ORIGINAL RESEARCH published: 23 February 2021 doi: 10.3389/fvets.2021.623666 Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization Godelind Alma Wolf-Jäckel 1*†, Mikael Lenz Strube 2, Kirstine Klitgaard Schou 1, Christiane Schnee 3, Jørgen S. Agerholm 4 and Tim Kåre Jensen 1 1 National Veterinary Institute, Technical University of Denmark, Kongens Lyngby, Denmark, 2 Department of Biotechnology Edited by: and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark, 3 Institute of Molecular Pathogenesis, Nick Wheelhouse, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany, 4 Section for Veterinary Reproduction Edinburgh Napier University, and Obstetrics, Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of United Kingdom Copenhagen, Taastrup, Denmark Reviewed by: Nicole Borel, Abortion in cattle causes significant economic losses for cattle farmers worldwide. University of Zurich, Switzerland Fabio S. Lima, The diversity of abortifacients makes abortion diagnostics a complex and challenging University of California, Davis, discipline that additionally is restrained by time and economy. Microbial culture has United States traditionally been an important method for the identification of bacterial and mycotic *Correspondence: abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture Godelind Alma Wolf-Jäckel [email protected] species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as †Present address: Godelind Alma Wolf-Jäckel, polymerase chain reaction (PCR) and next-generation sequencing have been established Section of Pathobiological Sciences, as alternatives to traditional microbial culturing methods in several diagnostic fields Department of Veterinary and Animal Sciences, University of Copenhagen, including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging Frederiksberg, Denmark microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in Specialty section: This article was submitted to several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA Animal Reproduction - amplicon sequencing, and FISH were applied separately and in combination in order Theriogenology, to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase a section of the journal Frontiers in Veterinary Science the diagnostic rate using combined molecular methods, (iii) investigate the presence of Received: 30 October 2020 the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira Accepted: 21 January 2021 spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses Published: 23 February 2021 and placentas submitted for routine diagnostics were screened for pathogenic bacteria Citation: Wolf-Jäckel GA, Strube ML, using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well Schou KK, Schnee C, Agerholm JS as of selection of bacterial abortifacients, was assessed using specific FISH assays. and Jensen TK (2021) Bovine The presence of Chlamydia spp. and chlamydia-like organisms was assessed using Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle Amplicon Sequencing and is free from most viral abortifacients. The 16S rDNA amplicon sequencing–guided FISH Fluorescence in situ Hybridization. Front. Vet. Sci. 8:623666. approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for doi: 10.3389/fvets.2021.623666 cases with tissue lesions (n = 115) was increased from 46 to 53% when compared Frontiers in Veterinary Science | www.frontiersin.org 1 February 2021 | Volume 8 | Article 623666 Wolf-Jäckel et al. Bovine Abortion Diagnostics NGS FISH to routine diagnostic methods. Identification of Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with an established etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacients were either not detected or not identified as abortifacients. Keywords: Chlamydiaceae, culture-independent, chlamydia-like organisms (CLO), deep sequencing, diagnostics, fluorescence in situ hybridization (FISH), lesion association, zoonosis INTRODUCTION zoonotic bacteria Chlamydia spp., C. burnetii, and Leptospira spp. PCR, sequencing, and FISH have lately been applied individually Abortion in cattle causes significant economic losses for farmers for the detection of these agents in bovine abortions (11, 13, worldwide. Bovine abortion diagnostics is a complex, expensive, 14). Denmark is in the fortunate position of being free from and time-consuming field, which inter alia is due to the variety of many important abortigenic pathogens, especially viruses. For abortifacients including bacteria, protozoa, viruses, and fungi. example, bovine herpesvirus type 1 has been eradicated, and Microbial culture continues to be an important diagnostic eradication of bovine viral diarrhea virus (BVDV) is almost tool in bovine abortion diagnostics (1, 2). However, because of complete (15). Further, Campylobacter fetus subsp. venerealis the time-consuming nature of the method, its costliness, and and Tritrichomonas foetus are not present anymore, and bovine inherent culturability bias, molecular, culture-independent brucellosis was eradicated with the last case diagnosed in 1962. methods are gaining importance for the detection and Chlamydia spp. and Leptospira spp. have never been diagnosed identification of pathogens in veterinary diagnostics including as cause of bovine abortion in Denmark; however, the routine bovine abortion diagnostics (3, 4). DNA and RNA recovery methods included in the national surveillance program neither methods, such as polymerase chain reaction (PCR) and next- specifically target these agents nor C. burnetii, and the current generation sequencing, come with the advantages of detecting prevalence of these infections is therefore uncertain. pathogens based on their nucleic acid sequences and thereby The diagnostic rate of most bovine abortion studies is allowing for an efficient, highly sensitive, and specific screening generally unsatisfyingly low and seldom reaches 50% (7, for a variety of pathogens including emerging, opportunistic, 16–19). Applying molecular high-throughput and in situ and “difficult-to-culture” species (5, 6). detection methods could enhance the diagnostic rate, e.g., by In bovine abortion diagnostics, the establishment of a detecting opportunistic pathogens that might be missed by causal relationship between the detected abortifacient and conventional methods. placental or fetal lesions is crucial for making an etiologic Here, we evaluate the diagnostic benefit of applying real- diagnosis, especially when facultative abortigenic pathogens time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, such as Coxiella burnetii and ubiquitous pathogens such as and FISH separately and in combination in bovine abortion Escherichia coli are involved (7). Moreover, abortion material diagnostics. The aims of the study were to (i) identify potentially is often considered as compromised because of autolysis abortifacient bacteria without the bias of culturability, (ii) and putrefaction, which further underlines the importance of increase the diagnostic rate using combined molecular methods, investigating the pathogen–lesion association. Fluorescence in (iii) investigate the presence of the difficult-to-culture zoonotic situ hybridization (FISH) is a bridging microscopy technique agents C. burnetii, Chlamydia spp., and Leptospira spp. in bovine that combines molecular accuracy with microbial culture- abortions in Denmark. independence and spatial resolution of the pathogen location within the tissue. It is therefore well-suited to evaluate the pathogen–lesion association and helps to enhance diagnoses MATERIALS AND METHODS (8–10). FISH visualizes microbial cells by fluorescently labeled nucleotide probes that bind complementarily to the ribosomal Molecular, culture-independent methods were applied to RNA (rRNA) of the target cell, a molecule present in large abortion material from 162 abortion cases from a previous numbers in cells with active or just recently terminated protein diagnostic study that mainly applied routine diagnostic methods expression. The rRNA’s composition of variable and conserved such as necropsy, bacterial culture, and histopathology (20). − ◦ regions makes it possible to find short sequence stretches that are Additional tissue samples were collected and stored at 80 C for unique to a species, genus, or a broader taxonomic unit, thereby the present study. Information about the animals, reproduction allowing for different levels of differentiation between pathogenic data, and necropsy, histopathology, and bacterial culture findings taxa, as well as species identification in samples containing as well the diagnostic criteria used in the routine diagnostic study multiple species (8). FISH has lately been applied successfully in are summarized in the Supplementary File 1. detecting and identifying