Early Detection of Mycobacterium avium subsp. paratuberculosis Infected Cattle: Use of Experimental Johnins and Innovative Interferon-Gamma Test Interpretative Criteria.

Sara Corneli,Alessandro Dondo,Carla Sebastiani,Massimo Biagetti,Paola Papa,Marcella Ciullo,Antonella Di Paolo,Piera Mazzone,Martina Torricelli,Nicoletta Vitale,Monica Cagiola,Linda Petrucci,Ludovica Curcio,Silva Costarelli

doi:10.3389/fvets.2021.638890

Abstract

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused by Mycobacterium avium subsp.paratuberculosis (MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted from Mycobacterium bovis (MB) and from M. avium (MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p < 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1–100%] with a specificity of 91.8% (CI 95%, 81.9–97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1–89.2%) with a specificity of 100% (CI 95%, 94.1–100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

Highlights

Mycobacterium avium subsp. paratuberculosis (MAP) is a slowgrowing mycobacterium [1] and the causal agent of Johne’s disease (JD) or paratuberculosis (PTB), a chronic inflammatory bowel disease seen in farmed ruminants and wildlife species worldwide [2, 3]
For the 10th criterion, we compared the ratio between purified protein derivatives (PPDs) (PPDB/PPDA > PPDs extracted from MB AN5 (PPDB)/PPDJ = MAP infection); and for the 11th and 12th criteria, we considered the difference between PPDA and PPDJs and two different cutoff values (PPDJ – PPDA > 0.05; 0.1 = MAP infection)
One subject became positive for the culture test 3 months later, one was positive for enzyme-linked immunosorbent assay (ELISA) and MAP isolation 10 months later, and two cattle became positive for ELISA (Supplementary Figure 1)

Summary

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP) is a slowgrowing mycobacterium [1] and the causal agent of Johne’s disease (JD) or paratuberculosis (PTB), a chronic inflammatory bowel disease seen in farmed ruminants and wildlife species worldwide [2, 3]. Paratuberculosis (MAP) is a slowgrowing mycobacterium [1] and the causal agent of Johne’s disease (JD) or paratuberculosis (PTB), a chronic inflammatory bowel disease seen in farmed ruminants and wildlife species worldwide [2, 3]. MAP may act as a zoonotic agent in some human diseases [7,8,9,10]. For more than a century, it has been associated with Crohn’s disease, a chronic inflammatory bowel disease characterized by transmural inflammation and granuloma formation. Other diseases have been associated with MAP, such as sarcoidosis, Blau syndrome, type 1 diabetes, Hashimoto’s thyroiditis, and multiple sclerosis [9, 11,12,13,14,15,16]. MAP may contaminate food for human consumption, such as dairy and meat products, infant formula [17,18,19], and water [6, 20]; and MAP seems to resist pasteurization treatment of milk at 72◦C for 15 s [high-temperature, short-time (HTST) pasteurization] [21,22,23]

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Conclusion