Novel recombinant Mce-truncated protein based ELISA for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in domestic livestock

Zahra Hemati,Kundan Kumar Chaubey,Masoud Haghkhah,Abdollah Derakhshandeh,Shoor Vir Singh,Hasnain Seyed Ehtesham

doi:10.1371/journal.pone.0233695

Zahra Hemati, Kundan Kumar Chaubey + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0233695

Copy DOI

Abstract

Johne’s disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.

Highlights

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of chronic wasting disease in domestic livestock species commonly known as paratuberculosis or Johne’s disease (JD) [1, 2]
Based on Bioinformatics analysis, MAP2191 gene from MAP was of 1065bp and encodes for a typical linear Mammalian cell entry (Mce) protein that has 354 amino acids with an estimated molecular weight of 37.510 kDa
Results were comparable; only 3 (2.5%) positive serum in i-ELISA were missed by Mce-ELISA (Table 4). These results showed higher specificity and lowered sensitivity in case of Mce-ELISA as compared to i-ELISA in buffaloes (Table 5)

Summary

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of chronic wasting disease in domestic livestock species commonly known as paratuberculosis or Johne’s disease (JD) [1, 2]. The disease inflicts major economic losses to livestock production system and dairy industry by lowering productivity, both in terms of quality and quantity, of milk, meat, fiber and skin, by way of increased morbidity, mortality and early cullings [1, 6]. With the scale and complexity of the expanding problem of MAP, pressure is increasing to find a way to control MAP infections in animals in order to rescue high milk yielding breeds of domestic livestock, and reduce production and economic losses in the livestock, dairy industry and animal farming, and impending threat for large scale human infection by consumpton of milk and milk products contaminated with MAP bacilli [6, 11, 12]

Objectives

Methods

Results

Conclusion