Borrelia miyamotoi and Candidatus Neoehrlichia mikurensis in Ixodes ricinus Ticks, Romania

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To the Editor: Ixodes spp. ticks are vectors for human and animal pathogens. Ix. ricinus ticks are widely distributed, frequently reported to feed on humans, and the main vector for a large variety of tickborne pathogens ( 1 ). The effect of ticks and tickborne diseases on public health, animal health and welfare, and animal production appears to be an increasing global problem, which will lead to considerable economic costs ( 2 ). Borrelia miyamotoi is a spirochete that belongs to the relapsing fever group and causes symptoms similar to those of other relapsing fever group pathogens and Lyme borreliosis, including erythema migrans−like skin lesions ( 3 ). The geographic distribution of B. miyamotoi is sporadic; it has been detected in Ixodes spp. ticks in many countries in Europe and in North America and Asia. In Russia, the United States, and recently in the Netherlands, B. miyamotoi was detected in humans and confirmed to cause disease ( 4 , 5 ). In Romania, pathogens that cause Lyme borreliosis and reptile-associated borreliae were identified in different tick populations ( 6 , 7 ). However, no information is available on the presence of relapsing fever group borreliae in this country. Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum are obligate, intracellular, tickborne pathogens of the family Anaplasmataceae; both are emerging zoonotic agents. Candidatus N. mikurensis causes monocytotropic ehrlichiosis in canids and humans and granulocytic anaplasmosis in humans and domestic animals ( 8 ). These 2 pathogens are found throughout Europe in Ix. ricinus ticks ( 8 ). A. phagocytophilum has been reported in questing Ix. ricinus ticks, dogs, wild boars, hedgehogs, and tortoises in Romania ( 9 ). Recently, Candidatus N. mikurensis was detected in an Ix. ricinus tick that had bitten a human in Romania ( 10 ). This recently discovered tickborne agent was shown to be a risk for disease in humans and has been detected in questing Ix. ricinus ticks throughout Europe and in animal tissue samples and human patients ( 8 ). Relapsing fever spirochetes and potential public health risks associated with tickborne pathogens are a serious medical problem. Thus, we assessed the presence of B. miyamotoi, A. phagocytophilum, and Candidatus N. mikurensis in questing Ix. ricinus ticks in Romania. Questing Ix. ricinus ticks were available from previous studies conducted by our research group. A random sampling approach was used as described ( 7 ). To detect potentially pathogenic bacteria, 468 questing Ix. ricinus ticks were collected from 4 regions from Romania, randomly selected, and analyzed. Detection of pathogens was performed by using multiplex quantitative PCRs (qPCRs) specific for the flaB and ospA genes of B. miyamotoi, the msp2 gene of A. phagocytophilum, and the groEL gene of Candidatus N. mikurensis. We used IQ Multiplex Powermix (Bio-Rad, Carlsbad, CA, USA) and a final reaction volume of 20 μL ( 8 ). For detection of A. phagocytophilum and Candidatus N. mikurensis, we also performed multiplex qPCR as described ( 8 ). For detection of B. miyamotoi, a specific region of the flab gene was targeted by using multiplex qPCR according to a previous described protocol ( 1 ). For quality control of qPCRs, we included positive and negative controls. Sequences of qPCR products were analyzed and compared with sequences available in GenBank. B. miyamotoi was detected in 7 ticks: 2 (1.59%) of 126 males, 2 (0.68%) of 296 females, and 3 (6.52%) of 46 nymphs. A. phagocytophilum was detected in 16 ticks: 1 (0.79%) of 126 males, 11 (3.72%) of 296 females, and 4 (8.70%) of 46 nymphs. Candidatus N. mikurensis was detected in 25 ticks: 5 (3.97%) of 126 males, 18 (6.08%) of 296 females, and 2 (4.35%) of 46 nymphs. Overall prevalences were 1.50% for B. miyamotoi, 3.42% for A. phagocytophilum, and 5.34% for Candidatus N. mikurensis. Prevalences of each pathogen in specific varied by locality (Table). No co-infections were detected. Table Prevalence of 3 bacterial species in questing Ixodes ricinus in 14 localities, Romania Locality (county) No. ticks tested (nymphs, males, females) No. (%) ticks positive, by bacterial species Borrelia miyamotoi Anaplasma phagocytophilum Candidatus Neoehrlichia mikurensis Cugir (Alba) 19 (8, 4, 7) 0 2 (10.53) 1 (5.26) Vladimirescu (Arad) 17 (0, 5, 12) 0 2 (11.76) 0 Bicaci (Bihor) 23 (12, 5, 6) 0 4 (17.4) 0 Bistrița (Bistriţa-Năsăud) 30 (0, 10, 20) 0 0 0 Poiana Mărului (Brașov) 66 (0,10, 56) 2 (3.03) 0 0 Vultureni (Cluj) 44 (3, 10, 31) 1 (2.27) 3 (6.82) 2 (4.55) Micești (Cluj) 62 (0, 15, 47) 1 (1.62) 0 2 (3.23) Reșița (Caraș-Severin) 21 (0, 10, 11) 0 0 0 Corund (Harghita) 59 (7, 17, 37) 0 1 (1.7) 4 (6.78) Bistra (Maramureș) 26 (1, 10, 15) 0 0 0 Icland (Mureș) 37 (6, 5, 26) 2 (5.41) 4 (10.81) 8 (21.62) Mediaș (Sibiu) 12 (2, 4, 6) 1 (8.33) 0 2 (16.67) Rătești (Satu Mare) 22 (7, 10, 15) 0 0 1 (4.55) Lugoj (Timiș) 30 (0, 11, 19) 0 0 5 (16.67) Total 468 (46, 126, 296) 7 (1.5) 16 (3.42) 25 (5.34) We analyzed flab, msp2, and groEL gene sequences obtained by qPCR. These sequences showed 99%–100% identities with gene sequences of B. miyamotoi (GenBank accession no. KJ847050), A. phagocytophilum (accession no. KP164415), and Candidatus N. mikurensis (accession no. FJ966365). In Romania, the density of Ix. ricinus ticks is high and their host diversity is extensive ( 7 ). However, data for effects of tickborne pathogens on public health are scarce in this country. In this study, we detected B. miyamotoi, A. phagocytophilum, and Candidatus N. mikurensis in questing Ix. ricinus ticks in Romania, which confirms the emerging trend of these pathogens in Europe. Because of the scarcity of information on human infections with these pathogens in Romania, serologic and molecular investigations and their implementation are needed for diagnosis, which might help in assessing the effect of these pathogens on public health.

