H. L. Neibergs

Resolving the evolution of extant and extinct ruminants with high-throughput phylogenomics

The Pecorans (higher ruminants) are believed to have rapidly speciated in the Mid-Eocene, resulting in five distinct extant families: Antilocapridae, Giraffidae, Moschidae, Cervidae, and Bovidae. Due to the rapid radiation, the Pecoran phylogeny has proven difficult to resolve, and 11 of the 15 possible rooted phylogenies describing ancestral relationships among the Antilocapridae, Giraffidae, Cervidae, and Bovidae have each been argued as representations of the true phylogeny. Here we demonstrate that a genome-wide single nucleotide polymorphism (SNP) genotyping platform designed for one species can be used to genotype ancient DNA from an extinct species and DNA from species diverged up to 29 million years ago and that the produced genotypes can be used to resolve the phylogeny for this rapidly radiated infraorder. We used a high-throughput assay with 54,693 SNP loci developed for Bos taurus taurus to rapidly genotype 678 individuals representing 61 Pecoran species. We produced a highly resolved phylogeny for this diverse group based upon 40,843 genome-wide SNP, which is five times as many informative characters as have previously been analyzed. We also establish a method to amplify and screen genomic information from extinct species, and place Bison priscus within the Bovidae. The quality of genotype calls and the placement of samples within a well-supported phylogeny may provide an important test for validating the fidelity and integrity of ancient samples. Finally, we constructed a phylogenomic network to accurately describe the relationships between 48 cattle breeds and facilitate inferences concerning the history of domestication and breed formation.

A whole genome association analysis identifies loci associated with Mycobacterium avium subsp. paratuberculosis infection status in US holstein cattle

The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis (Map) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont enrolled in longitudinal herd studies between January 1999 and November 2007 were assessed for the presence of Map in both faecal and tissue samples. An animal was considered tissue infected if any sample contained at least one colony forming unit of Map per gram of tissue (CFU/g) and the same definition was employed for faecal samples. Each animal was genotyped with the Illumina BovineSNP50 BeadChip and after quality assurance filtering, 218 animals and 45 683 SNPs remained. We sought to identify loci associated with four different case/control classifications: presence of Map in the tissue, presence of Map in faeces, presence of Map in both tissue and faeces and presence of Map in tissue but not faeces. A case-control genome wide association study was conducted to test the four different classifications of Map infection status (cases) when compared with a Map-negative control group (control). Regions on chromosomes 1, 5, 7, 8, 16, 21 and 23 were identified with moderate significance (P < 5 x 10(-5)). Two regions, one on chromosome 3 (near EDN2) and another on chromosome 9 (no positional gene candidates), were identified with a high level of association to the presence of Map in tissue and both tissue and faeces respectively (P < 5 x 10(-7), genome-wide Bonferonni P < 0.05).

A Metagenomics and Case-Control Study To Identify Viruses Associated with Bovine Respiratory Disease

ABSTRACT Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirty-eight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P < 0.0001), bovine rhinitis A virus (P = 0.005), and the recently described bovine influenza D virus (P = 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and real-time PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies. IMPORTANCE Bovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact.

Identification of loci associated with tolerance to Johne’s disease in Holstein cattle

