Experimental infection sera
Swine serum samples originated from an experimental co-infection of pigs with T. gondii and T. spiralis . Before infection, animals used in the experiment were assumed T. gondii and T. spiralis free on basis of the post partum determined negative serological status of sows which gave birth to these animals . Briefly, eight to nine week old animals had been singly (T. gondii n = 8, T. spiralis n = 10), simultaneously (n = 10), or successively (T. gondii/T. spiralis n = 9, T. spiralis/T. gondii n = 10) orally inoculated with either 2,700 or 2,000 T. gondii tissue cysts (strain DX) and/or 5,000 T. spiralis muscle larvae (strain ISS 14) per pig. Because two animals of the T. spiralis/T. gondii inoculated group did not seem to be infected, they were excluded from the experiment . A total of 444 serum samples were collected in series at 0, 5, 12, 19, 26, 33, 40, 47 and 54 days post infection (p.i.) from 45 pigs and four additional non-inoculated animals which served as negative control animals. This animal study, under number DEC 2008.III.03.023, was reviewed and approved by the local animal ethics committee according to the recommendations of the EU directive 86/609/EEC. Numbers of animals and their suffering were minimized.
Negative field sera
Blood samples of conventional finisher pigs were collected for Salmonella baseline monitoring at the abattoir in 2007 by the Dutch Food and Consumer Product Safety Authority (nVWA). The blood was left at room temperature with a minimum of 2 hours to clot and subsequently centrifuged for 10 minutes at 1,100 × g. Serum was drawn and dispensed in aliquots and kept at -20°C until further use.
Serum samples were analyzed by a commercially obtained T. gondii ELISA (ID Screen Toxoplasmosis Indirect, ID-VET, Montpellier, France; hereafter referred as E3-TOX). Serum samples remaining under the designated cut-off value of the ELISA were considered to originate from T. gondii infection negative pigs. Because during the sample period no pigs with T. spiralis infections were reported , all animals were considered T. spiralis infection negative.
Sera from the experimentally infected animals were tested by the bead-based assay for T. gondii and T. spiralis antibodies simultaneously (hereafter referred to as BBA-TOX and BBA-TRI, respectively), by an RIVM in-house T. gondii ELISA (hereafter referred to as E1-TOX) and two commercially available T. gondii ELISA kits (Safepath, Carlsbath, CA, USA, hereafter referred to as E2-TOX) and E3-TOX, and by one T. spiralis ELISA (Safepath, hereafter referred to as E-TRI). All T. gondii indirect tests used an antigen based on T. gondii tachyzoites, of which E3-TOX utilized a recombinant tachyzoite surface protein (SAG-1) as antigen. The T. spiralis tests were based on ES antigens. Bead-based assays were run according to the specifications described in the section bead-based assay. Testing with the in-house ELISA  was described earlier  and included an intra-plate correction of E1-TOX data. All commercial ELISAs were run according to the specification of the kit providers. For E3-TOX, normalization of data was included.
A bead-based assay was developed for simultaneous detection of specific antibodies which were captured by T. spiralis and T. gondii antigens on two different bead levels. NSB was recorded with reference beads, which is a bead level without coupled antigens. Each bead level was recognized via the emission of light with a unique intensity and wavelength of the beads intrinsic fluorescence. Specific and NSB in each individual serum sample were determined by the extrinsic response, which was generated by the emission of light by a fluorophore attached to the secondary antibody. Because NSB may vary between serum samples, the extrinsic response of reference beads was used to determine the non-specific response. To obtain a specific response per individual serum sample, this non-specific response was subtracted from the response of coupled beads. A BD Accuri flow cytometer was used for enumeration of micro-particles, excitation of fluorescent markers and measurement of emitted light from these markers.
Chemicals, materials and solutions
L4, L10 and L11 carboxylated Cyto-plex™ beads (cat# FM5CR04, FM5CR10 and FM5CR11, respectively) were purchased from Thermo Scientific (Waltham, MA, USA). T. gondii tachyzoite lysate, strain RH (cat#: R29123) was from Meridian Life Science Inc. (Saco, ME, USA). T. spiralis Excretory/Secretory antigen (ES) was obtained from Instituto Superiore Sanità (Rome, Italy). Microcentrifuge copolymer tubes (cat# 1415-2500) were acquired from Star Lab GmbH (Ahrensberg, Germany). N-hydroxysulfosuccinimide sodium salt (sNHS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC; cat#: 03449) and 2-(N-morpholino)ethanesulfonic acid hydrate (MES; cat#: M8250) were bought from Sigma-Aldrich Chemie B.V. (Zwijndrecht, the Netherlands). A 45 mM MES buffer was prepared and adjusted to pH 6.0 with sodium hydroxide. PBS at pH 7.2 consisted of 0.01 M sodium chloride (NaCl, Merck KGaA, Darmstadt, Germany), 1 mM di-sodium hydrogen phosphate (Merck) and 3 mM potassium dihydrogen phosphate (Merck). Water was of milliQ quality. Storage buffer and HNT-PBS solution were provided by RnAssays (Utrecht, the Netherlands). The 0.45 μm filter plates (cat#: MSHVN4550) were from Millipore (Amsterdam, the Netherlands). Goat anti-swine secondary antibody conjugated with fluorescent DyLight 488 was purchased from Jackson Immuno Research (West Grove, PA, USA).
