paratuberculosis (10-107 cells ml-1) were inoculated into MGIT and BACTEC media (containing recommended supplements) with and without prior chemical decontamination of the milk samples with 0.75% (w/v) cetylpyridinium chloride for 5 h. Time for the detection of growth in days was recorded for each culture system, and a M. paratuberculosis count for each milk sample was calculated from BACTEC readings using a published formula. Correlation between MGIT and BACTEC detection times was 0.6983. Both culture systems were capable of detecting 10-100 M. paratuberculosis cells ml-1 in milk within 30-40 days when no decontamination treatment was applied, but only 102-103 cells ml-1 or greater when chemical decontamination was applied before culture. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The non-radiometric MGIT system could be substituted for the radiometric BACTEC system for the culture of M. paratuberculosis from milk without loss of detection sensitivity. Chemical decontamination before culture caused a significant reduction in numbers of viable M. paratuberculosis in all spiked milk samples resulting in decreased detection capability for both culture systems.     

Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and L?wenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.     

Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis.

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the length of its R1 alkyl chain. No differences in the production of these novel mycobactin were observed among species. Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M. intracellulare M12. However, a sample of mycobactin J isolated by Merkal and McCullough (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.     

Inhibition of Fungal Growth in the Cultural Isolation of Mycobacteria

Antifungal antibiotics were compared to determine their usefulness in primary mycobacterial cultures. Amphotericin B was found to be more effective in preventing fungal growth from bovine fecal specimens than were cycloheximide, nystatin, and tetracycline. Amphotericin B did not affect the growth rate of the following Mycobacterium species: M. avium, M. bovis, M. intracellulare, M. paratuberculosis, M. phlei, or M. tuberculosis, but it inhibited the growth of M. fortuitum. There was no observable effect on numbers of colonies of M. paratuberculosis on primary isolation from fecal specimens. It is recommended that, for the primary isolation of pathogenic mycobacteria from specimens likely to contain fungi, the inoculum should be pretreated with benzalkonium chloride, followed by mixing with amphotericin B or inoculation onto media containing amphotericin B.     

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.     

Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows' milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73 degrees C for 15 s or 25 s with and without prior homogenization (2,500 lb/in(2) in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73 degrees C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73 degrees C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73 degrees C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

Description of the Infection Status in a Norwegian Cattle Herd Naturally Infected by Mycobacterium avium subsp. paratuberculosis

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-γ (IFN-γ) immunoassay, measurement of antibodies, and pathological and bacteriological examination.     

A model for analyzing growth kinetics of a slowly growing Mycobacterium sp.

This report describes a simple method for quantifying viable mycobacteria and for determining generation time. We used statistical models and computer analysis of growth curves generated for the slowly growing mycobacterium Mycobacterium paratuberculosis under controlled conditions to derive a mathematical formula relating the dependent variable, growth, to the independent variables, log10 number of organisms in the inoculum (inoculum size) and incubation time. Growth was measured by a radiometric method which detects 14CO2 release during metabolism of a 14C-labeled substrate. The radiometric method allowed for early detection of growth and detected as few as three viable bacteria. The coefficient of variation between culture vials inoculated with the same number of M. paratuberculosis was 0.083. Radiometric measurements were highly correlated to spectrophotometric and plate count methods for measuring growth (r = 0.962 and 0.992, respectively). The proportion of the total variability explained by the model in a goodness of fit test was 0.9994. Application of the model to broth cultures provided accurate estimates of the number of M. paratuberculosis (standard error = 0.21, log10 scale) and the growth rate (coefficient of variation, 0.03). Generation time was observed to be dependent upon the number of organisms in the inoculum. The model accurately described all phases of growth of M. paratuberculosis and can likely be applied to other slowly growing microorganisms.     

A model for analyzing growth kinetics of a slowly growing Mycobacterium sp

This report describes a simple method for quantifying viable mycobacteria and for determining generation time. We used statistical models and computer analysis of growth curves generated for the slowly growing mycobacterium Mycobacterium paratuberculosis under controlled conditions to derive a mathematical formula relating the dependent variable, growth, to the independent variables, log10 number of organisms in the inoculum (inoculum size) and incubation time. Growth was measured by a radiometric method which detects 14CO2 release during metabolism of a 14C-labeled substrate. The radiometric method allowed for early detection of growth and detected as few as three viable bacteria. The coefficient of variation between culture vials inoculated with the same number of M. paratuberculosis was 0.083. Radiometric measurements were highly correlated to spectrophotometric and plate count methods for measuring growth (r = 0.962 and 0.992, respectively). The proportion of the total variability explained by the model in a goodness of fit test was 0.9994. Application of the model to broth cultures provided accurate estimates of the number of M. paratuberculosis (standard error = 0.21, log10 scale) and the growth rate (coefficient of variation, 0.03). Generation time was observed to be dependent upon the number of organisms in the inoculum. The model accurately described all phases of growth of M. paratuberculosis and can likely be applied to other slowly growing microorganisms.     

Inactivation of Mycobacterium avium subsp. paratuberculosis in cow's milk by means of high hydrostatic pressure at mild temperatures

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.     

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.     

