Toxoplasmosis is a parasitic infection caused by the intracellular protozoa Toxoplasma gondii. This parasite has a complex lifecycle and affects its definitive host as well as various intermediate hosts, among which domestic and wild animals and humans (Dubey, 2010). During its distinct developmental phases, the parasite manifests itself as a tachyzoite, a bradyzoite or a sporozoite in the oocyst. The sexual multiplication proceeds only within the definitive hosts (domestic or wild members of the family Felidae), wherein five morphologically different generations of the gamonts develop in the enterocytes, leading to formation of the gametocytes. Following the fertilization of the micro- and the macrogamete and the rupture of the infected cell, the unsporulated oocysts are discharged into the intestinal lumen. The final hosts are responsible for the extensive shedding of oocysts in the environment (Dubey, 1995; Afonso et al., 2008). The sporulated oocysts containing eight sporozoites show a high resistance to different environmental factors and under convenient circumstances may remain infectious for 1.5 years (Dubey, 2010). On average, the final host sheds at least one million oocysts in the acute phase of the infection, resulting in a massive contamination of the environment. This explains the persistence of the parasite in wild reservoir and livestock (Black and Boothroyd, 2000; Afonso et al., 2008; Innes, 2010; Opsteegh et al., 2016). The sporulated oocysts release the sporozoites upon ingestion by the intermediate host, followed by a differentiation to tachyzoites, several cell divisions in the enterocytes, and eventually dissemination to the peripheral tissues. There, the fast-multiplying tachyzoites convert into tissue cysts with the slowly dividing bradyzoites, and remain there as the dormant stage of the infection. The predation of the intermediate host or its tissues by the Felidae leads to the new sexual reproduction cycle, in which the bradyzoites transform back to tachyzoites and merozoites (Dubey, 1995; Afonso et al., 2008).

Several infection routes have been described for the different hosts of T. gondii; the majority of the herbivorous animals acquires the infection through ingestion of water or plants contaminated with oocysts. The predation of other mammals or birds or ingestion of the placenta and/or the aborted offspring of small ruminants facilitates the transmission of toxoplasmosis to carnivores and omnivores (Black and Boothroyd, 2000; Innes, 2010). In humans, foodborne toxoplasmosis mainly results from the consumption of raw or undercooked meat from infected animals, like domestic pigs. The global prevalence of this parasite includes one third of the human population and as such represents one of the most common parasitic zoonosis worldwide (Tenter et al., 2000; Ajzenberg et al., 2002; Aspinall et al., 2002; Bosch-Driessen et al., 2002; Kijlstra and Jongert, 2008; Innes, 2010; Robert-Gangneux and Dardé, 2012; Torgerson and Mastroiacovo, 2013). Consequently, infection in human has a severe short- and long-term impact, ranging from congenital or adult toxoplasmosis in healthy individuals to T. gondii-induced encephalitis in immune-compromised patients. In particular congenital toxoplasmosis in seronegative pregnant women has a very severe clinical relevance for the fetus, since the infection may result in abortion, intracranial calcifications, mental retardation or chorioretinitis in the newborn (Peyron et al., 2017). Finally, acquired toxoplasmosis has recently been associated with an increase in suicide rates or Parkinson's disease (Israelski and Remington, 1993; Holland, 2003; Lester, 2010; Miman et al., 2010; Wang et al., 2017). Therefore, numerous preventive measures are recommended in an attempt to decrease the global infection burden in the human population. The commonly applied precautions include hygienic processing of water and meat, such as boiling of surface water and avoiding the consumption of raw or undercooked meat, as only long term freezing at −12°C or baking above 67°C can effectively deactivate tissue cysts. Especially, pork is often consumed undercooked and is processed in many other meat products, reaching on average 300 consumers per pig (Fehlhaber et al., 2003; Belluco et al., 2016). Additionally, direct contact with contaminated soil, plants or cat feces should be avoided by wearing gloves when gardening or emptying the litter box, and by thoroughly washing fresh vegetables and fruits. Providing clear information on these preventive measures to seronegative pregnant women, in combination with a frequent serological screening to detect the acute infection during pregnancy, has proven to be successful in decreasing the infection rate (Breugelmans et al., 2004; Peyron et al., 2017). In livestock, the preventive measures are predominantly focused on strictly indoor housing, preventing access for cats, rodent control and the appropriate carcass disposal (Jones and Dubey, 2012; Robert-Gangneux and Dardé, 2012). Whereas environmental contamination as well as the prevalence of toxoplasmosis in sheep is overall high, the situation in regard to pigs may vary per country or the farm management. Consequently, the lack of uniform validation of the variety of serological assays, and the missing gaps in the correlation between the persistence of antibodies and parasite in pork are still to be improved. Nevertheless, the estimated average prevalence in the pig population seems to be very low in European countries (2.2%) and the USA (2.7%), presumably due to a shift from small and less strictly confined to large scale facilities, implementing all-in-all-out or farrow-to-finish models (Hill et al., 2008; EFSA, 2012; Guo et al., 2015). However, the recent rise of organic or free-range farming in order to improve animal welfare seems to contribute to an increase in infection rate in pig livestock and, as such, to the incidence of foodborne human toxoplasmosis (Kijlstra et al., 2004; Dubey et al., 2012; EFSA, 2012). The risk for humans to become infected by consumption of undercooked or raw pork is also not clear. The knowledge of the parasite persistence in edible tissues of naturally infected pigs is limited, as are the role of strain or dose in the parasite survival in the host. Such information might be of pivotal importance for vaccine development.

