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Insight into Trichinella britovi Infection in Pigs: Effect of Various Infectious Doses on Larvae Density and Spatial Larvae Distribution in Carcasses and Comparison of the Detection of Anti-T. britovi IgG of Three Different Commercial ELISA Tests and Immunoblot Assay

There is limited information available on the Trichinella britovi (T. britovi) muscle larvae (ML) distribution in pig muscle and the humoral immune response of pigs infected with moderate doses of this parasite; therefore, this study investigated the infectivity of a Polish strain of T. britovi for pigs, the antibody response of this host to various doses of T. britovi, and the efficiency of three different commercial ELISA kits and an immunoblot assay at detecting anti-T. britovi IgG. No significant differences in terms of the infection level of particular muscles or of whole carcasses between pigs infected with 3000 and those infected with 5000 ML of T. britovi were observed. The highest intensity of T. britovi infection was reported in the diaphragm pillars. The larvae of T. britovi showed homogeneous distribution with respect to the muscle side. Statistically, specific IgG antibodies against excretory–secretory (ES) antigens of Trichinella ML were first detected by all ELISA protocols on day 36 post infection; however, individual pig results showed some differences between the three tests applied. A significant increase in the level of anti-T. britovi IgG was observed between days 36 and 41 post infection, and from day 45 until day 62 after T. britovi infection, production of these antibodies reached its plateau phase. No positive correlation was found between the anti-T. britovi IgG level and the larvae density in 15 different muscles. Sera of T. britovi-infected pigs showed reactivity with T. britovi ML ES antigens of 62, 55, and 52 kDa. The results provide novel information on spatial larvae distribution in muscles and the humoral immune response of pigs exposed to two different doses of a Polish strain of T. britovi, extend knowledge on serological diagnostic tools which may be introduced in veterinary practice for the detection of T. britovi infections in pig production, and offer practical solutions for meat hygiene inspectors in the field at sampling sites when examining pig carcasses for Trichinella.


Introduction
Trichinellosis is an emerging and re-emerging worldwide foodborne zoonotic disease caused by the consumption of raw or undercooked meat infected with parasitic nematodes of the genus Trichinella. Globally, cases and outbreaks of human trichinellosis are primarily associated with the consumption of infected meat and/or meat products derived from domestic pigs and wild boars [1].
The distribution of T. britovi ML in particular muscles and muscle sides of pigs experimentally infected with 3000 and 5000 ML of T. britovi is shown in Tables 1 and 2. Muscle larvae of T. britovi were isolated from all experimentally infected pigs and the presence of the larvae was confirmed in all muscles analyzed. The average larval load in the entire carcasses of pigs infected with 3000 ML was 13.84 lpg, while in pigs infected with a dose of 5000 ML it was 14.34 lpg.
Muscle larvae of T. britovi showed a homogeneous distribution with respect to the muscle side, since no statistically significant differences were found between the left and right sides of the 13 different laterally paired muscles in both experimental groups of pigs (Student's t-test, p > 0.05). The highest intensity of T. britovi ML infection was reported in the diaphragm pillars, and, with the exception of the tongue, it significantly differed from the intensity of infection of the remaining 13 muscles in the group of pigs infected with 3000 ML (one-way ANOVA followed by Tukey's post hoc test, p < 0.0001) or 5000 ML of T. britovi (Welch's ANOVA followed by Games-Howell's post hoc test, p < 0.0001).
