A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium

IF 2.9 Q2 PARASITOLOGY Food and Waterborne Parasitology Pub Date : 2022-06-01 DOI:10.1016/j.fawpar.2022.e00163
Biniam Hagos, Robert E. Molestina
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Abstract

Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.

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醇基卵囊灭活法用于隐孢子虫检测方法的建立
隐孢子虫是专性的细胞内寄生虫,可在儿童和免疫功能低下的成人中引起危及生命的腹泻。在摄入可在水和食物中污染、持续存在并抵抗消毒的厚壁卵囊后,通过粪-口途径传播。次氯酸钠、过氧化物、臭氧、甲醛和氨是隐孢子虫卵囊的适宜消毒剂。这些化学物质的有效浓度可能是有毒的,不适合下游研究使用无活力的卵囊。卵囊灭活方法,如紫外线、加热和乙醇或甲醇处理,通常更容易用于常规实验室使用,但它们在隐孢子虫检测开发中的适用性有限。本研究的目的是评价隐孢子虫卵囊的灭活方法,以便于在实验室中应用,并测试整个灭活卵囊在定量PCR (qPCR)中的实用性。实验是在加热(75 °C/10 min)或随时间增加乙醇和甲醇浓度的条件下进行的。采用碘化丙啶(PI)染色法、体外清除法和感染Hct-8细胞系的方法来评价处理的效果。将卵囊暴露于50%乙醇或甲醇中24 h,孢子子的脱落并未受到损害,尽管观察到明显的PI掺入。在体外实验中,这些化学物质的浓度≥70%才能完全抑制Hct-8细胞的清除和感染。通过光镜和免疫荧光观察,在4 °C下保存30 天的灭活卵囊保留了囊壁的完整性和抗原性。此外,直接应用于cop基因qPCR检测的非活卵囊为加标水样中隐孢子虫的鉴定和定量提供了有用的参考试剂。总之,我们已经建立了一种实用的方法来灭活在实验室的小C.卵囊,适用于开发针对寄生虫的检测或诊断分析。
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来源期刊
Food and Waterborne Parasitology
Food and Waterborne Parasitology Immunology and Microbiology-Parasitology
CiteScore
5.10
自引率
4.00%
发文量
38
审稿时长
13 weeks
期刊介绍: Food and Waterborne Parasitology publishes high quality papers containing original research findings, investigative reports, and scientific proceedings on parasites which are transmitted to humans via the consumption of food or water. The relevant parasites include protozoa, nematodes, cestodes and trematodes which are transmitted by food or water and capable of infecting humans. Pertinent food includes products of animal or plant origin which are domestic or wild, and consumed by humans. Animals and plants from both terrestrial and aquatic sources are included, as well as studies related to potable and other types of water which serve to harbor, perpetuate or disseminate food and waterborne parasites. Studies dealing with prevalence, transmission, epidemiology, risk assessment and mitigation, including control measures and test methodologies for parasites in food and water are of particular interest. Evidence of the emergence of such parasites and interactions among domestic animals, wildlife and humans are of interest. The impact of parasites on the health and welfare of humans is viewed as very important and within scope of the journal. Manuscripts with scientifically generated information on associations between food and waterborne parasitic diseases and lifestyle, culture and economies are also welcome. Studies involving animal experiments must meet the International Guiding Principles for Biomedical Research Involving Animals as issued by the Council for International Organizations of Medical Sciences.
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