title: Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization.
abstract: The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.
authors.affiliation: Division of Bacteriology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands.
authors.forename: Cornelis P J
authors.initials: CP
authors.lastname: Bekker
authors.forename: Sander
authors.initials: S
authors.lastname: de Vos
authors.forename: Amar
authors.initials: A
authors.lastname: Taoufik
authors.forename: Olivier A E
authors.initials: OA
authors.lastname: Sparagano
authors.forename: Frans
authors.initials: F
authors.lastname: Jongejan
mesh_terms: D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification*; D000712:Anaplasmosis / Q000175:diagnosis / Q000382:microbiology*; D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016995:Ehrlichia ruminantium / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D015511:Goat Diseases / Q000175:diagnosis / Q000382:microbiology*; D006041:Goats; D006357:Heartwater Disease / Q000175:diagnosis / Q000382:microbiology*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000382:microbiology*; D013987:Ticks / Q000382:microbiology*
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S