Qiumei You

A principal component regression based genome wide analysis approach reveals the presence of a novel QTL on BTA7 for MAP resistance in holstein cattle.

Bovine Johnes disease (JD), caused by Mycobacterium avium spp. paratuberculosis (MAP), causes significant losses to the dairy and beef cattle industries. Effective vaccination or therapeutic strategies against this disease are currently unavailable and infected animals either get culled or die due to clinical disease. An alternative strategy to manage the disease is to selectively breed animals with enhanced resistance to MAP infection. Therefore, the objective of this study was to identify genetic loci putatively associated with MAP infection in a resource population consisting of Holstein cattle using a genome-wide association approach. The BovineSNP50 BeadChip, containing 54,001 single nucleotide polymorphisms (SNPs), was used to genotype 232 animals with known MAP infection status. Since, traditional case-control analytical techniques are based on single-marker analysis and do not account for the existence of linkage disequilibrium (LD) between markers, we used a novel principal component regression approach, where each SNP was fit in a logistic regression model, along with principal components of other SNPs on the same chromosome showing association with the trait, as covariates. Such an approach allowed us to account for the LD that exists between multiple markers showing an association on the same chromosome. Our analysis revealed the presence of at least 12 genomic regions on BTA1, 5, 6, 7, 10, 11 and 14 that were associated with the MAP infection status of our resource population. A brief description of these genomic regions, and a discussion of the analysis used in this study, have been presented.

Gene expression profiling of PBMCs from Holstein and Jersey cows sub-clinically infected with Mycobacterium avium ssp. paratuberculosis

Infection of calves with intracellular Mycobacterium avium ssp. paratuberculosis (MAP) commonly results in a granulomatous, chronic inflammatory bowel disease known as Johnes disease. The asymptomatic stage of this infection can persist for the entire production life of an adult cow, resulting in reduced performance and premature culling, as well as transmission of MAP to progeny and herd-mates. It has been previously shown that the gene expression profiles of peripheral blood mononuclear cells (PBMCs) of healthy cows, and those chronically infected with MAP are inherently different, and that these changes may be indicative of disease progression. Since resistance to MAP infection is a heritable trait, and has been proposed to differ amongst domestic dairy cattle breeds, the objective of the present study was to compare gene expression profiles of PBMCs from healthy adult Holstein and Jersey cows to those considered to be sub-clinically infected with MAP, as indicated by serum ELISA. Microarray analysis using a platform containing more than 10,000 probes and ontological analysis identified differences in gene expression between a) healthy and infected cows, including genes involved in the inflammatory response, and calcium binding, and b) infected Holsteins and Jerseys, including genes involved in the immune response, and antigen processing and presentation. These results suggest a mixed pro- and anti-inflammatory phenotype of PBMCs from MAP-infected as compared to healthy control animals, and inherently different levels of immune and inflammatory-related gene expression between MAP-infected Holsteins and Jerseys.

Proteomic analysis of plasma from Holstein cows testing positive for Mycobacterium avium subsp. paratuberculosis (MAP).

Johnes disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johnes antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & β (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.

Polymorphisms in the gene encoding bovine interleukin-10 receptor alpha are associated with Mycobacterium avium ssp. paratuberculosis infection status

BackgroundJohnes disease is a chronic inflammatory bowel disease (IBD) of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Since this pathogen has been implicated in the pathogenesis of human IBDs, the goal of this study was to assess whether single nucleotide polymorphism (SNPs) in several well-known candidate genes for human IBD are associated with susceptibility to MAP infection in dairy cattle.MethodsThe bovine candidate genes, interleukin-10 (IL10), IL10 receptor alpha/beta (IL10RA/B), transforming growth factor beta 1 (TGFB1), TGFB receptor class I/II (TGFBR1/2), and natural resistance-associated macrophage protein 1 (SLC11A1) were sequenced for SNP discovery using pooled DNA samples, and the identified SNPs were genotyped in a case-control association study comprised of 242 MAP negative and 204 MAP positive Holstein dairy cattle. Logistic regression was used to determine the association of SNPs and reconstructed haplotypes with MAP infection status.ResultsA total of 13 SNPs were identified. Four SNPs in IL10RA (984G > A, 1098C > T, 1269T > C, and 1302A > G) were tightly linked, and showed a strong additive and dominance relationship with MAP infection status. Haplotypes AGC and AAT, containing the SNPs IL10RA 633C > A, 984G > A and 1185C > T, were associated with an elevated and reduced likelihood of positive diagnosis by serum ELISA, respectively.ConclusionsSNPs in IL10RA are associated with MAP infection status in dairy cattle. The functional significance of these SNPs warrants further investigation.

Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

BackgroundDuring systemic gram-negative bacterial infections, lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg) using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR).ResultsTwelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg) and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep.ConclusionWe have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

Variation in the ovine cortisol response to systemic bacterial endotoxin challenge is predominantly determined by signalling within the hypothalamic–pituitary–adrenal axis

Bi-directional communication between the neuroendocrine and immune systems is designed, in part, to maintain or restore homeostasis during physiological stress. Exposure to endotoxin during Gram-negative bacterial infection for example, elicits the release of pro-inflammatory cytokines that activate the hypothalamic-pituitary-adrenal axis (HPAA). The secretion of adrenal glucocorticoids subsequently down regulates the host inflammatory response, minimizing potential tissue damage. Sequence and epigenetic variants in genes involved in regulating the neuroendocrine and immune systems are likely to contribute to individual differences in the HPAA response, and this may influence the host anti-inflammatory response to toxin exposure and susceptibility to inflammatory disease. In this study, high (HCR) and low (LCR) cortisol responders were selected from a normal population of 110 female sheep challenged iv with Escherichia coli endotoxin (400 ng/kg) to identify potential determinants that contribute to variation in the cortisol response phenotype. This phenotype was stable over several years in the HCR and LCR animals, and did not appear to be attributed to differences in expression of hepatic immune-related genes or systemic pro-inflammatory cytokine concentrations. Mechanistic studies using corticotrophin-releasing factor (0.5 microg/kg body weight), arginine vasopressin (0.5 microg/kg), and adrenocorticotropic hormone (0.5 microg/kg) administered iv demonstrated that variation in this phenotype is largely determined by signalling within the HPAA. Future studies will use this ovine HCR/LCR model to investigate potential genetic and epigenetic variants that may contribute to variation in cortisol responsiveness to bacterial endotoxin.

Bovine IFNGR2, IL12RB1, IL12RB2, and IL23R polymorphisms and MAP infection status

Mycobacterium avium ssp. paratuberculosis (MAP) infection causes a chronic granulomatous inflammatory condition of the bovine gut that is characterized by diarrhea, progressive weight loss, and emaciation, and ultimately leads to loss in productivity and profitability of dairy operations. The host cytokine machinery is known to play an important role in protecting against MAP infection. Therefore, the goal of the present study was to assess whether polymorphisms in candidate genes encoding important cytokines and cytokine receptors are associated with MAP infection status of dairy cattle. MAP infection status was evaluated based on serum and milk enzyme-linked immunosorbent assays (ELISAs) for MAP-specific antibodies. Twenty previously reported polymorphisms in genes encoding bovine interferon gamma (IFNG), IFNGR1, IFNGR2, IL22, IL22RA1, IL12RB1, IL12RB2, and IL23R were genotyped in a resource population of 446 dairy Holsteins with known MAP infection status, and logistic regression was used to assess the statistical association with a binomial MAP infection status phenotype. Four SNPs in IFNGR2, IL12RB1, IL12RB2, and IL23R were found to be associated with the MAP infection status of the resource population. These results underscore the importance of cytokines and their receptors in conferring protection against MAP infection and warrant further functional characterization of these associations.

Enhanced cutaneous hypersensitivity reactions are associated with ovine high and low cortisol responsiveness to acute endotoxin challenge.

