Immunological analysis of the components of the antigen complex A60 of Mycobacterium bovis BCG.

The antigen complex of A60 of Mycobacterium bovis BCG was analyzed by different immunological techniques to assess its relevance to tuberculosis and the involvement of its components in the immune reactions elicited in humans by tuberculous infection. A60 is composed of about 30 components, of which 8 were identified by available monoclonal antibodies (lipoarabinomannan, a glycolipid, and proteins of 65, 40, 38, 35, 19, and 14 kDa). The majority (87.5%) of anti-mycobacterial antibodies in sera from tuberculosis patients was directed against A60. Western blot (immunoblot) analysis indicated that the majority of the highly antigenic proteins present in mycobacterial homogenates were components of the A60 complex. A small percentage (7.8%) of A60 epitopes proved to be species specific. Thus, A60 proteins of 66, 41, 38, 37, 35, 34, 32, and 22 kDa were found to contain B-cell epitopes specific for M. bovis and not shared by Mycobacterium leprae oR Mycobacterium avium

Intrathecal synthesis of anti-mycobacterial antibodies in patients with tuberculous meningitis. An immunoblotting study.

Cerebrospinal fluid (CSF) and serum samples from eight patients with bacteriologically proven (6) or clinically suspected (2) tuberculous meningitis were tested for the presence of anti-mycobacterial IgG antibodies by an affinity-mediated immunoblot technique. This technique is based on agarose gel isoelectric focusing of paired CSF and serum samples diluted to the same IgG concentration, and transfer of the specific IgG antibodies onto mycobacterial antigen-loaded nitrocellulose sheets. An intrathecal synthesis of anti-mycobacterial oligoclonal IgG antibodies, often superimposed on diffuse polyclonal production was shown in all patients but not in patients with tension headache or other neurological disorders. Similar results were obtained when a purified mycobacterial antigen, A60, was used for coating the nitrocellulose sheets in place of a whole mycobacterial homogenate, indicating that A60 was a major immunogen. The number of anti-mycobacterial oligoclonal IgG bands increased with time, and persisted for years even in clinically cured patients. Some IgG bands had no detectable anti-mycobacterial activity, at least with the antigens preparations used in this study. The demonstration of such anti-mycobacterial IgG bands in the CSF could be a useful adjunct for the diagnosis of tuberculous meningitis, especially in the case of negative cultures

Comparison of thermostable macromolecular antigens from leprosy-associated bacteria.

Three types of bacteria are associated with leprosy: Mycobacterium leprae, leprosy-derived corynebacteria (LDC), and armadillo-derived mycobacteria (ADM). The immunological relationships between these three types of bacteria and Mycobacterium bovis BCG, used as a reference, were determined by cross-immunoelectrophoresis. When compared with the reference, cross-reactions were observed with a variable number of antigens: 2 in the case of strain LDC 15, 4 with M. leprae, and from 1 to 10 in the case of the ADM, depending on their subgroup. Next, thermostable macromolecular antigens (TMAs), the major cross-reactive antigens of leprosy-associated bacteria, were compared by anti-TMA antibody ELISA tests. The LDC TMAs displayed high cross-reactivity between the subgroups and lower cross-reactivity with the TMAs of M. bovis BCG. Evidence for the presence of a species-specific moiety in TMA of the different LDC was obtained by using depleted anti-TMA antisera. Western blot analysis revealed the presence of many proteins in the TMAs of LDC and M. bovis BCG, some of them being species-specific and other cross-reactive

Le programme français d'éradication de la tremblante du cheptel ovin fondé sur l'utilisation de la génétique

