The development of vaccines and effective diagnostic methods for bovine tuberculosis requires an understanding of the immune response against its causative agent, Mycobacterium bovis. Although this disease is primarily investigated and diagnosed through the assessment of cell-mediated immunity, the role of B cells and antibodies in bovine tuberculosis has been relatively undervalued and understudied. Current evidence indicates that circulating M. bovis-specific antibodies are not effective in controlling the disease. However, local humoral immune responses may contribute to either defence or pathology. Recent studies in animal models and cattle vaccine trials suggest a potential beneficial role of B cells in tuberculosis control. This review discusses the role of B cells and antibodies in bovine tuberculosis and explores antibody-based diagnostics for the disease, including traditional techniques, such as different ELISA, new platforms based on multiple antigens and point-of-care technologies. The high specificity and sensitivity values achieved by numerous antibody-based tests support their use as complementary tests for the diagnosis of bovine tuberculosis, especially for identifying infected animals that may be missed by the official tests.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that mainly affects cattle. The traditional bTB control strategy is to slaughter animals positive for the official diagnostic test. This method, however, may not be economically feasible in developing countries and is less effective when cattle are in contact with wildlife reservoirs of infection. In these scenarios, cattle vaccination could be an important strategy to control bTB. In this review, we describe the diversified vaccine development against bTB and identify gaps in current knowledge. We discuss progress made in live attenuated (including BCG), inactivated and subunit vaccines tested in preclinical models and field trials. We also provide an overview of existing knowledge in the correlates and biomarkers of protection after vaccination against bTB, as well as the most recent advances in the understanding of the protective immune response against tuberculosis. The similarities between the mechanisms of the immune protection elicited by humans and cattle against TB and bTB, respectively, make any advance in human TB and bTB vaccines mutually beneficial. In accordance with the One Health concept, this mutual benefit is expected to progress in the future.

Background Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. Results To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes ( Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa ). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. Conclusions This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.

Mammalian cell entry (mce) genes are not only present in genomes of pathogenic mycobacteria, including Mycobacterium tuberculosis (the causative agent of tuberculosis), but also in saprophytic and opportunistic mycobacterial species. MCE are conserved cell-wall proteins encoded by mce operons, which maintain an identical structure in all mycobacteria: two yrbE genes (A and B) followed by six mce genes (A, B, C, D, E and F). Although these proteins are known to participate in the virulence of pathogenic mycobacteria, the presence of the operons in nonpathogenic mycobacteria and other bacteria indicates that they play another role apart from host cell invasion. In this respect, more recent studies suggest that they are functionally similar to ABC transporters and form part of lipid transporters in Actinobacteria. To date, most reviews on mce operons in the literature discuss their role in virulence. However, according to data from transcriptional studies, mce genes, particularly the mce1 and mce4 operons, modify their expression according to the carbon source and upon hypoxia, starvation, surface stress and oxidative stress; which suggests a role of MCE proteins in the response of Mycobacteria to external stressors. In addition to these data, this review also summarizes the studies demonstrating the role of MCE proteins as lipid transporters as well as the relevance of their transport function in the interaction of pathogenic Mycobacteria with the hosts. Altogether, the evidence to date would indicate that MCE proteins participate in the response to the stress conditions that mycobacteria encounter during infection, by participating in the cell wall remodelling and possibly contributing to lipid homeostasis.

Abstract Introduction Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) are cytokines widely used in ex vivo monocyte differentiation experiments, vaccine formulations and disease treatment. The aim of this study was to produce recombinant bovine GM-CSF and IL-4 in an episomal expression system that conserves the postransductional modification of the native proteins and to use the products to differentiate bovine monocytes into dendritic cells. Material and Methods The recombinant proteins rGM-CSF and rIL-4 were expressed in PEAKrapid CRL-2828 human kidney cells, ATCC CRL-2828. The functional activity of the recombinant cytokines was monitored by registering morphological changes in bovine monocytes and assessing the expression of CD14 upon incubation with them. Results Both recombinant proteins were detected in the cell culture supernatant of transfected cells. Culture supernatants of transfected cells induced in bovine monocytes morphological changes that resemble macrophages or dendritic cells. In addition, bovine cells treated with rGM-CSF and rIL-4 showed reduced expression of the macrophage surface marker CD14 compared with untreated cells. This effect indicates the expected differentiation. The expression of the cytokines was stable after many successive cell passages and a freeze/thaw cycle. Conclusions The semi-stable mammalian episomal expression system used in this study allowed us to easily produce functional bovine rGM-CSF and rIL-4 without the need for protein purification steps.