There were no significant differences in the infection level of particular muscles or entire carcasses between pigs infected with 3000 and those infected with 5000 ML of T. britovi when a Mann-Whitney U-test was applied (p > 0.05) ( Table 3). Table 1. Distribution and intensity of T. britovi larvae infection (lpg) in muscles of pigs (n = 6) experimentally infected with 3000 muscle larvae of T. britovi. SD-standard deviation; IQR-interquartile range. 1 The lpg value of each left muscle was compared with the lpg value of each right muscle. 2 The lpg value of diaphragm pillars was compared with the lpg value of the remaining 14 analyzed muscles (left and right combined). † Calculated using Student's t-test. ‡ Calculated using Mann-Whitney U-test. § Calculated using one-way ANOVA followed by Tukey's post hoc test. * The lpg value of a given muscle differs statistically from the lpg value of the diaphragm pillars. Table 2. Distribution and intensity of T. britovi larvae infection (lpg) in muscles of pigs (n = 8) experimentally infected with 5000 muscle larvae of T. britovi. SD-standard deviation; IQR-interquartile range. 1 The lpg value of each left muscle was compared with the lpg value of each right muscle. 2 The lpg value of diaphragm pillars was compared with the lpg value of the remaining 14 analyzed muscles (left and right combined). † Calculated using Student's t-test. ‡ Calculated using Mann-Whitney U-test. § Calculated using Welch's ANOVA followed by Games-Howell's post hoc test. * The lpg value of a given muscle differs statistically from the lpg value of the diaphragm pillars.

Detection of Anti-T. britovi IgG in Pigs Using Three Commercial ELISA Kits
The specific IgG antibody response against the ES antigens of Trichinella ML measured using different ELISA commercial kits in the group of pigs experimentally infected with 5000 ML of T. britovi is shown in Table 4, and a serological evaluation of individual T. britovi-infected pigs is presented in Table 5. Statistically, all three ELISA tests first detected IgG antibodies against the T. britovi ML ES antigens (PP ≥ 15% for the PrioCHECK ® Trichinella Ab ELISA (ThermoFisher Scientific, Schlieren, Switzerland); S/P% ≥ 60% for the ID Screen ® Trichinella Indirect Multi-species ELISA (ID Vet, Grabels, France); and S/P ≥ 0.3 for the Pigtype ® Trichinella Ab ELISA (Qiagen, Leipzig, Germany)) on day 36 post infection (pi) ( Table 4). Among eight infected pigs, four (pigs no. 1, 5, 6, and 8) were first found to be positive by all three tests on the same day after infection (36 or 41 dpi) ( Table 5). In the case of another two pigs (pigs no. 3 and 4), the PrioCHECK ® Trichinella Ab and ID Screen ® Trichinella Indirect Multi-species assays gave a positive result on the same day after inoculation (45 and 29 dpi, respectively), whereas the Pigtype ® Trichinella Ab ELISA classified these pigs as positive one blood draw earlier (41 and 27 dpi, respectively) ( Table 5). Consistency between the results given by the PrioCHECK ® Trichinella Ab test and the Pigtype ® Trichinella Ab test was observed in pigs no. 2 and 7, which were found to be positive on days 36 and 41 pi, respectively, while the ID Screen ® Trichinella Indirect Multi-species ELISA classified these pigs as positive one blood draw later (41 and 45 dpi, respectively) ( Table 5). In addition, two pig serum samples (from pigs no. 2 and 7 collected on days 36 and 41 pi, respectively) which gave doubtful results in the ID Screen ® Trichinella Indirect Multi-species ELISA with relatively high S/P% values (51.770 and 56.163%) were classified as positive by the other two tests on those days. Moreover, serum samples obtained from five pigs (pigs no. 1, 2, 3, 4, and 5) on days 27, 29, 36, and 41 pi were classified either by the PrioCHECK ® Trichinella Ab or Pigtype ® Trichinella Ab assay as negative, but their PP or S/P value reached a level close to the cut-off (i.e., PP = 13.057%, 14.248%, and 14.810%, respectively for pigs no. 3, 4, and 5 analyzed by the PrioCHECK ® Trichinella Ab assay and S/P = 0.287, 0.288, and 0.288 for pigs no. 1, 2, and 3 analyzed by the Pigtype ® Trichinella Ab kit). As expected, all these pigs, including those that generated doubtful results in the ID Screen ® Trichinella Indirect Multi-species ELISA, were detected as positive at the next blood collection time point (i.e., 2, 4, 5, or 7 days later) by all the ELISA kits applied.