National audienceStudies carried out since 1993 on scrapie infected flocks, have allowed to describe the frequency of susceptible alleles of the PrP gene in French dairy sheep breeds, to verify the greater risk of susceptible animals, and to test the selection feasibility and efficiency based on PrP genotyping. The idea of using a genetic strategy to eradicate scrapie in infected flocks was born as a result. Since 2002, the programme to eradicate scrapie in French sheep livestock, implemented by the Ministry of Agriculture, is rooted in a genetic strategy based on the genotyping of the PrP gene : its objective is aimed both at eradicating scrapie in infected flocks and reinforcing the resistance of the entire national flock. The fact that it was launched within the framework of the livestock selection organisation, defined in 1966 by a French law, is obviously a key point to explain its efficient and rapid implementation from the starting year. Moreover, it illustrates the mobilisation of all the people in charge of the sheep breeding programmes in France : the jointed evolutions observed for the ARR allelic frequency, the genetic merit for production traits and the maintenance of criteria describing the genetic variability are evidence of a good application of the objectives defined for the breeding programmes. The national supervision, carried out by INRA, the Institut de l’Elevage and France UPRa selection, will set out, in the next years, to reach all the objectives of this national programme, particularly by supplying scrapie infected flocks with resistant sheep, managing the genetic variability of the selection flocks and diffusing resistant germplasm towards the commercial flocks. This plan is based on the following crucial points : resistance of ARR/ARR is universal to natural infections and ARR/ARR sheep are not healthy carriers. It will be necessary to always check these crucial points and include any new scientific knowledge in the breeding programmes.Les travaux conduits en ovins laitiers dès 1993 dans des élevages ovins atteints de tremblante ont permis de connaître la fréquence des allèles sensibles du gène PrP selon les races considérées, vérifier le risque accru de tremblante pour ces génotypes, et tester la faisabilité d’une sélection sur le gène PrP et son efficacité sur le risque de tremblante dans les troupeaux. Ils ont ainsi contribué à l’émergence de l’outil génétique pour éradiquer la tremblante dans les élevages atteints. Depuis 2002, le programme d’éradication de la tremblante mis en place en France par le Ministère de l’Agriculture, est fondé sur le génotypage du gène PrP : il vise à éradiquer à court et moyen terme la tremblante dans les élevages atteints, tout en renforçant la résistance génétique à moyen et long terme de l’ensemble du cheptel national, compte tenu des délais de renouvellement des cheptels femelles. Avoir choisi d’asseoir le programme national de sélection sur le dispositif existant d’amélioration génétique du cheptel national, organisé dans le cadre de la loi de l’Elevage de 1966, est manifestement un point clé pour expliquer la mise en oeuvre rapide et efficace du programme dès la première année, confirmant la mobilisation massive de tous les maîtres d’oeuvre des schémas de sélection des ovins en France : les évolutions conjointes constatées pour les fréquences alléliques en faveur de l’allèle ARR, les index de sélection pour les caractères de production et les indicateurs de gestion de la variabilité génétique sont une bonne illustration de l’application des objectifs assignés aux programmes de sélection. L’encadrement national, conduit par l’INRA, l’Institut de l’Elevage et France UPRa Sélection (Unité de Promotion et de sélection de Race), s’attachera, dans les prochaines années, à vérifier et à aider à la mise en oeuvre des quatre objectifs du programme national de sélection, en particulier la fourniture de reproducteurs résistants pour les élevages atteints, la gestion de la variabilité génétique dans les noyaux de sélection et la diffusion vers les élevages de production. Ce programme repose sur les points clés d’universalité de la résistance génétique à la tremblante en situation de contamination naturelle et d’absence de porteurs sains, qu’il faudra en permanence continuer de valider

Comparative cutaneous testing with purified protein derivative and the antigen complex A60 in vaccinated subjects and tuberculosis patients.

Some 840 bacille Calmette-Guérin (BCG)-vaccinated healthy controls and tuberculosis patients from two Chinese hospitals were submitted to comparative skin tests with purified protein derivative of tuberculin (PPD; as reference) and with the antigen complex A60 from Mycobacterium bovis BCG. In a first trial, including 581 persons (185 healthy juveniles, 180 healthy adults and 216 tuberculosis patients), a limited dose of A60 (1 microgram) was used. Performance of the A60 test was similar to that of 5 I.U. PPD for controls (cut-off values = 5 mm induration diameter), but lower than that seen for tuberculosis patients (10 mm cut-off values). A second survey was conducted on 259 persons (109 recently revaccinated healthy persons, considered as tuberculin-negative in the first trial, and 150 tuberculosis patients), using a higher dose of A60 (2 micrograms) and the same dose of PPD (5 I.U.). Similar results were obtained with the two tests in all cases, thus supporting the possibility of PPD replacement by A60 in cutaneous testing. The pattern of induration diameter distribution in healthy subjects who took part in the first testing round (64% positively rate) was displaced to the inactivity side (with a peak at 5 to 9-mm diameter), in comparison with the second round (90% positivity rate and peak at 10-14 mm). This indicates a progressive fading of cellular immunity reactions after BCG vaccination. In tuberculosis patients, no correlation was found among the following three parameters: positivity at cutaneous testing (with PPD or A60), titer of anti-A60 mycobacterial immunoglobulins in blood (IgG titer higher than cut-off line) and presence of mycobacteria in sputum