Inbreeding and purging at the genomic Level: the Chillingham cattle reveal extensive, non-random SNP heterozygosity.

Local breeds of livestock are of conservation significance as components of global biodiversity and as reservoirs of genetic variation relevant to the future sustainability of agriculture. One such rare historic breed, the Chillingham cattle of northern England, has a 350-year history of isolation and inbreeding yet shows no diminution of viability or fertility. The Chillingham cattle have not been subjected to selective breeding. It has been suggested previously that the herd has minimal genetic variation. In this study, high-density SNP genotyping with the 777K SNP chip showed that 9.1% of loci on the chip are polymorphic in the herd, compared with 62-90% seen in commercial cattle breeds. Instead of being homogeneously distributed along the genome, these loci are clustered at specific chromosomal locations. A high proportion of the Chillingham individuals examined were heterozygous at many of these polymorphic loci, suggesting that some loci are under balancing selection. Some of these frequently heterozygous loci have been implicated as sites of recessive lethal mutations in cattle. Linkage disequilibrium equal or close to 100% was found to span up to 1350 kb, and LD was above r(2) = 0.25 up to more than 5000 kb. This strong LD is consistent with the lack of polymorphic loci in the herd. The heterozygous regions in the Chillingham cattle may be the locations of genes relevant to fitness or survival, which may help elucidate the biology of local adaptation in traditional breeds and facilitate selection for such traits in commercial cattle.

A single nomenclature and associated database for alleles at the major histocompatibility complex class II DRB1 locus of sheep.

The development of standardised nomenclatures with associated databases containing reference sequences for alleles at polymorphic loci within the major histocompatibility complex (MHC) has been facilitated by the development of the immuno polymorphism database (IPD). Recently, included within IPD-MHC is information on allelic diversity within sheep species (IPD-MHC-OLA). Here, we present the first report of progress in populating the sheep IPD-MHC database with alleles at the class II MHC DRB1 locus. The sequence of 63 Ovar-DRB1 alleles within 24 allelic families is now held within the database, each meeting the minimum requirement of a complete second exon. These sequences are derived from a combination of genomic and cDNA-based approaches and represent the most extensive collection of validated alleles at the sheep DRB1 locus yet described. Although these 63 alleles probably represent only a fraction of the DRB1 allelic diversity in sheep species worldwide, we encourage the research community to use the official allelic nomenclature and to contribute allelic sequences to the database via its web-based submission tool. In time, the IPD-MHC-OLA resource will underpin population-based MHC genotyping studies and help to simplify meta-analyses of multi-source data from wild and domestic sheep populations.

Lack of evidence for an association between MHC diversity and the development of bovine neonatal pancytopenia in Holstein dairy cattle.

Bovine neonatal pancytopenia (BNP), a disease of neonatal calves, has been described in a number of European countries since 2006. The disease results in high mortality of calves aged 1-4 weeks and is characterised by severe bone marrow pathology resulting in profound thrombocytopenia and consequent haemorrhagic diathesis. A number of hypotheses including a novel virus infection, plant toxins, a vaccine associated isoimmune disease, or a genetic defect have been suggested to explain the aetiology of this disease. However, as the number of cases in affected herds remains small, it is hypothesised that the genetic background of the calf may influence disease susceptibility. To test this we focused on the class II region of the major histocompatibility complex (MHC) which is often associated with variations in immune response and susceptibility to antibody mediated autoimmune disease. Forty-three cases of BNP and sixty-eight controls were genotyped at the polymorphic class II MHC-DRB3 locus. Twenty DRB3 alleles were identified with seven appearing at frequencies ≥ 0.05. A comparison of the allelic frequencies between diseased and control groups showed that there was no evidence for any significant differences, suggesting that the MHC does not appear to be a predisposing risk factor in the development of BNP in Holstein dairy cattle.

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells.

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.

Conservation of promoter, coding and intronic regions of the non-classical MHC class II DYA gene suggests evolution under functional constraints.

