Genome-wide associations with longevity and reproductive traits in U.S. rangeland ewes.

Introduction: Improving ewe longevity is an important breeding and management goal, as death loss and early culling of mature ewes are economic burdens in the sheep industry. Ewe longevity can be improved by selecting for positive reproductive outcomes. However, the breeding approaches for accomplishing this come with the challenge of recording a lifetime trait. Characterizing genetic factors underpinning ewe longevity and related traits could result in the development of genomic selection strategies to improve the stayability of sheep through early, informed selection of replacement ewes. Methods: Towards this aim, a genome-wide association study (GWAS) was performed to identify genetic markers associated with ewe longevity, reproductive, and production traits. Traits evaluated included longevity (i.e., length of time in the flock), parity and the lifetime number of lambs born, lambs born alive, lambs weaned, and weight of lambs weaned. Ewe records from previous studies were used. Specifically, Rambouillet (n = 480), Polypay (n = 404), Suffolk (n = 182), and Columbia (n = 64) breed ewes (N = 1,130) were analyzed against 503,617 SNPs in across-breed and within-breed GWAS conducted with the Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) model in R. Results: The across-breed GWAS identified 25 significant SNPs and the within-breed GWAS for Rambouillet, Polypay, and Suffolk ewes identified an additional 19 significant SNPs. The most significant markers were rs411309094 (13:22,467,143) associated with longevity in across-breed GWAS (p-value = 8.3E-13) and rs429525276 (2:148,398,336) associated with both longevity (p-value = 6.4E-15) and parity (p-value = 4.8E-15) in Rambouillet GWAS. Significant SNPs were identified within or in proximity (±50 kb) of genes with known or proposed roles in reproduction, dentition, and the immune system. These genes include ALPL, ANOS1, ARHGEF26, ASIC2, ASTN2, ATP8A2, CAMK2D, CEP89, DISC1, ITGB6, KCNH8, MBNL3, MINDY4, MTSS1, PLEKHA7, PRIM2, RNF43, ROBO2, SLCO1A2, TMEM266, TNFRSF21, and ZNF804B. Discussion: This study proposes multiple SNPs as candidates for use in selection indices and suggests genes for further research towards improving understanding of the genetic factors contributing to longevity, reproductive, and production traits of ewes.

Ecology and Chronic Wasting Disease Epidemiology Shape Prion Protein Gene Variation in Rocky Mountain Elk (Cervus elaphus nelsoni).

As chronic wasting disease (CWD) continues to spread across North America, the relationship between CWD and host genetics has become of interest. In Rocky Mountain elk (Cervus elaphus nelsoni), one or two copies of a leucine allele at codon 132 of the prion protein gene (132L*) has been shown to prolong the incubation period of CWD. Our study examined the relationship between CWD epidemiology and codon 132 evolution in elk from Wyoming, USA, from 2011 to 2018. Using PCR and Sanger sequencing, we genotyped 997 elk and assessed the relationship between genotype and CWD prevalence estimated from surveillance data. Using logistic regression, we showed that each 1% increase in CWD prevalence is associated with a 9.6% increase in the odds that an elk would have at least one copy of leucine at codon 132. In some regions, however, 132L* variants were found in the absence of CWD, indicating that evolutionary and epidemiologic patterns can be heterogeneous across space and time. We also provide evidence that naturally occurring CWD is not rare in 132L* elk, which merits the study of shedding kinetics in 132L* elk and the influence of genotype on CWD strain diversity. The management implications of cervid adaptations to CWD are difficult to predict. Studies that investigate the degree to which evolutionary outcomes are shaped by host spatial structure can provide useful epidemiologic insight, which can in turn aid management by informing scale and extent of mitigation actions.

Combined purebred and crossbred genetic evaluation of Columbia, Suffolk, and crossbred lamb birth and weaning weights: systematic effects and heterogeneous variances.