Variation in the Level of Anti-T. britovi IgG in the Serum of Pigs Infected with T. britovi
In the group of pigs experimentally infected with 5000 ML of T. britovi, a statistically significant increase in the level of anti-Trichinella IgG was observed between days 36 and 41 post infection when all three assays were performed. The significance is demonstrated for the ID Screen ® Trichinella Indirect Multi-species ELISA results by p < 0.0001 in Mauchly's sphericity test, p = 0.007 in Greenhouse-Geisser's correction, p = 0.005 in Huynh-Feldt's correction, p = 0.01 by lower bound estimation, and p = 0.002 in the Bonferroni post hoc test. For the Pigtype ® Trichinella Ab ELISA results, the corresponding values are p < 0.001 in Mauchly's sphericity test, p < 0.0001 in Greenhouse-Geisser's correction, p < 0.0001 in Huynh-Feldt's correction, p = 0.004 by lower bound estimation, and p < 0.0001 in the Bonferroni post hoc test. The PrioCHECK ® Trichinella Ab ELISA results yielded p < 0.0001 in Mauchly's sphericity test, p = 0.0006 in Greenhouse-Geisser's correction, p = 0.0002 in Huynh-Feldt's correction, p = 0.005 by lower bound estimation, and p = 0.006 in the Bonferroni post hoc test (Table 4). In general, from day 45 until the end of the experiment, i.e., day 62 after T. britovi infection, the production of these antibodies reached a plateau phase and persisted at a constant high level without any significant differences as measured by all three ELISA tests (Table 4).

Correlation between Number of Recovered Larvae (lpg) in Particular Muscles of Experimentally Infected Pigs and Anti-T. britovi IgG Level
No positive correlation was found between larvae density in 15 different muscles and OD level measured by three different ELISA kits on days 41, 51, and 62 post infection, except for one partial example at 41 dpi when such a correlation was shown between the intensity of T. britovi ML infection in forelimb extensors and the OD value generated by the Pigtype ® Trichinella Ab assay (Table 6). Table 6. Correlation between intensity of T. britovi ML infection (lpg) in muscles of pigs experimentally infected with 5000 ML of T. britovi and anti-T. britovi IgG level (OD) measured by three different commercial ELISA kits.  Table 7 summarizes the correlations between ELISA index values (PP, S/P%, and S/P) generated by three different ELISA kits in individual pigs experimentally infected with T. britovi as well as the entire experimental model. In all eight pigs infected with 5000 ML of T. britovi, a significant (p < 0.05) strong or very strong positive correlation with R correlation coefficients ranging from 0.725 to 0.895 and from 0.907 to 0.963, respectively, was found between the ELISA index values generated by the PrioCHECK ® Trichinella Ab ELISA (PP) and the ID Screen ® Trichinella Indirect Multi-species ELISA (S/P%); PrioCHECK ® Trichinella Ab ELISA (PP) and Pigtype ® Trichinella Ab ELISA (S/P); and ID Screen ® Trichinella Indirect Multi-species ELISA (S/P%) and Pigtype ® Trichinella Ab ELISA (SP). In addition, a high positive correlation with R correlation coefficient ranging from 0.903 to 0.912 was found between all three tests for the entire experimental model (Table 7).

