Age at Infection as a Key Predictor of Cyst Burden in Pigs Experimentally Infected with Taenia solium

Age at Infection as a Key Predictor of Cyst Burden in Pigs Experimentally Infected with Taenia solium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Age at Infection as a Key Predictor of Cyst Burden in Pigs Experimentally Infected with Taenia solium Eloy Gonzales-Gustavson, Francesco Pizzitutti, Gabrielle Bonnet, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5883272/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Parasites & Vectors → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Taenia solium cysticercosis is a zoonotic parasitic disease with significant public health implications, particularly in endemic regions of low- and middle-income countries. In pigs, cyst burden varies widely, with most harboring fewer than 10 cysts and only a small fraction carrying high cyst loads. Age has been identified as a key factor influencing infection susceptibility. However, inconsistencies in previous studies have hindered clear characterization of infection patterns and immunity. In this study, we conducted controlled experiments involving the infection of pigs with T. solium eggs to evaluate the relationship between pig age and susceptibility to infection. Methods: A total of 52 pigs from northern Peru, aged 4 to 22 weeks, were experimentally infected with T. solium eggs to examine age-related differences in cyst burden. Pigs were housed individually under controlled conditions and fed commercial pig diets. Infections were administered using an esophageal catheter, delivering 20,000 T. solium eggs encapsulated in gelatin capsules. Six age groups were studied using a standardized egg pool to ensure consistency across infection rounds. After 10 weeks, necropsies were performed to count cysts in all muscles, the brain, and other organs. Weekly serological tests monitored seroconversion. Statistical models were used to analyze cyst counts and assess the effects of age and other predictors. Results: The number of live, degenerated, and total cysts was overdispersed making a negative binomial model the most suitable choice to represent the data and their dependence on age at infection. Younger pigs showed low median live cyst count, similar to older pigs, while median cyst burden increased in pigs infected at intermediate ages, around natural weaning age. The negative binomial regression showed that age and a covariate inversely related to age at infection were significantly associated with cyst count at necropsy. Other covariates such as pool viability and sex did not significantly affect model performance. Serological tests confirmed seroconversion in all pigs. Conclusions: Our results show that younger pigs display partial protection against the development of cysticerci compared to those infected at the natural weaning age (around 9 to 12 weeks of age). Additionally, infection susceptibility then decreases with age in a way that is consistent with previous literature hypothesizing near-complete resistance by one year of age. Taenia solium pig cysticercosis age at infection innate immunity susceptibility at infection Figures Figure 1 Figure 2 Background Taenia solium cysticercosis is a zoonotic parasitic infection that poses significant public health challenges, particularly in low- and middle-income countries [ 1 ]. The parasite’s lifecycle involves humans as the definitive host, with the adult tapeworm residing in the intestine where it sheds proglottids and eggs in the host’s feces. When these eggs are consumed by free-roaming pigs, they develop into the larval stage, known as cysticercosis. Humans can also inadvertently ingest the eggs, leading to the development of cysticercosis in various tissues, most notably in the central nervous system, causing neurocysticercosis, a severe neurological condition [ 2 ]. In endemic regions, an estimated 1–3% of the human population harbors the adult T. solium tapeworm [ 3 , 4 ], serving as the primary source of cysticercosis and leading to a pig infection prevalence of approximately 10–30% [ 5 – 7 ]. Pigs with high cyst burdens, hundreds to thousands of cysts, readily visible in the carcass during slaughter, represent only a small minority of infections in the population. The majority of infected pigs harbor fewer than 10 cysts [ 8 ] and are less likely to be detected during slaughter. Typically, over 80% of the parasites are harbored by fewer than 20% of the hosts [ 9 ]. This phenomenon, known as parasite aggregation, is broadly attributed to several density-dependent mechanisms, both intrinsic and extrinsic to the pig, which contribute to the observed overdispersion in infection pattern [ 10 ]. Innate and acquired immunity are two such mechanisms that are known to play a critical role in limiting the number of cysts in pig populations infected with T. solium [ 11 ]. Past evidence suggests that age at infection, sex, and the genetic strain of the host animal are potential contributors to variability in infection with various taeniid cestodes, with age emerging as the most influential factor [ 12 , 13 ]. Age has been identified as contributing to innate resistance among naïve or previously unexposed animals challenged with different tapeworm species such as T. taeniaeformis in rats, T. pisiformis in rabbits, T. saginata in cattle and T. hydatigena in sheep [ 12 ]. It has further been suggested that immunity may not fully prevent the establishment of new cysts but likely modulates the infection’s progression over time [ 11 ]. A few studies have utilized controlled exposure to more accurately characterize resulting infection intensity at different ages [ 14 ]. However, methodological inconsistencies—such as insufficient standardization of the infective dose, differences in egg batches, pig breeds, incomplete organ examination, and the inability to detect cysts due to an inappropriately short time interval between infection and necropsy [ 12 , 13 ] limit the strength of the evidence. This significant variability in cyst burden presents challenges for implementing effective strategies for monitoring and evaluating the impact of potential control interventions [ 15 , 16 ]. While understanding pig immunity is critical to determining what control or elimination interventions are likely to be most effective, the evidence in this regard remains limited and insufficiently precise to feed into intervention modelling, even when considering studies with other taeniid cestodes. The objective of this study was to accurately characterize differences in infection burden across age groups after a single infection, to improve understanding of age-related and innate immunity, as well as to inform representation of pig immunity in transmission simulation models designed to guide interventions. We achieved this through a controlled experiment designed to minimize as many sources of variation as possible, allowing us to determine differences in cyst burden in pigs infected with T. solium at varying ages. Methods Animals The initial study design included 40 pigs, divided into four age groups: four, 10, 16, and 22-weeks of age (10 per age group). Due to space limitations, infections were conducted in six separate rounds. After the 4th round, interim results suggested that more data were needed to improve precision around 10 weeks of age. Therefore, we added 12 more pigs: we created two new age groups (seven and 13 weeks-old, with five pigs each) and included two additional 10-week-old pigs. This brought the final total to 52 pigs. The pigs used in this study were purchased from nine different commercial farms in northern Peru, selected based on the availability of pigs of the required ages for each experimental round. The acquired pigs were of mixed breeds commonly found in the region. While mixed-breed pigs are more genetically variable than pure breeds, they are more representative of the pigs that are used in pig farming, hence ensuring better generalizability in our results. Five pigs were excluded leaving an analytic sample of 47 pigs (Table 1 ); four died prior to the scheduled necropsy and one had an aberrant serologic response at baseline. Table 1 Median, minimum, and maximum numbers of live, degenerated, and total cysts by age at infection, along with the corresponding number of pigs per group Live cysts Degenerated cysts Total cysts Age at infection Number of pigs Median Minimum Maximum Median Minimum Maximum Median Minimum Maximum 4 weeks 7 103 0 270 63 5 490 192 71 514 7 weeks 5 642 172 1126 17 3 25 645 180 1143 10 weeks 11 66 0 1682 12 0 326 172 1 1705 13 weeks 5 216 152 552 10 5 15 231 165 557 16 weeks 10 118.5 1 809 58.5 1 260 286 30 833 22 weeks 9 7 0 171 71 1 217 75 2 394 The pigs were housed in individual pens at the specific pathogen-free facilities of the Center for Global Health at Cayetano Heredia University (UPCH) in Tumbes. The animals were fed exclusively with properly packaged commercial feed, stored at our facilities to prevent contamination. Water was provided ad libitum . Serological test All pigs underwent serologic screening to rule-out prior exposure to T. solium before purchase, using lentil lectin-bound glycoprotein enzyme-linked immunoelectrotransfer blot assay (LLGP-ETIB) [ 17 ] and antigen ELISA (TsW8/TsW5 mAb set) [ 18 ]. All pigs were required to be negative on both tests (absence of any of the 7 reactive bands on LLGP-EITB; optical density ratio < 1 on Ag-ELISA) to be included in the study. The mothers of all included pigs, except three due to owner refusal, were also screened to ensure negative status of the sow. Throughout the duration of the experiment, weekly blood samples were also taken to monitor the evolution of seroconversion with both tests. These results will feed into a separate paper. Preparation of Eggs Pool Tapeworms for this experiment were sourced from control interventions in which human stool was collected from the community setting and screened for T. solium taeniasis using a combination of microscopy, coproantigen ELISA, and molecular confirmation of species using PCR [ 19 ]. Stool was collected during bowel preparation prior to antiparasitic treatment. Gravid proglottids were collected and stored in saline solution at 4°C for a maximum of two weeks, ensuring that eggs used in the experiment had not been exposed to antiparasitic drugs or preservatives. To minimize variability between infections due to differences in egg viability, we pooled T. solium eggs from multiple tapeworms for each round of infections, using a new pool for each round. The number of tapeworms used to create each pool was 10, 3, 7, 8, 6 10 and 3, respectively. The number of tapeworms per round varied depending on tapeworm availability and the number of viable eggs available for each tapeworm, however, we managed to combine at least 3 tapeworms to prepare the pool used in each round. Each tapeworm originated from a