Development of a miniaturized protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs.

Katharina Loreck,Elke Mueller,Annett Reissig,Sylvia Mitrenga,Diana Meemken,Claudia Engemann,Ralf Ehricht,Matthias Greiner,Regina Heinze,Baochuan Lin

doi:10.1371/journal.pone.0217290

Abstract

In order to monitor the occurrence of zoonotic agents in pig herds as well as to improve herd health management, the development of new cost-effective diagnostic methods for pigs is necessary. In this study, a protein microarray-based assay for the simultaneous detection of immunoglobulin G (IgG) antibodies against different zoonotic agents and pathogens causing production diseases in pigs was developed. Therefore, antigens of ten different important swine pathogens (Toxoplasma gondii, Yersinia enterocolitica, Salmonella spp., Trichinella spp., Mycobacterium avium, Hepatitis E virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, the porcine reproductive and respiratory syndrome virus, Influenza A virus) were spotted and covalently immobilized as ‘antigen-spots’ on microarray chips in order to test pig serum for the occurrence of antibodies. Pig serum was sampled at three German abattoirs and ELISA tests for the different pathogens were conducted with the purpose of creating a panel of reference samples for microarray analysis. To evaluate the accuracy of the antigens on the microarray, receiver operating characteristic (ROC) curve analysis using the ELISA test results as reference was performed for the different antigens. High area under curve values were achieved for the antigens of two zoonotic agents: Toxoplasma gondii (0.91), Yersinia enterocolitica (0.97) and for three production diseases: Actinobacillus pleuropneumoniae (0.77), Mycoplasma hyopneumoniae (0.94) and the porcine reproductive and respiratory syndrome virus (0.87). With the help of the newly developed microarray assay, collecting data on the occurrence of antibodies against zoonotic agents and production diseases in pig herds could be minimized to one measurement, resulting in an efficient screening test.

Highlights

Due to the frequent occurrence of zoonotic agents in pig herds at slaughter, fast and economic monitoring tools to control these pathogens are in high demand in pork production [1, 2]
The objective of this study was to test whether a protein microarray with antigens of six different zoonotic agents (T. gondii, Y. enterocolitica, Salmonella spp., Trichinella spp., M avium, Hepatitis E virus) and four different production diseases (M. hyopneumoniae, A. pleuropneumoniae, porcine reproductive and respiratory syndrome virus (PRRSV), Influenza A virus) spotted and covalently immobilized on the microarray surface would allow the simultaneous testing of pigs for the presence of different antibodies
This study investigates the accuracy of antigens on a microarray by using established enzyme-linked immunosorbent assay (ELISA) tests with comparable antigens as reference tests

Summary

Introduction

Due to the frequent occurrence of zoonotic agents in pig herds at slaughter, fast and economic monitoring tools to control these pathogens are in high demand in pork production [1, 2]. In 2011, the European Food Safety Authority (EFSA) identified Salmonella spp., Yersinia (Y.) enterocolitica, Toxoplasma (T.) gondii and Trichinella spp. as the most relevant biological public health hazards in the context of meat inspection of swine [3]. These zoonotic agents as well as Mycobacterium (M.) avium [4] and Hepatitis E virus [5] from pig carcasses constitute a danger to human health, but can usually not be detected by the official post-mortem meat inspection due to the lack of macroscopically visible carcass alterations. A cost-efficient diagnostic method suitable for routine testing to accomplish a broad monitoring for multiple pathogens is missing

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