Proceedings of the 10th International Colloquium on Paratuberculosis

#175 Differential activation <strong>of</strong> γδ+ and CD8+ T cells in <strong>the</strong> early innate immune response to
MAP infection
Julianne Zickovich, Emily Kimmel, Philip Fox, C. Amy Eibs, kerri Rask, Kirk Lubick, Kathy Gauss, Mark Jutila,
Jay Radke, Colorado State University, USA
We used carboxyfluorescein succinimidyl ester (CFSE) labeling <strong>of</strong> bovine PBMCs isolated from naïve animals,
antibody staining <strong>of</strong> distinct T cell subsets and 2-color FACS to show suppression <strong>of</strong> innate proliferative
responses <strong>of</strong> gd + and CD8 + T cell subsets after exposure to bovine monocytes that have taken up M. avium
ssp. paratuberculosis. Consistent with differential activation, <strong>the</strong> CD8 T cell population showed little IFN-g
production when compared with cells exposed to monocytes that had taken up M. avium ssp. avium. The CD4 +
subset remained unchanged after exposure to ei<strong>the</strong>r subspecies. Results were similar when comparing T cell
responses to infected allogeneic or syngeneic (autologous) monocytes. We have fur<strong>the</strong>r shown that when
taken up in bovine monocytes, M. avium ssp. paratuberculosis suppressed mRNA(s) encoding select signaling
leukocyte activation molecules (SLAM) and confirmed by RT-PCR altered expression <strong>of</strong> mRNA encoding
SLAM F5, F7 and F8. SLAM proteins serve as TCR co-receptors that can modulate <strong>the</strong> production <strong>of</strong> IFN-g
in T cells. All nine receptors in <strong>the</strong> SLAM family (SLAM F1-9) are differentially expressed on hematopoietic
cells. In T cell activation, SLAM cross-linking with its homotypic ligand on <strong>the</strong> surface <strong>of</strong> an infected cell will
induce interaction <strong>of</strong> SAP with its cytoplasmic tail and induce IFN-g and IL-2. Increased macrophage SLAMF5
has previously been associated with increased T cell proliferation and secretion <strong>of</strong> IFN-g; and higher levels <strong>of</strong>
SLAMF7 with increased killing by natural killer (NK) cells and increased proliferation <strong>of</strong> B cells. There is less
known <strong>of</strong> effecter functions in cells where SLAMF8 is increased. The apparent suppression <strong>of</strong> SLAM molecules
when comparing virulent M. avium ssp. paratuberculosis infection with that <strong>of</strong> avirulent M. avium ssp. avium is
consistent with suppressed T cell activation and provides one potential mechanism for M. avium ssp. paratuberculosis
to avoid or dampen a robust early innate response.
#184 Development <strong>of</strong> an experimental infection protocol in cattle using a low passage
cultured strain <strong>of</strong> MAP
Karren Plain, Doug Begg, Anna Waldron, Kumi deSilva, Richard Whittington,University <strong>of</strong> Sydney, Australia
Experimental infection <strong>of</strong> animals permits <strong>the</strong> study <strong>of</strong> disease pathogenesis over time. However, many cattle
experimental infections use gut homogenates or large doses <strong>of</strong> an unknown passage strain <strong>of</strong> MAP. Though
ensuring infection, <strong>the</strong>se can have very different disease progression and clinical outcomes compared to natural
infection.
A pilot study was performed to assess <strong>the</strong> efficacy <strong>of</strong> a low passage laboratory seed stock culture
(CM00/416) <strong>of</strong> a MAP field isolate to infect calves. Highly passaged cultured strains <strong>of</strong> MAP can lose virulence
resulting in low numbers <strong>of</strong> infected and clinically diseased animals.
Calves were sourced from a dairy farm confirmed to be free <strong>of</strong> Johne’s disease in an area <strong>of</strong> Australia with
low disease prevalence in cattle. Three doses <strong>of</strong> MAP were given over 4 weeks with a total dose <strong>of</strong> 4.8 x 10E8
viable bacteria. Calves were necropsied 2-3months post-infection and infection status was assessed by faecal/
tissue culture, Mptb DNA detection in <strong>the</strong> gut tissue and associated lymph nodes, serum ELISA and histology.
Tissue culture identified MAP in <strong>the</strong> gut and lymph nodes <strong>of</strong> infected calves 2 months post-infection.
Quantitative PCR (QPCR) to detect IS900 MAP DNA was performed. This QPCR assay was designed such
that it does not detect environmental mycobacteria and has a high analytical sensitivity (0.001pg <strong>of</strong> MAP genomic
DNA). Mptb DNA was detectable in gut and lymph nodes <strong>of</strong> exposed calves 2-3 months post-infection.
No visible macroscopic or microscopic lesions were found.
This experimental model was designed to be a reproducible and standardizable technique for inducing infection,
with advantages over high dose regimes and gut or faecal homogenates that contain variable numbers
<strong>of</strong> MAP and contaminants. Confirmation <strong>of</strong> infection with this low passage cattle strain via QPCR and tissue
culture has enabled <strong>the</strong> initiation <strong>of</strong> a large scale experimental infection trial using this method.
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