different human donor from the North-West of Peru (Piura and Tumbes regions). Pools were prepared by mixing eggs harvested from gravid proglottids obtained from three to ten different tapeworms. Egg viability (Evans blue stain) and percentage of activated oncospheres using the enzyme method and movement of the hexacanth embryo [ 20 ] were assessed for each proglottid and for the entire pool. The mean percentage of activated oncospheres across all pools was 82% (range 63–97%). Experimental design In total, six different groups of pigs, corresponding to infections at four, seven, 10, 13, 16, and 22 weeks of age, were experimentally infected over six rounds (Fig. 1 ). The same pool of eggs was used for each round and pigs of different age groups were infected with eggs from the same pool. Infections were administered using an esophageal catheter, following a previously described method with slight modifications [ 21 ]. Briefly, each pig received 20,000 T. solium eggs suspended in olive oil, encapsulated in gelatin capsules, which were delivered through an esophageal catheter. The capsules were then flushed into the stomach with 20–100 mL of bottled drinking water. All pigs were anesthetized with a combined intramuscular dose of ketamine (20 mg/kg) and xylazine (2 mg/kg). After the infection procedure, the pigs were monitored by a veterinary team for stress reaction (vital signs) and for signs of emesis or regurgitation of the capsules. Pigs were then maintained in individual pens for a period of 10 weeks until necropsy. All pigs were weighed at the time of infection, and the median, minimum, and maximum weight per group are provided as Supplementary Information in Table S1 . For necropsy, pigs were induced to anesthesia with intramuscular ketamine (20 mg/kg) and xylazine (2 mg/kg) and then euthanized by intravenous sodium pentobarbital (100 mg/kg). The carcass was then dissected by trained staff using 3 to 5 mm slices of all muscles, brain, and other organs, and visually inspected to identify and count cysts, recording the number and type (viable or degenerated) of cysts for each animal. Data analysis We used Kruskal-Wallis as a preliminary test to evaluate differences in live cyst counts between pigs infected at different ages. This non-parametric test was chosen due to its robustness against violations of normality assumptions and its ability to handle skewed data distributions commonly observed in cyst count outcomes. Significant differences detected by the Kruskal-Wallis test indicated the need for further exploration of age-related effects through more advanced modeling techniques. We evaluated several statistical regression models, including Poisson, Negative Binomial, Zero-Inflated Poisson, Zero-Inflated Negative Binomial, and negative binomial family Generalized Estimating Equations, for their ability to fit the observed counts of live T. solium cysts in pigs infected at different ages. Each model was assessed for its ability to predict the number of live cysts while accounting for the distribution of the count data and potential overdispersion. Several predictors were considered and incorporated into the models if they improved their fit to the data. These predictors included the farm of origin of the pigs, the round of infection (to account for potential variation between infection periods), the viability of each egg pool (to adjust for differences in the proportion of viable oncospheres across rounds), the inverse of age and quadratic age (to capture potential non-linear effects of age on cyst counts), and the sex of the pigs. 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. Most of the analyses were performed using R [ 22 ] and the packages MASS [ 23 ], pscl [ 24 ] and ggplot2 [ 25 ], except for the Generalized Estimating Equations that was developed with the xtgee command in STATA [ 26 ]. Results The median number of live, degenerated and total cysts, along with their respective ranges across different ages at infection, are presented in Table 1 . A significant variation in the median number of live cysts was observed between age groups (Kruskal-Wallis H-test, H = 15.65, df = 5, P = 0.008), suggesting that the age at infection influences cyst burden in pigs. Specifically, younger pigs showed similarly low median cyst counts as the older ones, with a notable increase in cyst burden observed in pigs infected at intermediate ages. The extent of this variation is detailed in Table 1 . These results suggest that age plays an important role in the progression of cysticercosis following infection with T. solium . The pattern observed for degenerated cysts was the inverse of that seen for live cysts (Table 1 ). The number of degenerated cysts was higher in younger and older pigs compared to those infected at intermediate ages, with marginally significant differences between age groups (Kruskal-Wallis H-test, H = 10.29, df = 5, P = 0.07). The total number of cysts (live and degenerated combined) followed a trend comparable to that of live cysts but with less pronounced differences with age. These differences were marginally significant (Kruskal-Wallis H-test, H = 10.31, df = 5, P = 0.07). Median values for each group are provided in Table 1 . Only four pigs harbor cysts in the brain, with a total of seven cysts found in just two groups: pigs infected at 7 and 16 weeks old. Each pig had between one and two cysts, and all cysts were viable. Statistical model prediction All statistical models gave similar results. However, the negative binomial regression emerged as the most suitable model to predict the number of live cysts as a response variable based on age and other potential covariates. This conclusion was supported by the lower AIC, BIC, and by likelihood ratio comparisons. We considered a model linear in age as well as a model introducing additional quadratic or inverse terms. The inclusion of either a quadratic or inverse age term improved the model by providing a better representation of the observed cyst counts at younger ages. We selected the inverse of age as it has a more straightforward biological interpretation and provides credible figures (contrary to the introduction of a quadratic term) when extrapolating the model to pigs older than those in our experiment. Covariates such as farm of origin of the pig, pool viability and sex were also tested but did not significantly affect the model coefficients or improve model fit. The coefficients of the final model, along with their respective confidence intervals, are presented in Table 2 , while the model's predictions, along with their credible intervals and observed values, are depicted in Fig. 2 . Table 2 Coefficients, standard errors, z-values, p-values, and 95% confidence intervals for the negative binomial regression model predicting the number of live cysts. The model includes the intercept, age, and the inverse of age as predictors. Variable Estimate Standard Error z-value p-value 95% Confidence Interval Minimum Maximum (Intercept) 10.847 1.596 6.80 < 0.001 7.36 14.30 Age -0.269 0.074 -3.64 0.0003 -0.42 -0.10 Inverse of Age -20.335 6.585 -3.09 0.0022 -33.64 -5.51 Serological results All pigs used in this experiment started with no bands on LLGP-EITB and with antigen optical density ratio < 1. Additionally, all pigs exhibited seroconversion of at least one LLGP-EITB band after infection, occurring between three- and 9-weeks post-infection, with a median seroconversion time of five weeks. Following the initial seroconversion, all pigs, except for one (which had only one live cyst out of a total of two), maintained seroconversion and typically kept or increased the number of LLGP-EITB bands, which has been shown to be correlated with the presence and level of infection in prior literature [ 27 , 28 ]. Seroconversion on the antigen test consistently occurred before the LLGP-EITB test, between one- and 9-weeks post-infection, with a median time of 3 weeks. All animals exceeded the threshold of one (meaning they were positive on the antigen test) [ 18 ] at least once after infection; in 38 animals, the optical density ratio exceeded three at least at some point after infection, nine maintained values lower than three, and five had an initial increase followed by a progressive decrease until the time of necropsy. These five pigs all had fewer than 10 live cysts. These results, which are important to refine our understanding of how serology relates to infection, will be analyzed in detail in a future publication. Discussion There is high variability in parasite burdens in animals infected with cestodes, particularly T. solium . Innate immunity may contribute to these effects. Statistical analysis of the results of this study demonstrated a relationship between age and cyst burden. These results suggests that the immunity of naïve pigs (not previously exposed) is higher in younger and older pigs, and lowest around 9 weeks of age, with the relationship between age and the logarithm of the mean number of cysts best represented through the sum of two terms, one proportional to age, and one proportional to inverse age. There are many challenges in measuring the contribution of immunity to cyst development, including the effect of pig genetic lineage and variability in egg batches and numbers [ 12 ], and potentially host microbiome or stress. In this study, several of these variables were controlled for, including through the use of pooled eggs from different tapeworms with confirmed viability and hatching rates, as well as direct inoculation into the stomach to ensure consistent exposure. However, genetic variability among animals could not be controlled in the design of the experiment. The pigs we used came from commercial farms where standardized purebred lines are not commonly maintained. Instead, modern commercial pig production relies on crossbred lines selected by producers to optimize productivity based on growth rate, feed efficiency, and carcass quality. The specific mixed breeds used were typical of the northern Peru region, resulting from genetic improvement programs in rural areas, and commonly found in Peruvian villages. To meet the strict age group requirements for the study, pigs were sourced from multiple granges. It is plausible that granges may differ somewhat in the genetic makeup of their pigs, hence we included the farm of origin among the explanatory variables we considered in the statistical analysis and were able to demonstrate that inclusion of this variable did not improve the model. Note that, to our knowledge, no study has ever been undertaken regarding differences in susceptibility to infection among pig breeds or lines, so we could not rely on the literature to support or disprove a possible link between pig genetics and susceptibility. The relatively high variability observed within age groups highlights the complexity of the factors that determine cyst development, even under controlled