This peer-reviewed article has been accepted for publication but not yet copyedited or typeset, and so may be subject to change during the production process. The article is considered published and may be cited using its DOI 10.1017/S0950268820002897. Epidemiology & Infection is published by Cambridge University Press. Risk factors for Toxoplasma gondii seropositivity in the Old Order Amish Markon, A. O. 1 , Ryan K. A. 2,3 , Wadhawan A. 4,5 , Pavlovich M. 2,3 , Groer M.W. 6 , Punzalan C. 1 , Gensheimer K. 1 , Jones J. 4 , Daue M.L. 2,3,7 , Dagdag A. 4 , Donnelly P. 8 , Peng X. 4,5 , Pollin T. 2,3,7 , Mitchell B.D. 2,3,7 , Postolache T.T. 4,9,10 1 U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition (CFSAN), Office of Analytics and Outreach (OAO), Division of Public Health Informatics and Analytics (DPHIA), College Park, MD, USA. 2 Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA. 3 Program for Personalized and Genomic Medicine, University of Maryland School of Medicine, Baltimore, MD, USA. 4 Mood and Anxiety Program, Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, USA. 5 Saint Elizabeth’s Hospital, Psychiatry Residency Training Program, Washington, DC, USA. 6 College of Nursing, University of South Florida College of Nursing, Tampa, FL, USA. 7 Geriatrics Research and Education Clinical Center, Veterans Affairs Medical Center, Baltimore, MD, USA. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 2 8 Amish Research Clinic, University of Maryland School of Medicine, Lancaster, PA, USA. 9 Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 19, Military and Veteran Microbiome: Consortium for Research and Education (MVM-CoRE), Denver, CO, USA. 10 Mental Illness Research, Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 5, VA Capitol Health Care Network, Baltimore, MD, USA. Corresponding authors: Main: Teodor T. Postolache: tpostola@som.umaryland.edu André O. Markon andre.markon@fda.hhs.gov Disclaimer: The findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the U.S. Food and Drug Administration (FDA). Data Availability: The data cannot be shared because of the original protocol and Amish consent. A collaborative effort could be considered and interested individuals should contact the corresponding author. 40-word summary: Risk of infection with the major foodborne pathogen, Toxoplasma gondii, varies by individual, geographic, sociocultural, and economic factors. We assessed the risk of toxoplasmosis, focusing on food safety and environmental factors, among the Old Order Amish, a relatively homogenous population. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. mailto:tpostola@som.umaryland.edu mailto:andre.markon@fda.hhs.gov https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 3 Summary Toxoplasma gondii is an important human disease-causing parasite. In the US, T. gondii infects >10% of the population, accrues economic losses of US$3.6 billion/year, and ranks as the second leading culprit of foodborne illness-related fatalities. We assessed toxoplasmosis risk among the Old Order Amish, a mostly homogenous population with high prevalence of T. gondii seropositivity, using a questionnaire focusing on food consumption/preparation behaviours and environmental risk factors. Analyses were conducted using multiple logistic regression. Consuming raw meat, rare meat, or unpasteurized cow or goat milk products were associated with increased odds of seropositivity [unadjusted ORs: 2.192, 1.613, 1.718, and 1.741, respectively). In separate models by sex, consuming raw meat, or consuming unpasteurized cow or goat milk products, were associated with increased odds of seropositivity among women; washing hands and touching meat with decreased odds of seropositivity among women [adjusted OR (AOR): 0.462]; and cleaning cat litterbox with increased odds of seropositivity among men (AOR: 5.241). This is the first study to assess associations between behavioural and environmental risk factors and T. gondii seropositivity in a US population with high seroprevalence for T. gondii. Our study emphasizes the importance of proper food safety behaviours to avoid risk of infection. