Genomic analysis of an outbreak of bovine tuberculosis in a man-made multi-host species system: A call for action on wildlife in Brazil.

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from eight species, died due to TB, which is likely an underestimated number.

Genomic and temporal analyses of in southern Brazil.

is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. In this study, whole genome sequencing (WGS) of 74 .

Quantification of ovine herpesvirus 2 by digital PCR in an outbreak of malignant catarrhal fever.

Infection with ovine gammaherpesvirus 2 (OvHV-2) is generally asymptomatic in sheep; however, when it crosses the species barrier, it causes malignant catarrhal fever (MCF) in cattle. In the present study, we developed a real-time PCR assay and a droplet digital PCR assay and use both methods to study an outbreak caused by OvHV-2. Both PCR methods showed high sensitivity and specificity and were able to detect low copy numbers of OvHV-2 in sheep and cattle.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M.

ELISA using a recombinant chimera of ESAT-6/MPB70/MPB83 for Mycobacterium bovis diagnosis in naturally infected cattle.

Bovine tuberculosis (bTB) control programs generally rely on intradermal tuberculin tests for the antemortem diagnosis of Mycobacterium bovis infection in cattle, but these tests detect only a portion of the infected animals. The aim of the present study was to evaluate the diagnostic coverage of a combination of the bTB antemortem techniques known as the comparative intradermal tuberculin test (CITT) and an ELISA based on a recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen in cattle. The results were compared to postmortem findings based on M.

Effect of hyper or hypo-osmotic conditions on neutral amino acid uptake and oxidation in tissues of the crab Chasmagnathus granulata.

We investigated the transport of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) and (14)C-alanine oxidation in hepatopancreas and jaw muscle of Chasmagnathus granulata submitted to 24, 72, and 144 h of hypo- or hyperosmotic stress. While (14)C-MeAIB uptake increased in jaw muscle and hepatopancreas from crabs submitted to hyperosmotic stress, it did not change in tissues from animals submitted to hypo-osmotic stress. Incubation of jaw muscle and hepatopancreas from control groups with 1 mM ouabain did not decrease (14)C-MeAIB uptake.

Effect of hyperosmotic shock on phosphoenolpyruvate carboxykinase gene expression and gluconeogenic activity in the crab muscle.

Chasmagnathus granulata phosphoenolpyruvate carboxykinase (PEPCK) cDNA from jaw muscle was cloned and sequenced, showing a specific domain to bind phosphoenolpyruvate in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), respectively, specific for all PEPCKs. In the kinase-1 motifs the GK was changed to RK. The first 19 amino acids of the putative enzyme contain hydrophobic amino acids and hydroxylated residues specific to a mitochondrial type signal.

The crystal structure of the Leishmania major surface proteinase leishmanolysin (gp63).

Background: Despite their medical importance, there is little available structural information for the surface antigens of infectious protozoa. Diseases caused by the protozoan parasite Leishmania are common in many developing countries. Human infection occurs during the bite of infected sandfilies, when Leishmania promastigote cells from the insect gut enter the bloodstream.

Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions.

The study of experimental infection of inbred strains of mice with the intracellular protozoan parasite Leishmania major has contributed significantly not only to our understanding of this fascinating host/parasite relationship but also to that of many basic immunological phenomena. Much has been learned about the cognate interaction of antigen-specific T cells and antigen-presenting cells, about cytokine and T cell subset regulation, and the requirements for costimulation. Specifically, the immune response to experimental L.

Characterization of T cell-mediated responses in nonhealing and healing Leishmania major infections in the absence of endogenous IL-4.

IL-4 drives polarized Th2 responses, and differentiating Th2 cells down-regulate their sensitivity to IL-12. Therefore, the failure of BALB/c mice to heal Leishmania major infection could be due to an IL-4-dependent biased Th2 response or to a reduced capacity of Leishmania-specific Th cells to respond to IL-12. We examined the ability of CD4+ Th cells from L.

Crystallization and preliminary X-ray diffraction studies of leishmanolysin, the major surface metalloproteinase from Leishmania major.

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals.

Gamma interferon response in secondary Leishmania major infection: role of CD8+ T cells.

