Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN. USA.
Department of Microbiology, University of Tennessee, Knoxville, TN, USA.
Int J Parasitol. 2024 Mar;54(3-4):131-137. doi: 10.1016/j.ijpara.2023.12.002. Epub 2023 Dec 13.
Toxoplasma gondii is an apicomplexan protozoan parasite that can infect mammals and birds. The infection can cause acute toxoplasmosis and death in susceptible hosts. Bioassay using cats and mice has been the standard for the isolation of T. gondii from infected hosts for the past several decades. However, bioassay is labor-intensive, expensive, and involves using laboratory animals. To search alternative approaches and o work towards replacement of animal experiments, we summarized the key literature and conducted four experiments to isolate T. gondii in vitro by cell culture. A few heart tissue samples from animals with the highest antibody titers in a given collection were used for T. gondii isolation. These experiments included samples from five out of 51 wild ducks, four of 46 wild turkeys, six of 24 white-tailed deer, as well as from six kangaroos that had died with acute toxoplasmosis in a zoo. These experiments resulted in three isolates from five chronically infected wild ducks (60%), four isolates from four chronically infected wild turkeys (100%), one isolate from six chronically infected white-tailed deer (17%), and four isolates from six kangaroos with acute toxoplasmosis (67%). In addition, five isolates from the five chronically infected wild ducks were obtained by bioassay in mice, showing a 100% success rate, which is higher than the 60% rate by direct cell culture. These T. gondii isolates were successfully propagated in human foreskin fibroblast (HFF) or Vero cells, and genotyped by multilocus PCR-RFLP markers. The results showed that it is practical to isolate T. gondii directly in cell culture. Although the cell culture approach may not be as sensitive as the bioassay, it does provide an alternative that is simple, cost-effective, ethically more acceptable, and less time-sensitive to isolate T. gondii. In this paper we propose a procedure that may be applied and further optimized for isolation of T. gondii.
刚地弓形虫是一种顶复门的原生动物寄生虫，可感染哺乳动物和鸟类。感染可导致易感宿主发生急性弓形虫病和死亡。在过去几十年中，使用猫和老鼠进行生物测定一直是从感染宿主中分离弓形虫的标准方法。然而，生物测定劳动强度大、成本高，并且涉及使用实验室动物。为了寻找替代方法并努力替代动物实验，我们总结了关键文献，并进行了四项实验，通过细胞培养体外分离弓形虫。从给定集合中抗体滴度最高的动物的一些心脏组织样本被用于弓形虫分离。这些实验包括来自 51 只野鸭中的 5 只、46 只野火鸡中的 4 只、24 只白尾鹿中的 6 只以及动物园中死于急性弓形虫病的 6 只袋鼠的样本。这些实验导致从 5 只慢性感染的野鸭中分离出 3 株（60%）、从 4 只慢性感染的野火鸡中分离出 4 株（100%）、从 6 只慢性感染的白尾鹿中分离出 1 株（17%）和从 6 只患有急性弓形虫病的袋鼠中分离出 4 株（67%）。此外，通过小鼠生物测定从 5 只慢性感染的野鸭中获得了 5 株分离株，成功率为 100%，高于直接细胞培养的 60%。这些弓形虫分离株成功地在人包皮成纤维细胞（HFF）或vero 细胞中繁殖，并通过多位点 PCR-RFLP 标记物进行了基因分型。结果表明，直接在细胞培养中分离弓形虫是可行的。虽然细胞培养方法可能不如生物测定灵敏，但它确实提供了一种简单、经济高效、在伦理上更可接受且对时间不敏感的替代方法来分离弓形虫。在本文中，我们提出了一种可应用于进一步优化的分离程序。