experimental conditions, emphasizing the importance of accounting for such heterogeneity in future investigations. Several explanations have been put forward to justify the change in susceptibility due to age. With respect to younger ages, in rats infected with T. taeniaeformis , this change was attributed to little or no proteolytic activity in their intestines to assist in the hatching of eggs [ 29 ]. However, several other components could also be involved, such as innate immunity transferred by the mother, or pH or other differences in gastric fluids. We ruled out passive transfer of maternal acquired immunity because the mothers were free of infection. Evidence from other Taenia species also suggests that the passive transfer of maternal acquired antibodies does not play a role [ 13 , 30 ]. Our research group conducted a previous study of age on infection in which pigs were infected at one, three, and 5 months with a single proglottid each. While the current study has coherent results for older pigs, there are differences in the youngest group: in the earlier study, pigs infected at one month of age had the highest mean live cyst counts and percentage of viable cysts [ 14 ]. However, this method of infection is subject to the large variability in the number of eggs per proglottid, reported to range from 3,900 to 120,000 eggs [ 31 ], which is one of the main limitations we wanted to address in the current study. Given this important distinction, comparison of results across these two studies is therefore challenging. The increased susceptibility observed at the natural weaning age [ 32 , 33 ] and the progressive resistance to infection with age seen in this study are consistent with patterns commonly reported in other cestodes, though these trends have not been thoroughly described [ 12 , 13 ]. The peak susceptibility coincides with the natural weaning age, but we can rule out weaning itself as a causal factor, as the 4-week-old animals in this study were forcibly weaned. If immunosuppression or stress due to weaning had been significant, it would also have affected these animals. Regarding the increased resistance with age in older pigs, experimental infections with limited numbers of animals [ 34 , 35 ], as well as evidence from mass necropsies in endemic areas, suggest the potential for complete resistance to infection over time [ 36 , 37 ], even though this has not been completely demonstrated experimentally for T. solium . Given that the pigs had no prior exposure, as confirmed by two serological methods, this decrease in susceptibility can only be attributed to a progressive strengthening of innate immunity. Nonetheless, certain stress factors may influence susceptibility. For instance, farrowing has been reported to predispose sows to reinfection due to immunosuppressive effects during this period [ 38 ]. This phenomenon has been used to explain the presence of cysticerci with different microsatellite patterns in a naturally infected sow, suggesting infections by distinct tapeworms at different times [ 39 , 40 ]. Possibly the best description of a similar pattern of infection at different ages comes from two separate studies involving T. hydatigena in lambs grazed on pastures permanently contaminated by experimentally infected dogs. These studies reported a progressive increase in the number of live cysts from weeks one to 12 post-infection [ 41 ], followed by a progressive decrease from three to 6 months [ 42 ]. In this case, the total number of cysts also differed by age, with younger lambs showing a lower proportion. While these findings were partly attributed to lower ingestion of contaminated pasture, they were also linked to passive immunity transferred from the mother or a combination of these factors. Comparing these results with the experimental infection reported here, it is also plausible that susceptibility to infection is influenced by the age at which exposure occurs. Given the marked difference in the number of live cysts between ages, accompanied by an inverse pattern in degenerated cysts and a marginal difference in the number of total cysts (following the same pattern as live cysts), our results might be due to a pre-encystment immunity. This concept, introduced by Gemmell and Soulsby [ 43 ], describes the early-stage challenges faced by the oncosphere after ingestion. These challenges include exposure to gastric acid in the stomach, interaction with the intestinal epithelium, circulation through the bloodstream, and eventual implantation and growth within muscular tissue to form larvae. During the pre-encystment phase, something—either in transit or at the site of implantation—reduced their ability to develop into viable cysts. The exact mechanisms or factors responsible for this differential success rate remain unclear, highlighting a need for further research into the specific host or environmental factors affecting this process. One of our primary aims in undertaking this study was to use the results to inform inclusion of pig immunity into our agent-based model, CystiAgent (and associated neurocysticercosis model, CystiHuman) to improve simulation of endemic transmission and the effect of interventions [ 44 – 48 ]. The interaction of age-related pig susceptibility and the environmental exposure to T. solium eggs, which eventually leads to infection, depicts a complex scenario in which multiple processes and factors likely interact across varying scales of time and space. Among the practical implications are the optimal age at which to undertake interventions in pigs (e.g. vaccination/treatment of younger vs. older pigs) and the frequency of intervention that could be most cost-effective. Further investigation is needed to explore additional factors influencing the infection patterns of T. solium , particularly acquired immunity. This study has several limitations that should be considered when interpreting the findings. First, the pigs used in the experiments came from different farms, which may introduce variability in their baseline immunity, health status, or other intrinsic factors. Additionally, while our infection system ensures the controlled uptake of a precise number of eggs, it does not fully replicate natural infection dynamics. Natural infections are likely to occur gradually, with animals being exposed to varying doses of eggs over time, starting at a young age. In contrast, our experimental design involved a single, high-dose exposure (20,000 eggs), which may not fully reflect field conditions and could influence the immune response observed. Moreover, our method of delivering eggs directly into the stomach bypasses natural routes of exposure, such as oral ingestion, which could impact how the immune system is activated. While the difference between these methods may be minimal, it remains a factor to consider. Finally, as an experimental system, the controlled nature of our study does not fully replicate the complexity of real-world scenarios, such as continuous low-level exposure in highly endemic areas. Future studies should explore the effects of reinfections and different infection doses to better understand the natural immune response and its implications for disease control strategies. Conclusions This study represents the first controlled infection trial in pigs using T. solium eggs that effectively accounts for key confounding factors. Our findings reveal that younger pigs exhibit partial protection against developing cysticerci compared to those infected at natural weaning age. Moreover, beyond that age, susceptibility to infection progressively decreases with age in a way that is coherent with prior hypotheses in the literature of complete or near-complete resistance by approximately one year of age. Although our results demonstrate that higher susceptibility occurs around 7 and 16 weeks of age, the maximum susceptibility estimated by our model is likely to occur around 9 weeks of age. These results provide critical insights into the age-related dynamics of porcine susceptibility to T. solium and have significant implications for understanding the epidemiology and control of cysticercosis. Declarations Acknowledgements 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; Javier Bustos; 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; 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). 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, MM, ME, CM, RG, GA, 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, GA, HG and SO; data curation, EG, FP, MM, CM and RG; writing original draft preparation, EG; writing, review and editing, EG, FP, GB, 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. References WHO TEAM. 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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. 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. 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. 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/ Zeileis A, Kleiber C, Jackman S. Regression Models for Count Data in R. J Stat Softw [Internet]. 27. Available from: URL http://www.jstatsoft.org/v27/i08/ Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York; 2016. StataCorp. Stata 18: Data Analysis and Statistical Software. College Station TX: StataCorp LLC. 2023. Arroyo G, Lescano AG, Gavidia CM, Lopez-Urbina T, Ara-Gomez M, Gomez-Puerta LA, et al. Antibody Banding Patterns on the Enzyme-Linked Immunoelectrotransfer Blot (EITB) Assay Clearly Discriminate Viable Cysticercosis in Naturally Infected Pigs. Pathogens. 2023;13:15. Jayashi CM, Gonzalez AE, Castillo Neyra R, Rodríguez S, García HH, Lightowlers MW. Validity of the Enzyme-linked Immunoelectrotransfer Blot (EITB) for naturally acquired porcine cysticercosis. Vet Parasitol. 2014;199:42–9. Musoke AJ, Williams JF, Leid RW, Williams CS. The immunological response of the rat to infection with Taenia taeniaeformis . IV. Immunoglobulins involved in passive transfer of resistance from mother to offspring. Immunology. 1975;29:845–53. Lightowlers MW, Rickard MD, Mitchell GF. Taenia taeniaeformis in mice: Passive transfer of protection with sera from infected or vaccinated mice and analysis of serum antibodies to oncospheral antigens. Int J Parasitol. 1986;16:307–15. Ma Y, Su Y, Yan Q, He L, Yan X, Chen J, et al. [Study on number and mature rate of eggs in gravid proglottids of Taenia solium ]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2002;20:98–100. Stolba A, Wood-Gush DGM. The behaviour of pigs in a semi-natural environment. Anim Sci. 1989;48:419–25. Boe K. The process of weaning in pigs: when the sow decides. Appl Anim Behav Sci. 1991;30:47–59. 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. de Aluja AS, Martinez M JJ, Villalobos ANM. Taenia solium cysticercosis in young pigs: age at first infection and histological characteristics. Vet Parasitol. 