CD8+ T cells have been shown to contribute to the rapid resolution of secondary lesions developing in immune mice challenged with Leishmania major. In the present study, we assessed directly the participation of specific CD8+ T cells in the memory response induced in immune mice by reinfection. Lymphocyte populations from reinfected immune mice exhibit marked secondary gamma interferon (IFN-gamma) responses.

Structure of Leishmania mexicana lipophosphoglycan.

Lipophosphoglycan (LPG) was isolated from the culture supernatant of Leishmania mexicana promastigotes and its structure elucidated by a combination of 1H NMR, fast atom bombardment mass spectrometry, methylation analysis, and chemical and enzymatic modifications. It consists of the repeating phosphorylated oligosaccharides PO4-6Gal beta 1-4Man alpha 1- and PO4-6[Glc beta 1-3]Gal beta 1-4Man alpha 1-, which are linked together in linear chains by phosphodiester linkages. Each chain of repeat units is linked to a phosphosaccharide core with the structure PO4-6Gal alpha 1-6Gal alpha 1-3Galf beta 1- 3[Glc alpha 1-PO4-6]Man alpha 1-3Man alpha 1-4GlcNH2 alpha 1-6 myo-inositol, where the myo-inositol residue forms the head group of a lyso-alkylphosphatidylinositol moiety.

Identification of a surface metalloproteinase on 13 species of Leishmania isolated from humans, Crithidia fasciculata, and Herpetomonas samuelpessoai.

Promastigotes of thirteen species of Leishmania isolated from human patients, as well as L. enriettii, Crithidia fasciculata and Herpetomonas samuelpessoai, were examined for the expression of an amphiphilic, surface-oriented metalloproteinase by surface radioiodination of living cells, fractionation by Triton X-114 extraction and phase separation, and zymogram analysis by fibrinogen-SDS-PAGE. In all species of Leishmania, and the two monoxenous trypanosomatid parasites of insects, an ectoproteinase similar to the Promastigote Surface Protease, or PSP, was observed.

Secreted acid phosphatase of Leishmania mexicana: a filamentous phosphoglycoprotein polymer.

In the promastigote, or insect stage, most species of the parasitic protozoan Leishmania secrete an acid phosphatase. The enzyme purified from the culture medium of Leishmania mexicana is shown to be a complex [13.3% (wt/wt) protein, 74.

Monoclonal antibodies to Leishmania mexicana promastigote antigens. I. Secreted acid phosphatase and other proteins share epitopes with lipophosphoglycan.

The abundant surface glycolipid, lipophosphoglycan (LPG), of Leishmania promastigotes is composed of phosphosaccharide repeating units linked via a phosphosaccharide core to a conserved lyso alkylphosphatidylinositol membrane anchor. It is shown in this paper that monoclonal antibodies (mAbs) directed against LPG also react with an acid phosphatase secreted by L. mexicana promastigotes.

Genetic control of the immune response in mice to Leishmania mexicana surface protease.

Congenic mouse strains were tested in the lymphocyte proliferation assay for their response to the purified surface protease of Leishmania mexicana (gp63). The data obtained allow us to distinguish three different patterns of response, influenced both by H-2 (class II) and non-H-2 genes. Mice of the C57BL/10 (B10) background carrying H-2 haplotypes b,q, and r were found to be high responders; those carrying H-2 haplotypes d, j, v, and z were low responders; and those with H-2a, H-2f, H-2k, H-2p, and H-2u haplotypes were intermediate responders.

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence.

Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase.

The effects of a variety of inhibitors suggested that the promastigote surface protease (PSP) of Leishmania might be a zinc metalloprotease. To investigate this possibility, we conducted atomic emission and absorption spectroscopic analyses, which show that PSP contains 1 atom of zinc per 63-kDa monomer. Further studies showed that the enzyme can be biosynthetically labeled with 65ZnCl2.

Secondary structure of the promastigote surface protease of Leishmania.

By Raman spectroscopic analysis we have determined the secondary structure of the promastigote surface protease, named PSP or gp63, of Leishmania major. It consist of nearly 50% antiparallel beta-strand, and less than 20% alpha-helix. These results are contrasted with the predominantly alpha-helical VSGs of the African trypanosomes and the alpha-helical metalloprotease thermolysin.

Purification and characterization of a metabolite-regulated pyruvate kinase from Leishmania major promastigotes.

The pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.