Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Animal Science
Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act areas of plant and animal production, protection, and health and sustainable agriculture.Lameness and hoof health, though impacting dairy producers both as an animal welfare issue as well as being implicated in lowered milk production, is little studied. Further, it is one of the top three reasons dairy cattle are culled prematurely, only following infertility and mastitis. Currently dairy cattle in commercial herds are retained for an average of 2.5 lactations and are then removed from the herd. Premature culling represents a significant cost to the producer and contributes to the overall carbon footprint of the dairy due to the environmental costs associated with raising replacement dairy heifers. The goal of this work is to develop management strategies to reduce the incidence of lameness at the genetic selection level. By characterizing genomic regions associated with lameness, the impact of lameness can be addressed at the herd level (sire selection). This will improve the economic sustainability of dairy production, improve the welfare of the cattle, and reduce the environmental impact. We will be using genomic association studies to identify the particular genetic regions associated with two prevalent hoof lesions known to be under genetic control and result in lameness: sole ulcers and foot warts. Using DNA from cows with and without the conditions we will use the high density array to identify significant associations between each lesion and specific chromosomal regions. Those regions will then be fine mapped to determine maximal association and, if possible, through sequencing the DNA we will identify particular sequence alterations within specific genes. Associations of the hoof lesions with particular sequences will be further confirmed and then that information will be shared with researchers, cooperative extension specialists and county advisors, and dairy genomic testing companies for use as DNA-based markers in dairy breeding programs.The ultimate goal is to provide tools to enable producers to select against hoof lesions and thereby reduce lameness, improve productivity, and enhance dairy farm sustainability.
Animal Health Component
30%
Research Effort Categories
Basic
65%
Applied
30%
Developmental
5%
Goals / Objectives
Lameness and hoof health is a little studied though significant aspect of dairy cattle management. Lameness impacts dairy producers as an animal welfare issue and reduces milk production. It is one of the top three reasons dairy cattle are culled prematurely, following infertility and mastitis. The incidence of lameness in dairy cattle as reported in the literature ranges up to 50% though a more common estimate is between 15 -25%. The economic impact of each lameness case is estimated by Dr. Steven Berry, Emeritus Specialist in Cooperative Extension, to be $300 - $400/animal with overall annual costs of lameness exceeding $160,000,000 in California alone. The economic loss coupled with the animal health and welfare aspect of lameness is of enormous concern of the multi-billion dollar Dairy Industry.Selection schemes for dairy cattle focus on sire contribution to milk production with little consideration given to a cow's physical structure or disease probability. Our overall aim is to characterize the prevalence, the lacational stage of occurrence, and identify controlling genomic regions with the goal to mitigate the impact of lameness. In this way, lameness can be addressed twofold: addressing the condition on a per cow basis and at the herd level. Specifically, the goal of this work is to develop management strategies to reduce the incidence of lameness at the herd health, genetic selection level.We catalogued six phenotypic binary hoof traits that contribute to lameness on three commercial California dairies: white line disease, sole ulcer, other claw horn lesions, interdigital phlegmon (foot rot), digital dermatitis (foot warts), and other lesions. In this work we demonstrated that certain hoof lesions that contribute to lameness are prevalent: white line disease and foot warts (digital dermatitis) at ~8% and 17% of cows, respectively. Other hoof lesions are less prevalent with many associated with the transition period and farm hygiene suggesting management practices may assist in reducing the incidence. Most important, two of the lesions, sole ulcers (having a prevalence of ~4%) and foot warts have significant genetic contributions. Using a threshold model, heritabilities were estimated for each binary trait. The heritability for risk varied by lesion with the higher estimates being 0.40 (95% confidence range 0.20 - 0.67) for foot warts and 0.30 (95% confidence range 0.08 - 0.63) for sole ulcer. Including terms to account for cow productivity either on a 305 d mature equivalent (ME) or on a per lactation basis had minimal effects on the heritability estimates strongly suggesting that selection for hoof health is not correlated with response to selection for greater milk production and that improvement could be made for both traits simultaneously. In this project, we continue to build upon the findings noted above, and do an intensive interrogation of the genetics behind the risk for each of the hoof lesions. We are working with four commercial dairies within the central valley of California and they have provided us with blood samples daughters of those sires that do and do not have hoof lesions as well as from any cow over the age of 8 years that have not had any lameness episode during their lifetime (controls) and any cow that has had more than two lameness events with positive diagnosis of foot warts or sole ulcers. From those samples, DNA is extracted and subjected to a genome wide association approach (GWA) using the bovine SNP array available from Illumina, Inc (Illumina BovineSNP50 BeadChip) to identify chromosomal regions contributing to the expression of traits influencing lameness. In a preliminary study we identified suggestive linkage to chromosomal regions but the association did not reach statistical significance. We are expanding our samples incorporating additional cases of foot warts (for a total of 70), sole ulcers (for a total of 70) and control cows (for a total of 175). Power calculation indicates that the total number of foot wart and sole ulcer cases combined with the 175 controls proposed will provide >90% power to detect significant SNPs at p < 0.05, assuming the minor allele frequency of the SNPs is 3-4 times higher in cases. Thus, the enlarged sample size after adding new cows will be sufficient to power the GWAS and reveal SNPs significantly associated with the FW and SU phenotype. Importantly, both graduate and undergraduate students will assist in the execution of this project and we are working with the local county extension advisors.From this work we will identify quantitative trait loci (QTL) associated with sole ulcer and with foot wart phenotypes. We will further interrogate the genome in associated regions to uncover genetic polymorphisms that may underlie these deficits in hoof health. The end goal of this project is to develop genetic markers and then distribute that information to permit genetic selection to enhance the longevity and welfare of the cow in the production herd.
