Analysis of host gene expression profiles for identification of diagnostic biomarker in subclinical infection with Mycobacterium avium subspecies paratuberculosis in cattle

Abstract

Johnes disease (JD) is the chronic wasting disease of the ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) which cause major economic losses for dairy industry worldwide. JD is mainly distributed by fecal-oral route through contaminated materials such as feed, water, and milk with MAP. During the early stage of infection, infected cattle do not show clinical symptoms however, they shed low numbers of MAP into environment and MAP can be circulated in the herd and infect other cattle. Therefore, it is very important to detect infected cattle at early stage for successful eradication of the disease. However, current diagnostic methods, including fecal culture, fecal PCR, and ELISA are insufficient for diagnosis of subclinical stages of disease. Therefore, alternative diagnostic methods which enable to detect subclinical cattle have been requested.<br/> First, we described the gene expression profiles of MAP-infected cattle which were classified by the results of ELISA and fecal PCR. Six genes (LTF, HGF, HP, CXCR3, GBP6, and TFRC) were significantly up-regulated in subclinical cattle. These genes should be further evaluated to determine their suitability for diagnosis of subclinical infection of MAP. Various factors including infection dose, infected age, animal species, and coexistence of other disease might affect the accuracy of these prognostic biomarkers. Accordingly, field studies need to be conducted to determine the adequacy of these prognostic biomarkers for use as a diagnostic tool of subclinical stage of JD.<br/> Second, we demonstrated that manipulation of host responses for the survival of MAP that occurs during the subclinical phases of JD in cattle. Downregulation of IL-17A, IL-17F, IL-22, IL-26, HMGB1, and IRF4 and upregulation of PIP5K1C indicate suppression of the Th1 response due to MAP infection and loss of granuloma integrity. In addition, increased expression of IRF5 and IRF7 suggest activation of IFN-α/β signaling during subclinical stages, which induced indoleamine 2,3-dioxygenase mediated depletion of tryptophan metabolism. Increased expression of CORO1A indicate modulation of calcium signaling, which enhanced the survival of MAP. Taken together, distinct host gene expression induced by MAP infection indicates enhanced survival of MAP during subclinical stages.<br/> Third, we describes the response of eight host biomarkers (HP, TIMP1, MMP9, SERPINE1, TFRC, S100A8, DEFB1, and DEFB10) significantly discriminated MAP-infected and non-infected cattle. Moreover, these eight biomarkers showed good accuracy (AUC≥0.7) for diagnosis of subclinical animals. Additionally, four genes (TIMP1, S100A8, DEFB1, and DEFB10) showed sensitivity over 80% and specificity over 90%. Taken together, a real-time PCR method was developed based on eight biomarkers that can be used as a new diagnostic tool for JD with good diagnostic performance. <br/> In conclusion, these results demonstrate the possibility of diagnosing subclinical cases of paratuberculosis using biomarker-based diagnostic techniques and expected to contribute to the development of biomarker based diagnostic methods to replace the currently used diagnostic methods.<br/>