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Humans Infected with Relapsing Fever Spirochete Borrelia miyamotoi, Russia

Alexander E. Platonov, Ludmila S. Karan, Nadezhda M. Kolyasnikova … (2011)

Borrelia miyamotoi, discovered in Japan in 1995, belongs to the relapsing fever group of Borrelia ( 1 ). Relapsing fever borreliae infections are characterized by influenza-like illness and >1 relapse episode of bacteremia and fever. B. miyamotoi is more distantly related to B. burgdorferi, a group of spirochetes that includes B. burgdorferi s.l. strains (B. afzelii; B. garinii; and B. burgdorferi s.s., the causative agent of Lyme disease) ( 2 , 3 ). In Eurasia and North America, B. miyamotoi is found in a small percentage of all species of ixodid tick vectors of B. burgdorferi, including Ixodes persulcatus ( 1 , 3 , 4 ), I. ricinus ( 5 – 7 ), I. scapularis ( 2 , 3 , 8 , 9 ), and I. pacificus ( 10 ). It is transmitted transovarially and transstadially by ticks and coexists with B. burgdorferi ( 2 , 3 ). Recently, we discovered B. miyamotoi in I. persulcatus and I. ricinus ticks in the European and Asian regions of Russia. In these areas, human ixodid tick-borne infections, including those caused by B. afzelii, B. garinii, and viral tick-borne encephalitis virus (TBEV; genus Flavivirus) are endemic and transmitted by the same tick species. Despite the presence of B. miyamotoi in vector ticks, to our knowledge, human disease caused by this spirochete has not been definitively established. We previously noted presumptive B. miyamotoi infection in residents of central Russia with influenza-like illness but were uncertain whether their clinical manifestations were caused by co-infecting B. burgdorferi s.l. species ( 11 – 13 ). To confirm those findings and develop initial estimates of the prevalence and severity of B. miyamotoi infection, we conducted a comparative cohort study. We used improved antibody assays and PCRs to compare the relative frequency and clinical manifestations of B. miyamotoi infection with those of B. garinii infection in Russia and B. burgdorferi infection in the United States. Methods Study Design We enrolled patients admitted to Municipal Clinical Hospital No. 33 in Yekaterinburg City, Russia, from May 19 through August 25, 2009, for suspected tick-borne infection. Yekaterinburg is in the Asian part of Russia, ≈1,200 miles east of Moscow. Viral tick-borne encephalitis and acute borreliosis are highly endemic to this region. Patients with moderate or severe disease are usually hospitalized. We compared the clinical characteristics of patients experiencing laboratory-confirmed B. miyamotoi infection with those of patients experiencing B. garinii infections from the same area and with those of patients who experienced B. burgdorferi infection in the northeastern United States. The US data came from a tick-borne diseases study conducted during 1991–2008 ( 14 , 15 ). For each patient at all study sites, we recorded the presence or absence of a standard set of 11 clinical manifestations. All patients signed an informed consent form in accordance with the institutional review boards of the Municipal Clinical Hospital in Yekaterinburg City or the University of Connecticut School of Medicine. We also determined the frequency of B. garinii, B. afzelii, B. burgdorferi, and B. miyamotoi in I. persulcatus and I. ricinus ticks in Yekaterinburg and several additional regions of Russia (Figure 1). Ticks were collected by drag cloth, visually identified to species level, and analyzed by PCR to identify specific Borrelia species. Figure 1 Percentage of Ixodes persulcatus (I. p.) and I. ricinus (I. r.) ticks infected with Borrelia miyamotoi in Russia. The number of ticks that were tested is given in parenthesis. Star indicates study location of human B. miyamotoi infection. Case Definitions Diagnosis of B. miyamotoi infection required the report of a tick bite, presence of clinical manifestations consistent with borreliosis, and laboratory evidence of B. miyamotoi infection. Clinical manifestations included fever, headache, chills, fatigue, vomiting, and myalgia. Confirmation of active infection consisted of amplification of B. miyamotoi DNA/RNA in blood by species-specific PCR and detection of anti-borreliae immunoglobulin (Ig) M in acute- and/or convalescent-phase serum samples. In Russia, diagnosis of B. garinii infection required report of a tick bite, physician diagnosis of erythema migrans (EM; an expanding, ring-like erythematous rash >5 cm in diameter), or an influenza-like illness. Confirmation of infection consisted of amplification of B. garinii DNA/RNA in blood by specific PCR, followed by direct sequencing of 5S-23S ribosomal RNA (rRNA) intergenic spacers, and detection of anti-borreliae IgM in acute- and/or convalescent-phase serum samples. In the United States, diagnosis of B. burgdorferi infection required a physician’s diagnosis of EM or an influenza-like illness. For all cases, confirmation of infection consisted of a >4-fold increase in anti–B. burgdorferi antibody in acute- and convalescent-phase serum samples. The diagnosis of TBEV infection was based on a viral-like illness, including headache (with or without meningitis or encephalitis), amplification of TBEV RNA in blood by species-specific PCR, and/or detection of anti-TBEV IgM in an acute-phase serum sample. Laboratory Assays PCR The PCR we used enabled detection of DNA and RNA sequences. DNA/RNA was extracted from 2 mL of whole venous blood with EDTA or from tick suspensions by using an AmpliSens Riboprep Kit (Central Institute of Epidemiology, Moscow, Russia) according to the manufacturer’s instructions. Of the blood samples used for PCR, 81% were obtained at the time of hospital admission and 96% within 2 days of admission. To assay the inhibitory effect of blood and tick extracts on the PCR, all samples were spiked with a universal RNA recombinant control having a known number of RNA copies per milliliter. Reverse transcription of RNA to cDNA was performed by using an Amplisens Reverta-L Kit (Central Institute of Epidemiology). The cDNA samples were assayed for B. miyamotoi and other tick-borne pathogens by using real-time quantitative PCR (qPCR) assays in a Rotor Gene 6000 cycler (Corbett Life Science, Concorde, New South Wales, Australia). The cDNA samples were divided into 2 aliquots, and different types of real-time qPCR were performed on each. The first used in-house primers and a probe that targeted the 16S rRNA gene of B. miyamotoi. The inclusion of the reverse transcription procedure improved the detection sensitivity because the 16S rRNA that also became detectable is present in higher copy numbers than the 16S rRNA gene. The detection limit of at least 5 × 103 copies/mL was determined by using positive recombinant DNA of the B. miyamotoi 16S rRNA gene fragment with a known number of copies. The B. miyamotoi–specific forward and reverse primers at 360 nmol/L were, respectively, Brm1 5′-CGCTGTAAACGATGCACACTTGGTGTTAATC-3′ and Brm2 5′-CGGCAGTCTCGTCTGAGTCCCCATCT-3′. The corresponding dye-labeled probe (final concentration 100 nmol/L) was R6G-CCTGGGGAGTATGTTCGCAAGAATGAAACTC-BQH1. The PCR conditions were 95°C for 15 min; followed by 10 cycles at 95°C for 20 s, 67°C for 50 s, and 72°C for 20 s; then by 40 cycles at 95°C for 20 s, 60°C for 50 s, and 72°C for 20 s. The fluorescence signal was recorded at the 60°C step for the last 40 cycles. Each run included negative controls and positive recombinant control DNA of the B. miyamotoi 16S rRNA gene fragment as a standard. PCR-based detection of B. burgdorferi s.l., Anaplasma phagocytophilum, Ehrlichia chaffeensis, Ehrlichia muris, and TBEV was performed on the second cDNA aliquot by using a commercial multiplex PCR (AmpliSens TBEV, B. burgdorferi s.l., A. phagocytophillum, E. chaffeensis/E. muris-FL; Central Institute of Epidemiology) ( 16 ), according to the manufacturer’s instructions. This assay was designed to detect, but not discriminate between, B. afzelii, B. burgdorferi s.s., and B. garinii. The same assays were used to detect specific DNA/RNA in ticks and humans. The specificity of B. miyamotoi and B. burgdorferi s.l. assays was confirmed by direct sequencing of flagellin gene fragments and/or 16S rRNA gene fragments and/or 5S-23S rRNA intergenic spacer amplified from blood samples of the same patients or from the same ticks (GenBank accession nos. GU797331–GU797350, JF951378–JF951392). Of the 97 borreliae sequenced, results of DNA amplification using species-specific PCR were entirely consistent with the sequencing results. Absence of false-positive PCR results means that our PCRs were highly specific. Amplification and further direct sequencing of the B. miyamotoi flagellin gene were performed by using degenerate primers FLA120F 5′-AGAATTAATMGHGCWTCTGATGATG-3′ and FLA920R 5′-TGCYACAAYHTCATCTGTCATT-3′ ( 2 , 5 ). The 16S rRNA gene fragment was amplified and sequenced by using 2 primers pairs: first Bf1 5′-GCTGGCAGTGCGTCTTAAGC-3′ and Brsp2 5′-CCTTACACCAGGAATTCTAACTTCCYCTAT-3′, second Brsp1 5′-GGGGTAAGAGCCTACCAAGGCTATGATAA-3′ and Br1 5′-GCTTCGGGTACTCTCAACTC-3′ ( 5 ). Borrelial 5S-23S rRNA intergenic spacer was amplified and sequenced by using nested PCR with outer primers pairs IGSa 5′-CGACCTTCTTCGCCTTAAAGC-3′ and IGSb 5′-AGCTCTTATTCGCTGATGGTA-3′ and inner primers pair IGSe 5′-CCTTAAAGCTCCTAGGCATTCACCA-3′ and IGSd 5′-CGCGGGAGAGTARGTTATTGCGA-3′ ( 17 ). Nucleotide sequences were aligned, compared, and analyzed by using MEGA4.1 (www.megasoftware.net), ClustalW (www.clustal.org), and BLAST (www.ncbi.nlm.nih.gov/blast/Blast.cgi). ELISA Serum samples collected at the time of admission and 1–2 weeks later were tested for anti-borrelial IgM and IgG. Serologic evidence of exposure to borreliae was detected by ELISA EUROIMMUN EI 2132–9601 M and EI 2132–9601–2 G (EUROIMMUN AG, Lübeck, Germany). The ELISA consisted of a mixture of whole antigens from B. afzelii, B. burgdorferi, and B. garinii and thus could detect but not discriminate specific antibody against any of these species. Seroconversion in patients infected with the relapsing fever borrelia B. persica also has been detected by EUROIMMUN assay ( 18 ). Serum from most B. miyamotoi–positive patients reacted to the antigen(s) in this assay. Anti-TBE IgM was detected by the semiquantitative EUROIMMUN ELISA EI 2661–9601 M. Statistical Analyses Comparisons were performed by using the Mann-Whitney U test (independent numeric interval variables), χ2 test (categorical variables), and corresponding exact tests, if necessary; p 0.99 Multiple EM 0 14 7 0.03 0.18 0.36 Fever† 98 67 32 0.001 0.99 0.99 >0.99 >0.99 Neck stiffness 2 0 38 >0.99 37.2°C for patients in Russia and maximum oral temperature >37.7°C for patients in the United States. Although mean peripheral leukocyte and platelet counts were lower for patients with B. miyamotoi than B. garinii infection, they were within the reference range. Proteinuria and transient elevation of serum alanine aminotransferase and aspartate aminotransferase concentrations were found for 3× more B. miyamotoi patients than B. garinii patients (51% and 68% vs. 15% and 20%, respectively, p 1,000 B. miyamotoi cases might occur in Russia each year. More studies are necessary to determine if this projection is accurate. Acute B. miyamotoi infection was more severe than early stage B. burgdorferi infection. The time from symptom onset to hospital admission was shorter, and the number of clinical manifestations was greater for patients with B. miyamotoi infection than with B. garinii infection. Relapsing febrile episodes were only reported for B. miyamotoi patients. Such multiple disease episodes not only have an adverse effect on a patient’s health but also may result in costly medical bills, many days or weeks of lost wages, and medical misdiagnosis ( 19 – 22 ). Co-infection of B. miyamotoi with other ixodid tick–transmitted agents may increase disease severity ( 15 , 23 ). Additional problems that might occur with B. miyamotoi infection are ocular, neurologic, respiratory, cardiac, and pregnancy complications associated with relapsing fever ( 19 – 22 ). Our study had several limitations. Attempts to detect B. miyamotoi on blood smear or in culture were not successful, although we confirmed B. miyamotoi infection with a combination of qPCR, genetic sequencing, clinical, and seroconversion evidence. The comparison of clinical manifestations of Borrelia spp. infection of patients from Russia and the United States was complicated by enrollment at different times and from different locations, although we assessed the same 11 clinical manifestations at each location. The possibility that the clinical description of our B. miyamotoi cases was compromised by unrecognized co-infection with B. burgdorferi s.l. is unlikely. The expected number of cases of co-infection depends on the prevalence of the pathogens in ticks in the region ( 3 , 11 , 24 ), and this number is even fewer than the 4 B. miyamotoi patients with EM we found. Inclusion or exclusion of these 4 cases had no effect on our comparative analysis with patients who did not have B. miyamotoi infection. We limited our description of B. garinii cases to those that were confirmed by detection of amplifiable B. garinii DNA/RNA, although such cases may be more severe than those in which such DNA/RNA cannot be detected ( 25 , 26 ). Patients with B. burgdorferi s.l. PCR–negative results experienced fewer symptoms and milder fever than did patients with B. burgdorferi s.l. PCR–positive results. Our analysis of patients with B. miyamotoi and B. garinii infection was limited to those who were hospitalized, although hospital admission policy in these regions of Russia is liberal because of concern about TBE and problems associated with B. burgdorferi infection. The geographic dispersion and extent of B. miyamotoi disease in humans are unclear, but the infection probably occurs outside of Russia, given the comparative infection rates of vector ticks in Russia and at several locations in Europe and the United States ( 2 – 8 ). In the northeastern United States, ≈15% of all spirochetes carried by I. scapularis ticks are B. miyamotoi ( 2 ). Cases may remain undiagnosed because of the nonspecific nature of the illness, which might be confused with viral infections or such tick-borne infections as Lyme disease, babesiosis, anaplasmosis, or ehrlichiosis, and because of the lack of laboratory tests for confirmatory diagnosis ( 19 – 22 ). B. miyamotoi infection may have negative health consequences, including relapsing disease that may last for months and may not respond to inappropriate antimicrobial drug therapy. The discovery of a Borrelia sp. that is pathogenic in humans and transmitted by an array of ixodid ticks greatly expands the potential geographic distribution of this disease ( 1 – 11 ). Further investigation of possible B. miyamotoi infection in humans is warranted wherever I. pacificus, I. persulcatus, I. ricinus, and I. scapularis ticks are found.

