Newly attenuated Mycobacterium bovis mutants as vaccines against bovine tuberculosis, particularly for possums.

Bovine tuberculosis costs New Zealand more than $80 million per year, mostly because extensive areas of the country are occupied by brushtail possums infected with Mycobacterium bovis. AgResearch has a major programme to produce new live tuberculosis vaccines that can be delivered to possums. Primary work involved development of molecular biological methods to enable genetic manipulation of M. bovis, including the production of random and specific mutants. Many avirulent mutants of M. bovis have been produced and their vaccine efficacy has been compared to BCG in guinea pigs. Selected mutants that perform at least as well as BCG are retested in guinea pigs using an extended vaccination protocol in which animals are pre-sensitized to environmental mycobacteria to mimic natural exposure. Ten candidate vaccines that have induced good protection in guinea pigs have been subsequently tested as vaccines in possums. While the protective efficacy of an M. bovis mutant inoculated into guinea pigs reliably indicated that some protection would be induced in possums, the most protective mutant in guinea pigs was different from that in possums. This illustrates the importance of testing in the target species as part of new vaccine development. An important outcome of this work was the identification of an operon in M. bovis whose inactivation produced an avirulent M. bovis vaccine candidate that was better than BCG in protecting possums from experimental tuberculosis. Allelic exchange methods are now being used to produce vaccine strains with multiple specific mutations to improve safety and immunological characteristics.

The efficacy of live tuberculosis vaccines after presensitization with Mycobacterium avium.

The variable efficacy of BCG in humans has been extensively documented but its cause is still not well understood. One possible reason for this variation is the effect of presensitization with environmental mycobacteria. To investigate in guinea pigs the effects of presensitization with well characterized Mycobacterium avium strains on the vaccine efficacy of BCG and of two recently developed, avirulent strains of Mycobacterium bovis. Two strains of M. avium containing the DNA insertion element IS901 (M. avium+) and two strains not containing this element (M. avium-) were inoculated subcutaneously or by oral administration into guinea pigs to assess their virulence in these animals and their ability to induce delayed type hypersensitivity to tuberculins. Subsequently, groups of guinea pigs presensitized with orally administered M. avium+ and M. avium- and a control group were vaccinated with BCG, or one of two newly attenuated strains of M. bovis. All groups were then challenged by the aerosol route with virulent M. bovis. Vaccine efficacy was assessed 5 weeks later by the presence of macroscopic lesions and bacterial counts of spleen and lung. No macroscopic lesions were observed in any of the guinea pigs inoculated with strains of M. avium+ or M. avium- and all animals gave delayed-type hypersensitivity skin-test reactions to avian PPD. In the vaccine experiment, presensitization with orally administered M. avium+ alone produced a low level of protection against subsequent challenge with virulent M. bovis. In the absence of presensitization with M. avium or after presensitization with an M. avium- strain, BCG and two attenuated strains of M. bovis produced significant levels of protection. No additional protection was observed in lungs of guinea pigs presensitized with M. avium+ and subsequently vaccinated with BCG. In contrast, both newly attenuated strains of M. bovis induced significant protection in lungs after such presensitization. Presensitization of guinea pigs by the oral administration of M. avium+ provides a model for testing vaccines under conditions where the efficacy of BCG has been compromised by prior sensitization with environmental mycobacteria.

Vaccine and skin testing properties of two avirulent Mycobacterium bovis mutants with and without an additional esat-6 mutation.

