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Detection of Ehrlichia risticii from feces of infected horses by immunomagnetic separation and PCR.
Abstract: Potomac horse fever, caused by Ehrlichia risticii, is an important disease of equines. The major features of the disease are fever, leukopenia, and diarrhea. The organism has been detected from the blood mononuclear cells of infected horses, but its presence in the feces has not been known. A method for immunomagnetic separation of E. risticii from the feces of infected horses was developed, and the separated organisms were detected by PCR. Coating immunomagnetic beads (Dynabeads) with a 1:5 dilution of rabbit anti-E. risticii serum and incubating the Dynabeads with fecal samples for 25 min at room temperature gave optimum results. E. risticii was detected from the feces during the course of diarrhea from two experimentally infected horses. In horse 1, watery diarrhea occurred from days 11 to 16 postinfection (p.i.), after which the feces became soft on day 17 p.i. and then returned to normal. The organisms were first detected from the feces on day 11 p.i., peaked on day 13 p.i., and then gradually decreased until day 16 p.i., after which they became undetectable. In horse 2, first, on day 12 p.i., there was soft feces which continued and progressed to diarrhea on day 17 p.i. The feces became normal after day 18 p.i. The organisms in the feces of this horse were first detected on day 12 p.i. and peaked on day 14 p.i., after which they declined until day 16 p.i. and then became undetectable. In both horses, the number of organisms in the mononuclear cells peaked on days 10 and 11 p.i., respectively, 3 days prior to the respective peaks in the feces. E. risticii was not detected from the plasma samples obtained from these horses. There was a drastic reduction in PCR amplification of E. risticii DNA for fecal samples stored frozen at -20 degrees C in comparison with those stored at 4 degrees C. The presence of the organism in the feces only during the soft- or diarrheal-feces phase supports the previous hypothesis that the diarrhea is caused by the organisms replicating in cells lining the intestines. This rapid simple method of detection of the organisms from the feces will be helpful in diagnostic and epidemiologic studies of Potomac horse fever.
Publication Date: 1994-09-01 PubMed ID: 7814538PubMed Central: PMC263957DOI: 10.1128/jcm.32.9.2147-2151.1994Google 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
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The research article focuses on detecting Ehrlichia risticii, the bacteria responsible for Potomac horse fever, from the feces of infected horses through a technique involving immunomagnetic separation and Polymerase Chain Reaction (PCR).
Research Overview
In this study, the researchers have:
- Developed a method for immunomagnetic separation of E. risticii from the feces of infected horses,
- Detected the separated organisms through PCR,
- Studied the prevalence of the organism in the feces during the course of diarrhea in experimentally infected horses, and
- Evaluated the efficiency of PCR amplification of E. risticii DNA for fecal samples stored under different temperatures.
Methodology
The research showed that coating immunomagnetic beads (Dynabeads) with a 1:5 dilution of rabbit anti-E. risticii serum and incubating the Dynabeads with fecal samples for 25 minutes at room temperature yielded the best results.
Key Findings
- The organism was first detected in the feces of the infected horses during the diarrheal phase, supporting the previous hypothesis that diarrhea is caused by the organisms’ multiplication in intestinal lining cells.
- The number of E. risticii organisms peaked in the mononuclear cells three days prior to their peak in the feces, providing crucial timing information about the progression of the infection.
- The researchers could not detect E. risticii in the plasma samples taken from the horses, suggesting that the bacteria mainly confine to specific bodily systems.
- Fecal samples stored at -20 degrees Celsius showed a drastic reduction in PCR amplification of E. risticii DNA compared to those stored at 4 degrees Celsius, pointing to the importance of appropriate sample storage conditions for successful testing and detection.
Conclusion
The study suggests a quick and succinct way to detect Ehrlichia risticii, the bacteria causing Potomac horse fever in horses. Such detection aids diagnostic efforts and can provide valuable information for epidemiology studies related to the disease.
Cite This Article
APA
Biswas B, Vemulapalli R, Dutta SK.
(1994).
Detection of Ehrlichia risticii from feces of infected horses by immunomagnetic separation and PCR.
J Clin Microbiol, 32(9), 2147-2151.
https://doi.org/10.1128/jcm.32.9.2147-2151.1994 Publication
Researcher Affiliations
- Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park 20742.
MeSH Terms
- Animals
- DNA, Bacterial / isolation & purification
- Diarrhea / microbiology
- Diarrhea / veterinary
- Ehrlichia / genetics
- Ehrlichia / immunology
- Ehrlichia / isolation & purification
- Ehrlichiosis / microbiology
- Ehrlichiosis / veterinary
- Enzyme-Linked Immunosorbent Assay
- Feces / microbiology
- Fluorescent Antibody Technique
- Horse Diseases / microbiology
- Horses / microbiology
- Immunomagnetic Separation
- Leukocytes, Mononuclear / microbiology
- Polymerase Chain Reaction
References
This article includes 16 references
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Citations
This article has been cited 9 times.- 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.
- Baird JD, Arroyo LG. Historical aspects of Potomac horse fever in Ontario (1924-2010). Can Vet J 2013 Jun;54(6):565-72.
- Ancelin M, Buteau-Lozano H, Meduri G, Osborne-Pellegrin M, Sordello S, Plouët J, Perrot-Applanat M. A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus. Proc Natl Acad Sci U S A 2002 Apr 30;99(9):6023-8.
- Dutta SK, Vemulapalli R, Biswas B. Association of deficiency in antibody response to vaccine and heterogeneity of Ehrlichia risticii strains with Potomac horse fever vaccine failure in horses. J Clin Microbiol 1998 Feb;36(2):506-12.
- Gagné A, Lacouture S, Broes A, D'Allaire S, Gottschalk M. Development of an immunomagnetic method for selective isolation of Actinobacillus pleuropneumoniae serotype 1 from tonsils. J Clin Microbiol 1998 Jan;36(1):251-4.
- 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.
- Choi BK, Wyss C, Göbel UB. Phylogenetic analysis of pathogen-related oral spirochetes. J Clin Microbiol 1996 Aug;34(8):1922-5.
- Pfeffer M, Wiedmann M, Batt CA. Applications of DNA amplification techniques in veterinary diagnostics. Vet Res Commun 1995;19(5):375-407.
- Enroth H, Engstrand L. Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens. J Clin Microbiol 1995 Aug;33(8):2162-5.
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