Simple and rapid detection of Mycobacterium tuberculosis complex organisms in bovine tissue samples by PCR.

Mycobacterium bovis is a slowly growing microorganism, and confirmation of the diagnosis by conventional culture is a lengthy process. A simple, rapid method for the extraction of DNA from bovine tissue samples was developed and used in a PCR designed for the diagnosis of tuberculosis. Tissues from 81 cattle from tuberculosis-infected herds (group 1) and 19 cattle from tuberculosis-free herds (group 2) were tested in this PCR, and the results were compared with those of conventional culture. The PCR assay detected 71.4% of the culture-positive animals from group 1. Tissue from all animals in group 2 were negative in the PCR assay and by culture. The described method could be used as a rapid screening technique which would be complementary to culture of tissue specimens for the routine diagnosis of bovine tuberculosis. The PCR technique is much faster than culture and reduces the time for diagnosis from several months to 2 days. It also provides for the detection of M. bovis when rapidly growing Mycobacterium spp. are present in the sample and may be able to detect the presence of M. bovis in samples even when organisms have become nonviable.     

Recent Advances in Archaeological Science Techniques Special Feature: A Neandertal dietary conundrum: Insights provided by tooth enamel Zn isotopes from Gabasa,Spain

The characterization of Neandertals’ diets has mostly relied on nitrogen isotope analyses of bone and tooth collagen. However, few nitrogen isotope data have been recovered from bones or teeth from Iberia due to poor collagen preservation at Paleolithic sites in the region. Zinc isotopes have been shown to be a reliable method for reconstructing trophic levels in the absence of organic matter preservation. Here, we present the results of zinc (Zn), strontium (Sr), carbon (C), and oxygen (O) isotope and trace element ratio analysis measured in dental enamel on a Pleistocene food web in Gabasa, Spain, to characterize the diet and ecology of a Middle Paleolithic Neandertal individual. Based on the extremely low δ⁶⁶Zn value observed in the Neandertal’s tooth enamel, our results support the interpretation of Neandertals as carnivores as already suggested by δ¹⁵N isotope values of specimens from other regions. Further work could help identify if such isotopic peculiarities (lowest δ⁶⁶Zn and highest δ¹⁵N of the food web) are due to a metabolic and/or dietary specificity of the Neandertals.