Surveillance of bulk raw and commercially pasteurized cows' milk from approved Irish liquid-milk pasteurization plants to determine the incidence of Mycobacterium paratuberculosis

Over the 13-month period from October 2000 to November 2001 (inclusive), the Food Safety Authority of Ireland (FSAI) carried out surveillance of Irish bulk raw (n = 389) and commercially pasteurized (n = 357) liquid-milk supplies to determine the incidence of Mycobacterium paratuberculosis. The pasteurization time-temperature conditions were recorded for all pasteurized samples. Overall, 56% of whole-milk pasteurized samples had been heat treated at or above a time-temperature combination of 75 degrees C for 25 s. All analyses were undertaken at the Department of Food Science (Food Microbiology) laboratory at Queen's University Belfast. Each milk sample was subjected to two tests for M. paratuberculosis: immunomagnetic separation-PCR (IMS-PCR; to detect the presence of M. paratuberculosis cells, live or dead) and chemical decontamination and culture (to confirm the presence of viable M. paratuberculosis). Overall, M. paratuberculosis DNA was detected by IMS-PCR in 50 (12.9%; 95% confidence interval, 9.9 to 16.5%) raw-milk samples and 35 (9.8%; 95% confidence interval, 7.1 to 13.3%) pasteurized-milk samples. Confirmed M. paratuberculosis was cultured from one raw-milk sample and no pasteurized-milk samples. It is concluded that M. paratuberculosis DNA is occasionally present at low levels in both raw and commercially pasteurized cows' milk. However, since no viable M. paratuberculosis was isolated from commercially pasteurized cows' milk on retail sale in the Republic of Ireland, current pasteurization procedures are considered to be effective.     

Evaluation of an automated system for non-radiometric detection of Mycobacterium avium paratuberculosis in bovine feces

Cultivation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from feces remains the most reliable method to detect infected animals. The purpose of this study was to evaluate a broth-based automated system used for cultivation of mycobacteria such as M. tuberculosis from human hosts, for the detection of M. paratuberculosis in bovine feces. Bovine feces was spiked with tenfold serial dilutions of M. paratuberculosis (5x10(5) to 5x10(-1) organisms), then processed with a double-centrifugation technique that included disinfection prior to inoculation into broth tubes. The same pathogen dilution series was also inoculated directly into broth and broth with uninfected processed feces. All of the system signal-positive bottles were identified within 30 days, with the highest concentration of M. paratuberculosis detected by the system in as few as 8 days. The presence of the pathogen was confirmed with acid-fast staining and an IS900-based PCR assay when growth of M. paratuberculosis was indicated by the system. However, some of the signal-negative cultures inoculated with the equivalent of 0.5 organisms tested PCR-positive 56 days post-inoculation, indicating that longer culture periods may lead to detection of small quantities of the organisms. Additionally, it was indicated that the processing step had a detrimental effect on detection of the organism. Comparison of the broth- and Herrold's egg yolk medium (HEYM) solid media-based culture methods with defined check test specimens corroborated the experimental evaluation of this system, indicating that broth-based detection could provide a more rapid assay for M. paratuberculosis. These results suggest that this automated system could be used to detect this organism in bovine feces, but that new approaches to processing the feces for culture should be explored.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Etiology of Crohn's disease: the role of Mycobacterium avium paratuberculosis

Crohn's disease is a chronic inflammatory bowel disease characterized by transmural inflammation and granuloma formation. Several theories regarding the etiology of Crohn's disease have been proposed, one of which is infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), which causes a similar disease in animals, and is present in the human food chain. Considerable evidence supports the presence of M. paratuberculosis in the intestinal tissues of many patients with Crohn's disease including culture, detection of homologous mycobacterial DNA, detection of the mycobacterial insertion sequence IS900 by both PCR and in situ hybridization in tissues, and a serologic immune response to recombinant M. paratuberculosis antigens. Despite this evidence, and our personal belief that M. paratuberculosis is a cause of Crohn's disease, widespread acceptance of this hypothesis will require evidence that specific anti-mycobacterial chemotherapy will cure the disease.     

Gene encoded antimicrobial peptides, a template for the design of novel anti-mycobacterial drugs

Nisin A is the most widely characterized lantibiotic investigated to date. It represents one of the many antimicrobial peptides which have been the focus of much interest as potential therapeutic agents. This has resulted in the search for novel lantibiotics and more commonly, the engineering of novel variants from existing peptides with a view to increasing their activity, stability and solubility.The aim of this study was to compare the activities of nisin A and novel bioengineered hinge derivatives, nisin S, nisin T and nisin V. The microtitre alamar blue assay (MABA) was employed to identify the enhanced activity of these novel variants against M. tuberculosis (H37Ra), M. kansasii (CIT11/06), M. avium subsp. hominissuis (CIT05/03) and M. avium subsp. paratuberculosis (MAP) (ATCC 19698). All variants displayed greater anti-mycobacterial activity than nisin A. Nisin S was the most potent variant against M. tuberculosis, M. kansasii and M. avium subsp. hominissuis, retarding growth by a maximum of 29% when compared with nisin A. Sub-species variations of inhibition were also observed with nisin S reducing growth of Mycobacterium avium subsp. hominissuis by 28% and Mycobacterium avium subsp. paratuberculosis by 19% and nisin T contrastingly reducing growth of MAP by 27% and MAC by 16%.Nisin S, nisin T and nisin V are potent novel anti-mycobacterial compounds, which have the capacity to be further modified, potentially generating compounds with additional beneficial characteristics. This is the first report to demonstrate an enhancement of efficacy by any bioengineered bacteriocin against mycobacteria.