The major histocompatibility complex (MHC) in ruminants contains a unique pair of class II genes (DYA and DYB) of unknown function. As functional genes show higher levels of nucleotide conservation than pseudogenes we compared the DYA genes from sheep and cattle, two species which diverged from a common ancestor approximately 20 million years ago. Comparative analysis identified levels of nucleotide conservation in immediate promoter (97%), coding (94%) and intronic regions (91%) comparable with functional MHC genes. The Ovar-DYA transcript revealed an open reading frame encoding a 288 amino acid protein compared with a 253 amino acid protein associated with the BoLA-DYA transcript. A dinucleotide deletion in exon 4 of the Ovar-DYA transcript combined with alternative exon 5 splice sites introduces unusual diversity to the cytoplasmic domain of the Ovar-DYalpha polypeptide. The degree of conservation between these class II MHC genes is consistent with evolution under purifying selection suggesting that these genes retain a unique function in ruminants.

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection.

Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended coevolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.

A statistically derived index for classifying East Coast fever reactions in cattle challenged with Theileria parva under experimental conditions.

A statistically derived disease reaction index based on parasitological, clinical and haematological measurements observed in 309 5 to 8-month-old Boran cattle following laboratory challenge with Theileria parva is described. Principal component analysis was applied to 13 measures including first appearance of schizonts, first appearance of piroplasms and first occurrence of pyrexia, together with the duration and severity of these symptoms, and white blood cell count. The first principal component, which was based on approximately equal contributions of the 13 variables, provided the definition for the disease reaction index, defined on a scale of 0-10. As well as providing a more objective measure of the severity of the reaction, the continuous nature of the index score enables more powerful statistical analysis of the data compared with that which has been previously possible through clinically derived categories of non-, mild, moderate and severe reactions.

A highly sensitive, non-radioactive assay for T cell activation in cattle: applications in screening for antigens recognised by CD4(+) and CD8(+) T cells.

We describe a highly sensitive, non-radioactive assay for T cell activation, based on the rapid induction of class II MHC expression by constitutively negative bovine endothelial cells, when cultured in the presence of supernatants derived from activated bovine T cells. We demonstrate the effectiveness of this assay in detecting rBoIFNgamma and activation of immune CD4(+) and CD8(+) T cell lines and clones in response to specific antigen and transfected COS-7 cells, respectively. We also demonstrate its utility in identifying purified pathogen fractions that activate immune CD4(+) T cell clones.

Cattle MHC: evolution in action?

Because major histocompatibility complex (MHC) genes play a major role in the development of acquired immune responses, it is essential to obtain comparative information on their organisation, expression and possible functional dichotomies in different species. In human, three classical, polymorphic class I genes (HLA-A, B- and -C) and four expressed A/B class II gene pairs (HLA-DM, -DP, -DQ and -DR) are each present on all haplotypes. With the exception of the HLA-DRB loci, it has been assumed that a similar rigid organisational situation exists in other mammalian species. However, extensive analysis of the bovine MHC (BoLA) at both the genomic and transcriptional levels has revealed a degree of genetic fluidity not described in other species. None of the four (or more) classical class I genes identified is consistently expressed, and haplotypes differ from one another in both the number and composition of expressed class I genes. Similarly, in the class II region, the number of DQ genes varies between haplotypes in both number and composition. These variations in both class I and II (which appear to reflect differences at the genomic level) are likely to play an important role in cattle immune responses. The observed phenotypic differences in cattle demonstrate very clearly the dynamic nature of the MHC region. This review addresses the functional impact of such variation in different breeds and populations, and its significance in terms of MHC evolution.

Identification of diverse BoLA DQA3 genes consistent with non-allelic sequences.

Genetic diversity within the DQA genes of the major histocompatibility complex (Mhc) of cattle is characterised by multiple polymorphic loci that can vary in number between haplotypes. Previous analysis of the second exon sequences derived from genomic BoLA DQA3 genes identified two distinct families, DQA3*01 and DQA3*02. In this report, we describe the nucleotide and predicted amino acid sequences of the entire coding region of three transcribed BoLA DQA3 genes representing each of these families. These data provide additional evidence that the BoLA DQA3 locus is distinct from BoLA DQA1 and BoLA DQA2. In addition, the amino acid sequence of DQA3 genes from the two families is shown to differ by 35 out of the 254 amino acids. Putative locus-specific amino acid sequence motifs within the transmembrane and intracytoplasmic domains of DQA genes are shown to differ between the DQA3*01 and DQA3*02 genes. Phylogenetic analysis reveals a genetic distance that is considerably larger than that seen between orthologous Mhc allelic families. These data are consistent with either an extremely divergent family of DQA3 genes or an allele at an additional BoLA DQA4 locus.