In the last decades numerous potential vaccine candidates have been experimentally tested mainly in mice and to a lesser extent in pigs. The formulations varied between a single recombinant parasitic protein or a combination of antigens, among which surface antigens (SAGs) or excretion/secretion proteins (GRAs, ROPs, MICs), but also DNA vaccines encoding B or T cell epitopes have been evaluated. However, the degrees of success were variable and did not led to a commercial vaccine (Vercammen et al., 2000; Letscher-Bru et al., 2003; Jongert et al., 2008; Li et al., 2011; Cao et al., 2015; Wagner et al., 2015). Vaccination did move forward by the use of attenuated viable strains, which resulted with a single commercially available vaccine for sheep, but their efficiency is tested under experimental circumstances and strictly species-dependent, and cannot yet be extrapolated to other livestock species or humans (Katzer et al., 2014; Burrells et al., 2015). Nevertheless, several experimental data in pigs reported reduction in parasite burden in infected and subsequently heterologous challenged pigs, in which the choice of the strain had an important effect on the viability of the parasite (Solano Aguilar et al., 2001; Dawson et al., 2004, 2005; Kringel et al., 2004; Garcia et al., 2005; Verhelst et al., 2011, 2015). In these studies, the involvement of the innate and acquired immune system was observed, dominated by antibodies production against the parasitic antigens, and by the Th1-type of the immune response. Depending on the experimental model, high levels of anti-GRA7 alone or anti-GRA1, -GRA7 and –TLA IgG's were detected upon an inoculation with IPB-G strain or a DNA GRA1-GRA7 cocktail vaccination, followed by the RH-strain or IPB-G challenge, respectively (Jongert et al., 2008; Verhelst et al., 2011), whereas a challenge with heterologous M4-strain oocysts after an experimental inoculation with viable S48-strain tachyzoites elicited a high TLA-specific IgG production (Burrells et al., 2015). In addition to the enhanced humoral immunity, a polarized Th1-immune response was observed after inoculation with a variety of the infectious T. gondii strains (Solano Aguilar et al., 2001; Dawson et al., 2004, 2005; Jongert et al., 2008; Verhelst et al., 2015). The significantly increased IFN-γ protein concentration in serum and the supernatant from the cultured PBMCs, and IFN-γ mRNA or DNA expression in PBMCs and intestinal lymphoid tissues, appeared positively correlated with the duration of the experiments (Solano Aguilar et al., 2001; Dawson et al., 2004, 2005; Jongert et al., 2008; Verhelst et al., 2015). In parallel with IFN-γ, also other cytokines were involved in the immune response against the parasite, as shown in the infection with the VEG-strain oocysts and the increased secretion of IL-15 and TNF-α (Dawson et al., 2005). Subsequently, a Th-2 response profile with predominantly IL-10 as anti-inflammatory cytokine was observed after the early phase of the infection, dominated by IFN-γ production, as mentioned earlier (Solano Aguilar et al., 2001; Aliberti, 2005). In contrast, IL-12 (IL-12p35 and IL-12p40) mRNA expression was not detected in PBMCs shortly after inoculation (7 and 14 dpi) in another study in pigs (Dawson et al., 2005). Nonetheless, even an excessive production of parasite-specific antibodies or Th-1/Th2-response cytokines did not provide a full protection during the acute phase of the infection, preventing from the cysts formation. Despite the active role the different components of the host's immune system play in the early stage of T. gondii infection, it remains a subject of discussion and ongoing research, whether the intermediate host can clear the tissues from the cysts on long term. It is noteworthy, however, that several studies in pigs notified reduced or undetectable counts of the parasite DNA in multiple porcine tissues, and a decline in viability of the cysts, as tested by bio-assay in mice (Jongert et al., 2008; Verhelst et al., 2011, 2015; Burrells et al., 2015). Taking into account the lack of an obligatory screening of pigs or pork meat to prevent transmission to humans, knowledge on the pig as an intermediate host for T. gondii, and in particular strategies to reduce the amount of viable parasites in tissues, may contribute to diminishing the risk of zoonosis by consumption of porcine meat (EFSA, 2007, 2012; Opsteegh et al., 2016). In light of these data, the aim of this study was to confirm differences between T. gondii strains in persistence of the parasite in tissues of experimentally infected pigs and to relate the dose and strain to the immune responses in the pigs upon a single infection or a heterologous challenge.

