Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis

Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets

Genetic diversity and antimicrobial resistance in Staphylococcus aureus and coagulase‐negative Staphylococcus isolates from bovine mastitis in Minas Gerais, Brazil

Abstract The aims of this study were to determine the antimicrobial susceptibility profile and genetic diversity of Staphylococcus spp. isolated from dairy cows in Minas Gerais, Brazil, and to assess the relationship among the isolates’ susceptibility profiles and pulsed‐field gel electrophoresis (PFGE) genotypes. Seventy‐nine isolates were used, including S. aureus (n = 71) and coagulase‐negative staphylococci (CoNS) (n = 8). Susceptibility to 12 antimicrobial agents was performed. All Staphylococcus spp. were subjected to PFGE. Staphylococcus aureus and CoNS isolates exhibited full susceptibility only to cephalothin. The greatest percentages of resistance among Staphylococcus spp. were observed to penicillins, folate pathway inhibitors, and tetracyclines. Twelve S. aureus and four CoNS were classified as multidrug resistance strains. Percentage of MRSA was also higher among CoNS (75%), compared to S. aureus isolates (2.81%). Adopting 100% of similarity, 34 different genotypes were identified. Association of minimum‐spanning tree (MST) analysis with data from municipalities, herds, methicillin‐resistant S. aureus (MRSA), and resistance patterns for all isolates did not show any clustering. However, a clustering pattern of bacterial species was observed. Results from this study indicate a high frequency of antimicrobial resistance, especially among CoNS, and a high genetic diversity among Staphylococcus spp. isolated from dairy cows with mastitis in Minas Gerais, Brazil

Evaluation of ERIC-PCR as genotyping method for Corynebacterium pseudotuberculosis isolates.

The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations

Immune Response of Calves Vaccinated with <i>Brucella abortus</i> S19 or RB51 and Revaccinated with RB51

<div><p><i>Brucella abortus</i> S19 and RB51 strains have been successfully used to control bovine brucellosis worldwide; however, currently, most of our understanding of the protective immune response induced by vaccination comes from studies in mice. The aim of this study was to characterize and compare the immune responses induced in cattle prime-immunized with <i>B</i>. <i>abortus</i> S19 or RB51 and revaccinated with RB51. Female calves, aged 4 to 8 months, were vaccinated with either vaccine S19 (0.6–1.2 x 10¹¹ CFU) or RB51 (1.3 x 10¹⁰ CFU) on day 0, and revaccinated with RB51 (1.3 x 10¹⁰ CFU) on day 365 of the experiment. Characterization of the immune response was performed using serum and peripheral blood mononuclear cells. Blood samples were collected on days 0, 28, 210, 365, 393 and 575 post-immunization. Results showed that S19 and RB51 vaccination induced an immune response characterized by proliferation of CD4⁺ and CD8⁺ T-cells; IFN-ɣ and IL-17A production by CD4⁺ T-cells; cytotoxic CD8⁺ T-cells; IL-6 secretion; CD4⁺ and CD8⁺ memory cells; antibodies of IgG1 class; and expression of the phenotypes of activation in T-cells. However, the immune response stimulated by S19 compared to RB51 showed higher persistency of IFN-ɣ and CD4⁺ memory cells, induction of CD21⁺ memory cells and higher secretion of IL-6. After RB51 revaccination, the immune response was chiefly characterized by increase in IFN-ɣ expression, proliferation of antigen-specific CD4⁺ and CD8⁺ T-cells, cytotoxic CD8⁺ T-cells and decrease of IL-6 production in both groups. Nevertheless, a different polarization of the immune response, CD4⁺- or CD8⁺-dominant, was observed after the booster with RB51 for S19 and RB51 prime-vaccinated animals, respectively. Our results indicate that after prime vaccination both vaccine strains induce a strong and complex Th1 immune response, although after RB51 revaccination the differences between immune profiles induced by prime-vaccination become accentuated.</p></div

Biotyping and Genotyping (MLVA16) of <i>Brucella abortus</i> Isolated from Cattle in Brazil, 1977 to 2008

<div><p>Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of <i>Brucella</i> spp. is scarce or unavailable. This study aimed (<i>i</i>) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of <i>B. abortus</i> and (<i>ii</i>) to analyze their distribution. <i>B. abortus</i> biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian <i>B. abortus</i> strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian <i>B. abortus</i> isolates were useful to better understand the epidemiology of bovine brucellosis in the region.</p></div

IFN-ɣ and IL-17A production by CD4⁺ and CD8⁺ T-cell subsets in peripheral blood mononuclear cells of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle upon <i>in vitro</i> stimulation with ɣ-irradiated <i>B</i>. <i>abortus</i> 2308.

<p>Tendency (median) (a) and box plot (median, first and third quartiles) (b) charts of the results. Whiskers show the lower and upper 1.5 interquartile range. Vaccinations were indicated by arrows. Significant differences (P < 0.05) between vaccination regimens (on same day) are indicated by uppercase letters (Mann-Whitney-test), and lowercase letters indicate statistical difference between days in same group (Skillings Mack test followed by Wilcoxon signed rank test).</p

Granzyme B and perforin-expressing CD8⁺ T-cells in peripheral blood mononuclear cells of S19 and RB51 prime vaccinated, and RB51 revaccinated cattle upon <i>in vitro</i> stimulation with ɣ-irradiated <i>B</i>. <i>abortus</i> 2308.

<p>Tendency (median) (a) and box plot (median, first and third quartiles) (b) charts of the results. Whiskers show the lower and upper 1.5 interquartile range. Vaccinations were indicated by arrows. Significant differences (P < 0.05) between vaccination regimens (on same day) are indicated by uppercase letters (Mann-Whitney-test), and lowercase letters indicate statistical difference between days in same group (Skillings-Mack test followed by Wilcoxon signed rank test).</p