Additional file 3: of 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

The number of Ixodes ricinus and Ixodes hexagonus ticks infected with a certain pathogen, for all life stages together or for larvae (L), nymphs (N) or adults (A) separately. (CSV 719 bytes

Additional file 1: of 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

IGS sequences obtained in this study. (XLSX 13 kb

Molecular Detection of Tick-Borne Pathogens in Humans with Tick Bites and Erythema Migrans, in the Netherlands

<div><p>Background</p><p>Tick-borne diseases are the most prevalent vector-borne diseases in Europe. Knowledge on the incidence and clinical presentation of other tick-borne diseases than Lyme borreliosis and tick-borne encephalitis is minimal, despite the high human exposure to these pathogens through tick bites. Using molecular detection techniques, the frequency of tick-borne infections after exposure through tick bites was estimated.</p><p>Methods</p><p>Ticks, blood samples and questionnaires on health status were collected from patients that visited their general practitioner with a tick bite or erythema migrans in 2007 and 2008. The presence of several tick-borne pathogens in 314 ticks and 626 blood samples of this cohort were analyzed using PCR-based methods. Using multivariate logistic regression, associations were explored between pathogens detected in blood and self-reported symptoms at enrolment and during a three-month follow-up period.</p><p>Results</p><p>Half of the ticks removed from humans tested positive for <i>Borrelia burgdorferi</i> sensu lato, <i>Anaplasma phagocytophilum</i>, <i>Candidatus</i> Neoehrlichia mikurensis, <i>Rickettsia helvetica</i>, <i>Rickettsia monacensis</i>, <i>Borrelia miyamotoi</i> and several <i>Babesia</i> species. Among 92 <i>Borrelia burgdorferi</i> s. l. positive ticks, 33% carried another pathogen from a different genus. In blood of sixteen out of 626 persons with tick bites or erythema migrans, DNA was detected from <i>Candidatus</i> Neoehrlichia mikurensis (n = 7), <i>Anaplasma phagocytophilum</i> (n = 5), <i>Babesia divergens</i> (n = 3), <i>Borrelia miyamotoi</i> (n = 1) and <i>Borrelia burgdorferi</i> s. l. (n = 1). None of these sixteen individuals reported any overt symptoms that would indicate a corresponding illness during the three-month follow-up period. No associations were found between the presence of pathogen DNA in blood and; self-reported symptoms, with pathogen DNA in the corresponding ticks (n = 8), reported tick attachment duration, tick engorgement, or antibiotic treatment at enrolment.</p><p>Conclusions</p><p>Based on molecular detection techniques, the probability of infection with a tick-borne pathogen other than Lyme spirochetes after a tick bite is roughly 2.4%, in the Netherlands. Similarly, among patients with erythema migrans, the probability of a co-infection with another tick-borne pathogen is approximately 2.7%. How often these infections cause disease symptoms or to what extend co-infections affect the course of Lyme borreliosis needs further investigations.</p></div

Detected DNA sequences in 314 ticks obtained from 293 participants.

<p>The results on <i>B</i>. <i>burgdorferi</i> s. l. have been published by Hofhuis et al. 2013 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005042#pntd.0005042.ref029" target="_blank">29</a>].</p

Phylogenetic tree of the sequences obtained from human blood samples.

<p>PCR and sequencing was performed on the real-time PCR-positive blood samples. Sequences were clustered with (reference) sequences from Genbank. The evolutionary distance values were determined by Kimura method, and the tree was constructed according to the neighbor-joining method. A.) <i>Anaplasma phagocytophilum</i>: Phylogenetic tree of partial heat shock protein gene <i>groEL</i> of <i>Anaplasma phagocytophilum</i> of the four, one sequences is slightly different by couple of mismatches. All four are part of zoonotic variant of <i>Anaplasma phagocytophilum</i>. B.) <i>Babesia</i> genospecies: Three of the tested blood samples for <i>Babesia</i> genospecies yielded a sequence for the ribosomal <i>18S rRNA</i> gene, and showed to be identical to <i>B</i>. <i>divergens</i> sequences. C.) <i>Candidatus</i> Neoehrlichia mikurensis: Five out of seven <i>Candidatus</i> Neoehrlichia mikurensis yielded a partial sequence of the citrate synthase gene <i>gltA</i>. D.) <i>Candidatus</i> Neoehrlichia mikurensis: All seven <i>Candidatus</i> Neoehrlichia mikurensis yielded a partial sequence of the heat shock protein gene <i>groEL</i>.</p

Characteristics of participants with DNA of tick-borne pathogens detected in blood.

<p>Characteristics of participants with DNA of tick-borne pathogens detected in blood.</p

Prevalence of DNA detection of tick-borne pathogens in blood of persons with tick bites or erythema migrans (EM), as determined by PCRs.

<p>Prevalence of DNA detection of tick-borne pathogens in blood of persons with tick bites or erythema migrans (EM), as determined by PCRs.</p

<i>H. aegyptium</i> ticks used in this study together with the PCR and cultivation results.

<p><i>H. aegyptium</i> ticks used in this study together with the PCR and cultivation results.</p

Oligonucleotide primer pairs used.

<p>(* primers used in sequence analysis)</p><p>Oligonucleotide primer pairs used.</p

Phylogenetic analysis based on <i>glpQ</i> (A) and <i>gyrB</i> (B) genes of the genus <i>Borrelia</i>.

<p>Phylogenetic analysis based on <i>glpQ</i> (A) and <i>gyrB</i> (B) genes of the genus <i>Borrelia</i>.</p