A genome-wide association study for natural antibodies measured in blood of Canadian Holstein cows.

BACKGROUND: Natural 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. RESULTS: In 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. CONCLUSIONS: This 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.

The effect of immunological status, in-vitro treatment and culture time on expression of eleven candidate reference genes in bovine blood mononuclear cells.

BACKGROUND: Technical 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. METHODS: The 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. RESULTS: Our findings showed that the expression of the most common reference genes, ß-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, ß2-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. CONCLUSIONS: The 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.

Genetic selection of cattle for improved immunity and health.

The immune system is a sensing structure composed of tissues and molecules that are well integrated with the neuroendocrine system. This integrate system ensures non-self from self-discrimination. In this capacity the immune system provides detection and protection from a wide range of pathogens. In mammals, the immune system is regulated by several thousand genes (8-9% of the genome) which indicate its high genetic priority as a critical fitness trait providing survival of the species. Identifying and selectively breeding livestock with the inherent ability to make superior immune responses can reduce disease occurrence, improve milk quality and increase farm profitability. Healthier animals also may be expected to demonstrate improvements in other traits, including reproductive fitness. Using the University of Guelph's patented High Immune Response technology it is possible to classify animals as high, average, or low responders based on their genetic estimated breeding value for immune responsiveness. High responders have the inherent ability to produce more balanced and robust immune responses compared with average or low responders. High responders dairy cattle essentially have about one-half the disease occurrence of low responders, and can pass their superior immune response genes on to future generations thereby accumulating health benefits within the dairy herd.

Bovine mastitis: frontiers in immunogenetics.

Mastitis is one of the most prevalent and costly diseases in the dairy industry with losses attributable to reduced milk production, discarded milk, early culling, veterinary services, and labor costs. Typically, mastitis is an inflammation of the mammary gland most often, but not limited to, bacterial infection, and is characterized by the movement of leukocytes and serum proteins from the blood to the site of infection. It contributes to compromised milk quality and the potential spread of antimicrobial resistance if antibiotic treatment is not astutely applied. Despite the implementation of management practises and genetic selection approaches, bovine mastitis control continues to be inadequate. However, some novel genetic strategies have recently been demonstrated to reduce mastitis incidence by taking advantage of a cow's natural ability to make appropriate immune responses against invading pathogens. Specifically, dairy cattle with enhanced and balanced immune responses have a lower occurrence of disease, including mastitis, and they can be identified and selected for using the high immune response (HIR) technology. Enhanced immune responsiveness is also associated with improved response to vaccination, increased milk, and colostrum quality. Since immunity is an important fitness trait, beneficial associations with longevity and reproduction are also often noted. This review highlights the genetic regulation of the bovine immune system and its vital contributions to disease resistance. Genetic selection approaches currently used in the dairy industry to reduce the incidence of disease are reviewed, including the HIR technology, genomics to improve disease resistance or immune response, as well as the Immunity(+)™ sire line. Improving the overall immune responsiveness of cattle is expected to provide superior disease resistance, increasing animal welfare and food quality while maintaining favorable production levels to feed a growing population.

A genome-wide association study of immune response traits in Canadian Holstein cattle.

BACKGROUND: Breeding for enhanced immune response (IR) has been suggested as a tool to improve inherent animal health. Dairy cows with superior antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) have been demonstrated to have a lower occurrence of many diseases including mastitis. Adaptive immune response traits are heritable, and it is, therefore, possible to breed for improved IR, decreasing the occurrence of disease. The objective of this study was to perform genome-wide association studies to determine differences in genetic profiles among Holstein cows classified as High or Low for AMIR and CMIR. From a total of 680 cows with immune response phenotypes, 163 cows for AMIR (81 High and 82 Low) and 140 for CMIR (75 High and 65 Low) were selectively genotyped using the Illumina Bovine SNP50 BeadChip. Results were validated using an unrelated population of 164 Holstein bulls IR phenotyped for AMIR and 146 for CMIR. RESULTS: A generalized quasi likelihood score method was used to determine single nucleotide polymorphisms (SNP) and chromosomal regions associated with immune response. After applying a 5% chromosomal false discovery rate, 186 SNPs were significantly associated with AMIR. The majority (93%) of significant markers were on chromosome 23, with a similar peak found in the bull population. For CMIR, 21 SNP markers remained significant. Candidate genes within 250,000 base pairs of significant SNPs were identified to determine biological pathways associated with AMIR and CMIR. Various pathways were identified, including the antigen processing and presentation pathway, important in host defense. Candidate genes included those within the bovine Major Histocompatability Complex such as BoLA-DQ, BoLA-DR and the non-classical BoLA-NC1 for AMIR and BoLA-DQ for CMIR, the complement system including C2 and C4 for AMIR and C1q for CMIR, and cytokines including IL-17A, IL17F for AMIR and IL-17RA for CMIR and tumor necrosis factor for both AMIR and CMIR. Additional genes associated with CMIR included galectins 1, 2 and 3, BCL2 and ß-defensin. CONCLUSIONS: The significant genetic variation associated with AMIR and CMIR in this study may imply feasibility to include immune response in genomic breeding indices as an approach to improve inherent animal health.

