BACKGROUND: Anaplasma phagocytophilum is currently regarded as a single species. However, molecular studies indicate that it can be subdivided into ecotypes, each with distinct but overlapping transmission cycle. Here, we evaluate the interactions between and within clusters of haplotypes of the bacterium isolated from vertebrates and ticks, using phylogenetic and network-based methods. METHODS: The presence of A. phagocytophilum DNA was determined in ticks and vertebrate tissue samples. A fragment of the groEl gene was amplified and sequenced from qPCR-positive lysates. Additional groEl sequences from ticks and vertebrate reservoirs were obtained from GenBank and through literature searches, resulting in a dataset consisting of 1623 A. phagocytophilum field isolates. Phylogenetic analyses were used to infer clusters of haplotypes and to assess phylogenetic clustering of A. phagocytophilum in vertebrates or ticks. Network-based methods were used to resolve host-vector interactions and their relative importance in the segregating communities of haplotypes. RESULTS: Phylogenetic analyses resulted in 199 haplotypes within eight network-derived clusters, which were allocated to four ecotypes. The interactions of haplotypes between ticks, vertebrates and geographical origin, were visualized and quantified from networks. A high number of haplotypes were recorded in the tick Ixodes ricinus. Communities of A. phagocytophilum recorded from Korea, Japan, Far Eastern Russia, as well as those associated with rodents had no links with the larger set of isolates associated with I. ricinus, suggesting different evolutionary pressures. Rodents appeared to have a range of haplotypes associated with either Ixodes trianguliceps or Ixodes persulcatus and Ixodes pavlovskyi. Haplotypes found in rodents in Russia had low similarities with those recorded in rodents in other regions and shaped separate communities. CONCLUSIONS: The groEl gene fragment of A. phagocytophilum provides information about spatial segregation and associations of haplotypes to particular vector-host interactions. Further research is needed to understand the circulation of this bacterium in the gap between Europe and Asia before the overview of the speciation features of this bacterium is complete. Environmental traits may also play a role in the evolution of A. phagocytophilum in ecotypes through yet unknown relationships.

Biology Centre Institute of Parasitology Czech Academy of Sciences České Budějovice Czech Republic

Centre for Ecological and Evolutionary Synthesis Department of Biosciences University of Oslo P O Box 1066 Blindern 0316 Oslo Norway

Centre for Infectious Disease Control Bilthoven The Netherlands

Department for Bacteriology and Parasitology Croatian Veterinary Institute Savska Cesta 143 Zagreb Croatia

Department of Parasitology and Zoology University of Veterinary Medicine Budapest Budapest Hungary

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Department of Parasitology Faculty of Veterinary Medicine University of Zaragoza Zaragoza Spain

Department of Pathology and Parasitology Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Czech Republic

Department of Veterinary Sciences University of Turin Via L da Vinci 44 Grugliasco 10095 Turin Italy

Department of Wildlife Fish and Environmental Studies Swedish University of Agricultural Sciences Umeå Sweden

Diergeneeskundig Centrum Zuid Oost Drenthe Coevorden The Netherlands

Dutch Wildlife Health Centre Utrecht University Utrecht The Netherlands

Emerging Zoonoses Research Group Instituto Agroalimentario de Aragón Zaragoza Spain

Evolutionary Systems Research Group Centre for Ecological Research Hungarian Academy of Sciences Tihany Hungary

Friedrich Loeffler Institut Federal Research Institute for Animal Health Greifswald Insel Riems Germany

Institute of Vertebrate Biology v v i Medical Zoology Laboratory Academy of Sciences of the Czech Republic Brno Czech Republic

Institute of Zoology Slovak Academy of Sciences Bratislava Slovakia

Laboratory of Entomology Wageningen University Wageningen The Netherlands

Zobrazit více v PubMed

Ehrlich PR, Raven PH. Butterflies and plants: a study in coevolution. Evolution. 1964;18:586–608. doi: 10.1111/j.1558-5646.1964.tb01674.x. DOI

Thompson JN. The geographic mosaic of coevolution. Chicago: The University of Chicago Press; 2005.

