BCG Vaccine Strains Research Articles

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

Gene expression profiling and antigen mining of the tuberculin production strain Mycobacterium bovis AN5

Control of bovine tuberculosis (bTB) relies on regular testing of cattle with a crude preparation of mycobacterial antigens termed purified protein derivative (PPD). Worldwide production of bovine PPD uses the Mycobacterium bovis AN5, a strain that was originally isolated circa 1948 in Great Britain (GB). Despite its worldwide use, the AN5 strain is poorly characterised. AN5 was adapted to grow on glycerol in a process similar to that used for the derivation of the BCG vaccine strains; during this process, it is known that BCG deleted the genes for some potent antigens. Our previous analysis of the genome of M. bovis AN5 showed that it had not suffered extensive gene deletion events during in vitro adaptation. However, glycerol adaptation of AN5 strain may have caused differences in its global gene expression profile that could affect antigen expression. To assess this, we determined the transcriptome profile of AN5 and compared it to expression data for two endemic GB strains of M. bovis that account for ∼61% of all GB bTB cases. Genes expressed at lower levels in AN5 compared to M. bovis field isolates were then screened for antigenicity in naturally infected animals. Using this approach a number of genes were found to be expressed at lower levels in AN5, including those for known antigens. Our results show that field strains of M. bovis show some significant differences in gene expression to AN5, and that this differential gene expression may impact on the antigen profiles expressed by AN5 during in vitro culture.

Tuberculosis vaccine strain Mycobacterium bovis BCG Russia is a natural recA mutant

BackgroundThe current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy.ResultsAs a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA.ConclusionAccording to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type.

QuantiFERON®-TB Gold testing for tuberculosis in psoriasis patients commencing anti-tumour necrosis factor α therapy

Conflicts of interest: none declared. Sir, The National Institute for Health and Clinical Excellence (NICE) guidelines published in July 2006 advise etanercept, a TNF‐α antagonist, as a treatment for severe plaque psoriasis in patients who have not responded to other therapies.1 Patients embarking on any immunosuppressive treatment require screening for tuberculosis (TB). This has traditionally consisted of Mantoux skin testing alongside risk stratification for exposure from history.2 The problem with skin testing and screening is cross‐reactivity with the BCG vaccine, which often gives false positive results. This may lead to unnecessary chemoprophylaxis, not only delaying the start of immunosuppressive therapy but also carrying with it possible dangerous side‐effects. There are also practical difficulties in reading the Mantoux on patients with widely affected skin in severe psoriasis and the cost implications of unnecessary TB treatment. The QuantiFERON®‐TB Gold test (QFT‐G; Cellestis Ltd, Carnegie, Australia) is a novel TB enzyme‐linked immunosorbent assay, based on detection of interferon‐γ release from sensitized leucocytes exposed to the synthetic Mycobacterium tuberculosis antigens, ESAT‐6 and CFP‐10. These antigens are secreted by M. tuberculosis but are absent from BCG vaccine strains and from most nontuberculous mycobacteria. There is no cross‐reactivity in previously vaccinated patients. The test is reported to have greater sensitivity and specificity than Mantoux testing (QFG‐T: 89% and 98·1%; Mantoux: 72% and 82%).3 4–5

Tuberculosis vaccine strain Mycobacterium bovis BCG Russia is a natural recA mutant

AbstractThe current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy. As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was successful in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA. According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. Our data suggest that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild type.

Differential productions of lipid virulence factors among BCG vaccine strains and implications on BCG safety

Safety of BCG is a major concern in countries with a high burden of HIV/AIDS. Current BCG vaccine comprises of a heterogeneous group of substrains showing genotypic differences. The impact of these differences on BCG efficacy and safety remains unknown. Here we show that three BCG substrains, BCG-Japan, -Moreau, and -Glaxo, do not produce phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), two cell wall lipids known to be important for the virulence of Mycobacterium tuberculosis and Mycobacterium bovis, suggesting that these BCG strains are more attenuated than others. We found that there is a good correlation between the ability of BCG strains to produce these two lipids and the propensity of BCG to induce complications following vaccination in children, which provides a partial explanation for the molecular mechanisms of BCG reactogenicity. Our finding has important implications for national immunization programmes particularly in HIV endemic countries. We suggest that PDIMs/PGLs analysis could offer a practical means for assessing the safety of various BCG vaccine strains currently used in the world.

