UNDERSTANDING PARASITE RESISTANCE IN ORGANIC LIVESTOCK AND USING A SYSTEMS APPROACH FOR CONTROL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1010329
• Grant No.: 2016-51300-25723
• Project No.: MISW-2016-04406
• Proposal No.: 2016-04406
• Program Code: 113.A
• Project Start Date: Sep 1, 2016
• Project End Date: Aug 31, 2021
• Grant Year: 2016

Project Director
Burke, J. M.

Recipient Organization
USDA-ARS, GENETICS AND PRECISION AGRICULTURE UNIT
810 HIGHWAY 12 EAST
MISSISSIPPI STATE,MS 39762

Performing Department
(N/A)

Non Technical Summary
One of the greatest barriers to organic production of ruminant livestock is the control of gastrointestinal nematodes (GIN) or parasites. The goals of this project are to 1) understand host mechanisms involved in GIN resistance/resilience by examining differences in immune response among susceptible, resilient, and resistant individuals and breed types, 2) identify genetic loci associated with resistance/resilience, 3) further examine successful systems approaches of GIN control, including fall lambing to minimize summer exposure of GIN to lambs, diverse forage and grazing systems to minimize GIN exposure and use secondary plant compounds for control, and finally, 4) work with farmers enrolled in the National Sheep Improvement Program (NSIP) to understand tools for selection for GIN resistance. Participating farmers will be involved by providing 5000 DNA samples and phenotypes on GIN resistance on lambs in NSIP to use in our objectives. We will educate organic farmers on the importance of NSIP's breeding values and elite resistant sires to improve flock genetics for GIN resistance. These goals address OREI Priorities #6 by improving systems based animal production, animal health, and pest (parasite) management practices in the areas of grazing and pasture to improve animal productivity, health and welfare while retaining or enhancing economic viability; and #7 by selecting animal genotypes adapted to organic systems specifically to GIN resistance while grazing organic pasture.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
313	3610	1060	100%

Knowledge Area
313 - Internal Parasites in Animals;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1060 - Biology (whole systems);

Keywords

genetics

holistic farming systems

immunology

organic

parasites

sheep

Goals / Objectives
One of the greatest barriers to organic production of ruminant livestock is the control of internal parasites. The long term goals of the project are to 1) understand host mechanisms involved in GIN resistance (the ability to completely resist infection) or resilience (the ability to tolerate an infection without detriment to production) by examining differences in immune response between susceptible, resilient, and resistant individuals and breed types, 2) utilize genomics to identify genetic loci associated with resistance and/or resilience, 3) continue to examine successful approaches of GIN control, specifically lambing in the fall compared with winter/spring, and manipulate forage and grazing systems to minimize GIN exposure, and 4) work with farmers currently enrolled in the National Sheep Improvement Program (NSIP) to understand selection for GIN resistance, and organic farmers on education and adaptation of NSIP in their flocks to enable them to improve genetics for GIN resistance to minimize the need for deworming. These goals address OREI Priorities #6 by improving systems based animal production, animal health, and pest (parasite) management practices in the areas of grazing and pasture to improve animal productivity, health and welfare while retaining or enhancing economic viability; and #7 by selecting animal genotypes adapted to organic systems specifically to GIN resistance while grazing organic pasture.

