Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis.

Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis), responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cell mediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex and poorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) has been crucial to develop a more proper antigen. In the present study, we selected six M. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by in-silico analysis and evaluated them in experimental and natural infection; none of these antigens had been previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animals with both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected with Mycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually induced an IFN-γ response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the most valuable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAP infected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B among TST and MTC specific-PCR positive animals, although this result needs to be proven in further studies with a higher sample size. Our data confirm the feacibility to implement bioinformatic screening tools and suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate their contribution to bTB diagnosis.

Study of the immunological profile towards Mycobacterium bovis antigens in naturally infected cattle.

A number of studies have determined the contribution of Th1 and Th2 responses to the protective immunity and pathology of Mycobacterium bovis infection. However, much of that information is derived from experimentally infecting cattle with M. bovis and few data from naturally infected animals are available. The aim of this study was to characterize the immunological profile towards M. bovis antigens of naturally infected cattle by measurement of cytokine mRNA expression in PBMC, and to determine which lymphocyte subsets are involved in recall responses of PBMC from M. bovis infected cattle to M. bovis antigens. Consistent with data from cattle experimentally infected with M. bovis, naturally infected animals were found to display a Th1 cytokine profile in response to M. bovis PPDB stimulation. Production of IFN-gamma mRNA by PBMC after PPDB stimulation statistically distinguishes between infected and healthy herds, suggesting that this molecule is usable as an M. bovis-infection marker. As happens in experimentally infected cows, CD4, CD8 and gammadeltaTCR cells from a herd naturally infected with M. bovis are the predominant T cell subsets expanded in response to PPDB.

Mce2R from Mycobacterium tuberculosis represses the expression of the mce2 operon.

The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586, which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter-lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end (Rv0670) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative -10 and -35 promoter boxes. M. tuberculosis carrying mce2 and end promoter-lacZ fusions were used to infect J774 macrophage-like cells. Expression of beta-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.

The knockout of the lprG-Rv1410 operon produces strong attenuation of Mycobacterium tuberculosis.

P27 lipoprotein was previously described as an antigen in the Mycobacterium tuberculosis complex, encoded by the lprG gene, also named Rv1411 in the TubercuList (http://genolist.pasteur.fr/TubercuList) gene bank. It forms an operon with Rv1410 that encodes for an efflux pump, P55. A mutant of the H37Rv strain of M. tuberculosis not producing P27 (strain DeltaP27) was obtained by two-step mutagenesis using the counterselectable marker sacB and a thermosensitive origin of replication in the shuttle plasmid pPR27. By RT-PCR, we observed no lprG or Rv1410 mRNA in the DeltaP27 mutant strain compared with the wild type and complemented strains. Western blot experiments using anti-P27 polyclonal sera showed that the P27 protein was present both in the parental and in a complemented strain, in which the entire lprG-Rv1410 operon was reintroduced, but absent in the mutant strain. The three strains showed similar growth kinetics and characteristics in culture broth. To study the effect of the lprG mutation on M. tuberculosis virulence, BALB/c mice were inoculated to determine bacterial loads in spleens. At days 15 and 35 after infection, decreases of 1.5 and 2.5 logs in the bacterial load were found, respectively, in animals inoculated with the DeltaP27 mutant strain or with the wild type. This attenuation was reverted in the complemented strain. These results demonstrated that lprG gene is required for growth of M. tuberculosis in immunocompetent mice. The reversion of attenuation in the complemented strain indicates that the attenuated phenotype resulted from disruption of the lprG-Rv1410 operon.

Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis.

mce3 is one of the four mce operons in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence of this bacterium. Upstream of mce3 there is a putative regulatory gene (Rv1963) that harbours a double tetR-family signature. To study the role of this putative regulatory gene in the transcriptional regulation of the mce3 operon, Mycobacterium smegmatis mc(2)155 and M. tuberculosis H37Rv strains that harboured gene fusions between the mce3 promoter region and the Escherichia coli lacZ gene, either containing or not containing the Rv1963 gene, were used. The presence of the Rv1963 gene in the strains greatly reduced beta-galactosidase activity, suggesting that the Rv1963-encoded protein is a transcriptional repressor of the mce3 operon. Expression of mce3 by recombinant M. tuberculosis was increased when it was grown in a macrophage-like cell line (J774), compared to the level of expression seen when the recombinant bacterium was grown under in vitro conditions. However, no lifting of repression was induced. The mce3 promoter was defined by deletion and cloning of the Rv1963-Rv1964 intergenic region in a 200 bp DNA fragment harbouring the region upstream of the Rv1964 start codon. Gel-shift experiments determined that the Rv1963-binding site was located in this region. These results indicate that the mce3 operon is transcriptionally regulated and that under certain, unknown, conditions repression of gene expression could be lifted.