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 4 INTRODUCTION The apicomplexan parasite Toxoplasma gondii (T. gondii) infects one-third of the world’s population, disproportionately affects socioeconomically disadvantaged groups, and ranks among five neglected parasitic infections targeted in the United States (US) by the Centers for Disease Control (CDC) for public health action[1, 2]. Some estimates indicate that in the US ~170,000 new toxoplasmosis cases occur annually and that 1.1 million are currently infected, amounting to infection among 13.2% (age-adjusted: 12.4%) of the population [3]. Other sources note that although >40 million men, women, and children in the US may harbour the parasite without symptoms because the immune system prevents occurrence of the full-blown illness, consequences of infection can be severe for the immunocompromised and for women infected during or shortly before pregnancy [2]. The burden of T. gondii infection by foodborne routes, which is estimated to account for about half of all US cases, exceeds $3.6 billion and equates to the loss of 459,481 quality-adjusted lifedays (QALDs)[4]. According to Scallan at al. (2011), 2.6% of US-acquired foodborne T. gondii infections will result in hospitalizations, and 0.02% will result in death, leading to an estimated 327 deaths annually, making toxoplasmosis the second leading cause of foodborne illness fatality in the US [5]. Sexual reproduction of T. gondii occurs in its definitive felid hosts, including cats, while asexual reproduction occurs in intermediate hosts, including humans and nearly all warm-blooded vertebrates. The parasite has three infectious stages[6] -15 : sporulated oocysts that contain sporozoites, tachyzoites that invade and multiply rapidly within the https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 5 cells of intermediate hosts, and bradyzoites that slowly replicate in tissue cysts (life cycle detailed in Figure 1). Tissue cysts are most often found in the central nervous system; the eyes; and smooth, skeletal, or cardiac muscles. While bradyzoites in tissue cysts characterize chronic infection, tachyzoites characterize primary acute or reactivated infections, which can trigger adaptive IgG immune responses[7, 8]. In reactivation, tissue cysts rupture and release bradyzoites that transform back into destructive tachyzoites 11,13 . Toxoplasmosis may present in immunocompetent individuals with painless lymphadenopathy; mononucleosis-like symptoms, including fever, malaise, sore throat, and maculopapular rash; ocular infections in some older populations; or no symptoms in pregnant women[9] ,12 . Severe clinical manifestations, such as pneumonitis, chorioretinitis, or multi-organ involvement, occur most often in reactivated and primary infections among immunocompromised individuals and in congenital infections[9, 10]. HIV-positive individuals often present with life-threatening toxoplasmic encephalitis, characterized by headache, confusion, weakness, focal neurologic involvement, and seizures[11, 12]. Previously, chronic infections were not associated with clinical manifestations[9], but recent studies have linked chronic infection to schizophrenia[13-15] and suicidal behaviour [16, 17]. Multiple behavioural, socioeconomic, and demographic factors may contribute to the risk of toxoplasmosis. Studies focusing on behavioural risk factors often use a combination of immunoassays to determine infection and questionnaires to conduct epidemiologic assessment of potential exposures. While the questionnaires themselves are often tailored to specific populations, many ask similar questions regarding behaviour-related exposures: most notably, eating different meats[18-22] (porcine, https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 6 bovine, lamb/mutton, other); handling or eating raw or undercooked meat[18, 22]; consuming dairy/ dairy products[22-24]; eating unwashed or improperly washed fruits and/or vegetables[22, 25, 26]; gardening and/or engaging in contact with soil[18, 21, 22, 26]; living with pets, such as cats and/or dogs at home[18, 19, 26]; drinking water (treatment, source, other characteristics)[19, 22, 27]; and even hunting/consuming wild game (venison, boar, other)[18, 27]. In the US, those below the federal poverty threshold have higher T. gondii seropositivity[3], while higher early-life socioeconomic status (SES) has been shown to be related to seronegativity[28]. Other risk factors for higher seroprevalence, specifically in the US, include foreign country of birth, Hispanic and non-Hispanic Black race/ethnicity, lower than high school education levels, crowded housing conditions, rurality/urbanicity, and employment type[3, 22]. The Old Order Amish (OOA) population in the US, by virtue of its homogeneous characteristics, provides a unique opportunity to assess relationships between T. gondii seropositivity and specific behavioural and environmental (including food-related) risk factors. T. gondii seropositivity prevalence levels among the OOA exceed those found among the general US population[29], and the geographic, ethnic, and socioeconomic homogeneity of the OOA population [30] helps to control for potential confounding that can otherwise complicate risk factor assessment. In addition, the Amish lifestyle