Development of polymorphic markers in the immune gene complex loci of cattle.

The addition of cattle health and immunity traits to genomic selection indices holds promise to increase individual animal longevity and productivity, and decrease economic losses from disease. However, highly variable genomic loci that contain multiple immune-related genes were poorly assembled in the first iterations of the cattle reference genome assembly and underrepresented during the development of most commercial genotyping platforms. As a consequence, there is a paucity of genetic markers within these loci that may track haplotypes related to disease susceptibility. By using hierarchical assembly of bacterial artificial chromosome inserts spanning 3 of these immune-related gene regions, we were able to assemble multiple full-length haplotypes of the major histocompatibility complex, the leukocyte receptor complex, and the natural killer cell complex. Using these new assemblies and the recently released ARS-UCD1.2 reference, we aligned whole-genome shotgun reads from 125 sequenced Holstein bulls to discover candidate variants for genetic marker development. We selected 124 SNPs, using heuristic and statistical models to develop a custom genotyping panel. In a proof-of-principle study, we used this custom panel to genotype 1,797 Holstein cows exposed to bovine tuberculosis (bTB) that were the subject of a previous GWAS study using the Illumina BovineHD array. Although we did not identify any significant association of bTB phenotypes with these new genetic markers, 2 markers exhibited substantial effects on bTB phenotypic prediction. The models and parameters trained in this study serve as a guide for future marker discovery surveys particularly in previously unassembled regions of the cattle genome.

An analysis of effects of heterozygosity in dairy cattle for bovine tuberculosis resistance.

Genetic selection of cattle more resistant to bovine tuberculosis (bTB) may offer a complementary control strategy. Hypothesising underlying non-additive genetic variation, we present an approach using genome-wide high density markers to identify genomic loci with dominance effects on bTB resistance and to test previously published regions with heterozygote advantage in bTB. Our data comprised 1151 Holstein-Friesian cows from Northern Ireland, confirmed bTB cases and controls, genotyped with the 700K Illumina BeadChip. Genome-wide markers were tested for associations between heterozygosity and bTB status using marker-based relationships. Results were tested for robustness against genetic structure, and the genotypic frequencies of a significant locus were tested for departures from Hardy-Weinberg equilibrium. Genomic regions identified in our study and in previous publications were tested for dominance effects. Genotypic effects were estimated through ASReml mixed models. A SNP (rs43032684) on chromosome 6 was significant at the chromosome-wide level, explaining 1.7% of the phenotypic variance. In the controls, there were fewer heterozygotes for rs43032684 (P < 0.01) with the genotypic values suggesting that heterozygosity confers a heterozygote disadvantage. The region surrounding rs43032684 had a significant dominance effect (P < 0.01). SNP rs43032684 resides within a pseudogene with a parental gene involved in macrophage response to infection and within a copy-number-variation region previously associated with nematode resistance. No dominance effect was found for the region on chromosome 11, as indicated by a previous candidate region bTB study. These findings require further validation with large-scale data.

Fine-mapping host genetic variation underlying outcomes to Mycobacterium bovis infection in dairy cows.

BACKGROUND: Susceptibility to Mycobacterium bovis infection in cattle is governed in part by host genetics. However, cattle diagnosed as infected with M. bovis display varying signs of pathology. The variation in host response to infection could represent a continuum since time of exposure or distinct outcomes due to differing pathogen handling. The relationships between host genetics and variation in host response and pathological sequelae following M. bovis infection were explored by genotyping 1966 Holstein-Friesian dairy cows at 538,231 SNPs with three distinct phenotypes. These were: single intradermal cervical comparative tuberculin (SICCT) test positives with visible lesions (VLs), SICCT-positives with undetected visible lesions (NVLs) and matched controls SICCT-negative on multiple occasions. RESULTS: Regional heritability mapping identified three loci associated with the NVL phenotype on chromosomes 17, 22 and 23, distinct to the region on chromosome 13 associated with the VL phenotype. The region on chromosome 23 was at genome-wide significance and candidate genes overlapping the mapped window included members of the bovine leukocyte antigen class IIb region, a complex known for its role in immunity and disease resistance. Chromosome heritability analysis attributed variance to six and thirteen chromosomes for the VL and NVL phenotypes, respectively, and four of these chromosomes were found to explain a proportion of the phenotypic variation for both the VL and NVL phenotype. By grouping the M. bovis outcomes (VLs and NVLs) variance was attributed to nine chromosomes. When contrasting the two M. bovis infection outcomes (VLs vs NVLs) nine chromosomes were found to harbour heritable variation. Regardless of the case phenotype under investigation, chromosome heritability did not exceed 8% indicating that the genetic control of bTB resistance consists of variants of small to moderate effect situated across many chromosomes of the bovine genome. CONCLUSIONS: These findings suggest the host genetics of M. bovis infection outcomes is governed by distinct and overlapping genetic variants. Thus, variation in the pathology of M. bovis infected cattle may be partly genetically determined and indicative of different host responses or pathogen handling. There may be at least three distinct outcomes following M. bovis exposure in dairy cattle: resistance to infection, infection resulting in pathology or no detectable pathology.

