Nested polymerase chain reaction for detection of Ehrlichia risticii genomic DNA in infected horses.
Abstract: A nested polymerase chain reaction was developed for amplifying a 529-bp segment of the 16S ribosomal RNA gene of Ehrlichia risticii from equine buffy coat cells. Confirmation of identity of the amplified bands was accomplished by Southern hybridization and DNA sequencing. The study indicated a detection limit of > 10 copies of the target gene, and specificity for E. risticii as based on a panel of test rickettsiae. Ticks (Ixodes pacificus) collected in an area of northern California enzootic for equine monocytic ehrlichiosis were found to be negative for E. risticii DNA.
Publication Date: 1997-03-01 PubMed ID: 9106958DOI: 10.1016/s0304-4017(96)01083-7Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research focuses on using a nested polymerase chain reaction to identify the presence of the Ehrlichia risticii DNA in horses, with a specific focus on equine buffy coat cells. The method was found to be sensitive and specific, but ticks from an area prone to equine ehrlichiosis did not show E. risticii DNA.
Objective and Methodology
- The main aim of the study was to develop a nested polymerase chain reaction (PCR) for amplifying a specific portion (529-bp segment) of the 16S ribosomal RNA gene of a bacterium known as Ehrlichia risticii. This bacterium usually exists in the buffy coat cells of horses.
- This nested PCR detection method was validated through Southern hybridization and DNA sequencing techniques to confirm the identity of the amplified bands. These methods serve to characterize the target DNA fragment obtained after amplification.
Findings and Implications
- The study determined that the nested PCR method could identify more than 10 copies of the target gene, implying a high sensitivity for detecting a small number of E. risticii bacteria.
- The testing also indicated that this method is specific to E. risticii by testing it against a panel of rickettsiae. Rickettsiae are a group of bacteria that also include E. risticii.
- This specificity for E. risticii is an extremely valuable indicator for researchers and clinicians in avoiding false positives, leading to improved disease diagnosis and subsequent treatment.
- As part of the study, ticks of the species Ixodes pacificus, collected in a region of northern California known to be enzootic for equine monocytic ehrlichiosis (a disease caused by E. risticii), were examined for the presence of E. risticii DNA.
- Interestingly, these ticks were found to be negative for E. risticii DNA. This implies that in this particular region, horses might not contract the bacteria from these kinds of ticks, contradicting previous beliefs about possible sources of infection.
Cite This Article
APA
Barlough JE, Rikihisa Y, Madigan JE.
(1997).
Nested polymerase chain reaction for detection of Ehrlichia risticii genomic DNA in infected horses.
Vet Parasitol, 68(4), 367-373.
https://doi.org/10.1016/s0304-4017(96)01083-7 Publication
Researcher Affiliations
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616, USA. jebarlough@ucdavis.edu
MeSH Terms
- Animals
- Blotting, Southern
- DNA Primers
- DNA, Bacterial / blood
- Ehrlichia / genetics
- Ehrlichia / isolation & purification
- Ehrlichiosis / diagnosis
- Ehrlichiosis / veterinary
- Horse Diseases
- Horses
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
- Sensitivity and Specificity
Citations
This article has been cited 10 times.- Kopper JJ, Willette JA, Kogan CJ, Seguin A, Bolin SR, Schott HC 2nd. Detection of pathogens in blood or feces of adult horses with enteric disease and association with outcome of colitis.. J Vet Intern Med 2021 Sep;35(5):2465-2472.
- Greiman SE, Tkach M, Vaughan JA, Tkach VV. Laboratory maintenance of the bacterial endosymbiont, Neorickettsia sp., through the life cycle of a digenean, Plagiorchis elegans.. Exp Parasitol 2015 Oct;157:78-83.
- Greiman SE, Tkach VV, Pulis E, Fayton TJ, Curran SS. Large scale screening of digeneans for Neorickettsia endosymbionts using real-time PCR reveals new Neorickettsia genotypes, host associations and geographic records.. PLoS One 2014;9(6):e98453.
- Greiman SE, Tkach VV, Vaughan JA. Transmission rates of the bacterial endosymbiont, Neorickettsia risticii, during the asexual reproduction phase of its digenean host, Plagiorchis elegans, within naturally infected lymnaeid snails.. Parasit Vectors 2013 Oct 22;6:303.
- Vieira RF, Vieira TS, Nascimento Ddo A, Martins TF, Krawczak FS, Labruna MB, Chandrashekar R, Marcondes M, Biondo AW, Vidotto O. Serological survey of Ehrlichia species in dogs, horses and humans: zoonotic scenery in a rural settlement from southern Brazil.. Rev Inst Med Trop Sao Paulo 2013 Sep-Oct;55(5):335-40.
- Pusterla N, Madigan JE, Chae JS, DeRock E, Johnson E, Pusterla JB. Helminthic transmission and isolation of Ehrlichia risticii, the causative agent of Potomac horse fever, by using trematode stages from freshwater stream snails.. J Clin Microbiol 2000 Mar;38(3):1293-7.
- Pusterla N, Huder JB, Leutenegger CM, Braun U, Madigan JE, Lutz H. Quantitative real-time PCR for detection of members of the Ehrlichia phagocytophila genogroup in host animals and Ixodes ricinus ticks.. J Clin Microbiol 1999 May;37(5):1329-31.
- Barlough JE, Reubel GH, Madigan JE, Vredevoe LK, Miller PE, Rikihisa Y. Detection of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Pleuroceridae: Juga spp.) from northern California.. Appl Environ Microbiol 1998 Aug;64(8):2888-93.
- Reubel GH, Barlough JE, Madigan JE. Production and characterization of Ehrlichia risticii, the agent of Potomac horse fever, from snails (Pleuroceridae: Juga spp.) in aquarium culture and genetic comparison to equine strains.. J Clin Microbiol 1998 Jun;36(6):1501-11.
- Mott J, Rikihisa Y, Zhang Y, Reed SM, Yu CY. Comparison of PCR and culture to the indirect fluorescent-antibody test for diagnosis of Potomac horse fever.. J Clin Microbiol 1997 Sep;35(9):2215-9.
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