presents the prospect of evaluating potential associations between T. gondii seropositivity and culturally specific meal-preparation practices, such as slaughtering, smoking, curing, and canning meats[31]. These distinctive characteristics, in conjunction with antibody testing and data collection using OOA lifestyle-specific questions, allowed this study to pursue the objective of advancing understanding of https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 7 relationships between the prevalence of T. gondii seropositivity and behavioural and environmental (including food-related) risk factors of infection, while minimizing confounding by other potential sociodemographic and behavioural determinants. METHODS STUDY POPULATION THE AMISH WELLNESS STUDY The Amish Wellness Study (AWS), an ongoing community-based investigation conducted by the University of Maryland Baltimore and approved by the University of Maryland Baltimore Institutional Review Board, was established in 2010 to offer health screening and collection of DNA, blood samples, and basic medical information to address specific research questions. Criteria for participation included membership in the OOA community of Lancaster County, Pennsylvania; age of ≥18 years; and an informed consent. A registered nurse obtained multiple clinical measures of general health (e.g., weight, height, waist and hip circumference, blood pressure, measures of respiratory health). An Amish liaison facilitated administration of the study questionnaire in person to assess several aspects of each participant’s health and well-being, including mood, sleep, and medical, personal and family histories; as well as to evaluate life-style factors. A University of Maryland Amish Research Clinic research nurse confirmed completion of each questionnaire. A fasting blood sample was drawn for measurement of lipids and glucose, and from this sample, we also measured T. gondii IgG antibodies for this study. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 8 For the present study, we sent the Amish Toxoplasma Risk Factor Questionnaire (ATRFQ) to a convenience sample from the AWS (n = 3551, nonrandomly selected, based on availability to participate), between December 30, 2015 and January 3, 2016. We evaluated participant responses to the ATRFQ, along with their demographic information and T. gondii antibody results from the AWS. IMMUNOGLOBULIN G SEROINTENSITY AND SEROPOSITIVITY TESTING For this study, the laboratory of Maureen Groer at the University of South Florida in Tampa determined T. gondii antibody seroprevalence using the enzyme-linked immunosorbent assay (ELISA) (IBL International, Männedorf, Switzerland) that tests for RH factor common to all T. gondii serotypes. Concentrations of ≥12 IU/ml were defined as seropositive, and of 8-12 IU/m as equivocal. The equivocal samples were tested a second time, and if the second testing yielded samples that tested positive for concentrations of ≥12 IU/ml on the second test were also considered positive as previously reported [32]. AMISH TOXOPLASMA INFECTION RISK FACTOR QUESTIONNAIRE (ATRFQ) The ATRFQ was developed by researchers at the University of Maryland Baltimore with input from epidemiologists at FDA’s Center for Food Safety and Applied Nutrition (CFSAN) and co-author Jeffrey Jones, based on current risk factor literature and subject matter expertise. The questionnaire was adapted after incorporating input from 3 sessions of review and feedback by a group of Amish liaisons, 3 sessions of feedback by nurses experienced in working with the Amish, and individual researcher comments and questions to maximize comprehension and cultural sensitivity. The https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 9 questionnaire consisted of 30 questions focusing primarily on food consumption and preparation, as well as other environmental risk factors, such as water sources, pet ownership and contact, and contact with soil. The pet component specifically asked about cat/ kitten ownership, cat breeding, cat litter cleaning, where the cats live (outdoors vs indoors), where the cats/ kittens eat, type of food they eat, visits of not- owned cats, type of cats seen in the garden or yard, cats for breeding, feral cats (domesticated and returned to the wild), and stray cats (lost or abandoned). . It was mailed to study participants between December 30, 2015 and January 3, 2016 with a cover letter, response envelope, and US$2 compensation. The cover letter informed participants that they could keep the $2 even if they decided not to participate. Responses from the returned questionnaire received by April 11, 2016 were entered into an Access database. Returned envelopes received after April 11, 2016 