Project Methods
Objective 1A: Determine effect of Katahdin sire EBV on H. contortus infection in Katahdin lambs (WVU): In Yr 1 and 2, lambs from high and low GIN resistant sires previously exposed to GIN then dewormed, will be infected with H. contortus to examine infection measures, antibody analysis, and peripheral cell counts for 7 wk post-infection. A subset of lambs will be slaughtered to enumerate worm burden, and run RNA-sequencing, qPCR analysis, and gene expression analysis on abomasal tissue.Objective 1B: Evaluate breed differences in immune responses to different life stages of H. contortus. GIN-free lambs will receive a priming infection of H. contortus, dewormed, rested and circulating immune cells collected and exposed to larval adult and egg stage of H. contortus. Damage to larvae and adults will be assessed by measuring ATP in larvae and adults after exposure to peripheral blood mononuclear cells derived from naïve and primed sheep. Damage to eggs will be assessed by determining egg ability to develop into infective larval stages. In another study, GIN-free lambs will be maintained as naïve lambs to serve as uninfected controls. Primed lambs will be euthanized at either 7 or 28 d following H. contortus infection. Infection measures will be collected and after euthanasia, abomasal contents will be collected and H. contortus larvae and adults will be counted, adult parasites will be sexed and length measurements will be taken. Abomasal tissue will be used for kinomic and RNA-seq analysis, and gene expression.Objective 2. Identification of genetic loci associated with resistance to GIN. DNA from lambs will be run on the OvineSNP50 BeadChip. The genotypes from approximately 54,000 SNP spaced across the genome for all of the selected animals will be collected. We will use PLINK whole genome association analyses software tools and perform a series of genotyping data quality control measures. This includes, but is not limited to, the removal any SNP probes with low genotype quality scores, minor allele frequency below 0.05, as well as any samples not in Hardy Weinberg equilibrium. Following data quality assurances, we will then perform genome association analyses.Objective 3A. Integration of resistant genetics, grazing systems, and season of lambing to control GIN (ARS). Using resistant sires, fall and winter lambing, and a combination of forage systems and management aimed at minimizing off-farm inputs, GIN infection will be determined that will begin in May 2016 (breeding ewes) and continue for the life of this project. In each breeding season, at least 6 rams will be exposed to a minimum of 20 ewes each (n = 120 ewes/season) for 30 d. FEC and body condition score will be determined on ewes at birth, 30 and 60 d post-lambing, body weight at breeding and 30 and 60 d post-lambing, and additional FEC, packed cell volume, and FAMACHA scores on lambs determined every 14 d between 90 and 210 d of age.Objective 3B. Dissecting the forage system to understand minimization of GIN infectivity (FVSU, ARS). Small plots of native legumes and forbs will be established. In mid-July of Yr 3, ungrazed established plots will be used to determine viability of GIN eggs in fecal pellets placed on plots. The aliquots will be scattered in three 30 cm2 random locations within each plot. Herbage samples around fecal material will be collected every 2-3 d. Herbage will be divided into forage species, each cut at 10 cm intervals from base to top of plant to recover, speciate and count larvae. Pioneering effects of the native forage system at the soil level, soil samples at the site of fecal deposition will be collected, baermmanized to collect larvae, and identification of non-GIN and GIN larvae will occur. Concentration and chemical structure of condensed tannins will be characterized in all of the legumes during their vegetative growth period using the thiolysis method.In order to understand how often the native mixes can be grazed, animal performance (GIN infection and growth), native plant mixes of 50% grass will be established in 12 individual 0.4 ha plots. Forages will be grazed at a height of at least 25 cm and will rest for 30 to 45 d or longer. In June of Yr 3, weaned lambs will be rotationally grazed and compared with a conventional rotational pasture system with predominantly tall fescue and/or bermudagrass. FEC, packed cell volume, incidence of deworming, body weight will be measured every 14 d for 120 d. Plant persistence, flowering, pollinator behavior will be observed throughout the study. Forage quality and mass will be determined every 28 d, and soil quality will be determined every 12 mo.Objective 3C. Utilization of Texel sires to improve parasite resistance in an organic farming operation (WVU). We will compare the impact on parasitism of grazing lambs sired by Texel or Suffolk rams over 3 years. Two Texel and 2 Suffolk rams each year would be used to mate 50 ewes per sire breed (25 ewes/sire). All lambs generated by these matings will be evaluated for parasite resistance and gain while grazing. Weight, blood and fecal samples will be collected weekly from June to October and pooled fecal samples will be collected monthly to determine specie of trichostrongylid parasite present on the pastures. At the completion of the study, ultrasonic measurement of fat depth and rib eye area will be performed on all lambs.Objective 4A. Data Collection from the Sheep Industry. Over the four years of the project, pedigree and production data, including EBV on FEC and other production traits will be provided on 5,000 lambs through NSIP on the collaborating Katahdin flocks. Those data will be used to identify (i) high and low FEC EBV rams for use in studies conducted at WVU for defining the mechanisms of immuno-response to a parasite challenge (Objective 1), and (ii) the 1,250 lambs for genotyping based on their parasite resistance, resilience or susceptibility (Objective 2). Of the animal phenotypes collected, 1250 lambs will be selected for genotyping (for Objective 2B) based on the highest (1/3 of lambs) and lowest (1/3 of lambs) EBV for the Wfec (FEC at weaning or approximately 90 days of age). Another 1/3 of lambs considered to be resilient based on having FAMACHA scores of 1 at sample collection and moderate FEC/Wfec will be included at the resilient group.

