A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Serum prevalence of anti-Trypanosoma vivax antibodies in cattle in Tapira-MG, Brazil / Soroprevalência de anticorpos anti-Trypanosoma vivaxem bovinos em Tapira-MG, Brasil

Trypanosoma vivax is considered the most important pathogenic Trypanosoma for cattle and causes great damage to the dairy and beef cattle industries. This study aimed to evaluate the prevalence of anti-T. vivax antibodies in dairy cattle from the municipality of Tapira, located in the Alto Paranaíba region, Minas Gerais, Brazil. The 74 blood serum samples from dairy cattle were analyzed using an indirect immunofluorescence reaction. The seroprevalence was 82.4 % (61/74), and the highest incidence observed can be correlated with the transit of untested animals, the presence of vectors, and needle sharing by owners. The data allowed defining Tapira as an area of expansion of T. vivax epizootic infections in the state of Minas Gerais.

O Trypanosoma vivax é considerado o mais importante trypanosoma patogênico para bovinos e causa grandes prejuízos na pecuária de corte e leite. Este estudo teve como objetivo avaliar a prevalência anticorpos de anti-Trypanosoma vivax em bovinos leiteiros do município de Tapira, localizado na região do Alto Paranaíba, Minas Gerais, Brasil. As 74 amostras de soro sanguíneo de bovinos leiteiros foram analisadas por meio de reação de imunofluorescência indireta. A soroprevalência foi de 82,4% (61/74), que pode estar relacionada ao trânsito de animais não testados, presença de vetores e compartilhamento de agulhas pelos proprietários. Os dados permitiram definir Tapira como uma área de expansão das infecções epizoóticas por Trypanosoma vivax no estado de Minas Gerais.

Identification of Leptospiral Protein Antigens Recognized by WC1+ γδ T Cell Subsets as Target for Development of Recombinant Vaccines.

Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. γδ T cells play a role in the protective immune response in livestock species against Leptospira, while human γδ T cells also respond to Leptospira. Thus, activation of γδ T cells has emerged as a potential component in the optimization of vaccine strategies. Bovine γδ T cells proliferate and produce gamma interferon (IFN-γ) in response to vaccination with inactivated leptospires, and this response is mediated by a specific subpopulation of the WC1-bearing γδ T cells. WC1 molecules are members of the group B scavenger receptor cysteine-rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and γδ TCR coreceptors, the WC1 system has been proposed as a novel target to engage γδ T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ γδ T cells and identify two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific γδ T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+ γδ T cells respond. Identification of protein antigens will enhance our understanding of the role γδ T cells play in the leptospiral immune response and in recombinant vaccine development.

Histophilus somni stimulates bovine monocyte-derived macrophages to release microparticles that increase fibrin clot formation in vitro.

Histophilus somni is a Gram-negative coccobacillus that causes diffuse vasculitis and intravascular thrombosis that can lead to multiple organ failure in cattle. Macrophages are important cellular mediators of fibrin deposition and removal at sites of inflammation. It has become evident that macrophages and other cells release microparticles (MPs) that have an array of biological activities, including pro-coagulant activity. We sought to determine whether monocyte-derived macrophages exposed to H. somni in vitro release MPs that activate the clotting cascade in a manner that could lead to thrombus formation. Bovine monocyte-derived macrophages were incubated with H. somni (at a 10:1 ratio) in RPMI with 10% heat inactivated fetal bovine serum for 6 h at 37 °C with 5 % CO2. Membrane-shed MPs were isolated from the conditioned media, washed twice with Ca2+ and Mg2+ free HBSS, and pro-coagulant activity assessed by a one-step plasma clotting assay. We observed greater pro-coagulant activity for MPs from H. somni stimulated macrophages than from unstimulated controls. Microparticle pro-coagulant activity was inhibited by addition of an anti-tissue factor antibody. We also observed co-localization of fluorescein-labeled H. somni cells and annexin V staining as evaluated by confocal microscopy. These results demonstrate that exposure to H. somni cells causes bovine monocyte-derived macrophages to release MPs that contain tissue factor, the first such report for bovine macrophages. We infer that if similar events occur in vivo they could amplify thrombus formation in bovine histophilosis.

Effects of inflammatory stimuli on responses of macrophages to Mycoplasma bovis infection.