1998;76:71–9. Dixon MA, Winskill P, Harrison WE, Whittaker C, Schmidt V, Sarti E, et al. Force-of-infection of Taenia solium porcine cysticercosis: a modelling analysis to assess global incidence and prevalence trends. Sci Rep. 2020;10:17637. Poudel I, Sah K, Subedi S, Kumar Singh D, Kushwaha P, Colston A, et al. Implementation of a practical and effective pilot intervention against transmission of Taenia solium by pigs in the Banke district of Nepal. Fuehrer H-P, editor. PLoS Negl Trop Dis. 2019;13:e0006838. Dixon MA, Winskill P, Harrison WE, Whittaker C, Schmidt V, Flórez Sánchez AC, et al. Global variation in force-of-infection trends for human Taenia solium taeniasis/cysticercosis. eLife. 2022;11:e76988. Pajuelo MJ, Eguiluz M, Roncal E, Quiñones-García S, Clipman SJ, Calcina J, et al. Genetic variability of Taenia solium cysticerci recovered from experimentally infected pigs and from naturally infected pigs using microsatellite markers. Winkler A, editor. PLoS Negl Trop Dis. 2017;11:e0006087. Pajuelo MJ, Eguiluz M, Dahlstrom E, Requena D, Guzmán F, Ramirez M, et al. Identification and Characterization of Microsatellite Markers Derived from the Whole Genome Analysis of Taenia solium . Brehm K, editor. PLoS Negl Trop Dis. 2015;9:e0004316. Gemmell MA. Factors regulating tapeworm populations: the changing opportunities of lambs for ingesting the eggs of Taenia hydatigena . Res Vet Sci. 1976;21:223–6. Gemmell MA. HYDATIDOSIS AND CYSTICERCOSIS.: 4. Acquired Resistance to Taenia hydatigena under Conditions of a Strong Infection Pressure. Aust Vet J. 1972;48:26–8. 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. 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. 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. 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. 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. 2020;13:372. Copado F, de Aluja AS, Mayagoitia L, Galindo F. The behaviour of free ranging pigs in the Mexican tropics and its relationships with human faeces consumption. Appl Anim Behav Sci. 2004;88:243–52. Pray IW, Swanson DJ, Ayvar V, Muro C, Moyano LM, Gonzalez AE, et al. GPS Tracking of Free-Ranging Pigs to Evaluate Ring Strategies for the Control of Cysticercosis/Taeniasis in Peru. PLoS Negl Trop Dis [Internet]. 2016 [cited 2018 Nov 3];10. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818035/ Additional Declarations No competing interests reported. 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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-5883272","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430906615,"identity":"220f4c73-7aca-447e-9c39-31af56bc7cd9","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":430906616,"identity":"56c17592-dd4f-42e1-991f-f47cc81dd50d","order_by":1,"name":"Francesco Pizzitutti","email":"","orcid":"","institution":"Universidad San Francisco de Quito","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Pizzitutti","suffix":""},{"id":430906617,"identity":"579b1ac9-0187-4864-81e8-88fb30b67f65","order_by":2,"name":"Gabrielle Bonnet","email":"","orcid":"","institution":"London School of Hygiene and Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gabrielle","middleName":"","lastName":"Bonnet","suffix":""},{"id":430906618,"identity":"e05feb6e-c865-4a41-bf81-fa8068dfefac","order_by":3,"name":"Miguel Muro","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Muro","suffix":""},{"id":430906619,"identity":"7ea12fc6-8301-469a-932a-48ad698bb88d","order_by":4,"name":"Mayra Elizalde","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Mayra","middleName":"","lastName":"Elizalde","suffix":""},{"id":430906620,"identity":"e2a6a489-9767-483c-a149-02b35dc76dba","order_by":5,"name":"Claudio Muro","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Claudio","middleName":"","lastName":"Muro","suffix":""},{"id":430906621,"identity":"b4e35468-0f22-4516-b1e7-333174241dcd","order_by":6,"name":"Ricardo Gamboa","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Gamboa","suffix":""},{"id":430906622,"identity":"24a6cb21-700d-4f81-9fc4-2e868b25c953","order_by":7,"name":"Gianfranco Arroyo","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Gianfranco","middleName":"","lastName":"Arroyo","suffix":""},{"id":430906623,"identity":"155ac2d5-7b11-4d9f-8c5e-c34f2dffc543","order_by":8,"name":"Sarah Gabriël","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Gabriël","suffix":""},{"id":430906624,"identity":"8f36a71a-e418-473d-9ebf-ac4d48040921","order_by":9,"name":"William K. Pan","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"K.","lastName":"Pan","suffix":""},{"id":430906625,"identity":"156074cd-0b51-4923-b02e-778217d721d5","order_by":10,"name":"Héctor H. Garcia","email":"","orcid":"","institution":"Universidad Peruana Cayetano Heredia","correspondingAuthor":false,"prefix":"","firstName":"Héctor","middleName":"H.","lastName":"Garcia","suffix":""},{"id":430906626,"identity":"0c235953-4379-4dc2-af82-cff41ff81b5e","order_by":11,"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":"2025-01-22 19:23:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5883272/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5883272/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06844-6","type":"published","date":"2025-09-24T15:58:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78943918,"identity":"ffaa5137-abbd-4ca7-93da-7185849cf2d6","added_by":"auto","created_at":"2025-03-21 07:30:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137960,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental design for the pig study: Pigs of different ages at infection, indicated by the circles, were experimentally infected with 20,000 \u003cem\u003eT. solium\u003c/em\u003e eggs to assess infection patterns and overdispersion across age at infection groups.\u003c/p\u003e","description":"","filename":"Fig1New.png","url":"https://assets-eu.researchsquare.com/files/rs-5883272/v1/550114b68a8c10c45fb735d3.png"},{"id":78943916,"identity":"95776bbb-240e-462e-bd7f-e8438df7d12e","added_by":"auto","created_at":"2025-03-21 07:30:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81116,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of live cysts (y axis) in pigs infected at different ages (x axis). Blue dots represent individual cyst counts, while the red line illustrates the predicted values from the negative binomial regression model, showing differences across ages. The shaded area represents the 95% credible intervals from the model, indicating the range of expected values.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5883272/v1/808e222c638e9defa592996f.png"},{"id":92430610,"identity":"d99cf83a-3607-49b6-ae10-2d2cc2faeb19","added_by":"auto","created_at":"2025-09-29 16:06:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":801166,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5883272/v1/0c355d79-bbb2-46ad-bb8c-ab712eb7b4fe.pdf"},{"id":78943917,"identity":"c3ac9109-4e6e-4d00-9cfc-38f103c7518d","added_by":"auto","created_at":"2025-03-21 07:30:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15880,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5883272/v1/ee1ef59c2fa39389319e7908.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Age at Infection as a Key Predictor of Cyst Burden in Pigs Experimentally Infected with Taenia solium","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eTaenia solium\u003c/em\u003e cysticercosis is a zoonotic parasitic infection that poses significant public health challenges, particularly in low- and middle-income countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The parasite\u0026rsquo;s lifecycle involves humans as the definitive host, with the adult tapeworm residing in the intestine where it sheds proglottids and eggs in the host\u0026rsquo;s feces. When these eggs are consumed by free-roaming pigs, they develop into the larval stage, known as cysticercosis. Humans can also inadvertently ingest the eggs, leading to the development of cysticercosis in various tissues, most notably in the central nervous system, causing neurocysticercosis, a severe neurological condition [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn endemic regions, an estimated 1\u0026ndash;3% of the human population harbors the adult \u003cem\u003eT. solium\u003c/em\u003e tapeworm [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], serving as the primary source of cysticercosis and leading to a pig infection prevalence of approximately 10\u0026ndash;30% [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Pigs with high cyst burdens, hundreds to thousands of cysts, readily visible in the carcass during slaughter, represent only a small minority of infections in the population. The majority of infected pigs harbor fewer than 10 cysts [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and are less likely to be detected during slaughter. Typically, over 80% of the parasites are harbored by fewer than 20% of the hosts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This phenomenon, known as parasite aggregation, is broadly attributed to several density-dependent mechanisms, both intrinsic and extrinsic to the pig, which contribute to the observed overdispersion in infection pattern [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Innate and acquired immunity are two such mechanisms that are known to play a critical role in limiting the number of cysts in pig populations infected with \u003cem\u003eT. solium\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePast evidence suggests that age at infection, sex, and the genetic strain of the host animal are potential contributors to variability in infection with various taeniid cestodes, with age emerging as the most influential factor [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Age has been identified as contributing to innate resistance among na\u0026iuml;ve or previously unexposed animals challenged with different tapeworm species such as \u003cem\u003eT. taeniaeformis\u003c/em\u003e in rats, \u003cem\u003eT. pisiformis\u003c/em\u003e in rabbits, \u003cem\u003eT. saginata\u003c/em\u003e in cattle and \u003cem\u003eT. hydatigena\u003c/em\u003e in sheep [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has further been suggested that immunity may not fully prevent the establishment of new cysts but likely modulates the infection\u0026rsquo;s progression over time [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A few studies have utilized controlled exposure to more accurately characterize resulting infection intensity at different ages [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, methodological inconsistencies\u0026mdash;such as insufficient standardization of the infective dose, differences in egg batches, pig breeds, incomplete organ examination, and the inability to detect cysts due to an inappropriately short time interval between infection and necropsy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] limit the strength of the evidence.