In vitro infection with Theileria parva is associated with IL10 expression in all bovine lymphocyte lineages.

The protozoan parasite Theileria parva infects and transforms bovine lymphocytes, giving rise to a fatal lymphoproliferative condition known as East Coast fever. Although immune cattle mount strong cytolytic T lymphocyte responses to the parasite, naive animals appear unable to respond and develop severe immunopathological lesions. We have investigated the patterns of cytokine mRNA expressed by 19 bulk and cloned parasite-infected lymphoblast cell lines using a multiplex PCR system. Considerable variation was observed in the cytokine profiles of these lines and only IL10 was universally expressed. Investigation of cloned lines representing the major bovine lymphocyte populations failed to reveal a lineage-specific pattern of cytokine mRNA expression that could be associated with infection. Nonetheless, analysis of a CD4+ T cell clone before and after transformation with the parasite indicated that infection does alter the pattern of cytokine expression, with apparent upregulation of IL10. These observations raise the possibility that IL10 derived from infected cells may influence the immune responses of naive cattle to challenge.

Analysis of genetic diversity at the DQA loci in African cattle: evidence for a BoLA-DQA3 locus.

We describe the development of a polymerase chain reaction (PCR)-based approach for analysis of genetic diversity at the DQA loci in African Bos indicus and Bos taurus cattle. This approach, equally effective in European and Asian cattle breeds, detects the presence or absence of DQA1 and most duplicated DQA2 genes. Nucleotide and predicted amino acid sequence analysis of the highly polymorphic second exons, in addition to analysis of the locus-specific and relatively non-polymorphic transmembrane, cytoplasmic, and 3-prime untranslated regions, has provided evidence for considerable diversity between each of the duplicated DQA2 genes. Therefore, we propose the designation BoLA-DQA3 for the previously unpublished alleles at the second DQA2 locus. Fourteen distinct PCR restriction fragment length polymorphism (RFLP) patterns, each identifying families of alleles at three DQA loci, can be distinguished. Nucleotide sequence analysis of new PCR-RFLP patterns from 193 Kenyan Boran, Ethiopian Arsi (B. indicus), and Guinean N'Dama (B. taurus) cattle identified 13 DQA1 alleles within eight major allelic families, five DQA2 alleles within a single allelic family, and seven DQA3 alleles within three major allelic families.

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells.

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

Analysis of the fine specificities of sheep major histocompatibility complex class II-specific monoclonal antibodies using mouse L-cell transfectants.

The fine specificities of two panels of monoclonal antibodies (mAbs) for sheep major histocompatibility complex (MHC) class II molecules were determined using five mouse L-cell transfectants, each expressing a defined sheep DQ or DR MHC class II A/B gene pair. Using the transfectants in an indirect fluorescence antibody assay, previous immunochemical characterization of the mAbs was confirmed for 16 of 23 mAbs tested. The MHC class II subtype specificity (DQ or DR) of each mAb was assigned without interference from the products of other expressed class II loci. This allowed the identification of both cross-locus specificities as well as defining fine specificities of mAbs previously only partially characterized by immunochemical techniques.

Mapping and characterization of the DQ subregion of the ovine MHC.

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11 kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.

Evidence for the expression of two distinct MHC class II DR beta like molecules in the sheep.

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DR beta chains are expressed in the sheep. Two anti-beta chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DR beta chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 beta chain. Amino-terminal sequence analysis of the alpha chain associated with VPM37 beta chain shows that this alpha chain is homologous to the human DR alpha chain strongly indicating that the beta chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of post-translational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DR beta chains in the sheep.

Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes.

Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines.