Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/12/2007
Publication Date: 10/28/2007
Citation: Stabel, J.R., Robbe-Austerman, S., Davis, W.C. 2007. Immunologic Responses to Mycobacterium avium subsp. paratuberculosis in Neonatal Calves After Oral or Intraperitoneal Experimental Infection [abstract]. 9th International Colloquium on Paratuberculosis. Abstract No. 29A, p. 114.
Technical Abstract: Infection models are useful for studying host responses to infection to aid in the development of diagnostic tools and vaccines. The majority of experimental models for ruminants have utilized an oral inoculation of live Mycobacterium avium subsp. paratuberculosis (MAP) in order to establish infection, thereby mimicking the fecal-oral route of transmission generally observed in the field. The current study was designed to compare the effectiveness of oral and intraperitoneal inoculation on the host immune response to MAP infection. Twenty neonatal Holstein calves were obtained from status level 4 herds and randomly assigned to 5 treatment groups: 1) control noninfected (C), 2) oral (Oral), 3) oral with dexamethasone pretreatment (Oral/DXM), 4) intraperitoneal (IP), and 5) oral/mucosal (Oral/M). The oral group was fed milk replacer containing 1010 cfu of live MAP, strain K-10, 2x per day for 14 consecutive days. The Oral/DXM group were inoculated in the same manner as the Oral group but the calves were administered 0.25 mg/kg BW dexamethasone IV for 3 consecutive days prior to bacterial challenge, and again on days 28 and 56 post-challenge. Intraperitoneal inoculation of calves with 1010 cfu MAP, strain K-10, was performed on days 0, 7, 14, and 21 of the study. The Oral/M calves were inoculated by feeding milk replacer containing live MAP obtained by scraping the ileal mucosa from a clinically infected cow on days 0, 7, and 14. All calves were housed in AAALAC-accredited BSL-2 facilities during the study. Throughout the study, blood and fecal samples were obtained from calves on days -5 and -4 prior to the first inoculation of MAP, and then on days 7, 14, 21, 28, and monthly thereafter for the 12 month term of the study. Blood samples were processed for isolation of peripheral blood mononuclear cells (PBMC) followed by incubation with medium only (nonstimulated), concanavalin A (ConA), a whole cell sonicate of MAP (MpS), and johnin purified protein derivative (JPPD) for 24 and 48 hr for determination of cytokine secretion, lymphocyte proliferation, and flow cytometric analyses. Results demonstrated that oral inoculation of calves significantly increased lymphocyte proliferative responses to K-10 MpS at 12 months. Secretion of antigen-stimulated iNOS by PBMC was higher for oral infection groups at both 6 and 12 months post-infection compared to control calves. IP calves had the earliest antigen-specific IFN-gamma responses at 7 d post-infection, preceding responses noted for other infection groups that followed between 90 and 120 d. Average IL-10 responses to ConA and MPS were higher at 1 and 6 months and declined significantly by 12 months post-infection. At 1 month, Oral and Oral/M calves had higher MPS-stimulated IL-10 than other treatment groups. By 12 months only the Oral/M calves had higher IL-10 secretion than control calves. Intracellular IFN-gamma and IL-10 levels were measured for CD4+, CD8+, and gd T cell subpopulations. At 3 months post-infection, there was significantly higher IFN-gamma in CD4+ cells stimulated with MPS in the Oral treatment. Intracellular IL-10 was higher in CD4+ and CD8+ T cells in Oral and IP calves compared to the other treatments. These results demonstrate that exposure and infection to MAP will invoke early immunologic responses characterized by IFN-gamma, IL-10, and iNOS secretion.