Despite the benefits of crossbreeding on animal performance, genetic evaluation of sheep in the U.S. does not directly incorporate records from crossbred lambs. Crossbred animals may be raised in different environments as compared to purebreds. Systemic factors such as age of dam and birth and rearing type may, therefore, affect purebred and crossbred performance differently. Furthermore, crossbred performance may benefit from heterozygosity, and genetic and environmental variances may be heterogeneous in different breeds and their crosses. Such issues must be accounted for in a combined (purebred and crossbred) genetic evaluation. The objectives of this study were to i) determine the effect of dam age and birth type on birth weight, and dam age and birth-rearing type on weaning weight, in purebred and crossbred lambs, ii) test for heterogeneous genetic and environmental variances in those weights, and iii) assess the impact of including weights on crossbred progeny on sire estimated breeding values (EBV). Performance records were available on purebred Columbia and Suffolk lambs. Crossbred information was available on lambs sired by Suffolk, Columbia or Texel rams mated to Columbia, Suffolk, or crossbred ewes. A multiple-trait animal model was fitted in which weights from Columbia, Suffolk, or crossbred lambs were considered different traits. At birth, there were 4,160, 2,356, and 5,273 Columbia, Suffolk, and crossbred records, respectively, with means (SD) of 5.14 (1.04), 5.32 (1.14), and 5.43 (1.23) kg, respectively. At weaning, on average at 122 (12) d, there were 2,557, 980, and 3,876 Columbia, Suffolk, and crossbred records, respectively, with corresponding means of 39.8 (7.2), 40.3 (7.9), and 39.6 (8.0) kg. Dam age had a large positive effect on birth and weaning weight in pure and crossbred lambs. At birth, however, the predicted effect was larger in crossbred and Suffolk lambs. While an increase in a number of lambs born and reared had a strong and negative influence on birth and weaning weight, the size of the effect did not differ across-breed types. Environmental variances were similar at birth and weaning, but additive variances differed among breed types for both weights. Combining purebred and crossbred information in the evaluation not only improved predictions of genetic merit in purebred sires but also allowed for direct comparisons of sires of different breeds. Breeders thus can benefit from an additional tool for making selection decisions.

Combining multiple breeds in a genetic evaluation allows for their direct comparison. However, differences in management and other systematic effects among breeds may affect the evaluation. Estimates of genetic merit of sires may also be biased by heterosis in crossbred progeny. We examined genetic and environmental factors that affect the efficacy of a multi-breed genetic evaluation. Birth and weaning weights of Columbia, Suffolk, and their cross, were available. Depending on the breed type, the systematic effects of dam age and either birth or birth-rearing type on weights differed. Separately for birth and weaning, weights were defined as a different trait for each breed type. A multi-breed, multi-trait model was fitted that accounted for systematic effects unique to a breed type, and heterosis. Estimated direct and maternal heritabilities were moderate. Genetic correlations between breeds were moderate to high. Estimates of genetic merit of Columbia and Suffolk sires were unaffected by bias due to heterosis and environmental effects when crossbred lambs were included in a purebred or a combined Columbia, Suffolk, and crossbred evaluation. For direct across-breed comparisons, breed type-specific adjustments for systematic effects are necessary when combining weight data on pure and crossbred lambs in a joint genetic evaluation.

Thrombospondin-Related Anonymous Protein (TRAP) Family Expression by Babesia bovis Life Stages within the Mammalian Host and Tick Vector.

The tick-transmitted disease bovine babesiosis causes significant economic losses in many countries around the world. Current control methods include modified live-attenuated vaccines that have limited efficacy. Recombinant proteins could provide effective, safe, and low-cost alternative vaccines. We compared the expression of the Babesia bovis thrombospondin-related anonymous protein (TRAP) family from parasites in bovine blood, in vitro induced sexual stages, and kinetes from tick hemolymph. Quantitative PCR showed that in blood and sexual stages, TRAP3 was highly transcribed as compared to the other TRAPs. In contrast, the TRAP1 gene was highly transcribed in kinetes as compared to the other TRAPs. Fixed immunofluorescence assays showed that TRAP2, 3, and 4 proteins were expressed by both blood and sexual stages. Conversely, TRAP1 protein, undetected on blood and induced sexual stages, was the only family member expressed by kinetes. Live IFA revealed that TRAP2, 3, and 4 proteins were expressed on the surface of both B. bovis blood and sexual stages. Modeling of B. bovis TRAP1 and TRAP4 tertiary structure demonstrated both proteins folded the metal-ion-dependent adhesion site (MIDAS) domain structure of Plasmodium TRAP. In conclusion, TRAP proteins may serve as potential vaccine targets to prevent infection of bovine and ticks with B. bovis essential for controlling the spread of bovine babesiosis.

Model definition for genetic evaluation of purebred and crossbred lambs including heterosis.

