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Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization

Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for th...

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Autores principales: Wolf-Jäckel, Godelind Alma, Strube, Mikael Lenz, Schou, Kirstine Klitgaard, Schnee, Christiane, Agerholm, Jørgen S., Jensen, Tim Kåre
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940352/
https://www.ncbi.nlm.nih.gov/pubmed/33708810
http://dx.doi.org/10.3389/fvets.2021.623666
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author Wolf-Jäckel, Godelind Alma
Strube, Mikael Lenz
Schou, Kirstine Klitgaard
Schnee, Christiane
Agerholm, Jørgen S.
Jensen, Tim Kåre
author_facet Wolf-Jäckel, Godelind Alma
Strube, Mikael Lenz
Schou, Kirstine Klitgaard
Schnee, Christiane
Agerholm, Jørgen S.
Jensen, Tim Kåre
author_sort Wolf-Jäckel, Godelind Alma
collection PubMed
description Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture 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 polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing–guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared 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.
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spelling pubmed-79403522021-03-10 Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization Wolf-Jäckel, Godelind Alma Strube, Mikael Lenz Schou, Kirstine Klitgaard Schnee, Christiane Agerholm, Jørgen S. Jensen, Tim Kåre Front Vet Sci Veterinary Science Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture 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 polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing–guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared 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. Frontiers Media S.A. 2021-02-23 /pmc/articles/PMC7940352/ /pubmed/33708810 http://dx.doi.org/10.3389/fvets.2021.623666 Text en Copyright © 2021 Wolf-Jäckel, Strube, Schou, Schnee, Agerholm and Jensen. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Veterinary Science
Wolf-Jäckel, Godelind Alma
Strube, Mikael Lenz
Schou, Kirstine Klitgaard
Schnee, Christiane
Agerholm, Jørgen S.
Jensen, Tim Kåre
Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization
title Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization
title_full Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization
title_fullStr Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization
title_full_unstemmed Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization
title_short Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization
title_sort bovine abortions revisited—enhancing abortion diagnostics by 16s rdna amplicon sequencing and fluorescence in situ hybridization
topic Veterinary Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940352/
https://www.ncbi.nlm.nih.gov/pubmed/33708810
http://dx.doi.org/10.3389/fvets.2021.623666
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