Immunity against reinfection in pigs following Taenia solium infection and a quantitative dose-response model

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Methods: This study investigates the effect of T. solium infection and reinfection on cyst development in pigs and evaluates how acquired immunity constrains parasite burden. A total of 116 pigs were purchased from commercial farms in northern Peru and housed in pathogen-free facilities under controlled conditions. Of these, 110 pigs were allocated to 18 experimental groups to (1) evaluate the impact of infection and reinfection with varying doses of T. solium eggs and (2) generate a model to predict the number of live cysts produced, given the dose and age at infection. Gravid proglottids collected from human cases of T. solium taeniasis were used to prepare egg pools, ensuring viability consistency. Infections were administered orally using gelatin capsules via esophageal catheterization, followed by necropsy 10 weeks after the final infection event to quantify cysts. A negative binomial regression model was used to analyze cyst burden dependence on infection dose, past infection, age, and other factors. Results: No statistically significant differences in cyst counts were observed between pigs infected once and those that were reinfected, regardless of the initial dose (as low as 100 eggs) or reinfection dose (up to 20,000 eggs). This finding highlights that infection results in strong acquired immunity, effectively blocking subsequent infections. A quantitative dose-response model suggests that the relationship between egg dose and the number of viable cysts is best described by a power relationship. Combining data from single-infection and reinfected pigs into a unified model improved prediction precision. Finally, incorporating age at infection results in a model of the number of viable cysts in pigs depending on dose and age that combines acquired and innate immunity effects, i.e. changes in susceptibility with age. Conclusions: Initial exposure to T. solium eggs induces strong acquired immunity in pigs, effectively preventing reinfection. Our quantitative dose-response model predicting live cyst counts based on egg dose and pig age offers valuable insights for integrating immunity processes into models of T. solium transmission. Taenia solium experimental infection acquired immunity reinfection quantitative dose-response live cysts prediction Figures Figure 1 Figure 2 Background Taenia solium is a zoonotic parasite causing an estimated 2.8 million disability-adjusted life years (DALYs) globally [ 1 ]. It also leads to significant economic losses, particularly in low- and middle-income countries [ 2 ]. The parasite is listed among the 20 diseases that disproportionately affect marginalized populations in tropical and subtropical regions and are targeted for control, elimination, or eradication by 2030 [ 3 ]. Humans serve as the definitive host, harboring the adult tapeworm, which sheds proglottids and eggs in feces. These eggs are ingested by the intermediate host, the pig, in which cysts develop in muscle and other tissues. Humans can also act as accidental intermediate hosts when they ingest eggs, sometimes leading to the development of neurocysticercosis, a severe condition characterized by cysts in the central nervous system [ 4 ]. Understanding the dynamics of transmission—how, when, where, and why pigs become infected with different numbers of cysts in rural areas—is critical for designing effective control strategies [ 5 ]. One of the challenges relates to understanding the high variability in the number of cysts in infected pigs, with most infections involving fewer than 10 cysts (making them often hard to identify through meat inspection) while some involve thousands of cysts [ 4 ]. These infection patterns are influenced by a complex interplay of parasite, host, and environmental factors, which result in the overdispersion of the larval population within the host population [ 9 ]. Both egg and intermediate host populations exhibit considerable heterogeneity. Eggs vary significantly in quantity and infectivity, while intermediate hosts display a wide range of resistance to infection [ 5 ]. A key factor driving resistance to infection is acquired immunity [ 6 – 8 ], yet, our understanding of this phenomenon is still incomplete. Prior studies have suggested that animals exposed to two doses of T. solium eggs may not have more cysts at necropsy than those exposed to a single dose [ 9 – 11 ]. However, low sample sizes and heterogeneity in animal, egg viability and numbers used in experimental studies have made it hard to confirm and quantify the impact of immunity. In the most recent of those studies [ 9 ], pigs were first infected with 100,000 eggs and later reinfected with the same dose from a different tapeworm, with reinfected pigs harboring no more cysts than those infected once. However, interpretation of the results was complicated by the fact that pigs infected only with the second tapeworm developed very few cysts, making it unclear whether the reinfected pigs were truly resistant or if the second batch of eggs had lower infectivity. Additionally, the study only examined 2 kg of muscle per pig, potentially misrepresenting the actual cyst burden [ 9 ]. There is therefore a need to build on and improve on this early literature with a study design that will address most or all of the limitations of prior studies. Greater certainty regarding immunity mechanisms may further feed into mathematical models of T. solium , which are often employed to simulate disease transmission. In these models, the variability in parasite burdens is often simulated through scenarios where pigs are infected at low and high intensities, sometimes with infection burdens represented by a categorization of pigs between “lightly” and “heavily” infected pigs [ 12 – 15 ]. Those models generally do not integrate any immunity mechanism, and simulated heterogeneities in pig infection loads may be driven by geographical dispersion of eggs, a phenomenon that remains insufficiently understood, despite recent advances [ 16 ]. However, accurate simulation of how pigs with different profiles (age, past exposure) get infected with different parasite loads – and particularly the impact of immunity in disparities in cyst burdens – is critical to developing better informed models and more accurately simulating the impact of possible disease control mechanisms. Previous studies by our team have looked into geographical dispersion mechanisms for eggs [ 16 ] and the effect of host age as a determinant of variation in infection burden [ 17 ]. In the present study, we focused on assessing the role of acquired immunity by infecting and reinfecting pigs in highly controlled conditions and evaluating the total cyst burden across the entire carcass. Additionally, we integrated these results with our earlier findings on innate immunity to develop a model that predicts cyst numbers as a result of infection dose and age at infection. This study aimed to provide a comprehensive and quantified description of the mechanisms by which the host might regulate parasite populations. Methods This study investigates: (1) the effect of reinfection on the development of cysticercosis following an initial infection with varying doses of T. solium eggs, and (2) the influence of a primary infection with different egg doses on the number of cysts that develop in infected pigs. These analyses are part of a broader investigation, which also examines the effect of pig age on cyst development (reported in a separate manuscript [ 17 ]). Since some pigs contributed to multiple analyses, we begin by detailing the overall experimental design before describing the statistical methods applied to address the two objectives outlined above. Animals A total of 116 pigs were purchased from 13 commercial farms in northern Peru. Pigs raised in such commercial farms are expected to be unexposed to T. solium . Farms were selected based on the availability of pigs within the required age range and the capacity of our corrals to accommodate them. Six pigs were excluded from the study: one had an aberrant serologic response at baseline while five died prior to the scheduled necropsy, leaving a final cohort of 110 pigs. These pigs were divided into 18 groups to address two primary objectives: (1) to evaluate the effects of acquired immunity by subjecting different groups to varying infection and reinfection doses, along with their respective infection controls (14 groups described in Fig. 1 A, this study); and (2) to assess the effects of innate immunity by infecting pigs of different ages with a fixed dose (six groups described in Fig. 1 B, also reported in a previous study [ 17 ]). Two groups were common for both experiments. In the present study, we first describe and analyze the effect of reinfection. This is followed by a complementary analysis aimed at predicting the number of viable cysts based on dose, as well as other relevant factors such as sex, age, effect of reinfection, and viability of the egg pool administered. The animals were housed in individual pens at the specific pathogen-free facilities of the Center for Global Health at Cayetano Heredia University (UPCH) in Tumbes. They were fed exclusively with properly packaged commercial feed, stored at our facilities to prevent contamination, while water was provided ad libitum . Serological testing In line with the method described in [ 17 ], all pigs underwent serologic screening with antigen ELISA (TsW8/TsW5 mAb set) [ 18 ] and lentil lectin-bound glycoprotein enzyme-linked immunoelectrotransfer blot assay (LLGP-ETIB) [ 19 ] to rule out T. solium exposure prior to purchase. To be included in the study, pigs had to be negative on both tests (optical density ratio < 1 on Ag-ELISA and absence of any reactive band on LLGP-EITB). The mothers of all animals (except three due to owner refusal) were screened to check that they were also negative. We took weekly blood samples throughout the experiment to monitor the evolution of the serological response using both tests. Preparation of Egg Pools In line with the method described in [ 17 ], tapeworms for this experiment were obtained from human stool collected in community settings as part of control interventions. Stool was collected during bowel preparation prior to antiparasitic treatment. It was screened for T. solium taeniasis using a combination of microscopy, coproantigen ELISA, and molecular confirmation of species using PCR [ 20 ]. Gravid proglottids were collected and stored in a saline solution at 4°C for up to two weeks. This design ensured that the eggs used in this experiment had not been exposed to any antiparasitic drug or preservative. Our study consisted of three waves of infection and reinfection, each comprising two rounds: one for the initial infection and another for reinfection, totaling six rounds. For each round, we created a pool of T. solium eggs from at least three different tapeworms (each coming from a different donor from the North-West of Peru – Piura and Tumbes regions) to minimize variability between infections due to differences in egg viability. The exact number of tapeworms used to create the pool utilized in each round (10, 3, 7, 8, 6, 10, and 3 respectively) varied depending on tapeworm availability and the number of viable eggs available for each tapeworm. Egg viability was assessed for each proglottid and for the entire pool using Evans blue stain before mixing the eggs harvested from the gravid proglottids obtained from the tapeworms used in each pool. We also assessed the percentage of activated oncospheres in each proglottid and pool using the enzyme method and movement of the hexacanth embryo [ 21 ]. The percentage of activated oncospheres was 82%, on average, across all pools (range 63–97%). Infection procedure and Necropsy Infections and reinfections were administered using an esophageal catheter, following a method previously described in [ 22 ] with slight modifications described in [ 17 ]. The assigned dose of T. solium eggs, suspended in olive oil and encapsulated in gelatin capsules, was delivered to each pig through an esophageal catheter. The capsules were flushed into the pigs’ stomach using 20–100 mL of mineral water. An intramuscular combination of ketamine (20 mg/kg) and xylazine (2 mg/kg) was used to anesthetize the pigs. After the procedure, a