Seroprevalence of Toxoplasma gondii and Toxocara canis in a human rural population of Southern Rio Grande do Sul

Due to a growing population of homeless pets, especially dogs and cats, zoonoses continue to represent a significant public health problem. The most common intestinal parasites in dogs and cats are protozoa from the genera Giardia, Cystoisospora, Sarcocystis, Cryptosporidium , and nematodes from the genera Toxocara, Toxascaris, Ancylostoma, Uncinaria, Capillaria, and Trichuris . In addition, cats are definitive hosts of the cosmopolitan protozoan Toxoplasma gondii . All of the above parasites are zoonotic agents, with Toxoplasma gondii , Toxocara canis , Toxocara cati having the highest epidemiological importance, as they are the etiological agents of toxoplasmosis and toxocariasis, respectively 1 . Human toxoplasmosis is a disease with worldwide distribution and high prevalence 2 , 3 , as is human toxocariasis, also known as visceral larva migrans. Both infectionsmay present as ocular disease 4 . Even though these are important parasitic diseases, they remain neglected public health issues. Their clinical presentation is diverse as related to the immune response of the host to the parasite load and the affected organ 5 . Both parasites have similar routes of infection that include consumption of contaminated drinking water, raw fruits or vegetables, raw or undercooked meat, viscera contaminated with oocysts excreted by infected cats ( T. gondii ) or eggs eliminated by dogs ( T. canis ). The risk factors and behaviors in children include frequent contact with soil in playgrounds and schools, recreation rooms or sandboxes containing dog and cat feces, poor personal hygiene, geophagia, and the lack of parental supervision 5 , 6 . There are also other forms of transmission such as organ transplantation, blood transfusion, and vertical transmission in humans 7 - 10 . After invading the hosts, these parasites reach the liver or the eye producing ocular lesions, or neuronal manifestations. In Brazil, the seroprevalence of these pathogens varies according to geographic regions and biology of the local environment 11 . The rural population is more susceptible to infection by these parasites 12 , since most of them live in poor conditions of sanitary infrastructure and hygiene, do not have adequate information on these diseases, as prevention methods. Moreover, inadequate child care an low rates of antiparasitic drugs in dogs and cats are important factores for the dissemination of these diseases. Seroepidemiological studies in rural populations help in collecting information on parasitic diseases providing information to the affected population and helping in determining control measures and appropriate treatment regimens. This study, therefore, aimed to investigate the seroprevalence of T. gondii and T. canis in the rural population of Cerrito Alegre, a district of Pelotas, located in the Southern region of Rio Grande do Sul State, RS, Brazil.

Of the 344 samples tested, 53.2% (n=183) were positive for T. gondii. Among the 225 individuals having cats, 54.7% (n=123) were seropositive for T. gondii while 50.4% (n=60) of seropositive individuals did not have any pets in the residence. Most of the patients habitually consumed meat (79.4%), and their seropositivity was determined to be 53.5%, compared to 52.1% in those who did not consume canned meats; this difference was not significant. Habitual consumption of undercooked meat was the only variable that was statistically associated with T. gondii seropositivity ( p = 0.04) ( Table 1 ). Table 1 - Socio-demographic characteristics of the study population for the presence of anti- Toxoplasma gondii antibodies in a rural area of Southern Rio Grande do Sul region Variables Presence of anti- T. gondii antibodies P Unadjusted OR (CI95%) Negative (%) Positive (%) Age 