Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe
© Jahfari et al.; licensee BioMed Central Ltd. 2012
Received: 20 November 2011
Accepted: 19 April 2012
Published: 19 April 2012
Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis.
Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test.
The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis.
Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis- infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.
The most prevalent tick-borne infection of humans in the Northern hemisphere is Lyme  The same tick species transmitting the etiologic agents of Lyme disease also serve as the vector of pathogens causing tick-borne encephalitis and several forms of rickettsioses, anaplasmoses and ehrlichioses . Members of the family Anaplasmataceae are obligatory intracellular bacteria that reside within membrane-enclosed vacuoles. Human ehrlichiosis and anaplasmosis are two closely related diseases caused by various members of the genera Ehrlichia and Anaplasma. A major difference between these two members is their cellular tropism. Ehrlichia chaffeensis, the etiologic agent of human monocytotropic ehrlichiosis (HME), is an emerging zoonosis that causes clinical manifestations ranging from a mild febrile illness to a fulminant disease characterized by multi-organ system failure . Anaplasma phagocytophilum causes human granulocytotropic anaplasmosis (HGA), previously known as human granulocytotropic ehrlichiosis . Despite the presence of Anaplasma phagocytophilum in questing Ixodes ricinus ticks in the Netherlands , only one human case has been reported . Seropositivity against anaplasmosis was observed in risk groups, such as foresters and suspected Lyme disease patients, but not in control groups . Still, the incidence of these tick-borne diseases and the associated public health risks remain largely unknown.
A novel candidate species in the family of Anaplasmataceae, called Candidatus Neoehrlichia mikurensis (N. mikurensis), was first isolated from wild rats and was also found in I. ovatus in Japan . Neoehrlichia mikurensis can be distinguished from other genera based on sequence analysis of 16S rDNA, citrate synthase (gltA) and heat shock protein GroEL genes . This recently identified bacterium is detected in several tick species and rodents in different parts of the world under different names [7–11]. The N. mikurensis found in I. ricinus ticks in Italy has been referred to as Candidatus Ehrlichia walkerii and the Ehrlichia species isolated from a rat in China was called “Rattus strain” . Furthermore, a N. mikurensis has been described in I. persulcatus in Russia  and I. ovatus from China and Japan . In the US, an Ehrlichia-like organism, closely related to N. mikurensis, was previously detected in raccoons. This variant is called Candidatus Neoehrlichia lotoris. The Asian N. mikurensis isolates showed a 99% similarity based on the 16S rDNA to the Ehrlichia schotti. Ehrlichia schotti was first described in 1999 in I. ricinus in the Netherlands by Leo Schouls and was named after his technician . Later this species was reported in I. ricinus in Russia  and subsequently in Germany and Slovakia . These findings raised the question whether Ehrlichia schotti is the same as N. mikurensis.
Reported human cases of N. mikurensis infection (until October 2011)
Symptoms and clinical signs
Episodes of fever, nonproductive cough, left thoracic pain, vein thrombosis, hypochromic anemia, reduced numbers of leukocytes, decreased percentage of lymphocytes, increased proportion of monocytes and elevated levels of CRP, microbiological analysis were negative.
Headaches, fever, intracerebral and subarachnoid hemorrhage, aneurysm, elevated CRP, pulmonary infiltration, microbiological analyses were negative, elevated infection parameters. Patient died from septic multi-organ failure.
Chronic lymphocytic leukemia
Transitory ischemic attack, hemolytic anemia, fever, erysipelas-like rash, transitory weakness of the left side of face and arm, hemolytic anemia, thrombocytopenia, thrombosis, pulmonary infiltration, increased proportion of monocytes and elevated levels of CRP, blood and other cultures were negative
Malaise, fever, moderate dyspnea, elevated leukocytes/neutrophils, elevated CRP, microbiological analysis were negative
Female 55 yr
Mantle Cell Lymphoma
Spiking fever, myalgias, arthralgias, erthema nodosum, elevated CRP, blood-, urine culture and pharyngeal swabs were negative. Antinuclear-, antinucleolar antigens and rheumatoid factor screens were negative.
Liver transplantation and splenectomy
Spiking fever, extreme fatigue, joint pain, skin erythema, painful and stiffened subcutaneous veins, mild leukocytosis and elevated CPR, blood and urine cultures and pharyngeal swab were negative.