veterinary team monitored the pigs for vital signs and for any signs of emesis or capsule regurgitation. The reinfection procedure was performed 12 weeks after the initial infection. We kept all pigs in individual pens for 10 weeks following the last infection, until necropsy. For necropsy, pigs were anesthetized as per the procedure described above then euthanized via intravenous sodium pentobarbital (100 mg/kg). Trained personnel then meticulously examined all muscles, the brain and other organs using 3–5 mm slicing to obtain accurate cysts counts [ 23 ]. The team identified and counted cysts and recorded the number, type (viable or degenerated) and location of cysts individually for each pig. Experimental designs Infection-reinfection experiment A total of 65 piglets (four weeks old) and 19 pigs (16 weeks old) were used in the infection-reinfection experiment, corresponding to the groups described in Fig. 1 A. Initially, the four-week-old piglets were divided into four clusters of 15 each and infected with 100, 1,000, 5,000, or 20,000 T. solium eggs, respectively. After 10 weeks (the time necessary to get fully developed cysts [ 24 ]), each cluster was subdivided into three groups of five pigs: One group was necropsied to determine the cyst burden resulting from a single infection and was considered as a positive control of single infection at four weeks old. The remaining two groups were reinfected after two weeks, at 12 weeks of age, with either 5,000 or 20,000 eggs, respectively, to evaluate the additional cyst burden following reinfection with each of these reinfection doses. The remaining five piglets were also infected with 20,000 eggs and correspond to the group included in the age-related infection experiment, in which a total of 10 pigs were used. In addition to the piglets, 16-week-old pigs were included as secondary positive controls for single infection at that age and to verify the effectiveness of the egg pool used during reinfection. These control pigs were infected once with either 5,000 or 20,000 eggs at the same time as the reinfected subgroups received their second infection. All animals infected at 16 weeks of age, whether initially infected or reinfected, were necropsied 10 weeks later, at 26 weeks of age. Additional details of each experimental group are provided in Table 1 . Table 1 Median, minimum, and maximum counts of live, degenerated, and total cysts across the different groups involved in the infections at 4 weeks of age, reinfections at 16 weeks of age, and controls (infected only once at 16 weeks of age), along with the corresponding number of pigs per group. Clusters Infection at 4 weeks Infection at 16 weeks Number of pigs Live cysts Degenerated cysts Total Cysts Median Min Max Median Min Max Median Min Max Pigs infected once 100 0 5 0 0 1 0 0 7 1 0 8 1,000 0 4 15.5 1 51 6 3 31 33 4 59 5,000 0 5 143 0 348 7 3 158 163 88 355 20,000 0 7 103 0 270 63 5 490 192 71 514 Pigs reinfected with 5000 eggs 100 5,000 5 0 0 2 22 0 197 22 0 198 1,000 5,000 5 29 1 105 4 0 15 29 8 110 5,000 5,000 5 34 0 695 1 0 15 35 0 710 20,000 5,000 5 187 7 565 55 1 490 199 132 731 Pigs reinfected with 20000 eggs 100 20,000 4 6 0 12 15 0 33 25.5 1 36 1,000 20,000 5 14 4 88 15 2 34 36 16 103 5,000 20,000 5 58 0 658 22 2 244 111 22 699 20,000 20,000 5 134 2 236 30 0 187 189 119 238 Controls 0 5,000 9 43 0 205 28 4 555 90 14 556 0 20,000 10 118.5 1 809 58.5 1 260 286 30 833 Due to logistical and operational constraints, the experiments were carried out in three waves of infection, each spaced approximately six months apart. Each wave included two rounds: an initial infection round, where all pigs in that wave were exposed to T. solium eggs, followed by a reinfection round, where a subset of the previously infected pigs was exposed again. A new pool of T. solium eggs was used for each infection round to ensure consistency across exposures (see Preparation of Egg Pools for details). This design allowed for a controlled evaluation of infection and reinfection dynamics. Quantitative Dose-Response We conducted multiple analyses of the number of cysts developed by pigs as a function of the infective dose. First, we considered only pigs infected once at 4 weeks with varying doses. In a second stage, we incorporated pigs infected twice (at 4 and 16 weeks) and used the results of the analysis of the effects of prior infection on pigs’ response to infection to combine those pigs with those that were infected only once. Finally, we included all pigs, regardless of their age at first infection, using evidence on the effect of age, as described in a previous paper [ 17 ], to build a comprehensive model. Data analysis For descriptive statistics, the median, minimum, and maximum values were used to summarize data. To analyze differences between infection and reinfection, a negative binomial regression model was developed, with the number of live cysts as the response variable and the infection doses at 4 weeks old and 16 weeks old as predictors, respectively. Other covariables, such as sex, the pigs' farm of origin, the infection round (to consider possible variations between infection periods) and pool viability, were included to identify potential confounding effects. A negative binomial regression model was also developed to create a quantitative dose-response model using data on the number of live cysts that developed following a single infection at different doses. This model was then expanded, incorporating additional animals with multiple infections and infected at different ages, by including the effect of age as described in a prior manuscript. Model fit was evaluated using criteria such as Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC), and the best-fitting model was selected based on these indices and residual diagnostics. All the analyses were performed using R [ 25 ] and the packages MASS [ 26 ], ggplot2 [ 27 ] and plotly [ 28 ]. Results Infection-Reinfection Experiment The median, minimum, and maximum numbers of viable, degenerated, and total cysts observed in each of the 14 groups—either infected once or reinfected—are summarized in Table 1 , along with the number of pigs per group. We found that reinfected pigs consistently exhibited similar cyst counts compared to pigs infected only once at different doses (Fig. 2 ). To compare cyst counts between the major clusters (pigs infected once versus pigs reinfected with 5,000 or 20,000 eggs) across the four different initial doses (logarithmic transformed), a negative binomial regression model with a two-way factorial 4 x 3 design (four different doses at four weeks old: 100, 1000, 5000, 20000 vs three clusters: infected once, reinfected with 5000 and reinfected with 20000) was applied using data from 12 of the groups (Table 2 ). Covariables such as sex, pool viability and/or dose adjusted for pool viability were tested but did not significantly improve the model. The analysis showed no statistically significant differences in cyst counts between pigs infected once at 4 weeks old and those reinfected with either 5,000 or 20,000 eggs, regardless of the initial infection dose, including cases where pigs initially infected with as few as 100 eggs were later reinfected with 20,000 eggs. Table 2 Negative binomial regression model describing the cyst burden as a function of the log of the infection dose at 4 weeks of age and dummies reflecting single infection vs. reinfection at 16 weeks with either 5,000 or 20,000 eggs. The table includes parameter estimates, standard errors, z-values, p-values, and 95% confidence intervals for each variable in the model. Variable Estimate Standard Error z-value p-value 95% Confidence Interval Minimum Maximum (Intercept) -3.59 0.88 -4.06 0.00005 -5.73 -1.28 Log (Infection dose at 4 weeks) 0.92 0.10 8.92 < 0.000001 0.65 1.18 Reinfected at 16 weeks with 5,000 vs infected once 0.57 0.49 1.18 0.24 -0.38 1.53 Reinfected at 16 weeks with 20,000 vs infected once 0.59 0.49 1.21 0.23 -0.41 1.62 Two groups of pigs, infected once at 16 weeks of age with either 5,000 or 20,000 eggs, served as controls for the reinfection process. Both groups developed a substantial number of cysts (median: 43 live cysts, 90 total for 5,000 eggs and 118.5 live cysts and 286 in total for 20,000 eggs). Those cyst counts were similar to those of pigs infected at 4 weeks old with the same doses. This suggests that the eggs used in this second infection had infective potential (at least when used to infect naïve pigs of the same age) and that, should there be no immunity or partial immunity after a first infection, the statistical analysis would have been able to capture the difference in cyst numbers between pigs infected once at four weeks of age and pigs infected twice (see Table 1 for the full summary statistics of the controls cluster). While degenerated cysts are of lesser importance (given that they do not contribute to transmission), we also sought to evaluate whether the number of degenerated cysts was higher in reinfected pigs than in pigs infected once. We therefore conducted a Kruskal-Wallis to compare both the number and the proportion of degenerated cysts. Neither comparison showed statistical significance (Kruskal-Wallis H-test: H = 2.14, df = 2, P = 0.34 for the number of degenerated cysts; H = 1.04, df = 2, P = 0.59 for the proportion of degenerated cysts). Quantitative Dose-Response In the previous analysis, the dose given to pigs at first infection had a high significant association with the number of viable cysts observed after infection (Table 2 ), contrary to reinfection status or dose. To describe the relationship between dose and the number of viable cysts, a quantitative dose-response model was developed using negative binomial regression. A first model (referred to as the "Single Model") includes only pigs infected once at four weeks of age and analyzes the relationship between the number of viable cysts and the infection dose. Its results are provided in the first lines of Table 3 below and depicted in Fig. S1 in supplements, where it is represented in pink. Table 3 Negative binomial regression model describing the cyst burden as a function of the log of the infection dose. The “Single model” only includes data from pigs infected once at 4 weeks of age with different doses, the “Reinfected model” also includes data from pigs reinfected once after initial infection at 4 weeks old. Finally, the full combined “Exposure response model” also includes information from pigs infected at different ages. The table includes parameter estimates, standard errors, z-values, p-values, and 95% confidence intervals for each variable in the model. Variable Estimate Standard Error z-value p-value 95% Confidence Interval Minimum Maximum Single model (only includes pigs infected once at 4 weeks of age) - Theta (θ) value: 0.42; standard error: 0.13 (Intercept) -4.58 1.45 -3.17 0.0015 -8.23 0.44 Log of Infection dose 1.04 0.17 5.97 < 0.000001 0.55 1.51 Reinfected model (includes all pigs infected at 4 weeks of age) - Theta (θ) value: 0.43; standard error: 0.08 (Intercept) -2.97 0.82 -3.60 0.0003 -4.83 -0.87 Log of Infection dose 0.89 0.10 8.63 < 0.000001 0.64 1.14 Exposure response model (includes all pigs) - Theta (θ) value: 0.43; standard error: 0.05 (Intercept) 0.56 1.91 0.29 0.77 -3.95 4.97 Age -0.21 0.07 -2.87 0.004 -0.37 -0.04 Inverse of age -10.21 5.26 -1.94 0.05 -22.10 1.72 Log of Infection dose 0.88 0.10 8.90 < 0.000001 0.63 1.10 Covariates farm of origin, the infection round, sex and pool viability were tested but were not significant. Since no significant differences were observed between infection and reinfection (Table 2 ), reinfection data were combined with data on pigs infected once into a second model (the "Reinfected Model"). This model only includes pigs initially infected at 4 weeks of age. This model preserved the trend of the initial single-infection model while reducing the width of the confidence intervals, enhancing the precision of predictions. Finally, based on findings from a previous manuscript that described the effect of age and included both an inverse age effect and a term proportional to age [ 17 ] a comprehensive model, referred to as the "exposure response model" was developed. In the “age” model, pigs infected at younger ages are less susceptible than those at the natural weaning age (approximately nine weeks old), with susceptibility then progressively decreasing as age increases. The "exposure