Elsheikha H M, Rosenthal B M, Murphy A J, Dunams D B, Neelis D A, Mansfield L S
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
Vet Parasitol. 2006 Feb 18;135(3-4):223-34. doi: 10.1016/j.vetpar.2005.09.014. Epub 2005 Nov 8.
The objective of this study was to evaluate the utility of a simple, efficient, and rapid method for the isolation of Sarcocystis neurona merozoites and Besnoitia darlingi tachyzoites from cultured cells. The efficacy of this purification method was assessed by microscopy, SDS-PAGE, Western blotting, immuno-fluorescence, and three novel quantitative PCR assays. Culture medium containing host cell debris and parasites was eluted through PD-10 desalting columns. This purification method was compared to alternatives employing filtration through a cellulose filter pad or filter paper. The estimated recovery of S. neurona merozoites purified by the column method was 82% (+/-3.7) of the original merozoites with 97.5% purity. In contrast, estimated recovery of S. neurona merozoites purified by filter pad and filter paper was 40% and 30% with 76% and 83% purity, respectively. The same procedures were applied to purify B. darlingi tachyzoites from cultured cells. Of the original cultured B. darlingi tachyzoites, 94% (+/-2.5) were recovered from the PD-10 column with 96.5%, purity whereas percentage recovery of B. darlingi tachyzoites purified by filter pad and filter paper were 51% and 35% with 84% and 88% purity, respectively. All described methods maintained sterility so that purified parasites could be subsequently cultured in vitro. However, purification using a PD-10 column minimized parasite loss and the loss of viability as determined by the trypan blue dye exclusion assay, the rate of parasite production, and plaque forming efficiency in cell culture. Moreover, column-purified parasites improved the sensitivity of an immuno-fluorescent (IFA) analysis and real-time quantitative PCR assays targeted to parasite 18S ribosomal DNA and hsp70 genes. This technique appears generally applicable for purifying coccidia grown in cell cultures.
本研究的目的是评估一种从培养细胞中分离肉孢子虫神经元裂殖子和达林贝斯诺孢子虫速殖子的简单、高效且快速的方法的实用性。通过显微镜检查、SDS-PAGE、蛋白质印迹、免疫荧光以及三种新型定量PCR检测方法评估了这种纯化方法的效果。含有宿主细胞碎片和寄生虫的培养基通过PD-10脱盐柱洗脱。将这种纯化方法与使用纤维素滤垫或滤纸过滤的替代方法进行了比较。通过柱法纯化的肉孢子虫神经元裂殖子的估计回收率为原始裂殖子的82%（±3.7），纯度为97.5%。相比之下，通过滤垫和滤纸纯化的肉孢子虫神经元裂殖子的估计回收率分别为40%和30%，纯度分别为76%和83%。相同的程序用于从培养细胞中纯化达林贝斯诺孢子虫速殖子。在原始培养的达林贝斯诺孢子虫速殖子中，从PD-10柱中回收了94%（±2.5），纯度为96.5%，而通过滤垫和滤纸纯化的达林贝斯诺孢子虫速殖子的回收率分别为51%和35%，纯度分别为84%和88%。所有描述的方法都保持了无菌状态，以便纯化后的寄生虫能够随后在体外培养。然而，使用PD-10柱进行纯化可将寄生虫损失和活力损失降至最低，这通过台盼蓝染料排除试验、寄生虫产生率以及细胞培养中的噬斑形成效率来确定。此外，柱纯化的寄生虫提高了针对寄生虫18S核糖体DNA和hsp70基因的免疫荧光（IFA）分析和实时定量PCR检测的灵敏度。该技术似乎普遍适用于纯化在细胞培养中生长的球虫。
