Introduction: Toxoplasmosis is a zoonosis that represents a serious public health problem, caused by Toxoplasma gondii, which affects 20-90% of the world human population [1,2]. It is a serious problem especially when considering the congenital transmission due to congenital sequels. Treatment with highly diluted substances is one of the alternative/complementary medicines most employed in the world [3,4]. The current ethical rules regarding the number of animals used in animal experimental protocols with the use of more conservative statistical methods [5] can not enhance the biological effects of highly diluted substances observed by the experience of the researcher.
Aim: To evaluate the minimum number of animals per group to achieve a significant difference among the groups of animals treated with biotherapic T. gondii and infected with the protozoan regarding the number of cysts observed in the brain.
Material and methods: A blind randomized controlled trial was performed using eleven Swiss male mice, aged 57 days, divided into two groups: BIOT-200DH - treated with biotherapic (n=6) and CONTROL - treated with hydroalcoholic solution 7% (n=7).The animals of the group BIOT-200DH were treated for 3 consecutive days in a single dose 0.1ml/dose/day. The animals of BIOT – 200DH group were orally infected with 20 cysts of ME49-T. gondii. The animals of the control group were treated with cereal alcohol 7% (n=7) for 3 consecutive days and then were infected with 20 cysts of ME49 -T. gondii orally. The biotherapic 200DH T. gondii was prepared with homogenized mouse brain, with 20 cysts of T. gondii / 100μL according to the Brazilian Homeopathic Pharmacopoeia [6] in laminar flow. After 60 days post-infection the animals were killed in a chamber saturated with halothane, the brains were homogenized and resuspended in 1 ml of saline solution. Cysts were counted in 25 ml of this suspension, covered with a 24x24 mm coverglass, examined in its full length. This study was approved by the Ethics Committee for animal experimentation of the UEM - Protocol 036/2009. The data were compared using the tests Mann Whitney and Bootstrap [7] with the statistical software BioStat 5.0.
Results and discussion: There was no significant difference when analyzed with the Mann-Whitney, even multiplying the "n" ten times (p=0.0618). The number of cysts observed in BIOT 200DH group was 4.5 ± 3.3 and 12.8 ± 9.7 in the CONTROL group. Table 1 shows the results obtained using the bootstrap analysis for each data changed from 2n until 2n+5, and their respective p-values. With the inclusion of more elements in the different groups, tested one by one, randomly, increasing gradually the samples, we observed the sample size needed to statistically confirm the results seen experimentally. Using 17 mice in group BIOT 200DH and 19 in the CONTROL group we have already observed statistical significance. This result suggests that experiments involving highly diluted substances and infection of mice with T. gondii should work with experimental groups with 17 animals at least.
Despite the current and relevant ethical discussions about the number of animals used for experimental procedures the number of animals involved in each experiment must meet the characteristics of each item to be studied. In the case of experiments involving highly diluted substances, experimental animal models are still rudimentary and the biological effects observed appear to be also individualized, as described in literature for homeopathy [8]. The fact that the statistical significance was achieved by increasing the sample observed in this trial, tell us about a rare event, with a strong individual behavior, difficult to demonstrate in a result set, treated simply with a comparison of means or medians.
Conclusion: Bootstrap seems to be an interesting methodology for the analysis of data obtained from experiments with highly diluted substances. Experiments involving highly diluted substances and infection of mice with T. gondii should be better work with experimental groups using 17 animals at least.
Toxoplasma gondii GRA15 DNA Vaccine with a Liposomal Nanocarrier Composed of an SS-Cleavable and pH-Activated Lipid-like Material Induces Protective Immunity against Toxoplasmosis in Mice