response model" incorporated data from all pigs infected during the experiment, using both dose and age at infection as predictors. The coefficients of this model are presented in Table 3 (bottom section). The coefficient for the logarithm of the first infective dose is similar to what it was in each of the prior models and the confidence interval is similar to that of the “Reinfected” model. The blue curve in Fig. S1 represents the predicted values from this model for pigs infected at four weeks of age. Serological results Results were similar to those already described for pigs involved in the “age” experiment in [ 17 ]. Briefly, all pigs used in this experiment initially tested negative on the LLGP-EITB and had antigen optical density ratios below 1. After infection, all pigs seroconverted on the LLGP-EITB between three- and nine-weeks post-infection (median: five weeks). Pigs also seroconverted on the antigen test (this consistently happened before seroconversion on the LLGP-EITB) between one- and nine-weeks post-infection (median: three weeks). Cysts in the brain Out of the 110 pigs necropsied during the experiment, 16 had cysts in the brain. One of those harbored six degenerated cysts and 34 live cysts; the remaining animals presented only live cysts. Among the animals with only live brain cysts, six had only one cyst, another six had two cysts, two animals had three cysts, and one animal had nine cysts. The pig that developed 34 live cysts was infected with 5,000 eggs and reinfected with 20,000 eggs. Discussion In this controlled experimental infection, we aimed to determine the impact of prior exposure to T. solium eggs and to assess how the number of viable cysts in pigs infected twice compares with the number of such cysts developed by pigs infected only once. This study addresses several limitations of previous studies on experimental infections with taeniids by implementing measures to reduce variability. Specifically, we aimed to ensure greater uniformity and control over host and parasite populations, as well as egg doses, to improve reproducibility and comparability of results. The present study overcame those challenges by using tapeworm pools with controlled infectivity, larger numbers of pigs, and whole-carcass cyst counts. We did not seek to control for pig genetic background as having only pure-bred animals in our experiment would have increased homogeneity but decreased the generalizability of our results, given that the general practice in commercial farming worldwide is to use mixed-breed pigs like the ones in our experiment. The study, despite its tight control of most sources of variability, still found substantial differences in infection outcomes within groups (which was to a certain extent expected [ 5 ]) but was nevertheless able to demonstrate the existence of a strong acquired immunity that effectively protects pigs from reinfection. While the exact timing of immunity development remains unclear, our results indicate that the period during which pigs remain susceptible is relatively short in relation to their overall lifespan (less than 12 weeks – as the pigs in our experiment were reinfected after 12 weeks). The effect of acquired immunity had been hypothesized previously for taeniids [ 6 ] and several reviews had discussed how primary infections can block subsequent exposures, with multiple authors examining this phenomenon [ 5 , 7 , 8 ]. Although some experimental studies had reported contradictory results, such as calves being reinfected with T. saginata 10 weeks after the first infection, most experiments had produced opposite outcomes [ 29 ]. The main conclusion from these investigations had been that a form of acquired immunity likely develops following initial exposure [ 5 – 8 , 10 , 30 ]. These findings ultimately led to the development of the first successful vaccine against a parasitic infection: T. ovis , in sheep, the intermediate host [ 31 ]. There are now also commercially available vaccines against Echinococcus granulosus and T. solium [ 31 , 32 ]. Our study contributed to this body of knowledge by demonstrating that immunity acquired during the initial T. solium infection is highly protective against infection with subsequent exposure, at least in the timeframe we evaluated. Unlike vaccines, which prime the immune system by exposing it to the TSOL18 oncosphere protein before an infection occurs [ 31 ], immunity in our study was triggered by actual exposure to eggs. As a consequence of the required timeframe to verify reinfection effects at necropsy—given that cyst development takes approximately 10 weeks [ 24 ]—the interval between infection and reinfection in this study was necessarily long. Therefore, we could not determine the minimum time required for acquired immunity to become fully active and block reinfection. Studies on other tapeworm species suggest that this occurs progressively. For instance, experiments with T. hydatigena in sheep have shown that partial resistance can develop as early as seven days post-infection, with complete immunity established by 14 days [ 7 , 33 ]. Another important consideration is the minimum infective dose required to trigger an effective immune response. While our results demonstrate that as few as 100 eggs can block a reinfection with 20,000 eggs, the precise threshold remains undetermined. Studies on T. hydatigena and T. ovis suggest that an effective acquired immunity can be achieved with as few as 10 eggs [ 34 ], indicating that even minimal exposure may be sufficient to induce protection. Further experiments would be needed to confirm whether this is also true with T. solium . This study also showed how cyst burden relates to the egg dose at first infection. As expected, the size of the initial egg dose was found to be a significant positive predictor of the number of viable cysts, enabling the development of a model to predict live cyst numbers. Similar correlation with dose has been reported with other tapeworms and a rate of 1% of egg success was proposed for T. solium [ 7 , 10 ]. However, our results indicate that the relationship between infection dose and the number of viable cysts is best described by a power-law pattern, rather than a strictly linear one. Specifically, the expected number of cysts increases with the dose raised to the power of 0.88, suggesting a sublinear relationship. While this estimate implies that the proportion of eggs developing into cysts may decrease at higher doses—consistent with the idea of reduced infection efficiency—we note that the 95% confidence interval includes 1, meaning this trend is biologically plausible but not statistically conclusive. This interpretation aligns with prior observations that extremely high doses (e.g., 100,000 eggs) do not produce proportionally more cysts [ 22 ]. Such sublinear dynamics complicate the explanation of superinfected pigs found in natural settings, where individuals harbor more than 10,000 cysts [ 35 ], raising the possibility that additional biological or ecological mechanisms may contribute to extreme infection outcome. We sought to validate our exposure-response model against data from a previous study that compiled results from multiple experimental infections conducted by different research groups worldwide [ 35 ]. The model’s predictions aligned well with these independent datasets (considering expected variability in individual pig burden), further supporting its ability to describe T. solium cyst development across various experimental conditions. This analysis is presented in supplementary material (Fig. S2). In particular, the prior study suggested that pigs infected with 100 eggs had a [62–100%] probability of developing cysts, a range consistent with our experimental findings, where 11 out of 14 pigs (79%) receiving a first or single infective dose of 100 eggs developed cysts [ 35 ]. The results described in this paper, combined with those described in our earlier paper on the effects of age at infection [ 17 ], allowed us to leverage this unique experimental dataset to develop an overall exposure-response statistical model predicting the number of live cysts in pigs depending on both the dose of the primary infection and the age at infection. The model performed well in our dataset, capturing the observed effects of both dose and age on cyst burden. Significant variability in cyst counts is a common observance in experimental infections with T. solium and other tapeworm species. We implemented several measures in an attempt to limit sources of variability in our study, including serological monitoring before, during, and after infection; housing pigs in individual pens throughout the experiment; infecting them with pools of several tapeworms using oroesophageal catheters; maintaining a controlled environment to minimize exposure to insects and other pests; and exclusively using properly stored commercial feed. Despite these precautions, we observed significant variability among infected animals, even within the same group. This outcome is not novel and is a characteristic commonly observed in experimental infections with this and other tapeworm species [ 5 , 7 ]. In our study, the negative binomial dispersion parameter (θ) was estimated at 0.43 (Table 3 ), indicating a high level of overdispersion in cyst counts. This suggests that infection burdens vary substantially among pigs, with a greater variance than expected under a Poisson distribution. The substantial variability in the infection patterns of pigs in endemic areas could be explained in part by this infection pattern. It is also likely that pigs encounter high parasite loads when ingesting proglottids and lower loads when ingesting eggs. Finally, mechanical vectors may play a role in egg dispersion, however, studies in endemic areas have not confirmed a significant impact from this mode of transmission [ 16 ]. Conclusions Initial exposure of pigs to T. solium eggs provides strong acquired immunity against infection upon subsequent re-exposure, with protection evident even when the initial dose is as low as 100 eggs and the reinfection dose as high as 20,000 eggs. Despite minimizing variability during infection—using eggs with high in vitro viability, pooling eggs instead of entire proglottids, accurately counting eggs in infective doses, and administering infections intra-esophageally—significant variability in cyst counts was still observed within groups, suggesting that other factors may influence individual responses. Using data from these experiments, we developed a quantitative dose-response model to predict the number of live cysts in infected pigs based on egg exposure dose and age at infection. This model provides essential insights for incorporating pig immunity processes into CystiAgent [ 36 – 39 ], our agent-based model that simulates T. solium transmission and associated human neurocysticercosis model, CystiHuman [ 40 ], and evaluates the impact of control interventions in rural northern Peru. Declarations Funding This study was funded by the US National Institutes of Health National Institute of Allergy and Infectious Disease, grant number NIH R01AI141554, and received partial support through Fogarty International Center—US National Institutes of Health, grant number NIH D43TW001140. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials The data collected for this study are available from the corresponding author upon request. Authors` contributions Conceptualization, EG, FP, GB, SG, WP, HG and SO; methodology EG, CM, RG, JB, and SO; validation, EG, FP, GB, SG, and SO; formal analysis, EG, FP, GB, WP, and SO; investigation, EG, FP, GB, SG, and SO; resources, EG, JB, HG and SO; data curation, EG, FP, CM and RG; writing original draft preparation, EG; writing, review and editing, EG, FP, GB, JB, SG, WP, HG and SO; supervision, SO; project administration, HG and SO; funding acquisition, SO. All authors have read and agreed to the published version of the manuscript. Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards at the Universidad Peruana Cayetano Heredia (UPCH) and at Oregon Health & Science University (OHSU). The study was reviewed by the Institutional Ethics Committee for the Use of Animals at UPCH as well as the Institutional Animal Use and Care Committee at OHSU. Treatment of animals adhered to the Council for International Organizations of Medical Sciences (CIOMS) International Guiding Principles for Biomedical Research Involving Animals (Constancy N° 036-10-20, inscription code 103275 and approval date 16 September 2020). Consent for publication Consent for publication is not applicable as the study adhered to institutional and legal standards for animal research, and no individual approvals were required. Competing Interests The authors declare that they have no competing interests. Acknowledgement We would like to express our gratitude for the helpful support received from all the staff at the Center for Global Health at Cayetano Heredia University in Tumbes, Peru. Other members of the Cysticercosis Working Group in Peru include: Robert H. Gilman; Manuela Verastegui; Mirko Zimic; and Victor C. W. Tsang, (Coordination Board); Silvia Rodriguez; Isidro Gonzalez; Herbert Saavedra; Sofia Sanchez; Manuel Martinez, (Instituto Nacional de Ciencias Neurologicas, Lima, Peru); Saul Santivanez; Holger Mayta; Yesenia Castillo; Monica Pajuelo; Luz Toribio; Gianfranco Arroyo; Miguel Angel Orrego, (Universidad Peruana Cayetano Heredia, Lima, Peru); Maria T. Lopez; Cesar M. Gavidia; Luis Gomez-Puerta (School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru); Luz M. Moyano; Sukwan Handali; John Noh (Centers for Disease Control, Atlanta, GA); Theodore E. Nash, (NIAID, NIH, Bethesda, MD); Jon Friedland (Imperial College, London, United Kingdom). References World Health Organization. WHO estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007–2015 [Internet]. Geneva: World Health Organization; 2015 [cited 2025 Jan 24]. Available from: https://iris.who.int/handle/10665/199350 De Coster T, Van Damme I, Baauw J, Gabriël S. Recent advancements in the control of Taenia solium : A systematic review. Food Waterborne Parasitol. 2018;13:e00030. WHO TEAM. Ending the neglect to attain the Sustainable Development Goals: A road map for neglected tropical diseases 2021–2030 [Internet]. WHO; 2020. Available from: https://www.who.int/publications/i/item/9789240010352 Garcia HH, Gonzalez AE, Gilman RH. Taenia solium Cysticercosis and Its Impact in Neurological Disease. Clin Microbiol Rev. 2020;33:e00085-19, /cmr/33/3/CMR.00085 – 19.atom . Lawson JR, Gemmell MA. Hydatidosis and Cysticercosis: The Dynamics of Transmission. Adv Parasitol [Internet]. Elsevier; 1983 [cited 2021 Feb 14]. p. 261–308. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0065308X08604649 Lightowlers MW. Fact or hypothesis: concomitant immunity in taeniid cestode infections. Parasite Immunol. 2010;32:582–9. Gemmell MA, Johnstone PD. Experimental epidemiology of hydatidosis and cysticercosis. Adv Parasitol. 1977;15:311–69. Rickard MD, Williams JF. Hydatidosis/Cysticercosis: Immune Mechanisms and Immunization Against Infection. Adv Parasitol [Internet]. Elsevier; 1982 [cited 2021 May 3]. p. 229–96. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0065308X08602778 de Aluja AS, Villalobos ANM, Plancarte A, Rodarte LF, Hernandez M, Zamora C, et al. Taenia solium cysticercosis: immunity in pigs induced by primary infection. Vet Parasitol. 1999;81:129–35. Flisser A, Pérez-Montfort R, Larralde C. The immunology of human and animal cysticercosis: a review. Bull World Health Organ. 1979;57:839–56. de Aluja AS, Villalobos ANM, Plancarte A, Rodarte LF, Hernández M, Sciutto E. Experimental Taenia solium cysticercosis in pigs: characteristics of the infection and antibody response. Vet Parasitol. 1996;61:49–59. Braae UC, Devleesschauwer B, Gabriël S, Dorny P, Speybroeck N, Magnussen P, et al. CystiSim – An Agent-Based Model for Taenia solium Transmission and Control. Liang S, editor. PLoS Negl Trop Dis. 2016;10:e0005184. Winskill P, Harrison WE, French MD, Dixon MA, Abela-Ridder B, Basáñez M-G. Assessing the impact of intervention strategies against Taenia solium cysticercosis using the EPICYST transmission model. Parasit Vectors. 2017;10:73. Pray IW, Wakeland W, Pan W, Lambert WE, Garcia HH, Gonzalez AE, et al. Understanding transmission and control of the pork tapeworm with CystiAgent: a spatially explicit agent-based model. Parasit Vectors. 2020;13:372. Pizzitutti F, Bonnet G, Gonzales-Gustavson E, Gabriël S, Pan WK, Gonzalez AE, et al. Spatial transferability of an agent-based model to simulate Taenia solium control interventions. Parasit Vectors. 2023;16:410. Gonzales-Gustavson E, Pray IW, Gamboa R, Muro C, Vilchez P, Bonnet G, et al. Evaluating the role of corrals and insects in the transmission of porcine cysticercosis: a cohort study. MD; 2021. Gonzales-Gustavson E, Pizzitutti F, Bonnet G, Muro M, Elizalde M, Muro C, et al. Age at Infection as a Key Predictor of Cyst Burden in Pigs Experimentally Infected with Taenia solium [Internet]. In Review; 2025 [cited 2025 Mar 21]. Available from: https://www.researchsquare.com/article/rs-5883272/v1 Arroyo G, Toribio L, Garrido S, Chile N, Lopez-Urbina T, Gomez-Puerta LA, et al. Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena . Parasit Vectors. 2024;17:172. Gonzalez AE, Cama V, Gilman RH, Tsang VCW, Pilcher JB, Chavera A, et al. Prevalence and Comparison of Serologic Assays, Necropsy, and Tongue Examination for the Diagnosis of Porcine Cysticercosis in Peru. Am J Trop Med Hyg. 1990;43:194–9. Mayta H, Talley A, Gilman RH, Jimenez J, Verastegui M, Ruiz M, et al. Differentiating Taenia solium and Taenia saginata Infections by Simple Hematoxylin-Eosin Staining and PCR-Restriction Enzyme Analysis. J CLIN MICROBIOL. 2000;38:133–7. Wang IC, Ma YX, Kuo CH, Fan PC. A comparative study on egg hatching methods and oncosphere viability determination for Taenia solium eggs. Int J Parasitol. 1997;27:1311–4. Santamaria E, Plancarte A, De Aluja AS. The Experimental Infection of Pigs with Different Numbers of Taenia solium Eggs: Immune Response and Efficiency of Establishment. J Parasitol. 2002;88:69. Lightowlers MW. Diagnosis of Porcine Cysticercosis at Necropsy: When Is Enough, Enough? Trends Parasitol. 2020;36:575–8. García HH, Gonzalez AE, Evans CA, Gilman RH. Taenia solium cysticercosis. The Lancet. 2003;362:547–56. R Core Team. R: A Language and Environment for Statistical Computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing; 2024. Available from: https://www.R-project.org/ Venables WN, Ripley BD. Modern Applied Statistics with S [Internet]. Fourth. New York: Springer; 2002. Available from: https://www.stats.ox.ac.uk/pub/MASS4/ Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York; 2016. Sievert C. Interactive Web-Based Data Visualization with R, plotly, and shiny [Internet]. Chapman and Hall/CRC; 2020. Available from: https://plotly-r.com Gallie G, Sewell M. The survival of cysticercus bovis in resistant calves. Vet Rec. 1972;91:481–2. Gemmell MA, Soulsby EJL. The development of acquired immunity to tapeworms and progress towards active immunization, with special reference to Echinococcus spp. Bull World Health Organ. 1968;39:45–55. Lightowlers M, Gauci C, Jabbar A, Alvarez C. Vaccines Against Cestodes Parasites. Parasit Helmiths Target Screens Drugs Vaccines. Conor R. Caffrey (Editor), Paul M. Selzer (Series Editor). First Edition. Wiley-VCH Verlag GmbH & Co. KGaA; 2012. p. 485–503. Flisser A, Gauci CG, Zoli A, Martinez-Ocana J, Garza-Rodriguez A, Dominguez-Alpizar JL, et al. Induction of protection against porcine cysticercosis by vaccination with recombinant oncosphere antigens. Infect Immun. 2004;72:5292–7. Gemmell MA, Blundell SK, Macnamara FN. Immunological responses of the mammalian host against tapeworm infections. VII. The effect of the time interval between artificial immunization and the ingestion of egg on the development of immunity by sheep to Taenia hydatigena . Exp Parasitol. 1968;23:83–7. Gemmell MA, Lawson JR. The ovine cysticercosis as models for research into the epidemiology and control of the human and porcine cysticercosis Taenia solium : I. Epidemiological considerations. Acta Leiden. 1989;57:165–72. Andrade-Mogrovejo DA, Gonzales-Gustavson E, Ho-Palma AC, Prada JM, Bonnet G, Pizzitutti F, et al. Development of a dose-response model for porcine cysticercosis. Devleesschauwer B, editor. PLOS ONE. 2022;17:e0264898. Pizzitutti F, Bonnet G, Gonzales-Gustavson E, Gabriël S, Pan WK, Pray IW, et al. Non-local validated parametrization of an agent-based model of local-scale Taenia solium transmission in North-West Peru. Sato MO, editor. PLOS ONE. 2022;17:e0275247. Pray IW, Pizzitutti F, Bonnet G, Gonzales-Gustavson E, Wakeland W, Pan WK, et al. Validation of a spatial agent-based model for Taenia solium transmission (“CystiAgent”) against a large prospective trial of control strategies in northern Peru. Torgerson PR, editor. PLoS Negl Trop Dis. 2021;15:e0009885. Pray IW, Wakeland W, Pan W, Lambert WE, Garcia HH, Gonzalez AE, et al. Understanding transmission and control of the pork tapeworm with CystiAgent: a spatially explicit agent-based model. Parasit Vectors [Internet]. 2020 [cited 2020 Oct 1];13. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379812/ Pizzitutti F, Pan W, Barbieri A, Miranda JJ, Feingold B, Guedes GR, et al. A validated agent-based model to study the spatial and temporal heterogeneities of malaria incidence in the rainforest environment. Malar J [Internet]. 2015 [cited 2018 Nov 15];14. Available from: http://www.malariajournal.com/content/14/1/514 Bonnet G, Pizzitutti F, Gonzales-Gustavson EA, Gabriël S, Pan WK, Garcia HH, et al. CystiHuman: A model of human neurocysticercosis. Perkins A, editor. PLOS Comput Biol. 2022;18:e1010118. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6496988","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445814878,"identity":"3177f60d-4c7b-41b3-aa74-8a58b9ca46da","order_by":0,"name":"Eloy Gonzales-Gustavson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYPACCyBmPgDjGRCjRQKI2RJI1sIDV4lfCz//4mMPf9RIyMn3r/kmXZhTF83A3rxNgnGHDU4tkjOepRtIHJMwNrjxdpv0zG2Hcxt4jpVJMJ5Jw6nF4MYZMwkDNonEDRJnt0nzbjuQ2yCRYybB2HYYpxZ7kJaEfxL182eceQbUUpfbIP8GpOU/blv4e8wkDrZJJDCc72EDamEG2sID0nIApxaJG2xpko19EoYbbrAZW/MC/dLGk1ZskXgmGacW/v7DxyR/fLORl+8//PA2yGH97Ic33vi4ww6nFgagk1AZbCAisQG3Dgb+A+gMEGDEp2UUjIJRMApGGgAA0eVSWS4tenYAAAAASUVORK5CYII=","orcid":"","institution":"Universidad Nacional Mayor de San Marcos","correspondingAuthor":true,"prefix":"","firstName":"Eloy","middleName":"","lastName":"Gonzales-Gustavson","suffix":""},{"id":445814879,"identity":"61805a98-968b-4e07-b2d2-b8e3617a6c79","order_by":1,"name":"Francesco Pizzitutti","email":"","orcid":"","institution":"Universidad San Francisco de Quito","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Pizzitutti","suffix":""},{"id":445814880,"identity":"05946484-2bb9-4f59-94c9-bf77a5eb3a9f","order_by":2,"name":"Gabrielle Bonnet","email":"","orcid":"","institution":"London School of Hygiene \u0026 Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gabrielle","middleName":"","lastName":"Bonnet","suffix":""},{"id":445814881,"identity":"4091895f-6d3d-4479-9a9b-209ceee25022","order_by":3,"name":"Claudio Muro","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Claudio","middleName":"","lastName":"Muro","suffix":""},{"id":445814882,"identity":"d8bac8ed-69fa-439e-bc59-4fe17274240f","order_by":4,"name":"Ricardo Gamboa","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Gamboa","suffix":""},{"id":445814883,"identity":"e72891fb-71ae-43ab-bd7d-bb5cd007626e","order_by":5,"name":"Javier A. Bustos","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"A.","lastName":"Bustos","suffix":""},{"id":445814885,"identity":"b54dd0af-1cc9-4d1c-b40f-c430b1a65cf7","order_by":6,"name":"Sarah Gabriël","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Gabriël","suffix":""},{"id":445814886,"identity":"f8e533fe-0659-44e0-a182-99a5e85e9c8e","order_by":7,"name":"William K. Pan","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"K.","lastName":"Pan","suffix":""},{"id":445814888,"identity":"f3a6de93-c2d6-4279-8df7-aec9918ad384","order_by":8,"name":"Héctor H. Garcia","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Héctor","middleName":"H.","lastName":"Garcia","suffix":""},{"id":445814890,"identity":"b7724530-4014-4395-a510-57fee545f405","order_by":9,"name":"Seth O´Neal","email":"","orcid":"","institution":"Oregon Health \u0026 Science University and Portland State University","correspondingAuthor":false,"prefix":"","firstName":"Seth","middleName":"","lastName":"O´Neal","suffix":""}],"badges":[],"createdAt":"2025-04-21 14:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6496988/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6496988/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81199419,"identity":"ca3a0cb9-3211-438b-851f-cc5af4176c5f","added_by":"auto","created_at":"2025-04-23 11:00:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147375,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design of the study, indicating the dose, age at infection/reinfection, number of animals per group planned and achieved, and necropsy. The groups are subdivided as follows: A. Infection and reinfection design: This includes 14 groups of pigs infected with four different doses (corresponding to group “clusters”): 100, 1,000, 5,000, and 20,000 eggs given at four weeks old. Each of the doses is represented in a different color. Each cluster was divided into three groups: one necropsied after 10 weeks and the other two reinfected with 5,000 or 20,000 eggs at 16 weeks old. Two additional groups were infected once at 16 weeks old with each of the reinfection doses, representing reinfection controls. Groups marked with * were also used in the age experiment described in panel B. B. Age experiment: Pigs were infected once with 20,000 eggs at six different ages: four, seven, 10-, 13-, 16-, and 22-weeks old A complete description is available in [17].