were not included in the analytical sample used for this study. STATISTICAL ANALYSES The analytical sample included those with both T. gondii serological measures and ATRFQ responses. Analyses were performed to describe the analytical sample (Table 1) and to assess the strength and significance of associations with examined behavioural and environmental (including food-related) risk factors through models estimating odds of T. gondii seropositivity (Tables 2-4). In addition to age and sex derived from the parent AWS survey, the ATRFQ variables addressed contact with soil, pets (including cats and litter boxes), and shoes/feet before touching food; farm vs. other-than-farm residence and time at residence; drinking untreated or treated water and drinking water source/filtration; working with animals/raw meat/horses; consuming https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 10 locally-produced, cooked, cured, frozen, canned, raw and/or rare meats; consuming raw oysters; consuming unpasteurized goat and/or cow milk/products; washing produce; washing hands/utensils after raw meat contact; and eating outside the home. Bivariate analyses, including chi-square (Χ 2 ) and t-tests, compared seropositive to seronegative to identify significant or borderline significant (P value <0.05 and <0.20) variables. For those variables, we then specified comparison reference groups and estimated unadjusted odds ratios (UORs), 95% confidence intervals (CIs), and P values (Tables 2 and 3). We included the variables that exhibited significance in the bivariate UOR analysis (P value <0.05) in the final unconditional multiple logistic regression models by sex to estimate adjusted odds ratios (AORs), 95% CIs, and P values, evaluating the associations between each of the predictor variables and the outcome of T. gondii seropositivity as defined above (Table 4). These analyses were performed using SAS 9.4 (Cary, NC), as well as the X 2 calculator in McDonald (2014)[33]. We also adjusted the results for relatedness (heritability) and shared households by applying a variance component approach using SOLAR 6.6.2 software (San Antonio, TX) to assess significance (P value <0.05) [34]. RESULTS Of the 3551 questionnaires mailed to study participants, 1518 (43%) were returned. T. gondii serology was assessed in 55.5% (n=843) of those who returned questionnaires. We limited our analytical sample to the 843 Amish subjects with both serology test results and questionnaire response data (23.7% of the 3551 who were mailed and returned the questionnaire also had serology results). https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 11 More than half (56.7%) of the 843 study participants tested positive for T. gondii IgG antibodies (Table 1). Nearly two-thirds of participants (60.1%) were female among both the seropositive and seronegative groups. The age distribution/n was similar among seropositive individuals, but not among seronegative individuals or the whole study sample. Mean and median ages were higher among seropositive than among seronegative individuals, and there were more seropositive than seronegative males. In addition to sex, the Χ 2 test results from the first step of the bivariate analysis indicated significant/borderline significant associations between T. gondii seropositivity (P values <0.20) and the category-based variables capturing farm vs. other-than-farm residence and time at residence; working with horses; consuming unpasteurized cow milk/products; consuming cured, rare, and raw meats; pet contact; and washing hands/utensils after raw meat contact, as well as the frequency-based variables addressing how often unpasteurized cow milk/products, raw meats, rare meats, and meals outside the home were consumed. Of those variables, the UOR estimates from the second step of the bivariate analysis showed significance (P value <0.05) for pet contact, consumption of meat and unpasteurized milk/products, and handwashing behaviours (Table 2), as well as for frequency of consumption of unpasteurized cow milk/ yoghurt, rare meat, raw meat, and meals outside the home (Table 3). The four meat-eating behaviours, as well as the two variables addressing the frequency of consumption, were all associated with T. gondii seropositivity. The odds of seropositivity from eating rare and raw meat compared to not eating rare and raw meat https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 12 were 1.613 (95% CI: 1.017, 2.560) and 2.192 (95% CI: 1.172, 4.101), respectively, while seropositivity among those who reported eating compared to not eating raw ground beef was greater (OR: 2.568, 95% CI: 0.996, 6.623). Only one individual reported eating raw ground pork and was seropositive. Odds