Bascompte J, Jordano P, Olesen JM. Asymmetric coevolutionary networks facilitate biodiversity maintenance. Science. 2006;312:431–433. doi: 10.1126/science.1123412. PubMed DOI

Thompson JN. The coevolutionary process. Chicago: University of Chicago Press; 1994.

Bascompte J, Jordano P, Melián CJ, Olesen JM. The nested assembly of plant-animal mutualistic networks. Proc Natl Acad Sci USA. 2003;100:9383–9387. doi: 10.1073/pnas.1633576100. PubMed DOI PMC

Jordano P, Bascompte J, Olesen JM. Invariant properties in coevolutionary networks of plant-animal links. Ecol Lett. 2003;6:69–81. doi: 10.1046/j.1461-0248.2003.00403.x. DOI

Vázquez DP, Aizen MA. Asymmetric specialization: a pervasive feature of plant-pollinator interactions. Ecology. 2004;85:1251–1257. doi: 10.1890/03-3112. DOI

Streicker DG, Fenton A, Pedersen AB. Differential sources of host species heterogeneity influence the transmission and control of multihost parasites. Ecol Lett. 2013;16:975–984. doi: 10.1111/ele.12122. PubMed DOI PMC

Newman MEJ. Networks: an introduction. Oxford-New York: Oxford University Press; 2010.

Rigaud T, Perrot-Minnot M-J, Brown MJF. Parasite and host assemblages: embracing the reality will improve our knowledge of parasite transmission and virulence. Proc R Soc London B Biol Sci. 2010;277:3693–3702. doi: 10.1098/rspb.2010.1163. PubMed DOI PMC

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;32:589–595. PubMed PMC

Stuen S, Granquist E, Silaghi C. Anaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strategies. Front Cell Infect Microbiol. 2013;3:31. doi: 10.3389/fcimb.2013.00031. PubMed DOI PMC

Bakken JS, Dumler JS. Human granulocytic ehrlichiosis. Clin Infect Dis. 2000;31:554–560. doi: 10.1086/313948. PubMed DOI

André MR. Diversity of Anaplasma and Ehrlichia/Neoehrlichia agents in terrestrial wild carnivores worldwide: implications for human and domestic animal health and wildlife conservation. Front Vet Sci. 2018;5:293. doi: 10.3389/fvets.2018.00293. PubMed DOI PMC

Bown KJ, Lambin X, Ogden NH, Begon M, Telford G, Woldehiwet Z, et al. Delineating Anaplasma phagocytophilum ecotypes in coexisting, discrete enzootic cycles. Emerg Infect Dis. 2009;15:1948–1954. doi: 10.3201/eid1512.090178. PubMed DOI PMC

Baldridge GD, Scoles GA, Burkhardt NY, Schloeder B, Kurtti TJ, Munderloh UG. Transovarial transmission of Francisella-like endosymbionts and Anaplasma phagocytophilum variants in Dermacentor albipictus (Acari: Ixodidae) J Med Entomol. 2009;46:625–632. doi: 10.1603/033.046.0330. PubMed DOI PMC

Woldehiwet Z. Anaplasma phagocytophilum in ruminants in Europe. Ann N Y Acad Sci. 2006;1078:446–460. doi: 10.1196/annals.1374.084. PubMed DOI

Hofmeester TR, Coipan EC, van Wieren SE, Prins HHT, Takken W, Sprong H. Few vertebrate species dominate the Borrelia burgdorferi s.l. life cycle. Env Res Lett. 2016;11:043001. doi: 10.1088/1748-9326/11/4/043001. DOI

Barbour A, Fish D. The biological and social phenomenon of Lyme disease. Science. 1993;260:1610–1616. doi: 10.1126/science.8503006. PubMed DOI

