Stephen N. White

The sheep genome illuminates biology of the rumen and lipid metabolism

A genome for ewe and ewe Sheep-specific genetic changes underlie differences in lipid metabolism between sheep and other mammals, and may have contributed to the production of wool. Jiang et al. sequenced the genome of two Texel sheep, a breed that produces high-value meat, milk, and wool. The genome information will provide an important resource for livestock production and aid in the understanding of mammalian evolution. Science, this issue p. 1168 A genomic analysis of sheep explains specializations in digestive system physiology and wool production. Sheep (Ovis aries) are a major source of meat, milk, and fiber in the form of wool and represent a distinct class of animals that have a specialized digestive organ, the rumen, that carries out the initial digestion of plant material. We have developed and analyzed a high-quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants compared with nonruminant animals.

Haplotype variation in bovine Toll-like receptor 4 and computational prediction of a positively selected ligand-binding domain

Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Because it recognizes a broad class of pathogen-associated molecular patterns presented by lipopolysaccharides and lipoteichoic acid, TLR4 is a candidate gene for resistance to a large number of diseases. In particular, mouse models suggest TLR4 as a candidate gene for resistance to major agents in bovine respiratory disease and Johnes disease. The coding sequence of bovine TLR4 is divided into three exons, with intron/exon boundaries and intron sizes similar to those of human TLR4 transcript variant 1. We amplified each exon in 40 individuals from 11 breeds and screened the sequence for single-nucleotide polymorphisms (SNPs). We identified 32 SNPs, 28 of which are in the coding sequence, for an average of one SNP per 90 bp of coding sequence. Eight SNPs were nonsynonymous and potentially alter specificity of pathogen recognition or efficiency of signaling. To evaluate the functional importance of these SNPs, we used codon-substitution models to detect diversifying selection in an extracellular region that may physically interact with ligands. One nonsynonymous SNP is located within this region, and other substitutions are in adjacent regions that may interact with coreceptor molecules. The 32 SNPs were found in 20 haplotypes that can be assigned to geographic ranges of origin. Haplotype-tagging SNP analysis indicated that 12 SNPs need to be genotyped to distinguish these 20 haplotypes. These data provide a basic understanding of bovine TLR4 sequence variation and supply haplotype markers for disease association studies.

Genome-wide association identifies a deletion in the 3' untranslated region of striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins. We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine mapping and direct DNA sequencing identified an 8-bp deletion in the 3′ untranslated region (UTR) of the Striatin gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3′ UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in Striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized Striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, Plakophilin-2, Plakoglobin and Desmoplakin, all involved in the pathogenesis of ARVC in human beings. We suggest that Striatin may serve as a novel candidate gene for human ARVC.

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

Reduced Lentivirus Susceptibility in Sheep with TMEM154 Mutations

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10−9). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5–1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36–3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1

Previous studies initiated defining the role of host genetics in influencing the outcome of exposure to ovine progressive pneumonia virus. However, specific genes influencing host control of virus replication and disease progression have not been identified. This study, using 383 ewes of the Columbia, Polypay, and Rambouillet breeds, tested the hypothesis that host control of OPPV as measured by provirus levels in the peripheral blood associates with certain breeds and MHC class II Ovis aries (Ovar)-DRB1 expressed alleles. Rambouillet ewes were less likely to have measurable provirus levels as compared to Columbia ewes at ages 5 and 6 (P value < 0.02), and they exhibited lower provirus levels when compared to both Columbia and Polypay ewes of the same ages (P value < 0.05). The presence of DRB1*0403- or DRB1*07012-expressed alleles were significantly associated (P value = 0.019 and 0.0002, respectively) with lower OPP provirus levels but only were only found in 11% of the ewe flock. Analysis of each segregating amino acid in the β1 domain of DR β-chain revealed that amino acids Y31, T32, N37, T51, Q60, or N74 significantly associated (P value range = 0.0003–0.018) with lower OPP provirus levels, whereas amino acids H32, A38, or I67 associated (P value range = 0.013–0.043) with higher OPP provirus levels. These results suggest that Ovar-DRB1 contributes as one host genetic factor that controls OPP provirus levels, but does not fully account for the breed-specific OPP proviral differences.

Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.

Re-Emergence of the Apicomplexan Theileria equi in the United States: Elimination of Persistent Infection and Transmission Risk

Arthropod-borne apicomplexan pathogens that cause asymptomatic persistent infections present a significant challenge due to their life-long transmission potential. Although anti-microbials have been used to ameliorate acute disease in animals and humans, chemotherapeutic efficacy for apicomplexan pathogen elimination from a persistently infected host and removal of transmission risk is largely unconfirmed. The recent re-emergence of the apicomplexan Theileria equi in U.S. horses prompted testing whether imidocarb dipropionate was able to eliminate T. equi from naturally infected horses and remove transmission risk. Following imidocarb treatment, levels of T. equi declined from a mean of 104.9 organisms/ml of blood to undetectable by nested PCR in 24 of 25 naturally infected horses. Further, blood transfer from treated horses that became nested PCR negative failed to transmit to naïve splenectomized horses. Although these results were consistent with elimination of infection in 24 of 25 horses, T. equi-specific antibodies persisted in the majority of imidocarb treated horses. Imidocarb treatment was unsuccessful in one horse which remained infected as measured by nested PCR and retained the ability to infect a naïve recipient via intravenous blood transfer. However, a second round of treatment eliminated T. equi infection. These results support the utility of imidocarb chemotherapy for assistance in the control and eradication of this tick-borne pathogen. Successful imidocarb dipropionate treatment of persistently infected horses provides a tool to aid the global equine industry by removing transmission risk associated with infection and facilitating international movement of equids between endemic and non-endemic regions.

Development and Validation of an Ovine Progressive Pneumonia Virus Quantitative PCR

ABSTRACT Ovine progressive pneumonia virus (OPPV) infects at least one sheep in 81% of U.S. sheep flocks, as determined by serology, and can cause viral mastitis, arthritis, dyspnea, and cachexia. Diagnostic tests that quantify OPPV proviral load in peripheral blood leukocytes (PBL) provide an additional method for identification of infected sheep and may help to further understanding of the pathogenesis of OPPV-induced disease. In this study, we compared a new OPPV real-time quantitative PCR (qPCR) assay specific for the transmembrane region of the envelope gene (tm) with a competitive inhibition enzyme-linked immunosorbent assay (cELISA) using 396 PBL samples and sera from Idaho sheep. The OPPV qPCR had a positive concordance of 96.2% ± 2.3% and a negative concordance of 97.7% ± 2.5% compared to the cELISA, with a kappa value of 0.93, indicating excellent agreement between the two tests. In addition, the presence of tm in the three OPPV qPCR-positive and cELISA-negative sheep and in 15 sheep with different OPPV proviral loads was confirmed by cloning and sequencing. These data indicate that the OPPV qPCR may be used as a supplemental diagnostic tool for OPPV infection and for measurement of viral load in PBLs of infected sheep.

Identification of genetic variation and putative regulatory regions in bovine CARD15

Mutations in caspase recruitment domain 15 (CARD15) are associated with susceptibility to Crohn’s disease and Blau Syndrome. We performed comparative analyses of the bovine, murine, and human CARD15 transcripts to elucidate functionality of bovine CARD15 and examine its potential role in bovine disease resistance. Comparative analyses of intronic sequence across seven divergent species were performed to identify putative regulatory element binding motifs. High levels of interspecies conservation in sequence, genomic structure, and protein domains were detected indicating common functionality for CARD15 in cattle, human, and mouse. We identified species-specific regulatory elements in the 5′ and 3′ untranslated regions, suggesting that modes of regulation may have diverged across species. Thirty-one conserved putative regulatory element binding motifs were identified in the CARD15 intronic sequence of seven species. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified. Finally, 20 subspecies-specific haplotypes were predicted with 7 and 13 unique haplotypes explaining the diversity within B. taurus taurus and B. taurus indicus animals, respectively. Strong evidence for a simple causal relationship between these SNP loci and their haplotypes with Johne’s disease was not detected.