Molecular cloning of Ehrlichia risticii and development of a gene probe for the diagnosis of Potomac horse fever.
Abstract: A gene bank of Ehrlichia risticii was constructed in plasmid vector pUC13. Five clones representing discrete regions of the E. risticii genome were tested for their ability to hybridize specifically to E. risticii DNA. None of the clones cross-hybridized with Ehrlichia equi DNA, whereas four of these clones cross-hybridized with Ehrlichia canis and Ehrlichia sennetsu DNAs. However, one clone carrying a 1-kilobase HindIII fragment of E. risticii DNA failed to cross-react with the genomes of E. sennetsu, E. canis, and E. equi in dot blot hybridization assays. The sensitivity of this probe for the detection of E. risticii DNA was approximately 0.5 pg. By using this probe, the E. risticii DNA was detected in the peripheral blood mononuclear cells of 30 experimentally infected horses by 7 days postinfection (p.i.); the detection of E. risticii DNA peaked between 14 and 17 days p.i., a period immediately after the peak of the second rise in body temperature, during leukopenia and at the onset of diarrhea. E. risticii DNA was not detectable by 25 to 30 days p.i. E. risticii DNA was not detected in noninfected control horses.
Publication Date: 1990-09-01 PubMed ID: 2229378PubMed Central: PMC268087DOI: 10.1128/jcm.28.9.1963-1967.1990Google 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
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research paper discusses the construction of a gene bank of Ehrlichia risticii, a bacteria causing Potomac Horse Fever, and the subsequent development of a gene probe to facilitate its detection. The researchers also investigate the effectiveness of this probe for diagnosing infected horses.
Construction of a Gene Bank
- The researchers built a gene bank of Ehrlichia risticii DNA within a plasmid vector known as pUC13.
- This vector was used because it’s a popular choice for molecular cloning due to its ability to replicate within host cells.
- The Ehrlichia risticii gene bank was composed of multiple clones representing different regions of the bacterial genome.
Identification of Specific Probes
- The team tested five clones from the gene bank for their ability to specifically bind (or “hybridize”) to E. risticii DNA.
- Out of the five clones tested, one, carrying a 1-kilobase HindIII fragment of E. risticii DNA, didn’t cross-react with the genomes of E. sennetsu, E. canis, and E. equi. This suggests a high level of specificity, making this clone an ideal candidate for a diagnostic probe.
Probe Sensitivity and Specificity
- The sensitivity of the selected gene probe was evaluated to be approximately 0.5 picograms, indicating that the probe can detect E. risticii even in small amounts.
- In the absence of E. risticii DNA, there were no false positives, which showcases the probe’s specificity.
Probe Applications and Observations
- When the probe was used in experimentally infected horses, E. risticii DNA was successfully detected within a week post-infection.
- The highest detection occurred between 14 and 17 days post-infection, during the second rise in body temperature, leukopenia (a decrease in white blood cells), and the onset of diarrhea. This information contributes to understanding the timeline and progression of an E. risticii infection in horses.
- No E. risticii DNA was detected in non-infected control horses, further confirming the probe’s specificity.
- The inability to detect E. risticii DNA in the period of 25-30 days post-infection may indicate either the clearing of the bacteria by the horse’s immune system, or a limitation in the detectability of the probe at later stages of infection.
Cite This Article
APA
Thaker SR, Dutta SK, Adhya SL, Mattingly-Napier BL.
(1990).
Molecular cloning of Ehrlichia risticii and development of a gene probe for the diagnosis of Potomac horse fever.
J Clin Microbiol, 28(9), 1963-1967.
https://doi.org/10.1128/jcm.28.9.1963-1967.1990 Publication
Researcher Affiliations
- Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park 20742.
MeSH Terms
- Animals
- Cloning, Molecular
- DNA Probes
- DNA, Bacterial / genetics
- DNA, Bacterial / isolation & purification
- Ehrlichia / genetics
- Genes, Bacterial
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horses
- Rickettsiaceae Infections / diagnosis
- Rickettsiaceae Infections / microbiology
- Rickettsiaceae Infections / veterinary
References
This article includes 15 references
- Gribble DH. Equine ehrlichiosis.. J Am Vet Med Assoc 1969 Jul 15;155(2):462-9.
