Parasite strains and experimental infections
All infection experiments in cats, chickens and CD-1 mice had been ethically approved (Landesdirektion Leipzig, Germany, trial no. TVV 29/10). Care and maintenance of animals were in accordance with governmental and institutional guidelines.
We used samples from chickens (breed ISA JA 757) that had been experimentally infected with oocysts, tissue cysts or tachyzoites as reported in detail in a previous study [22]. Regardless of oocyst, tissue cyst or tachyzoite infections, the observation period usually lasted 5 weeks in all infected groups. In the case of tachyzoite infection, six inoculated and six non-inoculated birds were included and observed for a total of 10 weeks [22]. At the end of the observation period, blood was collected for serological analysis, the animals were euthanized and tissues (brain, heart, breast, thigh and drumstick musculature) were stored frozen at –20 °C until further use. A total of 23 non-infected control chickens and 66 inoculated chickens were used, which were orally inoculated with oocysts or brains of chronically infected mice or by intravenous injection of in vitro-cultivated tachyzoites [22].
Three different T. gondii strains were used: the type II T. gondii strain CZ-Tiger [25], type II T. gondii ME49 [26] and type III T. gondii NED [27]. The CZ-Tiger strain parasites were already available as oocysts while ME49 and NED parasites were initially cultivated as tachyzoites [28] and passaged via CD-1 mice and cats to generate tissue cysts and oocysts, respectively [22].
For infecting chickens, three different doses of oocysts were applied, i.e. 1x103 (CZ-Tiger, ME49, NED), 1x105 (CZ-Tiger, ME49), or 1x106 oocysts per bird (CZ-Tiger, ME49) [22]. For tissue cyst infection, one microscopically-positive mouse brain per bird was inoculated orally [22]. In vitro cultivated tachyzoites (T. gondii NED, 1x106 tachyzoites in 0.1 ml of sterile isotonic saline solution (B. Braun Melsungen AG, Melsungen, Germany)) were inoculated i.v. into the wing vein of each bird.
Polymerase chain reaction
MC-qPCR was essentially performed as described [29] with some slight modifications [22].
For the PD-qPCR, tissues were digested [11, 30] and the qPCR performed on digests as described [31, 32] using primers and a probe targeting the 529 bp repeat of T. gondii [33].
Sera and serological tests
Sera
Sera from experimentally and naturally exposed chickens were collected as detailed previously [9, 22]. When the chickens were sacrificed, blood was collected and allowed to clot. The samples were then centrifuged, sera collected and stored frozen at -20° C until further use.
MAT
The MAT for the detection of T. gondii-specific IgY antibodies was performed as previously described [34]. Each serum or fluid sample was two-fold serially diluted. A titre of 1:1 was applied as the positive cut-off.
IFAT
The IFAT was performed as reported previously [9]. Only complete peripheral fluorescence of the tachyzoite was considered specific. A titre of 1:50 was used as the positive cut-off.
TgSAG1-ELISA
Chicken sera were tested for antibodies against the native T. gondii tachyzoite surface antigen TgSAG1 as described [9] using affinity purified TgSAG1 of T. gondii tachyzoites [35, 36]. A cut-off optimized for maximum diagnostic specificity was applied (ELISA index 0.242) as previously described for the TgSAG1-ELISASH [9]. The subscript SH indicates “specificity high”. Moreover, a less-stringent cut-off optimized for Youden’s index was used (ELISA index 0.104) for the TgSAG1-ELISASL [9]. Here, the subscript “SL” indicates “specificity low”.
Luminex TgSAG1
Recombinant production of biotinylated TgSAG1 (TgSAG1bio) and coupling of the antigen to Luminex MagPlex® beads (Luminex Cooperation, ‘s-Hertogenbosch, The Netherlands) has been described recently [37]. In brief, the entire mature coding region of TgSAG1 (aa 31-289) was expressed as an N-terminal fusion with maltose binding protein (MBP), which enhances solubility during translation. MBP can be cleaved-off in situ by TEV protease, with recognizes its cleavage sequence and thus separates MBP from TgSAG1 in the engineered protein [37]. After the putative GPI-attachment site (Gly289 of TgSAG1) at the C-terminus, a 4 kDa peptide sequence (AviTag) and a six histidine-tag were added and used for purification. The AviTag is recognized by Echerichia coli biotin ligase BirA, resulting in the C-terminal in situ biotinylation of TgSAG1 at a unique lysine residue within the tag sequence. Subsequently, biotinylated TgSAG1bio was purified by metal chelate affinity chromatography using an Äkta Purifier system [37].