Crossbreeding is a common practice among commercial sheep producers to improve animal performance. However, genetic evaluation of U.S. sheep is performed within breed type (terminal sire, semi-prolific, and western range). While incorporating crossbred records may improve assessment of purebreds, it requires accounting for heterotic and breed effects in the evaluation. The objectives of this study were to: 1) describe the development of a paternal composite (PC) line, 2) determine the effect of direct and maternal heterosis on growth traits of crossbred lambs, 3) estimate (co)variance components for direct and maternal additive, and uncorrelated maternal environmental, effects, and 4) provide an interpretation of the estimates of random effects of genetic groups, and to use those solutions to compare the genetic merit of founding breed subpopulations. Data included purebred and crossbred records on birth weight (BN; n = 14,536), pre-weaning weight measured at 39 or 84 d (WN; n = 9,362) depending on year, weaning weight measured at 123 d (WW; n = 9,297), and post-weaning weight measured at 252 d (PW; n = 1,614). Mean (SD) body weights were 5.3 (1.1), 16.8 (3.9) and 28.0 (7.6), 39.1 (7.2), and 54.2 (8.7) kg for BN, WN (at the two ages), WW, and PW, respectively. In designed experiments, the Siremax, Suffolk, Texel, Polypay, Columbia, Rambouillet, and Targhee breeds were compared within the same environment. Estimates of heterotic effects and covariance components were obtained using a multiple trait animal model. Genetic effects based on founders' breeds were significant and included in the model. Percent estimates of direct heterosis were 2.89 ± 0.61, 2.60 ± 0.65, 4.24 ± 0.56, and 6.09 ± 0.86, and estimates of maternal heterosis were 1.92 ± 0.87, 4.64 ± 0.80, 3.95 ± 0.66, and 4.04 ± 0.91, for BN, WN, WW, and PW, respectively. Correspondingly, direct heritability estimates were 0.17 ± 0.02, 0.13 ± 0.02, 0.17 ± 0.02, and 0.46 ± 0.04 for BN, WN, WW, and PW. Additive maternal effects accounted for trivial variation in PW. For BN, WN, and WW, respectively, maternal heritability estimates were 0.16 ± 0.02, 0.10 ± 0.02, and 0.07 ± 0.01. Uncorrelated maternal environmental effects accounted for little variation in any trait. Direct and maternal heterosis had considerable impact on growth traits, emphasizing the value of crossbreeding and the need to account for heterosis, in addition to breed effects, if crossbred lamb information is included in genetic evaluation.

Crossbreeding is common in commercial sheep enterprises. It allows breeds with different attributes to be combined to generate crossbred progeny tailored to production environments and customer preferences. Additionally, crossbreds often benefit from heterosis, performing at levels above the average of their parental breeds. Over two decades, body weights were collected at birth and at pre-weaning, weaning, and post-weaning ages on purebred and crossbred lambs from semi-prolific (Polypay), western range (Columbia, Rambouillet, Targhee), and terminal sire (Siremax, Suffolk, Texel) breeds at the U.S. Sheep Experiment Station. When combined, the value of direct heterosis­that due to a lamb being crossbred­and maternal heterosis­that due to the lamb's dam being crossbred­increased birth (5%) and post-natal (up to 10%) weights in crossbred lambs. This highlights the value of crossbreeding to the U.S. sheep industry, especially in western range production systems. Genetic variation between and within breeds also was detected for the purebred parental breeds. Such heterotic and breed effects must be accounted for if crossbred performance is to be incorporated in genetic evaluation of purebreds. Therefore, these results provide the foundation for utilizing crossbred information in the evaluation and selection of purebred sheep in the United States.

A high-density genome-wide association with absolute blood monocyte count in domestic sheep identifies novel loci.

Monocytes are a core component of the immune system that arise from bone marrow and differentiate into cells responsible for phagocytosis and antigen presentation. Their derivatives are often responsible for the initiation of the adaptive immune response. Monocytes and macrophages are central in both controlling and propagating infectious diseases such as infection by Coxiella burnetii and small ruminant lentivirus in sheep. Genotypes from 513 Rambouillet, Polypay, and Columbia sheep (Ovis aries) were generated using the Ovine SNP50 BeadChip. Of these sheep, 222 animals were subsequently genotyped with the Ovine Infinium® HD SNP BeadChip to increase SNP coverage. Data from the 222 HD genotyped sheep were combined with the data from an additional 258 unique sheep to form a 480-sheep reference panel; this panel was used to impute the low-density genotypes to the HD genotyping density. Then, a genome-wide association analysis was conducted to identify loci associated with absolute monocyte counts from blood. The analysis used a single-locus mixed linear model implementing EMMAX with age and ten principal components as fixed effects. Two genome-wide significant peaks (p < 5x10-7) were identified on chromosomes 9 and 1, and ten genome-wide suggestive peaks (p < 1x10-5) were identified on chromosomes 1, 2, 3, 4, 9, 10, 15, and 16. The identified loci were within or near genes including KCNK9, involved into cytokine production, LY6D, a member of a superfamily of genes, some of which subset monocyte lineages, and HMGN1, which encodes a chromatin regulator associated with myeloid cell differentiation. Further investigation of these loci is being conducted to understand their contributions to monocyte counts. Investigating the genetic basis of monocyte lineages and numbers may in turn provide information about pathogens of veterinary importance and elucidate fundamental immunology.

