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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
Background: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice. Material and methods: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms. Results: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens. Conclusion: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials. © 2020 The Author(s).
Development of a miniaturized protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs
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.
Characteristics of methicillin-resistant Staphylococcus aureus from broiler farms in Germany are rather lineage- than source-specific
Methicillin-resistant Staphylococcus aureus (MRSA) are a major concern for public health, and broiler farms are a potential source of MRSA isolates. In this study, a total of 56 MRSA isolates from 15 broiler farms from 4 different counties in Germany were characterised phenotypically and genotypically. Spa types, dru types, SCCmec types, and virulence genes as well as resistance genes were determined by using a DNA microarray or specific PCR assays. In addition, PFGE profiles of isolates were used for analysis of their epidemiological relatedness. While half of the isolates belonged to spa type t011, the other half was of spa types t1430 and t034. On 3 farms, more than 1 spa type was found. The most common dru type was dt10a (n = 19), followed by dt11a (n = 17). Susceptibility testing of all isolates by broth microdilution revealed 21 different resistance phenotypes and a wide range of resistance genes was present among the isolates. Up to 10 different resistance phenotypes were found on individual farms. Resistance to tetracyclines (n = 53), MLSB antibiotics (n = 49), trimethoprim (n = 38), and elevated MICs of tiamulin (n = 29) were most commonly observed. Microarray analysis detected genes for leucocidin (lukF/S), haemolysin gamma (hlgA), and other haemolysines in all isolates. In all t1430 isolates, the egc cluster comprising of genes encoding enterotoxin G, I, M, N, O, U, and/or Y was found. The splitstree analysis based on microarray and PCR gene profiles revealed that all CC9/SCCmec IV/t1430/dt10a isolates clustered apart from the other isolates. These findings confirm that genotypic patterns were specific for clonal lineages rather than for the origin of isolates from individual farms.