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

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.

Keywords: IFN-γ test; Johne's disease; Johnin PPD; Mycobacterium avium subsp. paratuberculosis; bovine paratuberculosis; cattle; interpretative criteria; purified protein derivatives.

Figure 1

IFN-γ production in lymphocytes of 71 PTB-positive animals. Values are expressed as the mean of the differences between the OD_450nm of PPDs and the OD_450nm of PBS (±SD). IFN, interferon; PTB, paratuberculosis; PBS, phosphate-buffered saline; PPD, purified protein derivative; SD, standard deviation; OD, optical density; AI, Italian avian PPD; BI, Italian bovine PPD; JA, JB, JC, Johnins produced by the three strains of MAP: A and B (field strains) and C [strain American Type Culture Collection (ATCC) 19698].

Figure 2

The graph shows the OD values distribution of PPD Johnins (A) for the two dilutions: 1:5 and 1:10 and (B) for strains A, B, and C. OD, optical density; PPD, purified protein derivative.

Figure 3

ROC curve for comparisons of PPDA-PBS, PPDJA-PBS, and PPDJB-PBS according to the first three interpretative criteria of the IFN-γ test applied in the study for the diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP)-infected cattle. PPDJA and PPDJB showed higher AUC than PPDA, and the difference is statistically significant (p < 0.05). ROC, receiver operating characteristic; IFN, interferon; PPD, purified protein derivative; PPDA, avian PPD; PPDJA, PPD Johnin strain A; PPDJB, PPD Johnin strain B; AUC, area under the ROC curve.