RNA in situ hybridization of Escherichia coli in equine endometrial biopsies.
Abstract: Endometritis is one of the major causes of infertility in mares. and -haemolytic streptococci are among the bacterial species most frequently isolated from the equine uterus. Some bacteria such as -hemolytic streptococci, can persist in dormant forms and cause prolonged, latent or recurrent infections. Dormant bacteria may be present despite negative bacterial cultures, and they are resistant to antimicrobial treatment due to their resting metabolic state. The purpose of this study was to study formalin-fixed paraffin-embedded equine endometrial biopsies for the presence and localization of -bacteria, with a chromogenic RNAscope-method for detection of -related 16S ribosomal RNA. Hematoxylin-eosin-stained endometrial biopsies were evaluated to determine the level of inflammation and degeneration. During estrus, samples were taken for endometrial culture and cytology with a double-guarded uterine swab. The samples included eight samples with moderate to severe endometrial inflammation detected in endometrial histopathology, and growth of in bacterial culture, six samples with moderate to severe endometrial inflammation but negative bacterial culture, and five samples with no endometrial pathology (grade I endometrial biopsy, negative endometrial culture and cytology) serving as controls. Positive and negative control probes were included in the RNA hybridization, and results were confirmed with a fluorescence detection method (fluorescence hybridization). Only unspecific signals of limited size and frequency of occurrence were detected in all samples, with random localization in the endometrium. No samples contained rod-shaped signals corresponding to bacterial findings. In conclusion, there was no evidence of bacterial invasion in the endometrium regardless of the inflammatory status of the biopsy or previous bacterial culture results. According to these findings on a small number of samples, invasion of is not a common finding in the lamina propria of mares, but these bacteria may also evade detection due to localized foci of infections, or supra-epithelial localization under the cover of biofilm. These bacteria and biofilm covering the epithelium may also be lost during formalin-fixation and processing.
Copyright © 2023 Tukia, Hallman, Penttilä, Hänninen and Kareskoski.
Publication Date: 2023-06-09 PubMed ID: 37360403PubMed Central: PMC10288147DOI: 10.3389/fvets.2023.1186051Google Scholar: Lookup
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Summary
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The study evaluates a method to detect dormant bacteria, potentially causing infertility in mares due to persistent infections, within equine endometrial tissues. The research found no evidence of bacterial invasion regardless of the inflammation status, suggesting that these bacteria or possible biofilms may evade detection in the procedures used.
Introduction and Background
- The purpose of the research was to investigate the presence and localization of beta-hemolytic streptococci, a kind of bacteria that can form dormant bacteria in equine endometrial biopsies.
- These dormant bacteria are tough to detect due to their metabolic state and are resistant to antimicrobial treatment, potentially causing prolonged or recurring infections.
- Endometritis, mainly caused by these bacteria, is a significant cause of infertility in mares, making this a critical area of study.
Methodology
- The study made use of formalin-fixed paraffin-embedded equine endometrial biopsies, evaluated through hematoxylin-eosin staining, to determine inflammation and degeneration levels.
- Biopsies taken during the mare’s estrus phase were examined for bacterial presence through endometrial cultures and cytology tests using a double-guarded uterine swab.
- To identify the RNA of beta-hemolytic streptococci, an RNAscope-method was applied, and results were affirmed using a fluorescence detection method, known as fluorescence in situ hybridization.
- The study included nineteen different samples, differentiated based on varying degrees of pathology/ inflammation and results from bacterial cultures. These served as the studies control and test samples.
Results and Conclusion
- The results showed unspecific, limited signals in all samples, implying no concrete evidence of bacterial invasions in the endometrium. The signals were randomly located within the endometrium.
- None of the samples contained rod-shaped signals that could resemble bacterial findings.
- Although these results suggest that bacterial invasion of the type studied here is not usually seen in mares, the research also poses the possibility of these bacteria avoiding detection due to localized infections or being shielded with biofilm.
- During the process of formalin-fixation, bacteria and biofilm could potentially be lost, leading to none being visible in the samples studied.
Cite This Article
APA
Tukia E, Hallman I, Penttilä M, Hänninen S, Kareskoski M.
(2023).
RNA in situ hybridization of Escherichia coli in equine endometrial biopsies.
Front Vet Sci, 10, 1186051.
https://doi.org/10.3389/fvets.2023.1186051 Publication
Researcher Affiliations
- Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Saarentaus, Saarbrücken, Finland.
- Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Saarentaus, Saarbrücken, Finland.
- Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Saarentaus, Saarbrücken, Finland.
- Department of Pathology, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
- Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Saarentaus, Saarbrücken, Finland.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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