Progress 09/01/16 to 08/31/21

Outputs
Target Audience:Target audience includes scientists (animal scientists, geneticists), veterinarians, extension specialists, and producers, which were reached through scientific publications and professional meetings, dissemination through websites (www.wormx.info; www.attra.ncat.org), and virtual meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We provided training to producers on collecting fecal and blood samples from their animals, which not only benefits data collection for the current project but allows them to collect and submit blood for other genotyping or diagnostic analyses. Graduate students at West Virginia University (4), University of Idaho (2), University of Nebraska (1), Fort Valley State University (1), and University of Arkansas in collaboration with USDA, ARS (1 plus undergraduate students) were mentored in techniques necessary to contribute to accomplishments for this project as well as their lifelong professional goals. A series of videos were developed to help producers with techniques for parasite control (www.wormx.info; www.attra.ncat.org ). Producers were educated on the benefits of genetic selection for parasite resistance and other important production traits. How have the results been disseminated to communities of interest?Results have been disseminated in scientific meetings (American Consortium for Small Ruminant Parasite Control, COMBAR - the EU equivalent of the ACSRPC, American Society of Animal Science, Southern Section and National; American Association of Veterinary Parasitologists, World Association for the Advancement of Veterinary Parasitology, International Society of Genetics Conference, Plant and Animal Genetic Conference, Entomology Society of America), producer meetings (American Sheep Industry Association Conference, Southern SAWG Organic Research Forum), and webinars (ASI Let's Grow webinar available through YouTube; NSIP webinars on the rollout of genomic enhanced estimated breeding values). Results presented to participating and ancillary Katahdin producers stimulated immense discussion and outcomes which will be used to formulate programs for a wider base of organic sheep and goat producers. Results on parasitology tools developed were presented on the ATTRA website (https://attra.ncat.org) which targets sustainable sheep and goat producers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1) Determine mechanism of action of resistance/resilience to GIN. Katahdin rams selected for high or low fecal egg count (FEC) estimated breeding values (EBV; or parasite) resistance bred to ewes at the Southwest Virginia Agriculture Research and Extension Center in collaboration with WVU, produced lambs from each phenotype without affecting body weight or carcass traits. There was an association between high FEC EBV and compromised immune function in lambs, in that lambs with higher FEC EBV (more susceptible to parasites) appeared to have decreased survival to weaning in lambs either from parasites or other disease. Immune responses differed between resistant and susceptible animals. Interleukin-13, an immune modulator that may be expressed differentially between sheep breeds susceptible or resistant to gastrointestinal (GIN) parasites, is involved in altering mobility of the infective stage larvae of the blood sucking worm, Haemonchus contortus. Monocytes are another immune cell involved in inflammation and when derived from resistant sheep compared with susceptible sheep reduced the energy output of H. contortus larvae. The Texel breed is used for meat production and is known to have establishment of adult GIN worms, but low FEC. This mechanism may be due to an immune response that limits egg laying capacity of the adult worm. These experiments support the use of resistant breeds of sheep for organic production which will greatly reduce the need for deworming and support growth of lambs. Objective 2) Use of industry data and identification of genetic loci associated with resistance to GIN. Collection of phenotypes (fecal egg counts to indicate parasite infection, FAMACHA scores to indicate degree of anemia, and body weights) and 6708 blood cards for DNA analyses on lambs from 23 farms between 2016-2019 occurred and nearly 5000 DNA samples from these lambs with performance data and pedigrees in NSIP (National Sheep Improvement Program) were genotyped using GeneSeek Genomic Profiler Ovine 50K array. We were able to analyze more than the 1250 we anticipated due to a reduction in pricing of the array chips. This also allowed us to genotype an additional 600 samples using the Illumina Ovine Hight Density array that has close to 600K genetic markers. This was used for genome-wide association study analyses (GWAS), the first occurred on a sub-population conducted with FEC EBV revealing significance on five chromosomes and included regions described in published parasite resistance research in sheep. The most significant marker was associated with a gene not previously reported in parasite resistance studies, but known to be under selection in some populations of sheep (DIS3L2). Another GWAS study on 583 sheep using the HD array identified four significant SNP located within the first intron of the gene EGF-like repeats and discoidin domains 3 (EDIL3; has a role in the immune response of inflammation possibly increasing susceptibility to GIN). Sheep with alternate homozygous genotypes had higher FEC compared with all other genotypes. Further studies are needed to understand the role of EDIL3 and genetic mechanisms of susceptibility to GIN. These SNP may contribute to genetic strategies for improving parasite resistance traits in sheep. In collaboration with the Animal Genetics and Breeding Unit (University of New England, Armidale, Australia) the process for using the genotypes collected to augment prediction of animals' genetic merit (genomic-enhanced Estimated Breeding Values) was validated. The single-step genetic evaluation method was used, which is the most advanced means available to generate genetic predictions. Through the validation process, it was necessary to update heritablities and genetic correlations used in the genetic evaluation. The combined changes led to revised genetic evaluation system delivered through NSIP to Katahdin breeders in October 2021. The genetic diversity of this important breed was captured beyond understanding the genetics of gastrointestinal parasitism and built the foundation of genomic technologies in the U.S. sheep industry. That evolution provides tremendous opportunity for establishing more effective, sustainable, and resilient breeding programs in the U.S. sheep industry which benefit organic production. Objective 3) Examine farm systems that minimize the need for deworming. A multi-year study was completed on finishing fall or winter born lambs on grass pastures either with or without supplement finished to a light weight typical for grass finishing. Winter born lambs required more supplementation due to the poorer quality forages and had greater morbidity related to parasites; fall born ram lambs finished on cool season grasses performed well with or without supplement and had minimal issues with parasites, requiring fewer deworming in fall compared with winter born lambs. Data was collected in multiple years in addition to this study to examine differences in dynamics of GIN in lambs born in fall or winter and seems to indicate that after intense selection for parasite resistance within the flock, GIN infection and amount of deworming may be similar between seasons. Analyses continue on the impact of genetics vs. season which will aid in decisions on parasite management of lambs. A pollinator study was conducted at ARS, Booneville on organic and conventional sheep pastures and pollinator species are being counted and speciated. Decline of insect pollinators which support food production has increased the importance of accurately monitoring pollinator diversity and abundance over time. In organic and conventional sheep pastures, it was determined that differences in color of a passive form of trap in attracting wild bees occurred; bright blue vane traps attracted the highest rate of bees with the greatest diversity relative to other colors (yellow, purple, red) and that bright compared with dark colors attracted more bees. In a related study, bee communities were more diverse and higher evenness observed in non-grazed compared to grazed pastures, possibly due to differences in availability of floral resources that sheep consumed. However, bee abundance and species richness did not significantly differ among grazing treatments. These studies enabled scientists to understand species diversity in sampling wild bees in a livestock pasture setting which is important to entomologists, food scientists, environmentalists, and ecologists. Objective 4) Work with the farmers through extension and NSIP to obtain EBVs on GIN resistance. Much of this was captured in Obj. 2. In addition, impact of our outreach and publications were far reaching. While much of the work focused around the Katahdin sheep breed, it served as a successful model in understanding parasite resistance in organic livestock and that using a system of control is most successful. We were able to convey valuable information to organic and conventional farmers, extension specialists, veterinarians, geneticists, ecologists, and many more through our website (www.wormx.info), ATTRA/NCAT (www.ncat.org), and publications.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. Weaver, A.R., J.J Garza, S.P. Greiner, S.A. Bowdridge. 2021. Immune response to Haemonchus contortus in Texel sheep. Parasite Immunology 43:e12876.

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Target audience includes scientists (animal scientists, geneticists), veterinarians, extension specialists, and producers, which were reached through scientific publications and professional meetings, dissemination through websites (www.wormx.info; www.attra.ncat.org), and virtual meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We provided training to producers on collecting fecal and blood samples from their animals, which not only benefits data collection for the current project but allows them to collect and submit blood for other genotyping or diagnostic analyses. New graduate students learned techniques necessary to contribute to accomplishments for this project as well as their lifelong accomplishments. In addition, a series of videos were developed to help producers with techniques for parasite control (www.wormx.info; www.attra.ncat.org). Producers were educated on the benefits of genetic selection for parasite resistance and other important production traits. ? How have the results been disseminated to communities of interest?Results have been disseminated in scientific meetings (American Society of Animal Science, Southern Section and National; American Association of Veterinary Parasitologists), producer meetings (American Sheep Industry Association Conference), and webinars (ASI Let's Grow webinar available through YouTube). Results presented to Katahdin producers stimulated immense discussion and outcomes will be used to formulate programs for a wider base of organic sheep and goat producers. ? What do you plan to do during the next reporting period to accomplish the goals?We will continue to work with farmers to clarify any questions on data submission, and disseminate results of the genomic analyses. Several manuscripts are in progress. We will continue with data analyses and educational and outreach programs as planned.

