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Genetic and Ecologic Variability among Anaplasma phagocytophilum Strains, Northern Italy - Volume 20, Number 6—June 2014 - Emerging Infectious Disease journal - CDC

Genetic and Ecologic Variability among Anaplasma phagocytophilum Strains, Northern Italy - Volume 20, Number 6—June 2014 - Emerging Infectious Disease journal - CDC

link to Volume 20, Number 6—June 2014

Volume 20, Number 6—June 2014


Genetic and Ecologic Variability among Anaplasma phagocytophilum Strains, Northern Italy

To the Editor: The tick-borne pathogen Anaplasma phagocytophilum is an increasing potential public health threat across Europe. Its intraspecific genetic variability is associated with different reservoir host and vector tick species (14); however, the roles of various vertebrates as competent reservoirs of A. phagocytophilum in Europe need clarification (1). During March 2011–June 2013, we studied the prevalence and genetic variability of A. phagocytophilumin 821 questing Ixodes ricinus ticks (155 adults [A], 666 nymphs [N] collected by standard blanket dragging) and 284 engorged ixodid ticks (61A, 191N, 21 larvae [L]) collected from humans, dogs, sheep, hunted wild ungulates, live-trapped birds, and rodents. Blood samples from 1,295 rodents (yellow-necked mice [Apodemus flavicollis]), bank voles [Myodes glareolus], and harvest mice [Moscardinus avellanarius]) were also analyzed. All animal-handling procedures and ethical issues were approved by the Provincial Wildlife Management Committee (authorization n. 595 issued on 04.05.2011). The study site, Valle dei Laghi (northeastern Italian Alps), is a well-studied focus of emerging tick-borne pathogens in northern Italy (4).
Tick species were identified morphologically and by molecular analyses by using 16SrRNA sequences. A. phagocytophilum was detected in questing and feeding I. ricinus ticks by using a nested PCR amplification of the partial 16S rRNA gene (546-bp fragment) as described (4,5) and in rodent blood by using a real time-PCR assay targeting the msp2 gene (77 bp) (6). All positive samples were confirmed by using Sanger sequencing.
Overall prevalence of A. phagocytophilum in questing I. ricinus ticks was 1.8% (6A, 9N of 821) (Table). Among engorged ticks, only I. ricinus ticks were found positive for A. phagocytophilum, although tick species such as I. hexagounus (20 ticks from dogs and birds), I. trianguliceps (11 from rodents), and I. turdus (1 from a bird) were also analyzed. Infection prevalence in ticks from various hosts was: 4.3% (5N/115) in ticks from humans, 9.1% (1N/30) in ticks from dogs, 14.3% (4A, 1N, 2L/49) in ticks from wild ungulates, 7.7% (1A/30) in ticks from sheep, 10.7% (3N/28) in ticks from birds, and 6.1% (3N/49) in ticks from rodents (Table). Prevalence in rodent blood samples (A. flavicolis mice, M. avellanarius mice, M. glareolus bank voles) was 0.3% (4/1,295); only bank voles had positive results. None of the feeding I. ricinus larvae collected from rodents were infected with A. phagocytophilum.
We amplified and sequenced 2 genetic loci, groEL and msp4, from samples that were positive forA. phagocytophilum, which are known to be useful for phylogenetic studies (4,7,8). MrBayes v3.1.2 ( Web Site Icon ) was used to construct Bayesian phylogenetic trees for each gene (9). We deposited 54 new A. phagocytophilumsequences in GenBank with accession numbers KF031380–KF031433. Fourteen and 9 uniquegroEL and msp4 A. phagocytophilum genotypes, respectively, were found to circulate in this alpine valley.
The phylogenetic trees for groEL (Technical Appendix Adobe PDF file [PDF - 194 KB - 2 pages] Figure) andmsp4 (Technical Appendix Adobe PDF file [PDF - 194 KB - 2 pages] Figure, panel B) loci have similar topologies with strong support for 2 main clades (Technical Appendix Adobe PDF file [PDF - 194 KB - 2 pages] Figure, panels A and B), each with different host and vector association. The first clade (clade 1) contained sequences from questing I. ricinus ticks and engorged ticks collected from humans, dogs, wild ungulates, rodents, sheep, and birds. Our findings suggest that humans are exposed to several A. phagocytophilum genotypes exclusively from clade 1 (Technical Appendix Adobe PDF file [PDF - 194 KB - 2 pages] Figure, panels A and B). Our 3 unique A. phagocytophilum sequences were from 3 I. ricinus nymphs that fed on the same human clustered within this clade, but no clinical symptoms were observed.
The second clade (clade 2) includes sequences from rodents, specifically, bank voles (M. glareolus), other voles and shrews. Among tick species we found I. persulcatus to belong to this clade (Technical Appendix Adobe PDF file [PDF - 194 KB - 2 pages] Figure, panels A and B). We have found no evidence of circulation of this genotype in other hosts or in questing or engorged I. ricinus ticks in previously published data or in this study (Technical Appendix Adobe PDF file [PDF - 194 KB - 2 pages]Figure, panels A and B, clade 2). This finding suggests that the A. phagocytophilum genotype associated with mice, voles, and shrews in Europe may be maintained in enzootic cycles by another tick vector, such as I. trianguliceps, as observed in the UK for the field vole (Microtus agrestis) (8). This so-called ecologic strain probably does not represent an immediate threat to humans in northern Italy, unlike the rodent strain reported in the USA, since it occurs in very low prevalence, and because I. trianguliceps is an endophilic tick species that is unlikely to come into contact with humans.
In 1 questing I. ricinus tick at the nymphal stage, we detected a groEL sequence (KF031399) identical to a sequence isolated from humans with human granulocytic anaplasmosis in Europe (AF033101). The msp4 sequence for the same sample (KF031406) belonged to clade 1, and contained sequences of a strain found in 96 infected persons in the United States. This suggests that >1 human pathogenic strain now circulates in the investigated area. However, we did not find this strain in any of the host-fed ticks analyzed, so the host responsible for maintaining the circulation of this pathogenic strain must be identified before any recommendation for preventive measures can be provided.
Ivana Baráková, Markéta Derdáková, Giovanna Carpi, Fausta Rosso, Margherita Collini, Valentina Tagliapietra, Claudio Ramponi, Heidi C. Hauffe, and Annapaola RizzoliComments to Author 
Author affiliations: Fondazione Edmund Mach, Trento, Italy (I. Baráková, Giovanna Carpi, F. Rosso, M. Collini, V. Tagliapietra, H.C. Hauffe, A. Rizzoli)Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovak Republic (I. Baráková, M. Derdáková)Institute of Parisitology SAS, Košice, Slovak Republic (M. Derdáková)Yale School of Public Health, New Haven, CT, USA (G. Carpi)Ospedale Santa Chiara, Trento (C. Ramponi)