Over the last 30 years, analyses of nitrogen isotopes in collagen (δ¹⁵N_collagen) have provided direct evidence for Neandertal diets across Europe and Asia. These studies all indicate a carnivorous (1–12), or at least a meat-heavy, diet for European Neandertals. However, one peculiarity of Neandertal δ¹⁵N_collagen remains the subject of an ongoing debate. From the one Siberian and eight western European sites, where both Neandertal and associated fauna have been analyzed, nitrogen isotope ratios in Neandertal collagen are systematically higher than that of other carnivores (3, 6–8, 10, 11, 13, 14). An explanation for such elevated values could be the consumption of herbivores, such as mammoths, which themselves exhibit elevated δ¹⁵N values due to the consumption of plants growing on arid soils (1, 2, 7). While mammoth remains are usually scarce at Neandertal fossil localities, they were nonetheless occasionally consumed, as suggested by remains with cut marks and other human butchery signatures (15). The absence of mammoth remains at Middle Paleolithic sites could be a result of 1) Neandertals chose to leave large bone elements at the kill site and transport other edible carcass products, mainly meat, back to the habitation site (15), or 2) mammoths were not frequently consumed, and the δ¹⁵N peculiarity consequently reflects the consumption of other resources enriched in ¹⁵N.Alongside this δ¹⁵N peculiarity, one major obstacle to our knowledge of Neandertals’ subsistence patterns is that the preservation of organic matter limits the application of collagen-bound nitrogen isotope analysis to fossil specimens. Collagen degrades over time at a varying speed depending on climatic and environmental conditions (16). The oldest hominin specimen in which bone protein is preserved is that of Scladina (Belgium), which dates to 90,000 cal BP (calibrated years before the present) (17), but most studied specimens are younger than 50,000 cal BP (1–3, 6–8, 10–13, 18). Furthermore, these specimens are only from sites in northwestern and central Europe and Siberia, where climatic conditions favored collagen preservation. As a result, the variability of Neandertals’ diet over time and between regions may not accurately be reflected by the currently available isotope data. In Iberia, where the latest surviving Neandertals have been discovered (19, 20), collagen was successfully extracted for only one site (21). Therefore, our knowledge of Iberian Neandertal diets mostly relies on zooarcheological and dental calculus data, which show some inconsistencies (21–25). For instance, similar to other western European sites, zooarcheological studies emphasize the consumption of terrestrial mammals and birds (21). In contrast, analysis of dental calculus for microremains and ancient DNA metagenomic analysis (26–28) highlight the frequent consumption of plants and mushrooms. Indeed, Weyrich et al. (26) even suggest that Neandertals at El Sidrón (Fig. 1) rarely consumed meat but often ate mushrooms, which would also result in elevated δ¹⁵N values (29). The consumption of marine foods is also attested for coastal Neandertals, but its frequency cannot be truly assessed in the absence of isotope studies (21, 23–25, 30). Finally, cannibalism has been documented at two Iberian sites (El Sidrón and Zafarraya) (22, 31) (Fig. 1), though such practices appear limited and most likely occurred only during periods of nutritional stress (32). Therefore, it is challenging to confirm the homogeneity of Neandertals’ diets across time and space, calling into question a direct link between their subsistence strategy and disappearance.Open in a separate windowFig. 1.(A) Location of the Gabasa site as well as other Neandertal sites mentioned in the text. (B) Detailed map of the Gabasa region. San Estaban de Litera and Benabarre are nearby modern cities.This study aims to investigate if the Zn isotope proxy could help elucidate the dietary behaviors of Neandertals and the source of their δ¹⁵N peculiarity, specifically by studying a Late Pleistocene Iberian food web where the presence of mammoth has not been documented (33). The development of Zn isotope analysis (⁶⁶Zn/⁶⁴Zn, expressed as δ⁶⁶Zn) has proven that trophic level information can be retrieved from mammalian tooth enamel (δ⁶⁶Zn_enamel) (34, 35), including fossil samples from Pleistocene food webs where organic matter is typically not preserved (36, 37). Previous studies have demonstrated that δ⁶⁶Zn_enamel decreases by ca. 0.30 to 0.60 ‰ with each step in archeological and modern food webs (34–38) and that δ⁶⁶Zn values associated with breastfeeding are higher than postweaning-associated values (39). While the main source of variation of δ⁶⁶Zn_enamel values is diet, local geology can also likely influence the isotope ratio of a given animal (36, 39). To date, three modern assemblages from Koobi Fora (Kenya), Kruger Park, and the western Cape (South Africa) (40), a few animals from a historical site (Rennes, France) (41), and three Late Pleistocene sites (Tam Hay Marklot, Nam Lot, and Tam Pa Ling, Laos) (36, 37) are the only terrestrial food webs for which Zn isotope data in teeth and/or bones have been published (SI Appendix, Fig. S14). In the modern Koobi Fora savannah food web, δ⁶⁶Zn_enamel differences have been observed between browsers and grazers (35), but this pattern was not seen in any of the three Pleistocene Asian forest food webs (36, 37). Among modern and historical human populations, historically documented diets relying on plants are associated with higher δ⁶⁶Zn values than those that include a substantial quantity of animal products (41–44). Zinc isotopes of ancient hominins have been analyzed only in one Pleistocene Homo sapiens individual (37) from Southeast Asia, but not yet in any Neandertal specimen.This current study contributes significantly to our understanding of Iberian Neandertal diets by providing information on their trophic ecology for a region where traditional nitrogen isotope analyses are unfeasible due to the poor preservation of organic matter. We use the Zn isotopic tool as well as other mobility, ecological, and dietary proxies applied on tooth enamel from hominin and animal remains from the cave site Cueva de los Moros 1 (Gabasa, Pyrenees, Spain; Fig. 1). The site, excavated in the 1980s, is very well documented [for stratigraphic context, see Montes and Utrilla (45) and SI Appendix, Section S1]. All remains come from layers e, f, and g of a single stratigraphic layer directly above layer h dated to 143 ± 43 ka. Numerous carnivore remains were recovered along with Neandertal remains (layers e and f), allowing for comparison of the different meat-eating taxa. Coexisting herbivores from three different types of environmental contexts are homogeneously represented in layers e, f, and g: mountains (Iberian ibex [Capra pyrenaica], chamois [Rupicapra rupicapra]), forest (cervids including red deer [Cervus elaphus]), and open environments (horses [Equus ferus], European wild asses [Equus hydruntinus]).     

Immune responses of a meningococcal A + W outer membrane vesicle (OMV) vaccine with and without aluminium hydroxide adjuvant in two different mouse strains

Meningococci (Neisseria meningiditis) of serogroups A and W have caused large epidemics of meningitis in sub‐Saharan Africa for decades, and affordable and multivalent vaccines, effective in all age groups, are needed. A bivalent serogroup A and W (A + W) meningococcal vaccine candidate consisting of deoxycholate‐extracted outer membrane vesicles (OMV) from representative African disease isolates was previously found to be highly immunogenic in outbred mice when formulated with the adjuvant aluminium hydroxide (AH). OMV has been shown to have inherent adjuvant properties. In order to study the importance of AH and genetical differences between mice strains on immune responses, we compared the immunogenicity of the A + W OMV vaccine when formulated with or without AH in inbred C57BL/6J and BALB/cJ mice (Th1 and Th2 dominant strains, respectively). The immunogenicity of the vaccine was found to be comparable in the two mice strains despite their different immune profiles. Adsorption to AH increased anti‐OMV IgG levels and serum bactericidal activity (SBA). The immune responses were increased by each dose for the adsorbed vaccine, but the third dose did not significantly improve the immunogenicity further. Thus, a vaccine formulation with the A and W OMV will likely benefit from including AH as adjuvant.     