In the performed experiments, we compared the single or subsequent infection in pigs inoculated with either a high or a low dose of the IPB-G and the IPB-LR strains for the tissue specific parasite load and the accompanying immune response. The IPB-G strain has a mixed type I/II genotype, while the IPB-LR strain has a classic type II genotype (Jongert et al., 2008; Dubey et al., 2012). The antibody response against GRA7 and TLA, which are frequently used in serological assays in different species, was monitored until 120 dpi to confirm the successful inoculation and persistence of the infection (Figures 2, 3). Overall, for both IgM and IgG, independent from the infection dose, the GRA7-specific antibodies were detected very soon after the initial infection, starting from 10 dpi (Figure 2). Similar to previous results (Verhelst et al., 2015), we detected a late TLA-specific IgG response from 28 to 35 dpi onwards (Figure 3). However, results of the present study demonstrated that strain and dose are important factors to consider, since primary GRA7- and TLA-specific antibody responses could be detected earlier during infection and were higher upon inoculation with a high dose of the IPB-LR strain as compared to the IPB-G strain. A low dose on the other hand resulted in a later and less prominent seroconversion. Interestingly, whereas primary antibody responses were comparable in the heterologous challenge model, a clear IgM response was seen after the challenge with the heterologous strains, indicating exposure to other antigens, presumably due to the genetic diversity of both strains (Figure 5). Burrells et al. (2015) described a significant TLA-specific IgG increase after challenge of pigs with the heterologous strain M4 upon inoculation with the S48 strain. Strikingly, the challenge was performed with oocysts, after an initial inoculation with tachyzoites, stressing the expression of related or identical variability antigens in correlation to the parasite stage and the strain.

Together with a robust humoral response following T. gondii infection, a strong innate and cellular immune reaction is well described in mouse and human models, involving several populations of immune cells as well as different activation pathways (Aliberti, 2005; Miller et al., 2009; Andrade et al., 2013; Gazzinelli et al., 2014; Sturge and Yarovinsky, 2014). To date, it is well known that innate immune cells (macrophages, dendritic cells (DC's) and neutrophils) are involved in the acute stage of the infection by triggering the myeloid-differentiation primary response protein 88 (MyD88) signaling pathway after uptake and intracellular recognition of the parasite by CC-receptor 5 (CCR5) or Toll-like receptor (TLR) 11 and 12 in mice; TLR7, 8, and 9 in human and TLR7 and 9 in other mammals like pigs (Miller et al., 2009; Andrade et al., 2013; Koblansky et al., 2013; Gazzinelli et al., 2014). In particular interferon regulatory factor 8 (IRF8)⁺ dendritic cells, activated by the uptake of the parasite's protein profilin, are crucial for the induction of IL-12 secretion in mice. Human and porcine DCs and monocytes are activated by the recognition of the parasite's ssRNA and DNA via TLR7 and TLR9, respectively, prior to their pro-inflammatory cytokine response (Uenishi et al., 2012; Andrade et al., 2013).