Inflammation in the respiratory tract is thought to worsen the disease response to Mycoplasma bovis infection. This study investigated the cells involved in this response with a focus on proteases and cytokines as harmful effector mechanisms. By immunohistochemistry, Mac387-positive macrophages were the main cell type comprising the foci of caseous necrosis in cattle with M. bovis pneumonia. Thus, the study evaluated how priming of different types of macrophages with bacterial lysate (or pro-inflammatory cytokines induced by the bacterial lysate) affected their responses to M. bovis infection. Inducible responses were detected in monocyte-derived macrophages (M1-MDMs and M2-MDMs), whereas pulmonary alveolar macrophages (PAMs) were minimally affected by priming or infection. M. bovis-infected MDMs secreted MMP-12 and SPLA2, and priming with pro-inflammatory cytokines increased the secretion of cathepsin B in response to M. bovis infection. Of these, there were higher concentrations of cathepsin B and SPLA2 in lungs with M. bovis pneumonia compared to healthy lungs, and these are potential mechanisms for macrophage-induced lung damage in M. bovis infection. Priming of MDMs with either bacterial lysate or with pro-inflammatory cytokines caused an enhanced response to M. bovis infection with respect to IL-8 and IL-1ß secretion. The findings of this study suggest proteases, lipases and cytokines derived from monocyte-derived macrophages as possible mediators by which prior inflammation in the respiratory tract worsen disease outcomes from M. bovis infection.

MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene.

Mastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3' untranslated region (3' UTR) of the NKIRAS2, but not the 3'UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

Changes in serum biomarkers of inflammation in bovine besnoitiosis.

BACKGROUND: Acute and chronic besnoitiosis in extensive natural-service herds can have relevant effects in the health of bulls and negative consequences in their productive performance. Recent progress has been made in order to elucidate the pathogenesis of this disease. In this context, the study of biomarkers of inflammation in serum would contribute to gaining knowledge about the physiopathology of bovine besnoitiosis. Serological biomarkers could help in early diagnosis and prognosis, as seropositive bulls may have mild or severe testicular lesions. METHODS: Herein, we have investigated the diagnostic and/or prognostic value of a panel of serum (serological) biomarkers related to inflammation, including total protein, globulin and albumin, haptoglobin (Hp), adenosine deaminase (ADA) paraoxonase-1 (PON-1) and acetylcholinesterase (AChE) in naturally and experimentally B. besnoiti-infected males classified according to different clinical phases of the disease (acute, chronic and subclinical besnoitiosis). RESULTS: Results showed a similar response pattern in these biomarkers for naturally and experimentally infected cattle, with a few relevant variations. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during the chronic infection. Haptoglobin, albumin, PON-1 and ADA were identified as the biomarkers that showed changes of higher magnitude in the acute phase of the infection, whereas high total protein and globulin values were found in chronically infected cattle. We have described the changes of a panel of inflammatory biomarkers of acute and chronic bovine besnoitiosis. CONCLUSIONS: In summary, several biomarkers with promising diagnostic value have been identified. The biomarkers associated with acute infection are related to previously reported molecular biomarkers in testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement results from specific immunoglobulin M (IgM) detection.

Involvement of the VEGF signaling pathway in immunosuppression and hypoxia stress: analysis of mRNA expression in lymphocytes mediating panting in Jersey cattle under heat stress.

