Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 http://www.parasitesandvectors.com/content/7/1/301 RESEARCH Open Access Seroprevalence of Toxoplasma gondii infection and associated risk factors in Huicholes in Mexico Cosme Alvarado-Esquivel1*, Sandy Janet Pacheco-Vega1, Jesús Hernández-Tinoco2, Luis Francisco Sánchez-Anguiano2, Luis Omar Berumen-Segovia1, Francisco Javier Imard Rodríguez-Acevedo3, Isabel Beristain-García4, Elizabeth Rábago-Sánchez1, Oliver Liesenfeld5,6, Federico Campillo-Ruiz3 and Oscar Alberto Güereca-García4 Abstract Background: Very little is known about the seroepidemiology of Toxoplasma gondii infection in ethnic groups in Mexico. Huicholes are an indigenous ethnic group living in a remote mountainous region in Mexico. We sought to determine the prevalence of anti-Toxoplasma IgG and IgM antibodies in Huicholes; and to determine the association of Toxoplasma seropositivity with socio-demographic, behavioral, and clinical characteristics of Huicholes. Methods: We performed a cross sectional survey in Huicholes from September 2013 to January 2014. A convenience sampling method was used. We investigated the prevalence of anti-Toxoplasma IgG and IgM antibodies in 214 Huicholes using enzyme-linked immunoassays. A standardized questionnaire was used to obtain the characteristics of the Huicholes. Bivariate and multivariate analyses were used to assess the association of Toxoplasma exposure and Huicholes’ characteristics. Results: Of the 214 Huicholes studied (mean age: 37.98 ± 15.80 years), 71 (33.2%) were positive for anti-T. gondii IgG antibodies and 47 (66.2%) of them were also positive for anti-T. gondii IgM antibodies. Seroprevalence of T. gondii infection did not vary with age, sex, or occupation. However, seroprevalence of anti-T. gondii IgM antibodies was significantly higher in female than in male Huicholes. Multivariate analysis of socio-demographic and behavioral characteristics showed that T. gondii exposure was associated with consumption of turkey meat (OR = 2.28; 95% CI: 1.16-4.46; P = 0.01). In addition, seroprevalence of T. gondii infection was significantly higher in Huicholes suffering from dizziness and memory impairment than those without such clinical characteristics. Conclusions: Our results demonstrate serological evidence of T. gondii exposure among Huicholes which may be impacting their health. Results of this first study of T. gondii infection in Huicholes may be useful for the design of optimal preventive measures against infection with T. gondii. Keywords: Toxoplasma gondii, Seroprevalence, Huicholes, Cross-sectional study Background Infections with the parasite Toxoplasma gondii (T. gondii) occur worldwide [1] and affect about one third of humanity [2]. Although most infections with T. gondii are asymptom- atic, some infected individuals may suffer from symptom- atic pathological changes in the lymph nodes, eyes, and central nervous system [3]. In addition, pregnant women * Correspondence: alvaradocosme@yahoo.com 1Biomedical Research Laboratory, Faculty of Medicine and Nutrition, Juárez University of Durango State, Avenida Universidad S/N, 34000 Durango, Dgo, Mexico Full list of author information is available at the end of the article © 2014 Alvarado-Esquivel et al.; licensee BioM Creative Commons Attribution License (http:/ distribution, and reproduction in any medium Domain Dedication waiver (http://creativecom article, unless otherwise stated. with primary infection with T. gondii may transmit the in- fection to the fetus leading to congenital disease [4]. Im- munocompromised individuals infected with T. gondii may develop a life-threatening disease [5]. Ingestion of food or water contaminated with oocysts shed by cats [3,6] and eat- ing undercooked or raw meat containing tissue cysts [3,7,8] are important routes of T. gondii transmission. Very little is known about the epidemiology of T. gondii infection in ethnic groups in Mexico. We have previously studied the seroepidemiology of T. gondii infection in Mennonites [9] and Tepehuanos [10] in Durango, Mexico. To the best of our knowledge, there is not any report ed Central Ltd. This is an Open Access article distributed under the terms of the /creativecommons.org/licenses/by/4.0), which permits unrestricted use, , provided the original work is properly credited. The Creative Commons Public mons.org/publicdomain/zero/1.0/) applies to the data made available in this mailto:alvaradocosme@yahoo.com http://creativecommons.org/licenses/by/4.0 http://creativecommons.org/publicdomain/zero/1.0/ Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 Page 2 