Immunization against a Conserved Surface Polysaccharide Stimulates Bovine Antibodies with Opsonic Killing Activity but Does Not Protect against Babesia bovis Challenge.

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.

Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages.

BACKGROUND: Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite's environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. RESULTS: In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. CONCLUSIONS: Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis.

A DNA Regulatory Element Haplotype at Zinc Finger Genes Is Associated with Host Resilience to Small Ruminant Lentivirus in Two Sheep Populations.

Small ruminant lentivirus (SRLV) causes Maedi-Visna or Ovine Progressive Pneumonia in sheep and creates insidious livestock production losses. This retrovirus is closely related to human immunodeficiency virus and currently has no vaccines or cure. Genetic marker assisted selection for sheep disease resiliency presents an attractive management solution. Previously, we identified a region containing a cluster of zinc finger genes that had association with ovine SRLV proviral concentration. Trait-association analysis validated a small insertion/deletion variant near ZNF389 (rs397514112) in multiple sheep breeds. In the current study, 543 sheep from two distinct populations were genotyped at 34 additional variants for fine mapping of the regulatory elements within this locus. Variants were selected based on ChIP-seq annotation data from sheep alveolar macrophages that defined active cis-regulatory elements predicted to influence zinc finger gene expression. We present a haplotype block of variants within regulatory elements that have improved associations and larger effect sizes (up to 4.7-fold genotypic difference in proviral concentration) than the previously validated ZNF389 deletion marker. Hypotheses for the underlying causal mutation or mutations are presented based on changes to in silico transcription factor binding sites. These variants offer alternative markers for selective breeding and are targets for future functional mutation assays.

Genes involved in immune, gene translation and chromatin organization pathways associated with Mycoplasma ovipneumoniae presence in nasal secretions of domestic sheep.

Mycoplasma ovipneumoniae contributes to polymicrobial pneumonia in domestic sheep. Elucidation of host genetic influences of M. ovipneumoniae nasal detection has the potential to reduce the incidence of polymicrobial pneumonia in sheep through implementation of selective breeding strategies. Nasal mucosal secretions were collected from 647 sheep from a large US sheep flock. Ewes of three breeds (Polypay n = 222, Rambouillet n = 321, and Suffolk n = 104) ranging in age from one to seven years, were sampled at three different times in the production cycle (February, April, and September/October) over four years (2015 to 2018). The presence and DNA copy number of M. ovipneumoniae was determined using a newly developed species-specific qPCR. Breed (P<0.001), age (P<0.024), sampling time (P<0.001), and year (P<0.001) of collection affected log10 transformed M. ovipneumoniae DNA copy number, where Rambouillet had the lowest (P<0.0001) compared with both Polypay and Suffolk demonstrating a possible genetic component to detection. Samples from yearlings, April, and 2018 had the highest (P<0.046) detected DNA copy number mean. Sheep genomic DNA was genotyped with the Illumina OvineHD BeadChip. Principal component analysis identified most of the variation in the dataset was associated with breed. Therefore, genome wide association analysis was conducted with a mixed model (EMMAX), with principal components 1 to 6 as fixed and a kinship matrix as random effects. Genome-wide significant (P<9x10-8) SNPs were identified on chromosomes 6 and 7 in the all-breed analysis. Individual breed analysis had genome-wide significant (P<9x10-8) SNPs on chromosomes 3, 4, 7, 9, 10, 15, 17, and 22. Annotated genes near these SNPs are part of immune (ANAPC7, CUL5, TMEM229B, PTPN13), gene translation (PIWIL4), and chromatin organization (KDM2B) pathways. Immune genes are expected to have increased expression when leukocytes encounter M. ovipneumoniae which would lead to chromatin reorganization. Work is underway to narrow the range of these associated regions to identify the underlying causal mutations.