The chemical coupling to beads of either recombinant streptavidin (Sav; Anaspec, Fremont, CA, USA; 16.67 μg/106 MagPlex® beads, region 34), chicken serum albumin (CSA, Sigma Aldrich, Darmstadt, Germany; 12 µg/106 MagPlex® beads, region 54) as a negative control, or chicken IgY (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 6.67 µg/106 MagPlex® beads, region 52) as a positive control followed the instructions of the xMAP® Cookbook [38, 39]. Prior to coupling, bead stocks were vortexed for 30 s and sonicated for 30 s in a water-bath. Beads (1.5 x 106 each) were transferred from the stock to individual reaction tubes for each of the three bead regions, i.e. dye signatures, washed with distilled water, vortexed and sonicated for a few seconds and incubated in 80 µl 0.1 M NaH2PO4, pH 6.2 tube. The tubes were again vortexed and sonicated for 10 s prior to addition of 500 µg N-hydroxysulfosuccinimide (Sulfo-NHS, Thermo Fisher, Waltham, MA, USA) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Thermo Fisher, Waltham, MA, USA). The beads were then incubated for 20 min on a horizontal shaker (300 rpm) and vortexed briefly after 10 min.
After incubation, the tubes were again placed in a magnetic separator for 2 min and the supernatant removed. The beads were washed twice with 250 µl of 0.05 M 2-(N-morpholino) ethanesulfonic acid (MES, Sigma Aldrich, Darmstadt, Germany) before addition of conjugates, and each tube was adjusted to 500 µl by adding 0.05 M MES. Tubes were briefly vortexed and then incubated for 2 h on a horizontal shaker at 300 rpm, with an intermittent brief vortexing step after 1 h. The tubes were then placed in a magnetic separator for 2 min and the supernatant removed. 500 µl of PBS containing 0.02 % Tween-20, 0.1 % BSA and 0.05 % sodium azide (PBS-TBN) were added and the beads incubated for 30 min on a horizontal shaker at 300 rpm, before the samples were placed in a magnetic separator for 2 min to remove the supernatant. The beads were washed twice with 1 ml PBS-TBN without sonication. For storage, the beads were resuspended in 500 µl Stabilguard (Surmodics, Inc., Eden Prairie, MN, USA). TgSAG1bio (10 ng/1,500 beads) was added to the Sav-coated bead mix as described elsewhere [37].
Testing by BBMA was performed as previously described for human sera [39]. The three bead mixes were adjusted to 1,000 beads per sample in PBS containing 1 % BSA (PBS-B). Twenty µl of each region were added to 100 µl of samples (sera diluted 1:200 in PBS-B) in a 96-well plate (Greiner Bio-One, Kremsmünster, Austria). The plate, protected from light, was shaken at room temperature for 60 min. Beads were then washed twice with PBS containing 0.1 % Tween-20 (PBS-T). 100 µl of rabbit-F(ab’)2 anti-chicken IgG-phycoerythrin (Rockland Immunochemicals, Limerick, PA, USA), diluted 1:333 in PBS-B, added to each sample and the plate shaken at room temperature for 30 min, protected from light. Beads were again washed twice, resuspended in 125 µl PBS-B and analysed with a Bio-Plex 200 reader (Bio-Rad, Hercules, CA, USA). The readout was set to 50 beads per region and the timeout was set to 90 s. The High RP1 Target option was activated (i.e., increasing the voltage on the photomultiplier tube) for increased sensitivity, allowing quantification of lower concentrations of analytes and three wells containing only beads and PBS-B were set as blank samples.
Mouse bioassay
The mouse bioassay was conducted as described [9]. Briefly, IFNɣ-knockout mice (GKO, IFNɣ -/-, C.129S7(B6)-Ifngtm1Ts/J) or IFNɣ-receptor-knockout mice (GRKO, IFNɣreceptor -/-; B6.129Sv/Ev-IfngrtmAgt) were used. The mice were inoculated with pepsin-digested [11, 30] heart and drumstick musculature (two mice for each kind of tissue, monitored for 42 days). All mouse experiments (bioassays) reported in this publication were approved by the “Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei” of the German Federal State of Mecklenburg-Western Pomerania (permit 7221.3-2.5-001/13). Care and maintenance of animals were in accordance with governmental and institutional guidelines.
Statistical analysis
R version 3.5.3 (R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org) and the R package “optimal.cutpoints” were used to define an optimal cut-off for the TgSAG1bio-BBMA and to determine diagnostic sensitivity, specificity, and positive and negative predictive values, including 95% confidence intervals (95% CI). In addition, diagnostic sensitivity and diagnostic specificity, including 95% confidence intervals (95% CI), were determined using tools that were available online (http://vassarstats.net/clin1.html). To assess the overall diagnostic performance of a test, Youden’s index was calculated by the following formula using EXCEL spreadsheet functions: Sensitivity + Specificity – 1 [40]. To determine the relatedness of values measured in various serological diagnostic tests, linear regression was performed using the “lm” command in R, version 3.5.3. For this analysis, median fluorescence intensity (MFI) values and titers in IFAT and MAT were log10-transformed. Sera for which no titre had been determined in IFAT or MAT (i.e. seronegative sera), arbitrary titres of 1:25 (IFAT) or 1:0.5 (MAT) were used to allow for the calculation of log10 values.
Figures were assembled using R, version 3.5.3 or 4.0.0 (packages “ggplot2”, “reshape” and “scales”).