Progress 10/01/07 to 09/30/09
Outputs
OUTPUTS: The primary goal of the project was to assess the in utero effects of BVDV infection on development of the ruminant immune system. To that end, we initially pursued a sheep model due to the fiscal and experimental limitations of cattle. Over the first year, we determined appropriate virus isolates for use in sheep, and submitted a proposal for funding to the USDA. In response to that review for the unfunded proposal, we focused our efforts over the next 6 months towards isolation of sheep-specific reagents to identify the cells of interest in our studies. Appropriate reagents were identified, as well as appropriate viral stocks for pursuit of further work in the sheep model. PARTICIPANTS: Christopher C. Chase, South Dakota State University Alan J. Young, South Dakota State University. TARGET AUDIENCES: Practitioners, corporate entities with interest in domestic animal vaccinology and therapeutics. PROJECT MODIFICATIONS: As a result of the USDA grant meeting last December in Chicago, we identified an ongoing NRI-funded BVDV persistent infection study in cattle at Colorado State University, which would satisfy the specific aims we had prepared for this proposal, namely the identification of changes in immune tissues associated with progression of disease in cattle. To that end, we chose to focus on collaboration with Colorado to collect sufficient tissues to form the basis of future studies. This study involved culturing fetal macrophages and obtaining fetal immune tissues from fetuses that were infected at 75 days of gestation (day 0), 82 days of gestation (day 7), 96 days of gestation (day 21), and day 120 (day 45. We have two additional times points in November and January where we will be collecting additional tissues. We have obtained new findings on persistent infection and the innate immune system indicating that activation rather than suppression occurs early in infection. We also developed a Kuppfer cell isolation and culture system, which will be useful for future studies to determine the effect of BVDV and other flaviviruses on liver function. We have submitted and abstract on this work to the Conference of Research Workers in Animal Disease and will present the results in December in Chicago. We also submitted a USDA grant to piggyback on the NRI-funded Colorado study that was not funded. We intend to submit an NIH R-15 this year to further continue these studies.
Impacts
As a result of these studies, we have developed two important outcomes. First, we have identified a number of reagents capable of identifying both CD4 and CD8 positive tolerogenic T cells in ruminants. Some of these reagents were already available in our laboratory, whereas others were obtained over the course of the study from collaborating investigators. These reagents were used to characterize the populations of so-called regulatory T cells in sheep, deer, and cattle, and are currently being used to further define the role(s) that these subsets play in regulating tolerance in adult animals. A graduate student is now employed full time towards defining the developmental appearance of tolerogenic T cells in sheep and cattle, and alterations in these subsets associated with chronic infections such as Bovine Viral Diarrhea Virus. In addition, we have forged collaboration with researchers at the Mayo Clinic with an interest in the role of tolerogenic T cells in viral infections. Our future work will be focused on defining the role(s) of these T cells in the early response of ruminants to acute infection with BVDV, their nature in persistently infected animals, and potential "reprogramming" of these cells to promote an immune response in persistently-infected animals.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: It is the focus of this application to characterize and ovine model of BVDV persistent infection. To accomplish this goal, we will establish appropriate infection protocols to establish persistent infection in an ovine model, map the anatomical distribution of BVDV throughout the developing ovine lymphoid system, and define specific immunological deficiencies in PI lambs associated with BVDV infection. These data will be used to develop a comprehensive research focus on fetal immunity to viral infection, capable of support through both the USDA and NIH-NIAID. Specific Aims and Progress over the past year: 1. To define an experimental model to produce persistent infection of sheep with BVDV. Progress: While we are limited by the temporal availability of ewes at the appropriate gestational age, we did accomplish initial studies over the past year. As a result of these studies, we confirmed that the BJ strain of BVDV is our most promising candidate strain for testing. When ewes were infected at 55-60d gestation, fetuses progressed to parturition, at which time the fetus was stillborn. While the fetuses were born dead, these results are in contrast to another BVD strain that induced abortion within 1 month of inoculation. Our current goal is to better define the precise timing of inoculation to permit live births of persistently-infected lambs. Our next group of susceptible ewes with appropriately-aged fetuses will be available in November 2008. 2. To define the specificity of lymphocyte immunosuppression in sheep fetuses and lambs following infection with non-cytopathic BVDV. Progress: As defined above, we did not succeed in our initial efforts at producing persistently-infected live lambs, and therefore no functional data is available to define B cell suppression. However, tissues were harvested from both ewe and the stillborne fetuses, and we are currently analyzing those tissues for the presence of both BVDV and the distribution of lymphocyte subsets within the ileal Peyers patches, mesenteric lymph nodes, and placental interface. These results will be available before the next NRI submission deadline. 3. To define the effects of in utero BVDV function on immune development in sheep. Progress: As discussed in more detail below, we are currently working to define our functional assays of bovine leukocyte function in the ovine model. To this end, 8 adult ewes have been infected with BVDV strain BJ and PA131, and blood samples are harvested and tested prior to and following infection to map changes in leukocyte phenotype and function associated with infection. These studies are currently underway, and results will be available prior to the next NRI deadline. PARTICIPANTS: Christopher Chase - collaborator Stacy Lindblom-Dreis - Technician Lyle Braun - Technician TARGET AUDIENCES: Producers, Stakeholders, other Basic researchers PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
As a result of the above experiments, we have determined the appropriate BVD strain to attempt persistent infection of pregnant ewes. We have furthermore determined the appropriate time frame in which to attempt infection. Finally, we have defined the use of bovine assays for analysis of ovine tissues, in order to adapt previous work on bovine persistently infected calves to sheep. As a further result of this work, we have established a collaboration with additional BVD researchers to take advantage of ongoing work on the effects of BVD infection on the developing cattle immune system. We anticipate submitting a grant application to the USDA during this funding cycle to specifically examine immune alterations in persistently infected cattle.
Publications
- No publications reported this period
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