Characterizing Genetic Regulatory Elements in Ovine Tissues.

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

Molecular and Serological Detection of Piroplasms in Horses from Nigeria.

Equine piroplasmosis, an economically important disease of equids caused by the hemoprotozoan parasites Theileria equi, T. haneyi, and Babesia caballi, has a worldwide distribution. These parasites are transmitted by ixodid ticks. To improve the detection of horses in Nigeria exposed to piroplasm parasites, 72 horses with variable clinical signs of piroplasmosis were sampled from Northwest and Northcentral Nigeria and tested by nPCR and cELISA. Blood and serum samples were collected from each horse via jugular venesection. Individually, nPCR or cELISA failed to identify all horses exposed to piroplasms. A combination of species-specific nPCR and the OIE-approved T. equi and B. caballi cELISAs enhanced the detection of horses exposed to parasites. The results also demonstrated horses showing abnormal hematology were positive for only T. equi, except for one sample that was coinfected with T. equi and T. haneyi. We also identified ticks collected from some of the horses, with Rhipicephalus evertsi evertsi being the most prevalent. This study shows that a larger proportion of horses in the sample set were exposed to T. equi than B. caballi or T. haneyi. Additionally, ticks that have been previously reported as potential vectors for these parasites were found to have infested sampled horses. Further studies are needed to investigate which tick species are competent vectors for Theileria spp. and Babesia caballi in Nigeria.

Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation.

Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis, a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis.

Transcriptome dataset of Babesia bovis life stages within vertebrate and invertebrate hosts.

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as "housekeeping" genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.

Association of TMEM8B and SPAG8 with Mature Weight in Sheep.

Signature of selection studies have identified many genomic regions with known functional importance and some without verified functional roles. Multiple studies have identified Transmembrane protein 8B (TMEM8B)rs426272889 as having been recently under extreme selection pressure in domesticated sheep, but no study has provided sheep phenotypic data clarifying a reason for extreme selection. We tested rs426272889 for production trait association in 770 U.S. Rambouillet, Targhee, Polypay, and Suffolk sheep. TMEM8Brs426272889 was associated with mature weight at 3 and 4 years (p < 0.05). This suggested selection for sheep growth and body size might explain the historical extreme selection pressure in this genomic region. We also tested Sperm-associated antigen 8 (SPAG8) rs160159557 encoding a G493C substitution. While this variant was associated with mature weights at ages 3 and 4, it was not as strongly associated as TMEM8Brs426272889. Transmembrane protein 8B has little functional information except as an inhibitor of cancer cell proliferation. To our knowledge, this is the first study linking TMEM8B to whole organism growth and body size under standard conditions. Additional work will be necessary to identify the underlying functional variant(s). Once identified, such variants could be used to improve sheep production through selective breeding.

Assay to compare cell- and antibody-mediated immune responses in domestic sheep and goats.

Assessment of immune fitness is valuable in many aspects of livestock management and research. Determining immune consequences of selection for increased disease resistance or inhabiting various environments or climates can lead to different management decisions. The ability to measure immune responses due to different diets, pregnancy status, or aging will increase insight about how these factors contribute to overall immune health. The main objective of these experiments was to adapt a methodology used in cattle and pigs to measure both the humoral and cell-mediated immune response in sheep and goats. The route of administration of two antigens, Candida albicans and hen egg white lysozyme, were compared in sheep to determine differences in antibody or cell-mediated immune response. Subcutaneous injection produced a larger (P < 0.001) cell-mediated response compared to intramuscular injection. Inoculation in the axillary space produced a larger (P = 0.0031) antibody response compared to neck region. Finally, methodology was confirmed in goats. Complete blood cell counts were compared and lymphocytes were highest in low cell-mediated responders while eosinophils were highest in average antibody-mediated responders. This work provides a means to measure immune fitness in sheep and goats allowing for future experiments examining environmental or genetic effects on the immune response.

Quantitative analysis of Anaplasma marginale acquisition and transmission by Dermacentor andersoni fed in vitro.