Johnes disease, caused by Mycobacterium avium subspecies paratuberculosis (Map), is a fatal disease in cattle. The objective of this study was to identify loci associated with tolerance in cows infected with Map. Tolerance was defined as a cows fitness at a given level of Map infection intensity. Fitness was measured by Map faecal cultures, and Map infection intensity was measured by culturing four gut tissues. The quantitative phenotype of tolerance was defined by numerical indexes of cultures of peak (peak tolerance, PT) and average (average tolerance, AT) faecal and tissue Map from 245 Holstein cows. The categorical phenotype was defined as: ≥ 100 cfu Map tissue infection, and faecal shedding ≥ 75 cfu (intolerant) or <10 cfu (tolerant cows). In 94 cows, Map was identified in ≥ 1 tissue, including 44 cows with ≥ 100 Map tissue cfu and 36 with ≥ 1 faecal cfu. A genome-wide association analysis was performed after filtering, leaving genotypes for 45,789 SNPs in 90 animals for the quantitative phenotype and 16 cases and 25 controls for the categorical analysis of tolerance. rs41748405:A>C (BTA15) was associated with PT (P = 1.12 × 10(-7)) and AT (P = 2.17 × 10(-6)). Associations were identified with PT and adjacent SNPs ss61512613:A>G and ss61530518:A>G (BTA6) (P < 3.0 × 10(-5)), and with AT for ss61469568:A>G (BTA 2) (P = 3.3 × 10(-5)) and ss86284768:A>G (BTA1) (P = 3.31 × 10(-5)). For the categorical phenotype, an association was found with ss8632653:A>G (BTA6) (P < 5.0 × 10(-5)). This is the first study to identify loci associated with tolerance to Johnes disease.

Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves.

BackgroundBovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High density SNP genotyping with the Illumina BovineHD BeadChip was conducted on 1257 male and 757 female Holstein calves from California (CA), and 767 calves identified as female from New Mexico (NM). Of these, 1382 were classified as BRDC cases, and 1396 were classified as controls, with all phenotypes assigned using the McGuirk health scoring system. During the acquisition of blood for DNA isolation, two deep pharyngeal and one mid-nasal diagnostic swab were obtained from each calf for the identification of bacterial and viral pathogens. Genome-wide association analyses were conducted using four analytical approaches (EIGENSTRAT, EMMAX-GRM, GBLUP and FvR). The most strongly associated SNPs from each individual analysis were ranked and evaluated for concordance. The heritability of susceptibility to BRDC in pre-weaned Holstein calves was estimated.ResultsThe four statistical approaches produced highly concordant results for 373 top ranked SNPs that defined 126 chromosomal regions for the CA population. Similarly, in NM, 370 SNPs defined 138 genomic regions that were identified by all four approaches. When the two populations were combined (i.e., CA + NM) and analyzed, 324 SNPs defined 116 genomic regions that were associated with BRDC across all analytical methods. Heritability estimates for BRDC were 21% for both CA and NM as individual populations, but declined to 13% when the populations were combined.ConclusionsFour analytical approaches utilizing both single and multi-marker association methods revealed common genomic regions associated with BRDC susceptibility that can be further characterized and used for genomic selection. Moderate heritability estimates were observed for BRDC susceptibility in pre-weaned Holstein calves, thereby supporting the application of genomic selection to reduce the prevalence of BRDC in U.S. Holsteins.

Prevalence of the prion protein gene E211K variant in U.S. cattle

BackgroundIn 2006, an atypical U.S. case of bovine spongiform encephalopathy (BSE) was discovered in Alabama and later reported to be polymorphic for glutamate (E) and lysine (K) codons at position 211 in the bovine prion protein gene (Prnp) coding sequence. A bovine E211K mutation is important because it is analogous to the most common pathogenic mutation in humans (E200K) which causes hereditary Creutzfeldt – Jakob disease, an autosomal dominant form of prion disease. The present report describes a high-throughput matrix-associated laser desorption/ionization-time-of-flight mass spectrometry assay for scoring the Prnp E211K variant and its use to determine an upper limit for the K211 allele frequency in U.S. cattle.ResultsThe K211 allele was not detected in 6062 cattle, including those from five commercial beef processing plants (3892 carcasses) and 2170 registered cattle from 42 breeds. Multiple nearby polymorphisms in Prnp coding sequence of 1456 diverse purebred cattle (42 breeds) did not interfere with scoring E211 or K211 alleles. Based on these results, the upper bounds for prevalence of the E211K variant was estimated to be extremely low, less than 1 in 2000 cattle (Bayesian analysis based on 95% quantile of the posterior distribution with a uniform prior).ConclusionNo groups or breeds of U.S. cattle are presently known to harbor the Prnp K211 allele. Because a carrier was not detected, the number of additional atypical BSE cases with K211 will also be vanishingly low.

