ITS2 REGION-BASED MOLECULAR IDENTIFICATION OF FUNGAL PATHOGENS IN EQUINE CORNEAL ULCERATION

Abstract

This study aimed to assess the utility of polymerase chain reaction (PCR) and DNA sequencing in diagnosing fungal ulcerative keratitis in horses and compare their sensitivity with conventional microbiologic techniques used in laboratories. Conjunctival swabs from 12 horses with corneal ulcerations admitted to the Veterinary Teaching Hospital, College of Veterinary Medicine, PurdueUniversity were collected for examinations. 14 conjunctival swabs were analyzed by the conventional culture method and by generic PCR using universal fungal primers to amplify the internal transcribed spacer 2 (ITS2) genetic region followed by DNA sequencing. The conventional culture method revealed that two samples exhibited fungal growth that identified as Aspergillus sp. and Penicillium sp. while three conjunctival samples contained bacterial growth which was identified as Staphylococcus intermedius, Staphylococcus epidermidis and Streptococcus zooepidemicus. Interestingly, PCR followed by DNA sequencing of the biological specimens on the swabs identified fungal pathogens in 9/14 samples and Ascomycete species in 3/14 samples. In 2/14 samples, no fungal pathogen was identified using PCR. Fungal pathogens detected included Aspergillus sp. in six eyes, Penicillium sp. in one eye, Fusarium sp. in one eye and Cladosporium sp. in one eye. Our findings demonstrate a limitation of the conventional culture method as a diagnostic tool as a fungal pathogen was detected in only two samples by this method as compared to nine samples using PCR and DNA sequencing. Therefore, PCR followed by DNA sequencing is able to identify a greater spectrum of agents including fastidious organisms or organisms present in lower numbers within mixed infections than can be identified by culture. In conclusion, PCR combined by DNA sequencing not only proved to be an effective and rapid method for the diagnosis of fungal keratitis, but was also a more sensitive diagnostic tool compared to the conventional culture method. Our findings demonstrate that PCR, in particular semi-nested PCR, is a promising tool for faster diagnoses of fungal keratitis in affected horses.