Gaur Shashank, Kuhlenschmidt Theresa B, Kuhlenschmidt Mark S, Andrade Juan E
Department of Food Science and Human Nutrition, the University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Department of Pathobiology, the University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Parasitol Int. 2018 Apr;67(2):170-175. doi: 10.1016/j.parint.2017.11.001. Epub 2017 Nov 15.
Cryptosporidium parvum is the second leading cause of persistent diarrhea among children in low-resource settings. This study examined the effect of oregano essential oil (OEO) and carvacrol (CV) on inhibition of C. parvum infectivity in vitro. HCT-8 cells were seeded (1×10) in 96-well microtiter plates until confluency. Cell viability and infectivity were assessed by seeding HCT-8 cell monolayers with C. parvum oocysts (1×10) in two modalities: 1) 4h co-culture with bioactive (0-250μg/mL) followed by washing and incubation (48h, 37°C, 5% CO) in bioactive-free media; and 2) 4h co-culture of C. parvum oocysts followed by washing and treatment with bioactive (0-250μg/mL) during 48-h incubation. Cell viability was tested using Live/Dead™ assay whereas infectivity was measured using C. parvum-specific antibody staining via immunofluorescence detection. Loss of cell viability was observed starting at 125μg/mL and 60μg/mL for OEO and CV, respectively. Neither OEO nor CV modulated the invasion of C. parvum sporozoites in HCT-8 cells. Treatment with bioactive after invasion reduced relative C. parvum infectivity in a dose-dependent manner to 55.6±10.4% and 45.8±4.1% at 60 and 30μg/mL of OEO and CV, respectively. OEO and CV are potential bioactives to counteract C. parvum infection in children.
微小隐孢子虫是资源匮乏地区儿童持续性腹泻的第二大主要病因。本研究检测了牛至精油（OEO）和香芹酚（CV）对体外抑制微小隐孢子虫感染性的作用。将HCT-8细胞（1×10）接种于96孔微量滴定板中，直至汇合。通过以下两种方式用微小隐孢子虫卵囊（1×10）接种HCT-8细胞单层来评估细胞活力和感染性：1）与生物活性物质（0 - 250μg/mL）共培养4小时，然后洗涤并在无生物活性物质的培养基中孵育（48小时，37°C，5% CO）；2）微小隐孢子虫卵囊共培养4小时，然后洗涤并在48小时孵育期间用生物活性物质（0 - 250μg/mL）处理。使用Live/Dead™检测法测试细胞活力，而通过免疫荧光检测使用微小隐孢子虫特异性抗体染色来测量感染性。分别在OEO浓度为125μg/mL和CV浓度为60μg/mL时观察到细胞活力丧失。OEO和CV均未调节微小隐孢子子孢子对HCT-8细胞的侵袭。侵袭后用生物活性物质处理以剂量依赖方式将微小隐孢子虫相对感染性分别降低至：在OEO浓度为60μg/mL时为55.6±10.4%，在CV浓度为30μg/mL时为45.8±4.1%。OEO和CV是对抗儿童微小隐孢子虫感染的潜在生物活性物质。