0.7925 18-40 years 30 (45.5) 36 (54.5) 1 41 or more 113 (47.3) 126 (52.7) 0.93 (0.54;1.61) Sex 0.2589 Female 102 (49.3) 105 (50.7) 1 Male 59 (43.1) 78 (56.9) 1.28 (0.83;1.98) Education 0.2850 None to 8 th grade 158 (46.5) 182 (53.5) 1 High School and University 3 (75) 1 (25) 0.29 (0.03;2.81) Consumes canned meats 0.8371 No 34 (47.9) 37 (52.1) 1 Yes 127 (46.5) 146 (53.5) 1.06 (0.63;1.78) Cat owner 0.4529 (no 119) 59 (49.6) 60 (50.4) 1 (yes 225) 102 (45.3) 123 (54.7) 1.19 (0.76;185) Preparation and ingestion of meats 0.0459 Well cooked (done) 158 (48.1) 171 (51.9) 1 Rare to medium/both 3 (20) 12 (80) 3.70 (1.02;13.3) Consumes viscera * 0.4609 No (191) 86 (45.1) 105 (54.9) 1 Yes (153) 75 (49.1) 78 (50.9) 0.85 (0.56;1.30) Meat utilized in alimentation Chicken 119 (46.1) 139 (53.9) 0.8689 1.11 (0.68;1.82) Pork 65 (42.5) 88 (57.5) 0.1512 1.37 (0.89;2.10) Lamb 26 (47.3) 29 (52.7) 0.9482 0.98 (0.55;1.74) Beef 128 (47.6) 141 (52.4) 0.8475 0.87 (0.52;1.45) * Liver, kidney, heart * Liver, kidney, heart Association analyses were performed using data from positive individuals alone to investigate the association between risk factor and an increase in titers of anti- T. gondii antibodies. These individuals were divided into two groups, namely: 1:32 -1:128 dilution (lower) and 1:512 - 1:1024 dilution (higher). This data classification enabled verification of the significance for the following risk factors, namely consumption of pork (OR = 2.29, CI 95% 1.10 - 4.78), beef (OR = 3.14, CI 95% 1.05 - 9.41) and chicken (OR = 3.37, CI 95% 1.12 - 10.08) and the results showed that individuals who habitually ingest these meats had higher titers ( Table 2 ). Table 2 - Association of anti- Toxoplasma gondii antibodies titers with the habit of ingesting bovine, porcine and chicken meat, in a rural population of Southern Rio Grande do Sul region Titrations Pork consumption Beef consumption Chicken consumption Yes (%) No (%) p (OR) Yes (%) No (%) p (OR) Yes (%) No (%) p (OR) 1:32 to 1:128 63 (43.8%) 81 (56.2%) 0.0261 (2.29) 106 (73.6%) 38 (26.4) 0.0414 (3.14) 104 (72.2%) 40 (27.8%) 0.0301 (OR=3.37) 1:256 to 1:1024 25 (64.1%) 14 (35.9%) 35 (89.7%) 4 (10.3%) 35 (89.7%) 4 (10.3%) Of the 344 samples tested, 71.8% (n=247) were positive for T. canis . No statistically significant differences were detected among the variables evaluated for seroprevalence of T. canis (71.8%) ( Table 3 ). Table 3 - Socio-demographic characteristics of the population studied for the presence of IgG antibodies anti- Toxocara canis in a rural area of Southern Rio Grande do Sul region Variables Presence of anti- T. canis antibodies P value Unajusted OR (CI95%) Negative(%) Positive(%) Age* 0.6676 18-40 years 20 (30.3) 46 (69.7) 1 41 or more 66 (27.6) 173 (72.4) 1.14 (0.63;2.07) Sex 0.6898 Female 60 (29.0) 147 (71.0) 1 Male 37(27.0) 100 (73.0) 1.10 (0.68;1.79) Garden 0.8545 No 34 (28.8) 84 (71.2) 1 Yes 63 (27.9) 163 (72.1) 1.05 (0.64;1.71) Dog owner 0.4334 No 10 (34.5) 19 (65.5) 1 Yes 87 (27.6) 228 (72.4) 1.38 (0.62;3.08) Cat owner 0.7159 No 35 (29.4) 84 (70.6) 1 Yes 62 (27.6) 163 (72.4) 1.10 (0.67;1.79) Source of water** 0.7556 Well or watering hole (322) 91 (28.3) 231 (71.7) 1 Tap water (19) 6 (31.6) 13 (68.4) 1.17 (0.43;3.18) * Could not get all ages. ** Not all survey participants responded to their source of water * Could not get all ages. ** Not all survey participants responded to their source of water Seropositivity for both T. gondii and T. canis was 38.3% (n=132), however no statistical difference was observed between the risk factors. In the population studied, a relationship between age and seropositivity of T. canis and T. gondii was found, most individuals older than 40 years presented a positive result for both parasites, and this index reached 73.8% and 51.4% in patients who were 60 years of age or older for toxocariasis and toxoplasmosis, respectively.

