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Home / Equine Research Bank / J Clin Microbiol / A 16S rRNA-based PCR assay for detection and identification ...
Journal of clinical microbiology1996; 34(9); 2170-2174; doi: 10.1128/jcm.34.9.2170-2174.1996

A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle.

Abstract: A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of granulocytes containing ehrlichiae (morulae). Thirty-four of 36 microscopically positive samples were also positive by PCR, and 6 microscopically negative samples were negative by PCR as well. Six samples, in which morulae-like structures had been seen, were negative by PCR, also at a lower annealing temperature and when a reamplification of the first PCR products was performed. The identities of the PCR products from some canine and equine isolates were verified by direct DNA sequencing and were found to be identical with the Ehrlichia sequence found in these animal species that had been obtained earlier. The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, whereas the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates. Serum samples were analyzed by indirect fluorescent-antibody testing. Seventy-three percent of the animals which were positive by microscopy and PCR also had positive antibody titers. However, it was not possible to rely on a single serological result for diagnosis of present infection. It was, therefore, concluded that PCR was the most reliable method, useful in the clinical laboratory for specific and early diagnosis of granulocytic ehrlichiosis in animals.
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Publication Date: 1996-09-01 PubMed ID: 8862579PubMed Central: PMC229211DOI: 10.1128/jcm.34.9.2170-2174.1996Google Scholar: Lookup
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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.

The research paper details the development of a PCR-based assay for the detection of a specific type of bacteria in animals such as dogs, horses, and cattle. The assay is reliable and provides a useful and specific way for diagnosis of granulocytic ehrlichiosis in these animals.

Methodology

In this study, the researchers:

  • Developed a PCR-based assay to detect DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle.
  • Designed primers from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species.
  • Compared the results of the PCR test with microscopic examinations of stained blood smears to detect granulocytes containing ehrlichiae.
  • Used indirect fluorescent-antibody testing to analyze serum samples.

Findings

The findings of the research include:

  • 34 of 36 microscopically positive samples were also PCR positive, implying a high concordance between the two methods.
  • The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, while the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates.
  • 73% of the animals which were positive by both microscopy and PCR also had positive antibody titers.

Conclusion

The study concluded that:

  • Due to its efficacy, the PCR test is the most reliable method for diagnosing granulocytic ehrlichiosis in animals.
  • While serological results were useful, a single serological result was insufficient for a definitive diagnosis of the condition.

Cite This Article

APA
Engvall EO, Pettersson B, Persson M, Artursson K, Johansson KE. (1996). A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. J Clin Microbiol, 34(9), 2170-2174. https://doi.org/10.1128/jcm.34.9.2170-2174.1996

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 34
Issue: 9
Pages: 2170-2174

Researcher Affiliations

Engvall, E O
  • National Veterinary Institute, Uppsala, Sweden. eva.olsson@sva.se
Pettersson, B
    Persson, M
      Artursson, K
        Johansson, K E

          MeSH Terms

          • Animals
          • Cattle
          • Dogs
          • Ehrlichia / genetics
          • Ehrlichia / isolation & purification
          • Horses / microbiology
          • Leukocytes / microbiology
          • Molecular Sequence Data
          • Polymerase Chain Reaction / methods
          • RNA, Bacterial / analysis
          • RNA, Ribosomal, 16S / analysis