- Dawson JE, Abeygunawardena I, Holland CJ, Buese MM, Ristic M. Susceptibility of cats to infection with Ehrlichia risticii, causative agent of equine monocytic ehrlichiosis.. Am J Vet Res 1988 Dec;49(12):2096-100.
- Scott JC, Dutta SK, Myrup AC. In vivo harboring of equine herpesvirus-1 in leukocyte populations and subpopulations and their quantitation from experimentally infected ponies.. Am J Vet Res 1983 Jul;44(7):1344-8.
- Bresser J, Doering J, Gillespie D. Quick-blot: selective mRNA or DNA immobilization from whole cells.. DNA 1983;2(3):243-54.
- Holland CJ, Ristic M, Cole AI, Johnson P, Baker G, Goetz T. Isolation, experimental transmission, and characterization of causative agent of Potomac horse fever.. Science 1985 Feb 1;227(4686):522-4.
- Rikihisa Y, Perry BD. Causative ehrlichial organisms in Potomac horse fever.. Infect Immun 1985 Sep;49(3):513-7.
- Dutta SK, Myrup AC, Rice RM, Robl MG, Hammond RC. Experimental reproduction of Potomac horse fever in horses with a newly isolated Ehrlichia organism.. J Clin Microbiol 1985 Aug;22(2):265-9.
- Jenkins SJ, Jones NK, Jenny AL. Potomac horse fever agent in mice.. Vet Rec 1985 Nov 23;117(21):556-7.
- Ristic M, Holland CJ, Dawson JE, Sessions J, Palmer J. Diagnosis of equine monocytic ehrlichiosis (Potomac horse fever) by indirect immunofluorescence.. J Am Vet Med Assoc 1986 Jul 1;189(1):39-46.
- Pretzman CI, Rikihisa Y, Ralph D, Gordon JC, Bech-Nielsen S. Enzyme-linked immunosorbent assay for Potomac horse fever disease.. J Clin Microbiol 1987 Jan;25(1):31-6.
- Dutta SK, Rice RM, Hughes TD, Savage PK, Myrup AC. Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay.. Vet Immunol Immunopathol 1987 Jan;14(1):85-92.
- Dutta SK, Penney BE, Myrup AC, Robl MG, Rice RM. Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever).. Am J Vet Res 1988 Oct;49(10):1747-51.
- Tenover FC. Diagnostic deoxyribonucleic acid probes for infectious diseases.. Clin Microbiol Rev 1988 Jan;1(1):82-101.
- Shankarappa B, Dutta SK, Sanusi J, Mattingly BL. Monoclonal antibody-mediated, immunodiagnostic competitive enzyme-linked immunosorbent assay for equine monocytic ehrlichiosis.. J Clin Microbiol 1989 Jan;27(1):24-8.
- Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis.. J Mol Biol 1975 Nov 5;98(3):503-17.
Citations
This article has been cited 6 times.- Zuo P, Rabie AB. Novel method of cell-free in vitro synthesis of the human fibroblast growth factor 1 gene.. J Biomed Biotechnol 2010;2010.
- Kakoma I, Hansen RD, Anderson BE, Hanley TA, Sims KG, Liu L, Bellamy C, Long MT, Baek BK. Cultural, molecular, and immunological characterization of the etiologic agent for atypical canine ehrlichiosis.. J Clin Microbiol 1994 Jan;32(1):170-5.
- Biswas B, Vemulapalli R, Dutta SK. Detection of Ehrlichia risticii from feces of infected horses by immunomagnetic separation and PCR.. J Clin Microbiol 1994 Sep;32(9):2147-51.
- Dutta SK, Shankarappa B, Mattingly-Napier BL. Molecular cloning and analysis of recombinant major antigens of Ehrlichia risticii.. Infect Immun 1991 Mar;59(3):1162-9.
- Biswas B, Mukherjee D, Mattingly-Napier BL, Dutta SK. Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).. J Clin Microbiol 1991 Oct;29(10):2228-33.
- Shankarappa B, Dutta SK, Mattingly-Napier B. Identification of the protective 44-kilodalton recombinant antigen of Ehrlichia risticii.. Infect Immun 1992 Feb;60(2):612-7.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