SETTING: Molecular techniques are now available to develop new live tuberculosis vaccines by producing avirulent strains of the Mycobacterium tuberculosis complex with known genes deleted. OBJECTIVES: Determine if removal of esat-6 from new live tuberculosis vaccines with known attenuating mutations affects their vaccine efficacy and if it could enable the development of discriminating diagnostic tests. DESIGN: Remove the esat-6 gene by allelic exchange from two illegitimate mutants of Mycobacterium bovis that had previously been shown to have similar vaccine efficacy to BCG in a guinea pig vaccination model. Determine the effect this removal has on virulence, vaccine efficacy and skin test reactivity in guinea pigs. RESULTS: Two double knockout strains of M. bovis were produced and their virulence and vaccine efficacy were compared to their parent strains. Removal of the esat-6 gene had no significant effect on vaccine efficacy. In skin tests, animals inoculated with the double knockout strains reacted to PPD but not ESAT-6, whereas those inoculated with the parent strains had similar skin test reactivity to both PPD and esat-6. CONCLUSION: Removal of esat-6 from new live tuberculosis vaccine candidates has no significant effect on vaccine properties but does enable the use of skin tests to distinguish between vaccination and infection.

Production of avirulent mutants of Mycobacterium bovis with vaccine properties by the use of illegitimate recombination and screening of stationary-phase cultures.

A better tuberculosis vaccine is urgently required to control the continuing epidemic. Molecular techniques are now available to produce a better live vaccine than BCG by producing avirulent strains of the Mycobacterium tuberculosis complex with known gene deletions. In this study, 1000 illegitimate recombinants of Mycobacterium bovis were produced by illegitimate recombination with fragments of mycobacterial DNA containing a kanamycin resistance gene. Eight recombinant strains were selected on the basis of their inability to grow when stationary-phase cultures were inoculated into minimal medium. Five of these recombinants were found to be avirulent when inoculated into guinea pigs. Two of the avirulent recombinants produced vaccine efficacy comparable to BCG against an aerosol challenge in guinea pigs with M. bovis. One of these recombinants had an inactivated glnA2 gene encoding a putative glutamine synthetase. Transcriptional analysis showed that inactivation of glnA2 did not affect expression of the downstream glnE gene. The other recombinant had a block of 12 genes deleted, including the sigma factor gene sigG. Two avirulent recombinants with an inactivated pckA gene, encoding phosphoenolpyruvate carboxykinase which catalyses the first step of gluconeogenesis, induced poor protection against tuberculosis. It is clear that live avirulent strains of the M. tuberculosis complex vary widely in their ability as vaccines to protect against tuberculosis. Improved models may be required to more clearly determine the difference in protective effect between BCG and potential new tuberculosis vaccines.

An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine.

SETTING: Strains of the Mycobacterium tuberculosis complex are being rationally attenuated in order to develop better tuberculosis vaccines than BCG, and it would be helpful if new vaccines lacked an immunogenic protein which could be used as a skin test reagent for determining infection status. OBJECTIVE: To delete the esat6 gene from a virulent Mycobacterium bovis strain and determine (i) whether this mutant sensitizes guinea pigs to a skin test based on ESAT6 and (ii) what effect this has on the virulence of M. bovis. DESIGN: An homologous recombination technique was used to produce an esat6 knockout mutant of a virulent strain of M. bovis. Guinea pigs were inoculated with either the mutant or parent strain and their reactivity in intradermal skin tests was determined to bovine purified protein derivative (PPD) and recombinant ESAT6 protein. RESULTS: Production of an esat6 knockout strain was demonstrated by Southern blot hybridization and the polymerase chain reaction. Guinea pigs inoculated with either the esat6 knockout strain or its virulent parent had positive skin test reactions to PPD but only animal inoculated with the parent strain had positive skin test reactions to ESAT6. Gross pathology, histopathology and mycobacterial culture of tissues indicated that the knockout strain was less virulent than its parent. CONCLUSION: If an effective live tuberculosis vaccine can be produced by inactivation of virulence genes in M. bovis, then prior or subsequent knockout of the esat6 gene could contribute to the loss of virulence and enable the development of a test to distinguish between vaccinated and infected animals.

Restriction endonuclease analysis and plasmid profiling of Actinobacillus pleuropneumoniae serotype 7 strains.