          References

          This article includes 19 references
          1. Brosius J, Palmer ML, Kennedy PJ, Noller HF. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.. Proc Natl Acad Sci U S A 1978 Oct;75(10):4801-5.
            pubmed: 368799doi: 10.1073/pnas.75.10.4801google scholar: lookup
          2. Munderloh UG, Madigan JE, Dumler JS, Goodman JL, Hayes SF, Barlough JE, Nelson CM, Kurtti TJ. Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.. J Clin Microbiol 1996 Mar;34(3):664-70.
            pubmed: 8904434doi: 10.1128/jcm.34.3.664-670.1996google scholar: lookup
          3. Hultman T, Ståhl S, Hornes E, Uhlén M. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.. Nucleic Acids Res 1989 Jul 11;17(13):4937-46.
            pubmed: 2668874doi: 10.1093/nar/17.13.4937google scholar: lookup
          4. Hultman T, Bergh S, Moks T, Uhlén M. Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA.. Biotechniques 1991 Jan;10(1):84-93.
            pubmed: 2003929
          5. Rikihisa Y. The tribe Ehrlichieae and ehrlichial diseases.. Clin Microbiol Rev 1991 Jul;4(3):286-308.
            pubmed: 1889044doi: 10.1128/CMR.4.3.286google scholar: lookup
          6. Anderson BE, Dawson JE, Jones DC, Wilson KH. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis.. J Clin Microbiol 1991 Dec;29(12):2838-42.
            pubmed: 1757557doi: 10.1128/jcm.29.12.2838-2842.1991google scholar: lookup
          7. Fox GE, Wisotzkey JD, Jurtshuk P Jr. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.. Int J Syst Bacteriol 1992 Jan;42(1):166-70.
            pubmed: 1371061doi: 10.1099/00207713-42-1-166google scholar: lookup
          8. Wahlberg J, Holmberg A, Bergh S, Hultman T, Uhlén M. Automated magnetic preparation of DNA templates for solid phase sequencing.. Electrophoresis 1992 Aug;13(8):547-51.
            pubmed: 1451691doi: 10.1002/elps.11501301112google scholar: lookup
          9. Sexton DJ, Kanj SS, Wilson K, Corey GR, Hegarty BC, Levy MG, Breitschwerdt EB. The use of a polymerase chain reaction as a diagnostic test for Rocky Mountain spotted fever.. Am J Trop Med Hyg 1994 Jan;50(1):59-63.
            pubmed: 8304573
          10. Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.. J Clin Microbiol 1994 Mar;32(3):589-95.
            pubmed: 8195363doi: 10.1128/jcm.32.3.589-595.1994google scholar: lookup
          11. Iqbal Z, Chaichanasiriwithaya W, Rikihisa Y. Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis.. J Clin Microbiol 1994 Jul;32(7):1658-62.
            pubmed: 7929754doi: 10.1128/jcm.32.7.1658-1662.1994google scholar: lookup
          12. Wen B, Rikihisa Y, Fuerst PA, Chaichanasiriwithaya W. Diversity of 16S rRNA genes of new Ehrlichia strains isolated from horses with clinical signs of Potomac horse fever.. Int J Syst Bacteriol 1995 Apr;45(2):315-8.
            pubmed: 7537065doi: 10.1099/00207713-45-2-315google scholar: lookup
          13. Johansson KE, Pettersson B, Uhlén M, Gunnarsson A, Malmqvist M, Olsson E. Identification of the causative agent of granulocytic ehrlichiosis in Swedish dogs and horses by direct solid phase sequencing of PCR products from the 16S rRNA gene.. Res Vet Sci 1995 Mar;58(2):109-12.
            pubmed: 7539151doi: 10.1016/0034-5288(95)90061-6google scholar: lookup
          14. Dumler JS, Asanovich KM, Bakken JS, Richter P, Kimsey R, Madigan JE. Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia.. J Clin Microbiol 1995 May;33(5):1098-103.
            pubmed: 7542262doi: 10.1128/jcm.33.5.1098-1103.1995google scholar: lookup
          15. Pancholi P, Kolbert CP, Mitchell PD, Reed KD Jr, Dumler JS, Bakken JS, Telford SR 3rd, Persing DH. Ixodes dammini as a potential vector of human granulocytic ehrlichiosis.. J Infect Dis 1995 Oct;172(4):1007-12.
            pubmed: 7561173doi: 10.1093/infdis/172.4.1007google scholar: lookup
          16. Madigan JE, Richter PJ Jr, Kimsey RB, Barlough JE, Bakken JS, Dumler JS. Transmission and passage in horses of the agent of human granulocytic ehrlichiosis.. J Infect Dis 1995 Oct;172(4):1141-4.
            pubmed: 7561199doi: 10.1093/infdis/172.4.1141google scholar: lookup
          17. Goodman JL, Nelson C, Vitale B, Madigan JE, Dumler JS, Kurtti TJ, Munderloh UG. Direct cultivation of the causative agent of human granulocytic ehrlichiosis.. N Engl J Med 1996 Jan 25;334(4):209-15.
            pubmed: 8531996doi: 10.1056/NEJM199601253340401google scholar: lookup
          18. Madigan JE, Barlough JE, Dumler JS, Schankman NS, DeRock E. Equine granulocytic ehrlichiosis in Connecticut caused by an agent resembling the human granulocytotropic ehrlichia.. J Clin Microbiol 1996 Feb;34(2):434-5.
            pubmed: 8789032doi: 10.1128/jcm.34.2.434-435.1996google scholar: lookup
          19. Madigan JE, Gribble D. Equine ehrlichiosis in northern California: 49 cases (1968-1981).. J Am Vet Med Assoc 1987 Feb 15;190(4):445-8.
            pubmed: 3558086