Consequently and irrespective of the activated TLRs, the DC-driven IL-12 production leads to the activation of T-helper 1 cells and Natural Killer (NK) cells (Sturge and Yarovinsky, 2014). The latter massively produce IFN-γ, which not only continuously activates macrophages via a positive feedback mechanism, but also elicits the expression of the GTPases. The GTPases family includes four subfamilies: the very large inducible GTPases (VLIG), the Mx proteins, the immunity-related GTPases (IRGs) such as p47 or p65, and the guanylate-binding proteins (GBPs). The p47 IRG offers a robust protection against intracellular pathogens, being recruited to the parasite attachment site at the host cell (MacMicking, 2004; Taylor et al., 2004; Liesenfeld et al., 2011). Subsequently, a lethal damage to the parasitophorus vacuole (PV) is induced, leading to the rupture of the infected cell and release of the parasite into the cytosol (Gazzinelli et al., 2014). The infected cell undergoes necrosis, simultaneously with an enhanced local immune response. It is important to mention that IFN-γ-inducible IRGs are well studied in murine models, where 23 different genes have been identified to date, but the corresponding genes are not present in the human genome, which includes only one gene and one pseudogene (Bekpen et al., 2005; Zhao et al., 2009). This implies that both species deploy other intracellular defense mechanisms against T. gondii. The data on the identification of porcine GTPases are scarce, but a high similarity to the human IRGs is mentioned (MacMicking, 2004). Only two porcine GBPs have been reported until now: GBP1 and GBP2, whereas in humans 7 different GBPs have been identified (MacMicking, 2004; Li et al., 2016). More IRG's have been found using Affymetrix GeneChip® Porcine Genome Array but a detailed study in pigs is lacking (Fossum et al., 2014).

Thus, the continuous production of IFN-γ seems to be necessary in maintaining a delicate balance between the host immune system and the parasite's evasion strategies. Additionally, this cytokine plays a pivotal role in controlling both the acute and chronic phase of infection, as it facilitates stage conversion from the tachyzoite to the bradyzoite in acute toxoplasmosis and suppresses the opposite conversion during chronic infection (Denkers, 1999). Likewise, we detected a significant increased IFN-γ production by PBMC's after inoculation with two different T. gondii strains (Figure 4), which corroborates our previous results when inoculating pigs with the IPB-G strain (Verhelst et al., 2015). Here, we demonstrated a time- and dose-dependent increase in IFN-γ mRNA expression upon infection with the IPB-G strain. Several studies focused on experimental infection in pigs reported a time-dependent increase of IFN-γ levels in serum, supernatant from cultured PBMCs and IFN-γ mRNA expression in PBMCs and intestinal lymphoid tissues (Solano Aguilar et al., 2001; Dawson et al., 2004, 2005; Verhelst et al., 2015).

On the contrary, the inoculation with the low dose of the IPB-LR strain was almost as potent in inducing a relatively fast and strong IFN-γ production by PBMCs as the high dose of the same strain, which resulted in high IFN-γ mRNA levels at already 2 mpi, that were maintained until 120 dpi. Interestingly, the IFN-γ mRNA production in the LR_high dose group did not show any increase over time, implying reaching the maximum capacity from 1 mpi onwards. In line with our findings, IL-12 (IL-12p35 and IL-12p40) mRNA expression was not detected in PBMCs in the acute phase of the infection (7 and 14 dpi) in an earlier study in pigs (Dawson et al., 2005).

