Bovine abortions 16S rRNA

Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortiondiagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture hastraditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with theinherent bias of favoring the easy to culture bacteria, e.g. those that do not require cell culture, pre-enrichment, a variety ofselective growth media, or different oxygen levels for in vitro growth. Molecular methods like polymerase chain reaction (PCR)and next generation sequencing have been established as alternatives to traditional microbial culturing methods in severaldiagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technology thatcombines molecular accuracy with culture-independence, and spatial resolution of the pathogen-lesion relation, is also gaininginfluence in several diagnostic fields. In this study, qPCR, 16S rDNA amplicon sequencing, and FISH were applied separately and incombination in order to: i) identify potentially abortifacient bacteria without the bias of culturability, ii) increase the diagnosticrate using combined molecular methods, iii) assess the role of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydiaspp., and Leptospira spp. as bovine abortifacients in Denmark.Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenicbacteria using 16S rDNA amplicon sequencing. Subsequently, species-specific FISH assays were used to assess the lesion associationof a selection of abortifacients in the tissues. The presence of Chlamydia spp. and Chlamydia-like organisms was assessed usingqPCR and the role of fungal infections was determined by FISH screening for lesion-associated fungal elements.The 16S rDNA amplicon sequencing-guided FISH approach was suitable for enhancing abortion diagnostics, i.e. the diagnostic ratefor cases with tissue lesions (n=115) was increased from 46% to 53% when compared to routine diagnostic methods. Identificationof Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with anestablished etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacientswere either not detected or not identified as abortifacients.