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Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar
Accepted Manuscript Title: Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar Authors: Ralf Matthias Hagen, Hagen Frickmann, Julian Ehlers, Andreas Kruger, ¨ Gabriele Margos, Cecilia Hizo-Teufel, Volker Fingerle, Raphael Rakotozandrindrainy, Vera von Kalckreuth, Justin Im, Gi Deok Pak, Hyon Jin Jeon, Jean Philibert Rakotondrainiarivelo, Jean No¨el Heriniaina, Tsiry Razafindrabe, Frank Konings, Jurgen ¨ May, Benedikt Hogan, J¨org Ganzhorn, Ursula Panzner, Norbert Georg Schwarz, Denise Dekker, Florian Marks, Sven Poppert PII: DOI: Reference:
S0001-706X(17)30720-9 https://doi.org/10.1016/j.actatropica.2017.10.002 ACTROP 4463
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
17-6-2017 25-9-2017 2-10-2017
Please cite this article as: Hagen, Ralf Matthias, Frickmann, Hagen, Ehlers, Julian, Kruger, ¨ Andreas, Margos, Gabriele, Hizo-Teufel, Cecilia, Fingerle, Volker, Rakotozandrindrainy, Raphael, Kalckreuth, Vera von, Im, Justin, Pak, Gi Deok, Jeon, Hyon Jin, Rakotondrainiarivelo, Jean Philibert, Heriniaina, Jean No¨el, Razafindrabe, Tsiry, Konings, Frank, May, Jurgen, ¨ Hogan, Benedikt, Ganzhorn, J¨org, Panzner, Ursula, Schwarz, Norbert Georg, Dekker, Denise, Marks, Florian, Poppert, Sven, Presence of Borrelia spp.DNA in ticks, but absence of Borrelia spp.and of Leptospira spp.DNA in blood of fever patients in Madagascar.Acta Tropica https://doi.org/10.1016/j.actatropica.2017.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar
Short title: Spirochaetes in Madagascar
Ralf Matthias Hagen1,2,#, Hagen Frickmann2,3,#, Julian Ehlers4, Andreas Krüger2, Gabriele Margos5, Cecilia Hizo-Teufel5, Volker Fingerle5, Raphael Rakotozandrindrainy6, Vera von Kalckreuth7, Justin Im7, Gi Deok Pak7, Hyon Jin Jeon7, Jean Philibert Rakotondrainiarivelo6, Jean Noël Heriniaina6, Tsiry Razafindrabe6, Frank Konings7, Jürgen May8, Benedikt Hogan8, Jörg Ganzhorn4, Ursula Panzner7, Norbert Georg Schwarz8, Denise Dekker8, Florian Marks7,°, Sven Poppert9,°,*
1
Department
of
Preventive
Medicine,
Bundeswehr
Medical
Academy
Munich,
Neuherbergstraße 11, 80937 Munich, Germany; 2Department of Tropical Medicine at the Bernhard Nocht Institute, Bundeswehr Hospital Hamburg, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; 3Institute for Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, 18057 Rostock, Germany; 4Biological Faculty, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany;
5
National Reference
Center for Borrelia, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), Branch Oberschleißheim, Veterinärstraße 2, 85764 Oberschleißheim, Germany; 6
Department of Microbiology and Parasitology, University of Antananarivo, BP 566,
Antananarivo
101,
Madagascar;
7
International
Vaccine
Institute,
1
Gwanak-ro,
8
Nakseongdae-dong, Gwanak-gu, Seoul, Republic Korea; Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; 9University Medical Center, University Hospital Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany
#
Both authors contributed equally to this work.
°Both authors also contributed equally to this work. Spirochaetes in Madagascar 1
*Corresponding author: Sven Poppert, M.D., 9University Medical Center, University Hospital Hamburg-, Martini-Straße 52, 20251 Hamburg, Germany, Email: [email protected], phone number: 0049-176-80106141
Conflicts of interest: None.
Highlights
Borrelia spp. DNA was detected in Amblyomma (A.) variegatum ticks and Rhipicephalus (R.) microplus ticks in Madagascar.
DNA of a Borrelia (B.) theileri-related borreliae was detected in R. microplus for the first time in Madagascar.