of 7 http://www.parasitesandvectors.com/content/7/1/301 about the epidemiology of T. gondii infection in Huicholes (an indigenous ethnic group living in a remote mountain- ous region (Sierra Madre Occidental) in the western central Mexican states of Nayarit, Durango, Jalisco and Zacatecas. Life style in Huicholes differs from that in other rural popu- lation groups in Durango; they live in marked poverty with very poor housing and sanitary conditions. They have lim- ited access to health care services, and Hospitals in their re- gion do not have a number of laboratory tests for diagnosis of infectious diseases i.e., infection with T. gondii. It is im- portant to study the epidemiology of T. gondii infection in Huicholes because they live in a climatic scenario that may favor T. gondii infection. Huicholes live in a warmer and more humid area than other population groups in the re- gion. Environmental factors may contribute to a higher seroprevalence of T. gondii infection [1,11]. In general, the seroprevalence of T. gondii infection is higher in humid cli- mates than in dry climates; and this is the case in humans [12-14] and animals [15-17]. Furthermore, Huicholes eat meat from wild animals that may be infected with T. gondii. Therefore, we sought to determine the seroprevalence of T. gondii exposure in Huicholes and the association of Toxoplasma seropositivity with socio-demographic, behav- ioral, and clinical characteristics of Huicholes. Methods Study design and study population We performed a cross sectional survey in Huicholes in Mexico from September 2013 to January 2014. Huicholes were sampled in the locality of Huazamota in the mu- nicipality of El Mezquital in Durango State, Mexico. Huazamota (23°28´N 104°24´W) has an altitude of 600 meters above sea level, a warm-sub-humid climate, and a mean annual temperature of 19.2°C. The south region of El Mezquital municipality has a mean annual rainfall varying from 800 to 1000 mm. Other ethnic groups live in the mountainous region including Mexicaneros and Tepehuanos. Inclusion criteria for the study subjects were: 1) Huichol ethnicity (people who speak the Huichol lan- guage and identify themselves as Huicholes); 2) aged 14 years and older; and 3) that voluntarily accepted to participate. Sample size and sampling method For calculation of the sample size, we used a reference seroprevalence of 22.4% [10] as expected frequency of the factor under study, 7,000 as the size of population from which the sample was selected, 16.9% as the least accept- able result, and a confidence level of 95%. The result of the calculation was 214 subjects. A convenience sampling method was used. Specifically, the authors approached Huicholes leaders for permission and support; each leader communicated and invited all people under his command; those who accepted the invitation gathered in a specific area to provide socio-demographic data and blood sample; 214 people who met the inclusion criteria were enrolled. Socio-demographic, clinical, and behavioral data We used a standardized questionnaire to obtain the socio- demographic, clinical and behavioral characteristics of the Huicholes. Socio-demographic items were age, sex, birth place, residence, educational level, occupation, and socio- economic status. Clinical data included the presence of underlying diseases, presence or history of lymphadenop- athy, frequent presence of headache, dizziness, impairments of memory, reflexes, hearing, and vision, and a history of surgery, blood transfusion or transplants. Clinical data in- cluding impairments was self-reported. Huicholes were con- sidered “ill” when they suffered from any disease either acute or chronic affecting any organ or system i.e. digestive, respira- tory, circulatory, endocrine, or nervous, and included any psychiatric, rheumatic, hematological or nutritional disorder and any type of morbidity. In Huichol women, obstetric data were also obtained. Behavioral items included animal con- tacts, contact with cat excrement, foreign travel, meat con- sumption (pork, beef, goat, lamb, boar, chicken, turkey, pigeon, duck, rabbit, venison, squirrel, horse, opossum, or other), frequency of meat consumption, consumption of raw or undercooked meat, unpasteurized milk, dried or processed meat (ham, sausages or chorizo), consumption of unwashed raw vegetables, fruits, or untreated water, frequency of eating away from home (in restaurants or fast food outlets), contact with soil (gardening or agriculture), and type of flooring at home from all participants were obtained. Serological examination for T. gondii antibodies Serum samples were obtained from about 3 ml of whole blood. Sera were kept frozen at −20°C until analyzed. All sera were analyzed by qualitative and quantitative methods for anti-T. gondii IgG antibodies with a commercially avail- able enzyme immunoassay “Toxoplasma IgG” (Diagnostic Automation Inc., Calabasas, CA, USA). Anti-T. gondii IgG antibody levels were expressed as International Units (IU)/ ml, and a cut-off of ≥ 8 IU/ml was used for seropositivity. In addition, sera positive for T. gondii IgG were further ana- lyzed for anti-T. gondii IgM antibodies by a commercially available enzyme immunoassay “Toxoplasma IgM” kit (Diagnostic Automation Inc., Calabasas, CA, USA). The cut-off for IgM seropositivity for each assay was obtained by multiplying the mean cut-off calibrator optical density by a correction factor (f = 0.35-0.40) printed on the label of calibrator. All assays were performed following the in- structions of the manufacturer and included positive and negative controls in each run. A positive IgG test and a negative IgM test in a participant was interpreted as a la- tent infection. A positive IgG test and a positive IgM test in a participant was interpreted as probability of a recent or acute infection. Table 1 Socio-demographic characteristics of Huicholes and seroprevalence of T. gondii infection Characteristic No. of subjects tested Prevalence of T. gondii infection P value No. % Age groups (years) 30 or less 80 21 26.3 0.09 31-50 90 30 33.3 >50 44 20 45.5 Sex Male 86 29 33.7 0.89 Female 128 42 32.8 Birth place Durango state 181 57 31.5 0.22 Other Mexican state 33 14 42.4 Residence Durango state 210 69 32.9 0.6 Other Mexican State or abroad 4 2 50.0 Educational level No education 87 35 40.2 0.02 1-6 years 77 27 35.1 >6 years 50 9 18.0 Occupation Labourera 107 34 31.8 0.66 Non-labourerb 107 37 34.6 Socio-economic level Low 201 69 34.3 0.16 Medium 13 2 15.4 aLabourer: Agriculture, construction, business, livestock raising, factory worker, other. bNon-labourer: student, housekeeping or none occupation. Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 Page 3 of 7 http://www.parasitesandvectors.com/content/7/1/301 Statistical analysis We used the Epi Info version 3.5.4 and SPSS version 15.0 software for the statistical analysis. The Pearson’s chi- square test and the Fisher exact test (when values were less than 5) were used for initial comparison of the fre- quencies among groups. Multivariate analysis was used to assess the association between the characteristics of the Huicholes and the seropositivity to T. gondii. Variables were included in the multivariate analysis if they had a P value equal to or less than 0.15 in the bivariate analysis. Odds ratio (OR) and 95% confidence interval (CI) were calculated by multivariate analysis using the Enter method. The Hosmer-Lemeshow goodness of fit test was used to assess the fitness of the regression model. A P value <0.05 was considered statistically significant. Ethical aspects The purpose and procedures of the survey were ex- plained to all Huicholes. This study was approved by the Ethical Committee of the General Hospital of the Secre- tary of Health in Durango City, Mexico. Participation in the study was voluntary and a written informed consent was obtained from all participants and from the next of kin of minor participants. All Huicholes were proficient in Spanish and understood explanations about the pur- pose and procedure of the survey as well as the in- formed consent provided by the interviewers. Results of the laboratory tests were sent to the Huicholes’ nearest Hospital (Huazamota) where health care providers could inform participants about their results and provide them with medical care if needed. Results In total, we enrolled 214 Huicholes in the study includ- ing 86 (40.2%) males and 128 (59.8%) females. Most Huicholes were born in Durango; their mean age was 37.98 ± 15.80 years (range 14–82 years). General socio- demographic characteristics of the 214 Huicholes stud- ied are shown in Table 1. Of the 214 Huicholes studied, 71 (33.2%) were positive for anti-T. gondii IgG antibodies and 47 (66.2%) of them had anti-T. gondii IgM antibodies. Of the 71 anti-T. gon- dii IgG positive participants, 26 (36.6%) had IgG levels higher than 150 IU/ml, 1 (1.4%) between 100 to 150 IU/ ml, and 44 (62.0%) between 9 to 99 IU/ml. Seroprevalence of T. gondii infection did not vary signifi- cantly with age, sex, birthplace, residence, occupation or socioeconomic level of Huicholes (Table 1). In contrast, seroprevalence varied significantly with educational level; Huicholes with no education had