Virulence and pathogenic mechanisms of <i>Legionella pneumophila</i>

Strains of Legionella pneumophila which were virulent for guinea pigs were passaged on laboratory media such that they became avirulent. Virulent/avirulent pairs of strains were compared in an attempt to identify potential virulence or pathogenic factors. Virulent forms multiplied within guinea pig alveolar macrophages maintained in tissue culture over 48 hours and phagosome lysosome fusion was not inhibited in these macrophages. Avirulent strains did not multiply but those produced by passage on supplemented Mueller Hinton agar (MHIH) maintained viability whereas those produced by passage on buffered charcoal yeast extract agar (BCYE) were killed. Virulent legionellae produced more flagella than their paired avirulent forms but it was considered unlikely that this was related to pulmonary virulence. Minor differences in outer membrane proteins and reactivity with various antisera were observed between virulent/avirulent pairs but these were not consistent between the L.pneumophila strains and no virulence-associated proteins were found. The lipopolysaccharide and extracellular enzyme activities of the pairs of strains were indistinguishable. The Corby strain of L.pneumophila(CV) was isolated from its intracellular in vivo environment and these bacteria compared with the same strain grown on BCYE. The in vivo-grown CV showed no change in uptake by, or intracellular replication within, alveolar macrophages but, when Western blotted and incubated with various antisera, the two types of bacteria reacted differently. There was a reduced reaction by the CV grown in vivo, suggesting a change in the number and/or type of proteins expressed when exposed to an intracellular environment. The cellular responses in the lungs of guinea pigs immediately following aerosol challenge with virulent and avirulent forms of the Corby strain were compared. The avirulent forms evoked little change in the alveolar cell populations whereas an inflammatory response to CV occurred. Polymorphonuclear Ieucocytes (PMNLs) were the principal cell type involved and the initial increase in numbers of this cell type corresponded with a transient decrease in the viablility of CV, providing evidence that virulent L.pneumophila are killed by PMNLs. The multiplication of CV which followed suggested a developing resistance to the killing. Numbers of avirulent Corby decreased rapidly and, for the MHIH passaged form (CA), this was in contrast to its intracellular survival in vitro. An attempt was made to follow the changes in cellular responses using immunolabelling techniques and flow cytometry. Difficulties with obtaining consistent, sequential samples did not allow a full interpretation of the results but the technique showed promise for such studies. The tissue destructive protease (TDP) of L.pneumophila was shown to degrade or inactivate gamma interferon, IgG and possibly interleukin 2, proteins of possible significance to the host in protection against infection with L.pneumophila. Inhibition of TDP by the protease inhibitor a2 macroglobulin was demonstrated and, prophylactic treatment of guinea pigs with the inhibitor resulted in prolonged survival (compared with untreated controls) following aerosol challenge with CV. Intracellular production of TDP by L.pneumophila multiplying within guinea pig alveolar macrophages was demonstrated by ELISA and immunogold labelling and functional activity of the enzyme purified from infected guinea pig lungs was shown. A mutant of L.pneumophila, deficient in TDP production, was compared in the guinea pig model with its TDP-producing parent and CV. The mutant and parent were considerably less virulent than CV but, despite the deficiency in TDP production, the mutant was lethal for guinea pigs. The lung damage caused by the mutant was less severe than that caused by the parent or CV and it was suggested that progression of disease and cause of death was not typical of Legionnaires' disease. An immunocompromised mouse model of Legionnaires' disease was investigated using aerosol infection of severe combined immune deficient (SeID) mice. SCID mice did not become ill when given a potentially lethal aerosol of L.pneumophila and alveolar macrophages of these mice did not support the growth of L.pneumophila in vitro. SCID mice which had been reconstituted with human leukocytes were susceptible to aerosol challenge and developed lung lesions similar to those seen in guinea pigs and humans

Comparison of Interferon-γ Release Assay to Two Cut-Off Points of Tuberculin Skin Test to Detect Latent Mycobacterium tuberculosis Infection in Primary Health Care Workers

BackgroundAn interferon-γ release assay, QuantiFERON-TB (QFT) test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Here, we compared the performance of QFT with tuberculin skin test (TST) measured at two different cut-off points among primary health care work (HCW) in Brazil.MethodsA cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points.ResultsWe enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31), and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22). We investigated possible factors of discordance of TST vs QFT. Compared to the TST-/QFT- group, risk factors for discordance in the TST+/QFT- group with TST cut-off of ≥5 mm included age between 41-45 years [OR = 2.70; CI 95%: 1.32-5.51] and 46-64 years [OR = 2.04; CI 95%: 1.05-3.93], BCG scar [OR = 2.72; CI 95%: 1.40-5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09-4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03-4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01-2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05-2.62], were significantly associated with the TST+/QFT- group. No statistically significant associations were found among the TST-/QFT+ discordant group with either TST cut-off value.ConclusionsAlthough we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed

Increased B and T Cell Responses in M. bovis Bacille Calmette-Guérin Vaccinated Pigs Co-Immunized with Plasmid DNA Encoding a Prototype Tuberculosis Antigen.

The only tuberculosis vaccine currently available, bacille Calmette-Guérin (BCG) is a poor inducer of CD8(+) T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation. As the induction of strong CD8(+) T cell responses is a hallmark of DNA vaccines, a combination of BCG with plasmid DNA encoding a prototype TB antigen (Ag85A) was tested. As an alternative animal model, pigs were primed with BCG mixed with empty vector or codon-optimized pAg85A by the intradermal route and boosted with plasmid delivered by intramuscular electroporation. Control pigs received unformulated BCG. The BCG-pAg85A combination stimulated robust and sustained Ag85A specific antibody, lymphoproliferative, IL-6, IL-10 and IFN-γ responses. IgG1/IgG2 antibody isotype ratio reflected the Th1 helper type biased response. T lymphocyte responses against purified protein derivative of tuberculin (PPD) were induced in all (BCG) vaccinated animals, but responses were much stronger in BCG-pAg85A vaccinated pigs. Finally, Ag85A-specific IFN-γ producing CD8(+) T cells were detected by intracellular cytokine staining and a synthetic peptide, spanning Ag85A131-150 and encompassing two regions with strong predicted SLA-1*0401/SLA-1*0801 binding affinity, was promiscuously recognized by 6/6 animals vaccinated with the BCG-pAg85A combination. Our study provides a proof of concept in a large mammalian species, for a new Th1 and CD8(+) targeting tuberculosis vaccine, based on BCG-plasmid DNA co-administration