Based on the results of the detection of IFN-γ and the parasite DNA in tissues (Table 3), a balance between the host defense mechanisms and the invasion of the parasite was probably established soon after inoculation with the IPB-LR strain. Namely, the high IFN-γ production during the infection study was associated with the high counts of parasite DNA in the animal tissues. On the contrary, in the IPB-G groups IFN-γ production was elevated in the late phase of the infection and resulted in a very low to undetectable parasite load in the tissues, implying that high IFN-γ levels can tip the balance in favor of the host. Intriguingly, based on our observations and unpublished data from the acute infection model with the same strains, we speculate that exposure to a high dose of the IPB-G strain is more effective in activating innate immunity than the low dose of the same strain or the inoculation with the IPB-LR strain. The strain-dependent differences in the IFN-γ production profile may result from the genetic and thus, biological features of the used strains, indicating expression of variable virulence factors toward the intermediate host. In our opinion and according to others (Hunter and Sibley, 2012), the IPB-LR as genotype II strain, activates other pathway than the IPB-G strain, which is of an atypical genotype (mixed genotype I and II). The virulence factors initiating a pathway would be GRA15 (via NF-κB) for the former, and ROP18 (via STAT3/STAT6 pathway) for the latter. Consequently, the IPB-LR induces Th1 type of protective immunity and remains persistent in the chronic phase. When looking at the IPB-LR infected groups, no substantial difference was noticed in the IFN-γ production pattern or in the amount of T. gondii DNA at the end of the experiment. In our view, the IPB-LR strain does not show an acute virulence but it is adapted to persist within the intermediate host and, as such, increase own survival. To support these speculations, we observed a much milder clinical manifestation upon inoculation with the IPB-LR strain (Jennes et al., unpublished data). We further hypothesize that resistance to the chronic infection in IPB-G model results from the high acute virulence and the subsequent fast elimination of the tachyzoites before they can successfully multiply and disseminate. Consequently, fewer parasites can survive the initial parasitemia, which eventually will lead to reduced numbers of cysts in the tissues. Importantly, we conducted the studies in a homogenous pig population in order to exclude host diversity; however, as the strains are maintained by serial passage in mice, their virulence might be altered compared to the original isolate. Therefore, it is tempting to speculate that the high IFN-γ production together with the lower parasite counts in the porcine tissues originate from a coevolution toward host tolerance and reduced virulence, as suggested earlier by others (Gazzinelli et al., 2014). Furthermore, looking at the total IFN-γ expression following reinoculation with the heterologous T. gondii strain in the second experiment, no obvious difference between the groups could be observed (Figure 6). In the G_high/LR_high group we detected an initial increase, which was followed by a steady decrease after the challenge. The IFN-γ production profile in the reversed infection model (LR_high/G_high) supports our previous findings, showing a constant IFN-γ detection over time. Interestingly, in some animals basal or low level of cytokine mRNA were detected at 1mpi, followed by a substantial increase at the later time points, similar to the single infection experiment (Figures 4A, 6A). However, the final IFN-γ concentration at the end of the experiment upon a heterologous challenge was 10 times lower than after a single high dose inoculation. We could speculate that the primary infection with a mixed genotype I/II strain, characterized by a high acute virulence and long-term STAT3 and STAT6 activation, partially modulates the immune response upon the challenge with genotype II strain. As the result, the initial impairment of the Th1 response after the challenge leads to a lower than in a single infection model IFN-γ production, and elimination of a certain fraction of the parasites. Consequently, a reduced amount of the tachyzoites disseminate to convert into bradyzoites. The latter has been shown by a lower parasite load 60 dpi challenge than in IPB-LR experiment (Table 3 and Figure 6).

