Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
MEDICINE & EPIDEMOLOGY
Non Technical Summary
Infection with Anaplasma marginale is common in the Western United States, and has been estimated to cost the cattle industry in the United States $300 million per year. The purpose of this study is to to better understand targets for control and management of anaplasmosis, such as potentially interfering with acquisition and dissemination of the pathogen in the early stages of infection at the time of tick bite.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The objectives of the current study are follow the kinetics of Anaplasma marginale infection in cattle by examining the clinical disease, tissue dissemination, and antigen presentation when infected via one of three different routes: infected via ticks, infected intradermally without ticks, and infected intravenously.
Project Methods
Dermacentor occidentalis adults will be reared on rabbits; larvae that are produced will be verified free of A. marginale infection by sub-sampling and PCR-testing 100 randomly chosen larvae using a previously published PCR assay. The remaining larvae will be allowed to molt to nymphs. A 4 month old steer, free of evidence of previous infection with Anaplasma, will be infected intra-dermally with Idaho-strain A. marginale in bovine blood. Our preliminary data has documented 100% efficacy inducing bacteremia within 2-6 weeks with this isolate and methods. The steer will be bled weekly for PCR-testing and nymphal ticks will be fed on the animal when TaqMan CT values fall below 25 (indicating strong infection). Cloth capsules will be sewn onto shaven steer skin in order to maintain ticks with 100 nymphs/bag for a total of 400 nymphs. After nymphs molt to adults, experimental challenge of cattle will be performed. Sixteen 6-month old steers will be divided into three
treatment groups: 4 for tick-transmitted infection, 4 for intra-dermal inoculation of infected bovine blood stabilate by needle only, 4 by IV inoculation of blood stabilate, and 4 to receive uninfected bovine blood as a control. Following inoculation, all cattle will be examined every 4 days by a veterinarian for attitude, rectal temperature, mucous membrane color, and capillary refill time. Blood will be collected weekly for CBC, serology, quantitative PCR for A. marginale MSP5, and assessment of the cytokines IL-1β, IL-2, IL-10, IL-12, TNF-α, and IFN-γ by ELISA. Bacteria from blood will further be characterized for differential expression of surface antigens. Additionally, skin near the injection site (for cattle receiving tick-transmitted or ID infection) will be biopsied weekly. Five mm full skin-thickness biopsies will be obtained at 1 cm, 2 cm, 4 cm, and 6 cm distant from the injection site. These biopsies will be submitted for histopathology and quantitative PCR
for A. marginale MSP5. For all quantitative PCR tests, CT values for bacteria will be standardized using an internal bovine calibrator. Differences in bacterial load will be compared among tissues and treatment groups by nested ANOVA. Time from infection of skin to blood will be calculated for each group and compared among groups by log rank test. Any differences in incidence of anaplasmosis will be compared by χ2. This study will define the kinetics of A. marginale clinical disease, tissue dissemination, and antigen presentation in cattle after infection by ticks, intradermally by needle, and intravenously. Statistical analysis of the results will clarify how the tick-bovine interface changes disease and spread of the bacteria, compared with infection in the absence of ticks. Additionally, the insight into antigen variation could be useful for understanding chronic infection. These findings are relevant for future management targeting vaccines preventing tick transmission or
early bacterial dissemination after inoculation, or potentially other management strategies that seek to prevent chronic infection.