Project Methods
Genome wide association studies have been successfully accomplished with relatively few numbers of individuals (Settles et al, 2009) and our power calculations suggest that our sample size goal is adequate. Evidence in the published literature demonstrates that using QTLs has improved health and productivity of cattle lending credibility to the success of both identifying genetic markers and then employing them to assist in selection of cattle with decreased susceptibility to hoof lesions. To identify chromosomal regions associated with the expression of lameness traits, we will start with employing a genome wide association approach using the bovine SNP array available from Illumina, Inc (Illumina BovineSNP50 BeadChip). a cow will be classified as a case for foot warts (FW, digital dermatitis) or sole ulcer (SU, pododermatitis circumscripta) based upon exhibiting the lesion ≥ 1 time(s) as diagnosed by the hoof trimmer and a control if the cow has not exhibited a lameness event due to either FW or SU and is at least 8 years of age (the average age of onset for FW and SU is ~5 years). Blood samples will be collected and extracted using the QIAGEN QIAamp® DNA Blood Mini Kit (QIAGEN Inc., Valencia, CA) and quantified using the NanoDrop® (ND-1000 v3.2.1) spectrophotometer (Thermo Scientific, Wilmington, DE). High quality, concentrated DNA samples will be genotyped using the BovineHD BeadChip (Illumina Inc., San Diego, CA) by GeneSeek (Lincoln, NE). Illumina's GenCall algorithm will be used to call genotypes. Quality filtering of SNPs and genome-wide association analysis will be performed using PLINK 1.9. Basic genotype statistics for each marker, including call rate, minor allele frequency, Hardy-Weinberg Equilibrium (HWE) pvalue, the correlation coefficient (R), and allele and genotype counts will be calculated. Quality filtering will remove from further analysis any cows having less than 5% of all SNPs genotyped and SNPs with an overall amplification less than 95%. SNPs with a minor allele frequency less than 0.05 will be removed to exclude rare variants. After quality filtering, cows will be clustered based on identity-by-state using the population concordance test (p < 0.05) with the added constraint that each cluster must contain at least one case and one control. These clusters will then be used as criteria for the association analysis to correct for population stratification, minimizing associations due to underlying population structure as opposed to the phenotype. The allelic stratified case-control association analysis will test for association of SNP genotypes with FW and SU status. Significance for the stratified association analysis will be quantified using the Cochran-Mantel-Haenszel (CMH) p-value. To reduce false positives due to multiple testing across many loci, significant SNPs will be defined as those with Bonferroni corrected CMH p-Bonferroni < 0.05, where p-Bonferroni = p-unadjusted/(number of quality-filtered SNPs), while suggestive SNPs will be defined as p-Bonferroni < 0.20. Haplotype analysis will be performed with Haploview (Barrett et al., 2004) and fastPHASE (Scheet and Stephens, 2006) programs across positive chromosomes. To relax the overly stringent Bonferroni threshold for significance, permutation testing (100,000 permutations) will be performed respectively for the allelic and haplotype analyses to generate an empirical p-value for each SNP, and significance will be defined as p-empirical < 0.05. In addition to the stratified allelic GWAS, a pedigree-based GWAS analysis will be performed to account for and take advantage of known familial relationships, providing a different statistical approach to identify SNPs that are associated with expression of the FW and SU phenotypes.Once this objective is completed and associations have been determined, likely candidate genes located in the associated regions will be explored to identify potential susceptibility genes. The SNP with the strongest association with each of the two conditions will be genotyped in additional cows. Associated regions will be fine mapped with additional SNPs in 5 to 8 Mb segments (goal 1 SNP per 100 Kb). Regions with continued high association will be amplified by the polymerase chain reaction. The PCR products will be purified with Qiagen DNA extraction kits, the product sequenced at a core facility, and specific polymorphisms recorded for samples from cows with and without the lesions. The ultimate objective is to use the identified polymorphisms as genetic markers of susceptibility to hoof lesions and lameness. Utilizing those markers will assist in selection of cattle with decreased susceptibility to hoof lesions, reduced incidence of lameness, and improved animal health.The information obtained by the studies will then be shared with the aforementioned audiences to emphasize the effectiveness of selection, the need to characterize sires with respect to these particular markers, and to result in the markers being used in selection decisions ultimately effecting change in the dairy population. The effect of the work will be evaluated by how readily the information is absorbed and utilized by academic peers, dairy sire associations, and dairy producers.Settles M, Zanella R, McKay SD, Schnabel RD, Taylor JF, Whitlock R, Schukken Y, Van Kessel JS, Smith JM, Neibergs H. A whole genome association analysis identifies loci associated with Mycobacterium avium subsp. paratuberculosis infection status in US holstein cattle.Anim Genet. 2009; 40(5):655-62