\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6496988/v1/101b1787529624cea2714520.png"},{"id":81198729,"identity":"7994506a-5445-4d6c-b0da-98f75cf42b61","added_by":"auto","created_at":"2025-04-23 10:52:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9907,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of live cysts (y-axis) found in pigs infected at 4 weeks old with four different doses (x-axis: 100, 1000, 5000, and 20000 eggs) [RED BAR], compared with pigs infected with the same initial dose and reinfected with 5000 eggs [GREEN] or 20000 eggs [BLUE], after 12 weeks.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6496988/v1/94935550e49998647461e246.png"},{"id":81279508,"identity":"4d050dd8-61d3-44f3-8676-be3175c8047a","added_by":"auto","created_at":"2025-04-24 09:53:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":971472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6496988/v1/b75997ac-fe0b-4aa0-94cc-6378c089edc4.pdf"},{"id":81200104,"identity":"55940039-3745-4031-bb99-0c1fd866644c","added_by":"auto","created_at":"2025-04-23 11:08:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":85466,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6496988/v1/3afdc00e6a6592f8a91ca5ec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunity against reinfection in pigs following Taenia solium infection and a quantitative dose-response model","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eTaenia solium\u003c/em\u003e is a zoonotic parasite causing an estimated 2.8\u0026nbsp;million disability-adjusted life years (DALYs) globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It also leads to significant economic losses, particularly in low- and middle-income countries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The parasite is listed among the 20 diseases that disproportionately affect marginalized populations in tropical and subtropical regions and are targeted for control, elimination, or eradication by 2030 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Humans serve as the definitive host, harboring the adult tapeworm, which sheds proglottids and eggs in feces. These eggs are ingested by the intermediate host, the pig, in which cysts develop in muscle and other tissues. Humans can also act as accidental intermediate hosts when they ingest eggs, sometimes leading to the development of neurocysticercosis, a severe condition characterized by cysts in the central nervous system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding the dynamics of transmission\u0026mdash;how, when, where, and why pigs become infected with different numbers of cysts in rural areas\u0026mdash;is critical for designing effective control strategies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. One of the challenges relates to understanding the high variability in the number of cysts in infected pigs, with most infections involving fewer than 10 cysts (making them often hard to identify through meat inspection) while some involve thousands of cysts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These infection patterns are influenced by a complex interplay of parasite, host, and environmental factors, which result in the overdispersion of the larval population within the host population [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Both egg and intermediate host populations exhibit considerable heterogeneity. Eggs vary significantly in quantity and infectivity, while intermediate hosts display a wide range of resistance to infection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A key factor driving resistance to infection is acquired immunity [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], yet, our understanding of this phenomenon is still incomplete.\u003c/p\u003e \u003cp\u003ePrior studies have suggested that animals exposed to two doses of \u003cem\u003eT. solium\u003c/em\u003e eggs may not have more cysts at necropsy than those exposed to a single dose [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, low sample sizes and heterogeneity in animal, egg viability and numbers used in experimental studies have made it hard to confirm and quantify the impact of immunity. In the most recent of those studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], pigs were first infected with 100,000 eggs and later reinfected with the same dose from a different tapeworm, with reinfected pigs harboring no more cysts than those infected once. However, interpretation of the results was complicated by the fact that pigs infected only with the second tapeworm developed very few cysts, making it unclear whether the reinfected pigs were truly resistant or if the second batch of eggs had lower infectivity. Additionally, the study only examined 2 kg of muscle per pig, potentially misrepresenting the actual cyst burden [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. There is therefore a need to build on and improve on this early literature with a study design that will address most or all of the limitations of prior studies.\u003c/p\u003e \u003cp\u003eGreater certainty regarding immunity mechanisms may further feed into mathematical models of \u003cem\u003eT. solium\u003c/em\u003e, which are often employed to simulate disease transmission. In these models, the variability in parasite burdens is often simulated through scenarios where pigs are infected at low and high intensities, sometimes with infection burdens represented by a categorization of pigs between \u0026ldquo;lightly\u0026rdquo; and \u0026ldquo;heavily\u0026rdquo; infected pigs [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Those models generally do not integrate any immunity mechanism, and simulated heterogeneities in pig infection loads may be driven by geographical dispersion of eggs, a phenomenon that remains insufficiently understood, despite recent advances [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, accurate simulation of how pigs with different profiles (age, past exposure) get infected with different parasite loads \u0026ndash; and particularly the impact of immunity in disparities in cyst burdens \u0026ndash; is critical to developing better informed models and more accurately simulating the impact of possible disease control mechanisms.\u003c/p\u003e \u003cp\u003ePrevious studies by our team have looked into geographical dispersion mechanisms for eggs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and the effect of host age as a determinant of variation in infection burden [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, we focused on assessing the role of acquired immunity by infecting and reinfecting pigs in highly controlled conditions and evaluating the total cyst burden across the entire carcass. Additionally, we integrated these results with our earlier findings on innate immunity to develop a model that predicts cyst numbers as a result of infection dose and age at infection. This study aimed to provide a comprehensive and quantified description of the mechanisms by which the host might regulate parasite populations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis study investigates: (1) the effect of reinfection on the development of cysticercosis following an initial infection with varying doses of \u003cem\u003eT. solium\u003c/em\u003e eggs, and (2) the influence of a primary infection with different egg doses on the number of cysts that develop in infected pigs. These analyses are part of a broader investigation, which also examines the effect of pig age on cyst development (reported in a separate manuscript [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). Since some pigs contributed to multiple analyses, we begin by detailing the overall experimental design before describing the statistical methods applied to address the two objectives outlined above.\u003c/p\u003e \u003cp\u003eAnimals\u003c/p\u003e \u003cp\u003eA total of 116 pigs were purchased from 13 commercial farms in northern Peru. Pigs raised in such commercial farms are expected to be unexposed to \u003cem\u003eT. solium\u003c/em\u003e. Farms were selected based on the availability of pigs within the required age range and the capacity of our corrals to accommodate them. Six pigs were excluded from the study: one had an aberrant serologic response at baseline while five died prior to the scheduled necropsy, leaving a final cohort of 110 pigs. These pigs were divided into 18 groups to address two primary objectives: (1) to evaluate the effects of acquired immunity by subjecting different groups to varying infection and reinfection doses, along with their respective infection controls (14 groups described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, this study); and (2) to assess the effects of innate immunity by infecting pigs of different ages with a fixed dose (six groups described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, also reported in a previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). Two groups were common for both experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the present study, we first describe and analyze the effect of reinfection. This is followed by a complementary analysis aimed at predicting the number of viable cysts based on dose, as well as other relevant factors such as sex, age, effect of reinfection, and viability of the egg pool administered.\u003c/p\u003e \u003cp\u003eThe animals were housed in individual pens at the specific pathogen-free facilities of the Center for Global Health at Cayetano Heredia University (UPCH) in Tumbes. They were fed exclusively with properly packaged commercial feed, stored at our facilities to prevent contamination, while water was provided \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSerological testing\u003c/p\u003e \u003cp\u003eIn line with the method described in [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], all pigs underwent serologic screening with antigen ELISA (TsW8/TsW5 mAb set) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and lentil lectin-bound glycoprotein enzyme-linked immunoelectrotransfer blot assay (LLGP-ETIB) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] to rule out \u003cem\u003eT. solium\u003c/em\u003e exposure prior to purchase. To be included in the study, pigs had to be negative on both tests (optical density ratio\u0026thinsp;\u0026lt;\u0026thinsp;1 on Ag-ELISA and absence of any reactive band on LLGP-EITB). The mothers of all animals (except three due to owner refusal) were screened to check that they were also negative. We took weekly blood samples throughout the experiment to monitor the evolution of the serological response using both tests.