Late production of CXCL8 in ruminant oro-nasal turbinate cells in response to Chlamydia abortus infection.

Chlamydia abortus is an obligate intracellular bacterium that is an important cause of ovine abortion worldwide. There are reports of abortions in cattle, but these are very rare compared to the reported incidence in sheep. The bacterium is transmitted oro-nasally and can establish a sub-clinical infection until pregnancy, when it can invade the placenta and induce an inflammatory cascade leading to placentitis and abortion. Early host-pathogen interactions could explain differential pathogenesis and subsequent disease outcome in ruminant species. In this study, we assessed the ability of sheep and cattle oro-nasal turbinate cells to sense and respond to C. abortus infection. The cells expressed toll like receptor (TLR) 2, TLR4, nucleotide oligomerization domain (NOD) 1 and NOD-like receptor pyrin domain containing 3 (NLRP3) mRNA. In response to C. abortus infection, both ovine and bovine turbinate cells produce CXCL8 mRNA and protein late in the bacterial developmental cycle, but do not produce IL-1ß or TNF-α. The UV-inactivated bacteria did not elicit a CXCL8 response, suggesting that intracellular multiplication of the bacteria is important for activating the signalling pathways. The production of innate immune cytokines from cattle and sheep turbinate cells in response to C. abortus infection was found to be largely similar.

Genome-wide association study identifies novel loci associated with resistance to bovine tuberculosis.

Tuberculosis (TB) caused by Mycobacterium bovis is a re-emerging disease of livestock that is of major economic importance worldwide, as well as being a zoonotic risk. There is significant heritability for host resistance to bovine TB (bTB) in dairy cattle. To identify resistance loci for bTB, we undertook a genome-wide association study in female Holstein-Friesian cattle with 592 cases and 559 age-matched controls from case herds. Cases and controls were categorised into distinct phenotypes: skin test and lesion positive vs skin test negative on multiple occasions, respectively. These animals were genotyped with the Illumina BovineHD 700K BeadChip. Genome-wide rapid association using linear and logistic mixed models and regression (GRAMMAR), regional heritability mapping (RHM) and haplotype-sharing analysis identified two novel resistance loci that attained chromosome-wise significance, protein tyrosine phosphatase receptor T (PTPRT; P=4.8 × 10(-7)) and myosin IIIB (MYO3B; P=5.4 × 10(-6)). We estimated that 21% of the phenotypic variance in TB resistance could be explained by all of the informative single-nucleotide polymorphisms, of which the region encompassing the PTPRT gene accounted for 6.2% of the variance and a further 3.6% was associated with a putative copy number variant in MYO3B. The results from this study add to our understanding of variation in host control of infection and suggest that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control.

Bovine tuberculosis: the genetic basis of host susceptibility.

The prevalence of bovine tuberculosis (BTB) in the UK remains a significant economic burden and problem for the agri-food industry. Much effort has been directed towards improving diagnostics, finding vaccine candidates and assessing the usefulness of badger culling. The contribution that host genotype makes to disease outcome has, until recently, been overlooked; yet, it is biologically untenable that genetic variation does not play a role. In this review, we highlight the evidence, past and present, for a role of host genetics in determining susceptibility to BTB in livestock. We then address some of the major issues surrounding the design of future studies tasked with finding the exact causative genetic variation underpinning the TB susceptibility phenotype. Finally, we discuss some of the potential future benefits, and problems, that a knowledge of the genetic component to BTB resistance/susceptibility may bring to the agricultural industries and the wider scientific community.

BoLA-DR peptide binding pockets are fundamental for foot-and-mouth disease virus vaccine design in cattle.

