Developmental age, physical fitness and Toxocara seroprevalence amongst lower-secondary students living in rural areas contaminated with Toxocara eggs
W. Jarosz1, H. Mizgajska-Wiktor1, P. Kirwan2, J. Konarski3, W. Rychlicki4, G. Wawrzyniak†5
1Department of Biology and Environmental Protection, University School of Physical Education, Królowej Jadwigi 27/39, 61-871 Poznań, Poland
2Department of Zoology, University of Dublin, Trinity College, Dublin 2, Ireland.
3Chair of Theory and Methodology of Sport, University School of Physical Education, Królowej Jadwigi 27/39, 61-871 Poznań, Poland
4Department and Clinic of Tropical and Parasitic Diseases, University of Medical Sciences, S. Przybyszewskiego 49, 60-355 Poznań, Poland
5Chair of Anthropology and Biometry, University School of Physical Education, Królowej Jadwigi 27/39, 61-871 Poznań, Poland
Running title:
Developmental age, physical fitness, children exposure to Toxocara
Corresponding author:
Wojciech Jarosz
Department of Biology and Environmental Protection, University School of Physical Education, Królowej Jadwigi 27/39, 61-871 Poznań, Poland
e-mail:
telephone +48618355246
fax +48618355247
summary
Scarce and inconclusive information on general biological impact of Toxocara invasion on paratenic hosts, and people in particular, has led us to undertake a comprehensive study of the problem. The study has been conducted in a rural environment, which is considered a toxocarosis risk factor. Two hundred soil samples have been screened for Toxocara eggs by floatation, of which 14.5% were positive. Backyards close to households were most heavily contaminated with infectious eggs – 21.7% of positive samples. ELISA serological tests performed on 242 lower-secondary students found 14.5% of the studied population definitely positive – 16.5% of boys and 12.8% of girls, respectively. The odds of being infected with Toxocara were 2 times (CI: 1.15 – 3.85) more likely for individuals who owned a cat than those who did not own a cat. Strong significant correlation between seropositivity and the presence of a dog in a household was found with boys. The level of developmental age was significantly higher in seropositive than in seronegative students. No significant correlation has been observed between the motor abilities and seropositivity of students. Seropositive boys had significantly lower end-of-year grades than their seronegative counterparts.
Key words: Toxocara, soil contamination, students seroprevalence, developmental age, physical fitness
Introduction
Toxocarosis caused by the Toxocara nematode is widely prevalent in cats and dogs and is a zoonotic disease. Man – a paratenic host of the parasite – can become infected with eggs from soil contaminated with infected animal faeces. Toxocara antibodiesare found in 3 – 7% of adults and in 15 – 23% of children (Gillespie et al. 1993), while in the tropics seropositivity is as high as 92.8% among individuals aged > 15 years (Magnaval et al. 1994b). Human exposure to toxocarosis is higher in rural than in urban areas (seropositivity of 14 – 37% versus 2 – 5% respectively) (Magnaval et al., 1994a). A relationship between the degree of soil contamination with Toxocara eggs and child exposure to infection has been suggested (Mizgajska, 1997; Łużna-Lyskov, 2000; Pawłowski and Mizgajska, 2002). Human toxocarosis usually gives no distinctive symptoms (CT – Covert Toxocarosis), yet it can cause severe health consequences for example, Visceral Larva Migrans (VLM) and Ocular Larva Migrans (OLM) (Glickman, 1993; Pawłowski, 2001).
The impact of asymptomatic Toxocara on the paratenic host has, for ethical reasons, been tested only on laboratory animals. Studies have demonstrated that feeding Toxocara larvae to mice, depending on the dose, can cause various behavioural changes in the host and sometimes leads to cognitive disorders (Cox and Holland, 1998; Holland and Cox, 2001). A comparative study of intelligence and physical activity levels in humans infected and not infected with Toxocara has been conducted and the results are not conclusive. Hay et al. (1986) observed hyperactivity in toxocarosis seropositive persons, which they suggest could be specific to individuals. Nelson et al.(1996) have revealed that Toxocara canis-infected people tend to have lower intelligence and comprehension. Moreover, Marmor et al. (1987) suggested that toxocarosis has subtle effects on children’s cognitive development. Although these studies have demonstrated that toxocarosis can manifest itself in different symptoms (not necessarily clinical ones) this subject is still not explored well enough because of it’s complexity and difficulties in planning and performing the studies on humans as well as interpretation of the results.
The present study aims to determine the consequences for the general biological condition of children playing in the open areas contaminated with infected Toxocara eggs. A comprehensive examination of the rural environment was carried out to establish the relationship between the degree of soil contamination with Toxocara eggs and the prevalence of Toxocara seropositivity among students; and to identify a relationship between toxocarosis and student’s developmental age, physical fitness and performance at school and pet ownership. This study has applied a novel broad approach to the investigation of toxocarosis and the better understanding of the reaction of the human body to a non-specific parasite and may allow us to plan and introduce more effective prevention measures to reduce Toxocara transmission to humans.
material and methods
The degree of soil contamination with Toxocara eggs in recreational areas was determined in the Polish rural village of Kołaczkowo, 60 km southeast of Poznań. A comprehensive examination of all students in the only local lower secondary school was also conducted, examining the prevalence of Toxocara antibodies, developmental age, physical fitness and end-of-year grades in individual subjects. Students were questioned about the presence of pets at home (dogs and/or cats). Prior to this, meetings with the parents were held to make them aware of the significance of zoonotic diseases, toxocarosis in particular, their sources and ways of preventing infection. The written consent of the parents had to be obtained before samples of epithelium from the mouth and blood could be taken from the children.
Soil sampling for Toxocara eggs
The sites included the areas most frequently visited by children during their leisure time, i.e. the backyards close to households, the school and kindergarten playgrounds and the village park. The material was soil samples of ca. 500 ml, collected according to rectangular distribution or at equal intervals along the diagonal, from the upper soil layer of 3 cm. After collection, the soil samples were dried in the laboratory for 24 – 48 hours, then sifted through a 2 mm sieve to isolate stones and larger organic particles. Forty grams of homogenous material was taken from every sample and screened for eggs by modified Dada flotation method (Mizgajska-Wiktor, 2005). Wet preparations were examined by microscope. When eggs were found, they were measured (Gonzalez-Ruiz and Bendal, 1995) and on the basis of their size and other morphological features such as thickness of eggshells, their transparency and visibility of semicircular cavities on the surface (Mizgajska and Rejmenciak, 1997). Genus of eggs was determined, as was their age of development and viability. Eggs with a moving larva were considered infectious, while immature eggs were stored in a humidity chamber (100% relative humidity) to investigate whether they were capable of further embryonic development. In the process of microscopic examination, photographs were taken.
Examination of students
Lower-secondary students were examined in the study. Most of the children were aged 14 – 16 years, with six individuals aged 13 and three aged 17. Calendar age was evaluated according to anthropological standards on the base of the decimal system (e.g. 13.50 – 14.49 means 14 years old). Their number varied between 183 and 277 throughout the week of the full range of the study. During serological examination 242 students were tested, 277 for developmental age, 183 for physical fitness, 236 for school performance and 204 for pet possession.
Serological tests
Venous samples of ca 10 ml were taken at the elbow pit into polythene test tubes. Toxocara antibodies were detected with the commercial ELISA test (Bordier Affinity Products, Crissier, Switzerland). Its sensitivity is estimated at more than 90% (Smith and Noordin, 2006) and specificity of 96% (Jacquier et al. 1991). According to the protocol optical density of 1.026 or more (OD405≥1.026) was considered high positive titre (> cut-off), the titre of 0.823 – 1.025 was considered borderline, while lower values were regarded as a negative result. Serological test were conducted in Department and Clinic of Tropical and Parasitic Diseases, University of Medical Sciences, Poznań, Poland. A medical interview of all students was conducted, when blood samples were taken, to determine whether any symptoms of infection have been noticed. The interview covered abdominal pain, hepatomegaly, symptoms of spastic bronchitis, rash and urticaria, allergies, visual disorders, fever, weakness and anorexia.