BACKGROUND: Extreme panting under heat stress threatens dairy cattle milk production. Previous research has revealed that the gas exchange-mediated respiratory drive in critically ill dairy cattle with low O2 saturation induces panting. Vascular endothelial growth factor (VEGF) signaling may play important roles in immunosuppression and oxidative stress during severe respiratory stress responses in heat-stressed cattle. The objectives of this study were to transcriptomically analyze mRNA expression mediating heat-induced respiratory stress-associated panting, evaluate gas exchange, screen hub genes, and verify the expression of proteins encoded by differentially expressed genes in lymphocyte pathways. RESULTS: Jersey cattle were naturally heat-exposed. Physiological data were collected for response evaluation, and blood was collected for gas exchange and gene expression assays at 06:00, 10:00 and 14:00 continuously for 1 week. Lymphocytes were isolated from whole-blood samples for mRNA-seq and expression analysis of key pathway genes/proteins. The cattle respiration rates differed with time, averaging 51 bpm at 06:00, 76 bpm at 10:00, and 121 bpm at 14:00 (p < 0.05). Gas exchange analysis showed that both pH and pCO2 differed with time: they were 7.41 and 41 mmHg at 06:00, 7.45 and 37.5 mmHg at 10:00, and 7.49 and 33 mmHg at 14:00, respectively (p < 0.01). Sixteen heat-related differentially expressed genes (DEGs; 13 upregulated and 3 downregulated) were screened between 212 DEGs and 1370 heat stress-affected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) hub gene functional analysis annotated eleven genes to signal transduction, six genes to the immune response, and five genes to the endocrine response, including both prostaglandin-endoperoxide synthase 2 (PTGS2) and VEGF. Gene Ontology (GO) functional enrichment analysis revealed that oxygen regulation was associated with the phosphorus metabolic process, response to oxygen levels, response to decreased oxygen levels, response to hypoxia and cytokine activity terms. The main signaling pathways were the VEGF, hypoxia inducible factor-1(HIF-1), cytokine-cytokine receptor interaction and TNF pathways. Four genes involved Integrin beta 3 (ITBG3), PTGS2, VEGF, and myosin light chain 9 (MYL9) among the 16 genes related to immunosuppression, oxidative stress, and endocrine dysfunction were identified as participants in the VEGF signaling pathway and oxygenation. CONCLUSION: These findings help elucidate the underlying immune and oxygen regulation mechanisms associated with the VEGF signaling pathway in heat-stressed dairy cattle.

Intramammary infection caused by Staphylococcus aureus increases IgA antibodies to iron-regulated surface determinant-A, -B, and -H in bovine milk.

The aim of this study was to identify virulence factors that have high immunogenicity. An in vivo-expressed Staphylococcus aureus antigen was identified by probing bacteriophage expression libraries of S. aureus with antibodies in bovine mastitis milk. Eighteen clones were isolated, and their proteins were identified as 5 characterised proteins (IsdA, Protein A, IsdB, autolysin, and imidazole glycerol phosphate dehydratase) and 13 hypothetical proteins. We focused on IsdA, IsdB, and IsdH as virulence factors that have a high immunogenicity and are capable of inducing a specific humoral immune response in S. aureus-infected quarters. The optical density (OD) values of IsdA and IsdB IgA and IgG antibodies in milk affected by naturally occurring mastitis caused by S. aureus increased significantly compared to those in healthy milk. In the experimental infection study, the OD values of IsdA- and B-specific IgA and IgG antibodies were significantly increased from 2 to 4 weeks after S. aureus infection compared to day 0 (P < 0.05). On the other hand, we demonstrated that milk from natural and experimental intramammary infections caused by S. aureus are associated with significantly higher IgA levels against IsdH (P < 0.05), but no significant change in IgG levels. Our findings facilitated our understanding of the pathogenicity of S. aureus in bovine mastitis, as well as the mechanisms by which specific humoral immune responses to S. aureus infection are induced. In addition, the results obtained could provide insight into how bovine mastitis can be controlled, for example, through vaccination.

Adjuvant effect of saponin in an oil-based monovalent (serotype O) foot-and-mouth disease virus vaccine on the antibody response in guinea pigs and cattle.

To enhance the potency of a foot-and-mouth disease (FMD) vaccine, saponin was included in the vaccine formula. In this study, the combined effect of Montanide ISA 50 and saponin was evaluated. Two experiments were performed in guinea pigs and one in cattle to determine the optimal antigen and saponin doses. Only serotype O of foot-and-mouth disease virus (O/PanAsia-2 of ME-SA topotype) was employed in preparation of the monovalent vaccine. All animals were immunized twice with a four-week interval, except for the negative controls. Blood was collected 10 days after the second booster, and the immune response was evaluated using a serum neutralization test. Oil-based FMD vaccines containing saponin induced higher neutralizing antibody levels than formulations lacking saponin. The addition of saponin to formulations with low antigen payload (2.5 µg of inactivated whole virus particles [146S particles] per dose) gave significantly higher neutralizing antibody levels (p < 0.005) than 5 µg of 146S without saponin, suggesting that it can be used to improve FMD vaccine potency in susceptible animals. No adverse effects were observed in vaccinated cattle or guinea pigs.

Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in Mycobacterium avium subsp. paratuberculosis clearance In Vitro.