This large study investigates the associations between bovine major histocompatibility complex DRB3 alleles and their binding pockets with the immune response to a 40-mer peptide derived from foot-and-mouth disease virus (FMDV) VP1. A crossbred (Charolais and Holstein) cattle population (n=197) was immunised with the FMDV peptide and specific IgG1 and IgG2 responses were measured. Eighteen different DRB3 alleles were detected in this population, with several exhibiting highly significant associations with antibody response. Allele DRB3*1601 was correlated with relatively low IgG1 and IgG2 responses (p<0.001), whereas DRB3*1001 was associated with relatively high IgG1 and IgG2 responses (p<0.001). In contrast the allele *0901 which ranked highest for IgG1 response, only came 14th for IgG2 response. The amino acids at several positions within the peptide binding cleft of the DR molecule showed significant associations (p<0.001) with the level of antibody response. Further analysis showed that specific residues within binding pockets are likely to be crucial to vaccine design. In particular, polymorphisms at position beta70 in pocket 4 were strongly linked to the magnitude of response and highly significant associations were found for position beta57 in pocket 9 and position beta56 in pocket 10. Glutamic acid at position beta70 was associated with low FMDV peptide specific IgG1 and IgG2 response, whereas arginine at beta70 was associated (p<0.001) with a high FMDV peptide specific IgG1 and IgG2 levels. The data indicates that the amino acids within the binding pockets of the DRB3 alleles are critical for determining the degree of immune response and in addition may affect the ratio of IgG1/IgG2, which in turn will influence the efficacy of the peptide to induce protective immunity.

A rapid and robust sequence-based genotyping method for BoLA-DRB3 alleles in large numbers of heterozygous cattle.

The BoLA-DRB3 gene is a highly polymorphic major histocompatibility complex class II gene of cattle with over one hundred alleles reported. Most of the polymorphisms are located in exon 2, which encodes the peptide-binding cleft, and these sequence differences play a role in variability of immune responsiveness and disease resistance. However, the high degree of polymorphism in exon 2 leads to difficulty in accurately genotyping cattle, especially heterozygous animals. In this study, we have improved and simplified an earlier sequence-based typing method to easily and reliably genotype cattle for BoLA-DRB3. In contrast to the earlier method, which used a nested primer set to amplify exon 2 followed by sequencing with internal primers, the new method uses only internal primers for both amplification and sequencing, which results in high-quality sequence across the entire exon. The haplofinder software, which assigns alleles from the heterozygous sequence, now has a pre-processing step that uses a consensus of all known alleles and checks for errors in base calling, thus improving the ability to process large numbers of samples. In addition, advances in sequencing technology have reduced the requirement for manual editing and improved the clarity of heterozygous base calls, resulting in longer and clearer sequence reads. Taken together, this has resulted in a rapid and robust method for genotyping large numbers of heterozygous samples for BoLA-DRB3 polymorphisms. Over 400 Holstein-Charolais cattle have now been genotyped for BoLA-DRB3 using this approach.

Pig peripheral blood mononuclear leucocyte subsets are heritable and genetically correlated with performance.

Indicator traits used to select pigs for increased resistance to infection or improved health must be heritable and, preferably, be associated with improved performance. We estimated the heritability of a range of immune traits and their genetic and phenotypic correlations with growth performance. We measured immune traits on 589 pigs and performance on 1941 pigs from six farms, three of which were classified as 'high health status' (i.e. specific pathogen-free) and three were of lower health status. All pigs were apparently healthy. Immune traits were total white blood cells (WBC), and peripheral blood mononuclear leucocyte (PBML) subsets positive for CD4, CD8α, gamma delta (γδ) T cell receptor, CD11R1 (natural killer cell marker), B cell and monocyte markers at the start and the end of standard growth performance tests. At both time points, all immune traits were moderately to highly heritable except for CD8α+ cells. At end of test, heritability estimates (h2) (±s.e.) were 0.18 (±0.11) for total WBC count. For PBML subset proportions, the heritabilities were 0.52 (±0.14) for γδ TCR+ cells, 0.62 (±0.14) for CD4+ cells, 0.44 (±0.14) for CD11R1+ cells, 0.58 (±0.14) for B cells and 0.59 (±0.14) for monocytes. Farm health status affected the heritabilities for WBC, being substantially higher on lower health status farms, but did not have consistent effects on heritabilities for the PBML subsets. There were significant negative genetic correlations between numbers and proportions of various PBML subsets and performance, at both start and end of test. In particular, the proportion of PBML cells that were CD11R1+ cells, at end of test, was strongly correlated with daily gain (rg = -0.72; P < 0.01). There were also weaker but significant negative phenotypic correlations between PBML subsets measured at end of test and performance, for γδ+ T cells, CD8α+, CD11R1+ cells, B cells or monocytes. Phenotypic correlations with daily gain were generally lower at the start of test than at the end of test. These results show that most of the major pig PBML subsets are heritable, and that systemic levels of several of these PBML subsets are genetically negatively correlated with performance. This approach provides a basis for using immune trait markers when selecting boars that can produce higher-performing progeny.