Inbred rodent studies have demonstrated that cutaneous hypersensitivity reactions are exacerbated in stress-susceptible, and attenuated in stress-resistant strains of mice. This physiological response was, in part, mediated by activation of the hypothalamic-pituitary-adrenal axis during the acute restraint stress. A study was conducted to examine whether or not cutaneous hypersensitivity reactions are also associated with variable cortisol responsiveness to inflammatory stress in an outbred ovine population. High (H), medium (M), and low (L) cortisol responsive sheep were identified from a population of 110 females based on their estimated breeding values for cortisol concentration measured 4 h post-systemic challenge with Escherichia coli endotoxin (400 ng kg(-1)). Cutaneous hypersensitivity reactions to phytohaemagglutinin (PHA), 1-chloro-2, 4-dinitrobenzene (DNCB), and Candida albicans cellular antigen (CAA) were measured in these variable cortisol-responding sheep, in addition to serum interleukin (IL)-6, interferon (IFN)-gamma, and ovalbumin (OVA)-specific IgG concentrations. When compared to the M cortisol responders, both H and L cortisol responders had significantly greater cutaneous swelling during the elicitation phase in response to DNCB (P < 0.05) and CAA (P < 0.05); a similar but not significant trend was observed during the PHA challenge. The primary, but not the secondary, IgG response to OVA was significantly lower in the H and L cortisol responders when compared to the M cortisol responders. Differences in serum IL-6 or IFN-gamma concentration were not observed across variable cortisol-responsive groups. Together, these results demonstrate that cutaneous hypersensitivity reactions are enhanced in outbred H and L cortisol-responding sheep, independent of systemic modulation by IL-6 and IFN-gamma.

Assessment of yeast Saccharomyces cerevisiae component binding to Mycobacterium avium subspecies paratuberculosis using bovine epithelial cells

BackgroundSince yeast Saccharomyces cerevisiae and its components are being used for the prevention and treatment of enteric diseases in different species, they may also be useful for preventing Johne’s disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium spp. paratuberculosis (MAP). This study aimed to identify potential yeast derivatives that may be used to help prevent MAP infection. The adherence of mCherry-labeled MAP to bovine mammary epithelial cell line (MAC-T cells) and bovine primary epithelial cells (BECs) co-cultured with yeast cell wall components (CWCs) from four different yeast strains (A, B, C and D) and two forms of dead yeast from strain A was investigated.ResultsThe CWCs from all four yeast strains and the other two forms of dead yeast from strain A reduced MAP adhesion to MAC-T cells and BECs in a concentration-dependent manner after 6-h of exposure, with the dead yeast having the greatest effect.ConclusionsThe following in vitro binding studies suggest that dead yeast and its’ CWCs may be useful for reducing risk of MAP infection.

Bovine CLEC7A genetic variants and their association with seropositivity in Johne's disease ELISA.

Mycobacterium avium ssp. paratuberculosis (MAP) infection in cattle causes significant economic losses to the dairy and beef industries resulting from reduced productivity, premature culling and mortality. Bovine Dectin-1, an important pattern recognition molecule that is able to generate a proinflammatory response by acting alongside Toll like receptor (TLR) signaling, is known to co-operate with TLR2 to specifically activate a macrophage proinflammatory response against mycobacterial infections. Therefore, the goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine Dectin-1 (CLEC7A) and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples, collected from commercial dairy operations, were tested for MAP infection using blood and milk ELISAs and a resource population consisting of 197 infected and 242 healthy cattle was constructed. Pooled DNA was used for sequencing and eight single nucleotide polymorphisms (SNPs) were identified. Identified SNPs were genotyped on the resource population using the iPLEX MassARRAY system and statistical analysis was performed using logistic regression fitting the additive and dominance effects of each SNP in the model. Out of a total of eight identified SNPs, five were successfully genotyped, and three out of these five SNPs were found to be in complete linkage. Statistical analysis revealed a strong association between a non-synonymous SNP c.589A>G (p=0.008), and MAP infection status of the resource population inferred by seropositivity in MAP antibody specific ELISAs. This SNP c.589A>G was located in the geneic region that encodes the carbohydrate recognition domain of bovine Dectin-1. Therefore, further investigation of its functional relevance is warranted.