\u003c/p\u003e \u003cp\u003ePreparation of Egg Pools\u003c/p\u003e \u003cp\u003eIn line with the method described in [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], tapeworms for this experiment were obtained from human stool collected in community settings as part of control interventions. Stool was collected during bowel preparation prior to antiparasitic treatment. It was screened for \u003cem\u003eT. solium\u003c/em\u003e taeniasis using a combination of microscopy, coproantigen ELISA, and molecular confirmation of species using PCR [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Gravid proglottids were collected and stored in a saline solution at 4\u0026deg;C for up to two weeks. This design ensured that the eggs used in this experiment had not been exposed to any antiparasitic drug or preservative. Our study consisted of three waves of infection and reinfection, each comprising two rounds: one for the initial infection and another for reinfection, totaling six rounds. For each round, we created a pool of \u003cem\u003eT. solium\u003c/em\u003e eggs from at least three different tapeworms (each coming from a different donor from the North-West of Peru \u0026ndash; Piura and Tumbes regions) to minimize variability between infections due to differences in egg viability. The exact number of tapeworms used to create the pool utilized in each round (10, 3, 7, 8, 6, 10, and 3 respectively) varied depending on tapeworm availability and the number of viable eggs available for each tapeworm.\u003c/p\u003e \u003cp\u003eEgg viability was assessed for each proglottid and for the entire pool using Evans blue stain before mixing the eggs harvested from the gravid proglottids obtained from the tapeworms used in each pool. We also assessed the percentage of activated oncospheres in each proglottid and pool using the enzyme method and movement of the hexacanth embryo [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The percentage of activated oncospheres was 82%, on average, across all pools (range 63\u0026ndash;97%).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInfection procedure and Necropsy\u003c/h2\u003e \u003cp\u003eInfections and reinfections were administered using an esophageal catheter, following a method previously described in [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] with slight modifications described in [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The assigned dose of \u003cem\u003eT. solium\u003c/em\u003e eggs, suspended in olive oil and encapsulated in gelatin capsules, was delivered to each pig through an esophageal catheter. The capsules were flushed into the pigs\u0026rsquo; stomach using 20\u0026ndash;100 mL of mineral water. An intramuscular combination of ketamine (20 mg/kg) and xylazine (2 mg/kg) was used to anesthetize the pigs. After the procedure, a veterinary team monitored the pigs for vital signs and for any signs of emesis or capsule regurgitation. The reinfection procedure was performed 12 weeks after the initial infection.\u003c/p\u003e \u003cp\u003eWe kept all pigs in individual pens for 10 weeks following the last infection, until necropsy. For necropsy, pigs were anesthetized as per the procedure described above then euthanized via intravenous sodium pentobarbital (100 mg/kg). Trained personnel then meticulously examined all muscles, the brain and other organs using 3\u0026ndash;5 mm slicing to obtain accurate cysts counts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The team identified and counted cysts and recorded the number, type (viable or degenerated) and location of cysts individually for each pig.\u003c/p\u003e \u003cp\u003eExperimental designs\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInfection-reinfection experiment\u003c/h3\u003e\n\u003cp\u003eA total of 65 piglets (four weeks old) and 19 pigs (16 weeks old) were used in the infection-reinfection experiment, corresponding to the groups described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Initially, the four-week-old piglets were divided into four clusters of 15 each and infected with 100, 1,000, 5,000, or 20,000 \u003cem\u003eT. solium\u003c/em\u003e eggs, respectively. After 10 weeks (the time necessary to get fully developed cysts [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]), each cluster was subdivided into three groups of five pigs: One group was necropsied to determine the cyst burden resulting from a single infection and was considered as a positive control of single infection at four weeks old. The remaining two groups were reinfected after two weeks, at 12 weeks of age, with either 5,000 or 20,000 eggs, respectively, to evaluate the additional cyst burden following reinfection with each of these reinfection doses. The remaining five piglets were also infected with 20,000 eggs and correspond to the group included in the age-related infection experiment, in which a total of 10 pigs were used.\u003c/p\u003e \u003cp\u003eIn addition to the piglets, 16-week-old pigs were included as secondary positive controls for single infection at that age and to verify the effectiveness of the egg pool used during reinfection. These control pigs were infected once with either 5,000 or 20,000 eggs at the same time as the reinfected subgroups received their second infection. All animals infected at 16 weeks of age, whether initially infected or reinfected, were necropsied 10 weeks later, at 26 weeks of age. Additional details of each experimental group are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMedian, minimum, and maximum counts of live, degenerated, and total cysts across the different groups involved in the infections at 4 weeks of age, reinfections at 16 weeks of age, and controls (infected only once at 16 weeks of age), along with the corresponding number of pigs per group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClusters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInfection at 4 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInfection at 16 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNumber of pigs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eLive cysts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eDegenerated cysts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003eTotal Cysts\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePigs infected once\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e514\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePigs reinfected with 5000 eggs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e710\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e731\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePigs reinfected with 20000 eggs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e658\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e699\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e238\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e118.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e58.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e833\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDue to logistical and operational constraints, the experiments were carried out in three waves of infection, each spaced approximately six months apart. Each wave included two rounds: an initial infection round, where all pigs in that wave were exposed to \u003cem\u003eT. solium\u003c/em\u003e eggs, followed by a reinfection round, where a subset of the previously infected pigs was exposed again. A new pool of \u003cem\u003eT. solium\u003c/em\u003e eggs was used for each infection round to ensure consistency across exposures (see \u003cb\u003ePreparation of Egg Pools\u003c/b\u003e for details). This design allowed for a controlled evaluation of infection and reinfection dynamics.\u003c/p\u003e\n\u003ch3\u003eQuantitative Dose-Response\u003c/h3\u003e\n\u003cp\u003eWe conducted multiple analyses of the number of cysts developed by pigs as a function of the infective dose. First, we considered only pigs infected once at 4 weeks with varying doses. In a second stage, we incorporated pigs infected twice (at 4 and 16 weeks) and used the results of the analysis of the effects of prior infection on pigs\u0026rsquo; response to infection to combine those pigs with those that were infected only once. Finally, we included all pigs, regardless of their age at first infection, using evidence on the effect of age, as described in a previous paper [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], to build a comprehensive model.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eFor descriptive statistics, the median, minimum, and maximum values were used to summarize data. To analyze differences between infection and reinfection, a negative binomial regression model was developed, with the number of live cysts as the response variable and the infection doses at 4 weeks old and 16 weeks old as predictors, respectively. Other covariables, such as sex, the pigs' farm of origin, the infection round (to consider possible variations between infection periods) and pool viability, were included to identify potential confounding effects.\u003c/p\u003e \u003cp\u003eA negative binomial regression model was also developed to create a quantitative dose-response model using data on the number of live cysts that developed following a single infection at different doses. This model was then expanded, incorporating additional animals with multiple infections and infected at different ages, by including the effect of age as described in a prior manuscript.\u003c/p\u003e \u003cp\u003eModel fit was evaluated using criteria such as Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC), and the best-fitting model was selected based on these indices and residual diagnostics. All the analyses were performed using R [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and the packages MASS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], ggplot2 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and plotly [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eInfection-Reinfection Experiment\u003c/p\u003e \u003cp\u003eThe median, minimum, and maximum numbers of viable, degenerated, and total cysts observed in each of the 14 groups\u0026mdash;either infected once or reinfected\u0026mdash;are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, along with the number of pigs per group. We found that reinfected pigs consistently exhibited similar cyst counts compared to pigs infected only once at different doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To compare cyst counts between the major clusters (pigs infected once versus pigs reinfected with 5,000 or 20,000 eggs) across the four different initial doses (logarithmic transformed), a negative binomial regression model with a two-way factorial 4 x 3 design (four different doses at four weeks old: 100, 1000, 5000, 20000 vs three clusters: infected once, reinfected with 5000 and reinfected with 20000) was applied using data from 12 of the groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Covariables such as sex, pool viability and/or dose adjusted for pool viability were tested but did not significantly improve the model. The analysis showed no statistically significant differences in cyst counts between pigs infected once at 4 weeks old and those reinfected with either 5,000 or 20,000 eggs, regardless of the initial infection dose, including cases where pigs initially infected with as few as 100 eggs were later reinfected with 20,000 eggs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNegative binomial regression model describing the cyst burden as a function of the log of the infection dose at 4 weeks of age and dummies reflecting single infection vs. reinfection at 16 weeks with either 5,000 or 20,000 eggs. The table includes parameter estimates, standard errors, z-values, p-values, and 95% confidence intervals for each variable in the model.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStandard Error\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ez-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e95% Confidence Interval\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Intercept)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-5.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLog (Infection dose at 4 weeks)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.000001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReinfected at 16 weeks with 5,000 vs infected once\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReinfected at 16 weeks with 20,000 vs infected once\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTwo groups of pigs, infected once at 16 weeks of age with either 5,000 or 20,000 eggs, served as controls for the reinfection process. Both groups developed a substantial number of cysts (median: 43 live cysts, 90 total for 5,000 eggs and 118.5 live cysts and 286 in total for 20,000 eggs). Those cyst counts were similar to those of pigs infected at 4 weeks old with the same doses. This suggests that the eggs used in this second infection had infective potential (at least when used to infect na\u0026iuml;ve pigs of the same age) and that, should there be no immunity or partial immunity after a first infection, the statistical analysis would have been able to capture the difference in cyst numbers between pigs infected once at four weeks of age and pigs infected twice (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for the full summary statistics of the controls cluster).