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United States Department of Agriculture

Agricultural Research Service

Research Project: USING GENOMICS TO DEFINE AND CONTROL PARASITIC INFECTIONS IN CATTLE
2007 Annual Report


1a.Objectives (from AD-416)
The goals of this research are to evaluate the host pathogen relationship using genomics and immunologically-based approaches, and use this information to develop methods that complement and/or reduce drug intervention as a means to control gastrointestinal (GI) nematodes of cattle. In this regard, we will identify structural variations in the bovine genome that influence host resistance to GI nematodes, pinpoint functional and/or structural variations in parasite genes associated with the drug resistant phenotype, identify and enhance immune factors that regulate the host-pathogen interface as an alternative to anthelmintic treatment, and finally use this collective knowledge to develop integrated parasite control programs that incorporate marker-assisted selection schemes that reduce economic loss caused by the parasites. These goals will be attained through a systems-based approach that will employ molecular, immunological, and genomic techniques to studies at the gene, cell and whole animal levels.


1b.Approach (from AD-416)
Studies will focus on using genomic approaches to develop novel means to control parasitic diseases in cattle, and on-farm application of these techniques. These studies will investigate both the parasite genome and the host genome. Studies focusing on the parasite will evaluate genetic factors associated with drug-resistant and drug-susceptible phenotypes in parasitic nematodes. Investigations of the host genome will focus on the identification of host genetic loci that affect resistance to parasite infection and will develop and implement genome-wide selection or marker-assisted selection programs based on high density SNP information. Additionally the project will use SNP haplotypes to refine and characterize QTL for parasite resistance that were identified in earlier studies. At the same time studies will continue to characterize the host immune response to parasitic infection using microarrays and real time PCR to discern gene expression patterns in cattle demonstrating different levels of resistance and/or immunity to the parasites. Finally, the information gleaned from these studies will be applied on-farm through co-operative research efforts with producers to establish practical management programs to control and diminish the effects of parasites on production effeiciency.


3.Progress Report
Identification of genes associated with drug resistance Numerous genes have been linked to microbial drug resistance. P-glycoprotein (P-gp) is a member of the ABC transporter family and is a cell membrane-associated protein that transports a variety of drug substrates out of the cell. It is present in biological systems associated with drug absorption (intestines) and distribution (central nervous system and leukocytes). P-gp inhibition is an attractive therapeutic target to reverse multi-drug resistance which has been observed in parasitic nematodes. Furthermore, some of the currently used anthelmintics are likely substrates for this transporter and thus contribute to the problem with resistance in nematodes. To this end, we have identified and sequenced the 4000 bp sequence encoding P-gp in the parasitic nematode Ostertagia. Given the similarity between Ostertagi and Cooperia, this will be used as a starting point to clone a homologue from Cooperia and begin evaluating the role of this gene in the resistance phenotype. The lack of acquired resistance in Ostertagia can provide yet another control to identify markers in the resistant form of Cooperia. This research supports the following National Program Components of NP103: #2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C, Mucosal Diseases of Livestock and Poultry; and Component #7 Countermeasures to prevent and control parasitic diseases, Problem Statement 7A, Drug Resistant Gastrointestinal (GI) Parasitic Diseases.

High energy phosphate in the control of drug resistance The function of P-glycoprotein (Pgp), which confers multi-drug resistance by active efflux of drug, is dependent upon phosphorylation. A larval gene that functions as an apyrase and thus important in ATP metabolism was also identified. Though the actual mechanism of the phosphorylation is unknown, apyrases play a role in controlling the local pool of high energy phosphate for use in cellular enzymatic reactions. The Ostertagia apyrase gene was functionally expressed and is capable of dephosphorylating a number of di- and trinucleotide phosphates. In addition, it is heavily present in the larval stages of the parasite which are involved early on in the development of drug resistance. Work will continue toward identifying a link between the apyrase activity and the functionality of ABC transporters in promoting drug resistance in nematodes. This research supports the following National Program Components of NP103: #2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C, Mucosal Diseases of Livestock and Poultry; and Component #7 Countermeasures to prevent and control parasitic diseases, Problem Statement 7A, Drug Resistant Gastrointestinal (GI) Parasitic Diseases.


4.Accomplishments
Isolation of genetic material for BAC and normalized cDNA libraries The importance of understanding genetic variation in the parasite populations has recently been heightened in light of the rise and persistence of nematode resistance to the most widely used anthelmintics, in particular, the avermectins and milbemycins (AM). An appreciation for the mechanisms by which parasites circumvent drug intervention can only be realized by identifying markers that differentiate the resistant and susceptible phenotypes. To this end, a pure population of drug resistant Cooperia was generated and sufficient genetic material was isolated to begin the construction of genomic and normalized cDNA libraries for identifying genes differentially expressed as a result of drug treatment. This is a critical first step to using population genetics for identifying genetic markers associated with drug resistant nematodes. This research supports the following National Program Components of NP103: #2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C, Mucosal Diseases of Livestock and Poultry; and Component #7 Countermeasures to prevent and control parasitic diseases, Problem Statement 7A, Drug Resistant Gastrointestinal (GI) Parasitic Diseases.

Development of a highly effective parasite control program for grazing dairies in the Northeastern U.S. that minimizes anthelmintic applications In the certain parts of the U.S., including the Northeast and Upper Midwest, there is an increasing number of small dairy operations that have moved from the feeding of a total daily ration to rotational grazing. Although this movement has many positive effects, some producers have experienced problems resulting from increased incidence of gastrointestinal nematode infections. For a number of years we have worked with one such dairy to optimize parasite control while minimizing the application of anthelmintics. Given the increased reports of anthelmintic resistance such programs are essential to insure the sustainability of these dairy operations. Optimal control was achieved by:.
1)applying the drugs to all grazing animals approximately one month after spring turnout for the control of stomach worms and Cooperia,.
2)application of a second treatment in mid-summer to the young replacement heifers for the control of Nematodirus, and.
3)the use of an endectocide at housing in the fall on all stock to primarily control louse infestations during the winter housing period. This program increased the overall milk production in the herd by about 2lbs/animal/day.


5.Significant Activities that Support Special Target Populations
None


6.Technology Transfer

Number of active CRADAs and MTAs1
Number of non-peer reviewed presentations and proceedings8
Number of newspaper articles and other presentations for non-science audiences8

Review Publications
Harmon, A., Williams, Z.B., Zarlenga, D.S., Hildreth, M. 2007. Real-time PCR for Quantifying Haemonchus Contortus Eggs and Potential Limiting Factors. Parasitology Research. 101:71-76.

Stromberg, B.E., Gasbarre, L.C. 2006. Gastrointestinal nematode control programs. Veterinary Clinics of North America. 22:543-565.

Li, R.W., Sonstegard, T.S., Van Tassell, C.P., Gasbarre, L.C. 2007. Local Inflammation as a Possible Mechanism of Resistance to Gastrointestinal Nematodes in Angus Heifers. Veterinary Parasitology. 145:100-107.

Last Modified: 11/28/2014
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