Sandpit contamination with protozoa and bacteria

建立了蔗糖溶液离心浮法检测土壤隐孢子虫卵囊的新方法。由于土样中的卵囊被许多颗粒砂成分包围，通常的离心浮选技术无法有效收集卵囊。通过在蔗糖溶液中加入浓度为0.1%的明胶，解决了这个问题。尽管该方法的采收率从10%到20%不等，但在我们的调查中使用了该技术。农场周围的土壤样本已被确认为隐孢子虫病的地方性动物，我们检查了16个样本，发现所有样本都呈阳性。目前正在开展公园沙坑隐孢子虫污染的研究。与隐孢子虫卵囊不同，弓形虫卵囊不能仅通过形态学方法进行鉴别。然后，我们尝试了一种新的检测方法，使用聚合酶链反应。土壤样品中弓形虫卵囊的纯化方法与隐孢子虫相同。纯化后的样品悬浮在400 μl的TNE缓冲液（含1% PVP和1% SDS）中，按标准程序提取DNA，并进行PCR检测。在检查的144份砂土样品中，有120份（80%）检测到大肠杆菌。同一沙坑检测的弓形虫卵污染率为30%（43/144）。这与大肠杆菌研究的结果不同。100 g阳性沙中平均检出大肠杆菌数为3400个，10个沙坑中大肠杆菌数超过了本次试验大肠杆菌的最大检出限（16万个）。在一个大肠杆菌检查的案例中，一些未使用的海砂样本显示大肠杆菌阳性，这表明对沙子进行大肠杆菌检查并不是粪便污染的良好指标。

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.