the highest seropreva- lence of T. gondii exposure (40.2%). In the 71 Huicholes seropositive for anti-T. gondii IgG antibodies, the preva- lence of anti-T. gondii IgM antibodies was significantly higher in female (33/42: 78.6%) than in male (14/29: 48.3%), Huicholes (P = 0.008). Prevalence of high (>150 IU/ ml) IgG antibody levels was similar in female (16/128: 12.5%) and male (10/86: 11.6%), Huicholes (P = 0.84). Concerning clinical characteristics, seroprevalence of anti-T. gondii IgG was significantly higher in Huicholes suf- fering from dizziness and memory impairment than those without such clinical characteristics (Table 2). The fre- quency of T. gondii exposure in subjects with dizziness and memory impairment did not vary with age (P = 0.19 and P = 0.48, respectively). The frequencies of other clinical characteristics including the presence of underlying dis- eases; suffering from frequent headaches; presence or his- tory of lymphadenopathy; reflexes, hearing and visual impairments; histories of surgery, blood transfusion, and transplant were similar among T. gondii positive and T. gondii negative individuals. Histories of miscar- riage and stillbirth in women were not associated with T. gondii seropositivity. Table 2 Bivariate analysis of clinical data and infection with T. gondii in Huicholes Characteristic Subjects tested* no. Prevalence of T. gondii infection P value No. % Clinical status Healthy 171 59 34.5 0.34 Ill 41 11 26.8 Lymphadenopathy ever Yes 71 28 39.4 0.17 No 143 43 30.1 Headache frequently Yes 154 57 37 0.05 No 60 14 23.3 Memory impairment Yes 123 48 39 0.03 No 91 23 25.3 Dizziness Yes 123 51 41.5 0.004 No 85 19 22.4 Reflexes impairment Yes 73 29 39.7 0.14 No 141 42 29.8 Hearing impairment Yes 19 8 42.1 0.38 No 195 63 32.3 Visual impairment Yes 56 24 42.9 0.07 No 158 47 29.7 Surgery ever Yes 26 8 30.8 0.78 No 188 63 33.5 Transplantation Yes 0 0 0 - No 214 71 33.2 Blood transfusion Yes 20 7 35 0.85 No 194 64 33 Pregnancies None 11 0 0 0.07 One to three 40 13 32.5 Four to six 44 15 34.1 More than six 33 14 42.4 Deliveries Zero 14 2 14.3 0.26 Table 2 Bivariate analysis of clinical data and infection with T. gondii in Huicholes (Continued) One to three 44 12 27.3 Four to six 44 16 36.4 Seven to nine 19 9 47.4 Nine to twelve 7 3 42.9 Cesarean sections No 115 37 32.2 0.64 Yes 13 5 38.5 Miscarriages No 97 32 33 0.51 Yes 25 10 40 Stillbirths No 115 37 32.2 0.22 Yes 7 4 57.1 *Subjects with available data. Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 Page 4 of 7 http://www.parasitesandvectors.com/content/7/1/301 With respect to behavioral characteristics, a number of variables showed P values lower than 0.15 in the bivariate analysis including presence of dogs at home (P = 0.06), consumption of raw dried meat (P = 0.14), and consump- tion of meat from goat (P = 0.14), turkey (P = 0.002), and pigeon (P = 0.04). A selection of behavioral characteristics and their correlation with T. gondii exposure are shown in Table 3. Other behavioral characteristics of Huicholes in- cluding contact with cats, cleaning cat excrement, raising animals, traveling, consumption of meat other than goat, turkey and pigeon meat, frequency of meat consumption, degree of meat cooking, consumption of unpasteurized milk, processed meat, unwashed raw vegetables or fruits, untreated water, frequency of eating out of home, soil con- tact, and soil flooring at home showed P values higher than 0.15 in the bivariate analysis. Further analysis by using logistic regression showed that T. gondii exposure was only associated with consumption of turkey meat (OR = 2.28; 95% CI: 1.16-4.46; P = 0.01) (Table 4). A P = 0.35 was obtained in the Hosmer-Lemeshow test indicat- ing an acceptable fit of our regression model. In the 71 Huicholes with anti-T. gondii IgG antibodies, seroprevalence of anti-T. gondii IgM antibodies was higher (P = 0.006) in Huicholes who raised animals (41/55; 74.5%) than in those without such practice (6/16: 37.5%), and in Huicholes with consumption of raw dried meat (35/46; 76.1%) than in those without such eating habit (11/24: 45.8%) (P = 0.01). Discussion The present study was performed to investigate the seroe- pidemiology of T. gondii infection in Huicholes in Mexico. Results indicate that Huicholes have one of the highest seroprevalences of T. gondii infection reported in the re- gion. The seroprevalence found in Huicholes (33.2%) is Table 3 Bivariate analysis of selected putative risk factors for infection with T. gondii in Huicholes Characteristic Subjects tested* no. Prevalence of T. gondii infection P value No. % Cats at home Yes 93 33 35.5 