Diferencias proteómicas entre una cepa argentina de Mycobacterium bovis con la cepa de referencia AN5 / Proteomic differences between an argentine mycobacterium bovis strain and the reference AN5 strain

La tuberculosis bovina es en Argentina una enfermedad que provoca graves pérdidas económicas y que afecta a un 5 por ciento del ganado. En un trabajo previo de tipificación de cepas por RFLP mediante el uso de las sondas PGRS y DR se identificó una cepa altamente predominante que se llamó AA. A diferencia de la cepa salvaje AA, la cepa de referencia AN5, de origen europeo, que se utiliza para elaborar la tuberculina, es una cepa adaptada al crecimiento en laboratorio, que puede haber sufrido mutaciones en genes de antígenos o de virulencia. Para ello se analizó la producción de proteínas secretadas y del extracto celular, de la cepa salvaje AA comparada con la cepa de referencia AN5, con el propósito de identificar diferencias que puedan dar cuenta de la virulencia y para identificar nuevos antígenos. Se utilizaron técnicas como electroforesis en geles de policrilamida, geles de 2 dimensiones y Western blot utilizando antisueros específicos contra antígenos ya caracterizados y sueros de bovinos infectados con tuberculosis confirmada por aislamiento de M. bovis, empleando proteínas celulares y secretadas (a los 25 y 100 días de cultivo) de ambas cepas. Se pudieron identificar, una proteína secretada de aproximadamente 29 kDa y otra de 28 kDa del estracto celular que parecen ser exclusivas o producidas en mayor cantidad por la cepa AA. También, se identificaron otras pero cuyas bandas eran más débiles. En conclusión, algunas de las proteínas identificadas pueden servir para mejorar el diagnóstico de la tuberculosis bovina (AU)

Diferencias proteómicas entre una cepa argentina de Mycobacterium bovis con la cepa de referencia AN5 / Proteomic differences between an argentine mycobacterium bovis strain and the reference AN5 strain

La tuberculosis bovina es en Argentina una enfermedad que provoca graves pérdidas económicas y que afecta a un 5 por ciento del ganado. En un trabajo previo de tipificación de cepas por RFLP mediante el uso de las sondas PGRS y DR se identificó una cepa altamente predominante que se llamó AA. A diferencia de la cepa salvaje AA, la cepa de referencia AN5, de origen europeo, que se utiliza para elaborar la tuberculina, es una cepa adaptada al crecimiento en laboratorio, que puede haber sufrido mutaciones en genes de antígenos o de virulencia. Para ello se analizó la producción de proteínas secretadas y del extracto celular, de la cepa salvaje AA comparada con la cepa de referencia AN5, con el propósito de identificar diferencias que puedan dar cuenta de la virulencia y para identificar nuevos antígenos. Se utilizaron técnicas como electroforesis en geles de policrilamida, geles de 2 dimensiones y Western blot utilizando antisueros específicos contra antígenos ya caracterizados y sueros de bovinos infectados con tuberculosis confirmada por aislamiento de M. bovis, empleando proteínas celulares y secretadas (a los 25 y 100 días de cultivo) de ambas cepas. Se pudieron identificar, una proteína secretada de aproximadamente 29 kDa y otra de 28 kDa del estracto celular que parecen ser exclusivas o producidas en mayor cantidad por la cepa AA. También, se identificaron otras pero cuyas bandas eran más débiles. En conclusión, algunas de las proteínas identificadas pueden servir para mejorar el diagnóstico de la tuberculosis bovina

PCR-Restriction fragment length polymorphism analysis (PRA) of mycobacterium leprae from human lepromas and from a natural case of an armadillo of Corrientes, Argentina

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.

A 12.7 kb fragment of the Mycobacterium tuberculosis genome is not present in Mycobacterium bovis.

Southern blotting, sequence analysis and PCR experiments showed that Mycobacterium bovis and Mycobacterium bovis BCG lack a 12.7 kb fragment present in the genome of Mycobacterium tuberculosis. This region is 337 bp downstream of the RD2 region, which was previously described as being absent from some M. bovis BCG strains. The 12.7 kb fragment should be useful as a target for a PCR test to differentiate M. tuberculosis and M. bovis. An analysis of the 12.7 kb region suggests that it represents a deletion in M. bovis rather than an insertion in M. tuberculosis. The deletion removes most of the mce-3 operon, one of four highly related operons which may be involved in cell entry, and therefore it may contribute to differences in virulence or host range in the two species.

