Detection of bacterial DNA in synovial fluid from horses with infectious synovitis.
Abstract: Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.
Publication Date: 2004-07-28 PubMed ID: 15276769DOI: 10.1016/j.rvsc.2004.04.004Google Scholar: Lookup
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- Journal Article
Summary
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The study aimed to develop a reliable method for detecting and identifying bacterial species in the synovial fluid (a type of fluid found in joints) of horses with infectious synovitis (inflamed joints due to infection) through the use of a polymerase chain reaction (PCR) technique.
Research Background
- The background of the research was the challenge in diagnosing synovial infection in horses using standard culture techniques, which often don’t give conclusive results.
- Previous studies in humans have shown the effectiveness of using sensitive PCR techniques to detect bacterial involvement in acute synovitis.
- However, there had been no reports of successful extraction of bacterial DNA directly from horse samples for confirmation without prior culture.
Research Process
- The researchers took synovial fluid samples from 6 horses with culture confirmed synovial infection. They used this sample to perform broad range 16S rRNA gene PCR.
- Synovial fluid from 2 healthy horses was used as a negative control.
- The synovial fluid DNA was extracted and purified, and processed by touchdown PCR. The amplicons (pieces of DNA) that were produced were then detected by reverse line blot hybridisation and visualised with chemiluminescence (a method that allows the detection of specific DNA sequences).
Research Findings
- The researchers successfully detected pathogen-specific 16S rRNA gene sequences in all 6 synovial fluid samples from horses with infectious synovitis.
- No bacterial DNA was detected in the synovial samples from the healthy control horses, even after additional amplifier cycles.
- The type of pathogen was confirmed through DNA sequencing of the amplicons.
Research Conclusion
- The conclusion of the research was that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation proved to be a promising technique for detecting bacterial DNA in synovial fluid samples.
- Further research was suggested to aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but showing negative culture results.
- If the 16S PCR technique can be proven to be reliable and more sensitive than standard culturing techniques, it could become a powerful tool in diagnosing synovial infection in horses.
Cite This Article
APA
Pille F, Martens A, Schouls LM, Peelman L, Gasthuys F, Schot CS, De Baere C, Desmet P, Vandenberghe F.
(2004).
Detection of bacterial DNA in synovial fluid from horses with infectious synovitis.
Res Vet Sci, 77(3), 189-195.
https://doi.org/10.1016/j.rvsc.2004.04.004 Publication
Researcher Affiliations
- Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. frederik.pille@ugent.be
MeSH Terms
- Animals
- DNA, Bacterial / genetics
- DNA, Bacterial / isolation & purification
- Female
- Horse Diseases / microbiology
- Horses
- Luminescent Measurements
- Male
- Nucleic Acid Hybridization / methods
- Oligonucleotide Probes / genetics
- Polymerase Chain Reaction / veterinary
- RNA, Ribosomal, 16S / genetics
- Sequence Analysis, DNA / veterinary
- Synovial Fluid / microbiology
- Synovitis / microbiology
- Synovitis / veterinary
Citations
This article has been cited 3 times.- Haralambus R, Florczyk A, Sigl E, Gültekin S, Vogl C, Brandt S, Schnierer M, Gamerith C, Jenner F. Detection of synovial sepsis in horses using enzymes as biomarkers. Equine Vet J 2022 May;54(3):513-522.
- Elmas CR, Koenig JB, Bienzle D, Cribb NC, Cernicchiaro N, Coté NM, Weese JS. Evaluation of a broad range real-time polymerase chain reaction (RT-PCR) assay for the diagnosis of septic synovitis in horses. Can J Vet Res 2013 Jul;77(3):211-7.
- Svraka S, Kuijper E, Duizer E, Bakker D, Koopmans M. Clostridium difficile is not associated with outbreaks of viral gastroenteritis in the elderly in the Netherlands. Eur J Clin Microbiol Infect Dis 2010 Jun;29(6):677-82.
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