In this study we aim to investigate (i) whether Ehrlichia schotti is similar to the described N. mikurensis family, (ii) the distribution and prevalence of N. mikurensis in the Netherlands, Belgium and the UK, (iii) possible transmission routes of N. mikurensis in non-experimental settings and (vi) its putative mammalian hosts.
Collection, identification and DNA extraction of ticks
Questing I. ricinus from all stages and Dermacentor reticulatus adults were collected in 2009 and 2010 by flagging the vegetation at geographically different locations in the Netherlands and Belgium. Ticks collected in the UK and Vrouwenpolder (NL) have been described before . For global geographic location, see Additional file 1: Figure S1. Questing I. arboricola were collected from bird nests in two different areas in Belgium. Ixodes hexagonus feeding on hedgehogs were collected in a hedgehog-shelter in 2010. Ixodes ricinus feeding on red deer (Cervus elaphus), European mouflon (Ovis orientalis musimon), wild boar (Sus scrofa) and sheep (Ovis aries) were collected. All the collected ticks were immersed in 70% alcohol and stored at −20°C until the DNA extraction. Based on morphological criteria, tick species and stages were identified to species level, with stage and sex recorded . In doubtful cases, sequencing of tick mitochondrial 16S rDNA confirmed the tick-species . DNA from questing ticks was extracted by alkaline lysis . DNA from engorged ticks was extracted using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s manual (Qiagen, 2006, Hilden; Germany) following the manufacturer’s protocol for the purification of total DNA from ticks.
Preparation of DNA lysates from wild rodents
Longworth traps (Bolton Inc., UK), baited with hay, apple, carrot, oatmeal and mealworm were used to capture different species of rodents and insectivores at 7 different locations in the Netherlands between 2007 and 2010. Animals were anaesthetized with isoflurane and euthanized by cardiac puncture. Serum was collected and stored at – 20°C. Spleen, liver, kidney, brain and other organs were collected and frozen at −80°C. DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s manual (Qiagen, 2006, Hilden; Germany). All animals were handled in compliance with Dutch laws on animal handling and welfare (RIVM/DEC permits).
Polymerase chain reactions
Primers used for amplification and sequencing of gltA and GroEL genes of N. mikurensis , and the amplification of the Msp2 gene of A. phagocytophilum
Multiplex real-time PCR
Oligonucleotide primer and probe sequences were designed to be specific for the N. mikurensis GroEL gene using Visual OMP DNA (Software, Inc., Ann Arbor, USA). Primer sequences for the N. mikurensis GroEL gene were NMikGroEL-F2a and NMikGroEL-R2b and generated a 99-bp fragment which was detected with the NMikGroEL-P2a TaqMan probe (Table 2). Sequences were evaluated on the basis of the following criteria: predicted cross-reactivity with closely related organisms, internal primer binding properties for hairpin and primer-dimer potential, length of the desired amplicon, G-C content, and melting temperatures (T m s) of probes and primers. The specificity of the N. mikurensis GroEL primers for N. mikurensis in the multiplex real-time PCR assay was tested with DNA extracted from the following microorganisms: Rickettsia rickettsii Anaplasma phagocytophilum R. helvetica Bartonella henselae Ehrlichia canis B. afzelii B. garinii, B. sensu stricto, Babesia microti, Candidatus Midichloria mitochondrii and tick lysates containing Wolbachia species[22, 25] None were amplified. Random samples of tick lysates which were N. mikurensis-positive in the Q-PCR were routinely confirmed by conventional PCR using NMik fo-gltA and NMik re-gltA primers, followed by DNA sequencing.
Optimized conditions for multiplex PCR
PCR was performed in a multiplex format with a reaction volume of 20 μl, using the iQ Multiplex Powermix (Bio-Rad Laboratories, Hercules, USA), in the LightCycler 480 Real-Time PCR System (F. Hoffmann-La Roche, Basel, Switzerland). Final PCR reaction concentrations were 1x iQ Powermix, primers ApMSP2F and ApMSP2R at 250 nM each, probe ApMSP2P-FAM at 125 nM, primers NMikGroEL-F2a and NMikGroEL-R2b at 250 nM each, probe NMikGroEL-P2a-RED at 250 nM, and 3 μl of template DNA. Cycling conditions were: 95°C for 5 min, followed by 60 cycles of a 5 sec denaturation at 95°C followed by a 35 sec annealing-extension step at 60°C. Ticks lysates were considered positive if the Ct-value of a proper sigmoid curve was maximally three cycles more than the highest dilution of the positive control sample.