0.53 No 121 38 31.4 Dogs at home Yes 165 60 36.4 0.06 No 49 11 22.5 Goat meat consumption Yes 193 67 34.7 0.14 No 21 4 19 Chicken meat consumption Yes 209 71 34 0.17 No 5 0 0 Turkey meat consumption Yes 106 46 43.4 0.002 No 108 25 23.1 Pigeon meat consumption Yes 176 63 35.8 0.04 No 37 7 18.9 Venison consumption Yes 203 69 34 0.34 No 11 2 18.2 Squirrel meat consumption Yes 63 25 39.7 0.19 No 151 46 30.5 Raw dried meat Yes 122 46 37.7 0.14 No 86 24 27.9 Floor at home Ceramic or wood 6 3 50 0.19 Concrete 102 28 27.5 Soil 106 40 37.7 *Subjects with available data. Table 4 Multivariate analysis of selected characteristics of Huicholes and their association with T. gondii infection Characteristic Odds ratio 95% confidence interval P value Age (years) 30 or less 1.00 31-50 0.73 0.32-1.66 0.45 > 50 1.23 0.44-3.47 0.68 Educational level No education 2.08 0.83-5.19 0.11 1-6 years 1.92 0.68-5.38 0.21 > 6 years 1.00 Contact with dogs 2.11 0.93-4.78 0.07 Consumption of: Goat meat 1.61 0.46-5.60 0.45 Turkey meat 2.28 1.16-4.46 0.01 Pigeon meat 1.35 0.51-3.55 0.53 Raw dried meat 1.20 0.63-2.30 0.56 Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 Page 5 of 7 http://www.parasitesandvectors.com/content/7/1/301 higher than the mean (23.8%) seroprevalence of T. gondii infection reported in the general population in rural areas in Durango State [18]. In addition, the seroprevalence in Huicholes is higher than the 6.1% seroprevalence of T. gondii infection reported in urban general population in the capital Durango City [19]. With respect to other ethnic groups in the region, the seroprevalence found in Huicholes is comparable with the 30.3% seroprevalence of T. gondii infection reported in Mennonites [9] but is higher than the 22.4% seroprevalence reported in Tepehuanos [10]. Huicholes and Tepehuanos live in the same mountains (Sierra Madre Occidental), however, Huicholes live in more remote places deeper into the mountainous region than Tepehuanos. It is known that the seroprevalence of T. gondii infection varies depending on the climate conditions in the communities, i.e., a low sero- prevalence in dry and hot climate [14], and high seropreva- lence in humid regions [12]. Huicholes communities are located in a lower region on the mountains with warmer and more humid climate than the one of the Tepehuanos settlements. However, difference in the seroprevalences among Huicholes and Tepehuanos should be interpreted with care because of an age limitation of the comparison: the mean age in Tepehuanos (31.03 ± 16.71 years old) was lower than the one (37.98 ± 15.80 years old) in Huicholes. Toxoplasma exposure has been linked to low socioeco- nomic status [20,21], and such characteristics might have contributed to the increased seroprevalence of T. gondii in- fection in Huicholes. The seroprevalence of T. gondii infec- tion in Huicholes with low socioeconomic status (34.3%) was higher than in those with medium socioeconomic sta- tus (15.4%). However, such increase was not statistically sig- nificant because of the limited number (n = 12) of subjects with medium socioeconomic status in the comparison. In the present study women outnumber men. The higher number of women than men in the study may be due to a number of factors including refusal of some men to partici- pate, and more migration and violent deaths in men than in women in the study region. However, the imbalance in sexes in this study is unlikely to affect the seroprevalence rate since seroprevalence of T. gondii infection has been found similar in men and in women of general populations in rural [18] and urban [19] Durango, Mexico. Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 Page 6 of 7 http://www.parasitesandvectors.com/content/7/1/301 Multivariate analysis of the socio-demographic and be- havioral characteristics of the Huicholes allowed us to identify that consumption of turkey meat was positively associated with T. gondii exposure in Huicholes. In a pre- vious study in the general population in rural Durango, consumption of turkey meat was also associated with T. gondii exposure [18]. In addition, in a study in pregnant women in the urban capital Durango City, consumption of turkey meat was associated with T. gondii infection too [22]. Turkey meat is a potential source for infection with T. gondii [23]. Experimental T. gondii oocyst infections in turkeys have shown the parasite spreading over the whole organism as determined by polymerase chain reaction [24]. In a previous study in birds in Durango, no serological evi- dence of T. gondii infection in 16 turkeys (Meleagris gallo- pavo) was found [25]. However, the number of studied turkeys was too small to exclude T. gondii infections in tur- keys in Durango. Turkey meat is frequently cooked in big pieces; therefore, it is likely that this meat remains under- cooked in some deep areas. Seroprevalence of T. gondii infection usually increases with age in our region [18,19,26]. In the present survey, seroprevalence tended to increase with age; however, such increase did not reach statistical significance (P = 0.09). It is likely that the small sample size of the oldest Huicholes subgroup prevented us obtaining a statisti- cally significant difference. Remarkably, in the present study we found an associ- ation of T. gondii exposure with the presence of dizziness and memory impairment in Huicholes. This finding may indicate a causal association of infection with T. gondii and central nervous system illness in Huicholes. The asso- ciation of T. gondii exposure with dizziness and memory impairment was also found in a recent study in migrant agricultural workers in Durango [21]. The association of memory impairment and T. gondii exposure was also assessed in other ethnic groups in Durango including Mennonites [9] and Tepehuanos [10]; however, no asso- ciation was found. The association of dizziness and T. gondii exposure was not assessed in Mennonites [9] and Tepehuanos [10]. We are not aware of further reports on the association of dizziness with T. gondii infection. On the other hand, the association of memory impair- ment with T. gondii infection found in the present study agrees with previous reports [27,28]. In a previous study in gardeners in Durango City, T. gondii seropositivity was associated with memory impairment [27]. In a recent study in seniors in Germany, researchers found that T. gondii seropositivity was associated with a reduction of about 35% in working memory, a lower performance in verbal memory, and a decreased quality of life [28]. A number of reports indicate that T. gondii infection may lead to neurological and behavioral changes. Experiments in adult mice have shown that infections with T. gondii cause neurological and behavioral abnormalities secondary to inflammation and loss of brain parenchyma [29]. In addition, chronic infections with T. gondii in mice can damage the spatial learning and memory capability [30]. The behavioral alterations associated with T. gondii infec- tion in humans and animals were recently reviewed by Flegr [31] and Webster et al., [32]. Intriguingly, latent T. gondii infection was associated with improvements in cognitive control processes in young healthy humans [33]. However, this effect might perhaps not be found or even reversed in old age. Beste et al. [34] found that latent T. gondii infection leads to deficits in goal-directed behav- ior in otherwise healthy elderly individuals. Anti-T. gondii IgM antibodies were present in a high number of anti-T. gondii IgG positive Huicholes. Such high frequency of IgM positive results was unexpected and should be interpreted with care since IgM ELISA kits have a high rate of false positive results [35]. There- fore, discrimination between recent and latent T. gondii infections is not accurately obtained by ELISA. Other methods such as IgG avidity [36,37] may aid in such discrimination. The present study has some limitations, including the sampling method and the small sample size of eld- erly Huicholes. We were unable to perform random sam- pling because participation of Huicholes in the study depended to a large extent on the permission of the Hui- choles leaders. Huicholes leaders asked their people to participate and only Huicholes who attended the invitation were sampled. A small number of elders attended the invitation. Conclusion Our results demonstrate serological evidence of T. gon- dii exposure in Huicholes and Toxoplasma may be impacting their health. This is the first report of T. gon- dii infection in Huicholes, and results should be useful for the optimal design of preventive measures against T. gondii infection. Competing interests The authors declare that they have no competing interest. Authors’ contributions CAE conceived and designed the study protocol, participated in the coordination and management of the study, performed the laboratory tests and the data analysis and wrote the manuscript. SJPV, LOBS, FJIRA, OAGG and FCR obtained blood samples, submitted the questionnaires and performed the data analysis. JHT, IBG, OL, LFSA and ERS performed the data analysis, and wrote the manuscript. All authors read and approved the final version of the manuscript. Author details 1Biomedical Research Laboratory, Faculty of Medicine and Nutrition, Juárez University of Durango State, Avenida Universidad S/N, 34000 Durango, Dgo, Mexico. 