Only low amounts of Borrelia spp. DNA were detected in the assessed ticks, indicating low numbers of organisms and indicating an at the most limited risk of transmission to humans.
Borrelia spp. DNA was absent in the blood of fever patients from the highlands in Madagascar.
Leptospira (L.) spp. DNA was absent in the blood of fever patients from the highlands in Madagascar.
Abstract The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flab sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1,009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target Spirochaetes in Madagascar 2
DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar.
Key words: Borrelia spp.; Leptospira spp.; tick-borne relapsing fever; leptospirosis; fever of unknown origin; Madagascar, tick
Introduction Data on the prevalence of the spirochaetes Borrelia spp. and Leptospira spp. in Madagascar and their health-related relevance on the human population are scarce. Previous reports suggested a complete absence of leptospirosis and tick-borne relapsing fever in humans in Madagascar (Rodhain & Fontenille, 1989, Desvars et al., 2013). However, most recently, we have identified Rickettsia spp. in Madagascan ticks (Keller et al., 2016) and Brucella spp. in human blood samples of Madagascan fever patients (Boone et al., 2017), although little was known on the local occurrence of these pathogens before. Concerning Leptospira, small mammals that can serve as reservoirs are present in Madagascar and rodents have been shown to be infected with Leptospira (Rahelinirina et al., 2010; Ralaiarijaona et al 2001, Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014). Data on humans are extremely scarce (Desvars et al., 2013). Concerning Borrelia, suitable vectors like Ornithodoros moubata ticks are present in Madagascar (Colas-Belcour et al., 1952, Rodhain & Fontenille, 1989). There is no reliable information on the frequency of tick bites in humans in Madagascar. However, tick bites in humans are reportedly frequently, especially in farmers (personal correspondence of the authors). Recent data on the frequency of relapsing fever in humans in Madagascar are lacking (Rodhain & Fontenille, 1989). In the here described investigation, we assessed tick samples that were collected previously from Zebu cattle in Madagascar for another study (Keller et al., 2016) for the presence of DNA of Borrelia spp. and identified borrelia DNA. In a next step, we investigated human blood samples, that were collected in the scope of a study on fever of unknown origin (FUO) (Boone et al., 2017, Marks et al., 2017) for the presence of DNA of the spirochaetes Borrelia spp. and Leptospira spp..
Materials and methods Four hundred and three ticks were included in the study. Sixteen ticks (A. variegatum, 6 males; R. microplus, 10 females) were collected in 2013 from 8 free-ranging Zebu cattle in the region Atsimo-Andrefana while working on a project on turtle ticks (Ehlers et al., 2016) and screened for borrelia as a proof-of-principle approach. In addition, 387 ticks were Spirochaetes in Madagascar 3
collected in 2012 at slaughter-houses located in Antananarivo, the capital of Madagascar, from 83 Zebu cattle from the regions of Bongolava, Haute Masiatra, Itasy, Menabe, Mampikony, and Vakinkaratra, Madagascar (Keller et al., 2016). Identification was performed according to the guides and keys of Voltzit & Keirans and Walker et al. (Voltzit & Keirans, 2003; Walker et al., 2003). Ticks species included Amblyomma variegatum (n=249, 81 females, 150 males, and 18 nymphs) and Rhipicephalus microplus (n=138, 111 females, 27 males). DNA from ticks was extracted as previously described using QIAamp DNA® Mini Kits (Qiagen) (Keller et al 2016). All nucleic acid extractions of ticks were screened for Borrelia spp.-specific DNA as described (Parola et al., 2011). Samples with positive results for Borrelia spp. were further characterized by PCR and Sanger sequencing of the target genes flaB (P41), uvrA, pepX, gyrB and a longer fragment of the 16S rRNA gene as described previously (Barbour et al, 1996; Schwan et al., 2005; Margos et al., 2009, Margos et al., 2015; Assous et al., 2016; Venczel et al., 2016). PCRs for p41, uvrA, and pepX were only performed if sequencing of flaB, the 16S rRNA gene and gyrB led to inconclusive results. In case the PCRs for flaB, p41, the 16S rRNA gene, uvrA, and pepX failed or led to inconclusive sequencing results, the less sensitive gyrB PCR was not performed (Table 1). Sequencing was conducted by Eurofins Genomics (Ebersberg, Germany). Sequence alignment, genetic distance analyses and construction of phylogenetic trees was