\u003c/p\u003e \u003cp\u003eThis significant variability in cyst burden presents challenges for implementing effective strategies for monitoring and evaluating the impact of potential control interventions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. While understanding pig immunity is critical to determining what control or elimination interventions are likely to be most effective, the evidence in this regard remains limited and insufficiently precise to feed into intervention modelling, even when considering studies with other taeniid cestodes.\u003c/p\u003e \u003cp\u003eThe objective of this study was to accurately characterize differences in infection burden across age groups after a single infection, to improve understanding of age-related and innate immunity, as well as to inform representation of pig immunity in transmission simulation models designed to guide interventions. We achieved this through a controlled experiment designed to minimize as many sources of variation as possible, allowing us to determine differences in cyst burden in pigs infected with \u003cem\u003eT. solium\u003c/em\u003e at varying ages.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e \u003cp\u003eThe initial study design included 40 pigs, divided into four age groups: four, 10, 16, and 22-weeks of age (10 per age group). Due to space limitations, infections were conducted in six separate rounds. After the 4th round, interim results suggested that more data were needed to improve precision around 10 weeks of age. Therefore, we added 12 more pigs: we created two new age groups (seven and 13 weeks-old, with five pigs each) and included two additional 10-week-old pigs. This brought the final total to 52 pigs. The pigs used in this study were purchased from nine different commercial farms in northern Peru, selected based on the availability of pigs of the required ages for each experimental round. The acquired pigs were of mixed breeds commonly found in the region. While mixed-breed pigs are more genetically variable than pure breeds, they are more representative of the pigs that are used in pig farming, hence ensuring better generalizability in our results. Five pigs were excluded leaving an analytic sample of 47 pigs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); four died prior to the scheduled necropsy and one had an aberrant serologic response at baseline.\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 numbers of live, degenerated, and total cysts by age at infection, along with the corresponding number of pigs per group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eLive cysts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eDegenerated cysts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003eTotal cysts\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at infection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of pigs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e514\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1705\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5\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\u003e231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e557\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.5\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\u003e260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e833\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71\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\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e394\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\u003eThe pigs were housed in individual pens at the specific pathogen-free facilities of the Center for Global Health at Cayetano Heredia University (UPCH) in Tumbes. The animals were fed exclusively with properly packaged commercial feed, stored at our facilities to prevent contamination. Water was provided \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSerological test\u003c/p\u003e \u003cp\u003eAll pigs underwent serologic screening to rule-out prior exposure to \u003cem\u003eT. solium\u003c/em\u003e before purchase, using lentil lectin-bound glycoprotein enzyme-linked immunoelectrotransfer blot assay (LLGP-ETIB) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and antigen ELISA (TsW8/TsW5 mAb set) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. All pigs were required to be negative on both tests (absence of any of the 7 reactive bands on LLGP-EITB; optical density ratio\u0026thinsp;\u0026lt;\u0026thinsp;1 on Ag-ELISA) to be included in the study. The mothers of all included pigs, except three due to owner refusal, were also screened to ensure negative status of the sow. Throughout the duration of the experiment, weekly blood samples were also taken to monitor the evolution of seroconversion with both tests. These results will feed into a separate paper.\u003c/p\u003e \u003cp\u003ePreparation of Eggs Pool\u003c/p\u003e \u003cp\u003eTapeworms for this experiment were sourced from control interventions in which human stool was collected from the community setting and 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=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Stool was collected during bowel preparation prior to antiparasitic treatment. Gravid proglottids were collected and stored in saline solution at 4\u0026deg;C for a maximum of two weeks, ensuring that eggs used in the experiment had not been exposed to antiparasitic drugs or preservatives. To minimize variability between infections due to differences in egg viability, we pooled \u003cem\u003eT. solium\u003c/em\u003e eggs from multiple tapeworms for each round of infections, using a new pool for each round. The number of tapeworms used to create each pool was 10, 3, 7, 8, 6 10 and 3, respectively. The number of tapeworms per round varied depending on tapeworm availability and the number of viable eggs available for each tapeworm, however, we managed to combine at least 3 tapeworms to prepare the pool used in each round. Each tapeworm originated from a different human donor from the North-West of Peru (Piura and Tumbes regions).\u003c/p\u003e \u003cp\u003ePools were prepared by mixing eggs harvested from gravid proglottids obtained from three to ten different tapeworms. Egg viability (Evans blue stain) and percentage of activated oncospheres using the enzyme method and movement of the hexacanth embryo [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] were assessed for each proglottid and for the entire pool. The mean percentage of activated oncospheres across all pools was 82% (range 63\u0026ndash;97%).\u003c/p\u003e \u003cp\u003eExperimental design\u003c/p\u003e \u003cp\u003eIn total, six different groups of pigs, corresponding to infections at four, seven, 10, 13, 16, and 22 weeks of age, were experimentally infected over six rounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The same pool of eggs was used for each round and pigs of different age groups were infected with eggs from the same pool. Infections were administered using an esophageal catheter, following a previously described method with slight modifications [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, each pig received 20,000 \u003cem\u003eT. solium\u003c/em\u003e eggs suspended in olive oil, encapsulated in gelatin capsules, which were delivered through an esophageal catheter. The capsules were then flushed into the stomach with 20\u0026ndash;100 mL of bottled drinking water. All pigs were anesthetized with a combined intramuscular dose of ketamine (20 mg/kg) and xylazine (2 mg/kg). After the infection procedure, the pigs were monitored by a veterinary team for stress reaction (vital signs) and for signs of emesis or regurgitation of the capsules. Pigs were then maintained in individual pens for a period of 10 weeks until necropsy. All pigs were weighed at the time of infection, and the median, minimum, and maximum weight per group are provided as Supplementary Information in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. For necropsy, pigs were induced to anesthesia with intramuscular ketamine (20 mg/kg) and xylazine (2 mg/kg) and then euthanized by intravenous sodium pentobarbital (100 mg/kg). The carcass was then dissected by trained staff using 3 to 5 mm slices of all muscles, brain, and other organs, and visually inspected to identify and count cysts, recording the number and type (viable or degenerated) of cysts for each animal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eWe used Kruskal-Wallis as a preliminary test to evaluate differences in live cyst counts between pigs infected at different ages. This non-parametric test was chosen due to its robustness against violations of normality assumptions and its ability to handle skewed data distributions commonly observed in cyst count outcomes. Significant differences detected by the Kruskal-Wallis test indicated the need for further exploration of age-related effects through more advanced modeling techniques.\u003c/p\u003e \u003cp\u003eWe evaluated several statistical regression models, including Poisson, Negative Binomial, Zero-Inflated Poisson, Zero-Inflated Negative Binomial, and negative binomial family Generalized Estimating Equations, for their ability to fit the observed counts of live \u003cem\u003eT. solium\u003c/em\u003e cysts in pigs infected at different ages. Each model was assessed for its ability to predict the number of live cysts while accounting for the distribution of the count data and potential overdispersion. Several predictors were considered and incorporated into the models if they improved their fit to the data. These predictors included the farm of origin of the pigs, the round of infection (to account for potential variation between infection periods), the viability of each egg pool (to adjust for differences in the proportion of viable oncospheres across rounds), the inverse of age and quadratic age (to capture potential non-linear effects of age on cyst counts), and the sex of the pigs. 