Sandpit contamination with protozoa and bacteria

沙坑被原生动物和细菌污染

• 批准号: 09670256
• 负责人: UGA Shoji
• 金额: $ 1.79万
• 依托单位: Kobe University School of medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670256/
• 关键词: Toxocara canis; Toxocara cati; Toxocara spp.; Escherichi coli; Sandpit; Zoonoses; Cryptosporidium; 犬蛔虫; トキソカラ; トキソプラズマ; 猫蛔虫

We established a new detection method of Cryptosporidium oocysts from the soil by a centrifugal floatation method using sucrose solution. Because the oocysts in the soil samples were surrounded by many of the particulate sand components, usual centrifugal floatation techniques could not be applied to effective collection of the oocysts. This problem was overcome by adding gelatin to the sucrose solution at a concentration of 0.1%. Although the recovery efficiency of the method varied from 10 to 20%, this technique was used for our survey. Soil samples around the farm, which had been confirmed to be enzootic for cryptosporidiasis, were examined and we found that all of 16 samples were positive. Studies on the contamination with Cryptosporidium in sandpits in public parks are being carried out at present.Different from Cryptosporidium oocyst, Toxoplasma oocyst could not be identified only by morphological approach. Then, we attempted a new detection method using a polymerase chain reaction. Purification method of Toxoca oocysts from soil sample was the same as that used for Cryptosporidium. Purified samples suspended in 400 μl of TNE buffer (containing 1% PVP and 1% SDS) were used for DNA extraction according to the standard procedure and were subjected to the PCR.Escherichia coli was detected from 120 out of 144 (80%) sand samples examined. The contamination rate of Toxocara egg examined at the same sandpits was 30% (43/144). which was different from the results obtained through E.coli study. Average number of E.coli found in 100 g of positive sand was 3400, and the numbers of E.coli in 10 sandpits were over the maximum detection limit of E.coli of this test (160,000). In a case of E.coli examination, some of the unused sea sand samples showed positive for E.coli showing that a E.coli examination of the sand will not be a good indicator for the fecal contamination.

我们建立了一种利用蔗糖溶液离心浮选法检测土壤中隐孢子虫卵囊的新方法。由于土壤样品中的卵囊被许多颗粒沙成分包围，通常的离心浮选技术无法应用于有效收集隐孢子虫卵囊。尽管不同方法的回收效率不同，但通过向蔗糖溶液中添加浓度为 0.1% 的明胶来解决这个问题。从10%到20%，我们对农场周围已被证实存在隐孢子虫病的土壤样本进行了检查，结果发现所有16个样本均呈隐孢子虫污染。目前正在开展公园沙坑清理工作。与隐孢子虫卵囊不同，弓形虫卵囊仅通过形态学方法无法识别。然后，我们尝试了一种新的检测方法，使用聚合酶链反应从土壤样品中纯化弓形虫卵囊，其纯化方法与悬浮在 400 μl TNE 缓冲液（含有 1% PVP 和 1% SDS）中的纯化样品相同。根据标准程序提取 DNA 并进行 PCR。144 份中的 120 份检测到大肠杆菌（80%）在同一沙坑中检测到的弓蛔虫卵污染率为30%（43/144），与100克阳性沙子中发现的大肠杆菌平均数量不同。为3400个，10个沙坑的大肠杆菌数量均超过本次检测的大肠杆菌最高检出限（16万个）。大肠杆菌检查，一些未使用的海沙样本显示大肠杆菌呈阳性，表明沙子的大肠杆菌检查不能成为粪便污染的良好指标。

项目成果

Zhiliang Wu,S.Uga et al.: "Specific PCR Primeng for C.Parvum with extra high sensitivity"Molecular and Cellular Probes. in press. (1999)

吴志良，S.Uga等人：“具有超高灵敏度的C. Parvum特异性PCR引物”分子和细胞探针。

小野一男,辻英高,増田邦義,ライタマラ,宇賀昭二: "水系腸管病原性原虫 Cryptosparidium paruum の検査法の検討" 兵庫県衛生研究所年報. 32. 100-106 (1997)

Kazuo Ono、Hidetaka Tsuji、Kuniyoshi Masuda、Tamara Rai 和 Shoji Uga：“水传播肠道致病原虫 Cryptosparidium paruum 检测方法的研究”兵库县卫生研究所年度报告 32. 100-106 (1997)。

宇賀昭二, 小野一男, 伯孝由美, 片岡陳正, ライSK: "兵庫県下における公園砂場の犬・猫蛔虫卵ならびに大腸菌汚染状況" 神戸大学医学部保健学科紀要. 13. 71-76 (1997)

Shoji Uga、Kazuo Ono、Yumi Haku、Chinmasa Kataoka 和 Rai SK：“兵库县公园沙箱中狗和猫蠕虫卵和大肠杆菌污染的情况”神户大学医学院健康科学系通报 13。 71-76（1997）

松村武男, ラ-マンS, ライSK, 斎藤あつ子, 宇賀昭二: "生物環境汚染とその対策-犬・猫と砂場汚染の実際-" 繊維社企画出版部. 第12章. 316-324 (1997)

Takeo Matsumura、Raman S、Lai SK、Atsuko Saito、Shoji Uga：“生物环境污染及其对策——狗、猫和沙箱污染的现实” Senshisha计划出版部第12章。316-324（1997年）。

宇賀昭二: "砂場、新しい暮らしの流れ"現代生活研究会(出版). 206-210 (1999)

Shoji Uga：“沙盒，新的生活流程”现代生活研究组（出版）206-210（1999）。