Parasites, animals and study design
Trichinella britovi ML were isolated in our laboratory during routine inspections of meat from a naturally infected wild boar and then identified at the species level by multiplex polymerase chain reaction (multiplex PCR) according to the method described by Zarlenga et al. . Muscle larvae (ML) displaying motility were counted and then suspended in 30 % gelatin blocks. Fourteen healthy young Pulawska/Polish Large White crossbreed pigs aged 10 weeks and of 20 kg average body weight were purchased from the Czesławice experimental farm belonging to the University of Life Sciences in Lublin. The animals were randomly divided into two experimental groups (A1, n=8 and A2, n=6) and subsequently infected by administering a single dose per os of 5000 T. britovi ML/pig to group A1 or 3000 T. britovi ML/pig to group A2. Blood samples from each pig in group A1 were drawn from the right external jugular vein at 4 days prior to T. britovi experimental infection (−4) and at 3, 6, 9, 13, 15, 17, 20, 24, 27, 29, 36, 41, 45, 51, 55, 59 and 62 days post infection (dpi). Blood samples from pigs in group A2 were collected in the same way at 5 days prior to the infection and at 6, 13, 20, 30, 36, 45, and 62 dpi, and then they were designated for other serological studies as a part of broader research project conducted by our laboratory. During the experiment, pigs were housed in separate units with free access to water. The pigs were fed twice daily. The feed was prepared at an experimental farm of the University of Life Sciences in Lublin and was based on wheat (50%) and barley (35%) supplemented (15%) with protein concentrate, vitamins and minerals.
Larval recovery and counting
Pigs were sacrificed 62 days after experimental inoculation by using a combination of intramuscular administration of azaperone (2mg/kg) followed by intravenous injection of sodium pentobarbital (40 mg/kg). The muscle larvae intensity was determined by the digestion procedure stipulated by European Commission Regulation EU 2015/1375 . The samples intended for the digestion assay were collected from the diaphragm pillars (crura diaphragmatica), tongue (lingua), and the following muscles or muscle groups from the left and right sides of each carcass: the masseter (M. masseter), lateral and medial pterygoid (M. pterygoideus lateralis et medialis), intercostals (Mm. intercostales), abdomen (Mm. abdominis), back (M. longissimus thoracis), neck (Mm. colli), sublumbar (M. psoas major et minor), triceps brachii (M. triceps brachii), femoral biceps (M. biceps femoris), forelimb extensors (Mm. extensores antebrachii), forelimb ﬂexors (Mm. flexores antebrachii), crus extensors (Mm. extensores cruris) and crus ﬂexors (Mm. flexores cruris). The diaphragm pillars, tongue and masseters were digested entirely. The remaining muscles of total weight under 50 g were digested entirely, whereas those over 50 g were cropped to form 50 g specimens and then digested. The number of muscle larvae per gram of muscle tissue (lpg) was presented as an average calculated separately for group A1 and group A2 and each muscle examined.
The serological analyses described below were performed for the group of pigs infected with 5000 ML of T. britovi (group A1). The serological results from pigs infected with 3000 ML of T. britovi (group A2) generated by using an in-house ELISA based on T. spiralis and T. britovi ML ES antigens and a PrioCHECK® Trichinella Ab ELISA were described in detail in our previous paper as a part of broader research cycle .
Commercial ELISA tests for anti-T. britovi IgG detection
Serum levels of specific IgG antibodies against Trichinella ML ES antigens were determined using three different commercial diagnostic ES ELISA kits which are available on the European Union market: the PrioCHECK® Trichinella Ab (ThermoFisher Scientific, Schlieren, Switzerland); the ID Screen® Trichinella Indirect Multi-species (ID.vet, Grabels, France) and the Pigtype® Trichinella Ab (Qiagen, Leipzig, Germany). All tests are based on Trichinella ES antigens. The PrioCHECK® Trichinella Ab ELISA is suitable only for the examination of serum or meat juice samples from pigs, and peroxidase -labeled anti-pig antibody serves as a secondary antibody in this kit. The ID Screen® Trichinella Indirect Multi-species and Pigtype® Trichinella Ab ELISAs allow testing swine and other species of animals, and anti-Trichinella antibodies are detected by a multi-species horseradish peroxidase (HRP) conjugate. The results of the PrioCHECK® Trichinella Ab and Pigtype® Trichinella Ab tests are interpreted as positive or negative only. The design of the ID Screen® Trichinella Indirect Multi-species ELISA allows doubtful results to be obtained as well. All analyses were performed according to the manufacturers’ instructions. The sera from uninfected and T. britovi-infected pigs and the controls (from the ELISA kits) were tested in duplicate; the final result was the mean of two measurements of optical density (OD) for each serum analyzed. All ELISA kits met the validation acceptance criteria which were set by the producer. Interpretation of the results for each particular ELISA kit was performed as follows:
PrioCHECK® Trichinella Ab: the results were graded by calculating the percentage of positivity (P/P) value according to the following formula:
Serum samples with a P/P value equal to or over 15% (P/P ≥ 15%) were classified as positive (trichinellosis detected), while those with P/P values lower than 15% (P/P ˂ 15%) were considered negative (trichinellosis not detected).
Pigtype® Trichinella Ab ELISA: the results were categorized by calculating the sample-to-positivity (S/P) value according to the following formula:
Serum samples with S/P value equal to or over 0.3 (S/P ≥ 0.3) were regarded as positive (trichinellosis detected), while those with S/P values lower than 0.3 (S/P ˂ 0.3) were noted as negative (trichinellosis not detected).