Walls JJ, Greig B, Neitzel DF, Dumler JS. Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis. J Clin Microbiol. 1997;35:853–855. PubMed PMC

Blaňarová L, Stanko M, Carpi G, Miklisová D, Víchová B, Mošanský L, et al. Distinct Anaplasma phagocytophilum genotypes associated with Ixodes trianguliceps ticks and rodents in central Europe. Ticks Tick Borne Dis. 2014;5:928–938. doi: 10.1016/j.ttbdis.2014.07.012. PubMed DOI

Estrada-Peña A, de la Fuente J, Ostfeld RS, Cabezas-Cruz A. Interactions between tick and transmitted pathogens evolved to minimise competition through nested and coherent networks. Sci Rep. 2015;5:10361. doi: 10.1038/srep10361. PubMed DOI PMC

Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‛HGE agentʼ as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol. 2001;51:2145–2165. doi: 10.1099/00207713-51-6-2145. PubMed DOI

Scharf W, Schauer S, Freyburger F, Petrovec M, Schaarschmidt-Kiener D, Liebisch G, et al. Distinct host species correlate with Anaplasma phagocytophilum ankA gene clusters. J Clin Microbiol. 2011;49:790–796. doi: 10.1128/JCM.02051-10. PubMed DOI PMC

de la Fuente J, Kocan KM, Blouin EF, Zivkovic Z, Naranjo V, Almazán C, et al. Functional genomics and evolution of tick-Anaplasma interactions and vaccine development. Vet Parasitol. 2010;167:175–186. doi: 10.1016/j.vetpar.2009.09.019. PubMed DOI

Chastagner A, Dugat T, Vourc’h G, Verheyden H, Legrand L, Bachy V, et al. Multilocus sequence analysis of Anaplasma phagocytophilum reveals three distinct lineages with different host ranges in clinically ill French cattle. Vet Res. 2014;45:114. doi: 10.1186/s13567-014-0114-7. PubMed DOI PMC

Huhn C, Winter C, Wolfsperger T, Wüppenhorst N, Strašek Smrdel K, Skuballa J, et al. Analysis of the population structure of Anaplasma phagocytophilum using multilocus sequence typing. PLoS ONE. 2014;9:e93725. doi: 10.1371/journal.pone.0093725. PubMed DOI PMC

Rymaszewska A. Divergence within the marker region of the groESL operon in Anaplasma phagocytophilum. Eur J Clin Microbiol Infect Dis. 2008;27:1025–1036. doi: 10.1007/s10096-008-0539-x. PubMed DOI

Petrovec M, Bidovec A, Sumner J, Nicholson W, Childs J, Avsic-Zupanc T. Infection with Anaplasma phagocytophila in cervids from Slovenia: Evidence of two genotypic lineages. Wien Klin Wochenschr. 2002;114:641–647. PubMed

Dugat T, Chastagner A, Lagrée A-C, Petit E, Durand B, Thierry S, et al. A new multiple-locus variable-number tandem repeat analysis reveals different clusters for Anaplasma phagocytophilum circulating in domestic and wild ruminants. Parasit Vectors. 2014;7:439. doi: 10.1186/1756-3305-7-439. PubMed DOI PMC

Jahfari S, Coipan EC, Fonville M, van Leeuwen AD, Hengeveld P, Heylen D, et al. Circulation of four Anaplasma phagocytophilum ecotypes in Europe. Parasit Vectors. 2014;7:365. doi: 10.1186/1756-3305-7-365. PubMed DOI PMC

Heylen D, Fonville M, van Leeuwen A, Stroo A, Duisterwinkel M, van Wieren S, et al. Pathogen communities of songbird-derived ticks in Europeʼs low countries. Parasit Vectors. 2017;10:497. doi: 10.1186/s13071-017-2423-y. PubMed DOI PMC