Linear chromosomes in bacteria: no straight edge advantage?

The beginning of 2007 brought us the complete genome of the yeast Pichia stipitis, five archaeal and more than 25 completely sequenced bacterial genomes (Table 1). In addition, there were two genomics-related papers that deserve a special discussion. One of them (Fuchs et al., 2007) described an unfinished genome of the ubiquitous marine phototrophic bacterium Congregibacter litoralis, while the other (Cui et al., 2007) examined the properties of the Escherichia coli K-12 strains with a linear chromosome.

Evaluation of the GenoType MTBC assay for differentiating 120 clinical Mycobacterium tuberculosis complex isolates

The majority of mycobacterial infections are caused by strains of the Mycobacterium tuberculosis complex (MTBC). MTBC includes the species M. tuberculosis, M. africanum, M. bovis, M. microti, M. canettii and M. pinnipedii. The M. bovis species can be further divided into M. bovis subsp. bovis, M. bovis subsp. caprae and the M. bovis-derived BCG vaccine strain, whereas M. africanum includes two subtypes, I and II [1–5]. Identification of mycobacteria to the species level on the basis of growth rate, phenotypic characteristics and biochemical tests is laborious and extremely timeconsuming. GenoType MTBC (Hain Lifescience, Nehren, Germany) is a recently developed commercial DNA-strip assay for differentiating MTBC strains isolated from cultured material. The procedure involves isolating DNA from cultured material, multiplex amplification with biotinylated primers and reverse hybridization of the single-stranded, biotin-labeled amplicons to membrane-bound probes. The resulting banding pattern indicates the species of the isolated mycobacterium. The assay permits genetic differentiation of the following species: M. africanum I, M. bovis BCG, M. bovis subsp. bovis, M. bovis subsp. caprae, M. microti and M. tuberculosis/M. africanum II. The aim of the current study was to evaluate the proficiency of this assay for identifying clinical MTBC isolates. The study was performed using 120 consecutive clinical MTBC strains isolated from different patients at our hospital over a 5-year period (2000–2004). Specimens are routinely processed in accordance with international guidelines, and when necessary, an N-acetyl-L-cysteine-NAOH decontamination procedure is used [6, 7]. Specimens are inoculated into BacTAlert 3D tubes (Organon Technika, Durham, NC, USA) and onto Lowenstein–Jensen agar (LJ; bioMerieux, Marcy l’ Etoile, France) and incubated at 37°C for up to 6 and 8 weeks, respectively. All 120 strains included in this study were identified as members of the MTBC using gene probes (AccuProbe; GenProbe, San Diego, CA, USA) and further differentiated to the species level by means of colony morphology and biochemical analysis [nitrate reduction (Nitrati Test; Liofilchem, Roseto, Italy), niacin accumulation (BBL Taxo TB Niacin Test Strips; Becton Dickinson, Sparks, MD, USA), and growth on LJ slants in the presence of 1 μg/ml thiophen2-carboxylic acid hydrazide (TCH; bioMerieux)]. Of the 120 isolates, 119 (99.17%) were identified asM. tuberculosis; the remaining isolate was presumptively identified as M. bovis (negative niacin accumulation and nitrate reductase tests and no growth on TCH). Given this strain distribution, our results can only be applied to M. tuberculosis. Prior to use in this study, the LJ slants of all 120 isolates had been stored in a dark environment at room temperature and a loopful of culture material suspended in 1 ml distilled water had been preserved at −20°C. Four control strains (two M. africanum and two M. bovis) obtained from Institute Pasteur (Paris, France) were used. In parallel, four non-MTBC microorganisms (one M. kansasii, one Corynebacterium jeikeium, one Nocardia abscessus and one Escherichia coli) were tested with GenoType for cross reactions. The GenoType MTBC assay has formerly been evaluated in two German laboratories using control strains along with clinical isolates that were mostly obtained from positive liquid cultures [8, 9]. In the present study, the clinical isolates had been obtained exclusively from solid media. Moreover, these positive LJ slants had been stored for a prolonged period of time (ranging from weeks to years). Eur J Clin Microbiol Infect Dis (2007) 26:151–152 DOI 10.1007/s10096-007-0255-y