Recognition of the T. britovi ML ES Antigens by Anti-T. britovi IgG in Western Blot
The electrophoretic pattern of the T. britovi ML ES antigen is shown in Figure 1, and the Western blot patterns of reactivity of the ES antigens with sera collected from individual pigs infected with 5000 ML of T. britovi at various time points after infection are presented in Figure 2. T. britovi ML ES antigens showed bands of molecular weights in the range of 20 to 95 kDa with the most intense signal between 30 and 48 kDa ( Figure 1). Anti-T. britovi IgG antibodies in individual pig serum samples were first detected by Western blot on day 29 (in pig no. 4), 36 (in pigs no. 1, 2, 6, and 8), or 41 (pigs no. 3, 5, and 7) post infection. Serum samples from infected pigs recognized T. britovi ML ES antigens of molecular weights of 62, 55, and 52 kDa. The immunoblot indicated that all the protein bands observed on the day of seroconversion were also observable on successive days until the end of the experiment. Representative patterns also showed the differences in signal intensity and relative migration value of T. britovi ML ES reactive proteins between serum samples from individual pigs on particular days after infection. In general, all recognized bands in all experimentally infected pigs were more intense in the late phase of infection (62 dpi) than on the day of seroconversion. Sera collected from pigs before experimental infection did not recognize Trichinella protein bands.
of molecular weights of 62, 55, and 52 kDa. The immunoblot indicated that all the protein bands observed on the day of seroconversion were also observable on successive days until the end of the experiment. Representative patterns also showed the differences in signal intensity and relative migration value of T. britovi ML ES reactive proteins between serum samples from individual pigs on particular days after infection. In general, all recognized bands in all experimentally infected pigs were more intense in the late phase of infection (62 dpi) than on the day of seroconversion. Sera collected from pigs before experimental infection did not recognize Trichinella protein bands.

Discussion
Trichinella britovi is widely distributed among wildlife and several studies have confirmed that this species predominates in red foxes, raccoon dogs, and wolves in Poland [9,[22][23][24]. The parasite has also been found in martens and badgers living in the northeast regions of this country [25], which only proves its circulation in a wide range of hosts. Moreover, according to the Polish General Inspectorate, Trichinella is regularly detected in wild boars [26] and, as recent data have shown, almost 20% of all Trichinella-infested Polish wild boars were found to be infected with the T3 genotype [9,10]. Therefore, it

Discussion
Trichinella britovi is widely distributed among wildlife and several studies have confirmed that this species predominates in red foxes, raccoon dogs, and wolves in Poland [9,[22][23][24]. The parasite has also been found in martens and badgers living in the northeast regions of this country [25], which only proves its circulation in a wide range of hosts. Moreover, according to the Polish General Inspectorate, Trichinella is regularly detected in wild boars [26] and, as recent data have shown, almost 20% of all Trichinella-infested Polish wild boars were found to be infected with the T3 genotype [9,10]. Therefore, it should be stated that such high infection pressure exerted by environmental T. britovi poses a significant risk to pigs, particularly those in small backyard/outdoor breeding systems that are typically operated to poor zoohygienic standards. If the misconduct of hunters, who are often also the pig breeders, and the feeding of pigs with the offal and remains of hunted animals are included, the risk of T. britovi pig infection rises higher. Therefore, to better understand the infectivity of T. britovi for native Polish pig breeds and the humoral immune response accompanying the invasion, as well as to determine the usefulness of various commercial ELISA kits in the diagnostics of swine infection induced by various doses of T. britovi, we decided to extend our previous research [18] and provide deeper insight into T. britovi infection mechanisms in pigs. To achieve these goals, we used native Polish pig breeds and a Polish strain of T. britovi that had been isolated from a naturally infected wild boar and passaged at least once through a domestic pig before this experiment. Our results showed that in the group of pigs infected with 3000 and in the group infected with 5000 ML of