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Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR.
Abstract: A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evaluated on horses with infectious colitis and on freshwater stream snails collected from regions with a history of PHF. E. risticii could be detected in 22 of 153 (14.4%) horses with infectious colitis and in 25 of 234 (10.7%) snails in the TaqMan PCR. The same results were obtained in the conventional nested PCR. The Ehrlichia-load was in the range of 10,000-9,000,000 and 35,000-680, 000,000 Ehrlichia equivalents per microg leukocyte DNA and snail DNA, respectively.
Publication Date: 2000-06-01 PubMed ID: 10828519DOI: 10.1016/s0304-4017(00)00227-2Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study developed a method using real-time PCR to identify the Ehrlichia risticii bacteria in horses and snails. The bacteria, which can cause Potomac horse fever, was detected in a small percentage of tested horses and snails, and the method’s sensitivity and specificity matched those of the conventionally used nested PCR.
Introduction
- The research focuses on the development and application of a real-time PCR assay for the detection of Ehrlichia risticii.
- Ehrlichia risticii is a bacteria that causes Potomac horse fever, a disease that affects horses and is transmitted by freshwater snails.
- The research was aimed at identifying and quantifying this bacterium in horses with infectious colitis and in freshwater snails collected from regions with a history of Potomac horse fever cases.
Method
- The researchers used a real-time quantitative PCR (Polymerase Chain Reaction) with the TaqMan fluorogenic detection system. This system can detect and quantify a specific DNA sequence associated with the bacterium.
- The TaqMan PCR system identifies an 85 base pair section of the 16S rRNA gene, which is specific to the Ehrlichia risticii bacteria. This is done using a special fluorogenic probe and two primers.
- In total, eight different strains of E. risticii were tested using this method to evaluate its specificity.
Results
- The sensitivity and specificity of the TaqMan PCR assay were found to be comparable to those of a conventional nested PCR method, making it a viable alternative.
- A total of 153 horses with infectious colitis and 234 stream snails were tested using the developed TaqMan PCR assay. It was observed that approximately 14.4 percent of horses and 10.7 percent of snails were carrying the bacterium.
- The same results were obtained when the samples were tested using a conventional nested PCR method, providing validation for the TaqMan PCR assay.
- The quantity of Ehrlichia risticii DNA per microgram of leukocyte DNA collected from horses and snail DNA was also estimated to be in the range of 10,000-9,000,000 and 35,000-680,000,000 respectively.
Conclusion
- The study concluded that the TaqMan PCR assay is a reliable and efficient tool for the identification and quantification of Ehrlichia risticii in infected horses and snails.
- Further research is required to understand the ecology and transmission of this bacterium to develop strategies for control and prevention of Potomac horse fever.
Cite This Article
APA
Pusterla N, Leutenegger CM, Sigrist B, Chae JS, Lutz H, Madigan JE.
(2000).
Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR.
Vet Parasitol, 90(1-2), 129-135.
https://doi.org/10.1016/s0304-4017(00)00227-2 Publication
Researcher Affiliations
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616, USA. npusterla@ucdavis.edu
MeSH Terms
- Animals
- Colitis / microbiology
- Colitis / veterinary
- Ehrlichia / genetics
- Ehrlichiosis / genetics
- Ehrlichiosis / veterinary
- Horse Diseases / genetics
- Horse Diseases / microbiology
- Horses
- Polymerase Chain Reaction
- RNA, Ribosomal, 16S / genetics
- Sensitivity and Specificity
- Snails / microbiology
Citations
This article has been cited 10 times.- Pinn-Woodcock TL, Aprea MS, Lejeune M, Tomlinson JE. Molecular detection of pathogens in an equine fever diagnostic panel: 2019-2023. Equine Vet J 2026 Mar;58(2):486-496.
- de Albuquerque CV, da Silva Andrade M, de Freitas MS, Paulino PG, Santos HA, de Tarso Landgraf Botteon P. Significance of Anaplasma phagocytophilum, Babesia caballi, and Theileria equi as etiologic agents in horses with clinical manifestations from the metropolitan area of Rio De Janeiro, Brazil. Trop Anim Health Prod 2024 Sep 21;56(8):268.
- Thirumalapura NR, Livengood J, Beeby J, Wang W, Goodrich EL, Goodman LB, Erol E, Tewari D. Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever. J Vet Diagn Invest 2023 Jan;35(1):62-66.
- Budachetri K, Lin M, Yan Q, Chien RC, Hostnik LD, Haanen G, Leclère M, Waybright W, Baird JD, Arroyo LG, Rikihisa Y. Real-Time PCR Differential Detection of Neorickettsia findlayensis and N. risticii in Cases of Potomac Horse Fever. J Clin Microbiol 2022 Jul 20;60(7):e0025022.
- Arroyo LG, Moore A, Bedford S, Gomez DE, Teymournejad O, Xiong Q, Budachetri K, Bekebrede H, Rikihisa Y, Baird JD. Potomac horse fever in Ontario: Clinical, geographic, and diagnostic aspects. Can Vet J 2021 Jun;62(6):622-628.
- Teymournejad O, Lin M, Bekebrede H, Kamr A, Toribio RE, Arroyo LG, Baird JD, Rikihisa Y. Isolation and Molecular Analysis of a Novel Neorickettsia Species That Causes Potomac Horse Fever. mBio 2020 Feb 25;11(1).
- Durán MC, Marqués FJ. Detection of Neorickettsia risticii, the agent of Potomac horse fever, in a Gypsy Vanner stallion from Manitoba. Can Vet J 2016 Mar;57(3):293-5.
- 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.
- Slovis NM, Elam J, Estrada M, Leutenegger CM. Infectious agents associated with diarrhoea in neonatal foals in central Kentucky: a comprehensive molecular study. Equine Vet J 2014 May;46(3):311-6.
- Heller MC, McClure J, Pusterla N, Pusterla JB, Stahel S. Two cases of Neorickettsia (Ehrlichia) risticii infection in horses from Nova Scotia. Can Vet J 2004 May;45(5):421-3.
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