Impacts
What was accomplished under these goals? Objective 1) Katahdin rams selected for high or low FEC EBV (parasite) resistance bred to ewes at the Southwest Virginia Agriculture Research and Extension Center in collaboration with WVU, produced lambs from each phenotype without affecting body weight or carcass traits. There was an association between high FEC EBV and compromised immune function in lambs, which will be explored further outside the scope of the current grant. Objective 2) Collection of phenotypes on lambs from 21 farms between 2017-2019 occurred and 5000 DNA samples from these lambs were genotyped using 50K SNP. An additional 600 samples, including sires of these lambs and select lambs will be genotyped using high density SNP. Farmers submitted phenotypic data on lambs including fecal egg counts to indicate parasite infection among lambs in a contemporary group, FAMACHA scores, and body weights. Analyses using GWAS will occur. In addition, genetic diversity and population structure in Katahdin sheep recorded in the National Sheep Improvement Program (NSIP) is being evaluated. Objective 3) A multi-year study (13 years) was completed on finishing fall or winter born lambs on predominantly grass pastures either with or without supplement finished to a light weight typical for grass finishing. Winter born lambs required more supplementation due to the poorer quality forages and had greater morbidity related to parasites, but fall born ram lambs finished on cool season grasses performed well with or without supplement and had minimal issues with parasites, requiring fewer deworming in fall compared with winter born lambs (Wood et al., 2019). Data has been collected in multiple years to examine differences in dynamics of GIN in lambs born in fall or winter, which will aid in decisions on parasite management of lambs. Results have been analyzed using SAS but several interactions exist that will need to be examined in detail and a manuscript is in preparation. Plots of mixed native forages were established at Fort Valley State University to examine its effects on GIN egg hatch and larval survival in association with more diverse microenvironments compared with monoculture grass plots typical of small ruminant grazing in the Southeastern U.S. Due to the pandemic, research on this objective could not be completed in 2020. A pollinator study was conducted at ARS, Booneville on organic and conventional sheep pastures and pollinator species are being counted and speciated. Manuscripts are expected by early 2021. Objective 4) Phenotypic data and DNA on lambs enrolled in the National Sheep Improvement Program from 21 farms across the U.S. was collected between 2017 and 2019. Data will be used to find genetic markers for parasite resistance, contributing to earlier work that included susceptible and resistant Katahdin sires. Selection of sheep for parasite resistance balanced with maternal traits using estimated breeding values (EBV) and phenotypic traits demonstrated that the ARS and U.S. flocks consistently showed a downward trend (lower fecal egg counts) toward improved parasite resistance over the last 10 years. These sheep are genetically connected to animals from several farms in the U.S. NCAT participated in outreach by producing short videos on what to look for in sheep regarding parasites and health. A Grazing Management video (Part 1 and 2) were available March 2020, a webinar on Managing Internal Parasites (Parts 1, 2, 3) were available on the on the ATTRA website (www.attra.ncat.org).

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Jacobs, J.R., D. Middleton, S.P. Greiner, S.A. Bowdridge. 2020. RNA-sequencing of ovine PBMC after exposure to Haemonchus contortus larval antigen. Parasit. Immunol. https://doi.org/10.1111/pim.12697
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Middleton, D., J.J. Garza, S.P. Greiner, S.A. Bowdridge. 2020. Neutrophils rapidly produce Th2 cytokines in response to larval but not adult helminth antigen. Parasit. Immunol. https://doi.org/10.1111/pim.12679
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Jacobs, J.R., S.P. Greiner, S.A. Bowdridge. 2018. Impaired interleukin-4 signaling promotes establishment of Haemonchus contortus in sheep. Parasit. Immunol. https://doi.org/10.1111/pim.12597
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Garza, J.J., S.P. Greiner, S.A. Bowdridge. 2018. Ovine vital neutrophil extracellular traps bind and impair Haemonchus contortus L3 in a breed-dependent manner. Parasit. Immunol. https://doi.org/10.1111/pim.12572
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Shepherd, E.A., J.J, Garza, S.P. Greiner, S.A. Bowdridge. 2017. The effect of ovine peripheral blood mononuclear cells on Haemonchus contortus larval morbidity in vitro. Parasit. Immunol. https://doi.org/10.1111/pim.12424
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Garza, J.J., S.P. Greiner, S.A. Bowdridge. 2017. Serum-mediated Haemonchus contortus larval aggregation differs by larval stage and is enhanced by complement. Parasit. Immunol. http://doi.org/10.1111/pim.12409
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Shepherd, E.A, B. Russ, S.P. Greiner, S.A. Bowdridge. 2020. Interleukin-13 induces paralysis of Haemonchus contortus larvae in vitro. Parasite Immunology
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Shepherd, E.A., S.P. Greiner, S.A. Bowdridge. 2020. Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro. Parasite Immunology
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Weaver, A.R., J.J Garza, S.P. Greiner, S.A. Bowdridge. 2020. Immune response to Haemonchus contortus in Texel sheep. Parasite Immunology
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Weaver, A.R., J.M. Burke, J. Morgan, D.L. Wright, S.P. Greiner, S.A. Bowdridge. 2020.Effect of sire fecal egg count estimated breeding value on Katahdin lamb pasture performance. J Anim. Sci.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Weaver, A.R., J.M. Burke, J. Morgan, D.L. Wright, S.P. Greiner, S.A. Bowdridge. 2020.Effect of sire fecal egg count estimated breeding value on Haemonchus contortus infection in Katahdin sheep. J Anim. Sci.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Becker GM, Job RJ, Davenport KM, Burke JM, Lewis RM, Miller JE, Morgan JL, Notter DR, and Murdoch BM. Genome-wide association study to identify loci associated with gastrointestinal nematode resistance in Katahdin. Animal Genetics (2020)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Davenport KM, Hiemke C, McKay SD, Thorne JW, Lewis RM, Taylor T, Murdoch BM. Genetic structure and admixture from terminal breeds in the United States. Animal Genetics (2020)
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Notter, D.R., Heidaritabar, M., Burke, J.M., Spangler, G.L., Murdoch, B.M., MacNeil, M.D., Morgan, J.L.M., Morota, G., VanTassell, C.P. et al., 2020. Association analysis of SNP effects on lamb fecal egg count EBV in progeny-tested Katahdin sires. Genetics
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Terrill, T.H., Whitley, N.C., Smith, E., Lyte, K., Burke, J.M., 2020. Determining the efficacy of Red Cell� in combination with anthelmintic drugs against indicators of gastrointestinal nematode parasitism in sheep and goats. Vet. Parasitol.
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Acharya, M., Burke, J.M., Miller, J.E., Terrill, T.H., Wood, E.L., Muir, J.P., 2020. Quebracho tannins aid in the control of Eimeria spp. in lambs and goat kids. Vet. Parasitol.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Burke, J.M., Wood, E. 2020. Estimating the value of parasite resistance in sheep. Southern SAWG (abstr.), Little Rock, AR
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Burke, J.M., 2020. Organic compared with conventional systems for lamb production yield differences in performance in Southeastern U.S. pastures. Southern SAWG Organic Research Forum
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Burke, J.M., 2020. Organic and conventional pasture systems for lamb production in Southeastern U.S. J. Anim. Sci. Suppl. 1 (abstr.)
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Jones, O., Burke, J.M., Miller, J.E., Rosenkrans, C., 2020. Use of diatomaceous earth and copper oxide wire particles to control gastrointestinal nematodes in lambs. J. Anim. Sci. 98 (Suppl.1 Abstr.).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Burke, J.M., Notter, D.R., Morgan, J.L.M., Miller, J.E., Lewis, R.M., Heidaritabar, M., 2019. Integrating genetic selection with selective anthelmintic treatment to manage gastrointestinal nematodes in Katahdin sheep in the United States. Joint COMBAR/ACSRPC meeting, Ghent, Belgium. P. 74. (abstr.).