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Prevalence of Borrelia miyamotoi in Ixodes Ticks in Europe and the United States

Chris D Crowder, Heather E. Carolan, Megan A. Rounds … (2014)

Ixodes ticks can transmit a variety of pathogens, including viruses, bacteria, and protozoa ( 1 ). Borrelia spirochetes are one of the genera of bacteria transmitted by Ixodes ticks. Most Borrelia that infect ticks belong to the Borrelia burgdorferi senso lato group and include B. burgdorferi senso stricto, B. garinii, and B. afzelii, all of which cause Lyme disease in humans ( 1 ). Borrelia miyamotoi has been found in a variety of Ixodes ticks and is more closely related to the relapsing fever spirochetes that infect soft ticks than to the bacteria that cause Lyme disease ( 2 ). B. miyamotoi found in Europe and the United States also cause disease in humans ( 3 – 5 ). A study in Russia has shown that the spirochete B. miyamotoi has the ability to infect humans; infections with B. miyamotoi cause symptoms similar to those seen with relapsing fever, as well as erythema migrans-like skin lesions on rare occasions ( 6 ). B. miyamotoi has been found in ticks of the following species: Ixodes scapularis and I. pacificus in the United States, I. persulcatus in Japan, and I. ricinus and I. persulcatus in Europe and Asia ( 2 , 7 – 11 ). In North America, B. miyamotoi has been found as far north as the Canadian provinces of Ontario and Nova Scotia ( 12 ). In the United States, the geographic range of B. miyamotoi is from the Northeast to California and has been reported as far south as Tennessee ( 7 , 8 , 13 – 15 ). Previous studies have shown that B. miyamotoi can be placed into different genetic groups based upon its geographic location and has some variation within the genographic groups ( 6 , 9 ). To examine the prevalence distribution and diversity of B. miyamotoi in Ixodes ticks, we screened individual ticks by PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to detect tickborne pathogens, including B. miyamotoi ( 16 ). This approach has been used to characterize tickborne microorganisms, including Ehrlichia and Borrelia, from clinical specimens, heartworms in canine blood, and naturally occurring tick endosymbionts ( 16 – 19 ). Ticks that tested positive for B. miyamotoi were further characterized by using a Borrelia genotyping assay to assess genetic diversity ( 20 ). Materials and Methods B. miyamotoi Culture Isolate The B. miyamotoi strain Fr74B was obtained by the Centers for Disease Control and Prevention (Fort Collins, CO, USA), as a culture isolate. This strain was originally isolated from an infected Apodemus argenteus field mouse from Japan. The DNA from this strain was isolated by diluting the culture 1:10 with phosphate-buffered saline and heating to 95°C for 10 min. The raw lysate was then used in the Borrelia PCR/ESI-MS genotyping assay (Abbott Laboratories, Des Plaines, IL, USA) at 1 μL per PCR well ( 20 ). Ixodes Tick Collection and Extractions Ticks were obtained from most locations by flagging during 2008–2012. In Germany, a subset of ticks were also obtained after they were removed from persons. The species of Ixodes tick was determined by an entomologist and confirmed by the detection of the species-specific endosymbionts ( 19 ). The numbers and locations of the collection sites are described in Table 1. Table 1 Prevelance of Borrelia miyamotoi in Ixodes ticks, Europe and the United States, 2008–2012* Region/subregion Species Total no. ticks tested (nymphs; adults) No. ticks positive for B. miyamotoi (% of total) Czech Republic Zavadilka I. ricinus 153 (153; 0) 4 (2.6) Blatna I. ricinus 100 (100; 0) 2 (2.0) Dacice I. ricinus 93 (93; 0) 3 (3.2) Netolice I. ricinus 89 (89; 0) 0 (0) Germany Constance I. ricinus 226 (0; 48)* 4 (1.8) United States Connecticut Fairfield County I. scapularis 322 (309; 13) 16 (5.0) Litchfield County I. scapularis 18 (18; 0) 0 New London County I. scapularis 29 (29; 0) 0 New York Dutchess County I. scapularis 357 (357; 0) 2 (0.56) Suffolk County I. scapularis 180 (24; 156) 2 (1.1) Westchester County I. scapularis 44 (0; 44) 3 (6.8) Pennsylvania Chester County I. scapularis 80 (79; 1) 2 (2.5) Indiana Pulaski County I. scapularis 81 (0; 81) 10 (12.3) California Alameda County I. pacificus 22 (0; 22) 1 (4.5) Del Norte County I. pacificus 33 (0; 33) 0 Glenn County I. pacificus 44 (0; 44) 0 Humbolt County I. pacificus 74 (0; 74) 0 Lake County I. pacificus 129 (0; 129) 0 Marin County I. pacificus 85 (0; 85) 1 (1.2) Mendocino County I. pacificus 57 (0; 57) 2 (3.5) Napa County I. pacificus 65 (0; 65) 10 (15.4) Orange County I. pacificus 15 (0; 15) 0 Placer County I. pacificus 250 (0; 250) 4 (1.6) San Bernardino County I. pacificus 18 (0; 18) 0 Santa Cruz County I. pacificus 64 (0; 64) 0 Sonoma County I. pacificus 126 (126; 0) 2 (1.6) *A total of 119 ticks were removed from humans, and the life stage of 178 of the 226 ticks tested was not recorded. Nucleic acids were extracted from ticks according to a published protocol by using bead-beating homogenization followed by isolation of RNA and DNA with DNeasy Blood and Tissue Kit