For each PCR and real-time multiplex PCR, positive, negative controls and blank samples were included. A 10-3 to 10-5 dilution of a mixture of sequencing-confirmed N. mikurensis-positive tick lysates were used as positive controls. In order to minimize contamination, the reagent setup, the extraction and sample addition, and the real-time PCR as well as sample analysis were performed in three separate rooms, of which the first two rooms were kept at positive pressure and had airlocks.
DNA sequencing and genetic analysis
PCR amplicons were sequenced using the described primers (Table 2) and the BigDye Terminator Cycle sequencing Ready Reaction kit (Perkin Elmer, Applied Biosystems). All sequences were confirmed by sequencing both strands. Sequences were compared with sequences in Genbank using BLAST after subtraction of the primer sequences (http://www.ncbi.nlm.nih.gov/genbank/). The collected sequences were assembled, edited, and analysed with BioNumerics version 6.5 (Applied Maths NV, Sint-Martens-Latem, Belgium). Resulting sequences were aligned with those from related organisms in Genbank. Phylogenetic analyses of the sequences and related organisms were conducted using the BioNumerics program using the neighbour-joining algorithm with Kimura's two-parameter model. Bootstrap proportions were calculated by the analysis of 1000 replicates for neighbour-joining trees. DNA sequences are available upon request.
Comparison of Ehrlichia schotti with N. mikurensis
Members of the N. mikurensis group are distinguished from other genera based on sequence analysis of 16S rDNA, citrate synthase (gltA) and heat shock protein GroEL genes
Ehrlichia-like ‘schotti variant’
Ehrlichia-like ‘schotti variant’
Ehrlichia-like ‘schotti variant’
C. Ehrlichia walkerii
C. Ehrlichia walkerii
Ehrlichia-like ‘Rattus variant’
C. N. mikurensis
(TK4456 and IS58)
A. speciosus and Eothenomys.smithii
C. N. mikurensis
(FIN686 and Nagano21)
Ehrlichia-like ‘schotti variant’
C. N. mikurensis
C. N. mikurensis
C. N. lotoris
C. N. mikurensis
C. N. mikurensis
C. N. mikurensis
C. N. mikurensis
C. N. mikurensis
Prevalence and distribution of N. mikurensis
The prevalence and distribution of N. mikurensis in questing I. ricinus in the Netherlands and Belgium
Duin en Kruidberg (2009)
Duin en Kruidberg (2010)
Kop van Schouwen
Pyramide van Austerlitz
Rijk van Nijmegen
Brussel-area, (Sonian forest), (Belgium)
Total of all ticks
Average of all areas
Role of ticks in the transmission of N. mikurensis
Prevalence of N. mikurensis in questing I. ricinus , divided by lifecycle stage
D. reticularis, I. hexagonus and I. arboricola tested in the multiplex real-time PCR for the N. mikurensis.
Potential reservoir hosts of N. mikurensis
Spleens of wild rodent and insectivore species were tested by PCR and sequencing using N. mikurensis specific primers
I. ricinus adults feeding on animals living in nature reserve areas in the Netherlands were tested by multiplex real-time PCR for the presence of N. mikurensis
Prevalence in ticks (%)
positive ticks (n)
Ovis orientalis musimon
Recently, six human and one canine case of N. mikurensis infection were reported in different locations in Europe. These reports advocate a re-assessment of the occurrence of this microorganism in questing ticks. Schouls and colleagues described an Ehrlichia-like organism (Ehrlichia schotti) in Dutch ticks . In our study, the three genetic markers 16S rDNA, gltA and GroEL of Ehrlichia schotti-positive field isolates turned out to be similar and identical to DNA sequences available from N. mikurensis. Thus, Ehrlichia schotti and N. mikurensis are most likely one and the same species. Previous findings on E. schotti can be interpreted as findings on N. mikurensis. Thus, N. mikurensis has already been present in the Netherlands in 1999 [25, 41]. Furthermore, 11% of 289 engorged I. ricinus removed from humans were N. mikurensis- positive, indicating that the Dutch population is being exposed to ticks infected with N. mikurensis. Remarkably, human and animal cases of N. mikurensis infection in the Netherlands have not yet been described.
