Breeding parasite-resistant sheep that are less dependent on the use of anthelmintics to maintain sustainable management and productivity. It would also assist farmers to manage the growing anthelmintic-resistance problem in U.S. Very limited research work has been devoted to selection for resistance to gastrointestinal nematode infections using genetic markers in Florida native sheep populations. Thus, it is proposed that the identification of DNA markers that are indicative of parasite resistance (or susceptibility) could improve selection programs. In order to determine the resistance or susceptibility status of sheep, the objective of this project is to develop genetic markers for parasite resistance in Florida native sheep populations.
1. Identify and validate DNA markers in 100 selected genes (including promoter regions, coding regions and non-coding regions) associated with resistance using targeted sequencing in 160 Florida native sheep, previously characterized with different levels of resistance to natural gastrointestinal nematode infections (extreme individuals: highest fecal egg counts and lowest fecal egg counts).
2. Evaluate gene expression of the significant DNA markers associated with resistance or susceptibility to support the results from Objective 1.
3. Disseminate to producers the potential benefits of this tool in the selection of breeding animals, using the genetic markers showing benefit in resistance level to gastrointestinal nematodes.
The research protocol for this study was approved by the University of Florida Institutional Animal Care and Use Committee. Two hundred 3 month old Florida native lambs corresponding to the 15% bottom and top individuals for fecal egg count performance within the population will be used for the study. Lambs will be treated with levamisole (7.5 mg/kg of live weight). Animals will be placed on pasture and exposed to natural infection (Trichostrongylus columbiformis and Haemonchus contortus).
Fecal samples will be taken at deworming, 10 and 31 days post deworming. Fecal egg counts will be determined following the McMaster technique with a level of detection of 50 eggs/g. An average of the FEC measurements will be used for the analysis. Famacha score will be recorded on 10 and 31 days post deworming. A blood sample will be taken from the jugular vein at 10 and 31 days post-deworming to measure hematological parameters such as package cell volume (PCV), eosinophil count, lymphocyte count, neutrophil count, basophil count and monocyte count. Hematological parameters will be evaluated by using ProCyte Dx Hematology Analyzer. Average daily gain will be measured during the study.
DNA isolation and targeted sequencing. DNA will be purified from blood samples from day 31 post- deworming using DNAzol® BD Reagent (Thermo Fisher Scientific) per manufacturer’s instructions. The yield and purity will be calculated using the NanoDrop1000 (Thermo Fisher Scientific) within a range of 260 to 280 nm. Integrity of DNA samples will be tested by agarose gels at 4% before DNA storage at -20°C. DNA samples will be used to perform targeted sequencing. The targeted sequencing panel will be custom designed and will use kits with probe sets focused on genes associated with the immune response against GNI in order to identify specific and de novo single nucleotide polymorphisms. A subset of 100 genes of the sheep genome will be isolated and sequenced by using Illumina Next Generation Sequencing System (NGS) in four steps as follow: 1) Library preparation, 2) Cluster generation and hybridization to biotinylated probes to targeted genes, 3) Enrichment using streptavidin beads, 4) Elution from beads and Sequencing, and 5) Data analysis. Targeted sequencing will allow to focus time, expenses, and data analysis on gastrointestinal nematode infections and will enable sequencing at much higher coverage levels. A typical whole genome sequencing study achieves coverage levels of 30×–50× per genome, while a targeted resequencing covers the target region at 500×–1000× or higher. This technology evaluates single nucleotide polymorphisms that are missed on the traditional genotyping methods (such as Illumina Ovine SNP50 BeadChip) and provides an increased sensitivity to detect unique and rare single nucleotide polymorphism variants, testing targeted genes. Moreover, the main advantage of this technology is the detection of mutations that are missed by currently exome sequencing and Sanger sequencing.
Data from sequencing will be demultiplexed using Illuminas BCLtofastq. Data will be cleaned and trimmed (3’ end will be trimmed by removing low quality bases with < 20 quality score reads). Clean reads will be aligned to genome with MOSAIK software. Freebayes will be used for identification of SNPs and VCFtools was used to generate VCF files.
Association of SNPs to ADG, FEC and hematological parameters. The Saphiro-Wilk test will be used to test all variables for normality. If necessary, The R ‘car’ library will be used to estimate the power parameter λ and carry out transformation of data. JMP Genomics from SAS (SAS Institute Inc., Cary, NC) and will be used for genomic data management, quality control, statistical analyses and graphics. For quality control, marker properties will be evaluated and SNPs will be discarded if call rate < 95%, minor allele frequency (MAF) ≤0.01. The population structure will be estimated based on the analysis of principal components to cluster genetically similar individuals using marker information. Identity by state (IBS) matrix will be calculated from marker information. Q-K association analysis will be used in order to perform association mapping while controlling for population structure and/or familial relatedness (from marker information). A mixed model will be used for the association analysis and will include the IBS matrix. Bonferroni correction will be used to control for multiple testing.
Isolation of RNA and cDNA synthesis. From blood samples from 10 and 31 days post-deworming, total RNA will be isolated from lymphocytes using TRIzol Reagent (Thermo Fisher Scientific). Total RNA samples will be reverse transcribed into cDNA using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s guidelines.
Real-Time PCR. Real-Time PCR conditions for each primer set will be optimized and standardized according to the manufacturer’s guidelines. Real-Time PCR assays will be performed on the Eco Real Time PCR System (Illumina) using SYBR® Green PCR Master Mix (Thermo Fisher Scientific). Forty cycles of amplification with an initial incubation (95°C 10 min), followed by denaturation at 95°C for 15 sec and annealing at 60°C for 30 sec will be performed. Ct and amplification values of each sample will be used for the expression analysis and the amount of target will be normalized with GAPDH and B-actin genes. All samples will be compared with the control data (10 days post- deworming). The method used for relative quantification will be the quantitative comparison method that calculates the reaction efficiency without a standard curve. The analysis involves a second derived of the amplification curve fluorescence to obtain an inflection point and the reaction efficiency is calculated in the exponential phase of the curve (Pfaffl, 2001).
Statistical analysis for gene expression results. Analysis will be carried out by using REST® (Qiagen) in RG mode. This statistical software analyze data with a non-parametric method, which tests the transcripts for significance using a Pair-wise Fixed Reallocations Randomization and Boot-strapping Tests.
A small ruminant production symposium will be organized at the Department of Animal Sciences in the University of Florida. Furthermore, abstracts will be developed for meetings such as Annual Meeting of the American Society of Animal Science and National Association of County Agricultural Agents on year two to present the results obtained from this project. Moreover, 3 articles will be developed and published in the following journals: Parasite Immunology, Journal of Animal Science and Immunogenetics.
From April 2nd 2018 until May 10th 2018, phenotype collection will be conducted. Preliminary results will be available on next SARE’s report.
Educational & Outreach Activities
Results will be available next SARE’s report and will be disseminated with farmers from Ocala, Florida. Two scientific articles will be published from this research and a field day will be organized at the University of Florida.