Jahfari S, Ruyts S, Frazer-Mendelewska E, Jaarsma R, Verheyen K, Sprong H. Melting pot of tick-borne zoonoses: the European hedgehog contributes to the maintenance of various tick-borne diseases in natural cycles urban and suburban areas. Parasit Vectors. 2017;10:134. doi: 10.1186/s13071-017-2065-0. PubMed DOI PMC

Hofmeester TR, Krawczyk AI, van Leeuwen AD, Fonville M, Montizaan MGE, van den Berge K, et al. Role of mustelids in the life-cycle of ixodid ticks and transmission cycles of four tick-borne pathogens. Parasit Vectors. 2018;11:600. doi: 10.1186/s13071-018-3126-8. PubMed DOI PMC

Hing M, Woestyn S, Van Bosterhaut B, Desbonnet Y, Heyman P, Cochez C, et al. Diagnosis of human granulocytic anaplasmosis in Belgium by combining molecular and serological methods. New Microbes New Infect. 2014;2:177–178. doi: 10.1002/nmi2.65. PubMed DOI PMC

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;11:238. doi: 10.1186/s13071-018-2806-8. PubMed DOI PMC

Jahfari S, Hofhuis A, Fonville M, van der Giessen J, van Pelt W, Sprong H. Molecular detection of tick-borne pathogens in humans with tick bites and erythema migrans, in the Netherlands. PLoS Negl Trop Dis. 2016;10:e0005042. doi: 10.1371/journal.pntd.0005042. PubMed DOI PMC

Krawczyk AI, van Leeuwen A, Jacobs-Reitsma W, Wijnands LM, Bouw E, Jahfari S, et al. Presence of zoonotic agents in engorged ticks and hedgehog faeces from Erinaceus europaeus in (sub) urban areas. Parasit Vectors. 2015;8:210. doi: 10.1186/s13071-015-0814-5. PubMed DOI PMC

Hofmeester TR, Jansen PA, Wijnen HJ, Coipan EC, Fonville M, Prins HHT, et al. Cascading effects of predator activity on tick-borne disease risk. Proc Biol Sci. 2017;284:20170453. doi: 10.1098/rspb.2017.0453. PubMed DOI PMC

Sprong H, Tijsse-Klasen E, Langelaar M, De Bruin A, Fonville M, Gassner F, et al. Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever. Zoonoses Public Health. 2012;59:69–75. doi: 10.1111/j.1863-2378.2011.01421.x. PubMed DOI

Stigum VM, Jaarsma RI, Sprong H, Rolandsen CM, Mysterud A. Infection prevalence and ecotypes of Anaplasma phagocytophilum in moose Alces alces, red deer Cervus elaphus, roe deer Capreolus capreolus and Ixodes ricinus ticks from Norway. Parasit Vectors. 2019;12:1. doi: 10.1186/s13071-018-3256-z. PubMed DOI PMC

Kazimírová M, Hamšíková Z, Špitalská E, Minichová L, Mahríková L, Caban R, et al. Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia. Parasit Vectors. 2018;11:495. doi: 10.1186/s13071-018-3068-1. PubMed DOI PMC

Santos AS, de Bruin A, Veloso AR, Marques C, da Fonseca I, de Sousa R, et al. Detection of Anaplasma phagocytophilum, Candidatus Neoehrlichia sp, Coxiella burnetii and Rickettsia spp in questing ticks from a recreational park, Portugal. Ticks Tick Borne Dis. 2018;9:1555–1564. doi: 10.1016/j.ttbdis.2018.07.010. PubMed DOI

Szekeres S, van Leeuwen A, Tóth E, Majoros G, Sprong H, Földvári G. Road-killed mammals provide insight into tick-borne bacterial pathogen communities within urban habitats. Transbound Emerg Dis. 2019;66:277–286. doi: 10.1111/tbed.13019. PubMed DOI