Deaths caused by infectious diseases continue to remain a public health problem worldwide and, particularly, in Brazil, despite a reduction in their numbers in recent decades. Only a few reports on the seroprevalence of infection by parasitic agents in rural areas are currently available. We found that 53.2% of the population were seropositive for T. gondii while 71.8% were seropositive for T. canis in the rural population of the city of Pelotas. An association between individuals living in the countryside and seropositivity for toxoplasmosis has already been demonstrated 14 . Seropositivity rates for toxoplasmosis (56.7%), similar to those found in our study, have been reported in riverine populations in Brazil 15 . Among the risk factors evaluated for T. gondii and T. canis seropositivity, habitual eating of undercooked meat was the only significant risk factor (p = 0.045, OR = 3.7). Similarly, a study in pregnant women in Pelotas also described raw meat consumption as a significant risk factor for T. gondii seropositivity 16 . This association was also evident in a study conducted in the city of Arak (Iran) with 400 women, where handling or eating raw or undercooked meat was significantly associated with T. gondii seropositivity 17 . Among the seropositive cases aged between 18-40 years (n=36), 22 were women of childbearing age. This observation is important as parasitic infection during pregnancy is a major problem, especially during the first few months, when infection may result in miscarriage or birth defects. Some authors also cite issues that go beyond the complications of congenital infection. These include infertility mechanisms associated with T. gondii , such as the development of endometriosis, fetal rejection due to release of latent tachyzoites cysts in the endometrium, follicular aberrations in the ovaries, uterine atrophy, and hypothalamic dysfunction affecting reproduction that results from chronic toxoplasmosis 18 . The study shows that most individuals older than 40 years presented a positive result for both parasites, and this index was higher in patients aged 60 years or older. It should be noted that the increase in the elderly population in developing countries is drastically faster than in industrialized ones. As aging is associated with a higher prevalence of chronic and debilitating diseases (even in need of transplants), the use of drugs and immunobiological agents capable of altering immunity in the elderly becomes more frequent, and this immunosuppression may result in the reactivation of cysts of T. gondii and T. canis , among other tropical diseases typical of developing countries 19 , 20 . Analysis of T. gondii seropositive individuals showed statistical significance for the following risk factors, namely pork (OR = 2.29, CI 95% 1.10 - 4.78), beef (OR = 3.14, CI 95% 1.05 - 9.41), and chicken consumption (OR = 3.37, CI 95% 1.12 - 10.08) in individuals with high antibody titers, i.e., from 1:256 to 1:1024. Although antibody titration analysis was not carried out, other authors have reported that consumption of pork and beef is a significant risk factor for the presence of T. gondii antibodies 21 , 22 . The seroprevalence rate for T. canis was 71.8%. Similar rates have been observed in other regions in Brazil (52% and 65.4%) 23 , while seropositivity in other countries has been reported to range from 12% to 86% 10 , 24 , 25 . These differences in seroprevalence are attributable to various reasons, such as location, sample size, and age of the study population, as well as the diagnostic tests used. A study carried out in the urban area, identified a seroprevalence rate between 52 and 65.4%, suggesting that country life with little health information increases the probability of T. canis infection 23 . The results presented here pertain to adults 24 and analyzed children 26 living in islands (86.75%) and urban zones (12.7% and 9.5%), and found completely divergent seroprevalence rates. It is interesting to note that the lowest seroprevalence rate was obtained in urban areas where health information is more available, while the higher seroprevalence rate was from a study in Majuro, one of the Marshall Republic Islands, where the population lives on agriculture, fishing, and the United States aid. Contact with dogs did not affect T. canis seropositivity, as it was 72.4% in those with contact and 65.5% in those without. Similar figures of 60%, 62%, 66%, and 73.6% have been reported in studies from Brazil and Ghana on T. canis infection and its association with having pet dogs 23 , 25 , 27 . However, a study in rural Turkey found a significant association between having dogs at home and anti- T. canis antibodies 28 . The sources of water as an infection risk were also investigated as 231 seropositive patients (67%) consumed water from wells. Other studies have reported seropositivity rates of 13% and 88% 10 , 27 when unboiled and untreated water, respectively, was consumed; this may have contributed to infection with T. canis . The lack of significant association between risk factors and T. canis seropositivity may be related to the fact that more than 70% of the participants were seropositive, making it difficult to obtain statistical significance. A study on children in Pelotas, Rio Grande do Sul, Brazil, found that more than 50% of the children were seropositive for T. canis 29 . It is noteworthy that T. canis eggs are frequently found in places where children assemble and play, such as parks, squares, and student-leisure areas in Pelotas and the Southern Rio Grande do Sul 11 , 30 , 31 . Since our study investigated adults, the chances of already having been in contact with infectious forms of the helminth tend to increase, due to the “exposure time” factor. Concomitantly, the seropositivity of T. gondii and T. canis was 38.3%, and as already mentioned, both parasites have similar infection forms. Some studies indicate that the presence of antibodies to T. canis increases the risk of infection by T. gondii 32 . In the countryside of Sao Paulo, simultaneous infection rates by T. canis and T. gondii were found to be 14% 11 . We conclude that the rural population studied here displays high seropositivity for T. gondii and T. canis and that among the risk factors evaluated, only consumption of undercooked meat was a significant risk factor for T. gondii infection. Additionally, individuals who ingested pork, beef, or chicken meat had higher titers of anti- T gondii antibodies.