2Institute for Scientific Research “Dr. Roberto Rivera-Damm”, Juárez University of Durango State, Avenida Universidad S/N, 34000 Durango, Mexico. 3Servicios de Salud de Durango, Cuauhtémoc 225 norte, 34000 Durango, Mexico. 4Facultad de Enfermería y Obstetricia, Juárez University of Alvarado-Esquivel et al. Parasites & Vectors 2014, 7:301 Page 7 of 7 http://www.parasitesandvectors.com/content/7/1/301 Durango State, Cuauhtémoc 223 norte, 34000 Durango, Mexico. 5Institute for Microbiology and Hygiene, Campus Benjamin Franklin, Charité Medical School, Hindenburgdamm 27, D-12203 Berlin, Germany. 6Present address: Roche Molecular Diagnostics, Pleasanton, CA, USA. Received: 21 March 2014 Accepted: 26 June 2014 Published: 1 July 2014 References 1. Dubey JP: Toxoplasmosis of animals and humans. 2nd edition. Boca Raton, Florida: CRC Press; 2010. 2. Hill DE, Chirukandoth S, Dubey JP: Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev 2005, 6:41–61. 3. Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet 2004, 363:1965–1976. 4. Weiss LM, Dubey JP: Toxoplasmosis: A history of clinical observations. Int J Parasitol 2009, 39:895–901. 5. Munoz M, Liesenfeld O, Heimesaat MM: Immunology of Toxoplasma gondii. Immunol Rev 2011, 240:269–285. 6. Lilly EL, Wortham CD: High prevalence of Toxoplasma gondii oocyst shedding in stray and pet cats (Felis catus) in Virginia, United States. Parasit Vectors 2013, 6:266. doi:10.1186/1756-3305-6-266. 7. Alvarado-Esquivel C, Torres-Castorena A, Liesenfeld O, Estrada-Martínez S, Urbina-Álvarez JD: High seroprevalence of Toxoplasma gondii infection in a subset of Mexican patients with work accidents and low socioeconomic status. Parasit Vectors 2012, 5:13. doi:10.1186/1756-3305-5-13. 8. Walle F, Kebede N, Tsegaye A, Kassa T: Seroprevalence and risk factors for Toxoplasmosis in HIV infected and non-infected individuals in Bahir Dar, Northwest Ethiopia. Parasit Vectors 2013, 6:15. doi:10.1186/1756-3305-6-15. 9. Alvarado-Esquivel C, Rojas-Rivera A, Estrada-Martínez S, Sifuentes-Álvarez A, Liesenfeld O, García-López CR, Dubey JP: Seroepidemiology of Toxoplasma gondii infection in a Mennonite community in Durango State, Mexico. J Parasitol 2010, 96:941–945. 10. Alvarado-Esquivel C, Estrada-Martínez S, García-López CR, Rojas-Rivera A, Sifuentes-Álvarez A, Liesenfeld O: Seroepidemiology of Toxoplasma gondii infection in Tepehuanos in Durango, Mexico. Vector Borne Zoonotic Dis 2012, 12:138–142. 11. Meerburg BG, Kijlstra A: Changing climate-changing pathogens: Toxoplasma gondii in North-Western Europe. Parasitol Res 2009, 105:17–24. doi:10.1007/s00436-009-1447-4. 12. Julvez J, Magnaval JF, Meynard D, Perie C, Baixench MT: Seroepidemiology of toxoplasmosis in Niamey, Niger. Med Trop (Mars) 1996, 56:48–50. 13. Assmar M, Amirkhani A, Piazak N, Hovanesian A, Kooloobandi A, Etessami R: Toxoplasmosis in Iran. Results of a seroepidemiological study. Bull Soc Pathol Exot 1997, 90:19–21. 14. Markovich MP, Shohat T, Riklis I, Avni R, Yujelevski-Rozenblit D, Bassal R, Cohen D, Rorman E: Seroepidemiology of Toxoplasma gondii infection in the Israeli population. Epidemiol Infect 2014, 142:149–155. doi:10.1017/S0950268813000903. 15. Almería S, Calvete C, Pagés A, Gauss C, Dubey JP: Factors affecting the seroprevalence of Toxoplasma gondii infection in wild rabbits (Oryctolagus cuniculus) from Spain. Vet Parasitol 2004, 123:265–270. 16. Alvarado-Esquivel C, Silva-Aguilar D, Villena I, Dubey JP: Seroprevalence of Toxoplasma gondii infection in dairy goats in Michoacán State, Mexico. J Parasitol 2013, 99:540–542. doi:10.1645/12-103.1. 17. Alvarado-Esquivel C, Romero-Salas D, García-Vázquez Z, Crivelli-Diaz M, Barrientos-Morales M, Lopez-de-Buen L, Dubey JP: Seroprevalence and correlates of Toxoplasma gondii infection in domestic pigs in Veracruz State, Mexico. Trop Anim Health Prod 2014, 46:705–709. doi:10.1007/s11250-014-0551-3. 18. Alvarado-Esquivel C, Cruz-Magallanes HM, Esquivel-Cruz R, Estrada-Martínez S, Rivas-González M, Liesenfeld O, Martínez-García SA, Ramírez E, Torres-Castorena A, Castañeda A, Dubey JP: Seroepidemiology of Toxoplasma gondii infection in human adults from three rural communities in Durango State, Mexico. J Parasitol 2008, 94:811–816. 19. Alvarado-Esquivel C, Estrada-Martínez S, Pizarro-Villalobos H, Arce-Quiñones M, Liesenfeld O, Dubey JP: Seroepidemiology of Toxoplasma gondii infection in general population in a northern Mexican city. J Parasitol 2011, 97:40–43. 