The influence of genetic background versus commercial breeding programs on chicken immunocompetence.

Immunocompetence of livestock plays an important role in farm profitability because it directly affects health maintenance. Genetics significantly influences the immune system, and the genotypic structure of modern fast-growing chickens has been changed, particularly after decades of breeding for higher production. Therefore, this study was designed to help determine if intensive breeding programs have adversely affected immunocompetence or whether the immune response profiles are controlled to greater extent by genetic background. Thus, 3 indigenous chicken populations from different genetic backgrounds and 2 globally available modern broiler strains, Ross 308 and Cobb 500, were evaluated for various aspects of immune response. These included antibody responses against sheep red blood cells and Brucella abortus antigen, as well as some aspects of cell-mediated immunocompetence by toe web swelling test and in vitro blood mononuclear cell proliferation. Significant differences (P < 0.05) in antibody responses to both antigens and cellular proliferation were observed among populations but not consistently between modern commercial strains versus the indigenous populations. In fact, the immune response profiles of Cobb 500 were similar to the indigenous populations, but varied compared with the other commercial strain. In addition, considerable variation was recorded between indigenous populations for all responses measured in this study. The results of this study suggest that the variation observed in immune responses between these strains of chickens is most likely due to differences in the genetic background between each strain of chicken rather than by commercial selection programs for high production.

Incidence rates of clinical mastitis among Canadian Holsteins classified as high, average, or low immune responders.

The objective of this study was to compare the incidence rate of clinical mastitis (IRCM) between cows classified as high, average, or low for antibody-mediated immune responses (AMIR) and cell-mediated immune responses (CMIR). In collaboration with the Canadian Bovine Mastitis Research Network, 458 lactating Holsteins from 41 herds were immunized with a type 1 and a type 2 test antigen to stimulate adaptive immune responses. A delayed-type hypersensitivity test to the type 1 test antigen was used as an indicator of CMIR, and serum antibody of the IgG1 isotype to the type 2 test antigen was used for AMIR determination. By using estimated breeding values for these traits, cows were classified as high, average, or low responders. The IRCM was calculated as the number of cases of mastitis experienced over the total time at risk throughout the 2-year study period. High-AMIR cows had an IRCM of 17.1 cases per 100 cow-years, which was significantly lower than average and low responders, with 27.9 and 30.7 cases per 100 cow-years, respectively. Low-AMIR cows tended to have the most severe mastitis. No differences in the IRCM were noted when cows were classified based on CMIR, likely due to the extracellular nature of mastitis-causing pathogens. The results of this study demonstrate the desirability of breeding dairy cattle for enhanced immune responses to decrease the incidence and severity of mastitis in the Canadian dairy industry.

Type 1 and type 2 immune response profiles of commercial dairy cows in 4 regions across Canada.

Diseases of dairy cattle have adverse implications for both the dairy industry and animal welfare. Understanding adaptive immune response profiles of cattle on a national scale will provide insight into the potential for improving health and decreasing disease. The objectives of the present study were to evaluate immune response phenotypes of Holstein cows outside the peripartum period and to determine if antibody isotype bias to putative type 1 and type 2 test antigens is maintained. The cows, housed on commercial farms in 4 key dairy regions across Canada, were immunized with test antigens to measure their ability to mount cell-mediated immune responses (CMIR) and antibody-mediated immune responses (AMIR). Delayed-type hypersensitivity (DTH) was used as an indicator of CMIR and primary and secondary serum antibodies of the immunoglobulin (Ig) G1 and IgG2 isotypes were used to determine AMIR to the test antigens. Immune response phenotypes varied significantly among regions, herds, and cows. Cows in Alberta had significantly higher DTH responses and secondary responses to the type 2 test antigen than those in other regions. However, cows in Alberta had significantly lower primary antibody responses. It was found that Alberta had the lowest incidence of mastitis caused by Escherichia coli and Staphylococcus aureus compared with other regions. The IgG1/IgG2 antibody isotype ratio confirmed the nature of the test antigens. This was the first study to evaluate adaptive immune response profiles and disease incidence of dairy cows on a national scale and it therefore provides a glimpse of the current situation in Canada.