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

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.

Primary characterization of the immune response in pigs infected with Trichinella spiralis

“…This disease is distributed worldwide and has a notable impact on human health, with approximately 10 000 cases occurring each year [1]. The main causative agent of trichinellosis, Trichinella spiralis (T. spiralis), is usually undetected in pig carcasses due to the lack of macroscopically visible alterations, and pigs play an important role in the transmission of human trichinellosis [2][3][4]. Humans become infected via consumption of inadequately cooked or raw meat (usually pork) containing infective T. spiralis larvae [5][6][7].…”

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs

“…As respiratory pathogens such as the porcine reproductive and respiratory syndrome virus (PRRSV), A. pleuropneumoniae (APP) and Mycoplasma (M.) hyopneumoniae are of major economic concern in pork production worldwide [9], the antigen selection comprised not only zoonotic agents, but also antigens for these pig-specific pathogens. Antigens for two zoonotic agents (T. gondii, Y. enterocolitica) and three respiratory pathogens (APP, PRRSV, M. hyopneumoniae) showed highly promising test accuracy on the new microarray [5]. This result has encouraged the development of a pig-specific microarray including functionality for more antigens.…”

“…In parallel to the meat sampling, 184 blood samples from exactly the same pigs had been taken for the preceding study (ArrayTube platform with serum [5]). These blood serum samples were analysed with ten different ELISA tests (pigtype Toxoplasma Ab, pigtype Trichinella Ab, pigtype Yersinia Ab, pigtype Hepatitis E Virus Ab, pigtype Mycobacterium Ab, pigtype Swine Influenza Virus Ab, pigtype Salmonella Ab, pigtype PRRSV Ab (all Indical Bioscience GmbH, Leipzig, Germany), ID Screen Mycoplasma hyopneumoniae Indirect, ID Screen APP Screening Indirect serotypes 1-12 (both IDvet, Grabels, France)).…”

“…To overcome this obstacle, we previously reported the development of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs [5]. Protein microarrays are excellently suited for the simultaneous detection of different analytes and have already been used for the simultaneous detection of different antibodies [6][7][8].…”

“…By coupling different antigens on the microarray chip surface, the respective antibodies can be detected in a joint test run, which not only saves costs, but also analysis time. The recently described microarray-based assay was produced with 12 different antigens and validated on ELISA pretested serum samples [5]. As respiratory pathogens such as the porcine reproductive and respiratory syndrome virus (PRRSV), A. pleuropneumoniae (APP) and Mycoplasma (M.) hyopneumoniae are of major economic concern in pork production worldwide [9], the antigen selection comprised not only zoonotic agents, but also antigens for these pig-specific pathogens.…”