The development of a Q-PCR specific for N. mikurensis allowed us to test significant numbers of ticks without having to perform the labour-intensive Reverse-Line blotting. These analyses showed that the N. mikurensis is present in vegetation ticks throughout the Netherlands and Belgium. No N. mikurensis-positive ticks were found in one location in Belgium. One possible explanation is that this location in the Brussels-area is exceptional due to its reduced fauna and flora caused by human interference. This forest in the Brussels-area is also highly fragmented because of a railroad and several major motorways that run through the forest. Several parts of it can be ecologically considered ‘islands’, which could -through isolation of mammal and tick populations- explain the absence of the pathogen in this forest. More ticks of this unique area need to be tested in order to address this hypothesis. Neoehrlichia mikurensis was also not detected in ticks from the UK. This could indicate that these species have not (yet) been established on this island.
The overall prevalence of N. mikurensis in questing nymphs and adults is approximately 7%. From the public health point of view, it indicates that a significant proportion of people contracting a tick bite are exposed to N. mikurensis. Transmission of the N. mikurensis in ticks appears to occur horizontally rather than vertically. None of the tested larvae were found positive, even though the prevalence of nymphs is approximately 7% and 11% for adults. Other tick species, with more restricted host preference than I. ricinus, were also tested for the presence of N. mikurensis. Dermacentor reticulatus, I. hexagonus and questing I. arboricola were found negative. The data indicate that these tick species probably play insignificant roles in the transmission of N. mikurensis. In contrast, I. ricinus can be considered as its main vector in the Netherlands and Belgium.
A potential group of reservoir hosts for N. mikurensis are wild rodents. Indeed, spleen samples and other organs (kidney, liver and brain) of some rodent species turned out to be N. mikurensis-positive, which indicates a systemic infection of these rodents with N. mikurensis. The N. mikurensis isolates from ticks and wild rodents (Table 7) were genetically identical, indicating that rodents are potential reservoir hosts . However, the reservoir potential of rodents can only be by xenodiagnosis or experimental infection. The prevalence of N. mikurensis in I. ricinus ticks feeding on red deer, European mouflon, wild boar and sheep were comparable to the prevalence in questing ticks. From these prevalence data, it was not possible to infer the role of these animals in the transmission of N. mikurensis. However, it is clear that these animals are being exposed to the N. mikurensis through tick bites. Further experiments are necessary to determine whether there are other mammalian reservoirs than wild rodents.
Although human infection of the N. mikurensis has not been reported in the Netherlands, it is unclear to what extent N. mikurensis poses risks to public health. The symptoms described in all of the N. mikurensis infection cases were generally non-specific and usually seen in any other ordinary inflammatory reaction. What’s more, most of the Ehrlichia infections are known to be either asymptomatic or mild, self-limiting diseases . In other words, infection can occur without causing disease. So far, diagnosis has relied only on PCR amplification of the N. mikurensis. The lack of serological tests makes diagnosis particularly difficult. Against these backdrops, the actual incidence of human infection with Ehrlichia is likely to be much higher than currently reported in Europe. Thorough surveillance and improvement of diagnostic tools will probably increase the number of identified human cases, and consequently provide more insight in the public health relevance of N. mikurensis.
The authors are very grateful to the volunteers and co-workers of the Central Veterinary Institute of Wageningen University and Research Centre, particularly Cor Gaasenbeek Fred Borgsteede and Kitty Maassen who have dedicated much time and effort to monthly collections of ticks. We thank Francine Pacillij, Annette Benning and Frans Jacobs for having collected ticks on large herbivores. We are grateful to Ellen Tijsse-Klasen, Joke van der Giessen, Marieke Opsteegh and Ankje de Vries for collecting rodent samples, support and technical assistance. This study was financially supported by the Dutch Food and Consumer Product Safety Authority (VWA), Wageningen University and Research Centre and by the Ministry of Health, Welfare and Sport (VWS). Dieter Heylen was supported by a FWO postdoctoral fellowship.
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