done in MEGA5 (Kimura, 1980; Tamura et al., 2011). Basic local alignment search tool (BLAST) searches in GenBank were conducted. Genetic distance analyses were based on the Kimura 2-parameter model (Kimura, 1980). The evolutionary history was inferred using the Maximum Likelihood method based on the General Time Reversible model (Nei & Kumar, 2000). Initial trees for the heuristic search were obtained by applying Neighbour-Joining and BioNJ algorithms to a matrix of pair-wise distances estimated using the Maximum Composite Likelihood (MCL) approach, followed by selecting the topology with superior log likelihood value. To calculate node support values, 1,000 bootstrap repeats were chosen. A discrete Gamma distribution was used to model evolutionary rate differences among sites [+G]. The rate variation model allowed for some sites to be evolutionarily invariable [+I]. The trees were drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+noncoding for flaB and gyrB sequences. All positions containing gaps and missing data were eliminated. Further information is provided in the figure legends. Human EDTA-blood samples were collected between 09/2011 and 06/2013 from Madagascan patients with FUO (≥38.5°C) (von Kalckreuth et al., 2016; Boone et al 2017, Spirochaetes in Madagascar 4
Marks et al, 2017). Collection took place at paediatric wards as well as in inpatient or outpatient departments of the basic health centers Centre de Santé de Base (CSB) II Imerintsiatosika, Itasy Region, CSB II Fenoarivo and CSB II Isotry, Antananarivo, and of the clinic Centre Hospitalier Universitaire (CHU) Tsaralalana, Antananarivo, and included into the study. The median age of included patients was 21 years (IQR 11, 34). The age-distribution analysis indicated 48 (4.8%) of patients between 0 and 1 year of age, 73 (7.2%) patients between 2 and 4 years of age, 220 (21.8%) patients between 5 and 14 years of age, and 668 (66.2%) patients older than 14 years. Written informed consent was obtained from the patients or the next-of-kin in case of minors. All samples were stored at -20°C. We included all available samples of patients with body temperature above 38.5°C, that contained more than 1 ml. 1,009 of 1251 samples could be included, each sample representing one patient. 242 samples were not used, because of insufficient sample volume. The same samples were in parallel used for another study on brucellosis, Q fever and melioidosis (Boone et al 2017). In 174 (17.2%) cases, the corresponding patients had received an antibiotic drug before acquisition of the sample. Patients of all ages presenting with current fever or self-reported history of fever within the past 72 hours were eligible for enrolment. Blood was sampled from all patients meeting inclusion criteria and enrolled in the study. All health facilities recruiting patients represented primary level health services, therefore patients enrolled were assumed to present with early onset disease. Unfortunately no serum samples were collected for this study. Nucleic acid was extracted from 1 ml EDTA blood per patient sample using FlexiGene DNA® kits (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Following DNA extraction of human blood samples, real-time screening PCRs for Leptospira spp. and Borrelia spp. were performed as previously described (Stoddard et al., 2009; Parola et al., 2011). Positive controls were based on designed plasmids with a pEX-A128 vector backbone (eurofins Genomics, Ebersberg, Germany) and PCR-specific inserts. For the Leptospira spp.-specific screening PCR, a 254-base-pair insert of the lipL32 gene of L. interrogans serovar Icterohaemorrhagiae (Genbank: GU183106.1) was chosen (sequence: 5’TCATACGAACTCCCATTTCAGCGATTACGGCAGGAATCCAAACATAGAGATAGTATGCTT TTTTGTTTCCATCGACTAAACCGTCCGGCGCTTGTCCTGGCTTTACGTATCCGTAATAGT TGATCACAGATCCGTAGGGAAGTAACGTTTTTACGGTTTCGTTTGTCCCTGGGATTGTGT CCTCGCTCAGAACAAAAGAGCTTTTTAGGCTTGGCAGACCACCGAAAGCACCACAAGC GGTAATGCTTGCAAAG-3’). For the Borrelia spp.-specific PCR, a 148-base-pair fragment of the 16S rRNA gene of Borrelia burgdorferi (Genbank: L39081.1) was used (sequence: 5’AGCCTTTAAAGCTTCGCTTGTAGATGAGTCTGCGTCTTATTAGTTAGTTGGTAGGGTAAA Spirochaetes in Madagascar 5
TGCCTACCAAGGCGATGATAAGTAACCGGCCTGAGAGGGTGAACGGTCACACTGGAAC TGAGATACGGTCCAGACTCCTACGGGAGGC-3’). The plasmids were adjusted to cycle threshold (Ct) values of about 30 by serial dilutions. Ethical clearance was obtained from the Malagasy Ethical Committee; for patients recruited at the CSB-II in Imerintsiatosika and CSB-II Isotry ethical clearance was additionally obtained from the Institutional Review Board of the International Vaccine Institute.
