Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Animal Science
Non Technical Summary
Bovine leukemia virus (BLV) is a common infection in U.S. dairy herds. A 2007 USDA NAHMS study estimated that 83% of U.S. dairy herds are infected with BLV, and approximately 25%-50% of animals within aBLV-positiveherd are infected with BLV. Until recently, BLV was not considered to be economically important to dairy production in the U.S. In contrast, 21 European countries aggressively eradicated the disease and are now considered BLV-free. BLV infection rates in the U.S. are too high for aggressive strategies. Instead, lowering BLV infection rates will depend on producers and management strategies to reduce BLV infection.BLV infection rates in the U.S. are so high because the infection was thought to cause disease very rarely. Only 1%-10% of BLV-infected cows will develop cancer in their lifetime. However, it is likely that BLV has more negative effects on dairy herd health beyond cancer.BLV already costs the dairy industry $520 million every year, due to the lower milk production and shorter life expectancy of BLV-positivecows. BLV infection also likely causes immune suppression, which would put BLV-positivecows at risk for developing other infectious diseases that have a major economic impact on dairy production. The purpose of this project is to determineif BLV-positivecows are immune suppressed. Studying the immune system of BLV-positivecows is important for understanding overall herd health and production in U.S. dairy herds, which makes this project highly relevant to USDA-NIFA.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
The first goal of this project is to determineif bovine leukemia virus (BLV)-infected dairy cows are less able to mount adaptive immune responses to other stimuli when compared to healthy, BLV-uninfected cows. This goal is in line with the larger investigation into whether BLV increases the susceptibility of dairy cows to other infectious diseases of econoomic importance. Because BLV is so widespread in American dairy herds, any immune suppressive effects of BLV infection could have a large, negative impact on both the welfare and production of dairy herds.The second goal of this project is to develop the skills and career of the project director as a scientist. The demands on the project director will enable learning critical skills including, but not limited to, managing a grant, directing a scientific program, scientific writing andgiving scientific presentations.
Project Methods
Experiment 1To characterize the strength of the primary immune response, adult lactating Holstein dairy cattle will be divided into three cohorts: BLV-, BLV+ aleukemic (AL) and BLV+ persistent lymphocytosis (PL). Each cohort will contain 20 animals, based on previous results and our power analysis. Disease status will be determined via BLV ELISA for positive or negative status and relative percent B cell prevalence in peripheral blood for AL or PL status. Keyhold limpet hemocyanin (KLH), a potent, non-pathogenic immune stimulant,will be used with the synthetic adjuvant dimethyl dioctadecyl ammonium bromide (DDA) as a novel antigen to stimulate a primary immune response that will mimic a primary pathogen exposure without causing an infection. A subset of animals will receive only adjuvant (3 BLV-, 3 BLV+ AL and 3 BLV+ PL). Animals will receive a subcutaneous injection of KLH and DDA or DDA only in the neck. Peripheral blood will be collected from the coccygeal (tail) vein of cattle on day 0, prior to immunization with KLH, and on days 7, 14, 21, 28, 35 and 50 after immunization. Animals will be boosted with KLH or DDA only, as appropriate, on day 14 post-immunization. This timeline of blood collection is based upon a previous study using KLH in cattle.A serum ELISA will be used to measure KLH-specific IgM, IgG1 and IgG2. PBMCs will be isolated from whole blood and RNA will be extracted to measure expression of interferon-gamma(IFNg), tumor necrosis factor-alpha(TNFa) and interleukin-2 (IL-2) mRNA. On days 0, 14, 28 and 50 post-inoculation, PBMCs will be isolated and cultured in the presence of no stimulant, KLH or PWM to measure antigen-specific B or T cell activation. B cell activation will be measured using multi-color flow cytometry: surface IgM (SIgM) as a marker for mature B cells, BLV gp51 as a marker of BLV expression, CD45R0 as a memory marker and CD25 as a marker of activation. T cell activation will also be measured using CD4 and CD8 as lineage specific markers, CD45R0 for memory cells and CD25 as a marker of activation.Once the initial immune response has waned, as measured by a return of serum antibody levels close to baseline, cattle will receive a booster of KLH and DDA (the negative control animals will receive a booster of only DDA). Peripheral blood will be collected prior to immunization on day 0, as well on days 3, 5, 7, 14, 21 and 35 post-immunization. The accelerated blood collection timeline is due to the expectation of a more rapid secondary immune response to KLH. Blood will be processed and immune responses assessed exactly as described above for the primary immune response.Experiment 2As in experiment 1, age-matched, adult, lactating dairy cattle will be divided into three groups based upon BLV status: BLV-, BLV+ AL and BLV+ PL. Cattle will be assigned to their groups based upon criteria discussed in experiment 1. 10 cattle will be enrolled in each group. Cattle will receive a booster dose of BoviShield Gold FP4 + L5 on their regular schedule.Whole blood will be collected from the coccygeal vein. Blood collection will occur on day 0 prior to the boost, as well as on days 7 and 14 post-boost. BHV-1-, BVDV-, BRSV- and Leptospira-specific IgM, IgG1 and IgG2 antibodies will be quantified by commercial ELISA. PBMCs will be isolated and separate aliquots used for RNA extraction and quantification of IFNg, TNFaand IL-2 expression and cultured with appropriate antigens for measurement of B and T cell activation using multi-color flow cytometry, as described in experiment 1.