Avantages et limites des tests sanguins in vitro lymphocytes T/interféron gamma comparativement au test intradermique à la tuberculine pour le diagnostic de tuberculose ☆

The development of in vitro blood tests that measure the delayed hypersensitivity reaction developed after contact with Mycobacterium tuberculosis will change progressively the diagnosis of M. tuberculosis infection. These blood assays (Quantiferon TB Gold TM, Cellestis, Australia; T-SPOT.TB TM, Oxford Immunotec, United Kingdom) use specific, complex M. tuberculosis antigens (ESAT-6 and CFP-10), whereas the intra-dermal Mantoux test is done with tuberculin, a complex mixture of more than 200 antigens. ESAT-6 and CFP-10 are absent from all the BCG vaccine strains used throughout the world. Significant improvement in the specificity with equivalent or increased sensitivity of the in vitro tests compared to the Mantoux test will lead eventually to replacement of the latter.

Improved efficiency in detecting cellular immunity towards M. tuberculosis in patients receiving immunosuppressive drug therapy

Reactivation of a latent Mycobacterium tuberculosis infection in immunocompromised individuals is associated with significant morbidity and mortality. The limited sensitivity of the established tuberculin skin-test in identifying latently infected patients on immunosuppressive drug therapy represents a major obstacle to better tuberculosis control after transplantation. In this study, a quantitative flow-cytometric whole-blood assay and the skin-test were comparatively evaluated towards both diagnostic power and practicability in 117 long-term renal transplant recipients (age 53.1+/-14.8 years; 7.0+/-5.0 years after transplantation) in a low-prevalence region. Among the aforementioned renal transplant recipients, a high proportion (52.14%) had purified protein-derivative (PPD)-specific T-cell-immunity in vitro. Despite immunosuppression, prevalence as well as median frequencies of PPD-specific T-cells (0.22%; >0.05-4.71%) were as high as previously reported for immunocompetent individuals and haemodialysis patients. In contrast to in vitro testing, skin testing was less practicable in an ambulatory setting. Moreover, skin-test reactivity was significantly reduced as only 50.0% of patients with PPD-reactivity in vitro were skin-test positive. T-cell reactivity towards early secretory antigenic target-6 (ESAT-6), a protein specific for M. tuberculosis but absent from the bacillus Calmette-Guerin BCG-vaccine strain, was found in 52.9% of all individuals with PPD-reactivity in vitro. In conclusion, the whole-blood assay reveals a high prevalence of latent tuberculosis infection in renal transplant recipients. It may represent a valuable alternative to skin testing as the test result is not adversely affected by immunosuppression. Moreover, reactivity towards ESAT-6 allows the distinction of a latent infection from BCG-induced reactivity. The assay is well-suited for use in screening programmes and may facilitate the management of tuberculosis infection in immunocompromised individuals.

An In-House RD1-Based Enzyme-Linked Immunospot-Gamma Interferon Assay Instead of the Tuberculin Skin Test for Diagnosis of LatentMycobacterium tuberculosisInfection