          Citations

          This article has been cited 22 times.
          1. Seo HJ, Truong AT, Kim KH, Lim JY, Min S, Kim HC, Yoo MS, Yoon SS, Klein TA, Cho YS. Molecular Detection and Phylogenetic Analysis of Tick-Borne Pathogens in Ticks Collected from Horses in the Republic of Korea.. Pathogens 2021 Aug 24;10(9).
            doi: 10.3390/pathogens10091069pubmed: 34578102google scholar: lookup
          2. El Hamiani Khatat S, Daminet S, Duchateau L, Elhachimi L, Kachani M, Sahibi H. Epidemiological and Clinicopathological Features of Anaplasma phagocytophilum Infection in Dogs: A Systematic Review.. Front Vet Sci 2021;8:686644.
            doi: 10.3389/fvets.2021.686644pubmed: 34250067google scholar: lookup
          3. Springer A, Glass A, Topp AK, Strube C. Zoonotic Tick-Borne Pathogens in Temperate and Cold Regions of Europe-A Review on the Prevalence in Domestic Animals.. Front Vet Sci 2020;7:604910.
            doi: 10.3389/fvets.2020.604910pubmed: 33363242google scholar: lookup
          4. Hovius E, de Bruin A, Schouls L, Hovius J, Dekker N, Sprong H. A lifelong study of a pack Rhodesian ridgeback dogs reveals subclinical and clinical tick-borne Anaplasma phagocytophilum infections with possible reinfection or persistence.. Parasit Vectors 2018 Apr 12;11(1):238.
            doi: 10.1186/s13071-018-2806-8pubmed: 29650038google scholar: lookup
          5. Duscher GG, Fuehrer HP, Kübber-Heiss A. Fox on the run--molecular surveillance of fox blood and tissue for the occurrence of tick-borne pathogens in Austria.. Parasit Vectors 2014 Nov 21;7:521.
            doi: 10.1186/s13071-014-0521-7pubmed: 25413694google scholar: lookup
          6. Santos HA, Thomé SM, Baldani CD, Silva CB, Peixoto MP, Pires MS, Vitari GL, Costa RL, Santos TM, Angelo IC, Santos LA, Faccini JL, Massard CL. Molecular epidemiology of the emerging zoonosis agent Anaplasma phagocytophilum (Foggie, 1949) in dogs and ixodid ticks in Brazil.. Parasit Vectors 2013 Dec 11;6:348.
            doi: 10.1186/1756-3305-6-348pubmed: 24330631google scholar: lookup
          7. Dyachenko V, Geiger C, Pantchev N, Majzoub M, Bell-Sakyi L, Krupka I, Straubinger RK. Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20.. Vet Microbiol 2013 Mar 23;162(2-4):980-986.
            doi: 10.1016/j.vetmic.2012.10.021pubmed: 23146170google scholar: lookup
          8. Stefancíková A, Derdáková M, Lencáková D, Ivanová R, Stanko M, Cisláková L, Petko B. Serological and molecular detection of Borrelia burgdorferi sensu lato and Anaplasmataceae in rodents.. Folia Microbiol (Praha) 2008;53(6):493-9.
            doi: 10.1007/s12223-008-0077-zpubmed: 19381473google scholar: lookup
          9. Sirigireddy KR, Ganta RR. Multiplex detection of Ehrlichia and Anaplasma species pathogens in peripheral blood by real-time reverse transcriptase-polymerase chain reaction.. J Mol Diagn 2005 May;7(2):308-16.
            doi: 10.1016/S1525-1578(10)60559-4pubmed: 15858156google scholar: lookup
          10. Sarih M, M'Ghirbi Y, Bouattour A, Gern L, Baranton G, Postic D. Detection and identification of Ehrlichia spp. in ticks collected in Tunisia and Morocco.. J Clin Microbiol 2005 Mar;43(3):1127-32.
            doi: 10.1128/JCM.43.3.1127-1132.2005pubmed: 15750072google scholar: lookup
          11. Von Loewenich FD, Stumpf G, Baumgarten BU, Röllinghoff M, Dumler JS, Bogdan C. A case of equine granulocytic ehrlichiosis provides molecular evidence for the presence of pathogenic anaplasma phagocytophilum (HGE agent) in Germany.. Eur J Clin Microbiol Infect Dis 2003 May;22(5):303-5.
