Abstract. In murine models the inducible nitric oxide synthase (iNOS) and the natural resistance associated macrophage protein (NRAMP1) play major roles in host defense against mycobacteria. iNOS regulates nitric oxide (NO) production, which is noxious for ingested mycobacteria, and NRAMP1 displays pleiotropic antimicrobial effects, including upregulation of iNOS expression. Little is known about the role of these molecules in bovine tuberculosis (TB). In this work we demonstrate by Western blot a high expression of NRAMP1 in peripheral blood mononuclear cells (PBMCs), alveolar macrophages (obtained by bronchioalveolar lavage), and lymph node granulomas from 8 HolsteinFreisian cattle with autopsy-proven bovine TB. Immunohistochemistry revealed the abundant expression of NRAMP1 and iNOS in lymph node and lung granulomas. Immunoreactivity was abundant in the cytoplasm of many epithelioid macrophages and multinucleated giant cells of the Langhans type. A striking accumulation of nitrotyrosine (NT), an indicator of iNOS activity and local NO production, was observed in granuloma cells, particularly in multinucleated Langhans cells. This study shows that the expression of NRAMP1 and iNOS is costimulated in granulomas, which are protective T-cell reactions against mycobacteria.
Molecular typing of bacterial isolates provides a powerful approach for distinguishing Mycobacterium bovis (M. bovis) genotypes. It is known that M. bovis strain virulence plays a role in prevalence and spread of the disease, suggesting that strain virulence and prevailing genotypes are associated. However, it is not well understood whether strain virulence correlates with particular genotypes. In this study, we assessed the in vitro intracellular growth of 18 M. bovis isolates in bovine macrophages as an indicator of bacterial virulence and sought a relationship with the genotype identified by spoligotyping. We found 14 different spoligotypes—11 were already known and three spoligotypes had never been reported before. We identified 2 clusters that were phylogenetically related, containing 10 and 6 strains, respectively, and 2 orphan strains. Intracellular growth and phagocytic rates of 18 M. bovis strains were heterogeneous. Our results suggest that M. bovis intracellular growth and phagocytosis are independent of the bacterial lineage identified by spoligotyping.
Parasitic infection by the Fasciola hepatica (F. hepatica) promotes susceptibility towards other infections, such as Mycobacterium bovis. As consequence, could affect diagnostic tests for this disease. Hence, the objective of this study was to assess the impact of F. hepatica coinfection on the most commonly used immunodiagnostic bovine tuberculosis (bTB) tests in field conditions in an enzootic area for both diseases. Thus, from a dairy herd located in Hidalgo State, México, displaying a 59.2% and 28% prevalence of fascioliasis and bTB, respectively. Sixty-one cows were analyzed based on their response towards bTB immunodiagnostic tests, such as Single Intradermal Comparative Tuberculin Test (SICTT), gamma-interferon test (BOVIGAM) and enzyme-linked immunosorbent assay (ELISA), along with the assessment of the F. hepatica parasite load and serodiagnosis by ELISA. Three study groups were formed according to test results. Group 1: coinfected (n=22). Group 2: non-parasitized cows, and positive for bTB tests (n=13) and Group 3: parasitized cows without tuberculosis (n=26). In addition, a group of cows kept in fascioliasis - and tuberculosis-free zones were included (Group 4, n=10). A non-parametric Kruskal-Wallis test and a Dunn test were applied to analyze the results. In Group 1, significant differences were observed regarding IFN-γ production, but not for antibody levels to M. bovis or reactivity towards bovine PPD in relation Group 2. While, Groups 1 and 3 did not display difference in antibody levels against F. hepatica. Differences were observed regarding tuberculosis and Fasciola diagnostic tests when both coinfected and infected groups were compared to controls. It is concluded that F. hepatica coinfection in tuberculous animals studied, depressed the production of IFN-γ towards bovine PPD under in vitro conditions, but its reactivity to the SICTT not show to be altered.
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