Progress 09/01/18 to 08/31/19

Outputs
Target Audience:Target audience was farmers, educators, scientists and extension specialists associated with organic livestock production, genetics, genomics and parasite control. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development included participation in coordinating committees (NCERA-214, SCC-81) with collaborators in this grant, an International meeting in Ghent, Belgium (ACSRPC and the European COMBAR) focused on dewormer resistance and how to counter act it.In addition, a series of videos were developed (and continue to be developed) to help producers with techniques for parasite control (www.wormx.info). How have the results been disseminated to communities of interest?Results have been disseminated in scientific meetings (American Society of Animal Science, Southern Section and National; American Association of Veterinary Parasitologists), producer meetings (American Sheep Industry Association Conference; Katahdin Hair Sheep Int. Annual Meeting), and webinars (ASI Let's Grow webinar). Results presented to Katahdin producers stimulated immense discussion and outcomes will be used to formulate programs for a wider base of organic sheep and goat producers. ? What do you plan to do during the next reporting period to accomplish the goals?We will continue to work with farmers to clarify any questions on data submission, and collect remaining DNA samples. Samples will be submitted to GeneSeek for genotyping. Several manuscripts are in progress. We will continue with unfinished research experiments, and educational and outreach programs as planned.

Impacts
What was accomplished under these goals? Objective 1) Katahdin rams selected for high or low FEC EBV (parasite) resistance bred to ewes at the Southwest Virginia Agriculture Research and Extension Center in collaboration with WVU, produced lambs from each phenotype without affecting body weight or carcass traits. There was an association between high FEC EBV and compromised immune function in lambs, which will be explored further. Objective 2) We have nearly completed the third year of phenotype and DNA collection from participating farms (21 farms included across the U.S. in 2017, 2018, 2019). Farmers submitted phenotypic data on lambs including fecal egg counts to indicate parasite infection among lambs in a contemporary group, FAMACHA scores, and body weights. Farmers collected blood samples for DNA isolation for genotyping and GWAS analyses. We have more than 3840 samples collected (additional samples pending; includes sires and lambs), with 1863 blood cards in 2017 and 1926 in 2018. The total number of female samples is 1,968, and total number of male samples is 1,872. The total number of samples from lambs (male and female) is 3,775. Samples have been collected from 65 sires in total, however only 58 of these sires currently meet the criteria for having at least 10 offspring. Seven sires with samples collected currently have fewer than 10 offspring. Genetic diversity and population structure in Katahdin sheep (UNL). By understanding the level of genetic diversity present in a population, better breeding and conservation decisions can be made. It also aids in assessing the potential value of incorporating genomic information in the genetic evaluation orchestrated by NSIP. Our objective was to use pedigree analysis to assess the population structure, and potential losses in genetic diversity due to unequal contributions, bottlenecks and genetic drift, in Katahdin sheep. Despite some lessening in genetic diversity, the losses observed in the Katahdin was still less than that reported in other sheep breeds. To search for genetic loci associated with GIN resistance, a pilot genome-wide association study (GWAS) was conducted with 40 animals. A Short communication regarding these data has been submitted to Animal Genetic. Work was continued from a previous OREI grant by PD and co-PI to test candidate regions associated with GIN using 87 proven Katahdin sires. Both weaning and post-weaning fecal egg count were used as indicators of host resistance to GIN. For WFEC 8.45% and for PFEC 6.45% of our regions were validated to be significantly associated with the traits of interest considering the previous studies were the testing populations. Objective 3) A multi-year study (12 years) was completed on finishing fall or winter born lambs on predominantly grass pastures either with or without supplement finished to a light weight typical for grass finishing. Winter born lambs required more supplementation due to the poorer quality forages and had greater morbidity related to parasites, but fall born ram lambs finished on cool season grasses performed well with or without supplement and had minimal issues with parasites, requiring fewer deworming in fall compared with winter born lambs (Wood et al., 2019). Data has been collected in multiple years to examine differences in dynamics of GIN in lambs born in fall or winter, which will aid in decisions on parasite management of lambs. Results have been analyzed using SAS but several interactions exist that will need to be examined in detail and a manuscript is in preparation. Plots of mixed native forages were established at Fort Valley State University to examine its effects on GIN egg hatch and larval survival in association with more diverse microenvironments compared with monoculture grass plots typical of small ruminant grazing in the Southeastern U.S. A pollinator study was conducted at ARS, Booneville on organic and conventional sheep pastures. Establishment of native grasses and forbs was low on organic plots due to weed competition. Insect samples (numbers, species/classification) collected from plots are being analyzed. Objective 4) Phenotypic data and DNA on lambs enrolled in the National Sheep Improvement Program from 21 farms across the U.S. was collected between 2017 and 2019. Data will be used to find genetic markers for parasite resistance, contributing to earlier work that included susceptible and resistant Katahdin sires. Selection