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. Most of the analyses were performed using R [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and the packages MASS [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], pscl [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and ggplot2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], except for the Generalized Estimating Equations that was developed with the xtgee command in STATA [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe median number of live, degenerated and total cysts, along with their respective ranges across different ages at infection, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A significant variation in the median number of live cysts was observed between age groups (Kruskal-Wallis H-test, H\u0026thinsp;=\u0026thinsp;15.65, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, P\u0026thinsp;=\u0026thinsp;0.008), suggesting that the age at infection influences cyst burden in pigs. Specifically, younger pigs showed similarly low median cyst counts as the older ones, with a notable increase in cyst burden observed in pigs infected at intermediate ages. The extent of this variation is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These results suggest that age plays an important role in the progression of cysticercosis following infection with \u003cem\u003eT. solium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe pattern observed for degenerated cysts was the inverse of that seen for live cysts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of degenerated cysts was higher in younger and older pigs compared to those infected at intermediate ages, with marginally significant differences between age groups (Kruskal-Wallis H-test, H\u0026thinsp;=\u0026thinsp;10.29, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, P\u0026thinsp;=\u0026thinsp;0.07). The total number of cysts (live and degenerated combined) followed a trend comparable to that of live cysts but with less pronounced differences with age. These differences were marginally significant (Kruskal-Wallis H-test, H\u0026thinsp;=\u0026thinsp;10.31, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, P\u0026thinsp;=\u0026thinsp;0.07). Median values for each group are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eOnly four pigs harbor cysts in the brain, with a total of seven cysts found in just two groups: pigs infected at 7 and 16 weeks old. Each pig had between one and two cysts, and all cysts were viable.\u003c/p\u003e \u003cp\u003eStatistical model prediction\u003c/p\u003e \u003cp\u003eAll statistical models gave similar results. However, the negative binomial regression emerged as the most suitable model to predict the number of live cysts as a response variable based on age and other potential covariates. This conclusion was supported by the lower AIC, BIC, and by likelihood ratio comparisons. We considered a model linear in age as well as a model introducing additional quadratic or inverse terms. The inclusion of either a quadratic or inverse age term improved the model by providing a better representation of the observed cyst counts at younger ages. We selected the inverse of age as it has a more straightforward biological interpretation and provides credible figures (contrary to the introduction of a quadratic term) when extrapolating the model to pigs older than those in our experiment. Covariates such as farm of origin of the pig, pool viability and sex were also tested but did not significantly affect the model coefficients or improve model fit. The coefficients of the final model, along with their respective confidence intervals, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, while the model's predictions, along with their credible intervals and observed values, are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eCoefficients, standard errors, z-values, p-values, and 95% confidence intervals for the negative binomial regression model predicting the number of live cysts. The model includes the intercept, age, and the inverse of age as predictors.\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\u003e10.847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14.30\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.10\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-20.335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-33.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-5.51\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\u003e \u003c/p\u003e \u003cp\u003eSerological results\u003c/p\u003e \u003cp\u003eAll pigs used in this experiment started with no bands on LLGP-EITB and with antigen optical density ratio\u0026thinsp;\u0026lt;\u0026thinsp;1. Additionally, all pigs exhibited seroconversion of at least one LLGP-EITB band after infection, occurring between three- and 9-weeks post-infection, with a median seroconversion time of five weeks. Following the initial seroconversion, all pigs, except for one (which had only one live cyst out of a total of two), maintained seroconversion and typically kept or increased the number of LLGP-EITB bands, which has been shown to be correlated with the presence and level of infection in prior literature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Seroconversion on the antigen test consistently occurred before the LLGP-EITB test, between one- and 9-weeks post-infection, with a median time of 3 weeks. All animals exceeded the threshold of one (meaning they were positive on the antigen test) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] at least once after infection; in 38 animals, the optical density ratio exceeded three at least at some point after infection, nine maintained values lower than three, and five had an initial increase followed by a progressive decrease until the time of necropsy. These five pigs all had fewer than 10 live cysts. These results, which are important to refine our understanding of how serology relates to infection, will be analyzed in detail in a future publication.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is high variability in parasite burdens in animals infected with cestodes, particularly \u003cem\u003eT. solium\u003c/em\u003e. Innate immunity may contribute to these effects. Statistical analysis of the results of this study demonstrated a relationship between age and cyst burden. These results suggests that the immunity of na\u0026iuml;ve pigs (not previously exposed) is higher in younger and older pigs, and lowest around 9 weeks of age, with the relationship between age and the logarithm of the mean number of cysts best represented through the sum of two terms, one proportional to age, and one proportional to inverse age.\u003c/p\u003e \u003cp\u003eThere are many challenges in measuring the contribution of immunity to cyst development, including the effect of pig genetic lineage and variability in egg batches and numbers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and potentially host microbiome or stress. In this study, several of these variables were controlled for, including through the use of pooled eggs from different tapeworms with confirmed viability and hatching rates, as well as direct inoculation into the stomach to ensure consistent exposure. However, genetic variability among animals could not be controlled in the design of the experiment. The pigs we used came from commercial farms where standardized purebred lines are not commonly maintained. Instead, modern commercial pig production relies on crossbred lines selected by producers to optimize productivity based on growth rate, feed efficiency, and carcass quality. The specific mixed breeds used were typical of the northern Peru region, resulting from genetic improvement programs in rural areas, and commonly found in Peruvian villages. To meet the strict age group requirements for the study, pigs were sourced from multiple granges. It is plausible that granges may differ somewhat in the genetic makeup of their pigs, hence we included the farm of origin among the explanatory variables we considered in the statistical analysis and were able to demonstrate that inclusion of this variable did not improve the model. Note that, to our knowledge, no study has ever been undertaken regarding differences in susceptibility to infection among pig breeds or lines, so we could not rely on the literature to support or disprove a possible link between pig genetics and susceptibility.\u003c/p\u003e \u003cp\u003eThe relatively high variability observed within age groups highlights the complexity of the factors that determine cyst development, even under controlled experimental conditions, emphasizing the importance of accounting for such heterogeneity in future investigations.\u003c/p\u003e \u003cp\u003eSeveral explanations have been put forward to justify the change in susceptibility due to age. With respect to younger ages, in rats infected with \u003cem\u003eT. taeniaeformis\u003c/em\u003e, this change was attributed to little or no proteolytic activity in their intestines to assist in the hatching of eggs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, several other components could also be involved, such as innate immunity transferred by the mother, or pH or other differences in gastric fluids. We ruled out passive transfer of maternal acquired immunity because the mothers were free of infection. Evidence from other \u003cem\u003eTaenia\u003c/em\u003e species also suggests that the passive transfer of maternal acquired antibodies does not play a role [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur research group conducted a previous study of age on infection in which pigs were infected at one, three, and 5 months with a single proglottid each. While the current study has coherent results for older pigs, there are differences in the youngest group: in the earlier study, pigs infected at one month of age had the highest mean live cyst counts and percentage of viable cysts [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, this method of infection is subject to the large variability in the number of eggs per proglottid, reported to range from 3,900 to 120,000 eggs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which is one of the main limitations we wanted to address in the current study. Given this important distinction, comparison of results across these two studies is therefore challenging.\u003c/p\u003e \u003cp\u003eThe increased susceptibility observed at the natural weaning age [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and the progressive resistance to infection with age seen in this study are consistent with patterns commonly reported in other cestodes, though these trends have not been thoroughly described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The peak susceptibility coincides with the natural weaning age, but we can rule out weaning itself as a causal factor, as the 4-week-old animals in this study were forcibly weaned. If immunosuppression or stress due to weaning had been significant, it would also have affected these animals.