T. britovi, the diaphragm pillars were the most heavily parasitized muscle and, with the exception of the tongue, the infection level in the diaphragm pillars was significantly higher than the level in the other 13 muscles analyzed. This shows that the mechanism of T. britovi larvae deposition in swine muscles is rather similar to that of T. spiralis, and the diaphragm pillars should be considered the predilection site during official pig carcass examination for Trichinella. There are only three comprehensive studies on the distribution of T. britovi ML in pig muscles for comparison purposes. Nöckler et al. [16] showed that in pigs experimentally infected with 200, 1000, or 20,000 ML of T. britovi, the highest larvae burden was observed in the diaphragm. In contrast, in the studies performed by Pozio et al. [17], the tongue was the most highly parasitized muscle in swine experimentally infected with 10,000 ML of T. britovi. Other studies conducted by Kapel et al. [14] showed that, in pigs infected with 10,000 ML of T. britovi, the tongue tip presented the highest larvae density out of 18 muscles analyzed. Recent changes to the Regulation (EU) 1375/2015 [27] introduced the ISO 18743:2015 standard, in which the reference detection method of Trichinella larvae in meat as well as the sampling procedure are described in detail [28]. According to this document, in the case of official examination of domestic pigs for Trichinella, muscle samples should be taken from the diaphragm pillars or masseters. Our study results, however, showed that in pigs infected with 3000 ML and 5000 ML of T. britovi, the mean larval density in the masseters amounted to, respectively, 51.93% and 36.11% of that observed in the diaphragm pillars. In the studies performed by Nöckler et al. [16], this percentage ranged from 18.50 to 60.74 and in the research conducted by Pozio et al. [17] it was 70.59. It is difficult to speculate whether the same proportions would be observed with the intensity of infection of T. britovi in the diaphragm pillars at the level of the sensitivity of the artificial digestion method (i.e., one larva per 1 g of diaphragm pillars); however, Prost and Nowakowski [29] have shown that in pigs in which the intensity of infection in the diaphragm pillars ranged from 0.91 to 1.18 lpg of T. spiralis, the mean larval density in masseter muscles was only 21.2% of that observed in the pillars. This may suggest that the masseter muscles should not always be considered as equivalent to diaphragm pillars, and that the mass of the samples taken from masseters for official pig carcass examination should be proportionally increased, particularly when the maximum of 100 carcasses are tested in one batch and each animal is represented by a sample mass of 1 g. Furthermore, in all 13 laterally paired pig muscles analyzed, T. britovi showed a homogeneous distribution in terms of both anatomical location and the muscle side. Our study also showed that despite the use of two different doses of T. britovi, no exponential increase of larvae burden was observed in entire infected pig carcasses. In general, the Trichinella muscle larvae density correlates with the ingested dose of the parasites. However, such interdependencies are most apparent when the magnitude of the infective doses vary greatly and fall in different categories such as low (100-300 ML), moderate (2000-5000 ML), or high (10,000-20,000 ML) in terms of total number of larvae used for the infection. In other words, the use of doses of various categories induces significant differences in the muscle larval load, but various doses from one category do not always produce such results. The similar infection level in pigs infected with different doses of T. britovi detected in this study may be explained by a stronger local immune response elicited by the dose of 5000 ML and, as a consequence, more rapid expulsion of worms from the swine intestines. In the global literature, there are few data available that describe how different doses of T. britovi affect the larvae density in pig muscles. Nöckler et al. [16], for instance, showed that despite the use of two different infective doses, i.e., 200 and 1000 ML of T. britovi, the larvae burden in infected pig carcasses was rather similar and amounted to 1.82 and 0.98 lpg, respectively. In this context, important information was also provided by Franssen et al. [30], who showed that in rats experimentally infected with T. spiralis, increasing doses resulted in increasing numbers of recovered larvae only in a non-linear manner and doses above 10,000 ML even resulted in decreasing larvae density.