Identification of individuals infected with Mycobacterium tuberculosis is essential for the control of tuberculosis (TB). The specificity of the currently used tuberculin skin test (TST) is poor because of the broad antigenic cross-reactivity of purified protein derivative (PPD) with BCG vaccine strains and environmental mycobacteria. Both ESAT-6 and CFP-10, two secretory proteins that are highly specific for M. tuberculosis complex, elicit strong T-cell responses in subjects with TB. Using an enzyme-linked immunospot (ELISPOT)-IFN-gamma assay and a restricted pool of peptides derived from ESAT-6 and CFP-10, we have previously demonstrated a high degree of specificity and sensitivity of the test for the diagnosis of TB. Here, 119 contacts of individuals with contagious TB who underwent TST and the ELISPOT-IFN-gamma assay were consecutively recruited. We compared the efficacy of the two tests in detecting latent TB infection and defined a more appropriate TST cutoff point. There was little agreement between the tests (k = 0.33, P < 0.0001): 53% of the contacts with a positive TST were ELISPOT negative, and 7% with a negative TST were ELISPOT positive. Furthermore, respectively 76 and 59% of the ELISPOT-negative contacts responded in vitro to BCG and PPD, suggesting that most of them were BCG vaccinated or infected with nontuberculous mycobacteria. The number of spot-forming cells significantly correlated with TST induration (P < 0.0001). Our in-house ELISPOT assay based on a restricted pool of highly selected peptides is more accurate than TST for identifying individuals with latent TB infection and could improve chemoprophylaxis for the control of TB.

Induction of humoral immunity in response to immunization with recombinantMycobacterium bovisBCG expressing the S1 subunit ofBordetella pertussistoxin

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.

Strain-dependent suppressive effects of BCG vaccination on asthmatic reactions in BALB/c mice

The choice of BCG vaccine strain may play an important role in vaccination efficiency. To investigate whether the suppressive effects of BCG on asthma depend on the strain of BCG. Female BALB/c mice were injected intraperitoneally with 1 of 4 different strains of BCG (1 X 10(6) CFU): Pasteur F1173P2, Tokyo 172, Tice, and Connaught. Seven days later, the animals were sensitized by 2 injections of ovalbumin (20 microg) at 2-week intervals before being provoked with 1% ovalbumin aerosols on 3 successive days. Thereafter, the mice underwent a methacholine bronchial challenge and were killed to quantify the inflammatory cells and cytokines in bronchoalveolar lavage fluid and the supernatant of cultured splenocytes. The eosinophil proportion in the bronchoalveolar lavage fluid was significantly lower and the concentration of interferon-gamma and the interferon-gamma-interleukin 5 (IL-5) ratio in the supernatant of cultured splenocytes were significantly higher in each of the BCG-treated groups (n=10 per group) than in the asthma control group (n=15). However, the methacholine sensitivity (P < .05) and IL-5 concentration (P < .01) in the supernatant of cultured splenocytes were significantly lower only in the group treated with the Tokyo strain of BCG. There was a significant positive correlation between IL-5 and IL-10 concentrations (r = 0.79; P < .001). The 4 strains of BCG suppressed asthma to different degrees, but all strains induced a shift in the T(H)1/T(H)2 balance toward T(H)1 without increasing IL-10-related regulatory T-cell activity.

Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLS(B)) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLS(B) and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.

Molecular Typing of Mycobacterium bovis Isolates from South-east Brazil by Spoligotyping and RFLP

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

Evaluation of T-cell responses to novel RD1- and RD2-encoded Mycobacterium tuberculosis gene products for specific detection of human tuberculosis infection.

The tuberculin skin test for diagnosing Mycobacterium tuberculosis infection suffers from antigenic cross-reactivity of purified protein derivative with BCG, resulting in poor specificity in BCG-vaccinated populations. Comparative genomics has identified several genetic regions in M. tuberculosis and M. bovis that are deleted in M. bovis BCG. Proteins encoded in these regions will form the basis of new specific T-cell-based blood tests that do not cross-react with BCG, but only two, early secretory antigen target 6 and culture filtrate protein 10, have been studied in detail in humans. We investigated four novel gene products, encoded by RD2 (Rv1989c) and RD1 (Rv3873, Rv3878, and Rv3879c), that are absent from most or all of the vaccine strains of BCG, respectively. Sixty-seven overlapping peptides were tested in ex vivo gamma interferon enzyme-linked immunospot assays in 49 patients with culture-confirmed tuberculosis and 38 healthy BCG-vaccinated donors. Forty-five percent (95% confidence interval [CI], 31 to 57%) and 53% (95% CI, 39 to 67%) of the tuberculosis patients responded to Rv3879c and Rv3873, respectively, identifying these proteins as major M. tuberculosis T-cell antigens in humans, while 35 and 25% of the patients responded to Rv3878 and Rv1989c, respectively. Of the 38 BCG-vaccinated donors, 1 (2.6%) responded to peptides from Rv3878 and Rv3879c, 3 (7.9%) responded to Rv3873, and none responded to Rv1989c. Exclusion of cross-reactive peptides encoded in conserved motifs of Rv3873, a PPE family member, increased its specificity to 97.4%. The high specificity of Rv3879c peptides and nonconserved Rv3873 sequences, together with their moderate sensitivity in tuberculosis patients, identifies these peptides as candidates for inclusion in new T-cell-based tests for M. tuberculosis infection.

