【作者】 杜芬；

【导师】 赵俊龙；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2012， 博士

【摘要】 土壤生物污染是指一个或几个有害的生物种群从外界环境侵入土壤,引起土壤质量下降,不仅破坏了原有的生态平衡,还会对植物、动物、人类健康以及生态系统造成不良的影响。近年来,随着人感染禽流感、SARS、猪链球菌病、猪流感等突如其来的疫病灾害的发生,以及人民生活水平的不断提高和环境保护意识的逐渐增强,土壤环境的生物污染问题越来越受到大家的关注。为此本研究选取弓形虫和粪便污染指示菌为代表,首先建立其检测方法,然后对土壤的污染情况进行调查,紧接着针对猪场土壤环境中大肠杆菌的污染情况,通过土壤载体试验、盆栽试验、现场模拟试验从化学消毒剂、生物消毒剂、复合型消毒剂中筛选几种可行的消毒剂,并从戊二醛对土壤微生态环境的影响和残留两个方面进行了戊二醛对土壤的安全性评价。(1)土壤环境中弓形虫卵囊检测方法的建立和应用建立了土壤环境中弓形虫卵囊的PCR/B1、PCR/529和LAMP/MIC3检测方法,检测极限分别为50、5和5个速殖子/0.5g土壤,特异性试验说明仅弓形虫RH株和Prugniaud株能扩增得到特异性目的条带。PCR/B1和PCR/529扩增均为阳性的样品判定为PCR阳性。用PCR和LAMP方法分别检测来自公园、猪场和校园的483份土壤样品,结果表明,两种方法分别得到81(16.77%)份和133(27.54%)份阳性样品,其中猪场土壤的污染最为严重。武汉市6个公园的检测结果表明,所有的公园都受到了不同程度的污染(P>0.05)。在12个被调查的猪场中,仅1个猫低密度猪场的土壤未受到污染,PCR和LAMP检测结果显示猫高密度猪场土壤阳性率分别为低密度猪场的2.24和2.21倍,表明猫的数量与土壤的污染情况呈正相关。公园和校园土壤污染的季节性分析显示,土壤全年受到弓形虫卵囊的污染,各季节之间差异极显著(P<0.01),且从春天到冬天呈现逐渐下降的趋势。因此,土壤可能是人和动物感染弓形虫的传染源之一。另外,建立的PCR和LAMP方法能有效检测土壤中弓形虫卵囊。(2)猪场土壤环境细菌性污染的调查以细菌总数和粪便污染指示菌(大肠杆菌、大肠菌群)为指标,采集湖北省17个规模化猪场共86份土壤样品进行检测与计数,并评价了土壤的污染程度。采用的方法分别为：细菌总数—涂布牛肉膏蛋白胨平板培养；大肠杆菌—PCR方法和涂布麦康凯平板培养；大肠菌群—MPN法。结果显示,PCR方法检测所有土样均为大肠杆菌阳性,土壤中细菌总数、大肠杆菌和大肠菌群分别为4.5×104～6.9×107cfu/g、O～1.2×106cfu/g.1.2×104～5.3×107cfu/g.根据细菌总数判定有72.09%的土壤污染严重。根据大肠杆菌判定有41.86%的土壤被粪便污染严重,大肠菌群已超出我国畜禽养殖业污染物排放标准规定的10-10,000倍,表明土壤中有肠道致病菌存在且已严重超标。秋季土壤大肠杆菌污染最为严重,其次是春天、冬天和夏天。(3)土壤中大肠杆菌消毒剂的筛选本研究选取依绿笑(二氧化氯)、安必杀(有效碘)、戊二醛、申嗪霉素(M18)、天然净等五种消毒剂对土壤中大肠杆菌进行消毒效果评价,从而筛选最佳消毒剂。大肠杆菌稀释液的筛选试验结果表明,0.1％Tween80/PBS的分散性最好。化学消毒剂的中和剂鉴定试验表明,0.5%Na2S203+1%Tween80可有效中和200mg/L的二氧化氯,0.5%Na2S203+1%卵磷脂+1%Tween80可有效中和2000mg/L的有效碘,1%甘氨酸+1%卵磷脂+1％Tween80可有效中和2%的戊二醛。土壤载体试验结果表明：2000mg/L有效碘作用30min,200mg/L二氧化氯作用2min或100mg/L作用1h,0.05%戊二醛作用15min或0.01%作用30min,100mg天然净添加于5g土壤中作用48h可完全杀灭土壤中大肠杆菌。但申嗪霉素对土壤中大肠杆菌和细菌总数均无影响。现场模拟试验结果表明：400mg/L二氧化氯作用24h或600mg/L二氧化氯作用12h,0.1%戊二醛作用12h可杀灭土壤中95%以上的大肠杆菌。(4)戊二醛处理对土壤的安全性评价戊二醛处理后,通过对土壤中可培养微生物进行平板计数,发现戊二醛对土壤中大肠杆菌作用明显,高浓度对细菌、真菌影响较小,但对放线菌影响较大,中、低浓度对细菌、真菌、放线菌的冲击小,且都能在一段时间内恢复正常水平。Biolog的结果显示,低浓度的戊二醛处理有轻微提高微生物活性的作用,中、高浓度处理则有抑制作用,但经过一段时间后微生物活性均能恢复正常。总而言之,虽然戊二醛处理对土壤中微生物对单一碳源的利用能力产生了影响,但各处理间无明显差异。另外,建立了土壤中戊二醛的高效液相色谱检测方法,戊二醛采用2,4-二硝基苯肼柱前衍生后检测,戊二醛在土壤中的最低检测限和最低定量限分别为0.01mg/kg和0.02mg/kg。土壤中戊二醛的消解测定结果表明,在第1d可消除70%及以上,第2d可消除97%以上。总之,戊二醛对土壤微生态环境的影响比较柔和,且不会因残留导致二次污染,是一种安全、高效的土壤生物污染消毒剂。更多还原

【Abstract】 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.更多还原