The study of outer membrane vesicles (OMVs) became relevant because of their probable important role in the transfer of virulence factors to host cells. Campylobacter fetus is mainly a mammal pathogen whose virulence characterization is still limited. The aim of this study was to evaluate and to characterize the secretion of OMVs in this bacterium. By transmission electron microscopy, we confirmed the production of OMVs in all the strains assayed. Purified OMVs showed a spherical shape and variable size, although comparable to those of other gram-negative bacteria. We also confirmed the presence of the S-layer on the surface of the OMVs of all the strains assayed with the exception of those derived from the NTCC reference strain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C. fetus OMVs could contribute to the modulation of the host response and constitute a candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field. This work represents a platform to drive future studies towards the role of these subcellular structures in C. fetus-host interaction.

The gastrointestinal tract of chickens harbors a highly diverse microbiota contributing not only to nutrition, but also to the physiological development of the gastrointestinal tract. Microbiota composition depends on many factors such as the portion of the intestine as well as the diet, age, genotype, or geographical origin of birds. The aim of the present study was to demonstrate the influence of the geographical location over the cecal microbiota from broilers. We used metabarcoding sequencing datasets of the 16S rRNA gene publicly available to compare the composition of the Argentine microbiota against the microbiota of broilers from another seven countries (Germany, Australia, Croatia, Slovenia, United States of America, Hungary, and Malaysia). Geographical location played a dominant role in shaping chicken gut microbiota (Adonis R2 = 0.6325, P = 0.001; Mantel statistic r = 0.1524, P = 4e-04) over any other evaluated factor. The geographical origin particularly affected the relative abundance of the families Bacteroidaceae, Lactobacillaceae, Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. Because of the evident divergence of microbiota among countries we coined the term "local microbiota" as convergent feature that conflates non-genetic factors, in the perspective of human-environmental geography. Local microbiota should be taken into consideration as a native overall threshold value for further appraisals when testing the production performance and performing correlation analysis of gut microbiota modulation against different kind of diet and/or management approaches. In this regard, we described the Argentine poultry cecal microbiota by means of samples both from experimental trials and commercial farms. Likewise, we were able to identify a core microbiota composed of 65 operational taxonomic units assigned to seven phyla and 38 families, with the four most abundant taxa belonging to Bacteroides genus, Rikenellaceae family, Clostridiales order, and Ruminococcaceae family.

In recent years, foliar inoculation has gained acceptance among the available methods to deliver plant beneficial micro-organisms to crops under field conditions. Colonization efficiency by such micro-organisms largely depends on their ability to survive when applied on the leaves. In this work, we evaluated the survival and localization of Azospirillum brasilense Az39 (Az39) in excised soybean leaves. Scanning electron microscopy and confocal laser scanning microscopy of a red fluorescent-transformed variant of Az39 were used to determine bacterial localization, while the most probable number and plate count methods were applied for bacterial quantification. Microscopic observations indicated a decrease in the number of Az39 cells on the leaf surface at 24 h after treatment, whereas midribs and cell-cell junctions of the inner leaf epidermis became highly populated zones. The presence of Az39 inside xylem vessels was corroborated at 6 h after bacterization. Az39 population did not significantly decrease throughout 24 h. We could visualize Az39 cells on the surface and in internal tissues of soybean leaves and recover them through culture methodologies. These results evidence the survival capacity of Az39 on and inside leaves and suggest a previously unnoticed endophytic potential for this well-known plant growth-promoting rhizobacteria strain.

The morphology and physiology of the oogenesis have been well studied in the vector of Chagas disease Rhodnius prolixus. However, the molecular interactions that regulate the process of egg formation, key for the reproductive cycle of the vector, is still largely unknown. In order to understand the molecular and cellular basis of the oogenesis, we examined the function of the gene Bicaudal C (BicC) during oogenesis and early development of R. prolixus. We show that R. prolixus BicC (Rp-BicC) gene is expressed in the germarium, with cytoplasmic distribution, as well as in the follicular epithelium of the developing oocytes. RNAi silencing of Rp-BicC resulted in sterile females that lay few, small, non-viable eggs. The ovaries are reduced in size and show a disarray of the follicular epithelium. This indicates that Rp-BicC has a central role in the regulation of oogenesis. Although the follicular cells are able to form the chorion, the uptake of vitelline by the oocytes is compromised. We show evidence that the polarity of the follicular epithelium and the endocytic pathway, which are crucial for the proper yolk deposition, are affected. This study provides insights into the molecular mechanisms underlying oocyte development and show that Rp-BicC is important for de developmental of the egg and, therefore, a key player in the reproduction of this insect.

