Detection of bacterial DNA in synovial fluid from horses with infectious synovitis.
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.
Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. frederik.pille@ugent.be
F
F
Pille
A
A
Martens
L M
LM
Schouls
L
L
Peelman
F
F
Gasthuys
C S
CS
Schot
C
C
De Baere
P
P
Desmet
F
F
Vandenberghe
D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006734:Horse Diseases / Q000382:microbiology*; D006736:Horses; D008163:Luminescent Measurements; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013582:Synovial Fluid / Q000382:microbiology*; D013585:Synovitis / Q000382:microbiology / Q000662:veterinary*
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S