Another aim of the current work was to assess the antibody response in pigs infected with 5000 ML of T. britovi by using various commercial ELISA tests. Statistically, specific IgG antibodies against the ES antigens of Trichinella ML were first detected by all ELISA protocols on day 36 pi; however, individual pig results differed between the three tests applied. It is well known that the sensitivity of the serological tests depends on the time that has elapsed from the infection. The sensitivity of the PrioCHECK ® Trichinella Ab and Pigtype ® Trichinella Ab ELISAs was 62.5% (95% CI: 24.49-91.48) on day 36 pi, while for ID Screen ® Trichinella Indirect Multi-species it was 50% (95% CI: 15.7-84.30). The highest sensitivity on day 41 pi was shown by the Pigtype ® Trichinella Ab ELISA (100%, 95% CI: 63.06-100), while the PrioCHECK ® Trichinella Ab and ID Screen ® Trichinella Indirect Multi-species assays showed 87.5 (95% CI: 47.35-99.68) and 75% (95% CI: 34.91-96.81), respectively. The variations in individual pig classification between tests as positive, doubtful, or negative may be associated with the differences in the ELISA reagents, i.e., the methods of the ES antigen production, the determined cut-off point, or the type of secondary antibody used. In this regard, our previous study showed that an in-house ELISA based on T. britovi ML ES antigens was able to detect anti-Trichinella IgG in a group of pigs experimentally infected with 3000 ML of T. britovi one blood sampling earlier (6 days) than both the commercial PrioCHECK ® Trichinella Ab ELISA and an in-house T. spiralis ML ES-antigen ELISA. In addition, from a practical point of view, special attention should be paid to doubtful results, or, if the ELISA is construed to yield only positive (specific anti-Trichinella IgG detected) or negative (specific anti-Trichinella IgG not detected) results, consideration should be given to the serum OD values that are close to the cut-off and, in particular, those that reach 90% of the cut-off value and above. In such a scenario, the pigs should be re-tested and this examination may be performed over a very short time interval, e.g., 2-5 days. The wisdom of treating certain results with caution is also supported by our trial study in which serum samples from 100 healthy fatteners weighing 80-100 kg and aged 6-9 months without Trichinella larvae in 1 g muscle generated ELISA index values of 15, 4.43, and 13.67% of the cut-off value in the PrioCHECK ® Trichinella Ab, ID Screen ® Trichinella Indirect Multi-species, and Pigtype ® Trichinella Ab ELISAs, respectively (data not shown). It should also be noted that the differences in the detection of T. britovi infection between the ELISA tests in individual pigs concerned no more than a single blood collection time point. As serum samples were drawn at 2-6-day intervals, it can be assumed that the ELISA kit that generated a negative result, while the other ELISA kit produced a positive one, would have yielded a positive result even on the next day of blood collection. Overall, the correlations between ELISA kits were high for both individual infected pigs and the entire experimental model. Our research results also fill a gap in the literature, as, in the few existing studies, most authors used high doses of T. britovi to examine the immune response of experimentally infected pigs. In pigs infected with high doses of T. britovi (10,000-20,000) of various larval densities (2.23-123.1), seroconversion was observed between day 21 and 42 after inoculation [14,16,17,31]. At the low end of the dose scale, pigs infected with 200 or 1000 ML (three pigs per group) of T. britovi seroconverted at 60 dpi [16].
Finally, our study showed that a statistically significant increase in the level of anti-T. britovi IgG was observed between days 36 and 41 post infection and that from day 45 until the end of the experiment, i.e., day 62 after T. britovi infection, production of these antibodies reached its plateau phase and persisted at a constant high level without any significant differences as measured by all three ELISA tests. Similar kinetics in antibody production were observed in our previous experiment, when pigs were infected with 3000 ML of T. britovi [18]. These results indicate that the anti-T. britovi IgG production kinetics are not dose-dependent in pigs infected with different doses in the range of 3000-5000 ML of T. britovi. Moreover, when these results are compared with our previous ones [18], there were no statistically significant differences in the level of anti-T. britovi IgG antibodies between the groups of pigs infected with a dose of 3000 and 5000 larvae on days 36 and 45 post infection. The day 36 PrioCHECK ® Trichinella OD values were 0.661 ± 0.190 and 1.034 ± 0.801 for pigs experimentally infected with 3000 and 5000 ML of T. britovi, respectively, and a Mann-Whitney U test gave a p value of 0.747. The day 45 values after the same ELISA were 1.404 ± 0.278 and 2.038 ± 0.653 for pigs experimentally infected with 3000 and 5000 ML of T. britovi, respectively, and a Mann-Whitney U test gave a p value of 0.081. Nevertheless, anti-T. britovi IgG antibody level differences were found on day 62 pi, when the ELISA mentioned previously returned OD values of 1.627 ± 0.358 and 2.434 ± 0.481 for pigs experimentally infected with 3000 and 5000 ML of T. britovi, respectively, and a Mann-Whitney U test produced a p value of 0.01. This phenomenon is difficult to explain, because the infection levels in the two groups of pigs did not differ significantly. In addition, the differences in absolute anti-T. britovi IgG level between the group of pigs infected with 3000 and the group infected with 5000 ML of T. britovi were found only in the late parenteral phase (62 dpi) of the infection, which, in fact, also excludes any dose effect. It can therefore be assumed that there are endogenous factors which regulate the amount of protein products excreted or secreted by the parasite into the host's bloodstream in an unknown manner and that, so regulated, these protein products stimulate the host's immune system with varying intensity. This is also supported by the lack of correlation between the larvae density in different muscles of the infected swine and the anti-T. britovi level on particular days after infection.