Multidrug resistant tuberculosis outbreak in Buenos Aires: DNA fingerprinting analysis of isolates

In order to determine the possible relationship among HIV patients coinfected with multidrug resistant tuberculosis strains who had been receiving clinical assistance in our Hospital, clinical and epidemiological information from 28 patients was collected. DNA fingerprinting by restriction fragment length polymorphism (RFLP) pattern was performed on the mycobacterial isolates from these patients, using the restriction enzyme Pvull and IS 6110 as genetic marker. A unique RFLP pattern was found in 10 isolates from 10 different patients who had a disease caused by a single strain. Our findings confirm RFLP as a reliable and useful tool to analyze TB transmission. (AU)

Multidrug resistant tuberculosis outbreak in Buenos Aires: DNA fingerprinting analysis of isolates

In order to determine the possible relationship among HIV patients coinfected with multidrug resistant tuberculosis strains who had been receiving clinical assistance in our Hospital, clinical and epidemiological information from 28 patients was collected. DNA fingerprinting by restriction fragment length polymorphism (RFLP) pattern was performed on the mycobacterial isolates from these patients, using the restriction enzyme Pvull and IS 6110 as genetic marker. A unique RFLP pattern was found in 10 isolates from 10 different patients who had a disease caused by a single strain. Our findings confirm RFLP as a reliable and useful tool to analyze TB transmission.

Autonomic nervous system regulation of murine immune responses as assessed by local surgical sympathetic and parasympathetic denervation

En ratones sometidos a gangliectomia simpática cervical superior (SCGx) unilateral se estudió el efecto de la desnervación local sobre la respuesta imune de ganglios linfáticos submaxilares (SmLN). El contenido de norepinefrina (NE) de SmLN disminuyó en un 90% a los 7-20 dias luego de la SCGx uni o bilateral. Los SmLN ipsilaterales de ratones SCGx unilateralmente 10-20 días antes e inyectados i.d. o i.p. con eritrocitos de carnero mostraron una respuesta de células formadoras de placa (PFC) significantivamente mayor que el control invervado contralateral. En ratones inyectados con eritrocitos de carnero en al fase temprana de parálisis neural simpática (2 h luego de la SCGx), se detectó una respuesta PFC aumentada mientras que durante la fase de degeneración anterógrada de los terminales (6-24 h luego de SCGx), se observó una disminucióm crecimente de PFC. En ratones crónicamente SCGx se detectó también un aumento significativo en la hipersensibilidad por contacto y reacción alogeneica retardada en al oreja ipsilateral a la operación. Al inyectar células de SmLN desnervados a F1 de (BALB/c x C57Bl/6) o de (BALB/c x AKR) se obtuvbieron índices esplénicos significativamente mayores que los observados luego de inyectar células controles. La estimulación mitogénica en diferentes protocolos experimentales no resultó en diferencias significativas entre SmLN desnervados e inervados. Luego de la descentralización local por sección unilateral del tronco lingual-cuerda del tímpano, los SmLN ipsilaterales exhibieron menor respuesta PFC 8-28 días luego de la cirugía. Estos resultados indican función modulatoria del sistema nervioso autónomo en la respuesta inmune (AU)

Autonomic nervous system regulation of murine immune responses as assessed by local surgical sympathetic and parasympathetic denervation

En ratones sometidos a gangliectomia simpática cervical superior (SCGx) unilateral se estudió el efecto de la desnervación local sobre la respuesta imune de ganglios linfáticos submaxilares (SmLN). El contenido de norepinefrina (NE) de SmLN disminuyó en un 90% a los 7-20 dias luego de la SCGx uni o bilateral. Los SmLN ipsilaterales de ratones SCGx unilateralmente 10-20 días antes e inyectados i.d. o i.p. con eritrocitos de carnero mostraron una respuesta de células formadoras de placa (PFC) significantivamente mayor que el control invervado contralateral. En ratones inyectados con eritrocitos de carnero en al fase temprana de parálisis neural simpática (2 h luego de la SCGx), se detectó una respuesta PFC aumentada mientras que durante la fase de degeneración anterógrada de los terminales (6-24 h luego de SCGx), se observó una disminucióm crecimente de PFC. En ratones crónicamente SCGx se detectó también un aumento significativo en la hipersensibilidad por contacto y reacción alogeneica retardada en al oreja ipsilateral a la operación. Al inyectar células de SmLN desnervados a F1 de (BALB/c x C57Bl/6) o de (BALB/c x AKR) se obtuvbieron índices esplénicos significativamente mayores que los observados luego de inyectar células controles. La estimulación mitogénica en diferentes protocolos experimentales no resultó en diferencias significativas entre SmLN desnervados e inervados. Luego de la descentralización local por sección unilateral del tronco lingual-cuerda del tímpano, los SmLN ipsilaterales exhibieron menor respuesta PFC 8-28 días luego de la cirugía. Estos resultados indican función modulatoria del sistema nervioso autónomo en la respuesta inmune