Bovine tuberculosis is an important worldwide disease mainly related to cattle, although it also affects other mammals, including humans. In recent years, there have been considerable advances in the knowledge of the immune response mechanisms underlying the interaction of Mycobacterium bovis, the main agent of bovine tuberculosis, with its hosts. In this review we describe the most recent findings on the cattle immune response to M. bovis, particularly regarding trained innate immune responses and γδ T cells, that could support the development of vaccines and diagnostic tools to control this disease.

Members of the Mycobacterium tuberculosis complex (MTBC) are responsible for tuberculosis in several mammals. In this complex, Mycobacterium tuberculosis and Mycobacterium bovis, which are closely related, show host preference for humans and cattle, respectively. Although human and bovine tuberculosis are clinically similar, M. tuberculosis mostly causes latent infection in humans, whereas M. bovis frequently leads to an acute infection in cattle. This review attempts to connect the pathology in experimental animal models as well as the cellular responses to M. bovis and M. tuberculosis regarding the differences in protein expression and regulatory mechanisms of both pathogens that could explain their apparent divergent latency behaviour. The occurrence of latent bovine tuberculosis (bTB) would represent a serious complication for the eradication of the disease in cattle, with the risk of onward transmission to humans. Thus, understanding the physiological events that may lead to the state of latency in bTB could assist in the development of appropriate prevention and control tools.

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AbstractThe morphology and physiology of the oogenesis have been well studied in the vector of Chagas diseaseRhodnius prolixus. However, the molecular interactions that regulate the process of egg formation, key for the reproductive cycle of the vector, is still largely unknown. In order to understand the molecular and cellular basis of the oogenesis we examined the function of the geneBicaudal C(BicC) during oogenesis and early development ofR. prolixus. We show thatR. prolixus BicC(Rp-BicC) gene is expressed in the germarium, with cytoplasmic distribution, as well as in the follicular epithelium of the developing oocytes. RNAi silencing ofRp-BicCresulted in sterile females that lay few, small, non-viable eggs. The ovaries are reduced in size and show a disarray of the follicular epithelium. This indicates thatRp-BicChas a central role in the regulation of oogenesis. Although the follicular cells are able to form the chorion, the uptake of vitelline by the oocytes is compromised. We show evidence that the polarity of the follicular epithelium and the endocytic pathway, which is crucial for the proper yolk deposition, are affected. This study provides insights into the molecular mechanisms underlying oocyte development and show thatRp-BicCis important for de developenta of the egg and, therefore, a key player in the reproduction of this Chagas disease vector.Author summaryThe oogenesis is the process of egg formation. It is essential to guarantee transgenerational inheritance. It implies the differentiation of the gamete (oocyte) from a niche of stem cells in the germ line, the accumulation of yolk, and the formation of the chorion. These events are entangled in a regulated manner by the concerted communication between the different cell types that form the ovary. It is regulated by endogenous gene networks and linked to the physiological state of the insect by hormonal clues. This timely orchestrated process represents the interaction of gene networks. The genetic regulation behind the oogenesis is largely unknown inRhodnius prolixus. Here we identified a gene required for egg formation that interferes the uptake of the yolk by affecting the functional integrity of the follicular epithelium. Our results are of interest for a better understanding of a complex process essential for the survival of vector populations and provide knowledge to envisage and design new strategies for vector control.

IntroductionTo date, most studies of natural variation and metabolite quantitative trait loci (mQTL) in tomato have focused on fruit metabolism, leaving aside the identification of genomic regions involved in the regulation of leaf metabolism.ObjectiveThis study was conducted to identify leaf mQTL in tomato and to assess the association of leaf metabolites and physiological traits with the metabolite levels from other tissues.MethodsThe analysis of components of leaf metabolism was performed by phenotypying 76 tomato ILs with chromosome segments of the wild species Solanum pennellii in the genetic background of a cultivated tomato (S. lycopersicum) variety M82. The plants were cultivated in two different environments in independent years and samples were harvested from mature leaves of non-flowering plants at the middle of the light period. The non-targeted metabolite profiling was obtained by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). With the data set obtained in this study and already published metabolomics data from seed and fruit, we performed QTL mapping, heritability and correlation analyses.ResultsChanges in metabolite contents were evident in the ILs that are potentially important with respect to stress responses and plant physiology. By analyzing the obtained data, we identified 42 positive and 76 negative mQTL involved in carbon and nitrogen metabolism.ConclusionsOverall, these findings allowed the identification of S. lycopersicum genome regions involved in the regulation of leaf primary carbon and nitrogen metabolism, as well as the association of leaf metabolites with metabolites from seeds and fruits.