In regard to the involvement of immune cells in controlling the parasite's dissemination to the tissues and the chronic phase of toxoplasmosis, different populations seem to play a role. As described earlier and analogous to the acute infection stage, the production of IFN-γ is gradually taken over from the innate immune cells by T lymphocytes (Guan et al., 2007). Experimental infections in mice (Jongert et al., 2010; Suzuki et al., 2012) demonstrated the importance of CD4⁺ and CD8⁺ IFN-γ producing T cells in maintaining a chronic T. gondii infection, but the exact contribution of each subset remains unknown. Miller et al. (2006) describes higher production of IFN-γ by murine CD4⁺ cells upon in vitro stimulation by infected macrophages or by TLA, but admits that the higher protective potential against dissemination of the parasite by CD8⁺ or CD4⁺ lymphocytes is not simply expressed by the amount of this cytokine. Indeed, it seems that IL-4 and IL-10 cytokines, produced by CD4⁺ lymphocytes in addition to IFN-γ, might down regulate this protective capacity against the parasite. In line with that, due to their IFN-γ-independent cytolytic activity, the role of primed CD8⁺ T cells in the host's immunity during chronic toxoplasmosis has been widely acknowledged (Wang et al., 2005; Suzuki et al., 2012; Sa et al., 2013). In pigs, only a few experiments identified CD8⁺ and CD4⁺CD8⁺ cells in the acute phase of the infection as the major source of the IFN-γ production (Solano Aguilar et al., 2001; Dawson et al., 2005). The additive or synergistic effect of CD4⁺ T cells on the activity of the CD8⁺ T cell population should not, however, remain neglected. In our study, regardless of the strain, the CD4⁻CD8α^bright T cell subset contained the most IFN-γ positive cells, followed by the CD4⁺CD8α⁻ subset, whereas the CD4⁺CD8α^dim T cell subset showed very few IFN-γ positive cells (Figure 8). Additionally, the CD4⁻CD8α^bright population showed a temporal increase in IFN-γ production in animals infected with IPB-LR, while the percentage of this subset was rather declining from 4 wpi onwards, when infected with the IPB-G. The IFN-γ production resulting from the induced toxoplasmosis in pigs and the involvement of the different lymphocyte populations are in line with other studies, where the in vitro cytokine profile was investigated until 14 (Dawson et al., 2005), 40 (Solano Aguilar et al., 2001), or 56 dpi (Verhelst et al., 2011). However, opposite to the pig model, in murine experiments only two T lymphocyte subsets were differentiated (CD4⁺ and CD8⁺). Furthermore, the extent of the cellular response was positively correlated with the infection dose and the time-interval from the inoculation and was higher when induced by the strain with a greater tissue persistence. In perspective of future experiments in pigs, it would be desirable to focus on other immune cells, involved in the responses throughout the infection such as the immunosuppressive T regulatory (Treg) or Th17 cells. As recently shown in mice, the robust immune reaction expressed by the high IFN-γ levels in the acute phase of the infection severely reduces the activity of Tregs in a IL-2 dependent and IL-10 independent manner (Tenorio et al., 2011; Olguin et al., 2015). In a longitudinal clinical case study of human acquired cerebral toxoplasmosis a dual function of the Treg population was described, by simultaneous down regulation of CD4⁺ and activation of pathogen-specific CD8⁺ T lymphocytes (Rb-Silva et al., 2017). In human congenital infections not only CD4⁺ Treg cell population seems to be involved in the immune reaction triggered by T. gondii, but also a different subset of CD4⁺ or CD8⁺, namely Th17. The activity of this population is independent from IFN-γ, IL-4 and perforin activation, as their migration to the inflammation sites in initiated by certain chemokines. Interestingly, the results of the in vitro PBMCs stimulation with tachyzoites showed a higher percentage of CD4⁺ IL17 producing cells above the CD8⁺ (Silva et al., 2014). By investigating the fluctuations of the activity of Tregs and Th17 cells during the acute and chronic phase of the infection, we might determine whether these populations are also involved in the persistent immunity toward the parasite.