of sheep for parasite resistance balanced with maternal traits using estimated breeding values (EBV) and phenotypic traits demonstrated that the ARS and U.S. flocks consistently showed a downward trend (lower fecal egg counts) toward improved parasite resistance over the last 10 years. These sheep are genetically connected to animals from several farms in the U.S. NCAT participated in outreach in the following ways. Assisted in producing short videos on the topics of: FAMACHA in a Nutshell, How to Use Copper Oxide Wire Boluses, How to Take a Fecal Sample, and How to Take a Blood Sample. These videos are in the video library on the Consortium website, www.wormx.info, and as of May 2019 had been viewed a total of 1,227 times. Produced and delivered a 3-part webinar series in partnership with Food Animal Concerns Trust (FACT). This series was live in February 2019 and is archived on the FACT website and is included on the NCAT/ATTRA YouTube page, so that it continues to be seen by producers into the future. All persons who registered for any of the webinars received an email that included links to publications to support the learning. The content of the webinars was as follows: Managing Internal Parasites in Sheep and Goats: Know Thy Enemy (about the parasite life cycle, impacts on the animal, symptoms, survival strategies of the internal parasites)--viewed by 546 persons as of May 2019. Managing Internal Parasites in Sheep and Goats: Outsmart the Enemy (preventive techniques, including grazing management, nutrition, and animal selection; discussion of periparturient rise and of immunity)--viewed by 268 persons in webinar form, and slides used to present to 50 other individuals at workshops in Arkansas and Oklahoma. Managing Internal Parasites in Sheep and Goats: Attack the Enemy (about treatment strategies and minimizing treatment while maximizing effectiveness)--viewed by 214 persons as of May 2019. These webinars have been seen so far 1,028 times online. They continue to be accessed; from April 23, 2019 to May 23, 2019, more than 150 persons viewed the webinars on the FACT website. Therefore, outreach continues with no additional funding. In total, 972 persons received emails from FACT with resources attached, furthering outreach to those who perhaps have not viewed the webinars yet. NCAT/ATTRA has noticed a boost in downloads of internal parasite management titles following the webinar. There are 19 titles related to internal parasite management of sheep and goats housed on the ATTRA website (www.attra.ncat.org). Many of these are also featured on the website www.wormx.info as PDF's, and those are not counted by ATTRA. Some are featured on other websites as well and are not counted by us. On the ATTRA site alone, from May 1, 2018 to May 1, 2019, these 19 publications were downloaded or viewed 48,725 times. In addition to being available on the ATTRA website, ATTRA sends paper copies out for use at conferences and trainings.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Howell, B., Burke, J.M., Lee, C., Wood, E., Rosenkrans, C.F. Jr., Rorie, R., 2019. Factors that limit out-of-season breeding success in ewes. J. Anim. Sci. 97 (Suppl. 1), 74 (Abstr.)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Burke, J.M., Miller, J.E., 2019. Investing in parasite resistance in sheep. J. Anim. Sci. 97 (Suppl. 1), 75-76 (Abstr.)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Becker, G.M., Sawyer, R.J., Davenport, K.M., Rodriguez, A.M., Burke, J.M., Lewis, R., Miller, J.E., Morgan, J., Murdoch, B.M., 2019. Understanding the molecular mechanisms for gastrointestinal parasite resistance in Katahdin sheep. Plant Anim. Gen. Conf., San Diego, CA, Jan 13, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sharma Acharya, R., Fitting, E., Burke, J., and Joshi, N. 2019. Different pan traps for sampling pollinators and beneficial arthropods in the livestock pasture ecosystem. Nov. 18, 2019, St. Louis, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Burke, J.M., Notter, D.R., Morgan, J.L.M., Miller, J.E., Lewis, R.M., Heidaritabar, M., 2019. Integrating genetic selection with selective anthelmintic treatment to manage gastrointestinal nematodes in Katahdin sheep in the United States. Joint COMBAR/ACSRPC meeting, Ghent, Belgium. P. 74. (abstr.).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Burke, J.M., 2020. Organic and conventional pasture systems for lamb production in Southeastern U.S. J. Anim. Sci. (Abstr.)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Burke, J.M., Miller, J.E., Acharya, M., Wood, E., 2020. Copper oxide wire particles to complement control of gastrointestinal nematodes with levamisole and/or albendazole in lambs. J. Anim. Sci. (Abstr.).
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Dykes, D.S., Terrill, T.H., Whitley, N.C., Singh, A.K., Mosjidis, J.A., Burke, J.M., Kouakou, B., Miller, J.E., 2019. Effect of ground and pelleted sericea lespedeza whole plant and leaf only on gastrointestinal nematode and coccidial infection in goats. J. Agric. Sci Tech A 9, 93-102.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bowdridge, S.A., S.P. Greiner, A.R. Weaver, D.L. Wright. 2019. Utilization of Katahdin rams with lower fecal egg count EBV improves parasite resistance and fitness of progeny. Southern Section ASAS Meeting, Oklahoma City, OK
• Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Burke, J.M., Miller, J.E. 2019. Sustainable approaches to parasite control in ruminant livestock. Vet. Clinics: Food Anim. Pract. (In Press)
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Becker, G.M., Davenport, K.M., Burke, J.M., Lewis, R.M., Miller, J.E., Morgan, J.L., Notter, D.L., Murdoch, B.M., 2019. Genome-wide association study to identify genetic loci associated with gastrointestinal nematode resistance in Katahdin sheep. Anim. Gen. (In Review).
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Gebremariam, W.Z., Burke, J.L., Morgan, Notter, D.R., Lewis, R.M., 2019. Genetic diversity and population structure in the Katahdin sheep. J. Anim. Sci. (In Review).