            doi: 10.1007/s10096-003-0935-1pubmed: 12740667google scholar: lookup
          12. Ogden NH, Casey AN, Woldehiwet Z, French NP. Transmission of Anaplasma phagocytophilum to Ixodes ricinus ticks from sheep in the acute and post-acute phases of infection.. Infect Immun 2003 Apr;71(4):2071-8.
            doi: 10.1128/IAI.71.4.2071-2078.2003pubmed: 12654828google scholar: lookup
          13. Stuen S, Van De Pol I, Bergström K, Schouls LM. Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway.. J Clin Microbiol 2002 Sep;40(9):3192-7.
            doi: 10.1128/JCM.40.9.3192-3197.2002pubmed: 12202552google scholar: lookup
          14. Bjöersdorff A, Bagert B, Massung RF, Gusa A, Eliasson I. Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin.. Clin Diagn Lab Immunol 2002 Mar;9(2):341-3.
            doi: 10.1128/cdli.9.2.341-343.2002pubmed: 11874874google scholar: lookup
          15. Baumgarten BU, Röllinghoff M, Bogdan C. Prevalence of Borrelia burgdorferi and granulocytic and monocytic ehrlichiae in Ixodes ricinus ticks from southern Germany.. J Clin Microbiol 1999 Nov;37(11):3448-51.
            doi: 10.1128/JCM.37.11.3448-3451.1999pubmed: 10523532google scholar: lookup
          16. Munderloh UG, Jauron SD, Fingerle V, Leitritz L, Hayes SF, Hautman JM, Nelson CM, Huberty BW, Kurtti TJ, Ahlstrand GG, Greig B, Mellencamp MA, Goodman JL. Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.. J Clin Microbiol 1999 Aug;37(8):2518-24.
            doi: 10.1128/JCM.37.8.2518-2524.1999pubmed: 10405394google scholar: lookup
          17. 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.
            doi: 10.1128/JCM.37.5.1329-1331.1999pubmed: 10203480google scholar: lookup
          18. Stuen S, Artursson K, Olsson Engvall E. Experimental infection of lambs with an equine granulocytic Ehrlichia species resembling the agent that causes human granulocytic ehrlichiosis (HGE).. Acta Vet Scand 1998;39(4):491-7.
            doi: 10.1186/BF03547775pubmed: 9926463google scholar: lookup
          19. Pusterla N, Huder JB, Feige K, Lutz H. Identification of a granulocytic Ehrlichia strain isolated from a horse in Switzerland and comparison with other rickettsiae of the Ehrlichia phagocytophila genogroup.. J Clin Microbiol 1998 Jul;36(7):2035-7.
            doi: 10.1128/JCM.36.7.2035-2037.1998pubmed: 9650957google scholar: lookup
          20. Cinco M, Padovan D, Murgia R, Maroli M, Frusteri L, Heldtander M, Johansson KE, Engvall EO. Coexistence of Ehrlichia phagocytophila and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Italy as determined by 16S rRNA gene sequencing.. J Clin Microbiol 1997 Dec;35(12):3365-6.
            doi: 10.1128/jcm.35.12.3365-3366.1997pubmed: 9399564google scholar: lookup
          21. Fingerle V, Goodman JL, Johnson RC, Kurtti TJ, Munderloh UG, Wilske B. Human granulocytic ehrlichiosis in southern Germany: increased seroprevalence in high-risk groups.. J Clin Microbiol 1997 Dec;35(12):3244-7.
            doi: 10.1128/jcm.35.12.3244-3247.1997pubmed: 9399527google scholar: lookup
          22. Nilsson K, Jaenson TG, Uhnoo I, Lindquist O, Pettersson B, Uhlén M, Friman G, Påhlson C. Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden.. J Clin Microbiol 1997 Jan;35(1):243-7.
            doi: 10.1128/jcm.35.1.243-247.1997pubmed: 8968916google scholar: lookup
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