# Interferon-gamma producing CD4+ T cells quantified by flow cytometry as early markers for Mycobacterium avium ssp. paratuberculosis infection in cattle

## TL;DR

This study shows that measuring IFN-gamma in CD4+ T cells using flow cytometry can detect Mycobacterium avium ssp. paratuberculosis infection in calves earlier than current methods.

## Contribution

The study introduces a flow cytometry-based method to detect early MAP infection by quantifying IFN-γ in CD4+ T cells.

## Key findings

- IFN-γ production in CD4+ T cells allowed differentiation of infected and control calves from 16 weeks post-inoculation.
- The method showed higher sensitivity and specificity than the IFN-γ release assay (IGRA) in early infection stages.
- No antigen-specific IFN-γ was detected in CD8+ T cells throughout the study.

## Abstract

Current diagnostic methods for Johne’s disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.

The online version contains supplementary material available at 10.1186/s13567-024-01324-8.