When considering the parasite load in the tissues and the viability of the cysts in the bio-assay, a clear correlation was found between the amount of detected DNA and the dose of the used strain (Table 3). In general, we observed a decline in the concentration of the parasite's DNA in animals when inoculated with a high dose of the IPB-G, but not the IPB-LR strain. This dose- and time-dependent decline is prominently present in different tissues in the G_high group in comparison with G_low and G_high1/2t groups, indicating that the effect of the high dose is particularly visible after a longer infection time. These results are in line with the findings of Verhelst et al. (2011), where neither parasite DNA nor viable parasites were detected in certain muscle tissues 6 months after initial infection with the IPB-G strain. In the same study, brain and heart of all animals remained infectious as determined by a bio-assay and qPCR. However, these findings are opposite to results obtained in rats and cats, showing that inoculation with increasing amounts of tachyzoites or bradyzoites resulted in a decreased survival rate or in a higher number of tissue cysts, respectively (De Champs et al., 1998; Cornelissen et al., 2014). In addition and similar to our results, others reported a reduction in parasite burden in strain-vaccinated and challenged pigs (Kringel et al., 2004; Garcia et al., 2005; Jongert et al., 2008; Burrells et al., 2015). In these experiments vaccination with oligonucelotides, antigens or infection with attenuated strains can enhance Th1 responses to elicit sufficient protection during the acute phase of the infection, resulting in a lower parasite burden in comparison with the infected control animals. Referring to that, the strains used in our study differ greatly in terms of genetic background and associated virulence, both in mice and in pigs. Therefore, we have grounded scientific reasons to believe that the observed differences in the parasite load upon infection with both strains, especially in the heterologous co-infection model, are not coincidental.

For most tissue samples this dose- and strain-dependent reduction in the number of the tissue cysts in qPCR is consistent with the bio-assay in mice. It is noteworthy that a few qPCR-positive samples were negative in the bio-assay, indicating that parasite DNA, but not viable parasites were present. Consequently, these results show a substantially higher sensitivity of the qPCR method used here above the bio-assay, since it has been optimized and successfully applied for the detection of the parasite in various human or animal tissues, with a detection limit of 2–4 tissue cysts in 100 g of sample (De Craeye et al., 2011). Conversely, both techniques gave positive results on all samples derived from animals upon single inoculation with the IPB-LR strain. Therefore, we can assume that the reduced parasite load occurred due to the earlier infection with the IPB-G strain in the group G_high/LR_high. This phenomenon does not seem to be limited to type II strains (Velmurugan et al., 2009; Suzuki et al., 2012), but is also common in type I strains (Burrells et al., 2015).

Summarizing, the groups infected with the IPB-LR strain can serve as a classical model of T. gondii persistence in its intermediate host. The prominent production of parasite-specific antibodies, consistent amounts of IFN-γ and activation of cytotoxic T lymphocytes on the one hand and well-distributed DNA concentration together with isolation of the viable parasite on the other hand, clearly prove an established balance between the host immunity and the pathogen's activity. The parasite's persistence appears to be beneficial for the two, under conditions that the immunocompetent host can resist the immunomodulation by T. gondii. On the contrary, as the partial or total removal of the tissue cysts was observed in the IPB-G infected animals together with the increasing IFN-γ production profile on both the mRNA and protein level, we propose that the IPB-G strain induces a robust immune reaction in the host in the early phase of the infection. This IFN-γ-mediated response in pigs can lead to the resistance of the host to parasite invasion by elimination of the tissue cysts during the chronic infection. Further experiments to unravel the nature of this resistance are warranted as it could serve an important role in vaccination strategies and in the risk assessment for food safety and human health.

This study was carried out in accordance with the recommendations of the ethical standards defined by the EU legislation on the use of laboratory animals (2010/63/EU) and in accordance with the Belgian law (the Royal Decree 29/5/2013 and the Royal Decree 30/11/2001). The protocol was approved by the Ethical Committee of the faculties Veterinary Medicine and Bioscience Engineering at Ghent University (approval number no. 176 2009/149) and by the Ethical Committee of the WIV-ISP Institute (approval no. 176 20140704-01). The Biosafety Level 2 permit for working with pathogens for Ugent, Merelbeke: AMV/11062013/SBB219.2013/0145. The Biosafety Level 2 permit for working with pathogens for WIV-IPH, Brussels: 415240.

MJ designed the study, acquired and analyzed the data and drafted the manuscript. SD designed the study, acquired, and analyzed data and revised the manuscript. BD helped to design the flow cytometry study and revised the manuscript. PD and KD helped to design the study, gave valuable input and revised the manuscript. EC designed the study, analyzed the data and wrote the manuscript.