Experimental modeling of Acanthamoeba keratitis in mice: Isolation and cultivation of Acanthamoeba castellanii.

Abstract

Free-living Acanthamoeba can lead to granulomatous amoebic encephalitis or Acanthamoeba keratitis (AK), an eye infection. AK is most common among contact lens wearers, and causes include long-term lens use, contamination of the contact lens, and corneal trauma. In vitro studies of AK, especially the development of therapeutic drugs, need to be confirmed by in vivo experiments. We developed our AK mouse model using a variety of methods. A. castellanii (AK/AJ1; 1 × 10 cells) were loaded onto 2 mm contact lens pieces for insertion into the scratched eyes of mice under anesthesia, and the eyelids were sutured. After infection (1 to 14 days), in the daily follow-up, it was observed that the AK lesion had progressed in the mouse eyes, and PCR confirmed the amplification of the Acanthamoeba DNA. This study investigated the effective cultivation method to certify PCR analysis in the AK mouse model. After inoculation at 1, 3, 7, and 14 days, PCR was performed on eyeball samples collected from the AK mouse model. Successful cultivation from homogenized eyeballs of A. castellanii was done in the non-nutrient agar with the lawn of Escherichia coli and scaled up with PYG medium. Furthermore, the PCR products obtained from the amoeba cultured in the AK model showed a match of over 99 % with the genetic information of the inoculated amoeba. We suggest that cultivation and PCR methods can confirm the development of AK in the AK mouse model.