The association between plasma levels of acute phase proteins, haptoglobin, alpha-1 acid glycoprotein (AGP), pig-MAP, transthyretin and serum amyloid A (SAA) in Large White and Meishan pigs.

During infection, the acute phase response triggers the release of acute phase proteins (APP), alpha-(1) acid glycoprotein (AGP), serum amyloid A (SAA) and Pig-MAP into the circulation, accompanied by a decrease in plasma levels of transthyretin. We quantified the association between these APP in 26 apparently healthy pigs from two breeds, 13 Large White and 13 Meishan (16 male; 10 female). There was a significant correlation between plasma levels of haptoglobin and Pig-MAP (r=0.57; p<0.05), but no significant associations between any of the other APP tested. We also measured the relationship between PigMAP, transthyretin and SAA, and the proportions of peripheral blood mononuclear sub-sets, CD8(+) cells, CD4(+) cells, CD11R1(+) cells, MHC DQ(+) cells, and monocytes. There were correlations between both plasma levels of Pig-MAP and the proportion of monocytes (r=0.55; p<0.05) and plasma levels of transthyretin and the proportion of MHC DQ(+) cells (r=0.40; p<0.01). Breed and sex influenced plasma levels of Pig-MAP but not plasma levels of transthyretin. Overall, these results suggest closer links between the mechanisms that regulate the release haptoglobin, Pig-MAP and monocytes compared to those that regulate the release of AGP, SAA and transthyretin.

Modeling immunocompetence development and immunoresponsiveness to challenge in chicks.

The purpose of this study was 2-fold: 1) to develop a deterministic model that describes the development of immunocompetence and the kinetics of immunoresponsiveness to a pathogenic challenge in chicks and 2) to use this model to illustrate the importance of factors in experimental design, such as type of variable measured, measurement timing, and challenge age. Difficulties in evaluating immunological variables hinder attempts to improve animal health through selection on immunological variables. In young chicks, evaluating immunological variables is additionally complicated by immune system development and maternal immunity. The evaluation of immunocompetence and immunoresponsiveness and the definition of appropriate challenge and measurement strategies may be enabled through a mathematical model that captures the key components of the immune system and its development. Therefore, a model was developed that describes the development of immunocompetence as well as the kinetics of immunoresponsiveness to a pathogenic extracellular bacterial challenge in an individual chick from 0 to 56 d of age. The model consisted of 4 components describing immunocompetence (maternal and baseline immunity) and immunoresponsiveness (acute phase and antibody response). Individual component equations generally fit published data adequately. Four scenarios that represented combinations of challenge age and measurement timing were simulated. In each scenario, the immunoresponsiveness to a particular challenge was compared for 3 different levels of baseline immunity, representing 3 broiler genotypes. It was illustrated that experimental design (type of immunoresponsiveness measured, measurement timing, and challenge age) can have an important effect on the ranking of genotypes, groups, or individuals and on the reliability of extrapolations based on this ranking. It is concluded that this model is a potentially useful tool in the definition of appropriate challenge and measurement strategies when evaluating immunocompetence and immunoresponsiveness. Further, it may be used as a generator of hypotheses on global immunological relationships to be tested experimentally.

Quantitative evaluation of genetic and environmental parameters determining antibody response induced by vaccination against bovine respiratory syncytial virus.

