Establishment of a cell culture-qPCR system to quantify early developmental stages of Eimeria zuernii

Abstract

Bovine coccidiosis is caused by apicomplexans of the genus Eimeria and results in significant economic losses in the cattle industry worldwide. Numerous anticoccidial drugs are available for the treatment of bovine Eimeria infections. However, many compounds have been on the market for decades, and multidrug resistance is commonly observed in avian Eimeria. Recent reports of anticoccidial resistance in ovine Eimeria indicate the need for a rapid and inexpensive in vitro method to assess drug efficacy against ruminant Eimeria. Currently, no such assay exists for bovine Eimeria. The aim of this study was to develop a Madin-Darby bovine kidney (MDBK) cell culture-qPCR model to support the development of Eimeria (E.) zuernii in laboratory settings. The established in vitro assay was applied on three field strains of E. zuernii from the western United States to identify its general suitability for a variety of field strains. Infected cells were observed microscopically and analyzed by quantitative PCR (qPCR) at 48 and 192 h post infection (hpi). Light microscopy observations demonstrated E. zuernii sporozoite invasion as early as 24 hpi, while confocal laser scanning microscopy revealed early meront formation by 48 hpi. Gene copy numbers displayed variations in parasite copy numbers directly after infection and over the observation period over 192 h. Based on these findings, this assay is suitable for detecting E. zuernii gene copies in MDBK cells over an experimental period of 192 h. Though total gene copy numbers did not increase over time, we conclude that this assay is a suitable for sustaining the growth and development of E. zuernii stages in vitro. This testing system will allow for further investigations of bovine Eimeria while reducing the use of animal experiments.