\u003c/p\u003e \u003cp\u003eWhile degenerated cysts are of lesser importance (given that they do not contribute to transmission), we also sought to evaluate whether the number of degenerated cysts was higher in reinfected pigs than in pigs infected once. We therefore conducted a Kruskal-Wallis to compare both the number and the proportion of degenerated cysts. Neither comparison showed statistical significance (Kruskal-Wallis H-test: H\u0026thinsp;=\u0026thinsp;2.14, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, P\u0026thinsp;=\u0026thinsp;0.34 for the number of degenerated cysts; H\u0026thinsp;=\u0026thinsp;1.04, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, P\u0026thinsp;=\u0026thinsp;0.59 for the proportion of degenerated cysts).\u003c/p\u003e \u003cp\u003eQuantitative Dose-Response\u003c/p\u003e \u003cp\u003eIn the previous analysis, the dose given to pigs at first infection had a high significant association with the number of viable cysts observed after infection (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), contrary to reinfection status or dose. To describe the relationship between dose and the number of viable cysts, a quantitative dose-response model was developed using negative binomial regression. A first model (referred to as the \"Single Model\") includes only pigs infected once at four weeks of age and analyzes the relationship between the number of viable cysts and the infection dose. Its results are provided in the first lines of Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e below and depicted in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in supplements, where it is represented in pink.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNegative binomial regression model describing the cyst burden as a function of the log of the infection dose. The \u0026ldquo;Single model\u0026rdquo; only includes data from pigs infected once at 4 weeks of age with different doses, the \u0026ldquo;Reinfected model\u0026rdquo; also includes data from pigs reinfected once after initial infection at 4 weeks old. Finally, the full combined \u0026ldquo;Exposure response model\u0026rdquo; also includes information from pigs infected at different ages. The table includes parameter estimates, standard errors, z-values, p-values, and 95% confidence intervals for each variable in the model.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStandard Error\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ez-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e95% Confidence Interval\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSingle model (only includes pigs infected once at 4 weeks of age) -\u003c/b\u003e Theta (θ) value: 0.42; standard error: 0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Intercept)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-8.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLog of Infection dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.000001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eReinfected model (includes all pigs infected at 4 weeks of age) -\u003c/b\u003e Theta (θ) value: 0.43; standard error: 0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Intercept)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLog of Infection dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.000001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExposure response model (includes all pigs) -\u003c/b\u003e Theta (θ) value: 0.43; standard error: 0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Intercept)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInverse of age\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-10.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLog of Infection dose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.000001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eCovariates farm of origin, the infection round, sex and pool viability were tested but were not significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSince no significant differences were observed between infection and reinfection (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), reinfection data were combined with data on pigs infected once into a second model (the \"Reinfected Model\"). This model only includes pigs initially infected at 4 weeks of age. This model preserved the trend of the initial single-infection model while reducing the width of the confidence intervals, enhancing the precision of predictions.\u003c/p\u003e \u003cp\u003eFinally, based on findings from a previous manuscript that described the effect of age and included both an inverse age effect and a term proportional to age [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] a comprehensive model, referred to as the \"exposure response model\" was developed. In the \u0026ldquo;age\u0026rdquo; model, pigs infected at younger ages are less susceptible than those at the natural weaning age (approximately nine weeks old), with susceptibility then progressively decreasing as age increases. The \"exposure response model\" incorporated data from all pigs infected during the experiment, using both dose and age at infection as predictors. The coefficients of this model are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (bottom section). The coefficient for the logarithm of the first infective dose is similar to what it was in each of the prior models and the confidence interval is similar to that of the \u0026ldquo;Reinfected\u0026rdquo; model. The blue curve in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e represents the predicted values from this model for pigs infected at four weeks of age.\u003c/p\u003e \u003cp\u003eSerological results\u003c/p\u003e \u003cp\u003eResults were similar to those already described for pigs involved in the \u0026ldquo;age\u0026rdquo; experiment in [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, all pigs used in this experiment initially tested negative on the LLGP-EITB and had antigen optical density ratios below 1. After infection, all pigs seroconverted on the LLGP-EITB between three- and nine-weeks post-infection (median: five weeks). Pigs also seroconverted on the antigen test (this consistently happened before seroconversion on the LLGP-EITB) between one- and nine-weeks post-infection (median: three weeks).\u003c/p\u003e \u003cp\u003eCysts in the brain\u003c/p\u003e \u003cp\u003eOut of the 110 pigs necropsied during the experiment, 16 had cysts in the brain. One of those harbored six degenerated cysts and 34 live cysts; the remaining animals presented only live cysts. Among the animals with only live brain cysts, six had only one cyst, another six had two cysts, two animals had three cysts, and one animal had nine cysts. The pig that developed 34 live cysts was infected with 5,000 eggs and reinfected with 20,000 eggs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this controlled experimental infection, we aimed to determine the impact of prior exposure to \u003cem\u003eT. solium\u003c/em\u003e eggs and to assess how the number of viable cysts in pigs infected twice compares with the number of such cysts developed by pigs infected only once. This study addresses several limitations of previous studies on experimental infections with taeniids by implementing measures to reduce variability. Specifically, we aimed to ensure greater uniformity and control over host and parasite populations, as well as egg doses, to improve reproducibility and comparability of results. The present study overcame those challenges by using tapeworm pools with controlled infectivity, larger numbers of pigs, and whole-carcass cyst counts. We did not seek to control for pig genetic background as having only pure-bred animals in our experiment would have increased homogeneity but decreased the generalizability of our results, given that the general practice in commercial farming worldwide is to use mixed-breed pigs like the ones in our experiment. The study, despite its tight control of most sources of variability, still found substantial differences in infection outcomes within groups (which was to a certain extent expected [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]) but was nevertheless able to demonstrate the existence of a strong acquired immunity that effectively protects pigs from reinfection. While the exact timing of immunity development remains unclear, our results indicate that the period during which pigs remain susceptible is relatively short in relation to their overall lifespan (less than 12 weeks \u0026ndash; as the pigs in our experiment were reinfected after 12 weeks).\u003c/p\u003e \u003cp\u003eThe effect of acquired immunity had been hypothesized previously for taeniids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and several reviews had discussed how primary infections can block subsequent exposures, with multiple authors examining this phenomenon [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although some experimental studies had reported contradictory results, such as calves being reinfected with \u003cem\u003eT. saginata\u003c/em\u003e 10 weeks after the first infection, most experiments had produced opposite outcomes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The main conclusion from these investigations had been that a form of acquired immunity likely develops following initial exposure [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings ultimately led to the development of the first successful vaccine against a parasitic infection: \u003cem\u003eT. ovis\u003c/em\u003e, in sheep, the intermediate host [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. There are now also commercially available vaccines against \u003cem\u003eEchinococcus granulosus\u003c/em\u003e and \u003cem\u003eT. solium\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study contributed to this body of knowledge by demonstrating that immunity acquired during the initial \u003cem\u003eT. solium\u003c/em\u003e infection is highly protective against infection with subsequent exposure, at least in the timeframe we evaluated. Unlike vaccines, which prime the immune system by exposing it to the TSOL18 oncosphere protein before an infection occurs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], immunity in our study was triggered by actual exposure to eggs.\u003c/p\u003e \u003cp\u003eAs a consequence of the required timeframe to verify reinfection effects at necropsy\u0026mdash;given that cyst development takes approximately 10 weeks [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u0026mdash;the interval between infection and reinfection in this study was necessarily long. Therefore, we could not determine the minimum time required for acquired immunity to become fully active and block reinfection. Studies on other tapeworm species suggest that this occurs progressively. For instance, experiments with \u003cem\u003eT. hydatigena\u003c/em\u003e in sheep have shown that partial resistance can develop as early as seven days post-infection, with complete immunity established by 14 days [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother important consideration is the minimum infective dose required to trigger an effective immune response. While our results demonstrate that as few as 100 eggs can block a reinfection with 20,000 eggs, the precise threshold remains undetermined. Studies on \u003cem\u003eT. hydatigena\u003c/em\u003e and \u003cem\u003eT. ovis\u003c/em\u003e suggest that an effective acquired immunity can be achieved with as few as 10 eggs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], indicating that even minimal exposure may be sufficient to induce protection. Further experiments would be needed to confirm whether this is also true with \u003cem\u003eT. solium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study also showed how cyst burden relates to the egg dose at first infection. As expected, the size of the initial egg dose was found to be a significant positive predictor of the number of viable cysts, enabling the development of a model to predict live cyst numbers. Similar correlation with dose has been reported with other tapeworms and a rate of 1% of egg success was proposed for \u003cem\u003eT. solium\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, our results indicate that the relationship between infection dose and the number of viable cysts is best described by a power-law pattern, rather than a strictly linear one. Specifically, the expected number of cysts increases with the dose raised to the power of 0.88, suggesting a sublinear relationship. While this estimate implies that the proportion of eggs developing into cysts may decrease at higher doses\u0026mdash;consistent with the idea of reduced infection efficiency\u0026mdash;we note that the 95% confidence interval includes 1, meaning this trend is biologically plausible but not statistically conclusive. This interpretation aligns with prior observations that extremely high doses (e.g., 100,000 eggs) do not produce proportionally more cysts [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Such sublinear dynamics complicate the explanation of superinfected pigs found in natural settings, where individuals harbor more than 10,000 cysts [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], raising the possibility that additional biological or ecological mechanisms may contribute to extreme infection outcome.