Blood samples were collected from 344 patients at a Basic Healthcare Unit (BHU) in the town of Cerrito Alegre, RS (the 3 rd district of the city of Pelotas) between March 2012 and February 2014, and the sera stored at -20 °C. The sample size was representative of all individuals in the study area. A survey of risk factors associated with the presence of parasitic infections was performed using a semi-structured questionnaire, that evaluated the following variables: gender, age, education, presence of cats and dogs at home, possessing or raising other animals (cattle, pigs, sheep, chickens, ducks), type of meat consumed, garden farming, habit of eating undercooked or raw meat, and consumption of processed meats or viscera. The inclusion criteria for the study subjects were residence in the 3 rd District of Pelotas, agreeing to participate in the study by signing the Informed Consent document (IC), allowing blood collection, and being at least 18 years of age. The project was approved by the Faculty of Medicine Ethics Committee UFPel (Nº OF.36/12). Seropositivity for T. gondii was tested using Imuno-Con Toxoplasmose kit (WAMA Diagnóstica) an indirect immunofluorescence assay (IFA), according to the manufacturer’s instructions. Briefly, wells of the IFA slides were filled with 20 µL of serum sample diluted in AD Buffer (1:30) and kept in a moist chamber for 30 min at 37 °C. Afterwards, the slides were washed three times for 10 min. with the kit buffer (100 mL of PBS diluted in 900 mL of distilled water) with stirring. The slides were then dried at 37 °C for 5 min, anti-human antibody conjugated to FITC added, and kept in a moist chamber for 30 min at 37 °C. The slides were washed thrice, each for 10 min. with stirring and subsequently dried at 37 °C for 5 min. Next, three drops of glycerin were added to each well, and a cover slip placed for fluorescence microscope reading. The samples that were positive in the lowest dilution (1:32) were subsequently tested at further dilutions of 1:64, 1:128, 1:256, 1:512, and 1:1024. Positive and negative control sera were used in all slides. Serology testing for T. canis used TES antigens that were produced according to Santos et al . 13 , but with modifications. Polystyrene 96-well plates were coated with 1 µg/mL of TES antigen dissolved in carbonate-bicarbonate buffer (pH 9.6) for 16 h. at 4 ºC. The free protein binding sites were blocked with skim milk powder (5% in PBS-T;0.05% Tween) for 1 h. at 37 °C, while sera were pre-adsorbed on total Ascaris lumbricoides antigen (AgSoAl; 23.7 mg/mL). All samples were evaluated in duplicate at a dilution of 1:100. Anti-mouse IgG human peroxidase conjugate (1: 5000 in PBS-T buffer; Sigma-Aldrich) was used as the secondary antibody. Both sera and conjugate were incubated for 1 h. at 37 °C. Plates were washed thrice with PBS-T between all test stages. Ortho-phenylenediamine (OPD) at a concentration of 0.4 mg/mL in citrate-phosphate buffer pH 4.0 with hydrogen peroxide (30v 0.01%) was used as the chromogen, the plate incubated at room temperature for 15 min in the dark and the reaction was quenched with 50 µL of 1N sulfuric acid. The reaction product was colorimetrically detected by taking absorbance at 450 nm using a plate reader VICTOR X5 Multilabel (Perkin Elmer) with a 492 nm wavelength filter. Positive and negative control sera were added to plate in duplicate as controls. The sample size was calculated based on the size of the adult population of the rural locality to be evaluated (2,000 individuals), considering a margin of error of 5% and a 95% confidence level. Thus, the sample size found was of 323 individuals. All epidemiological data obtained from questionnaires were analyzed using Epi Info (ver. 6.04, CDC). To calculate associations, a logistic regression (unadjusted logistic regression) was used to estimate the odds ratio and their respective 95% confidence intervals for each outcome and their exposure variables. These analyzes were performed in the statistical package STATA 12.1 (StataCorp LP, College Station, USA). No adjusted analysis was performed for lack of associations in the crude analysis.