Developmental age determination
Oral-cavity epithelial cells were collected with a haematological knife and placed into a 1.5 ml Eppendorf tube, which was half filled with 0.9% sodium chloride solution (NaCl). The material was taken to a laboratory for EMN testing (electrophoretical mobility of nuclei) described by Shakhbazov et al. (1986; 1996). This is a simple and universal method, which can be used in developmental age determination of any plant or animal. The average EMN for humans is consistent with the general model of human development, i.e. its value grows significantly during the early ontogenesis, to reach a peak around the age of 17 and decline later (Czapla and Cieślik, 1998). The method makes use of the nucleus mobility in a changing electrical field. Following a microscopic examination of 100 cells, EMN index was determined by calculating the proportion between cells with a mobile nucleus and those where the nucleus did not pulsate. Biotest apparatus was used, which automatically calculates EMN index when the observation data were entered.
Physical fitness determination
Physical fitness was assessed on the basis of test results for muscular strength, flexibility, agility and oxygen endurance (Wachowski et al. 1987). The tests were conducted in the gymnasium after the students were given instructions, a demonstration of tests and a 10-minute warm-up (conducted by a PE teacher).
Muscular strength was tested through vertical jump. The student stood by the side of a centimetre-scaled board and marked his/her outreach with an upright hand. Then they made a vertical jump – as high as they could – from a half-squatting position with a forward bend. The height was read from the board. The test result was the distance obtained between standing reach and jumping reach, measured with a 10-1 m accuracy.
The flexibility of the lower back and hamstring muscle test was about measuring the depth of the forward bend (sit and reach test). The student got on a centimetre-graduated platform his/her toes around the edge, the feet kept together, the legs straightened up. During the deepest possible bend, the lowest outreach was read with a 10-1 m accuracy.
During the agility (coordination) test, the time was measured of a run in a figure of eight delimited with poles placed at five-metre intervals. The student had to run around the “eight” three times and the time was measured with a 10-1 second accuracy.
The cardiovascular endurance test was a five-minute Montoye step test. During that time, the student climbed up and down a 0.3-m-high step, doing so 30 times a minute. The pace was controlled by metronome. The student's pulse was measured with electronic sensors and an amplifier, connected to a computer with appropriate software between 60 and 120 seconds after the test was completed. The results obtained were used to calculate the endurance index expressed in points, according to the following formula:
E – endurance index
t – strain time [s]
p – pulse value after the strain
Student Survey
The survey consisted of an interview, during which simple questions were asked about the number of dogs and cats in the household. The school reports, made available by the school, were used to calculate the average grade for every student and were entered into the survey results.
Statistical Analysis
For statistical reasons, the level of Toxocara antibodies was used to identify two groups: seropositive (OD405≥1.026) and seronegative (OD405<1.025). Analysis was undertaken using Statistica software (StatSoft).
Binary logistic regression was used to determine the explanatory factors (sex, age, grades, cat ownership, dog ownership, agility, flexibility, strength and endurance) that predicted the prevalence of Toxocara. Dog ownership was not included in the model because the data was overdispersed for this variable i.e. all individuals that were infected with Toxocara owned a dog. Multiple logistic regression analysis was run in SPSS 14.0. Model selection was performed using forward log ratio stepwise selection using the likelihood ratio test starting with the maximal model. All risk factors were entered as fixed-factors using reference category formulation. Comparisons of models by the likelihood ratio test requires that the exact same dataset is used throughout the selection process. Observations with missing values for any of the variables included in the maximal model were therefore excluded. See results section for ultimate sample sizes and the number of excluded observations.
The influence of factors (sex, age and infection with Toxocara) on EMN, agility, flexibility, strength, endurance, and grades were analysed by analysis of variance (ANOVA), using 3-way ANOVAs.
results
Biological soil contamination
Toxocara eggs were found in 29 of the 200 soil samples studied (14.5%). The total number of eggs recovered was 152 (13.1 eggs per 100 g of soil) and 85.5% of these were embryonated. In each 40g soil sample 1 to 22 eggs were found. Morphological features indicated that 106 of the Toxocara eggs found in soil were T. canis (69.6%), 40 were T. cati and only 6 had the properties characteristic of the genus (could not be identified to species). Contamination was the strongest in backyards close to households (21.7% of positive samples) and in the school playground (4.6% of positive samples) (Table 1).