In general, the Western blot showed high compliance with the PrioCHECK ® Trichinella Ab and Pigtype ® Trichinella Ab ELISA results. In the cases of three pigs, the Western blot detected anti-T. britovi IgG one blood collection earlier than the ID Screen ® Trichinella Indirect Multi-species ELISA kit. However, it should also be highlighted that in the case of these serum samples, the ID Screen ® Trichinella Indirect Multi-species assay generated doubtful results with relatively high ELISA index values. Moreover, our immunoblot results confirmed the differences in anti-T. britovi IgG level during the course of infection, which were observed in all three ELISA tests: the signal intensity of ES protein bands reacting with anti-T. britovi IgG varied and was significantly higher in the late phase of infection (62 dpi) than on the day of seroconversion. The swine sera reacted with T. britovi ML ES antigens with molecular weights of 62, 55, and 52 kDa. In fact, our results are partially in accordance with the comprehensive studies performed by Gómez-Morales et al. [32], who showed that Trichinella-infected pig sera recognized ES proteins with a three-band pattern of 64-72, 59-63, and 48-55 kDa. It should be noted, however, that all global studies on the use of Western blot for the detection of Trichinella infection in pigs were performed using T. spiralis ML ES antigens as well as serum samples collected from pigs infected with T. spiralis [32,33]; the results of this research have been discussed elsewhere [34]. It was also proved that all Trichinella species share some ES proteins and, as a consequence, an ES antigen obtained from one Trichinella species is able to detect infections induced by other Trichinella species and genotypes [31]; however, recently, Grzelak et al. [35] showed that sera from Trichinella-infected persons displayed some differences in terms of their specific band patterns when T. spiralis and T. britovi ML ES antigens were used. It was also found by using one-dimensional electrophoresis that the native T. spiralis ES antigen shows more protein bands than the T. britovi ML ES antigen [35]. However, on the basis of our results and studies conducted by Gómez Morales et al. [32], it should be stated that the use of T. britovi ML ES antigen and serum samples of pigs infected with T. britovi did not significantly affect the Western blot band pattern compared with the pattern generated by the serum of pigs infected with T. spiralis tested by using T. spiralis ES-WB. Our studies have also shown that the Western blot pattern of individual infected pigs was stable; ES proteins that reacted with anti-T. britovi IgG on the day of seroconversion were also visualized after 20-30 days (i.e., 62 days after infection). Likewise, there were no additional protein bands reacting with anti-T. britovi IgG in the late phase of infection (day 62 pi) which had not been observed on the day of seroconversion. This may indicate that the invasion phase in particular individuals infected with T. britovi has no effect on the protein band profile assessed by using one-dimensional electrophoresis and a Western blot based on ES antigens from the muscle larvae of T. britovi. Therefore, for the purpose of wide-scale method validation studies, in which a Western-blot pattern is established, it is sufficient to take the sample from particular individuals at one time point after infection, provided that seroconversion is known to have occurred.

