Targeted next-generation sequencing assay to detect 3spp. directly from bovine ocular swabs.

Abstract

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species ofand. A third novelspp., designated tentatively as, has been identified from the eyes of cattle with and without pinkeye. These 3spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessingdiversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3spp. Targeted NGS with sequencing ofspp. housekeeping genes appears to be a suitable method for speciation ofdirectly from ocular swabs.