Bovines are the primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the main source of its transmission to humans. Here, we present a one-year longitudinal study of fecal shedding of E. coli O157. E. coli O157 obtained from recto-anal mucosal samples were characterized by multiplex PCR. The E. coli O157 prevalence ranged from 0.84% in July to 15.25% in November. The confinement within pens resulted in prevalence of 11%. Most animals (61.86%; 75/118) shed E. coli O157 at least in one sampling occasion. Of the positive animals, 82.19%, 16.44%, and 1.37% were stx positive on one, two and three sampling occasions, respectively. All the E. coli O157 isolated strains carried the genes eae and rfbO157, whereas 11%, 33% and 56% contained stx1, stx2 and stx1/stx2, respectively. The stx1/stx2 and stx2 types were significantly higher during the grazing and finishing periods, respectively, in comparison with the rearing and grazing periods. The presence of stx2a subtype was evident in four isolates, whereas stx2c was present in at least seven. However, both subtypes were present simultaneously in two isolates. The stx1/stx2c, stx1/stx2d and stx1/stx2NT genotypes occurred in 24, 2 and 15 isolates, respectively. The simultaneous occurrence of stx1 and stx2c significantly increased during grazing. Some cases of within-pen and between-pen transmission occurred throughout the study. Contagion levels during in-field grazing were higher than during permanent confinement in the pens. Thus, the individual patterns of shedding varied depending on the proportion of animals shedding the bacteria within pens and the time of shedding.

The genetic diversity of 14 Japanese plum (Prunus salicina Lindl) landraces adapted to an ecosystem of alternating flooding and dry conditions was characterized using neutral simple sequence repeat (SSR) markers. Twelve SSRs located in six chromosomes of the Prunus persica reference genome resulted to be polymorphic, thus allowing identification of all the evaluated landraces. Differentiation between individuals was moderate to high (average shared allele distance (DAS) = 0.64), whereas the genetic diversity was high (average indices polymorphism information content (PIC) = 0.62, observed heterozygosity (Ho) = 0.51, unbiased expected heterozygosity (uHe) = 0.70). Clustering and genetic structure approaches grouped all individuals into two major groups that correlated with flesh color. This finding suggests that the intuitive breeding practices of growers tended to select plum trees according to specific phenotypic traits. These neutral markers were adequate for population genetic studies and cultivar identification. Furthermore, we assessed the SSR flanking genome regions (25 kb) in silico to search for candidate genes related to stress resistance or associated with other agronomic traits of interest. Interestingly, at least 26 of the 118 detected genes seem to be related to fruit quality, plant development, and stress resistance. This study suggests that the molecular characterization of specific landraces of Japanese plum that have been adapted to extreme agroecosystems is a useful approach to localize candidate genes which are potentially interesting for breeding.

The Mce proteins are encoded in a variable number of operons (from one to eight) in all Mycobacterium species. A role in the transport of host and mycobacterial lipids has been demonstrated for some Mce proteins in the pathogen Mycobacterium tuberculosis but little is known about these proteins in Mycobacterium smegmatis, a soil dweller species.

The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world.

Immunodominant MPB83 antigen from Mycobacterium bovis was expressed as a chimeric protein fused to either β-galacotosidase, outer membrane lipoprotein OMP19 or periplasmic protein BP26 in gram-negative Brucella abortus S19, in all cases driven by each gene’s own promoter. All fusion proteins were successfully expressed and localized in the expected subcellular fraction. Moreover, OMP19-MPB83 was processed as a lipoprotein when expressed in B. abortus. Splenocytes from BALB/c mice immunized with the recombinant S19 strains carrying the genes coding for the heterologous antigens in replicative plasmids, showed equally specific INF-γ production in response to MPB83 stimulation. Association to the lipid moiety of OMP19 presented no advantage in terms of immunogenicity for MPB83. In contrast, fusion to BP26, which was encoded by an integrative plasmid, resulted in a weaker immune response. None of the constructions affected the survival rate or the infection pattern of Brucella. We concluded that B. abortus S19 is an appropriate candidate for the expression of M. bovis antigens both associated to the membrane or cytosolic fraction and may provide the basis for a future combined vaccine for bovine brucellosis and tuberculosis.