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.

Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein

“…The specificity of the tests based on IFN- γ assessment, however, is low in animals below 16 months of age [ 21 ]. Moreover, the identification of IFN- γ -positive animals should be followed by other diagnostic methods such as ELISA or MAP fecal shedding to evaluate disease's progression in infected herds [ 22 , 23 ]. Among the diagnostic tests, culture of MAP from feces, milk, blood, and tissues of infected animals is considered “gold standard” for the detection of MAP infection [ 24 ].…”

Paratuberculosis Cases in the Livestock Industry in Nigeria: A Review

“…M. a. ptb, like other Mycobacteria, has a dense, waxy cell wall of about 60 % lipid. This confers the properties of acid fastness, hydrophobicity, resistance to chemicals such as chlorine, and physical process (pasteurization) to the bacteria (Cocito et al, 1994;Okuni et al, 2020;Corneli et al, 2021).…”

Detection of Low MAP Shedder Prevalence in Large Free-Stall Dairy Herds by Repeated Testing of Environmental Samples and Pooled Milk Samples

“…Even when tested individually for MAP or MAP-specific antibodies with negative results, purchased animals can be in the subclinical stage of the disease. The interferon gamma response test can be a useful method for detecting early stages of the disease, but the practicability of this test is hampered by its limited specificity, specific pre-analytical requirements, and its high costs [ 12 , 13 , 14 ]. Testing animals in the subclinical stage with ELISA is not sufficient because of the limited sensitivity of available tests.…”