\u003c/p\u003e \u003cp\u003eWe sought to validate our exposure-response model against data from a previous study that compiled results from multiple experimental infections conducted by different research groups worldwide [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The model\u0026rsquo;s predictions aligned well with these independent datasets (considering expected variability in individual pig burden), further supporting its ability to describe \u003cem\u003eT. solium\u003c/em\u003e cyst development across various experimental conditions. This analysis is presented in supplementary material (Fig. S2). In particular, the prior study suggested that pigs infected with 100 eggs had a [62\u0026ndash;100%] probability of developing cysts, a range consistent with our experimental findings, where 11 out of 14 pigs (79%) receiving a first or single infective dose of 100 eggs developed cysts [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results described in this paper, combined with those described in our earlier paper on the effects of age at infection [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], allowed us to leverage this unique experimental dataset to develop an overall exposure-response statistical model predicting the number of live cysts in pigs depending on both the dose of the primary infection and the age at infection. The model performed well in our dataset, capturing the observed effects of both dose and age on cyst burden.\u003c/p\u003e \u003cp\u003eSignificant variability in cyst counts is a common observance in experimental infections with \u003cem\u003eT. solium\u003c/em\u003e and other tapeworm species. We implemented several measures in an attempt to limit sources of variability in our study, including serological monitoring before, during, and after infection; housing pigs in individual pens throughout the experiment; infecting them with pools of several tapeworms using oroesophageal catheters; maintaining a controlled environment to minimize exposure to insects and other pests; and exclusively using properly stored commercial feed. Despite these precautions, we observed significant variability among infected animals, even within the same group. This outcome is not novel and is a characteristic commonly observed in experimental infections with this and other tapeworm species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In our study, the negative binomial dispersion parameter (θ) was estimated at 0.43 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating a high level of overdispersion in cyst counts. This suggests that infection burdens vary substantially among pigs, with a greater variance than expected under a Poisson distribution. The substantial variability in the infection patterns of pigs in endemic areas could be explained in part by this infection pattern. It is also likely that pigs encounter high parasite loads when ingesting proglottids and lower loads when ingesting eggs. Finally, mechanical vectors may play a role in egg dispersion, however, studies in endemic areas have not confirmed a significant impact from this mode of transmission [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eInitial exposure of pigs to \u003cem\u003eT. solium\u003c/em\u003e eggs provides strong acquired immunity against infection upon subsequent re-exposure, with protection evident even when the initial dose is as low as 100 eggs and the reinfection dose as high as 20,000 eggs. Despite minimizing variability during infection\u0026mdash;using eggs with high in vitro viability, pooling eggs instead of entire proglottids, accurately counting eggs in infective doses, and administering infections intra-esophageally\u0026mdash;significant variability in cyst counts was still observed within groups, suggesting that other factors may influence individual responses. Using data from these experiments, we developed a quantitative dose-response model to predict the number of live cysts in infected pigs based on egg exposure dose and age at infection. This model provides essential insights for incorporating pig immunity processes into CystiAgent [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], our agent-based model that simulates \u003cem\u003eT. solium\u003c/em\u003e transmission and associated human neurocysticercosis model, CystiHuman [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and evaluates the impact of control interventions in rural northern Peru.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was funded by the US National Institutes of Health National Institute of Allergy and Infectious Disease, grant number NIH R01AI141554, and received partial support through Fogarty International Center\u0026mdash;US National Institutes of Health, grant number NIH D43TW001140. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data collected for this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003eAuthors` contributions\u003c/p\u003e\n\u003cp\u003eConceptualization, EG, FP, GB, SG, WP, HG and SO; methodology EG, CM, RG, JB, and SO; validation, EG, FP, GB, SG, and SO; formal analysis, EG, FP, GB, WP, and SO; investigation, EG, FP, GB, SG, and SO; resources, EG, JB, HG and SO; data curation, EG, FP, CM and RG; writing original draft preparation, EG; writing, review and editing, EG, FP, GB, JB, SG, WP, HG and SO; supervision, SO; project administration, HG and SO; funding acquisition, SO. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards at the Universidad Peruana Cayetano Heredia (UPCH) and at Oregon Health \u0026amp; Science University (OHSU). The study was reviewed by the Institutional Ethics Committee for the Use of Animals at UPCH as well as the Institutional Animal Use and Care Committee at OHSU. Treatment of animals adhered to the Council for International Organizations of Medical Sciences (CIOMS) International Guiding Principles for Biomedical Research Involving Animals (Constancy N\u0026deg; 036-10-20, inscription code 103275 and approval date 16 September 2020).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eConsent for publication is not applicable as the study adhered to institutional and legal standards for animal research, and no individual approvals were required.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to express our gratitude for the helpful support received from all the staff at the Center for Global Health at Cayetano Heredia University in Tumbes, Peru. Other members of the Cysticercosis Working Group in Peru include: Robert H. Gilman; Manuela Verastegui; Mirko Zimic; and Victor C. W. Tsang, (Coordination Board); Silvia Rodriguez; Isidro Gonzalez; Herbert Saavedra; Sofia Sanchez; Manuel Martinez, (Instituto Nacional de Ciencias Neurologicas, Lima, Peru); Saul Santivanez; Holger Mayta; Yesenia Castillo; Monica Pajuelo; Luz Toribio; Gianfranco Arroyo; Miguel Angel Orrego, (Universidad Peruana Cayetano Heredia, Lima, Peru); Maria T. Lopez; Cesar M. Gavidia; Luis Gomez-Puerta (School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru); Luz M. Moyano; Sukwan Handali; John Noh (Centers for Disease Control, Atlanta, GA); Theodore E. Nash, (NIAID, NIH, Bethesda, MD); Jon Friedland (Imperial College, London, United Kingdom).\u003c/p\u003e\n\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. 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PLOS Comput Biol. 2022;18:e1010118.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Taenia solium, experimental infection, acquired immunity, reinfection, quantitative dose-response, live cysts prediction","lastPublishedDoi":"10.21203/rs.3.rs-6496988/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6496988/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e \u003cem\u003eTaenia solium\u003c/em\u003e is a zoonotic parasite causing significant health and economic burdens, with complex transmission dynamics requiring improved control strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThis study investigates the effect of \u003cem\u003eT. solium\u003c/em\u003einfection and reinfection on cyst development in pigs and evaluates how acquired immunity constrains parasite burden. A total of 116 pigs were purchased from commercial farms in northern Peru and housed in pathogen-free facilities under controlled conditions. Of these, 110 pigs were allocated to 18 experimental groups to (1) evaluate the impact of infection and reinfection with varying doses of \u003cem\u003eT. solium\u003c/em\u003e eggs and (2) generate a model to predict the number of live cysts produced, given the dose and age at infection. Gravid proglottids collected from human cases of \u003cem\u003eT. solium\u003c/em\u003e taeniasis were used to prepare egg pools, ensuring viability consistency. Infections were administered orally using gelatin capsules via esophageal catheterization, followed by necropsy 10 weeks after the final infection event to quantify cysts. A negative binomial regression model was used to analyze cyst burden dependence on infection dose, past infection, age, and other factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e No statistically significant differences in cyst counts were observed between pigs infected once and those that were reinfected, regardless of the initial dose (as low as 100 eggs) or reinfection dose (up to 20,000 eggs). This finding highlights that infection results in strong acquired immunity, effectively blocking subsequent infections. A quantitative dose-response model suggests that the relationship between egg dose and the number of viable cysts is best described by a power relationship. Combining data from single-infection and reinfected pigs into a unified model improved prediction precision. Finally, incorporating age at infection results in a model of the number of viable cysts in pigs depending on dose and age that combines acquired and innate immunity effects, i.e. changes in susceptibility with age.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Initial exposure to \u003cem\u003eT. solium\u003c/em\u003eeggs induces strong acquired immunity in pigs, effectively preventing reinfection. Our quantitative dose-response model predicting live cyst counts based on egg dose and pig age offers valuable insights for integrating immunity processes into models of \u003cem\u003eT. solium\u003c/em\u003e transmission.\u003c/p\u003e","manuscriptTitle":"Immunity against reinfection in pigs following Taenia solium infection and a quantitative dose-response model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 10:52:37","doi":"10.21203/rs.3.rs-6496988/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"abe82429-42aa-46f5-800c-8c5f2c7ff78b","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-24T09:53:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-23 10:52:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6496988","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6496988","identity":"rs-6496988","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}