Seventeen serotype 7 Actinobacillus pleuropneumoniae strains isolated in New Zealand and A. pleuropneumoniae serotypes 1-12 reference strains were typed by restriction endonuclease analysis of chromosomal DNA and plasmid profiling. All serotype 7 strains produced similar DNA cleavage patterns and were significantly different to other reference serotype strains. Minor differences in the cleavage patterns enabled the 17 serotype 7 strains to be grouped into seven profiles. Plasmids were identified in all but three strains but the banding patterns did not account for the differences in the chromosomal profiles. The study showed that restriction endonuclease analysis and plasmid profiling are useful in epidemiological studies of porcine pleuropneumonia.

Production of avirulent Mycobacterium bovis strains by illegitimate recombination with deoxyribonucleic acid fragments containing an interrupted ahpC gene.

SETTING: Molecular genetic techniques have been used to elucidate virulence determinants and to produce vaccines for many bacterial pathogens but reliable techniques for slow-growing mycobacteria have not been available. OBJECTIVE: Oxidative defence genes including ahpC are involved in isoniazid resistance of strains of the Mycobacterium tuberculosis complex. The aim of this study was to inactivate ahpC by allelic exchange and to screen the expected background of illegitimate recombinants for their inability to grow in minimal medium (auxotrophy). DESIGN: M. bovis ATCC35723 was electroporated with linear fragments of deoxyribonucleic acid (DNA) containing an ahpC gene interrupted by a kanamycin resistance gene. Kanamycin-resistant colonies were screened for allelic exchange and auxotrophy. RESULTS: Southern blotting of DNA from kanamycin-resistant colonies revealed that no allelic exchange had occurred. Four of these recombinants were auxotrophs and subsequently were found to be avirulent in guinea pigs. The fragment insertion sites in the chromosome of each auxotroph were determined by DNA sequencing. In three cases, large chromosomal deletions had occurred. CONCLUSION: The M. bovis ahpC gene was not inactivated by this linear fragment approach but illegitimate insertion of such a fragment can be successfully used to produce avirulent auxotrophs which have potential for vaccine development.

Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria.

The effects of electroporation temperature, biochemical pretreatment of cells and stage of culture on electroporation efficiency for slow-growing mycobacteria were investigated. The efficiency of transformation into Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium intracellulare increased markedly with temperature. In contrast, the efficiency of transformation into Mycobacterium smegmatis, a fast-growing species, was higher at 0 degree C and decreased with temperature. While stage of culture had little effect, a further increase in efficiency of 2-4-fold was obtained following glycine or ethionamide pretreatment. Electroporation at 37 degrees C has been chosen as a standard condition for slow-growing species as it usually resulted in a transformation efficiency several orders of magnitude higher than that obtained at 0 degree C.

Evaluation of defined media suitable for isolation of auxotrophic mutants of mycobacteria.

In order to investigate the production of auxotrophs of slow growing mycobacterial pathogens, an evaluation of defined liquid and solid media suitable for Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium paratuberculosis was undertaken. Five defined liquid media were evaluated. A number of solid support matrices was added to the liquid media to produce solid media for evaluation. Tween 80 was added to all media to produce diffuse homogeneous growth. Although satisfactory growth in defined liquid media was achieved with all strains tested, poor growth was generally observed in the equivalent defined solid medium for strains belonging to the tuberculosis complex. Only M. paratuberculosis produced satisfactory growth on defined solid media, especially on Middlebrook 7H9. The inhibitory effect of Tween 80 and the presence of inhibitory factor(s) in agar were confirmed. Partial removal or inactivation of these factors by the addition of absorbents to the media was achieved.

Mycobacterium avium infection in a farmed deer herd.

Tuberculous lesions were identified over a 2-year period in 36 clinically normal red deer from a single herd. The lesions were only present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract, indicating infection by the oral route. Mycobacterium avium was isolated from 27 of 29 lesions examined by bacterial culture. Grossly and histologically, the lesions were indistinguishable from those caused by Mycobacterium bovis. DNA restriction endonuclease analysis revealed that all the 26 M. avium isolates available for examination had identical cleavage patterns. These patterns were identical to a New Zealand M. avium serotype 2 isolate from a pig and were very similar to a reference strain of M. avium serotype 2. The DNA examinations indicated that the deer were infected from a common source that was not identified.