of seropositivity comparing consumption of unpasteurized milk to not consuming raw milk was 1.211 (OR: 1.211, 95% CI: 1.055, 1.391). Among the behaviours related to pets, only cleaning a cat’s litterbox was found statistically significantly associated with T. gondii seropositivity (OR: 2.451, 95% CI: 1.167, 5.143). Because of the significant difference found between sex and seropositivity in the bivariate analysis, separate models by sex were used to estimate the adjusted associations between the other variables also identified as significantly associated with seropositivity (Table 4). Several of the associations between T. gondii seropositivity and behavioural and environmental (including food-related) risk factors did not retain significance in the adjusted models by sex. However, the separate adjusted models by sex showed that significance persisted nominally for cleaning the litterbox (among men only), eating raw meat (among women only), and washing hands after touching raw meat (protective in women only). Consuming raw goat or cow milk was significantly associated with seropositivity among women only (OR: 1.863, 95% CI: 1.086, 3.197; OR: 1.930, 95% CI: 1.127, 3.304, respectively, where only 2 respondents indicated consumption of unpasteurized goat milk products, and 421 indicated intake of unpasteurized cow milk products). Washing versus not washing hands after touching raw meat was also significantly associated with seropositivity among women only (OR: https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 13 0.462, 95% CI: 0.260, 0.823). These adjusted significant results held, even after adjustment for heritability and shared household. DISCUSSION This study found that members of the OOA community of Lancaster, Pennsylvania have a high seroprevalence of T. gondii antibodies, with over 56% of study participants testing seropositive, exceeding the levels reported in many other US populations, although similar to levels reported in Java, Indonesia (62.5%)[24] and among the Nanvik Inuit of Canada (59.8%)[27]. The Jones et al. (2014) US-based analysis of the 2009-2010 National Health and Nutrition Examination Survey (NHANES) that included a wave of participants aged ≥6 years found a much lower seroprevalence of 13.2% [95% confidence limit (CL): 11.8%, 14.5%] prior to adjustment for age and an age-adjusted seroprevalence of 12.4% (95% CL: 11.1%, 13.7%)[3]. As these studies vary in terms of recruitment and analytical methodologies, we suggest caution when interpreting these differences. The uniqueness of our study population may further complicate comparisons—as previously mentioned, the OOA community is an ethnically, behaviourally, and culturally distinct group. We are not aware of any studies assessing the seroprevalence of T. gondii in other Amish populations and have found only one study focusing on a similar population. Alvarado-Esquivel et al. (2010) conducted a T. gondii risk factor and seroprevalence analysis among 152 Mennonites in Durango, Mexico, an isolated and mostly rural population of ethnic Germanic descent, with cultural and behavioural aspects that differ from the surrounding communities[35]. They found lower seroprevalence (30.3%) among the Mennonites of Durango[35], compared to the 56% seroprevalence rate that we have observed in the OOA https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 14 community of Lancaster, Pennsylvania. Also, the seroprevalence of T. gondii IgG antibodies in the general population of Durango City, Mexico was reported to be 6.1%, which was remarkably less than the seroprevalence in the Mennonite community living in the same city[36]. This difference in seroprevalence may relate to diverse factors, including food sources, diet, unique group practices, geographic characteristics, and other factors that differ between this Mennonite community and the OOA. The rural location of residence may contribute to the higher seroprevalence among the OOA compared to other populations. Seroprevalence studies often consider living in a rural environment a risk factor because of the presence in rural settings of potentially infected animals that can transmit the parasite to humans in a variety of ways[12]. The finding of higher seroprevalence among rural compared to urban populations is consistent with the results from the United Kingdom, Germany, and Ireland[21, 37, 38]. In addition, a 2013 study by Muñoz-Zanzi et al. found that children residing on farms in Wisconsin were more likely seropositive than those who did not reside on farms[39]. In their multivariate analysis of the 1988-94 wave of NHANES, Jones et al. 2001 reported significantly lower seropositivity among non-Hispanic Blacks living in metropolitan areas (>1 million population) (OR: 0.74, 95% CI: 0.59, 0.93) than among non-Hispanic Blacks living in non-metropolitan areas[40]. Yet, the risk of T. gondii infection was not different among residents of metropolitan and non-metropolitan areas for populations of non-Hispanic Whites or Mexican-Americans[40]. Our study population lacked a proper urban counterpart to assess the impact of rurality/urbanicity, so we cannot exclude the possibility that there is simply high seroprevalence in the general study area. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 15 Several of the food- and food safety-related risk factors found significantly associated with T. gondii seropositivity in this study are consistent with results from other studies. First, consumption and handling of raw and/or undercooked meats was associated with T. gondii seropositivity for both beef and pork, as previously seen in the literature[18, 21-26]. While the practice of cleaning a cat’s litterbox was also observed to be a risk factor among males in our study, our findings highlight the importance of the foodborne transmission route for T. gondii. In addition, washing hands was found protective of T. gondii seropositivity in our study, especially among women. Our findings emphasize the importance of proper food safety behaviours, such as cooking meat to a safe temperature prior to eating/ not consuming raw meat, which is effective at diminishing risk of contracting the parasite. Also, people should avoid accidental contamination by thoroughly washing hands before and after handling meats, and properly washing preparation surfaces and utensils (such as knives) with soap and hot water after use. Washing of hands and/or use of gloves during gardening, may also help prevent contamination[2]. Our study also found that consumption of unpasteurized milk products was associated with risk of seropositivity. While intake of these products provides a possible route of exposure to the parasite[22-24], consumption rates are low among the US population[22, 41, 42], and thus could explain, in part, the increased seropositivity among the Amish. This study provided novel insights into risk factors for infection with a highly prevalent parasite in a unique US population. Among several strengths, the study population is known for its ethnic and socioeconomic homogeneity, which may have helped to minimize confounding by unmeasured variables. Second, response rates https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 16 were high for both the AWS questionnaire and the ATRFQ. Third, to counteract the possibility that the participants’ close family and household aggregation could have produced spurious associations, the study adjusted post-hoc for heritability and household in the analytical sample, without any loss of statistical significance for any of the positive findings reported in our study. Among the study’s important limitations, the findings may not be generalizable to other populations in the US or elsewhere, given the unique characteristics of the culturally distinct OOA[30]. In addition, we saw different distributions between age groups, which may be due to cumulative seropositivity and may have biased some of the observed associations. Our study was also only included adults (age 18y or older), which means that we are unable to assess whether infections were acquired during childhood. Consequently, we are unable to access certain risk factors and are also unable to provide recommendations to prevent these potential exposures. This study is also limited by a relatively small sample size given the number of risk factors analysed. Although the high seropositivity in the OOA may have mitigated sample size issues, the low incidence of certain behaviours may have masked the statistical significance of their potential association with seropositivity. We presented our findings, both formally and informally, to the OOA community in Lancaster, PA. We discussed the different T. gondii risk factors identified in our analysis and specifically addressed the observed sex-based differences. Direct feedback from the OOA community and the findings themselves have and will continue to be used to develop content for future education efforts, including food safety. We plan on applying for grants to develop additional educational materials, train Amish https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 17 educators, and conduct future studies to assess the possible relationships between knowledge and practice among the OAA. For example, future research efforts include developing a study to assess Toxoplasma risk factors and foetal infection, morbidity, and mortality, and possibly establishing a longitudinal study to look