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The dynamic interaction between the host and pathogens, along with environmental factors, influences the regulation of mammalian immune responses. Therefore, comprehensive in vivo immune-phenotyping during an active response to a pathogen can be complex and prone to confounding effects. Evaluating critical fundamental aspects of the immune system at a cellular level is an alternative approach to reduce this complexity. Therefore, the objective of the current study was to examine an in vitro model for functional phenotyping of bovine monocyte-derived macrophages (MDM), cells which play a crucial role at all phases of inflammation, as well influence downstream immune responses. As indicators of MDM function, phagocytosis and nitric oxide (NO −) production were tested in MDM of 16 cows in response to 2 common bacterial pathogens of dairy cows, Escherichia coli and Staphylococcus aureus. Notable functional variations were observed among the individuals (coefficient of variation: 33% for phagocytosis and 70% in the production of NO −). The rank correlation analysis revealed a significant, positive, and strong correlation (rho = 0.92) between NO − production in response to E. coli and S. aureus, and a positive but moderate correlation (rho = 0.58) between phagocytosis of E. coli and S. aureus. To gain further insight into this trait, another 58 cows were evaluated solely for NO − response against E. coli. The pedigree of the tested animals was added to the statistical model and the heritability was estimated to be 0.776. Overall, the finding of this study showed a strong effect of host genetics on the in vitro activities of MDM and the possibility of ranking Holstein cows based on the in vitro functional variation of MDM.

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BackgroundNatural antibodies (NAb) are an important component of the innate immune system, and fight infections as a part of the first line defence. NAb are poly-reactive and can respond non-specifically to antigens. Therefore, NAb may be a key trait when evaluating an animal’s potential natural disease resistance. Variation in NAb is caused by both genetic and environmental factors. In this study genetic parameters of NAb were estimated and a genome-wide association study (GWAS) was performed to gain further understanding on the genes that are responsible for the observed genetic variation of NAb in Canadian Holsteins.ResultsIn total, blood samples of 1327 cows from 64 farms were studied. NAb binding to keyhole limpet hemocyanin (KLH) were determined via indirect ELISA. Immunoglobulin (Ig) isotypes, IgG and IgM, were evaluated. From the sample population, 925 cows were genotyped for 45,187 markers and each individual marker was tested to detect genetic variation in NAb levels. The relationships among animals was accounted for with genomic relationship. Results show heritabilities of 0.27 ± 0.064 (IgG) and 0.31 ± 0.065 (IgM). In total, 23 SNPs were found to be associated with IgG, but no SNPs were associated with IgM (FDR p-value < 0.05). The significant SNPs were located on autosomal chromosomes 1, 20 and 21 of the cow genome. Functional annotation analysis of the positional candidate genes revealed two sets of genes with biologically relevant functions related to NAb. In one set, seven genes with crucial roles in the production of antibody in B cells were associated with the trafficking of vesicles inside the cells between organelles. In the second set, two genes among positional candidate genes were associated with isotype class-switching and somatic hypermutation of B cells.ConclusionsThis study demonstrated the possibility of increasing NAb through selective breeding. In addition, the effects of two candidate pathways are proposed for further investigation of NAb production in Holsteins.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5062-6) contains supplementary material, which is available to authorized users.

Reactive Nitrogen Species (RNS) are a group of bactericidal molecules produced by macrophages in response to pathogens in a process called oxidative burst. Nitric oxide (NO −) is a member of RNS produced from arginine by inducible Nitric Oxide Synthase (iNOS) enzyme. The activity of iNOS and production of NO − by macrophages following stimulation is one of the indicators of macrophage polarization towards M1/proinflammatory. Production of NO − by bovine monocyte-derived macrophage (MDM) and mouse peritoneal macrophages has been shown to be strongly associated with host genetic with the heritability of 0.776 in bovine MDM and 0.8 in mouse peritoneal macrophages. However, the mechanism of genetic regulation of macrophage response has remained less explored. In the current study, the transcriptome of bovine MDMs was compared between two extreme phenotypes that had been classified as high and low responder based on NO − production. The results showed that 179 and 392 genes were differentially expressed (DE) between high and low responder groups at 3 and 18 hours after exposure to Escherichia coli, respectively. A set of 11 Transcription Factors (TFs) (STAT1,