Progress 09/01/17 to 08/31/18

Outputs
Target Audience:Target audience includes farmers, extension specialists, veterinarians and scientists. We are working with farmers from Washington to Georgia and Maine to Texas to collect phenotypic data and DNA from their sheep. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We provided training to producers on collecting fecal and blood samples from their animals, which not only benefits data collection for the current project but allows them to collect and submit blood for other genotyping or diagnostic analyses. Producers learned how to use the FAMACHA system, a means to estimate parasite infection of an animal by the color of the membrane around the eye. New graduate students learned techniques necessary to contribute to accomplishments for this project as well as their lifelong accomplishments. In addition, a series of videos were developed (and continue to be developed) to help producers with techniques for parasite control (www.wormx.info). How have the results been disseminated to communities of interest?Results have been disseminated in scientific meetings (American Society of Animal Science, Southern Section and National; American Association of Veterinary Parasitologists), producer meetings (American Sheep Industry Association Conference; Texas Sheep and Goat Expo, San Angelo, TX), and webinars (ASI Let's Grow webinar). What do you plan to do during the next reporting period to accomplish the goals?We will continue to work with farmers to collect data and samples to reach our goal of obtaining 5000 DNA samples from lambs with phenotypic data and genetic linkages among several flocks and will include additional farms next year. It is amazing how the National Sheep Improvement Program has grown in terms of determining parasite resistance among lambs within and between flocks to benefit seedstock and commercial farms. Several farmers are aware of how genetics can contribute to parasite management allowing more resistant animals to be free of parasite infections. We will continue with research experiments, and educational and outreach programs as planned.