We thank D. Arnoldi, A. Konečný, E. Gillingham, and F. Rizzolli for help with tick and blood sample collection, and N. Ricci for providing ticks collected from humans. We thank veterinarians A. Aloisi, M. Danielli, E. Lutteri, and R. Zampiccoli for providing ticks collected from dogs, and the Trentino Hunters Association (Districts of Sopramonte and Valle dei Laghi) and the Forestry Guards of the Autonomous Province of Trento for providing ticks collected from deer.
The study was funded by the European Union grant FP7-261504 EDENext (to AR) and is catalogued by the EDENext Steering Committee as EDENext 149 (http://www.edenext.euExternal Web Site Icon), by the Fondazione Edmund Mach (to IB, AR and HCH), partially by the Slovak Academy of Science grants VEGA - 2/0055/ and APVV-0267-10 (to MD), and by the Autonomous Province of Trento under the EU FP7 PEOPLE Programme, Marie Curie Actions Cofund Post-doctoral project GENOTICK (to GC). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission.


  1. Stuen SGranquist EGSilaghi CAnaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strategies. Front Cell Infect Microbiol. 2013;3:31.External Web Site IconPubMedExternal Web Site Icon
  2. Keesing FHersh MHTibbetts MMcHenry DJDuerr SBrunner JReservoir competence of vertebrate hosts for Anaplasma phagocytophilum. Emerg Infect Dis2012;18:20136DOIExternal Web Site IconPubMedExternal Web Site Icon
  3. Mantelli BPecchioli EHauffe HCRosa RRizzoli APrevalence of Borrelia burgdorferi s.l. and Anaplasma phagocytophilum in the wood tick Ixodes ricinus in the Province of Trento, Italy. Eur J Clin Microbiol Infect Dis2006;25:7379DOIExternal Web Site IconPubMedExternal Web Site Icon
  4. Carpi GBertolotti LPecchioli ECagnacci FRizzoli AAnaplasma phagocytophilum groEL gene heterogeneity in Ixodes ricinus larvae feeding on roe deer in Northeastern Italy.Vector Borne Zoonotic Dis2009;9:17984DOIExternal Web Site IconPubMedExternal Web Site Icon
  5. Massung RFSlater KOwens JHNicholson WLMather TNSolberg VBNested PCR assay for detection of granulocytic ehrlichiae. J Clin Microbiol1998;36:10905 .PubMedExternal Web Site Icon
  6. Courtney JWKostelnik LMZeidner NSMassung RFMultiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol.2004;42:31648 . DOIExternal Web Site IconPubMedExternal Web Site Icon
  7. de la Fuente JMassung RFWong SJChu FKLutz HMeli MSequence analysis of themsp4 gene of Anaplasma phagocytophilum strains. J Clin Microbiol2005;43:130917DOIExternal Web Site IconPubMedExternal Web Site Icon
  8. Bown KJLambin XOgden NHBegon MTelford GWoldehiwet ZDelineating Anaplasma phagocytophilum ecotypes in coexisting, discrete enzootic cycles. Emerg Infect Dis.2009;15:194854DOIExternal Web Site IconPubMedExternal Web Site Icon
  9. Ronquist FHuelsenbeck JPMrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics2003;19:15724DOIExternal Web Site IconPubMedExternal Web Site Icon


Technical Appendix

Suggested citation for this article: Baráková I, Derdáková M, Carpi G, Rosso F, Collini M, Tagliapietra V, et al. Genetic and ecologic variability among strains of Anaplasma phagocytophilum, northern Italy [letter]. Emerg Infect Dis. 2014 Jun [date cited]. Web Site Icon
DOI: 10.3201/eid2006.131023

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