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Innate responses provide the first line of defense against viral infections, including the influenza virus at mucosal surfaces. Communication and interaction between different host cells at the early stage of viral infections determine the quality and magnitude of immune responses against the invading virus. The release of membrane-encapsulated extracellular vesicles (EVs), from host cells, is defined as a refined system of cell-to-cell communication. EVs contain a diverse array of biomolecules, including microRNAs (miRNAs). We hypothesized that the activation of the tracheal cells with different stimuli impacts the cellular and EV miRNA profiles. Chicken tracheal rings were stimulated with polyI:C and LPS from Escherichia coli 026:B6 or infected with low pathogenic avian influenza virus H4N6. Subsequently, miRNAs were isolated from chicken tracheal cells or from EVs released from chicken tracheal cells. Differentially expressed (DE) miRNAs were identified in treated groups when compared to the control group. Our results demonstrated that there were 67 up-regulated miRNAs, 157 down-regulated miRNAs across all cellular and EV samples. In the next step, several genes or pathways targeted by DE miRNAs were predicted. Overall, this study presented a global miRNA expression profile in chicken tracheas in response to avian influenza viruses (AIV) and toll-like receptor (TLR) ligands. The results presented predicted the possible roles of some DE miRNAs in the induction of antiviral responses. The DE candidate miRNAs, including miR-146a, miR-146b, miR-205a, miR-205b and miR-449, can be investigated further for functional validation studies and to be used as novel prophylactic and therapeutic targets in tailoring or enhancing antiviral responses against AIV.

BackgroundTechnical feasibility of RNA quantification by real time RT-PCR has led to enormous utilization of this method. However, real time PCR results need to be normalized due to the high sensitivity of the method and also to eliminate technical variation. Normalization against a reference gene that is constitutively transcribed and has minimum variation among samples is the ideal method. Nevertheless, many studies have shown that there is no general reference gene(s) with ideal characteristics and candidate reference genes should be tested before being used as a “normalizer” in each study.MethodsThe current study investigated the effects of previous exposure of the host to experimental test antigens and culturing time on the expression of 11 candidate genes when blood mononuclear cells (BMCs) were cultured and treated in-vitro by hen egg white lysozyme, Candida albicans extract and a mitogen. Mononuclear cells were isolated and cultured from 12 bovine blood samples representing 3 different immunological statuses. The expression of candidate housekeeping genes were measured by real-time RT-PCR at 4 and 24 hours post culture. The expression of candidate genes were first compared between the two time points in untreated samples. Constitutively expressed genes were further tested in linear mixed effects models to examine the effect of previous host exposure and in-vitro treatments.ResultsOur findings showed that the expression of the most common reference genes, β-actin, and Glyceraldehydes-3-phosphate dehydrogenase (GAPDH), are significantly decreased at 24 hours after culturing BMCs, even without any treatment. The effect of culturing time was also significantly influenced the expression of 18s ribosomal RNA, β2-microglobulin, Tyrosine 3-monooxygenase/tryptophan 5-monoxygenase activation protein, zeta polypeptide (YWHAZ) in BMCs. Only the expression of C-terminal binding protein 1 (CTBP1) and RAD50 among all tested genes were consistent after treatment of cultured BMCs with C. albicans whole yeast extract and Hen Egg White Lysozyme (HEWL), respectively. In addition, expressions of CTBP1, and RAD50 were independent from previous exposure of the host to the antigen.ConclusionsThe results of this study demonstrated inconsistent expression of commonly used reference genes in untreated cultured BMCs over time. As this condition applies to negative controls in real time RT-PCR study designs, normalization against these genes can largely deceive the outcome, especially in kinetic studies. Moreover, the potential effects of immunological memory on the expression of reference genes should be considered if BMCs are collected from different individuals under different environmental conditions and if these cells are treated in-vitro by an antigen.