Szekeres S, Coipan EC, Rigó K, Majoros G, Jahfari S, Sprong H, et al. Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in natural rodent and tick communities in Southern Hungary. Ticks Tick Borne Dis. 2015;6:111–116. doi: 10.1016/j.ttbdis.2014.10.004. PubMed DOI

Földvári G, Jahfari S, Rigó K, Jablonszky M, Szekeres S, Majoros G, et al. Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in urban hedgehogs. Emerg Infect Dis. 2014;20:496–498. doi: 10.3201/eid2003.130935. PubMed DOI PMC

de Bruin A, van Leeuwen AD, Jahfari S, Takken W, Földvári M, Dremmel L, et al. Vertical transmission of Bartonella schoenbuchensis in Lipoptena cervi. Parasit Vectors. 2015;8:176. doi: 10.1186/s13071-015-0764-y. PubMed DOI PMC

Overzier E, Pfister K, Thiel C, Herb I, Mahling M, Silaghi C. Anaplasma phagocytophilum in questing Ixodes ricinus ticks: comparison of prevalences and partial 16S rRNA gene variants in urban, pasture, and natural habitats. Appl Environ Microbiol. 2013;79:1730–1734. doi: 10.1128/AEM.03300-12. PubMed DOI PMC

Silaghi C, Nieder M, Sauter-Louis C, Knubben-Schweizer G, Pfister K, Pfeffer M. Epidemiology, genetic variants and clinical course of natural infections with Anaplasma phagocytophilum in a dairy cattle herd. Parasit Vectors. 2018;11:20. doi: 10.1186/s13071-017-2570-1. PubMed DOI PMC

Schouls LM, Van De Pol I, Rijpkema SG, Schot CS. Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in Dutch Ixodes ricinus ticks. J Clin Microbiol. 1999;37:2215–2222. PubMed PMC

Courtney JW, Kostelnik LM, Zeidner NS, Massung RF. Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol. 2004;42:3164–3168. doi: 10.1128/JCM.42.7.3164-3168.2004. PubMed DOI PMC

Alberti A, Zobba R, Chessa B, Addis MF, Sparagano O, Pinna Parpaglia ML, et al. Equine and canine Anaplasma phagocytophilum strains isolated on the Island of Sardinia (Italy) are phylogenetically related to pathogenic strains from the United States. Appl Environ Microbiol. 2005;71:6418–6422. doi: 10.1128/AEM.71.10.6418-6422.2005. PubMed DOI PMC

Wolfram Research, Inc. Mathematica, version 11.3. Champaign, IL, USA: Wolfram Research, Inc; 2018.

Bastian M, Heymann S, Jacomy M. Gephi: an open source software for exploring and manipulating networks. In: International AAAI Conference on Weblogs and Social Media; 2009. gephi.org. Accessed 1 Feb 2019.

Bastolla U, Fortuna MA, Pascual-García A, Ferrera A, Luque B, Bascompte J. The architecture of mutualistic networks minimizes competition and increases biodiversity. Nature. 2009;458:1018. doi: 10.1038/nature07950. PubMed DOI

Csardi G, Nepusz T. The igraph software package for complex network research. InterJournal Complex Systems 1695; 2006.

R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2018.

Estrada-Peña A, Sprong H, Cabezas-Cruz A, de la Fuente J, Ramo A, Coipan EC. Nested coevolutionary networks shape the ecological relationships of ticks, hosts, and the Lyme disease bacteria of the Borrelia burgdorferi (s.l.) complex. Parasit Vectors. 2016;9:517. doi: 10.1186/s13071-016-1803-z. PubMed DOI PMC

Craft ME, Caillaud D. Network models: an underutilized tool in wildlife epidemiology? Interdiscip Perspect Infect Dis. 2011;2011:676949. doi: 10.1155/2011/676949. PubMed DOI PMC

Faith DP. Conservation evaluation and phylogenetic diversity. Biol Conserv. 1992;61:1–10. doi: 10.1016/0006-3207(92)91201-3. DOI