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guérin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1ß and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1ß and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.

Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells.

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1ß, TNF-α, and IL-8. In this study, the expression of IL-1ß, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1ß and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1ß and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.

Alterations in TNF-α and its receptors expression in cows undergoing heat stress.

Heat stress is one of the environmental factors that most severely affects milk industry, as it has impact on production, immune responses and reproductive performance. The present study was conducted with high-performance Holando-Argentino cows. Our objective was to study TNF-α and its receptors pattern expression in cows from a region characterized by extreme climatic seasonality. Animals were evaluated in three periods: spring (n = 15), summer (n = 14) and autumn (n = 11). Meteorological records from a local station were used to estimate the temperature and humidity index (THI) by means of an equation previously defined. A THI higher than 68 is indicative of stressing conditions. During the summer period, the animals were exposed to 8.5 ±â€¯1.09 h of heat stress, or THI > 68. In spring, stress hours were reduced to 1.4 ±â€¯0.5 every day, while during the autumn, there were no recorded heat stress events. Expression of TNF-α, and its receptors was determined by qPCR. During the summer, TNF-α and its receptors expression diminished drastically compared to the rest of the year, when stressful conditions were infrequent. We conclude that animals that are not physiologically prepared to resist high temperatures might have a less efficient immune response, reinforcing the need to develop new strategies to improve animal welfare.

Ostertagia ostertagi Mediates Early Host Immune Responses via Macrophage and Toll-Like Receptor Pathways.

Ostertagia ostertagi is an abomasal parasite with significant economic impact on the cattle industry. Early host immune responses are poorly understood. Here, we examined time course expression of Toll-like receptors (TLRs) in peripheral blood mononuclear cells (PBMC) during infection where PBMC macrophages (Mϕ) generated both pro- and anti-inflammatory responses when incubated with excretory/secretory products (ESP) from fourth-stage larvae (OoESP-L4) or adult worms (OoESP-Ad). First, changes in cell morphology clearly showed that both OoESP-L4 and OoESP-Ad activated PBMC-Mϕ in vitro, resulting in suppressed CD40 and increased CD80 expression. Expression of mRNAs for TLR1, -4, -5, and -7 peaked 7 days postinfection (dpi) (early L4), decreased by 19 dpi (postemergent L4 and adults) and then increased at 27 dpi (late adults). The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) (transcript and protein) increased in the presence of OoESP-Ad, and the anti-inflammatory cytokine interleukin 10 (IL-10) (protein) decreased in the presence of OoESP-L4 or OoESP-Ad; however, IL-10 mRNA was upregulated, and IL-6 (protein) was downregulated by OoESP-L4. When PBMC-Mϕ were treated with ligands for TLR4 or TLR5 in combination with OoESP-Ad, the transcripts for TNF-α, IL-1, IL-6, and IL-10 were significantly downregulated relative to treatment with TLR4 and TLR5 ligands only. However, the effects of TLR2 ligand and OoESP-Ad were additive, but only at the lower concentration. We propose that O. ostertagi L4 and adult worms utilize competing strategies via TLRs and Mϕ to confuse the immune system, which allows the worm to evade the host innate responses.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Interaction between transforming Theileria parasites and their host bovine leukocytes.

Theileria are tick-transmitted parasites that cause often fatal leuko-proliferative diseases in cattle called tropical theileriosis (T. annulata) and East Coast fever (T. parva). However, upon treatment with anti-theilerial drug-transformed leukocytes die of apoptosis indicating that Theileria-induced transformation is reversible making infected leukocytes a powerful example of how intracellular parasites interact with their hosts. Theileria-transformed leukocytes disseminate throughout infected cattle causing a cancer-like disease and here, we discuss how cytokines, noncoding RNAs and oncometabolites can contribute to the transformed phenotype and disease pathology.