Bead coupling procedure
T. gondii tachyzoite lysate and T. spiralis Excretory/Secretory antigen (ES) were coupled to carboxylated beads through an amine coupling procedure. Briefly, an equivalent of 1.4 × 108 carboxylated beads of L10 and L11 were transferred to two 1.5 ml copolymer tubes. The beads were washed by three repeats of following steps: a 3 minutes centrifugation at 9,000 ×g, removal of the supernatant, addition of 1 ml water per tube and resuspension of the beads on a vortex. After the third removal of supernatant, beads from both tubes were resuspended in 1.1 ml solution consisting of 12.5 mg sNHS and 12.5 mg EDC in MES buffer. This suspension was incubated for 20 minutes at room temperature on a gyro rocker at 70 rpm. Beads were washed 2 more times with 500 μl water as described above and after removal of the supernatant, 50 μg of T. gondii lysate and 10 μg T. spiralis ES dissolved in 200 μl PBS pH 7.4 were added to the activated L10 and L11 beads, respectively. Resuspended beads were left to incubate for 2 hours on a gyro rocker at 70 rpm, washed and stored in a storage buffer. A non-coupled L4 reference bead suspension was produced with the same protocol with exception of the protein incubation step which was substituted by PBS incubation. This L4 bead is referred to as the reference bead.
Two 0.45 μm filter plates were soaked with 150 μl of a 0.2 μm filtered solution of HNT-PBS, subsequently incubated for five minutes at ambient temperature, and emptied by vacuum filtration. Serum samples were diluted 1:50 in HNT-PBS, transferred to a soaked and aspirated 0.45 μm filter plate, filtered with the use of the vacuum manifold and collected in an empty 96-wells plate. Thereafter, in another soaked filter plate, a quantity of approximately 5 × 105 T. gondii and T. spiralis antigen coupled beads and reference beads were suspended in 50 μl of HNT-PBS per well. Subsequently, one equivalent volume of filtered diluted sera was mixed and incubated with the bead-mix per well for 15 minutes on an orbital shaker (1,050 rpm). Beads were washed with 200 μl HNT-PBS by aspiration and additionally incubated with 100 μl 1:300 in HNT-PBS diluted fluorescent secondary antibody for 15 minutes. Finally, beads were washed once more and suspended in 100 μl HNT-PBS. Due to light sensitivity of beads and fluorescent reporter the filter plates were protected from light during incubation steps.
Internal and external fluorescent detection
A total of 600 beads per serum sample were analyzed for the intrinsic bead label on the FL4 channel, and extrinsic fluorescence reporter label on the FL1 channel using a BD Accuri C6 flow cytometer (BD Accuri Cytometers, Inc. Ann Arbor, MI, USA). The detector was equipped with a CSampler liquid handler (BD Accuri) and operated through CFlow software (version 184.108.40.206, BD Accuri). Beads were transported at a flow rate of 35 μl/min. The emission of the intrinsic fluorescence of the three bead levels, measured by the FL4 filter at 675 nm, was used to distinguish the T. gondii (TOX), T. spiralis (TRI) and reference (REF) beads. The median extrinsic fluorescence intensity (MFI) of the secondary antibody per bead level was determined by measuring the emission via the FL1 filter at 530 nm.
Correction for non-specific binding
Reference beads were used to indicate the measure of NSB in the test. Differences in NSB on uncoupled or antigen coupled bead levels may be expected due to differences in affinity of beads for non-specific antibodies caused by the molecular structure of the antigen, its orientation and concentration on the bead surface. Therefore, to estimate the NSB on T. gondii and T. spiralis bead levels from the response of a reference bead, a correction factor was calculated by testing 932 T. gondii and 13 extra T. spiralis negative swine sera (section Negative field sera) in the bead-based assay. With the use of least square regression, linear relations, expressed with the formulae y = slope*x + intercept, the relation between responses of the reference beads (x value) and T. gondii and T. spiralis bead responses (y values) were calculated in SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Because the residuals of the linear relation between responses of T. gondii and T. spiralis bead levels and reference bead responses were not normally distributed, all responses were log transformed.
Normalization of responses
To compare results between 96-wells plates, serum samples responses were normalized. The percentage of normalized responses (%NR) was calculated as a percentage of sample responses (MFIS) of a positive control response (MFIPC), which was present in quadruplicate on each plate, after subtraction of NSB (sections Correction for non-specific binding and Results and Discussion).
where subscript T represents T. gondii or T. spiralis in the considered case.
All statistical evaluations were performed with SPSS.
To specify the performance of the bead-based assay, expressed in area under the curve (AUC), receiving operator characteristic (ROC) calculations were performed using the experimental infection status as gold standard. Analysis was performed on the %NR of the BBA and E3-TOX, and OD450nmof the other ELISAs. ROC calculations were also performed on limited sets of serum from the experimental infection. These sets consisted of all samples minus serum samples drawn 5 days after inoculation with T. gondii (n = 408) and all samples minus serum samples drawn 5, 12 and 19 days after inoculation with T. spiralis (n = 360), for the T. gondii and T. spiralis indirect tests, respectively. To further specify the tests, diagnostic Sensitivity (Se), Specificity (Sp) and cut-off values at maximum Youden Index were determined from ROC calculations .
To assess the agreement between tests, the marginal homogeneity of paired proportions  were tested by McNemar's in a 2 × 2 contingency table. Furthermore, inter-rater agreement was calculated using Cohen's Kappa. For this, serum responses of all tests were labelled 0 (negative), when they were below the cut-off value or 1 (positive) when they were equal or above cut-off value. These dichotomized outcomes where then evaluated against the dichotomized outcomes of the other tests. Kappa values between 0.40 - 0.59, 0.60 - 0.79 and ≥ 0.80 are interpreted as moderate, substantial and excellent agreement, respectively .
The apparent prevalence (AP) is the proportion of the population which tests positive in the test, which is a measure of true prevalence (TP) and the capability of the test to predict true positives and negatives, and it was calculated as :
where TP is the proportion of actual infected animals which was calculated by:
where n is the number of sera which originate from inoculated animals, and N is the total number of sera.