ID Screen® Trichinella Indirect Multi-species ELISA: the results were judged by calculating the S/P % value according to the following formula:
Serum samples with S/P % value equal to or over 60% (S/P% ≥ 60%) were logged as positive (trichinellosis detected), while those with S/P % values equal or lower than 50% (S/P% ≤ 50%) were taken to be negative (trichinellosis not detected). Serum samples with S/P % values lower than 60% but greater than 50% (50% ˂ S/P% ˂ 60%) were interpreted as doubtful. When doubtful results were obtained, the serum samples were retested and the results yielded after the second test were accepted for further analysis.
Production of the T. britovi ML ES antigen and Western blot
The T. britovi ML ES antigen was produced as described previously [33, 36] and kept frozen at −70 °C. Briefly, T. britovi ML were washed with Roswell Park Memorial Institute medium (RPMI)-1640 and resuspended at 5000 ML/ml in RPMI-1640 supplemented with 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 200 mM L-glutamine, 100 mM Na-pyruvate and 100 units each of penicillin and streptomycin. Larvae were then incubated in a T-75 culture flask in 5% CO2 at 37°C for 18 hours. Following incubation, the medium containing ML ES products was filtered through a 0.22 μm membrane and concentrated by lyophilization. The protein concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Concentrate in 100 µg amounts was loaded onto 12% acrylamide separating gels. Electrophoresis by SDS-PAGE was run using a Mini-PROTEAN Tetra Cell electrophoresis chamber (Bio-Rad Laboratories, Hercules, CA, USA) at 180 V for approximately 1 hour. PageRuler Unstained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) was loaded onto each gel as a weight marker. Fragments of gel were stained with a PlusOne Silver Staining Kit (GE Healthcare, Waukesha, WI, USA) according to the manufacturer’s protocol. The unstained gels were also used for immunoblotting. The ES antigens of T. britovi that had been separated on gels were transferred to nitrocellulose membranes (BioRad) through a wet transfer system (BioRad) at 95 V for 1 hour. Subsequently, the membranes were blocked with Pierce Protein-Free T20 (TBS) Blocking Buffer (Thermo Fisher Scientific) for 1 h at room temperature and then cut into strips and incubated overnight at 4 °C with a set of T. britovi-infected pigs’ sera diluted 1:100. The following serum samples were used in immunoblot analysis: pig sera from the day when the results generated by commercial ELISAs indicated seroconversion, pig sera obtained one sampling time point earlier than the day of seroconversion detected by the ELISA tests, and pig serum samples obtained on day 62 post infection. The secondary antibody, goat anti-pig IgG conjugated to HRP (Sigma-Aldrich, St. Louis, MO, USA), was diluted 1:20 000 in PBS with 5% skimmed milk, added to each strip, and incubated for 1 h at room temperature. The immunoreactive bands were visualized on membrane strips using Sigmafast 3,3’-Diaminobenzidine tablets (Sigma-Aldrich) according to the manufacturer’s protocol. The gel and the membranes were scanned with the ChemiDoc™ MP Imaging System (Bio-Rad). The immunoblots were performed using two replicate samples. Serum samples collected from pigs 4 days prior to T. britovi infection were used as negative controls.
Data are presented as means and standard deviations. Medians and interquartile ranges are also displayed, if necessary.
The obtained data were investigated for normality with a Shapiro–Wilk test and for homogeneity of variances with Levene’s test. In order to compare the intensity of infection in the left and right sides of the individual muscles or muscle groups within each group of pigs, Student’s t-test (all assumptions met) or a Mann–Whitney U-test (for non-normally distributed data) was used. In order to compare the intensity of infection in the 15 different muscles within each group of pigs, one-way analysis of variance (ANOVA) with a post-hoc Tukey’s test (all assumptions met) or one-way ANOVA with Welch’s correction followed by a Games–Howell post-hoc test (for data with unequal variances) were applied. Comparison of the intensity of T. britovi ML infection in particular muscles and the entire carcasses between the group of pigs infected with 3000 and the group infected with 5000 ML of T. britovi was performed using a Mann–Whitney U-test (where numbers of animals in groups were unequal).
The differences in anti-T. britovi IgG level during the course of infection within each ELISA test performed in the group of pigs infected with 5000 ML of T. britovi were calculated using repeated measures analysis of variance (ANOVA) with a post-hoc Bonferroni test. Previously, sphericity had been assessed using Mauchly’s test. If the assumption of sphericity was violated, the Greenhouse–Geisser correction (Greenhouse–Geisser ԑ ˂ 0.75) or Huynh–Fieldt correction (Greenhouse–Geisser ԑ ˃ 0.75) were introduced. Comparison of the anti-T. britovi level on particular days after infection between the group of pigs infected with 3000 and the group infected with 5000 ML of T. britovi was performed by using a Mann–Whitney U-test (where numbers of animals in groups were unequal).
Correlation between the intensity of T. britovi ML infection in the muscles of pigs infected with 5000 ML of T. britovi and the level of anti-T. britovi IgG on days 41, 51 and 61 post infection was calculated using Pearson’s correlation coefficient.
Finally, correlations between the ELISA index values (PP, SP% and SP) generated by the PrioCHECK® Trichinella Ab and ID Screen® Trichinella Indirect Multi-species ELISAs, PrioCHECK® Trichinella Ab and Pigtype® Trichinella Ab ELISAs and ID Screen® Trichinella Indirect Multi-species and Pigtype® Trichinella Ab ELISAs for each pig infected with 5000 ML of T. britovi and the entire experimental model were calculated using Spearman’s correlation coefficient.
For all these analyses, the level of significance was set at P < 0.05. All statistical calculations were performed with Statistica (StatSoft, Kraków, Poland) and SPSS (SPSS Inc, Chicago, IL, USA) software.