[Tab. 1]
Examination of students
Serological tests
Of the 242 students, high positive titre (OD≥1.026) was found in 35 (14.5%), while the borderline (OD: 0.823 – 1.025) was found in 12 (4.9%). Prevalence among boys was slightly higher than among girls being – 21.2% and 17.3% respectively. Of the 109 boys, only 18 showed high positive titre (16.5%), while 6 were in the borderline. Of the 133 girls, 17 showed high positive titre (12.8%) and, like in the case of boys, 6 were in the borderline. (Figure 1; Table 2).
Age category, using age 13 years as the reference category, was included as a fixed factor and with an interaction between the following variables sex and cat ownership to allow for non-linear interactions with children of different ages and the sex of the child and pet ownership. An interaction between sex and cat ownership was also included in the model. The maximal model comprised of 133 observations following exclusion of missing values (144 observations were removed). Stepwise linear regression selected a minimal adequate model (Table 3) with an overall improvement of likelihood ratio from 94.46 to 87.27. The odds of being infected with Toxocara were 2 times (CI: 1.15 – 3.85) more likely for individuals who owned a cat than those who did not own a cat. There was an interaction between age and sex; however, this was not significant. The odds of being infected with Toxocara increased for girls as they got older. Females aged 15 years and 16-17 years were 4.6 and 2.1 times, respectively, more likely to be infected with Toxocara than those aged 13-14 years.
Incidence of infection in specific age groups was different for boys and girls. In the 14 year age group seroprevalence was considerably higher among boys (30.3%) than among girls (8.0%). Incidence of infection for girls grew with age, reaching a peak at 16 years. No Toxocara antibodies were found in the four 13 year olds students or in the three aged 17 years students (Table 2).
[Tab. 2] [Fig. 1]
Cat or dog presence in the household and Toxocara seroprevalence
Of 115 interviewed girls, 71.2% had at least one dog in the household and 39.1% at least one cat. The respective figures for the 89 interviewed boys were 70.0% and 48.3%. The odds of being infected with Toxocara were 2 times more likely for individuals who owned a cat than those who did not own a cat, as written above. All individuals that were infected with Toxocara owned a dog and therefore the data was overdispersed. The correlation between seropositivity and the presence of a cat or/and a dog at home was examined additionally with the χ2 test. With all students (boys and girls together) statistically significant association was found only between seropositivity and cat possession (χ2 =4.940, df=1, P=0.026). With boys, there was a statistically significant correlation between toxocarosis seropositivity and the presence of a cat in the household (χ2 =4.522, df=1, P=0.033) and an even stronger correlation for dogs (χ2 =7.856, df=1, P=0.005). No such correlations have been found with girls (Figure 2).
[Fig. 2]
Developmental age and Toxocara seroprevalence
Individuals infected with Toxocara had a statistically significant higher mean EMN(64.39±1.07) than individuals not infected (60.32±0.61; 3-way ANOVA with age, sex and infection with Toxocara as factors, model R2adj = 0.009, main effect of Toxocara, F1, 224 = 4.578, P=0.033).
The level of physical fitness and Toxocara seroprevalence
No statistically significant differences have been revealed through the comparison of seropositive and seronegative students, both boys and girls, in terms of flexibility, agility, strength and endurance.
Girls (53.25±0.87) were significantly more flexible than boys (49.04±0.75; 3-way ANOVA with age, sex and infection with Toxocara as factors, model R2adj = 0.358, main effect of sex, F1, 144 = 6.590, P=0.011). Flexibility increased significantly as the children got older from 43.5± SE 1.19 in children aged 13-14 years to 55.98 ± SE 0.75 in children aged 16-17 years and (main effect of age, F2, 144 = 10.772, P<0.001).
Mean strength was significantly higher for boys (42.63±0.75) when compared to girls (36.07±0.59; 3-way ANOVA with age, sex and infection with Toxocara as factors, model R2adj = 0.247, main effect of sex, F1, 144 = 19.313, P<0.001).
Mean endurance was significantly higher for boys (52.08±0.97) when compared to girls (44.44±0.63; 3-way ANOVA with age, sex and infection with Toxocara as factors, model R2adj = 0.176, main effect of sex, F1, 176 = 10.967, P<0.001).