In this study, we describe a new in vitro tick feeding system that facilitates the study of ticks and tick-borne pathogens. To optimize the system, we used Dermacentor andersoni and Anaplasma marginale as a tick-pathogen interaction model. Ticks were fed on bovine blood containing 10-fold dilutions of the pathogen to determine the effect of dose on tick infection rate. After feeding on infected blood, ticks were transferred to uninfected blood to stimulate bacterial replication within the tick vector. During stimulation feeding, blood samples were collected daily to determine if infected ticks secreted viable A. marginale. The results demonstrated similar attachment rates between the first and second tick feeding. Tick midgut and salivary glands were infected with A. marginale. However, salivary gland infection rates decreased as the percentage of parasitized erythrocytes decreased during tick acquisition feeding. Bacteria recovered from the in vitro system were able to infect a naïve bovine host. Using the highly transmissible A. marginale St. Maries strain, we demonstrated that the artificial tick feeding system is a suitable tool to study tick-pathogen interactions and that A. marginale tick salivary gland infection is dose dependent. This work demonstrates the utility of an artificial tick feeding system to directly study the association between the number of acquired pathogens and transmissibility by ticks.

Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages.

Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 (primed and distal enhancers), and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.

Evaluation of Rambouillet, Polypay, and Romanov-White Dorper × Rambouillet ewes mated to terminal sires in an extensive rangeland production system: body weight and wool characteristics1,2.

Though lamb sales account for the majority of annual receipts on U.S. sheep operations, wool is an important income source for many Western flocks. Crossing fine-wool sheep with prolific or composite hair sheep breeds can increase lamb production, but fleece quality and marketability may be reduced by a greater content of nonwool fibers (e.g., med and kemp). The objectives of this study were to compare BW and wool characteristics of Rambouillet, Polypay, and Romanov-White Dorper × Rambouillet (RW-RA) ewes under extensive rangeland management conditions. Ewe BW was collected before mating (fall) and 30 d postlambing (spring) each year from 1 yr up to 4 yr of age. In spring and fall, Rambouillet and Polypay ewes were similar in BW (P ≥ 0.94). Spring BW did not differ (P = 0.13) between RW-RA and Polypay, but Rambouillet ewes were heavier than RW-RA in the spring (P = 0.02). Both Rambouillet and Polypay ewes were heavier (P < 0.07) than RW-RA in the fall. Greasy fleece weight and mid-side wool samples were collected from ewes at 1 and 4 yr of age. Clean fleece weights (CFW) were estimated from average laboratory scoured yield of mid-side wool samples composited within-breed. Average fiber diameter (AFD), SD of fiber diameter (SD-FD), and percentage med (%M), kemp (%K), and total medullated fibers (%T) were quantified on individual mid-side wool samples. There was no difference in 1-yr-old CFW among breed types (P ≥ 0.96). Four-yr-old Rambouillet ewes had heavier CFW (2.29 kg; P < 0.001) than 4-yr-old Polypay (1.83 kg) and RW-RA ewes (1.86 kg), which were not different (P > 0.99). Within 1- and 4-yr-olds, AFD differed among breed type (P < 0.001) and was the finest for Rambouillet (20.1 and 21.9 µm, respectively), intermediate for RW-RA (22.8 and 24.8 µm), and coarsest for Polypay (24.2 and 26.7 µm). Also within 1- and 4-yr olds, SD-FD was lowest in Rambouillet, intermediate in Polypay, and greatest in RW-RA (P < 0.01). Wool from RW-RA ewes had greater %M, %K, and %T (P < 0.001) than wool from Rambouillet and Polypay ewes, which were not different (P > 0.99). Results indicated superior wool production for Rambouillet compared with the coarser, more variable wool produced by Polypay and RW-RA. Still, past research reported greater lamb production in Polypay and RW-RA ewes which, under recent market conditions, would be associated with greater annual gross revenue for these breed types than for Rambouillet.

Tissue inhibitor of metalloproteinase-1 and interleukin-10 in serum from naïve and scrapie infected sheep.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-10 (IL-10) were identified as potential biomarkers for ovine scrapie in a mouse model. The development of novel diagnostic methods to identify pre-clinical scrapie-infected animals is needed. In this study, ELISA was used to assess TIMP-1 and IL-10 levels in 158 serum samples from naïve and preclinical scrapie-infected sheep. Young (≤ 18 months) naïve sheep had significantly lower TIMP-1 levels compared with old (≥ 20 months) naïve and old infected sheep (P<0.04). Young naïve sheep had lower IL-10 than old naïve sheep (P<0.001). Both cytokines tended to have lower levels in young naïve sheep compared to infected sheep but this did not reach significance. A larger sample size will be helpful in determining the potential of these cytokines as a diagnostic tool.