A non-allergenic Ole e 1-like protein from birch pollen as a tool to design hypoallergenic vaccine candidates

Recombinant DNA technology offers several approaches to convert allergens into hypoallergenic derivatives that can represent the basis of novel, safer and more effective forms of allergy vaccines. In this context, we used a new strategy for the design of a hypoallergenic derivative of Ole e 1, the main allergen of olive pollen. By screening a cDNA library from birch pollen, the clone BB18, encoding the birch counterpart of Ole e 1, was identified. In this study, BB18 has been produce in Pichia pastoris as a recombinant protein and immunologically characterized. The well-established non-allergenic properties of BB18 were used to generate a genetic variant of Ole e 1, named OB(55-58), by site-direct mutagenesis of four residues (E(55)V(56)G(57)Y(58)) in an IgE/IgG epitope of Ole e 1 by the corresponding ones in BB18 (SDSE). OB(55-58) was expressed in P. pastoris, purified to homogeneity and analyzed for IgE-reactivity by means of ELISA using sera from olive pollen allergic patients and rat basophil activation assay. T cell reactivity was assayed in a mouse model of Ole e 1 sensitization. The mutant OB(55-58) exhibited an impaired IgE reactivity, but not affected T cell reactivity, compared to wild type rOle e 1. This study emphasizes the usefulness of BB18 as a tool for epitope mapping and for engineering hypoallergenic derivatives of Ole e 1 as vaccine candidates for allergy prevention and treatment.     

IL-17 and IFN-γ expression in lymphocytes from patients with active tuberculosis correlates with the severity of the disease

Th1 lymphocytes are crucial in the immune response against Mycobacterium tuberculosis. Nevertheless, IFN-γ alone is not sufficient in the complete eradication of the bacteria, suggesting that other cytokines might be required for pathogen removal. Th17 cells have been associated with M. tuberculosis infection, but the role of IL-17-producing cells in human TB remains to be understood. Therefore, we investigated the induction and regulation of IFN-γ and IL-17 during the active disease. TB patients were classified as High and Low Responder individuals according to their T cell responses against the antigen, and cytokine expression upon M. tuberculosis stimulation was investigated in peripheral blood and pleural fluid. Afterwards, the potential correlation among the proportions of cytokine-producing cells and clinical parameters was analyzed. In TB patients, M. tuberculosis induced IFN-γ and IL-17, but in comparison with BCG-vaccinated healthy donors, IFN-γ results were reduced significantly, and IL-17 was markedly augmented. Moreover, the main source of IL-17 was represented by CD4(+)IFN-γ(+)IL-17(+) lymphocytes, a Th1/Th17 subset regulated by IFN-γ. Interestingly, the ratio of antigen-expanded CD4(+)IFN-γ(+)IL-17(+) lymphocytes, in peripheral blood and pleural fluid from TB patients, was correlated directly with clinical parameters associated with disease severity. Indeed, the highest proportion of CD4(+)IFN-γ(+)IL-17(+) cells was detected in Low Responder TB patients, individuals displaying severe pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4(+)IFN-γ(+)IL-17(+) T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.     

The diagnosis and management of allergic reactions in patients sensitized to non-specific lipid transfer proteins

Sensitization to one or more non-specific lipid transfer proteins (nsLTPs), initially thought to exist mainly in southern Europe, is becoming accepted as a cause of allergic reactions to plant foods across Europe and beyond. The peach nsLTP allergen Pru p 3 is a dominant sensitizing allergen and peaches a common food trigger, although multiple foods can be involved. A frequent feature of reactions is the requirement for a cofactor (exercise, alcohol, non-steroidal anti-inflammatory drugs, Cannabis sativa) to be present for a food to elicit a reaction. The variability in the food and cofactor triggers makes it essential to include an allergy-focused diet and clinical history in the diagnostic workup. Testing on suspected food triggers should also establish whether sensitization to nsLTP is present, using purified or recombinant nsLTP allergens such as Pru p 3. The avoidance of known trigger foods and advice on cofactors is currently the main management for this condition. Studies on immunotherapy are promising, but it is unknown whether such treatments will be useful in populations where Pru p 3 is not the primary sensitizing allergen. Future research should focus on the mechanisms of cofactors, improving diagnostic accuracy and establishing the efficacy of immunotherapy.