Mycobacterium bovis in Swine: Spoligotyping of Isolates from Argentina

A total of 143 Mycobacterium bovis isolates of pigs, from the most productive swine area in Argentina, were typed by spoligotyping. Twenty-two different spoligotypes were identified, and 133 (93%) isolates were grouped into 12 clusters. One of them, designed SB0140, was the most frequent because it held 83 (58%) isolates. This spoligotype also grouped 362 (43%) out of 841 isolates from previously typed cattle and, thus, constitutes the most frequent in our country. In addition, 135 (94%) isolates revealed spoligotypes identical to those of cattle, showing an epidemiological link. On the other hand, there were seven novel spoligotypes, six of which were also unique since they had only one isolate each. This study aimed to identify the spoligotypes of M. bovis isolated from pigs to contribute to a better understanding of the distribution of bovine tuberculosis in the main productive area of Argentina

Assessment of the Immune Responses Induced in Cattle after Inoculation of a Mycobacterium bovis Strain Deleted in Two mce2 Genes

The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis

Assessment of the Immune Responses Induced in Cattle after Inoculation of a Mycobacterium bovis Strain Deleted in Two mce2 Genes

The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis

Vaccination with a BCG Strain Overexpressing Ag85B Protects Cattle against <em>Mycobacterium bovis</em> Challenge

<div><p><em>Mycobacterium bovis</em> is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with <em>leuD</em>. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with <em>M. bovis</em>, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.</p> </div

Mean tuberculin skin test responses to PPDB.

<p>Animals were vaccinated with BCG (n = 5, squares), Δ<i>leuD</i> BCG-85B (n = 5, triangles) or inoculated with PBS (n = 6, circles) and challenged after eight weeks with <i>M. bovis</i>. Values indicate skin thickness at one month post-vaccination and prior to challenge (A), and three months post-challenge (B). Horizontal lines indicate mean values. Data were analysed using a Mann Whitney test,*<i>P</i><0.05, **<i>P</i><0.005.</p

Correlation between IL-17 and IFN-γ mRNA expression in PPDB-stimulated PBMCs and disease severity.

<p> Results are expressed as mean increases in IL-17 (A) and IFN-γ (B) transcription at 15 days post-vaccination and IFN-γ transcription at 40 days post-challenge (C) of individual animals in relation to the corresponding total gross pathology scores (head lymph nodes, respiratory tract-associated lymph nodes and lungs). Animals vaccinated with either BCG (squares) or Δ<i>leuD</i> BCG-85B (triangles) and nonvaccinated (circles) were infected eight weeks after vaccination and sacrificed sixteen weeks post-challenge. Solid lines in panels A and B indicate linear regression; dashed lines indicate 95% confidence intervals. Values for <i>r²</i> and <i>p</i> of linear regression analysis are indicated.</p

Protective efficacy as measured by gross pathology.

<p>The calves were euthanized sixteen weeks after infection and thin slices of lungs and lymph nodes were analysed looking for granuloma formations. Pathology scores were established using the scoring system described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051396#s2" target="_blank">Material and Methods</a> (A) Mean pathology scores of lungs in vaccinated and nonvaccinated groups. (B) Mean pathology scores of total lesions (head lymph nodes, respiratory tract-associated lymph nodes and lungs) in vaccinated and nonvaccinated groups. Pathology scores for individual animals are plotted. Horizontal lines indicate median values. Significance was determined by Mann Whitney test: * Statistically significantly different, <i>P</i><0.05.</p

Determination of lymphocyte subsets in PPDB-stimulated PBMCs.

<p>Percentages of lymphocyte cell subsets CD4+ (A) and CD8+ (B) expressing CD25 for PPDB stimulated-PBMCs from animals vaccinated with BCG (n = 5, triangles), Δ<i>leuD</i> BCG-85B (n = 6, circles) or nonvaccinated (n = 6, squares) at 15, 30 and 60 days after vaccination and 20, 40, 70 and 100 days after challenge. The arrow indicates the challenge time point. Data were analysed using Mann-Whitney test for comparison between groups. (Statistically significantly different to that for the nonvaccinated group *<i>P</i><0.05 and ** <i>P</i><0.01).</p

Relative cytokine gene expression.

<p>Gene expression of IFN-γ (A), IL-2 (B), IL-4 (C) and IL-17 (D) was measured in PPDB-stimulated PBMCs from animals vaccinated with BCG (gray), Δ<i>leuD</i> BCG-85B (white) or nonvaccinated (black) at different time points (15 and 30 days post-vaccination and 20, 40, 70 and 100 days post-challenge). Transversal line indicates the challenge time point. Relative gene expression was calculated using the 2-ΔΔCt method with E correction, using <i>gadph</i> mRNA expression as reference gene and the pre-immune condition as the calibrator. Data were analysed using Mann-Whitney test, statistically significantly different, * <i>P</i><0.05 and ** <i>P</i><0.01. The bars indicate the median fold change.</p