Progress 05/12/05 to 09/01/07
Outputs
OUTPUTS: Data were collected from 5 healthy adult horses and 16 foals. ACTIVITIES: Neutrophils were separated from blood samples collected from adults at a single timepoint and from foals within 48 hours of birth and at 2, 4, and 8 weeks of age. The mRNA expression of selected cytokines by adult neutrophils challenged with isogenic virulent and avirulent Rhodococcus equi was determined. Both isolates induced significantly increased expression of tumor necrosis factor (TNF)-alpha, and interleukins (ILs)-6, -8, -12p40, and -23p19, but not interferon (IFN)-gamma or IL-12p35. Virulent R. equi induced significantly greater IL-23p19 mRNA than avirulent R. equi. In foals, virulent R. equi induced significantly greater expression by neutrophils of IFN-gamma, TNF-alpha, IL-6, IL-8, IL-12p35, IL-12p40, and IL-23p19. Moreover, there were significant age-related differences in expression of IL-6, IL-8, IL-12p40, and IL-23p19. Results indicate that R. equi-stimulated neutrophils are a source of
pro-inflammatory cytokines and that age-related differences in expression exist that could influence the susceptibility of foals to infection. EVENTS: Research poster presented at 2007 Society for Leukocyte Biology Conference, Boston, Mass, October 2007.
PARTICIPANTS: Cohen ND - Principal Investigator/Project Director: oversight of study design, sample collection and other aspects of study conduct, data analysis, data interpretation, and reporting results. Martens RJ - Co-Investigator: Participated in study design, sample collection and other aspects of study conduct, and interpretation and reporting of results. Smith III R - Co-Investigator: Participated in study design Payne S - Collaborator: Participated in study design, helped oversee molecular techniques, and interpretation and reporting of results Nerren JR - Graduate Student: Completed PhD in Veterinary Pathobiology under Drs. Cohen and Payne; responsible for day-to-day activities needed to complete this work Link Equine Research Endowment: Provided additional funding Texas A&M University Horse Center: Provided access to mares and foals used in the study
TARGET AUDIENCES: Target audiences include the equine horse breeding industry and equine veterinarians concerned with foal health.
PROJECT MODIFICATIONS: Our bactericidal assays yielded highly variable, unreliable results and this aspect of work was abandoned.
Impacts
This is the first description of quantification of cytokine expression by neutrophils, and the first report of the response of equine neutrophils to R. equi. The finding that there are age-related differences in cytokine responses by foal neutrophils informs strategies for control and prevention of R. equi, as well as efforts to control and prevent other infectious diseases of foals by highlighting qualitative and quantitative differences in innate immune responses of foals.
Publications
- Nerren JR, Payne S, Halbert ND, Martens RJ, Murrell Jm Butler JL, Cohen ND. 2008. Cytokine expression by adult and juvenile equine neutrophils in response to stimulation with Rhodococcus equin in vitro. Infection and Immunity.
- Nerren JH, Payne S, Halbert ND, Martens RJ, Cohen ND. 2007. Cytokine expression by adult equine neutrophils following exposure to virulent and avirulent Rhodococcus equi in vitro. Society for Leukocyte Biology Annual Meeting, Cambridge, MA, October 11.
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Progress 01/01/06 to 12/31/06
Outputs
All animal experiments have been completed. The first experiments entailed validating the real-time PCR assays for expression of selected cytokines, tumor necrosis factor TNF, alpha, interferon, IFN, gamma, interleukin IL, 6, IL, 8, IL, 12p35, and IL-12p40, by equine neutrophils in response to stimulation with 2 Rhodococcus equi isolates, isogenic virulent and avirulent strains, human recombinant granulocyte-macrophage colony stimulating factor, rhGM, CSF, as positive control, and media only, negative control. Experiments also were conducted to evaluate appropriate times for co-cultivation of cells. Data from these experiments using neutrophils from mature horses have been analyzed, and a manuscript of the results is in the final stages of preparation for submission for publication. The second set of experiments entailed evaluating temporal changes in expression of cytokines and bactericidal capacity for R. equi by neutrophils of foals. Blood was collected from foals
at the following times: the first 48 hours of life, and at 2, 4, and 8 weeks of age. Expression of IFN gamma, TNF alpha, IL6, IL8, IL12p35, IL12p40 by neutrophils that were stimulated by either 2 isogenic strains of R. equi, one virulent and one avirulent, rhGM CSF, and media have been assessed by real time PCR. Bactericidal capacity of foal neutrophils at each age has been completed. Data will be analyzed during the spring of 2007, and a manuscript will be prepared and submitted for publication during the summer of 2007, prior to the termination date for the study.
Impacts
According to the National Animal Health Monitoring and Surveillance program's equine report, pneumonia is a major cause of disease and death among foals. Pneumonia caused by Rhodococcus equi is a major health concern for the equine industry, particularly breeding farms. Results of the study will inform strategies for control and prevention of R. equi, thus enabling reductions in the burden of this economically important disease.
Publications
- Harrington JR, Halbert ND, Payne S, Martens RJ, Cohen ND. Cytokine expression by neutrophils of adult horses stimulated with virulent and avirulent Rhodococcus equi in vitro. Manuscript in preparation to submit to Infection and Immunity, 2007.
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Progress 01/01/05 to 12/31/05
Outputs
The following aims have been accomplished: Development and validation of assays for quantifying expression by equine neutrophils of the following cytokines: TNF-alpha, IL-1beta, IL-6, IL-8, interferon-gamma, IL-12p40, and IL-12p35, using beta2 microglobulin as a housekeeping gene. A bactericidal assay for Rhodococcus equi by equine neutrophils has been developed. Equine neutrophils have been examined for their expression of the aforementioned cytokines either without stimulation and following stimulation with GM-CSF, LPS, virulent R. equi, and avirulent R. equi. Results indicate significant and similar stimulation by virulent and avirulent R. equi for some cytokines, particularly IL-6 and IL-8 relative to unstimulated neutrophils. Interestingly, stimulation with GM-CSF and LPS produced little stimulation relative to R. equi. Study of the temporal changes in expression of these cytokines and the bactericidal capacity by neutrophils from healthy foals is ongoing; data
collection will be completed by the end of July 2006.
Impacts
Pneumonia is a major cause of disease and death among foals, and pneumonia caused by the bacterium Rhodococcus equi is a major concern for the equine industry, particularly breeding farms. Results of the study will help to define the role of neutrophil function in protecting foals from infection with R. equi. It may be possible to intervene to reduce the burden of the clinically severe and economically important disease caused by this bacterium by improving neutrophil function of very young foals.
Publications
- No publications reported this period
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