\u003c/p\u003e \u003cp\u003eRegarding the increased resistance with age in older pigs, experimental infections with limited numbers of animals [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], as well as evidence from mass necropsies in endemic areas, suggest the potential for complete resistance to infection over time [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], even though this has not been completely demonstrated experimentally for \u003cem\u003eT. solium\u003c/em\u003e. Given that the pigs had no prior exposure, as confirmed by two serological methods, this decrease in susceptibility can only be attributed to a progressive strengthening of innate immunity. Nonetheless, certain stress factors may influence susceptibility. For instance, farrowing has been reported to predispose sows to reinfection due to immunosuppressive effects during this period [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This phenomenon has been used to explain the presence of cysticerci with different microsatellite patterns in a naturally infected sow, suggesting infections by distinct tapeworms at different times [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePossibly the best description of a similar pattern of infection at different ages comes from two separate studies involving \u003cem\u003eT. hydatigena\u003c/em\u003e in lambs grazed on pastures permanently contaminated by experimentally infected dogs. These studies reported a progressive increase in the number of live cysts from weeks one to 12 post-infection [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], followed by a progressive decrease from three to 6 months [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this case, the total number of cysts also differed by age, with younger lambs showing a lower proportion. While these findings were partly attributed to lower ingestion of contaminated pasture, they were also linked to passive immunity transferred from the mother or a combination of these factors. Comparing these results with the experimental infection reported here, it is also plausible that susceptibility to infection is influenced by the age at which exposure occurs.\u003c/p\u003e \u003cp\u003eGiven the marked difference in the number of live cysts between ages, accompanied by an inverse pattern in degenerated cysts and a marginal difference in the number of total cysts (following the same pattern as live cysts), our results might be due to a pre-encystment immunity. This concept, introduced by Gemmell and Soulsby [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], describes the early-stage challenges faced by the oncosphere after ingestion. These challenges include exposure to gastric acid in the stomach, interaction with the intestinal epithelium, circulation through the bloodstream, and eventual implantation and growth within muscular tissue to form larvae. During the pre-encystment phase, something\u0026mdash;either in transit or at the site of implantation\u0026mdash;reduced their ability to develop into viable cysts. The exact mechanisms or factors responsible for this differential success rate remain unclear, highlighting a need for further research into the specific host or environmental factors affecting this process.\u003c/p\u003e \u003cp\u003eOne of our primary aims in undertaking this study was to use the results to inform inclusion of pig immunity into our agent-based model, CystiAgent (and associated neurocysticercosis model, CystiHuman) to improve simulation of endemic transmission and the effect of interventions [\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The interaction of age-related pig susceptibility and the environmental exposure to \u003cem\u003eT. solium\u003c/em\u003e eggs, which eventually leads to infection, depicts a complex scenario in which multiple processes and factors likely interact across varying scales of time and space. Among the practical implications are the optimal age at which to undertake interventions in pigs (e.g. vaccination/treatment of younger vs. older pigs) and the frequency of intervention that could be most cost-effective. Further investigation is needed to explore additional factors influencing the infection patterns of \u003cem\u003eT. solium\u003c/em\u003e, particularly acquired immunity.\u003c/p\u003e \u003cp\u003eThis study has several limitations that should be considered when interpreting the findings. First, the pigs used in the experiments came from different farms, which may introduce variability in their baseline immunity, health status, or other intrinsic factors. Additionally, while our infection system ensures the controlled uptake of a precise number of eggs, it does not fully replicate natural infection dynamics. Natural infections are likely to occur gradually, with animals being exposed to varying doses of eggs over time, starting at a young age. In contrast, our experimental design involved a single, high-dose exposure (20,000 eggs), which may not fully reflect field conditions and could influence the immune response observed.\u003c/p\u003e \u003cp\u003eMoreover, our method of delivering eggs directly into the stomach bypasses natural routes of exposure, such as oral ingestion, which could impact how the immune system is activated. While the difference between these methods may be minimal, it remains a factor to consider. Finally, as an experimental system, the controlled nature of our study does not fully replicate the complexity of real-world scenarios, such as continuous low-level exposure in highly endemic areas. Future studies should explore the effects of reinfections and different infection doses to better understand the natural immune response and its implications for disease control strategies.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study represents the first controlled infection trial in pigs using \u003cem\u003eT. solium\u003c/em\u003e eggs that effectively accounts for key confounding factors. Our findings reveal that younger pigs exhibit partial protection against developing cysticerci compared to those infected at natural weaning age. Moreover, beyond that age, susceptibility to infection progressively decreases with age in a way that is coherent with prior hypotheses in the literature of complete or near-complete resistance by approximately one year of age. Although our results demonstrate that higher susceptibility occurs around 7 and 16 weeks of age, the maximum susceptibility estimated by our model is likely to occur around 9 weeks of age. These results provide critical insights into the age-related dynamics of porcine susceptibility to \u003cem\u003eT. solium\u003c/em\u003e and have significant implications for understanding the epidemiology and control of cysticercosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\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; Javier Bustos; 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; 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\u003cp\u003eFunding\u003c/p\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, MM, ME, CM, RG, GA, 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, GA, HG and SO; data curation, EG, FP, MM, CM and RG; writing original draft preparation, EG; writing, review and editing, EG, FP, GB, 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"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO TEAM. Ending the neglect to attain the Sustainable Development Goals: A road map for neglected tropical diseases 2021\u0026ndash;2030 [Internet]. WHO; 2020. Available from: https://www.who.int/publications/i/item/9789240010352\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a HH, Gonzalez AE, Evans CA, Gilman RH. \u003cem\u003eTaenia solium\u003c/em\u003e cysticercosis. The Lancet. 2003;362:547\u0026ndash;56. \u003c/li\u003e\n\u003cli\u003eGarcia HH, Gonzalez AE, Tsang VCW, O\u0026rsquo;Neal SE, Llanos-Zavalaga F, Gonzalvez G, et al. Elimination of \u003cem\u003eTaenia solium\u003c/em\u003e Transmission in Northern Peru. N Engl J Med. 2016;374:2335\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eMahanty S, Garcia HH. Cysticercosis and neurocysticercosis as pathogens affecting the nervous system. Prog Neurobiol. 2010;91:172\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a HH, Gilman RH, Gonzalez AE, Verastegui M, Rodriguez S, Gavidia C, et al. Hyperendemic human and porcine \u003cem\u003eTaenia solium\u003c/em\u003e infection in Per\u0026uacute;. Am J Trop Med Hyg. 2003;68:268\u0026ndash;75. \u003c/li\u003e\n\u003cli\u003eGulelat Y, Eguale T, Kebede N, Aleme H, F\u0026egrave;vre EM, Cook EAJ. Epidemiology of Porcine Cysticercosis in Eastern and Southern Africa: Systematic Review and Meta-Analysis. Front Public Health. 2022;10:836177. \u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Neal SE, Pray IW, Vilchez P, Gamboa R, Muro C, Moyano LM, et al. Geographically Targeted Interventions versus Mass Drug Administration to Control \u003cem\u003eTaenia solium\u003c/em\u003e Cysticercosis, Peru. Emerg Infect Dis. 2021;27:2389\u0026ndash;98. \u003c/li\u003e\n\u003cli\u003eGarcia HH, Gonzalez AE, Gilman RH. \u003cem\u003eTaenia solium\u003c/em\u003e Cysticercosis and Its Impact in Neurological Disease. Clin Microbiol Rev. 2020;33:e00085-19, /cmr/33/3/CMR.00085-19.atom. \u003c/li\u003e\n\u003cli\u003eWoolhouse MEJ, Dye C, Etard J-F, Smith T, Charlwood JD, Garnett GP, et al. 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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\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eArroyo 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 \u003cem\u003eTaenia solium\u003c/em\u003e and \u003cem\u003eTaenia hydatigena\u003c/em\u003e. Parasit Vectors. 2024;17:172. \u003c/li\u003e\n\u003cli\u003eMayta H, Talley A, Gilman RH, Jimenez J, Verastegui M, Ruiz M, et al. Differentiating \u003cem\u003eTaenia solium\u003c/em\u003e and \u003cem\u003eTaenia saginata\u003c/em\u003e Infections by Simple Hematoxylin-Eosin Staining and PCR-Restriction Enzyme Analysis. J CLIN MICROBIOL. 2000;38:133\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eWang IC, Ma YX, Kuo CH, Fan PC. A comparative study on egg hatching methods and oncosphere viability determination for \u003cem\u003eTaenia solium\u003c/em\u003e eggs. Int J Parasitol. 1997;27:1311\u0026ndash;4. \u003c/li\u003e\n\u003cli\u003eSantamaria E, Plancarte A, De Aluja AS. The Experimental Infection of Pigs with Different Numbers of \u003cem\u003eTaenia solium\u003c/em\u003e Eggs: Immune Response and Efficiency of Establishment. J Parasitol. 2002;88:69. \u003c/li\u003e\n\u003cli\u003eR 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/\u003c/li\u003e\n\u003cli\u003eVenables WN, Ripley BD. Modern Applied Statistics with S [Internet]. Fourth. New York: Springer; 2002. Available from: https://www.stats.ox.ac.uk/pub/MASS4/\u003c/li\u003e\n\u003cli\u003eZeileis A, Kleiber C, Jackman S. Regression Models for Count Data in R. J Stat Softw [Internet]. 27. Available from: URL http://www.jstatsoft.org/v27/i08/\u003c/li\u003e\n\u003cli\u003eWickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York; 2016. \u003c/li\u003e\n\u003cli\u003eStataCorp. Stata 18: Data Analysis and Statistical Software. College Station TX: StataCorp LLC. 2023. \u003c/li\u003e\n\u003cli\u003eArroyo G, Lescano AG, Gavidia CM, Lopez-Urbina T, Ara-Gomez M, Gomez-Puerta LA, et al. Antibody Banding Patterns on the Enzyme-Linked Immunoelectrotransfer Blot (EITB) Assay Clearly Discriminate Viable Cysticercosis in Naturally Infected Pigs. Pathogens. 