Detection of Mycobacterium bovis in tissues by polymerase chain reaction.

A polymerase chain reaction (PCR) test was developed to detect Mycobacterium bovis in tissues. The test was based on amplification of a 248 bp segment of the insertion sequence, IS1081, present in six copies in strains of M. bovis and other members of the tuberculosis complex. The procedure involved digestion with proteinase K, lysis with sodium dodecyl sulphate, and extraction with hexadecyl tetramethyl ammonium bromide and phenol:chloroform:iso-amyl alcohol. When agarose gel electrophoresis was used for detection, the method was able to detect 1 fg of pure DNA, or 0.2 genome equivalents. It could also detect as few as 10 organisms from pure cultures and between 200-500 organisms from tissues spiked with cultured organisms. Detection by hybridization was only marginally more sensitive. The method was tested on 110 selected tissues recovered post mortem from a variety of animals. Fifty three of 58 samples diagnosed as M. bovis culture positive, including all samples containing microscopically visible acid-fast bacilli, were positive on duplicate testing by PCR. Five of 52 culture negative samples were also positive by PCR including three which contained large numbers of acid-fast organisms. Ten of the culture negative samples came from animals in a herd known to be free of bovine tuberculosis and all these were negative by PCR.

Characterization by restriction endonuclease analysis and seroagglutination of strains of Mycobacterium avium and Mycobacterium intracellulare obtained from farmed deer.

Restriction endonuclease analysis and seroagglutination were used to characterize strains of Mycobacterium avium and Mycobacterium intracellulare (collectively, MAI) recovered from 1 local herd and 2 imported shipments of red deer (Cervus elaphus) that developed sensitization to bovine tuberculin during skin testing. A total of 31 MAI strains were isolated from lymph node pools (head, thorax, abdomen, and peripheral regions) of 21 of 29 local deer. Similarly, 15 MAI strains were isolated from the lymph node pools of 12 deer from the 2 imported shipments. Mycobacterial strains were isolated from more than 1 of the lymph node pools of 9 local and 2 imported deer. Most of the strains (59% from local deer, 46% from imported deer) were recovered from the lymph nodes of the head region. After restriction endonuclease analysis of these isolates using the enzymes Bcl I, BstEII, and Pvu II, 26 of the strains from the local herd were separated into 3 groups, each consisting of strains with indistinguishable or closely related patterns. Seroagglutination results indicated that the first of these groups contained strains belonging to serotype 1, the second group contained strains belonging to serotype 8; and the third group of strains belonged to serotype 3 and 9. The 5 remaining strains from the local herd had unrelated restriction patterns. One of these belonged to serotype 3, whereas the remaining 4 could not be serotyped. Restriction analysis of the 15 strains from the imported deer identified 2 groups. Seroagglutination results indicated that 1 group contained strains belonging to serotype 2 and the other group contained strains belonging to serotype 8.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction endonuclease analysis of members of the Mycobacterium avium-M. intracellulare-M. scrofulaceum serocomplex.

Organisms belonging to the Mycobacterium avium-M. intracellulare-M. scrofulaceum (MAIS) serocomplex were subjected to restriction endonuclease analysis (REA) with the enzymes BstEII, PvuII, and BclI. Substantial genetic heterogeneity was observed between members of an authenticated collection of the 31 serotypes. Serotypes 2 and 3 were indistinguishable, however, as were serotypes 5 and 10. No direct correlation could be made between restriction pattern and species identification. REA of serotype 2 and serotype 8 isolates from various geographic locations and animal origins showed that, within limits, the restriction pattern could be used as an index of serotype. Some isolates that were unable to be classified serologically exhibited restriction patterns identical to those of strains that were able to be classified by seroagglutination. The difficulty of interpreting much of the epidemiological data concerning MAIS organisms may be partially explained by the extent of heterogeneity observed by REA. These findings support the contention that the MAIS complex has a substantially greater degree of heterogeneity than has been revealed by traditional methods.