2010 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.
Nematode apyrase (AP) genes in Ostertagia and Trichostrongylus were isolated and used to express recombinant proteins capable of dephosphorylating di- and trinucleotide phosphates. Our data demonstrate that the parasite secretes these proteins to decrease the host inflammatory responses during infection of the gastric glands of the abomasum. This AP secretion may be a key method to escape host immunity prior to worm maturity. Because this apyrase resides primarily within the esophagus of the worm, it is also a prime candidate for vaccine development. We continued to evaluate worm TGF-beta homologues involved in the growth and development of parasitic nematodes as targets for parasite control. Sequencing of the Ostertagia genome continues with accompanying data being generated on all 3 key life-cycle stages of the parasite.
We completed an Ostertagia challenge experiment to study protective immunology in cattle, and identified host molecular mechanisms associated with long-term protective immunity. These results led us to investigate the bovine abomasal microbiota in response to Ostertagia infections. We found that cattle tolerant to infection showed minimal disruption of their abomasal microbiota, which may be part of long-term protective immunity in the host. Potentially this three-way interaction between the host, its microflora and the infecting parasites can be exploited to improve animal health. We also completed the characterizing genes of Angus cattle resistant to parasitic nematode infections using state of the art DNA sequencing technologies.
We continued to evaluate regions of 8 different chromosomes, which showed effects on host response to parasite infection by developing a new approach to determine associations between these chromosomes and parasite response based on haplotypes (blocks of the genome approximately 1 million base pairs in length). This approach was taken to avoid genome resequencing based on false associations between single DNA markers and phenotype effects. Haplotype analysis revealed that only a region of Chr 6 remained nominally significant for effects on parasite tolerance in Angus cattle, and also revealed that the data from birthyear 2006 were due to uncontrollable environmental conditions. Future analyses will be based on use of a bovine ultra high density DNA marker assay (>750,000 markers) to improve haplotype phasing and provide better power for detection of host genetic effects.
A survey was completed to identify the incidence of gastrointestinal nematode infections in U.S. cattle operations and to assess the effectiveness of commercial anthelmintics to control these parasites. Both parasitological and genetic analyses were used to determine the species of parasites resistant to drug treatment as part of the 2008-2009 NAHMS Beef Study. Results unequivocally demonstrated that overuse of anthelmintic treatments has resulted in increased treatment failures and have changed the distribution of parasite species that have historically believed to be innocuous in cattle.
A national survey of cattle GI nematodes and how they are affected by anthelmintic treatment has been completed. This work was performed in collaboration with APHIS and two University collaborators. To date, anthelmintic resistance has been observed predominantly in nematode parasites of sheep and goats; and only anecdotal information suggested resistance in cattle species. In our study cattle operations randomly selected throughout the U.S. participated in an effort to assess the mode, regiment and effectiveness of anthelmintic treatment and its relationship to clearing nematode infections. Results demonstrated a wide distribution of drug resistance in cattle nematodes. Genetic studies were performed to identify the species of parasites infecting these animals. In nearly all instances, resistance was the result of Cooperia species, in particular Cooperia punctata. Furthermore, where this parasite was once considered a minor species infecting cattle, we have now shown that it has become a predominant pathogen resulting from its resistance in the face of other nematodes that continue to respond to the presence of drug. As a result, overuse of anthelmintics has not only selected for drug resistant nematodes, but has also selected for species with elevated pathology in the host and changed the population dynamics of parasites on pasture. Our results clearly demonstrate that there has been a rapid rise in the prevalence of cattle GI nematodes that are resistant to the most commonly used anthelmintics.
A study evaluating the relationship between GI nematodes of cattle and other gut flora has been performed. As with all mammalian species, normal gut flora can affect the well being of an animal; however, the interplay between normal gut flora, host immunity and GI nematodes present in the gut has never been evaluated as it relates to the effects on the host. This prompted us to characterize the bovine abomasal microbiota in response to GI nematode infections and in particular, Ostertagia ostertagi. We found that Ostertagia infections in immune cattle induced minimal if not undetectable disruption in the abomasal microbiota as well as host physiology. We hypothesize that this is related to the development of long-term protective immunity in the host and as such has a positive influence on animal health.
The cattle genome has been further studied to identify additional sequence polymorphisms. A 12X coverage of bovine genome sequence has been generated to discover SNP for all eight chromosomal regions that are significantly associated with fecal egg count (FEC) traits as determined by genome wide single marker association analysis of Angus cattle at Beltsville. This sequencing effort led to development of a high density DNA marker assay (>750,000 markers) that will help not only facilitate mapping of traits like FEC in populations with small sample size, but also will be a commercial product for the research community to investigate other genetic traits of interest in cattle.
The cattle GI nematode, Ostertagia ostertagi, demonstrates a remarkable ability to circumvent the host immune response. This escape mechanism is believed to result in part from proteins that are secreted by the parasite during infection in an effort to modulate the host immune system. We have identified an apyrase enzyme secreted by the parasite that is capable of dephosphorylating nucleotide di- and tri-phosphates such as ATP and ADP. ATP and its derivatives are important in setting up a cascade of gene expression signaling events that are responsible for initiating the immune response within a cell; this only occurs if surrounding cells are previously damaged and release ATP in the process. While in the gastric glands of the abomasum, the parasite undergoes rapid growth and causes enormous cell damage that surprisingly results in no inflammation. Our data demonstrate that Ostertagia secretes apyrase while in the gastric glands and as such is capable of inhibiting the host’s inflammatory response which is required to counter the presence of the parasite; an inflammatory response that is dependent upon the presence of ATP or ADP. Attenuation of apyrase is now a potential target for protective immunization trials as a means to vaccinate animals from parasite infection.
Gasbarre, L.C., Smith, L.L., Hoberg, E.P., Pilitt, P.A. 2009. Further characterization of a cattle nematode population with demonstrated resistance to current anthelmintics. Veterinary Parasitology. 166:275-280.
Gasbarre, L.C., Smith, L.L., Lichtenfels, J.R., Pilitt, P.A. 2009. The development and identification of cattlenematode parasitesresistant to multiple classes of anthelmintics in a commercial cattle populationin the U.S. Veterinary Parasitology. 166:281-285.
Zarlenga, D.S., Gasbarre, L.C. 2009. From parasite genomes to one healthy world; are we having fun yet? Veterinary Parasitology. 163:235-249.
Mills, J.A., Zarlenga, D.S., Habecker, P.L., Dyer, R.M. 2009. Age, segment, and horn disease affect expression of cytokines, growth factors and receptors in the epidermis and dermis of the bovine claw. Journal of Dairy Science. 92:5977-5987.
Li, R.W., Li, C., Elsasser, T.H., Liu, G., Garrett, W.M., Gasbarre, L.C. 2009. Mucin biosynthesis in the bovine goblet cell induced by Cooperia oncophora infection. Veterinary Parasitology. PMID 19647371.