2023;13:15. \u003c/li\u003e\n\u003cli\u003eJayashi CM, Gonzalez AE, Castillo Neyra R, Rodr\u0026iacute;guez S, Garc\u0026iacute;a HH, Lightowlers MW. Validity of the Enzyme-linked Immunoelectrotransfer Blot (EITB) for naturally acquired porcine cysticercosis. Vet Parasitol. 2014;199:42\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eMusoke AJ, Williams JF, Leid RW, Williams CS. The immunological response of the rat to infection with \u003cem\u003eTaenia taeniaeformis\u003c/em\u003e. IV. Immunoglobulins involved in passive transfer of resistance from mother to offspring. Immunology. 1975;29:845\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eLightowlers MW, Rickard MD, Mitchell GF. \u003cem\u003eTaenia taeniaeformis\u003c/em\u003e in mice: Passive transfer of protection with sera from infected or vaccinated mice and analysis of serum antibodies to oncospheral antigens. Int J Parasitol. 1986;16:307\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eMa Y, Su Y, Yan Q, He L, Yan X, Chen J, et al. [Study on number and mature rate of eggs in gravid proglottids of \u003cem\u003eTaenia solium\u003c/em\u003e]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2002;20:98\u0026ndash;100. \u003c/li\u003e\n\u003cli\u003eStolba A, Wood-Gush DGM. The behaviour of pigs in a semi-natural environment. Anim Sci. 1989;48:419\u0026ndash;25. \u003c/li\u003e\n\u003cli\u003eBoe K. The process of weaning in pigs: when the sow decides. Appl Anim Behav Sci. 1991;30:47\u0026ndash;59. \u003c/li\u003e\n\u003cli\u003ede Aluja AS, Villalobos ANM, Plancarte A, Rodarte LF, Hern\u0026aacute;ndez M, Sciutto E. Experimental \u003cem\u003eTaenia solium\u003c/em\u003e cysticercosis in pigs: characteristics of the infection and antibody response. Vet Parasitol. 1996;61:49\u0026ndash;59. \u003c/li\u003e\n\u003cli\u003ede Aluja AS, Martinez M JJ, Villalobos ANM. \u003cem\u003eTaenia solium\u003c/em\u003e cysticercosis in young pigs: age at first infection and histological characteristics. Vet Parasitol. 1998;76:71\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eDixon MA, Winskill P, Harrison WE, Whittaker C, Schmidt V, Sarti E, et al. Force-of-infection of \u003cem\u003eTaenia solium\u003c/em\u003e porcine cysticercosis: a modelling analysis to assess global incidence and prevalence trends. Sci Rep. 2020;10:17637. \u003c/li\u003e\n\u003cli\u003ePoudel I, Sah K, Subedi S, Kumar Singh D, Kushwaha P, Colston A, et al. Implementation of a practical and effective pilot intervention against transmission of \u003cem\u003eTaenia solium\u003c/em\u003e by pigs in the Banke district of Nepal. Fuehrer H-P, editor. PLoS Negl Trop Dis. 2019;13:e0006838. \u003c/li\u003e\n\u003cli\u003eDixon MA, Winskill P, Harrison WE, Whittaker C, Schmidt V, Fl\u0026oacute;rez S\u0026aacute;nchez AC, et al. Global variation in force-of-infection trends for human \u003cem\u003eTaenia solium\u003c/em\u003e taeniasis/cysticercosis. eLife. 2022;11:e76988. \u003c/li\u003e\n\u003cli\u003ePajuelo MJ, Eguiluz M, Roncal E, Qui\u0026ntilde;ones-Garc\u0026iacute;a S, Clipman SJ, Calcina J, et al. Genetic variability of \u003cem\u003eTaenia solium\u003c/em\u003e cysticerci recovered from experimentally infected pigs and from naturally infected pigs using microsatellite markers. Winkler A, editor. PLoS Negl Trop Dis. 2017;11:e0006087. \u003c/li\u003e\n\u003cli\u003ePajuelo MJ, Eguiluz M, Dahlstrom E, Requena D, Guzm\u0026aacute;n F, Ramirez M, et al. Identification and Characterization of Microsatellite Markers Derived from the Whole Genome Analysis of \u003cem\u003eTaenia solium\u003c/em\u003e. Brehm K, editor. PLoS Negl Trop Dis. 2015;9:e0004316. \u003c/li\u003e\n\u003cli\u003eGemmell MA. Factors regulating tapeworm populations: the changing opportunities of lambs for ingesting the eggs of \u003cem\u003eTaenia hydatigena\u003c/em\u003e. Res Vet Sci. 1976;21:223\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eGemmell MA. HYDATIDOSIS AND CYSTICERCOSIS.: 4. Acquired Resistance to \u003cem\u003eTaenia hydatigena\u003c/em\u003e under Conditions of a Strong Infection Pressure. Aust Vet J. 1972;48:26\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eGemmell MA, Soulsby EJL. The development of acquired immunity to tapeworms and progress towards active immunization, with special reference to \u003cem\u003eEchinococcus\u003c/em\u003e spp. Bull World Health Organ. 1968;39:45\u0026ndash;55. \u003c/li\u003e\n\u003cli\u003ePizzitutti F, Bonnet G, Gonzales-Gustavson E, Gabri\u0026euml;l S, Pan WK, Pray IW, et al. Non-local validated parametrization of an agent-based model of local-scale \u003cem\u003eTaenia solium\u003c/em\u003e transmission in North-West Peru. Sato MO, editor. PLOS ONE. 2022;17:e0275247. \u003c/li\u003e\n\u003cli\u003ePizzitutti F, Bonnet G, Gonzales-Gustavson E, Gabri\u0026euml;l S, Pan WK, Gonzalez AE, et al. Spatial transferability of an agent-based model to simulate \u003cem\u003eTaenia solium\u003c/em\u003e control interventions. Parasit Vectors. 2023;16:410. \u003c/li\u003e\n\u003cli\u003eBonnet G, Pizzitutti F, Gonzales-Gustavson EA, Gabri\u0026euml;l S, Pan WK, Garcia HH, et al. CystiHuman: A model of human neurocysticercosis. Perkins A, editor. PLOS Comput Biol. 2022;18:e1010118. \u003c/li\u003e\n\u003cli\u003ePray IW, Pizzitutti F, Bonnet G, Gonzales-Gustavson E, Wakeland W, Pan WK, et al. Validation of a spatial agent-based model for \u003cem\u003eTaenia solium\u003c/em\u003e transmission (\u0026ldquo;CystiAgent\u0026rdquo;) against a large prospective trial of control strategies in northern Peru. Torgerson PR, editor. PLoS Negl Trop Dis. 2021;15:e0009885. \u003c/li\u003e\n\u003cli\u003ePray 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. \u003c/li\u003e\n\u003cli\u003eCopado F, de Aluja AS, Mayagoitia L, Galindo F. The behaviour of free ranging pigs in the Mexican tropics and its relationships with human faeces consumption. Appl Anim Behav Sci. 2004;88:243\u0026ndash;52. \u003c/li\u003e\n\u003cli\u003ePray IW, Swanson DJ, Ayvar V, Muro C, Moyano LM, Gonzalez AE, et al. GPS Tracking of Free-Ranging Pigs to Evaluate Ring Strategies for the Control of Cysticercosis/Taeniasis in Peru. PLoS Negl Trop Dis [Internet]. 2016 [cited 2018 Nov 3];10. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818035/\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Taenia solium, pig cysticercosis, age at infection, innate immunity, susceptibility at infection","lastPublishedDoi":"10.21203/rs.3.rs-5883272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5883272/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 cysticercosis is a zoonotic parasitic disease with significant public health implications, particularly in endemic regions of low- and middle-income countries. In pigs, cyst burden varies widely, with most harboring fewer than 10 cysts and only a small fraction carrying high cyst loads. Age has been identified as a key factor influencing infection susceptibility. However, inconsistencies in previous studies have hindered clear characterization of infection patterns and immunity. In this study, we conducted controlled experiments involving the infection of pigs with \u003cem\u003eT. solium\u003c/em\u003e eggs to evaluate the relationship between pig age and susceptibility to infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A total of 52 pigs from northern Peru, aged 4 to 22 weeks, were experimentally infected with \u003cem\u003eT. solium\u003c/em\u003e eggs to examine age-related differences in cyst burden. Pigs were housed individually under controlled conditions and fed commercial pig diets. Infections were administered using an esophageal catheter, delivering 20,000 \u003cem\u003eT. solium\u003c/em\u003eeggs encapsulated in gelatin capsules. Six age groups were studied using a standardized egg pool to ensure consistency across infection rounds. After 10 weeks, necropsies were performed to count cysts in all muscles, the brain, and other organs. Weekly serological tests monitored seroconversion. Statistical models were used to analyze cyst counts and assess the effects of age and other predictors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The number of live, degenerated, and total cysts was overdispersed making a negative binomial model the most suitable choice to represent the data and their dependence on age at infection. Younger pigs showed low median live cyst count, similar to older pigs, while median cyst burden increased in pigs infected at intermediate ages, around natural weaning age. The negative binomial regression showed that age and a covariate inversely related to age at infection were significantly associated with cyst count at necropsy. Other covariates such as pool viability and sex did not significantly affect model performance. Serological tests confirmed seroconversion in all pigs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur results show that younger pigs display partial protection against the development of cysticerci compared to those infected at the natural weaning age (around 9 to 12 weeks of age). Additionally, infection susceptibility then decreases with age in a way that is consistent with previous literature hypothesizing near-complete resistance by one year of age.\u003c/p\u003e","manuscriptTitle":"Age at Infection as a Key Predictor of Cyst Burden in Pigs Experimentally Infected with Taenia solium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 07:30:25","doi":"10.21203/rs.3.rs-5883272/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-23T13:26:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-16T22:18:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-31T10:00:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72471049345007681607564487374632421201","date":"2025-03-27T12:16:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259294114328593943038205680704177248875","date":"2025-03-24T15:45:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147890298512801076277310371814750925232","date":"2025-03-23T21:15:26+00:00","index":"hide","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-21T17:57:59+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-19T08:09:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-19T07:27:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-03-18T18:35:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"abe82429-42aa-46f5-800c-8c5f2c7ff78b","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:02:40+00:00","versionOfRecord":{"articleIdentity":"rs-5883272","link":"https://doi.org/10.1186/s13071-025-06844-6","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-09-24 15:58:22","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-03-21 07:30:25","video":"","vorDoi":"10.1186/s13071-025-06844-6","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06844-6","workflowStages":[]},"version":"v1","identity":"rs-5883272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5883272","identity":"rs-5883272","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}