Antigen recognition and immunomodulation by gamma delta T cells in bovine tuberculosis

This report describes the in vitro proliferative responses of peripheral blood gammadelta T cells to defined mycobacterial protein Ags and the immunomodulatory effect of gammadelta T cells in cattle infected with Mycobacterium bovis. gammadelta T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating gammadelta T cell cultures produced enhanced levels of IFN-gamma and TGF-beta, but not IL-2 in response to the more immunodominant mycobacterial AGS: Depletion of gammadelta T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of gammadelta T cells upon other (alphabeta) T cell responses. Because gammadelta T cells constitute a major T cell population in the peripheral blood of cattle, the activities of gammadelta T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.     

Vivid molecular divergence over volcanic remnants: the phylogeography of Megadromus guerinii on Banks Peninsula,New Zealand

Megadromus guerinii, an endemic carabid beetle (Carabidae), is the most common carabid throughout its restricted range on Banks Peninsula, a formation of extinct volcanoes in Canterbury, New Zealand. This study characterises the small-scale phylogeographic patterns of M. guerinii across the formerly volcanically active Banks Peninsula using mitochondrial and ribosomal genes. Between the eastern and western areas of the peninsula, the mitochondrial, but not nuclear, DNA has a well-defined geographic distribution. Specifically, mitochondrial cytochrome c oxidase subunit I (CO1) identifies two distinct groups (> 6% divergence between eastern and western beetles) while ribosomal genes show no discernible pattern. Whether such a pattern represents male-biased dispersal, Wolbachia infection, a recent range expansion of a divergent lineage, or a deeper historic separation is explored. There is potential that male-biased dispersal could have occurred. Wolbachia infection was not detected. We conclude that historical processes have likely separated taxa in the eastern and western peninsula.     

Association between chloroplast and mitochondrial lineages in oaks

Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based on controlled crosses.      

gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.     

Immunohistochemical localization of irisin in mole rats (Spalax leucodon)

Irisin was first identified in skeletal muscle cells. It is an exercise protein that is secreted into the circulation; it causes conversion of white adipose tissue to brown adipose tissue. We investigated irisin immunoreactivity in mole rat (Spalax leucodon) tissues. We examined cerebellum, pituitary, heart, liver, pancreas, spleen, uterus, kidney and striated muscle in female adult mole rats. Tissues were processed, embedded in paraffin, sectioned at 5 μm and stained immunohistochemically for irisin. Irisin immunostaining was detected in the cytoplasm of stained cells; the cytoplasm of Purkinje cells was unstained. We found that irisin may be synthesized in many tissues. The function of locally synthesized irisin currently is unknown.     

Signal Regulatory Protein α (SIRPα)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria

Background

Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)α (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation.

Methodology/Principal Findings

In the present study, ESAT-6/CFP-10 complex and SIRPα interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a⁺ (SIRPα-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis ΔRD1). Sorted CD172a⁺ cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8α). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c⁺, CD172a⁺, and CD3⁺ cells, including CD172a-expressing multi-nucleated giant cells.

Conclusions/Significance

These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a⁺ cells, bind to CD172a⁺ cells, and induce multi-nucleated giant cells expressing CD172a.     

A Multi-Antigenic Adenoviral-Vectored Vaccine Improves BCG-Induced Protection of Goats against Pulmonary Tuberculosis Infection and Prevents Disease Progression

The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine.Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes.The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions.Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.     

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

Background

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia.

Methodology/Principal Findings

A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively.

Conclusions/Significance

The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.