A second-generation anti TB vaccine is long overdue

Mycobacterium bovis BCG vaccine significantly reduces the risk of tuberculosis by 50% and continues to be used to prevent tuberculosis around the world. However, it has been shown to be ineffective in some geographical regions. The existence of different BCG strains was described more than 60 years ago, these vary in their antigenic content but the genetic mutations in BCG strains have yet been shown to affect their protection. After the declaration of tuberculosis as a global emergency in 1993, current research attempts to develop a novel more-effective vaccine. Using new technologies, recombinant, auxotroph, DNA, subunit and phylogenetically closely related mycobacteria, naturally or genetically attenuated, have been used as vaccines in animal models, but their protective efficacy, is less than that offered by the current BCG vaccine. Today it is mandatory that a major effort be made to understand how different BCG vaccine strains influence immune response and why in some cases vaccines have failed, so we can rationally develop the next generation of tuberculosis vaccines to reduce the prevalence from 10% to less than 2 % for developed countries.

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization.

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.

Molecular epidemiology of bovine tuberculosis in the Czech Republic and Slovakia in the period 1965-2001 studied by spoligotyping

Spoligotyping was used to examine IS6110-positive DNA of 26 Mycobacterium bovis, M. bovis BCG and M. bovis subsp. caprae non-viable isolates stored up to 10 years. All of these isolates were previously identified by biochemical tests and all 17/17 tested isolates were earlier found virulent for guinea pigs. In total seven spoligotypes, designated S1&amp;ndash;S7, were detected and compared with the spoligotypes of 3 176 isolates in the database of the National Institute of Public Health and the Environment (RIVM) in Bilthoven, the Netherlands. A Neotype M. bovis strain, isolated in 1965 in the USA and thereafter stored in The Czechoslovak National Collection of Type Cultures (My 310/87) since 1987 was of an identical spoligotype S4 with the original reference M. bovis strain from the USA. The M. bovis isolates from capybara&amp;rsquo;s (Hydrochoerus hydrochaeris) imported from Germany to the Czech Republic in 1989, as well as cattle isolates from 1966, 1991 and 1994, were of the most common type S1. Also a human isolate from 1981, a M. bovis BCG vaccine strain and clinical M. bovis BCG isolates from three children with post-vaccinal complications were of this most predominant spoligotype. e four unique spoligotypes S2, S3, S5 and S6 were identified in M. bovis isolates from cattle in the years 1965, 1996 and 1967 in the CzechRepublic, respectively, but also in isolates from farmed red deer (Cervus elaphus) from 1991 and in cattle isolates from Slovakia from the year 1992. The scarcely occurring spoligotype S7, which is typical for M. b. caprae was detected in the Czech Republic from farmed red deer (1999), cattle isolates (1966, 1991, 1995) and in a strain isolated from an 80-year-old man (1999). Several strains isolated in each of three outbreaks in cattle herds were examined. Identical spoligotypes were detected in two outbreaks and different causal agents (M. bovis of spoligotype S1 and M. b. caprae of spoligotype S7) were identified in two cows from the third outbreak. e results confirm an effective control of bovine tuberculosis in the CzechRepublic and Slovakia during 1959&amp;ndash;1968, because previously circulating spoligotypes were successfully eradicated. e data also suggest other reservoirs of bovine tuberculosis may exist among free-living wild animals.

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4 + and CD8 + T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.