Effects of Fertility on Gene Expression and Function of the Bovine Endometrium

Infertility and subfertility are important and pervasive reproductive problems in both domestic animals and humans. The majority of embryonic loss occurs during the first three weeks of pregnancy in cattle and women due, in part, to inadequate endometrial receptivity for support of embryo implantation. To identify heifers of contrasting fertility, serial rounds of artificial insemination (AI) were conducted in 201 synchronized crossbred beef heifers. The heifers were then fertility classified based on number of pregnancies detected on day 35 in four AI opportunities. Heifers, classified as having high fertility, subfertility or infertility, were selected for further study. The fertility-classified heifers were superovulated and flushed, and the recovered embryos were graded and then transferred to synchronized recipients. Quantity of embryos recovered per flush, embryo quality, and subsequent recipient pregnancy rates did not differ by fertility classification. Two in vivo-produced bovine embryos (stage 4 or 5, grade 1 or 2) were then transferred into each heifer on day 7 post-estrus. Pregnancy rates were greater in high fertility than lower fertility heifers when heifers were used as embryo recipients. The reproductive tracts of the classified heifers were obtained on day 14 of the estrous cycle. No obvious morphological differences in reproductive tract structures and histology of the uterus were observed in the heifers. Microarray analysis revealed differences in the endometrial transcriptome based on fertility classification. A genome-wide association study, based on SNP genotyping, detected 7 moderate associations with fertility across 6 different chromosomes. Collectively, these studies support the idea that innate differences in uterine function underlie fertility and early pregnancy loss in ruminants. Cattle with defined early pregnancy success or loss is useful to elucidate the complex biological and genetic mechanisms governing endometrial receptivity and uterine competency for pregnancy.

Meta-Analysis of Two Genome-Wide Association Studies of Bovine Paratuberculosis

Background Bovine paratuberculosis (ParaTB) also known as Johnes disease, is a contagious fatal disease resulting from infection by Mycobacterium avium subspecies paratuberculosis (MAP). Previous studies have identified loci associated with ParaTB using different measurements to define cases and controls. The objective of this study was to combine the data from two recent studies to identify genetic loci associated with MAP tissue infection and humoral immune response, defined by MAP ELISA-positive cattle, by comparing cases and control animals for one or both measures of infection. Methodology/Principal Findings The two populations used for the association analyses were a cohort of MAP tissue infected animals and control Holstein cows from the USA and the second cohort composed of ELISA-positive and ELISA-negative Holstein cows from Italy. Altogether 1190 cattle were genotyped with the Illumina BovineSNP50 BeadChip. SNP markers were removed if the minor allele frequency <0.01 or genotyping failure was >5%. Animals were removed with >5% genotyping failure. Whole genome association analyses were conducted with the GRAMMAR-CG method using two different definitions of control populations. Conclusion/Significance The analyses identified several loci (P<5 e-05) associated with ParaTB, defined by positive ELISA and presence of bacteria in tissue compared to ELISA and tissue negative animals, on chromosomes 1, 12 and 15 and one unassigned SNP. These results confirmed associations on chromosome 12 and the unassigned SNP with ParaTB which had been found in the Italian population alone. Furthermore, several additional genomic regions were found associated with ParaTB when ELISA and tissue positive animals were compared with tissue negative samples. These loci were on chromosomes 1, 6, 7, 13, 16, 21,23 and 25 (P<5 e-05). The results clearly indicate the importance of the phenotype definition when seeking to identify markers associated with different disease responses.