Regionally Distinct Immune and Metabolic Transcriptional Responses in the Bovine Small Intestine and Draining Lymph Nodes During a Subclinical Mycobacterium avium subsp. paratuberculosis Infection.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative infectious agent of Johne's disease (JD), an incurable granulomatous enteritis affecting domestic livestock and other ruminants around the world. Chronic MAP infections usually begin in calves with MAP uptake by Peyer's patches (PP) located in the jejunum (JE) and ileum (IL). Determining host responses at these intestinal sites can provide a more complete understanding of how MAP manipulates the local microenvironment to support its long-term survival. We selected naturally infected (MAPinf, n=4) and naive (MAPneg, n=3) cows and transcriptionally profiled the JE and IL regions of the small intestine and draining mesenteric lymph nodes (LN). Differentially expressed (DE) genes associated with MAP infection were identified in the IL (585), JE (218), jejunum lymph node (JELN) (205), and ileum lymph node (ILLN) (117). Three DE genes (CD14, LOC616364 and ENSBTAG00000027033) were common to all MAPinf versus MAPneg tissues. Functional enrichment analysis revealed immune/disease related biological processes gene ontology (GO) terms and pathways predominated in IL tissue, indicative of an activated immune response state. Enriched GO terms and pathways in JE revealed a distinct set of host responses from those detected in IL. Regional differences were also identified between the mesenteric LNs draining each intestinal site. More down-regulated genes (52%) and fewer immune/disease pathways (n=5) were found in the ILLN compared to a higher number of up-regulated DE genes (56%) and enriched immune/disease pathways (n=13) in the JELN. Immunohistochemical staining validated myeloid cell transcriptional changes with increased CD172-positive myeloid cells in IL and JE tissues and draining LNs of MAPinf versus MAPneg cows. Several genes, GO terms, and pathways related to metabolism were significantly DE in IL and JE, but to a lesser extent (comparatively fewer enriched metabolic GO terms and pathways) in JELN suggesting distinct regional metabolic changes in IL compared to JE and JELN in response to MAP infection. These unique tissue- and regional-specific differences provides novel insight into the dichotomy in host responses to MAP infection that occur throughout the small intestine and mesenteric LN of chronically MAP infected cows.

Methods in isolation and characterization of bovine monocytes and macrophages.

Monocytes and macrophages belong to the mononuclear phagocyte system and play important roles in both physiological and pathological processes. The cells belonging to the monocyte/macrophage system are structurally and functionally heterogeneous. Several subsets of monocytes have been previously identified in mammalian blood, generating different subpopulations of macrophages in tissues. Although their distribution and phenotype are similar to their human counterpart, bovine monocytes and macrophages feature differences in both functions and purification procedures. The specific roles that monocytes and macrophages fulfil in several important diseases of bovine species, including among the others tuberculosis and paratuberculosis, brucellosis or the disease related to peripartum, remain still partially elusive. The purpose of this review is to discuss the current knowledge of bovine monocytes and macrophages. We will describe methods for their purification and characterization of their major functions, including chemotaxis, phagocytosis and killing, oxidative burst, apoptosis and necrosis. An overview of the flow cytometry and morphological procedures, including cytology, histology and immunohistochemistry, that are currently utilized to describe monocyte and macrophage main populations and functions is presented as well.

Bovine Adenovirus-3 Tropism for Bovine Leukocyte Sub-Populations.

A number of characteristics including lack of virulence and the ability to grow to high titers, have made bovine adenovirus-3 (BAdV-3) a vector of choice for further development as a vaccine-delivery vehicle for cattle. Despite the importance of blood leukocytes, including dendritic cells (DC), in the induction of protective immune responses, little is known about the interaction between BAdV-3 and bovine blood leukocytes. Here, we demonstrate that compared to other leukocytes, bovine blood monocytes and neutrophils are significantly transduced by BAdV404a (BAdV-3, expressing enhanced yellow green fluorescent protein [EYFP]) at a MOI of 1-5 without a significant difference in the mean fluorescence of EYFP expression. Moreover, though expression of some BAdV-3-specific proteins was observed, no progeny virions were detected in the transduced monocytes or neutrophils. Interestingly, addition of the "RGD" motif at the C-terminus of BAdV-3 minor capsid protein pIX (BAV888) enhanced the ability of the virus to enter the monocytes without altering the tropism of BAdV-3. The increased uptake of BAV888 by monocytes was associated with a significant increase in viral genome copies and the abundance of EYFP and BAdV-3 19K transcripts compared to BAdV404a-transduced monocytes. Our results suggest that BAdV-3 efficiently transduces monocytes and neutrophils in the absence of viral replication. Moreover, RGD-modified capsid significantly increases vector uptake without affecting the initial interaction with monocytes.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786 as a fusion ('MAP fusion protein particle'). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.