Washington State University Immunology of MAP infection in cattle with implications for infection in humans WC Davis Washington State University Pullman Washington

Our research has been focused on use of the natural host to gain insight into early and late stages of Map infection in humans Questions addressed Are the Map isolates causing disease in humans the same as in cattle? Is the immune response to Map in cattle the same as the immune response in humans ? Is the immune response to Map similar to the immune response to Mtb? Is it possible to make a vaccine to clear Map from the food stream?

Use of DNA short sequence repeats (SSRs) to compare Map isolates Ghadiali et al. J. Clin. Microbiol. 42:5345, 2004 Cattle 8g-4ggt 9g-4ggt 13g-5ggt 14g-5ggt 11g-5ggt 12g-5ggt Sheep 7g-4ggt 7g-5gt Goats Same DNA short sequence repeats (SSRs) present in human and ruminant isolates

We developed a cannulated ileum model to study immunopathogenesis of Johne’s disease Allowed for: Direct inoculation of ileum with Map. Constant access for biopsies. Tracking pathological changes over time.

Endoscopic evaluation of early and late stages of infection Uninfected/infected Naturally Infected No alterations evident during early stages of infection with Map Map undetectable in ileum during early stages of infection Alterations evident during late stages of infection with Map Enteritis similar to enteritis in humans

Summary of 27 yrs of study Bacteria are rapidly taken up and disseminated to peripheral tissues. Shedding of bacteria stops within a few days following exposure. No lesions or Map detectable in the ileum 18 months post exposure. Bacteria detectable in mesenteric and ileocecal lymph nodes by PCR. Immune response develops that controls but does not eliminate Map. Animals develop latent infection similar to latent infection with Mtb. Immune response to bovine and human isolates of Map similar. (Linda vs K10) CD4 and CD8 T cell response detectable ex vivo using flow cytometry. Immune response to Map resilient, not disrupted by: a. massive doses of Map 108 – 4 x 108. b. methotrexate immunosuppression. c. depletion of CD4 T cells at time of exposure.

Compared effect of deleting virulence genes on Map survival in vivo Development of a vaccine: Progress Compared effect of deleting virulence genes on Map survival in vivo pknG, slr2, relA

Immune response develops that clears infection We found that deletion of relA abrogates capacity of Map to survive in vivo Immune response develops that clears infection

We developed an assay to analyze the immune response to ΔrelA The assay distinguishes live from dead bacteria using a single gene probe F57 and qPCR We found that vaccination with Map/ΔrelA elicits development of cytotoxic T cells (CTL) with ability to kill intracellular bacteria

Further analysis showed the response directed towards a 35 kD membrane protein (MMP) MMP stimulation of monocyte-depleted PBMC (mdPBMC) from a Map/ΔrelA a vaccinated steer elicits an equivalent recall CTL response 2 cycles of ex vivo stimulation of mdPBMC from unvaccinated cattle with MMP elicits an equivalent recall CTL response

CT value Map infected MoΦ Gene quantity D Intracellular killing by MMP primed CTL ex vivo Map-DNA live dead ratios D100% L100% LD75:25% DL75:25% LD50:50% 4 DNA copies L = live D = dead MoΦ +MMP stimulated mdPBMC MoΦ + Unstimulated mdPBMC 24 hr MoΦ-T24 hr Standard DNA curve 4 x 107 copies MoΦ-T0 CT value Map infected MoΦ Gene quantity D

Summary Gained insight into early stages of infection that may occur in humans Infection under immediate control by innate immune mechanisms Adaptive immunity controls but does not eliminate Map The immune response involves both CD4 and CD8 T cells Event that disrupts protective immunity remains unknown Peptide based vaccine possible

Questions Support Laboratory team Kun Taek Park Andy Allen George Barrington Mahmoud Elnaggar Gaber Abdellrazeq Victoria Hulubei John Bannatine Support Johne’s disease integrated Program NIH Water Born Diseases subcontract WSU mAb Center WSU intramural grants

Generation of primed effector T cells Developed assays to analyze immune response to ΔrelA Intracellular Killing assay Ag PBMC Primed T cells 6 days Overlay MoΦ with primed T cells Monocytes 6 days Map 3 hrs wash MoΦ MoΦ infected 6 hrs Lyse Propidium monoazide Halogen light Isolate Map DNA qPCR