Impacts
What was accomplished under these goals? Objective 1) An experiment was conducted to determine differences in tissue gene expression at 42-d post-infection between high and low fecal egg count estimated breeding value sired-lambs. Lambs (n = 60) were processed and tissues samples are being analyzed using RNA-seq. In addition, 3 manuscripts were published and 2 submitted for publication. Objective 2) We have nearly completed the second year of phenotype and DNA collection from participating farms (18 farms included across the U.S.) nearly reaching half our targeted goal of 5,000 samples. Farmers submitted phenotypic data on lambs including fecal egg counts to indicate parasite infection among lambs in a contemporary group, FAMACHA scores, and body weights. Farmers collected blood samples for DNA isolation for genotyping and GWAS analyses (details below). At the University of Idaho, a preliminary trial was conducted on a subset of DNA samples from high and low parasite resistant lambs based on their estimated breeding values and fecal egg counts. The 50K chip worked well with these samples. GWAS analyses showed significant associations or differences between groups of lambs. These types of analyses will be used on 1,250 DNA samples from high and low parasite resistant lambs in the final year. Objective 3) We have 11 years of data to utilize to examine gastrointestinal nematode infection between fall and spring born lambs in Arkansas. Data will be analyzed and a manuscript prepared. Forage systems were established that included native grasses and/or forbs and legumes in Arkansas and Georgia. Experiments will be conducted in Year 3 to examine differences between monoculture grass vs. a diverse forage system on gastrointestinal egg hatch and nematode development. 4) We have worked with farmers involved in data/sample collection in Objective 2, helping them to understand data generated on their farm. In addition, collaborating farms have shared rams so that the farms contributing data to NSIP and this project will have connected genotypes, strengthening the estimated breeding values and genetics to be evaluated in Objective 2. Outreach has been conducted to educate farmers, extension specialists, scientists and veterinarians on systems approaches to control gastrointestinal nematodes. A manuscript was published on evaluation of parasite resistance traits and reproductive and growth traits, demonstrating little or no antagonisms on multiple selection of traits on-farm using NSIP data. The two main activities over the reporting period undertaken at the University of Nebraska-Lincoln (UNL) were (i) collecting and inventorying DNA samples (blood cards) on animals from stakeholder Katahdin flocks; and (ii) developing and implementing a strategy for identifying animals for genotyping for use in a preliminary Genome Wide Association Study to identify genetic markers associated with gastrointestinal nematode resistance/susceptibility. DNA sample collection and inventorying As a key element of the project, over four years pedigree and production data are being collected, including estimated breeding values (EBV) on fecal egg counts (FEC) and other production traits, on 5,000 lambs from collaborating Katahdin flocks participating in the National Sheep Improvement Program (NSIP; Objective 4). In addition to these data, FTA blood cards are being collected on these lambs (and their sires) to establish a repository of DNA samples for use in genotyping to potentially identify genetic markers for parasite resistance or resilience (Objective 2). Ultimately, 1,250 lambs will be genotyped. Over the past two years, stakeholder flocks have been provided with supplies for collecting blood samples, FTA blood cards, and labels. Once collected, blood cards are being sent to UNL for storage. A Microsoft Access database was developed to inventory the blood cards as they arrive. Beyond facilitating tracking the samples, animal IDs are being validated to ensure their integrity (uniqueness). For lambs born in 2017, 1,830 blood cards have been received from 17 stakeholders. Although sampling is not yet complete, 1,241blood cards have already been received from 10 stakeholders for lambs born in 2018. In just the first two years of the study, a large repository of DNA samples - over 61% of the target number - has been accumulated. The next step is to cross-check the IDs on these samples with those of animals recorded in the NSIP database in order to link the genomic with pedigree and performance information available. Identifying animals for genotyping for use in Genome Wide Association Study (GWAS) Objective 2B of the project entails identification of possible genetic loci associated with resistance to gastrointestinal nematodes (GIN). An opportunity arose to genotype approximately 40 Katahdin sheep using the Ovine Single Nucleotide Polymorphism (SNP) 50K BeadChip. As a preliminary step toward identifying genomic regions associated with GIN resistance, a strategy was established to identify animals with either high or low EBV for FEC at weaning (WFEC) for genotyping. As the first step in this strategy, animals with FEC EBV reported in NSIP with accuracies of greater than 50%, and with a DNA sample, were identified. There were 1,111 animals that met these criteria. Within this subset, animals were categorized as having either low (less than -81) or high (greater than 40) WFEC EBV, which coincides with either greater resistance or greater susceptibility to GIN, respectively. This captured approximately the top and bottom 10% of the animals when ranked on WFEC EBV. As a second step in the strategy, the strength of genetic relatedness (connectedness) was assessed among the animals in these distinct categories based on pedigree information. The outcome of this analysis is shown in Figure 1 for the low WFEC EBV category; the rams (sires) and lambs selected for genotyping are identified. The plot, which is based on a principal component analysis, reflects the genetic diversity observed among these animals. Greater distances coincide with less ancestral relatedness. Rams and one of their progeny were chosen for genotyping. The remaining lambs genotyped were drawn from other sire families.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Notter, D.R., Ngere, L., Burke, J.M., Morgan, J.L.M., Miller, J.E., 2018. Genetic parameters for ewe reproductive performance and peri-parturient fecal egg counts and their genetic relationships with lamb body weights and fecal egg counts in Katahdin sheep. J. Anim. Sci. 96, 1579-1589. Ngere, L., Burke, J.M., Morgan, J.L.M., Miller, J.E., Notter, D.R., 2018. Genetic parameters for fecal egg counts and their relationship with body weights in Katahdin lambs. J. Anim. Sci.96, 1590-1599. doi: 10.2527/jas2017.2000 Garza, J.J., S.P. Greiner, S.A. Bowdridge. 2018. Ovine vital NETs bind and impair H. contortus L3 in a breed-dependent manner. Parasit. Immunol. https://doi.org/10.1111/pim.12572 Shepherd, E.A., J.J. Garza, S.P Greiner, S.A. Bowdridge. 2017. Effects of ovine peripheral blood mononuclear cells on Haemonchus contortus larval death in vitro. Parasit. Immunol. 39:e12424 Garza, J.J., S.P. Greiner, S.A. Bowdridge. 2017. Serum-mediated H. contortus larval aggregation differs by larval stage and is enhanced by complement. Parasit. Immunol. 39:e12409. Weaver, A., J.J. Garza, S.A. Bowdridge. 2018. Immune response to various life stages of Haemonchus contortus differentiates resistance among sheep breeds. 63rd Annual Meeting AAVP Denver, CO. Middleton, D., J.J. Garza, S.A. Bowdridge. 2018. Neutrophils rapidly produce Th2 cytokines in response to larval but not adult Haemonchus contortus antigen. 63rd Annual Meeting, AAVP Denver, CO. Shepherd, E. S.A. Bowdridge. 2018. Characterization of ovine monocytes that induce Haemonchus contortus morbidity in vitro. 63rd Annual Meeting, AAVP Denver, CO. Garza, J.J., S.A. Bowdridge. 2018. Neutrophils from Suffolk sheep exhibit impaired chemotaxis in response to Haemonchus contortus antigen. 63rd Annual Meeting, AAVP Denver, CO.

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:Target audience includes farmers andscientists. We are working with farmers from Washington to Georgia and Maine to Texas to collect phenotypic data and DNA from their sheep. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We provided training to producers on collecting fecal and blood samples from their animals, which not only benefits data collection for the current project but allows them to collect and submit blood for other genotyping or diagnostic analyses. Producers learned how to use the FAMACHA system, a means to estimate parasite infeciton of an animal by the color of the membrane around the eye. New graduate students learned techniques necessary to contribute to accomplishments for this project as well as their lifelong accomplishments. How have the results been disseminated to communities of interest?It is too early in the project to disseminate any results. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work with farmers to collect data and samples to reach our goal of obtaining 5000 DNA samples from lambs with phenotypic data and genetic linkages among several flocks and will include additional farms next year. It is amazing how the National Sheep Improvement Program has grown in terms of determining parasite resistance among lambs within and between flocks to benefit seedstock and commercial farms. Several farmers are aware of how genetics can contribute to parasite management allowing more resistant animals to be free of parasite infections. Projects have begun at West Virginia University, Fort Valley State University, University of Idaho, and ARS according to plan in Objectives 1, 2, and 3 which will allow us to meet goals in Year 2. In addition, graduate students were hired at University of Nebraska, West Virginia University, University of Idaho, Fort Valley State University, and in assocation with ARS to work toward objectives of the project. We will continue with educational and outreach programs as planned.

Impacts
What was accomplished under these goals? While there are no completed accomplishments to date, we began the first year of data and sample collection from 18 farmers from Washington to Georgia and Maine to Arkansas. There will be up to three additional farms next year. Farmers submitted phenotypic data on lambs including fecal egg counts to indicate parasite infection among lambs in a contemporary group, FAMACHA scores, and body weights. Farmers collected blood samples for DNA isolation for genotyping and GWAS analyses.

Publications