Webb CO, Ackerly D, Mcpeek MA, Donoghue MJ. Phylogenies and community ecology. Annu Rev Ecol Syst. 2002;33:475–505. doi: 10.1146/annurev.ecolsys.33.010802.150448. DOI

Kembel SW, Cowan PD, Helmus MR, Cornwell WK, Morlon H, Ackerly DD, et al. Picante: R tools for integrating phylogenies and ecology. Bioinformatics. 2010;26:1463–1464. doi: 10.1093/bioinformatics/btq166. PubMed DOI

Marsot M, Henry PY, Vourc’h G, Gasqui P, Ferquel E, Laignel J, et al. Which forest bird species are the main hosts of the tick, Ixodes ricinus, the vector of Borrelia burgdorferi sensu lato, during the breeding season? Int J Parasitol. 2012;42:781–788. doi: 10.1016/j.ijpara.2012.05.010. PubMed DOI

de la Fuente J, Estrada-Peña A, Cabezas-Cruz A, Brey R. Flying ticks: anciently evolved associations that constitute a risk of infectious disease spread. Parasit Vectors. 2015;8:538. doi: 10.1186/s13071-015-1154-1. PubMed DOI PMC

Dinnis R, Seelig F, Bormane A, Donaghy M, Vollmer S, Feil E, et al. Multilocus sequence typing using mitochondrial genes (mtMLST) reveals geographic population structure of Ixodes ricinus ticks. Ticks Tick Borne Dis. 2014;5:152–160. doi: 10.1016/j.ttbdis.2013.10.001. PubMed DOI

Estrada-Peña A, Naranjo V, Acevedo-Whitehouse K, Mangold AJ, Kocan KM, de la Fuente J. Phylogeographic analysis reveals association of tick-borne pathogen, Anaplasma marginale, MSP1a sequences with ecological traits affecting tick vector performance. BMC Biol. 2009;7:57. doi: 10.1186/1741-7007-7-57. PubMed DOI PMC

Santos AS, Santos-Silva MM. Ixodes ventalloi Gil Collado, 1936: a vector role to be explored. London: Intechopen; 2018.

Estrada-Peña A, Venzal JM, Nava S. Redescription, molecular features, and neotype deposition of Rhipicephalus pusillus Gil Collado and Ixodes ventalloi Gil Collado (Acari, Ixodidae) Zootaxa. 2018;4442:262–276. doi: 10.11646/zootaxa.4442.2.4. PubMed DOI

Estrada-Peña A, Villar M, Artigas-Jeronimo S, López V, Alberdi P, Cabezas-Cruz A, et al. Use of graph theory to characterize human and arthropod vector cell protein response to infection with Anaplasma phagocytophilum. Front Cell Infect Microbiol. 2018;8:265. doi: 10.3389/fcimb.2018.00265. PubMed DOI PMC

de la Fuente J, Estrada-Peña A, Cabezas-Cruz A, Kocan KM. Anaplasma phagocytophilum uses common strategies for infection of ticks and vertebrate hosts. Trends Microbiol. 2016;24:173–180. doi: 10.1016/j.tim.2015.12.001. PubMed DOI

Nylin S, Agosta S, Bensch S, Boeger WA, Braga MP, Brooks DR, et al. Embracing colonizations: a new paradigm for species association dynamics. Trends Ecol Evol. 2018;33:4–14. doi: 10.1016/j.tree.2017.10.005. PubMed DOI

Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids

Circulation of Anaplasma phagocytophilum among invasive and native carnivore species living in sympatry in Poland

Role of invasive carnivores (Procyon lotor and Nyctereutes procyonoides) in epidemiology of vector-borne pathogens: molecular survey from the Czech Republic

Distant genetic variants of Anaplasma phagocytophilum from Ixodes ricinus attached to people

Presence of Roe Deer Affects the Occurrence of Anaplasma phagocytophilum Ecotypes in Questing Ixodes ricinus in Different Habitat Types of Central Europe