columns (QIAGEN, Valencia, CA, USA) instead of the published QiaAmp Virus Elute Kits ( 21 ). A negative control consisting of a lysis buffer without a tick was with each set of extractions. Ticks from the United States were processed at Ibis Biosciences (Carlsbad, CA, USA). Ticks collected from the European countries were isolated at their respective sources. Nucleic acid samples from Germany and the Czech Republic were shipped to Ibis at ambient temperatures; those from Czech Republic were shipped after being stabilized by RNAstable (Biomatrica, San Diego, CA, USA) per the manufacturer’s instructions. Molecular Detection and Genotyping of B. miyamotoi from Nucleic Acid Extracts B. miyamotoi was detected and identified by using a previously described broad-range PCR/ESI-MS assay designed to detect tickborne pathogens ( 16 ). For each set of samples analyzed with the assay, an extraction negative control sample as well as a PCR plate negative-control sample of water was included. A PCR-positive control was already built into the plate for each well in the form of a calibrant ( 20 ). Amplicons were analyzed by using a research use only PLEX-ID system (Abbott Laboratories). Samples positive for B. miyamotoi were further characterized by using a Borrelia PCR/ESI-MS genotyping assay as described that is designed to differentiate between Borrelia species and genotypes ( 20 ). PCR/ESI-MS assay provides genetic information about the PCR amplicon in the form of A, G, C, and T basecounts, and B. miyamotoi detection was defined as positive when one or more primer pairs produced an amplicon basecount signature that was unique to B. miyamotoi. Although most researchers agree that the nymphal stage of Ixodes ticks is the most epidemiologically essential life stage for transmission of B. burgdorferi sensu lato, because little is known about the transmission of B. miyamotoi from Ixodes ticks to humans, the data for both nymphs and adults were combined. Sequence Confirmation of B. miyamotoi Detections Representative samples positive for B. miyamotoi were selected for 16S Sanger sequencing. Primers were designed to amplify a 676-bp region of the 16S rRNA gene for Borrelia. A M13 tag was added to each primer for sequencing. The M13 forward sequence tag was 5′-CCC AGT CAC GAC GTT GTA AAA CG-3′, and the reverse tag was 5′-AGC GGA TAA CAA TTT CAC ACA GG-3′. The forward primer used was 5′-M13-CGC TGG CAG TGC GTC TTA AG-3′, and the reverse primer was 5′-M13-GCG TCA GTC TTG ACC CAG AAG TTC-3′. The amplification of the 16S rRNA genes was performed in a 50 μL reaction containing 1 μL nucleic acid extract, 1 unit of Platinum Taq High Fidelity polymerase (Invitrogen, Carlsbad, CA, USA) or Immolase Taq (Bioline, Randolph, MA, USA), the manufacturer’s PCR buffer, 2.0 mmol/L MgSO4, 200 μmol/L dATP, 200 μmol/L dCTP, 200 μmol/L dTTP, 200 μmol/L dGTP (Bioline), and 250 nmol/L of each primer. The following PCR cycling conditions were used on an MJ Dyad 96-well thermocycler (Bio-Rad Inc., Hercules, CA, USA): 95°C for 2 min, followed by 8 cycles of 95°C for 15 s, 50°C for 45 s, and 68°C for 90 s, with the 50°C annealing temperature increasing 0.6°C for each cycle. PCR was continued for 37 additional cycles of 95°C for 15 s, 60°C for 15 s, and 68°C for 60 s. The PCR cycle ended with a final extension of 4 min at 72°C. Reactions were visualized by electrophoresis on 1% agarose gels to ensure the presence of appropriately-sized products before being sent to SeqWright (Houston, TX, USA) for purification and sequencing with M13 primers. Resulting sequences were trimmed of primer sequences and a consensus created. The consensus sequence was analyzed with NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the nucleotide database to determine the species. Results Multilocus PCR/ESI-MS Genotyping of B. miyamotoi The multilocus Borrelia PCR/ESI-MS genotyping assay differentiates strains and species of Borrelia by their unique combination of basecount signatures. To characterize the prevalence of B. miyamotoi in Ixodes ticks we examined the basecount signatures from ticks that were positive for B. miyamotoi. Positive specimens from each of the 3 regions (United States, Europe, and Japan) typically produced basecount signatures at 5 of the 8 loci evaluated in the Borrelia genotyping assay. Based upon these 5 signatures, B. miyamotoi from the United States, Europe, and Japan are distinct genotypes (Table 2). All the specimens from North America had the same basecount signatures for the 5 detecting primer pairs. A separate signature combination was found for all of the European isolates detected in ticks from Germany and the Czech Republic. A third signature was observed from the CDC culture isolate from the Japanese strain. Although all 3 genotypes shared the same basecount for the locus BCT3515, the European genotype did not have any other basecount signatures in common with the other 2 genotypes. The North American and Japanese genotypes had the same signatures for 2 of the 4 remaining loci, BCT 3519 and BCT3511. We detected B. miyamotoi with 3 or more primers in the Borrelia genotyping assay in all but 4 