20. Yahaya N: Review of toxoplasmosis in Malaysia. Southeast Asian J Trop Med Public Health 1991, 22:102–106. 21. Alvarado-Esquivel C, Campillo-Ruiz F, Liesenfeld O: Seroepidemiology of infection with Toxoplasma gondii in migrant agricultural workers living in poverty in Durango, Mexico. Parasit Vectors 2013, 6:113. 22. Alvarado-Esquivel C, Sifuentes-Alvarez A, Narro-Duarte SG, Estrada-Martínez S, Díaz-García JH, Liesenfeld O, Martínez-García SA, Canales-Molina A: Seroepidemiology of Toxoplasma gondii infection in pregnant women in a public hospital in northern Mexico. BMC Infect Dis 2006, 6:113. 23. Koethe M, Pott S, Ludewig M, Bangoura B, Zöller B, Daugschies A, Tenter AM, Spekker K, Bittame A, Mercier C, Fehlhaber K, Straubinger RK: Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8. Vet Parasitol 2011, 180:179–190. 24. Bangoura B, Zöller B, Koethe M, Ludewig M, Pott S, Fehlhaber K, Straubinger RK, Daugschies A: Experimental Toxoplasma gondii oocyst infections in turkeys (Meleagris gallopavo). Vet Parasitol 2013, 196:272–277. 25. Alvarado-Esquivel C, Rajendran C, Ferreira LR, Kwok OC, Choudhary S, Alvarado-Esquivel D, Rodríguez-Peña S, Villena I, Dubey JP: Prevalence of Toxoplasma gondii infection in wild birds in Durango, Mexico. J Parasitol 2011, 97:809–812. 26. Alvarado-Esquivel C, Liesenfeld O, Burciaga-López BD, Ramos-Nevárez A, Estrada-Martínez S, Cerrillo-Soto SM, Carrete-Ramírez FA, López-Centeno Mde L, Ruiz-Martínez MM: Seroepidemiology of Toxoplasma gondii infection in elderly people in a northern Mexican city. Vector Borne Zoonotic Dis 2012, 12:568–574. doi:10.1089/vbz.2011.0875. 27. Alvarado-Esquivel C, Liesenfeld O, Márquez-Conde JA, Estrada-Martínez S, Dubey JP: Seroepidemiology of infection with Toxoplasma gondii in workers occupationally exposed to water, sewage, and soil in Durango, Mexico. J Parasitol 2010, 96:847–850. 28. Gajewski PD, Falkenstein M, Hengstler JG, Golka K: Toxoplasma gondii impairs memory in infected seniors. Brain Behav Immun 2014, 36:193–199. 29. Hermes G, Ajioka JW, Kelly KA, Mui E, Roberts F, Kasza K, Mayr T, Kirisits MJ, Wollmann R, Ferguson DJ, Roberts CW, Hwang JH, Trendler T, Kennan RP, Suzuki Y, Reardon C, Hickey WF, Chen L, McLeod R: Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection. J Neuroinflammation 2008, 5:48. 30. Wang HL, Bao AY, Wang GH, Jiang MS, Liu ZC, Dong HF, Guo Y: Effect of chronic Toxoplasma infection on the spatial learning and memory capability in mice. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 2006, 24:114–118. 31. Flegr J: Influence of latent Toxoplasma infection on human personality, physiology and morphology: pros and cons of the Toxoplasma-human model in studying the manipulation hypothesis. J Exp Biol 2013, 216:127–133. doi:10.1242/jeb.073635. 32. Webster JP, Kaushik M, Bristow GC, McConkey GA: Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J Exp Biol 2013, 216:99–112. doi:10.1242/jeb.074716. 33. Stock AK, Heintschel von Heinegg E, Köhling HL, Beste C: Latent Toxoplasma gondii infection leads to improved action control. Brain Behav Immun 2014, 37:103–108. doi:10.1016/j.bbi.2013.11.004. 34. Beste C, Getzmann S, Gajewski PD, Golka K, Falkenstein M: Latent Toxoplasma gondii infection leads to deficits in goal-directed behavior in healthy elderly. Neurobiol Aging 2014, 35:1037–1044. doi:10.1016/j. neurobiolaging.2013.11.012. 35. Liesenfeld O, Press C, Montoya JG, Gill R, Isaac-Renton JL, Hedman K, Remington JS: False-positive results in immunoglobulin M (IgM) Toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test. J Clin Microbiol 1997, 35:174–178. 36. Alvarado-Esquivel C, Sethi S, Janitschke K, Hahn H, Liesenfeld O: Comparison of two commercially available avidity tests for Toxoplasma-specific IgG antibodies. Arch Med Res 2002, 33:520–523. 37. Alvarado-Esquivel C, Niewiadomski A, Schweickert B, Liesenfeld O: Antiparasitic treatment suppresses production and avidity of Toxoplasma gondii-specific antibodies in a murine model of acute infection. Eur J Microbiol Immunol (Bp) 2011, 1:249–255. doi:10.1556/EuJMI.1.2011.3.9. doi:10.1186/1756-3305-7-301 Cite this article as: Alvarado-Esquivel et al.: Seroprevalence of Toxoplasma gondii infection and associated risk factors in Huicholes in Mexico. Parasites & Vectors 2014 7:301. Abstract Background Methods Results Conclusions Background Methods Study design and study population Sample size and sampling method Socio-demographic, clinical, and behavioral data Serological examination for T. gondii antibodies Statistical analysis Ethical aspects Results Discussion Conclusion Competing interests Authors’ contributions Author details References