Genome-Wide Association Identifies Multiple Genomic Regions Associated with Susceptibility to and Control of Ovine Lentivirus

Background Like human immunodeficiency virus (HIV), ovine lentivirus (OvLV) is macrophage-tropic and causes lifelong infection. OvLV infects one quarter of U.S. sheep and induces pneumonia and body condition wasting. There is no vaccine to prevent OvLV infection and no cost-effective treatment for infected animals. However, breed differences in prevalence and proviral concentration have indicated a genetic basis for susceptibility to OvLV. A recent study identified TMEM154 variants in OvLV susceptibility. The objective here was to identify additional loci associated with odds and/or control of OvLV infection. Methodology/Principal Findings This genome-wide association study (GWAS) included 964 sheep from Rambouillet, Polypay, and Columbia breeds with serological status and proviral concentration phenotypes. Analytic models accounted for breed and age, as well as genotype. This approach identified TMEM154 (nominal P = 9.2×10−7; empirical P = 0.13), provided 12 additional genomic regions associated with odds of infection, and provided 13 regions associated with control of infection (all nominal P<1×10−5). Rapid decline of linkage disequilibrium with distance suggested many regions included few genes each. Genes in regions associated with odds of infection included DPPA2/DPPA4 (empirical P = 0.006), and SYTL3 (P = 0.051). Genes in regions associated with control of infection included a zinc finger cluster (ZNF192, ZSCAN16, ZNF389, and ZNF165; P = 0.001), C19orf42/TMEM38A (P = 0.047), and DLGAP1 (P = 0.092). Conclusions/Significance These associations provide targets for mutation discovery in sheep susceptibility to OvLV. Aside from TMEM154, these genes have not been associated previously with lentiviral infection in any species, to our knowledge. Further, data from other species suggest functional hypotheses for future testing of these genes in OvLV and other lentiviral infections. Specifically, SYTL3 binds and may regulate RAB27A, which is required for enveloped virus assembly of human cytomegalovirus. Zinc finger transcription factors have been associated with positive selection for repression of retroviral replication. DLGAP1 binds and may regulate DLG1, a known regulator of HIV infectivity.

Loci on Bos taurus chromosome 2 and Bos taurus chromosome 26 are linked with bovine respiratory disease and associated with persistent infection of bovine viral diarrhea virus 1

The objective of this study was to identify loci linked with bovine respiratory disease (BRD) and subsequently to determine if these same loci were associated with bovine viral diarrhea virus persistent infection (BVD-PI) in affected calves or their dams. A genome-wide linkage study using 312 microsatellites was conducted to identify loci linked with BRD in a Brahman × Hereford sire half-sib family. Disease incidence was recorded from birth to slaughter by daily monitoring. Linkage was suggestive for a QTL on BTA2 (F = 7.31, P = 0.007) and BTA26 (F = 10.46, P = 0.001). Six and 7 markers were added and genotyped between 110 and 126 cM on BTA2 and between 42 and 72 cM on BTA26, respectively, in the intervals where linkage was found. These markers were used to reevaluate the Brahman × Hereford family and to evaluate 3 additional crossbred half-sib families. Linkage was found with BRD on BTA2 (F = 4.94, P < 0.01), with a peak at 110 cM, and on BTA26 (F = 4.03, P < 0.05), with peaks at 42 and 52 cM. The same markers were then tested for an association with BVD-PI in 1) BVD-PI calves compared with age-matched unaffected calves from the same herd or 2) dams with BVD-PI compared with age-matched unaffected calves. Sixty commercial beef cow-calf herds were tested for BVD-PI, and 79 calves from 8 ranches had BVD-PI. Four of 6 markers were associated (P = 4.8 × 10(-9) to P = 0.01) with BVD-PI on BTA2, and 4 of 7 markers were associated (P = 0.008 to P = 0.04) with BVD-PI on BTA26 when BVD-PI calves were compared with unaffected calves. The comparison of BVD-PI dams with unaffected calves detected associations with BVD-PI for all markers tested on BTA2 (P = 3 × 10(-9) to P = 0.005) and for 3 of 7 markers on BTA26 (P = 1.4 × 10(-6) to P = 0.006).