of the 68 positive specimens. Several factors may explain why all 5 primers did not detect the bacteria, including nucleic acid quality and quantity or differences in primer sensitivities. Table 2 Borrelia miyamotoi PCR/ESI-MS basecount signatures* Region Genotype BCT3515 (rplB) BCT3517 (flaB) BCT3519 (hbb) BCT3520 (hbb) BCT3511 (gyrB) Europe 1 A13G22C15T18 A41G30C23T27 A41G29C19T46 A52G29C13T47 A36G32C13T35 North America 2 A13G22C15T18 A43G28C23T27 A40G30C18T47 A52G30C13T46 A37G31C13T35 Japan 3 A13G22C15T18 A41G29C23T28 A40G30C18T47 A53G29C13T46 A37G31C13T35 *PCR/ESI-MS, PCR and electrospray ionization mass spectrometry. Prevalence of B. miyamotoi in Europe and the United States I. ricinus ticks from the Czech Republic and Germany in Europe and I. scapularis and I. pacificus ticks from 5 states in the United States were screened for B. miyamotoi by PCR/ESI-MS. B. miyamotoi was found in all regions examined in varying degrees (Table 1) and in all 3 Ixodes species examined. Germany had a low incidence rate; only 4 of the 226 ticks tested were infected (1.8%). Incidence of B. miyamotoi infection of ticks from the Czech Republic varied by region and ranged from 0% to 3.2% with an average infection rate of 2%. In North America, the infection rates of ticks varied from 0% to 15.4%. All negative controls were negative and all positive controls were positive. Sequence Confirmation of B. miyamotoi detections Representative samples were selected for 16S rRNA sequencing: 1 sample from Pennsylvania in the United States, 1 from Germany, and 1 from the Czech Republic. The samples from Germany and the Czech Republic were identical (KF740842 and KF740841, respectively) and matched 99.11% (669 bp out of 675 bp) of the B. miyamotoi LB-2001 sequence, a North American isolate from the East Coast (GenBank accession no. NC_022079). The sample from Pennsylvania (KF740843) was identical (675 bp of 675 bp) to the B. miyamotoi LB-2001 sequence. Discussion In this study, we identified 3 distinct B. miyamotoi genotypes in the United States, Europe, and Japan. Results show that B. miyamotoi is widely distributed across North America and Europe.We observed no genotypic differences using this PCR/ESI-MS assay between the B. miyamotoi detected in I. scapularis from the eastern US states and the midwest or between these bacteria and the B. miyamotoi detected in I. pacificus from California. In a study by Mun et al., a 766-bp region of the flagellin gene sequence were shown to have and a 0.9% difference between B. miyamotoi found in I. pacificus and those found in I. scapularis in the United States ( 8 ). However, our flagellin primers targeted a region of the flagellin gene that does not contain the differences identified by Mun et al., thus explaining why we found a single North American genotype. Previous studies that examined the sequence of the 16S rRNA gene from multiple B. miyamotoi strains indicated that strains from the United States and Europe were located in their own clusters ( 6 ). The Japanese strain FR64b grouped with isolates found in infected humans and I. persulcatus ticks in Russia, whereas the B. miyamotoi found in I. ricinus ticks from Russia grouped with those found in Europe ( 6 ). In our genetic analysis, the Japanese strain also differed from that found in I. ricinus in Europe. Our study demonstrates that the presence of B. miyamotoi in Ixodes ticks is widespread across the regions examined and was observed in all 3 species of field-collected Ixodes ticks. In Europe we observed B. miyamotoi in ≈2.0% of I. ricinus ticks tested, consistent with the detection rates in other studies examining I. ricinus prevalence at other locations in Europe ( 9 , 10 ). Our detection rates were also similar to those seen in an earlier study on ticks from Mendocino County, California ( 8 ). I. scapularis ticks from the East Coast region (New York, Connecticut, and Pennsylvania) were found to have infection rates ranging from 0% to 6.8% for ticks. In Indiana, however, a much higher percentage, ≈12%, of I. scapularis ticks examined were infected with B. miyamotoi. Other studies have also shown that local site-to-site prevalence of B. miyamotoi can vary greatly from the overall regional mean ( 13 ). Our study indicates that B. miyamotoi is likely present in any region where Ixodes ticks reside but that infection rates can vary greatly by region. Since the original description of B. miyamotoi as a human pathogen, studies have shown clinical infection in both healthy and immunocompromised patients in both Europe and the United States ( 3 – 6 , 22 ). If physicians know the regional infection rate in ticks, they will be alert for possible exposure risks for their patients. Standard Lyme borreliosis serologic tests offered by commercial laboratories cannot be relied on to detect B. miyamotoi infection in patients. B. miyamotoi has been shown to have transovarial transmission, suggesting that larval ticks may also pose a risk ( 7 ). Little is yet known about the transmission rates to humans, and further studies are required to better gauge the risk to humans in these B. miyamotoi-endemic regions.

