A novel bead-based assay to detect specific antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera of experimentally infected swine

BackgroundA novel, bead-based flow cytometric assay was developed for simultaneous determination of antibody responses against Toxoplasma gondii and Trichinella spiralis in pig serum. This high throughput screening assay could be an alternative for well known indirect tests like ELISA. One of the advantages of a bead-based assay over ELISA is the possibility to determine multiple specific antibody responses per single sample run facilitated by a series of antigens coupled to identifiable bead-levels. Furthermore, inclusion of a non-coupled bead-level in the same run facilitates the determination of, and correction for non-specific binding. The performance of this bead-based assay was compared to one T. spiralis and three T. gondii ELISAs. For this purpose, sera from T. gondii and T. spiralis experimentally infected pigs were used. With the experimental infection status as gold standard, the area under the curve, Youden Index, sensitivity and specificity were determined through receiver operator curve analysis. Marginal homogeneity and inter-rater agreement between bead-based assay and ELISAs were evaluated using McNemar's Test and Cohen's kappa, respectively.ResultsResults indicated that the areas under the curve of the bead-based assay were 0.911 and 0.885 for T. gondii and T. spiralis, respectively, while that of the T. gondii ELISAs ranged between 0.837 and 0.930 and the T. spiralis ELISA was 0.879. Bead-based T. gondii assay had a sensitivity of 86% and specificity of 96%, while the ELISAs ranged between 64-84% and 93-99%, respectively. The bead-based T. spiralis assay had a sensitivity of 68% and specificity of 100% while the ELISA scored 72% and 95%, respectively. Marginal homogeneity was found between the T. gondii bead-based test and one of the T. gondii ELISAs. Moreover, in this test combination and between T. spiralis bead-based assay and respective ELISA, an excellent inter-rater agreement was found. When results of samples before expected seroconversion were removed from evaluation, notably higher test specifications were found.ConclusionsThis new bead-based test, which detects T. gondii and T. spiralis antibodies simultaneously within each sample, can replace two indirect tests for the determination of respective antibodies separately, while performing equally well or better.

[1]  E. Pozio,et al.  Epidemiology, Diagnosis, Treatment, and Control of Trichinellosis , 2009, Clinical Microbiology Reviews.

[2]  Moez Sanaa,et al.  Review of the Community Summary Report on Trends and Sources of Zoonoses, Zoonotic agents and Antimicrobial Resistance in the European Union in 2005 Scientific Opinion of the Scientific Panel on Biological Hazards (BIOHAZ) and Animal Health and Welfare (AHAW) , 2007 .

[3]  Efsa Journal,et al.  European Food Safety Authority, European Centre for Disease Prevention and Control; The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2009 , 2011 .

[4]  F. Knapen.  Control of trichinellosis by inspection and farm management practices. , 2000 .

[5]  W. Campbell.  Trichinella and Trichinosis , 2011, Springer US.

[6]  J. R. Landis,et al.  The measurement of observer agreement for categorical data. , 1977, Biometrics.

[7]  J. Dame,et al.  Evaluation of an enzyme-linked immunosorbent assay for diagnosis of trichinellosis in swine. , 1992, American journal of veterinary research.

[8]  Roger M. Cooke,et al.  Prioritizing Emerging Zoonoses in The Netherlands , 2010, PloS one.

[9]  G. Köhler,et al.  Comparison of four methods for early detection of experimental Trichinella spiralis infections in pigs. , 1981, Veterinary parasitology.

[10]  A. Henken,et al.  Measurement of disease frequency , 2001 .

[11]  F. van Knapen,et al.  Specific serum antibody responses following a Toxoplasma gondii and Trichinella spiralis co-infection in swine. , 2012, Veterinary parasitology.

[12]  A. Kijlstra,et al.  Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR. , 2010, International journal of food microbiology.

[13]  M. Greiner,et al.  Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. , 2000, Preventive veterinary medicine.

[14]  D. Hill,et al.  PREVALENCE OF VIABLE TOXOPLASMA GONDII IN BEEF, CHICKEN, AND PORK FROM RETAIL MEAT STORES IN THE UNITED STATES: RISK ASSESSMENT TO CONSUMERS , 2005, The Journal of parasitology.

[15]  A. Semple.  Measurement of Disease , 1964 .

[16]  E. Ruitenberg,et al.  Control III Surveillance in Swine by Immunodiagnostic Methods , 1983 .

[17]  Martijn Bouwknegt,et al.  Seroprevalence of Trichinella spiralis and Toxoplasma gondii in pigs from different housing systems in The Netherlands. , 2007, Veterinary parasitology.

[18]  Sandra Hoffmann,et al.  Ranking the Risks: The 10 PaThogen-Food CombinaTions WiTh The greaTesT burden on PubliC healTh , 2011 .

[19]  A. Wingstrand,et al.  The time course of the specific antibody response by various ELISAs in pigs experimentally infected with Toxoplasma gondii. , 1997, Veterinary parasitology.

[20]  Valentina Rizzi,et al.  The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010. , 2012, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[21]  J. Noordhuizen.  Application of Quantitative Methods in Veterinary Epidemiology , 2001 .

[22]  R. Ko,et al.  The distribution of excretory/secretory antigens during the muscle phase of Trichinella spiralis and T. pseudospiralis infections , 1999, Parasitology Research.

[23]  Olivier Andreoletti,et al.  Scientific Opinion of the Panel on Biological Hazards , 2007 .

[24]  D. Despommier,et al.  Trichinella spiralis: secreted antigen of the infective L1 larva localizes to the cytoplasm and nucleoplasm of infected host cells. , 1990, Experimental parasitology.