Establishment of a germ carrier assay to assess disinfectant efficacy against oocysts of coccidian parasites

Abstract

Parasites are a common threat to human and animal health. One option to combat parasites that produce infective environmental stages is to inactivate them by chemical disinfection. Standardised laboratory assays that enable proper evaluation of products suspected to be efficient are highly desirable to allow prudent selection and use of such potentially hazardous agents. Here, we present a newly developed in vitro germ carrier assay to evaluate inactivation of oocysts of the model organism Cryptosporidium parvum by chemical disinfectants. Stainless steel discs were used as carrier to mimic surface contamination by C. parvum oocysts. The germ carriers were incubated with approved chemical disinfectant for the specified time (2 h) and rinsed thereafter to remove the disinfectant and recover the exposed oocysts. Recovered oocysts were transferred to HCT-8 monolayers, and 48 h later, genomic DNA was extracted and quantified by real-time PCR targeting the hsp70 gene to estimate parasite reproduction. A panel of commercially available and approved disinfectants were examined and data compared with those of suspension assays and historical data obtained from efficacy assays based on infection of chicken with oocysts of Eimeria tenella. Altogether, data achieved by these divergent assays allowed similar conclusions although the sensitivity of the in vitro assay was higher. Consequently, a threshold of 99.5 % inactivation is proposed to evaluate disinfectants in vitro using C. parvum as model organism as compared to the E. tenella animal infection assay (95 %).