The interactions of M. avium ssp. paratuberculosis (MAP) with host macrophages and the development of immune responses are not well understood. The pathogenicity of MAP is directly related to its ability to persist in host macrophages. Our overall aim is to study these mechanisms of survival at the cellular level.The aim of this work is to identify the specific MAP and macrophage proteins produced at immediate, early and sustained stages of MAP/ macrophage interaction and study the resultant changes in macrophage function.To perform these studies, a reliable, reproducible MAP/ macrophage engulfment assay was developed and optimised. In a series of pulse-chase experiments, the uptake of MAP, protein expression, engulfment dynamics, incubation times, multiplicity of infection (MOI), period of macrophage adherence and prestimulation of primary cells.The effects of bacterial growth phases, culture medium, and presentation at engulfment were also studied. Ziehl-Nielsen staining was used to evaluate uptake and survival of MAP within host macrophages. A MAP/ host cell MOI of 10 was the engulfment ratio of choice. Below a MOI of 10, engulfment was reduced.This preliminary work highlights the variability in engulfment of MAP by primary cells and the need to strictly standardise assay procedures to achieve uniform uptake of MAP. Studies of protein and gene expression at the cellular level require reproducible conditions and engulfment. Variability can be introduced through cell type, period of attachment of cells and variance in MAP culture parameters.Macrophages will be used for proteomic analysis and RNA extraction for micro array analysis through metabolic isotope-labelling (³⁵S and ¹⁴C) of bacteria and macrophages.