Detection And Control Of Toxoplasma Gondii And Bacteria In Soil Environment

Soil biological contamination refers to the decline of soil quality by one or more harmful organisms from the external environment intrude into soil. It does not only destroy the original ecological balance, but also has adverse effects on plants, animals, human health and ecosystem. In recent years, with the outbreaks of sudden epidemic disasters, such as human infection with avian influenza, SARS, swine streptococosis and swine flu, and also the improvement of people's living standards and the gradual increase of environmental protection awareness, the biological contamination of soil has been more and more subjected to everyone's attention.In our research, Toxoplasma gondii and fecal contamination indicator bacteria (Escherichia coli and coliform) were selected as the representative pathogens. Firstly, the detection methods were established, and investigated the soil contamination status. Secondly, according to the soil contamination status by E. coli, the best disinfectants were screened from chemical disinfectants, biological disinfectants and compound disinfectants using the soil carrier test, pot experiment and site simulation test. Lastly, the soil safety of glutaraldehyde treatment was evaluated by investigating the influence of soil ecosystems and the residues of glutaraldehyde.(1) Development and application of the detection methods for T. gondii oocysts in soilPCR/B1, PCR/529and LAMP/MIC3methods were developed for the detection of T. gondii oocysts in soil environment. The detection limit of the methods was determined to be50,5, and5tachyzoites per0.5g soil, respectively. The specificity assay showed that these methods were specific for T. gondii. The samples were considered as PCR positive when the results of both PCR/B1and PCR/529were positive. Four hundred and eighty-three soil samples were collected from public parks, pig farms and campus, and then detected for T. gondii oocysts contamination. Eighty-one (16.77%) and133(27.54%) soil samples were positive for T. gondii by PCR and LAMP, respectively. In addition, the soil of pig farms was the highly contaminated.Overall, all of six public parks were contaminated by different levels on both analyses (P>0.05). On the farm level,11of the12pig farms were T. gondii positive, while only one pig farm with low cat density was negative. The PCR and LAMP results suggested that the ratios for positive soil samples detected by PCR and LAMP between pig farms with dual cat density were2.24and2.21, respectively. It suggested that there was a positive relationship between the number of cats present on the farm and the contamination status of T. gondii oocysts in soil. Contamination of public parks and campus soil was found significantly different among the four seasons (P<0.01). The soil was found to be contaminated throughout the year, with a gradual decrease in the prevalence from spring to winter. Therefore, soil may be an important source in the transmission of Toxoplasma for animals and humans. In addition, the conventional PCR and LAMP developed in the present study are applicable to detect T. gondii oocysts in soil samples.(2) Survey on the contamination of bacteria in the soil of pig farmsA total of86soil samples from17large scale pig farms were collected to detect and count E. coli, coliform and bacteria in order to evaluate the contamination status. MacConkey plates and beef extract peptone plates were used to count E. coli and bacteria, respectively. MPN method was used to count coliform. E. coli was also detected by PCR. All the soil samples were E. coli-positive by PCR. The counting results showed that the number of E. coli, coliform and bacteria in one gram soil were0～1.2x106cfu,1.2x104～5.3×107cfu and4.5×104～6.9×107cfu, respectively. Out of total samples analyzed,72.09%soil samples were highly contaminated with the total number of bacteria, and41.86%with E. coli. Additionally, coliform had exceeded10to10,000times to China's livestock industry pollutant discharge standards. It suggested that the existence of intestinal pathogens in soil may be high. Soil contaminated with E. coli was high in autumn, followed by spring, winter and summer.(3) Screening of the disinfectants for E. coli in soilFive disinfectants were selected to evaluate their efficacy against E. coli in soil. The disinfectants were Yilvxiao (chlorine dioxide), Anbisha (available iodine), glutaraldehyde, Shenqinmycin (M18) and Tianranjing. The screening tests for the best dilution against E. coli showed that the spreading of0.1%Tween80/PBS was the most appropriate. The chemical disinfectants of200mg/L chlorine dioxide,2000mg/L available iodine and2%glutaraldehyde were neutralized effectively by using0.5%Na2S2O3+1%Tween80,0.5%Na2S2O3+1%lecithin+1%Tween80,1%glycine+1%lecithin+1%Tween80as neutralizers, respectively.In the soil carrier test, the doses of different disinfectants required for complete eradication of E. coli in soil were as follows:2000mg/L available iodine with30min contact,200mg/L chlorine dioxide with2min contact or100mg/L chlorine dioxide with1h contact,0.05%glutaraldehyde with15min contact or0.01%glutaraldehyde with30min contact,100mg Tianranjing in5g soil with48h contact. But M18had no effect on E. coli and the total bacteria in soil. The results of site simulation test showed that400mg/L chlorine dioxide with24h contact or600mg/L chlorine dioxide with12h contact,0.1%glutaraldehyde with12h contact can eradicate more than95%E. coli in soil.(4) Safety evaluation of soil by glutaraldehyde treatmentThe responses of soil microbes to glutaraldehyde were investigated by monitoring the culturable populations of E. coli, bacteria, fungi, and actinomycete. E. coli was not detected in the un-inoculated control soil throughout the duration of the experiment. In non-glutaraldehyde-treated and E. coli-inoculated soil, the population remained relatively stable. Glutaraldehyde application resulted in a significant decrease of E. coli population. All the treatments had little impact on bacteria and fungi population, and they quickly recovered to their normal levels within a short period of time. But high concentration had great impact on actinomycetes population. The results of Biolog showed that the microbial activity was slightly increased after low concentration of glutaraldehyde treatment, and it was inhibited after the medium and high concentrations of treatment, but activity was returned to normal in a short time. In short, the ability of using sole carbon source by soil microorganisms was changed after glutaraldehyde treatment, however, no significant difference was observed among the treatments. A method for the determination of glutaraldehyde residue in soil by high performance liquid chromatography (HPLC) with pre-colum derivatization by2,4-dinitrophenylhydrazine was developed. The limit of detection (LOD) and limit of quantitation (LOQ) for glutaraldehyde in soil were0.01mg/kg and0.02mg/kg. Glutaraldehyde in soil was dissipated nearly70%at the first day, and more than97%at the second day. The ultimate residues of glutaraldehyde in soil were not detectable. In conclusion, the impact of glutaraldehyde on soil ecosystems was relatively low and did not cause secondary pollution suggesting that it is a safe and efficient disinfectant for soil biological contamination.