Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Wolf-Jackel GA

PROVIDER: S-EPMC7940352 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Bovine Abortions Revisited-Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence <i>in situ</i> Hybridization.

Wolf-Jäckel Godelind Alma GA Strube Mikael Lenz ML Schou Kirstine Klitgaard KK Schnee Christiane C Agerholm Jørgen S JS Jensen Tim Kåre TK

Frontiers in veterinary science 20210223

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 ...[more]

PMID: 33708810

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Project description:Bovine mycotic abortion is sporadic and caused by different ubiquitous and opportunistic fungi. Recently, a broad spectrum of bacterial opportunists involved in bovine abortion was revealed by 16S rRNA gene amplicon sequencing. We hypothesized that fungal organisms potentially involved in bovine abortion also might remain undetected by conventional culture. In this retrospective study, we therefore applied fungal internal transcribed spacer 2 (ITS2) region amplicon sequencing to 74 cases of bovine abortion submitted to our diagnostic service. The investigation was complemented by fungal culture and, retrospectively, by data from bacteriological, virological and parasitological analyses and histopathological examination of placentas. Fungal DNA was found in both the placentas and abomasal contents, with 92 fungal genera identified. In 18 cases, >75% of the reads belonged to one specific fungal genus: Candida (n = 7), Malassezia (n = 4), Cryptococcus (n = 3), unidentified Capnodiales (n = 3), Actinomucor (n = 1), Cystofilobasidium (n = 1), Penicillium (n = 1), Verticillum (n = 1) and Zymoseptoria (n = 1) with one case harboring two different genera. By culture, in contrast, fungal agents were detected in only 6 cases. Inflammatory and/or necrotizing lesions were found in 27/40 histologically assessed placentas. However, no lesion-associated fungal structures were detected in HE- and PAS-stained specimens. Complementary data revealed the presence of one or more non-fungal possible abortifacient: Chlamydiales, Coxiella burnetii, Leptospira spp., Campylobacter fetus subsp. fetus, Streptococcus uberis, Escherichia coli, Streptococcus pluranimalium, Bacillus licheniformis, Campylobacter fetus subsp. fetus, Serratia marcescens, Trueperella pyogenes, Schmallenbergvirus, Neospora caninum. The mycobiota revealed by sequencing did not differ between cases with or without a possible infectious etiology. Our study suggests that amplicon sequencing of the ITS2 region from DNA isolated from bovine abortion does not provide additional information or new insight into mycotic abortion and without complementary analyses may easily lead to a false interpretation of the role of fungal organisms in bovine abortion.

Project description:Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.

Project description:Meat and seafood spoilage ecosystems harbor extensive bacterial genomic diversity that is mainly found within a small number of species but within a large number of strains with different spoilage metabolic potential. To decipher the intraspecies diversity of such microbiota, traditional metagenetic analysis using the 16S rRNA gene is inadequate. We therefore assessed the potential benefit of an alternative genetic marker, gyrB, which encodes the subunit B of DNA gyrase, a type II DNA topoisomerase. A comparison between 16S rDNA-based (V3-V4) amplicon sequencing and gyrB-based amplicon sequencing was carried out in five types of meat and seafood products, with five mock communities serving as quality controls. Our results revealed that bacterial richness in these mock communities and food samples was estimated with higher accuracy using gyrB than using16S rDNA. However, for Firmicutes species, 35% of putative gyrB reads were actually identified as sequences of a gyrB paralog, parE, which encodes subunit B of topoisomerase IV; we therefore constructed a reference database of published sequences of both gyrB and pare for use in all subsequent analyses. Despite this co-amplification, the deviation between relative sequencing quantification and absolute qPCR quantification was comparable to that observed for 16S rDNA for all the tested species. This confirms that gyrB can be used successfully alongside 16S rDNA to determine the species composition (richness and evenness) of food microbiota. The major benefit of gyrB sequencing is its potential for improving taxonomic assignment and for further investigating OTU richness at the subspecies level, thus allowing more accurate discrimination of samples. Indeed, 80% of the reads of the 16S rDNA dataset were represented by thirteen 16S rDNA-based OTUs that could not be assigned at the species-level. Instead, these same clades corresponded to 44 gyrB-based OTUs, which differentiated various lineages down to the subspecies level. The increased ability of gyrB-based analyses to track and trace phylogenetically different groups of strains will generate improved resolution and more reliable results for studies of the strains implicated in food processes.

Dataset Information

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

Publications

Bovine Abortions Revisited-Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence <i>in situ</i> Hybridization.

Similar Datasets

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