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. [36]. Muscle larvae (ML) displaying motility were counted and then suspended in 30% gelatin blocks. Fourteen healthy young Puławska/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. The muscle larval burden was determined by the digestion procedure laid down in ISO 18743:2015 standard [28]. 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.  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.

Serological Analyses
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 [18].

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 that 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 that 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 (PP) value according to the following formula: Serum samples with a PP value equal to or over 15% (PP ≥ 15%) were classified as positive (specific anti-Trichinella IgG detected), while those with PP values lower than 15% (PP < 15%) were considered negative (specific anti-Trichinella IgG not detected).
Pigtype ® Trichinella Ab ELISA: the results were categorized by calculating the sampleto-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 (specific anti-Trichinella IgG detected), while those with S/P values lower than 0.3 (S/P < 0.3) were noted as negative (specific anti-Trichinella IgG 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 (specific anti-Trichinella IgG detected), while those with S/P% values equal or lower than 50% (S/P% ≤ 50%) were taken to be negative (specific anti-Trichinella IgG 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 [35,37] 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 Lglutamine, 100 mM Na-pyruvate, and 100 units each of penicillin and streptomycin. Larvae were then incubated in a T-75 culture flask in 5% CO 2 at 37 • C for 18 h. 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 Nan-oDrop 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 h. 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 h. 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. britoviinfected pig sera diluted 1:100. In order to examine the protein band pattern at different time of infection, 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 or two sampling time points 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.

Statistical Analysis
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) were 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 ε < 0.75) or the Huynh-Feldt correction (Greenhouse-Geisser ε > 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.

Conclusions
Our data indicate that an inoculation dose in the range of 3000-5000 ML of a Polish strain of T. britovi is sufficient to induce infection in pigs with an infection level that may pose a threat to public health and that enables this parasite to spread in swine herds. Our results also confirmed that the diaphragm pillars should be considered a predilection site for sampling during routine post-mortem examination of pigs for Trichinella. The results also showed that the muscle side sampled has no relevance for the intensity of T. britovi ML determined, and they imply that in cases of epidemiological study or limited sample availability it is sufficient to take a sample from one side of an individual muscle and/or muscle group to obtain reliable results. Within the range of the doses of 3000-5000 ML of T. britovi used for pig infection, there are no clear interdependencies between the dose and the larval density in the muscles and this suggests that higher doses are effective in inducing the immune system to reduce the number of newborn larvae or to remove the parasites from the intestines in a more efficient manner. Further, in pigs inoculated with doses of 3000-5000 ML of T. britovi, an anti-T. britovi specific IgG response appears on day 36 pi, reaches a plateau on day 45 pi, and remains stable until 2 months pi. It should also be concluded that in pigs infected with moderate doses of T. britovi, there are other factors driving the specific anti-T. britovi production than the dose and the density of the larvae in muscles, because there were no differences in the kinetics of antibody production over the 62-day period of infection nor in the absolute levels of antibodies on days 36 and 45 pi between the group of pigs exposed to the dose of 3000 and the group exposed to 5000 ML; neither was there any correlation between the lpg value and the absolute level of antibodies on individual days after inoculation. The detectability of T. britovi infection in individual pigs differed slightly between the commercial ELISA kits on days 36 and 41 pi, which may be due to differences in the production of the kits' ES antigens, the method of determining the cut-off value, or the type of secondary antibody used. Finally, we conclude that when an ELISA yields doubtful results or the generated ELISA index values are close to the cut-off level (90% of cut-off or above), the pigs must be retested; this can be carried out after a short time interval such as 2-5 days. A Western blot test based on T. britovi ML ES antigens confirmed the ELISA results, and the sera of T. britovi-infected pigs showed reactivity with T. britovi ML ES antigens of 62, 55, and 52 kDa.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.