at a variety of other important genetic, epigenetic, proteomic, clinical, and environmental factors, with sampling data for T. gondii in food, soil, water, and farm and domestic animals, as well as neuroimaging and other markers of physical and neuropsychiatric health and functioning. CONCLUSION This study found T. gondii seropositivity among more than half of the participants from a unique OOA community with culturally specific meal preparation practices. The study highlights significant behavioural and environmental (including food-related) risk factors associated with infection, such as consuming undercooked meats and unpasteurized cow milk products, as well as protective behaviours, such as eating meals outside of the home and washing hands after contact with raw meat, showing the importance of proper food safety practices for meal preparation, which is of growing relevance to the consumers in the current general US population, as well. Future research will focus on characterizing specific microorganism traits, including serotypes, markers of infection, and genetic, physiological, and clinical associations with T. gondii seropositivity, as well as advancing understanding of education and outreach needed to https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 18 promote protective behaviours and reduce the burden of toxoplasmosis from foodborne and other routes. Acknowledgments: The authors thank Gursharon Nijjar, Christopher Chen, Phillip Miljanic, Hassan Yousufi, Nupur Hegde, Winny Mwaura, Hira Mohyuddin, Sonia Y. Postolache, Thea Postolache, Iqra Mohyuddin, Sunghee Flores, Hanna King, Christopher A. Lowry, Lisa A. Brenner, Andrew Hoisington, Jeffrey O’Connell, Amish Liaisons and nurses and staff of the Amish Research Clinic of the University of Maryland, Lancaster, PA, who went out of their way to support this project. The authors also thank the entire Amish community for collaborating in this project. We further would like to thank Dolores Hill, Dietmar Fuchs and Christopher Hunter for their expert advice on this project. This work was supported by the U.S. Food and Drug Administration through the cooperative agreement FDU.001418 (TTP). Additional funding for this study was provided by the Mid-Atlantic Nutrition Obesity Research Center (NORC) Pilot & Feasibility Project (Postolache, PI), a sub-award of the parent grant P30DK072488 (Simeon I. Taylor, Program Director), and, in part, by the VA Merit Review CSR&D grant 1I01 CX001310-01A1 (Postolache, PI). The views, opinions and findings contained in this article belong to the authors and should not be construed as an official position of the US Food and Drug Administration, the US Department of Veterans Affairs, or the National Institutes of Health. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 19 Conflicts of interest: The authors declare that they have no conflict of interest related to the publication of this paper. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 20 REFERENCES (1) Jones JL, Parise ME, Fiore AE. Neglected parasitic infections in the United States: toxoplasmosis. American Journal of Tropical Medicine & Hygiene 2014; 90(5): 794-799. (2) Centers for Disease Control and Prevention (CDC). Parasites - Toxoplasmosis (Toxoplasma infection) (https://www.cdc.gov/parasites/toxoplasmosis/index.html). Accessed June 3, 2019. (3) Jones JL, et al. Toxoplasma gondii seroprevalence in the United States 2009-2010 and comparison with the past two decades. American Journal of Tropical Medicine & Hygiene 2014; 90(6): 1135-1139. (4) Minor T, et al. The Per Case and Total Annual Costs of Foodborne Illness in the United States. Risk Analysis 2015; 35(6): 1125-1139. (5) Scallan E, et al. 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Commentary on: "Detection of Toxoplasma gondii in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran". Frontiers in Microbiology 2015; 6: 215. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 22 Table 1. Demographic Characteristics of Old Order Amish Participants, Toxoplasma gondii Serostatus and Risk Factor Study, 2015-2016. Characteristic Total n (%) 843 (100%) Seropositive n (%) 478 (56.7) Seronegative n (%) 365 (43.3) P value* Age (y) 18-29 180 (21.4) 77 (16.1) 103 (28.2) <0.0001 30-39 145 (17.2) 77 (16.1) 68 (18.6) 40-49 153 (18.2) 90 (18.8) 63 (17.3) 50-59 138 (16.4) 75 (15.7) 63 (17.3) 60-69 136 (16.1) 92 (19.3) 44 (12.1) 70+ 91 (10.8) 67 (14.0) 24 (6.6) Mean (SD) 46.6/45.0 (16.8) 49.3/49.0 (16.7) 43.2/41.0 (16.3) <0.0001 Sex Female 507 (60.1) 262 (54.8) 245 (67.1) <0.0003 Male 336 (39.9) 216 (45.2) 120 (32.9) Body mass index Mean (SD) 26.5 (4.9) 26.7 (5.0) 26.3 (4.8) 0.24 T. gondii titre concentration (IU/ml) ** Median (SD) 15.6 (3.4, 77.2) 67.9 (24.6, 113.6) 2.8 (1.5, 4.9) <0.0001 SD, standard deviation; T. gondii, Toxoplasma gondii. *P value from X 2 test of independence for age and sex[33]; from SAS 9.4 (Cary, NC) for t-test; italics denote significance at P <0.05. **T. gondii titre concentration: seropositive ≥12 IU/ml; seronegative <12 IU/ml. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 23 Table 2. Bivariate Analysis of Unadjusted Associations (Unadjusted Odds Ratios) of Serostatus and Category-based Behavioural Risk and Protective Factors of Old Order Amish Participants, Toxoplasma gondii Serostatus and Risk Factor Study, 2015-2016. Behavioural Risk or Protective Factor Seropositive of those positive for behaviour n/n (%) Seropositive of those negative for behaviour n/n (%) Unadjusted Odds Ratio 95% CI P value Pets (ref: no pets) Has pets 340/622 (54.7) 127/204 (62.3) 0.959 0.670-1.372 0.818 Has cats 267/455 (58.7) 211/388 (54.4) 1.209 0.912-1.603 0.187 Has kittens 164/287 (57.1) 314/556 (56.5) 1.096 0.811-1.481 0.549 Has contact with cats 364/638 (51.1) 114/205 (55.6) 1.134 0.818-1.571 0.452 Cleans the litterbox 30/40 (75.0) 396/703 (56.3) 2.451 1.167-5.143 0.018 Meat consumption (ref: no consumption) Ate rare meat 62/95 (65.3) 416/748 (55.6) 1.613 1.017-2.560 0.042 Ate raw meat 57/96 (59.4) 421/747 (56.4) 2.192 1.172-4.101 0.014 Ate raw ground pork* 1/1 (100.0) 471/836 (56.3) ∞ ∞ <0.05* Ate rare ground beef 19/25 (0.76) 459/818 (56.1) 2.568 0.996-6.623 0.051 Unpasteurized milk consumption** (ref: no consumption) Consumed unpasteurized cow or goat milk products* 423/735 (57.6) 55/108 (50.9) 1.718 1.116-2.643 0.01 Washing hands (ref: not always washing hands after touching raw meat) Always washing hands after touching raw meat 348/636 (54.7) 80/125 (64.0) 0.642 0.426-0.950 0.035 *Only 1 respondent indicated raw ground pork intake. **Only 2 respondents indicated consumption of unpasteurized goat milk products; 421 indicated intake of unpasteurized cow milk products. Italics denote significance at P <0.05. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 24 Table 3. Bivariate Analysis of Associations (Unadjusted Odds Ratios) of Serostatus and Frequency- based Behavioural Risk and Protective Factors of Old Order Amish Participants, Toxoplasma gondii Serostatus and Risk Factor Study, 2015-2016. Behavioural risk or protective factor Never <1/week ≥1 week Daily Unadjusted odds ratio 95% CI P value Consumption of: Unpasteurized cow milk/ yoghurt 57/112 (50.9) 47/82 (64.0) 77/131 (58.8) 297/ 518 (57.3) 1.211 1.055-1.391 0.007 Rare meat 416/748 (55.6) 14/20 (70.0) 2/3 (66.7) 1/1 (100.0) 1.434 1.023-2.011 0.037 Raw meat 440/790 (55.7) 2/3 (66.7) 1/1 (100.) 0/0 (n/a) 2.003 1.120-3.584 0.019 Less than once a month Once a month or more Unadjusted odds ratio 95% CI P value Location of meal consumption: Outside the home 334/570 (58.6) 75/148 (50.7) 0.611 0.418-0.984 0.01 Italics denote significance at P <0.05. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms 25 Table 4. Adjusted Associations (Adjusted Odds Ratios) of Toxoplasma gondii Seropositivity and Age and Behavioural Risk and Protective Factors by Sex of Old Order Amish Participants, Toxoplasma gondii Serostatus and Risk Factor Study, 2015-2016 Male Female Factor Adjusted odds ratio 95% CI P value Adjusted odds ratio 95% CI P value Age Female 1.027 1.016 – 1.038 <0.0001 Male 1.015 1.001 – 1.030 0.0348 Pets Has pets 0.819 0.442 – 1.520 0.5273 1.048 0.673 – 1.632 0.8364 Has kittens 1.408 0.882 – 2.247 0.1511 0.917 0.617 – 1.363 0.6684 Has cats 1.112 0.704 – 1.758 0.6481 1.278 0.892 – 1.830 0.1814 Cleaned the litterbox 5.241 1.165 – 23.584 0.0309 1.466 0.578 – 3.723 0.4205 Meat consumption* Ate rare meat 1.765 0.901 – 3.458 0.0978 1.45 0.762 – 2.760 0.2579 Ate raw meat 1.111 0.446 – 2.77 0.8216 3.536 1.469 – 8.510 0.0048 Ate rare ground beef 2.988 0.640 – 13.96 0.164 2.327 0.687 – 7.889 0.175 Unpasteurized milk consumption** Consumed unpasteurized cow or goat products** 1.481 0.719- 3.050 0.2872 1.863 1.086- 3.197 0.0239 Consumed unpasteurized cow milk products 1.450 0.719- 2.926 0.2996 1.930 1.127- 3.304 0.0166 Washing hands Always washing hands after touching raw meat 0.931 0.519 – 1.672 0.8115 0.462 0.26 – 0.823 0.0087 Italics denote significance at P <0.05. *Only 1 respondent indicated raw ground pork intake; results, therefore, not shown. **Only 2 respondents indicated consumption of unpasteurized goat milk products; 421 indicated intake of unpasteurized cow milk products. https://doi.org/10.1017/S0950268820002897 Downloaded from https://www.cambridge.org/core. Carnegie Mellon University, on 06 Apr 2021 at 01:38:24, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002897 https://www.cambridge.org/core https://www.cambridge.org/core/terms