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Borrelia miyamotoi in host-seeking Ixodes ricinus ticks in England.

K Hansford, M Fonville, S Jahfari … (2015)

This paper reports the first detection of Borrelia miyamotoi in UK Ixodes ricinus ticks. It also reports on the presence and infection rates of I. ricinus for a number of other tick-borne pathogens of public health importance. Ticks from seven regions in southern England were screened for B. miyamotoi, Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum and Neoehrlichia mikurensis using qPCR. A total of 954 I. ricinus ticks were tested, 40 were positive for B. burgdorferi s.l., 22 positive for A. phagocytophilum and three positive for B. miyamotoi, with no N. mikurensis detected. The three positive B. miyamotoi ticks came from three geographically distinct areas, suggesting a widespread distribution, and from two separate years, suggesting some degree of endemicity. Understanding the prevalence of Borrelia and other tick-borne pathogens in ticks is crucial for locating high-risk areas of disease transmission.

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Journal ID (nlm-ta): Emerg Infect Dis

Journal ID (iso-abbrev): Emerging Infect. Dis

Journal ID (publisher-id): EID

Title: Emerging Infectious Diseases

Publisher: Centers for Disease Control and Prevention

ISSN (Print): 1080-6040

ISSN (Electronic): 1080-6059

Publication date (Print): March 2016

Volume: 22

Issue: 3

Pages: 550-551

Affiliations

[1]University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania (Z. Kalmár, A.D. Mihalca, C.M. Gherman, M.O. Dumitrache, V. Cozma);

[2]National Institute of Public Health and Environment, Bilthoven, the Netherlands (H. Sprong, E.C. Coipan, M. Fonville)

Author notes

Address for correspondence: Zsuzsa Kalmár, University of Agricultural Sciences and Veterinary Medicine, Calea Mănăștur 3–5, Cluj-Napoca 400372, Romania; email: zsuzsa_kalmar@ 123456yahoo.com

Article

Publisher ID: 15-0140

DOI: 10.3201/eid2203.150140

PMC ID: 4766875

PubMed ID: 26889789

SO-VID: 0ccb7e43-d964-4f78-b651-dff837f3223d

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Borrelia miyamotoi and Candidatus Neoehrlichia mikurensis in Ixodes ricinus Ticks, Romania

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Ticks and tick-borne pathogens

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Humans Infected with Relapsing Fever Spirochete Borrelia miyamotoi, Russia

Prevalence of Borrelia miyamotoi in Ixodes Ticks in Europe and the United States

Borrelia miyamotoi in host-seeking Ixodes ricinus ticks in England.

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