The parameters controlling IgG antibody responses induced by vaccination against bovine respiratory syncytial virus (BRSV) were investigated in 463 Holstein-Charolais crossbred cattle. Pre- and post-vaccination sera were tested by enzyme linked immunosorbent assays (ELISA) for BRSV-specific IgG and IgG2. Year-of-birth, age, sex and pre-existing antibody were significant sources of variation for IgG responses. Pre-vaccination, progeny with a higher proportion of Holstein genes had higher total BRSV-IgG. By Day 35 post-vaccination, heritabilities peaked at 0.26 for total BRSV-IgG and 0.36 for BRSV-IgG1. There was no evidence for interbreed differences between Holstein and Charolais calves, post-vaccination. These results suggest that calf-sire has a major heritable influence on serum IgG levels following BRSV immunisation.

Radiation hybrid mapping of all 10 characterized bovine Toll-like receptors.

Toll-like receptors (TLRs) are cell-surface signalling molecules that recognize a range of highly conserved pathogen molecules and instigate the appropriate immune response. Here, we report the mapping of all 10 characterized bovine TLR genes using a radiation hybrid panel. The genomic organization of the bovine TLRs is similar to that of humans and mice. TLR1, TLR6 and TLR10 map closely together on Bos taurus chromosome 6 (BTA6), while TLR7 and TLR8 map to the X chromosome. TLR2, TLR3, TLR4, TLR5 and TLR9 map to BTA17, BTA27, BTA8, BTA16 and BTA22 respectively. Our increased knowledge of the genomic organization of the bovine TLR genes may promote our understanding of their evolution and help in the identification of bovine genes underlying disease-resistance traits.

In vitro peripheral blood mononuclear cell proliferation in a crossbred cattle population.

Immune function measured by Staphylococcus aureus- and phytohemagglutinin- (PHA-) induced cell proliferation was assessed in a population of 445 genetically defined, F2 and backcross Charolais-Holstein crossbred cattle when the animals were approximately 5 mo of age. Variation in Staph. aureus-induced, PHA-induced, and control proliferation [peripheral blood mononuclear cell (PBMC) and media only] was observed at d 2, 3, 4, 5, 9, and 10 of in vitro culture. The levels of Staph. aureus-induced, PHA-induced, and control proliferation were strongly positively correlated between days of culture within-assay (e.g., between d 2 and d 3 or between d 4 and d 5). Responses were also positively correlated when the same individuals were resampled and the assay repeated within 3 mo. Analyses fitting linear mixed models using REML showed that Staph. aureus-induced and PHA-induced proliferation was significantly associated with control proliferation and the year of birth. The age of the animal at sampling influenced only Staph. aureus-induced proliferation, with Staph. aureus-induced proliferation increasing with the age of the animal. Control proliferation was influenced by a sex x cross interaction, although in this study, sex was confounded by management, as female cattle were housed and reared differently from male cattle. All 3 measures of immune function were influenced by sire, demonstrating that these traits are partially under genetic control, and indicating that it may ultimately be possible to identify quantitative trait loci for these measures of immunity.

Innate immune traits differ between Meishan and Large White pigs.

A panel of innate immune traits were compared between Meishan and Large White pigs. These pigs were of similar age and kept under the same environmental conditions to reduce non-genetically derived variation in immune traits. The animals were all apparently healthy and were not experimentally challenged with any pathogen during the study. The measures only required a small blood sample. Total white cell counts were similar between the pig breeds. However, the numbers of lymphocytes, neutrophils and monocytes differed significantly, with Meishans having higher neutrophil and monocyte counts and lower lymphocyte counts. Flow cytometric methods were used to determine quantitatively the characteristics and function of neutrophils and monocytes. Meishan neutrophils were smaller and less complex than Large White neutrophils, and phagocytosis of Escherichia coli and the ensuing oxidative burst was lower in Meishan neutrophils compared to Large White neutrophils. Monocyte phagocytosis of E. coli was significantly less than that of neutrophils in both breeds but the function of Meishan monocytes as measured by phagocytosis and tumour necrosis factor alpha (TNFalpha) release did not differ from that of Large White monocytes. Levels of acute phase proteins also differed between the breeds with a significantly higher proportion of Meishans having elevated serum amyloid A levels. However, Meishans had lower alpha(1)-acid glycoprotein levels than Large Whites and haptoglobin levels were similar. Such differences in innate immune traits may have implications in the resistance to infection by a broad range of pathogens and subsequent disease effects in these breeds. Further studies are warranted to investigate the genes underlying these traits.

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line.

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.

Proinflammatory cytokine expression by Theileria annulata infected cell lines correlates with the pathology they cause in vivo.

Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.