Proceedings of the 10th International Colloquium on Paratuberculosis

#28 Intestinal MAP Infection via Peyer’s Patch Inoculation in a Calf Model
Jesse Hostetter, Brandon Plattner, James Roth, Jean Zylstra, Ratree Platt, Yu-Wei Chiang, Iowa State
University, USA; Fort Dodge Animal Health, USA
The objective <strong>of</strong> this project was to develop an intestinal model <strong>of</strong> MAP infection in <strong>the</strong> calf for evaluation <strong>of</strong>
mucosal pathology, and local and systemic immunologic responses. Methods: Our approach was to directly
inoculate MAP into <strong>the</strong> Peyer’s patches <strong>of</strong> <strong>the</strong> ileocecal junction in 40 five week old calves. We used right flank
laparotomy to identify and exteriorize <strong>the</strong> ileocecal junction. MAP suspended in saline was injected through
<strong>the</strong> serosal surface <strong>of</strong> <strong>the</strong> intestine into <strong>the</strong> Peyer’s patch region and <strong>the</strong> incision closed. We tested inoculum
doses ranging from 10 3 to 10 9 CFU, and each dose for 7, 30, 60 and 90 days post infection with 2 calves per
group. Feces was collected from each calf weekly. At each time point we necropsied calves in each dose group
and collected <strong>the</strong> inoculation site, lymph nodes (ileocecal, mesenteric, prescapular), spleen, and peripheral
blood. Results: The ileocecal valve and mesenteric lymph nodes were consistently colonized with MAP in <strong>the</strong>
10 7 and 10 9 groups from 7-90 days post infection, and this correlated with fecal shedding <strong>of</strong> <strong>the</strong> organisms.
Histologically we identified consistent granulomatous lesions with numerous acid fast bacilli in <strong>the</strong> 109 group
at each time point. These microscopic features were found in Peyer’s patches, distal villi, and in <strong>the</strong> ileocecal
lymph nodes. Using immunohistochemistry we identified mycobacteria within <strong>the</strong> villi and ileocecal lymph
nodes in <strong>the</strong> 10 7 group at 30, 60, and 90 days. In <strong>the</strong> 10 7 and 10 9 groups we identified significant production
<strong>of</strong> IFN-g and high expression <strong>of</strong> <strong>the</strong> activation marker CD25 in peripheral blood and lymph node mononuclear
cells using antigen recall assays. Conclusion: These data demonstrate that inoculation <strong>of</strong> MAP into <strong>the</strong> Peyer’s
patches results in infection that shares features with natural MAP infection including mucosal colonization,
fecal shedding, mucosal pathology, and local and systemic immunologic responses.
#64 Association between paratuberculosis infection and general immune status in dairy cattle
Pablo J Pinedo, Art Donovan, Owen Rae, Jason De la Paz, College <strong>of</strong> Veterinary Medicine, University <strong>of</strong> Florida, USA
The objective was to investigate <strong>the</strong> association between paratuberculosis infection as determined by serum
ELISA and <strong>the</strong> general humoral and cellular immune status <strong>of</strong> adult cows in a dairy herd in Florida (USA). A
total <strong>of</strong> 781 Holstein cows were tested for paratuberculosis infection by use <strong>of</strong> a USDA-licensed ELISA
(IDEXX-Laboratories). Optical densities were used to obtain sample-to-positive (S/P) ratios that were
converted to 4 S/P-ratio categories: negative (0-0.49); inconclusive (0.50-0.99); positive (1-3.49); strong
positive (≥3.5). Simultaneously, individuals were categorized for <strong>the</strong>ir ability to mount a general antibody
(AMIR) and cell-mediated immune response (CMIR). Ovalbumin and killed, whole cell Candida albicans were
antigens used to stimulate AMIR and CMIR, respectively. AMIR response was measured using an ELISA.
CMIR was evaluated by a delayed-type hypersensitivity reaction using an intradermal injection <strong>of</strong> candin
(C. albicans antigen) in <strong>the</strong> tail skin-fold and measuring <strong>the</strong> inflammatory response 24 hours later. Levels for
immune response were categorized as low, medium, or high for each <strong>of</strong> <strong>the</strong> two variables. The statistical
analysis was based on logistic regression (cumulative logit model), and considered <strong>the</strong> 4 S/P-ratio categories
for paratuberculosis ELISA as <strong>the</strong> dependent variable. The independent variables were AMIR, CMIR,
and parity. Given <strong>the</strong> potential association among variables, two way interactions were included in <strong>the</strong> model.
Parity and CMIR were significantly associated with paratuberculosis infection status. Cows with higher levels
<strong>of</strong> cell-mediated immunity were less likely to demonstrate evidence <strong>of</strong> paratuberculosis infection. For each
category increase in CMIR (low, moderate, high), <strong>the</strong> odds <strong>of</strong> being above any given ELISA S/P-ratio category
decreased by 68%. Fur<strong>the</strong>rmore, older cows were more likely to show paratuberculosis infection. For each
increase in parity, <strong>the</strong> likelihood <strong>of</strong> a higher level <strong>of</strong> infection increased by 75%. It is concluded that higher cellmediated
immune status was associated with a lower probability <strong>of</strong> paratuberculosis seropositivity.
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