Results In a very first step, we investigated 16 ticks (A. variegatum, 6 males; R. microplus, 10 females) that were collected in 2013 from 8 free-ranging Zebu cattle in the region AtsimoAndrefana while working on a project on turtle ticks (Ehlers et al., 2016). This was done as a proof-of-principle approach. Unexpectedly, one R. microplus tick turned out to be positive for borreliae. In a next step, we subsequently assessed 387 further ticks that were collected for a study on rickettsiae in a slaughter house in the capital Antananarivo (Keller at al 2016) (Table 1) and performed additional PCRs and sequencing of the positive samples for species identification. A total of 23 out of 403 ticks were positive for Borrelia spp. The positive ticks comprised 21 A. variegatum (21/255; 8.2%, 18 males, 2 females, 1 nymph) and 2 R. microplus (2/148; 1.4%, 2 females). Only real-time PCR curves with characteristic sigmoid shapes in the screening PCR (Parola et al., 2011) were considered positive. The mean cycle-threshold (Ct) value was 38.2 (± standard deviation (SD) 4.1). If questionable PCR curves were taken into account, the number of positive results would rise to 57 out of 255 A. variegatum ticks (22.4%, 56 adults (8 females, 48 males), 1 nymph) and 5 out of 148 R. microplus ticks (3.4%, all female). The Ct-values would be similar with 37.9 ± 2.9 (mean value ± standard deviation). PCRs of 23 ticks showed no atypical curves and were considered truly positive for Borrelia spp. DNA and were thus further analyzed (Table 1). One of these was the above mentioned tick from Southern Madagascar, all other positive ticks were from the slaughterhouse of Antananarivo. Species identification of Borrelia spp. by typing PCRs with subsequent Sanger sequencing failed for 22 out of 23 samples as shown in Table 1. Although 8 out of 110 typing PCRs were positive in these 22 ticks, low sequence quality did not allow further analyses. DNA detected in the R. microplus tick collected from a free-ranging Zebu cattle in the southwest of the island gave suitable sequencing results for 16S rRNA (GenBank accession KY066479), flaB (KY070335) and gyrB (KY070336). BLAST searches using the 16S rRNA PCR fragments revealed two hits with 100% identity with non-specified Borrelia spp. detected in R. microplus from Cote d'Ivoire (GenBank accession KT364342.1) and Brazil (EF141021.1), respectively Spirochaetes in Madagascar 6
(Yparraguirre et al., 2007; Ehounoud et al., 2016). Both strains also showed the highest identity when the flaB fragment was assessed (KT364345.1, EF141022.1), but were not considered for phylogenetic analysis due to short sequence length (query coverage 38% and 61%, respectively). Phylogenetic and genetic distance analyses were performed using the 16S rRNA, flaB and gyrB fragments in MEGA5 (Kimura, 1980). These analyses showed that - when available - Borrelia theileri formed a sister clade to the strain investigated here (Fig. 1, 2). For gyrB, no sequences for B. theileri and B. lonestari were found in GenBank and the strain clustered next to B. miyamotoi (Fig 3). However, the result is consistent with the phylogenies for 16S rRNA and flaB as B. miyamotoi fell into the clade that contained strain Mo063b and B. theileri. Genetic distance values obtained for the 16S rRNA fragment, for flaB and gyrB confirmed the close relatedness of Mo063B with B. theileri (Tables 2-4). Since B. theileri has the potential to infect humans and vectors of relapsing fever-related borreliae are present in Madagascar, we assessed 1,009 human blood samples which were collected for a study on FUO (Boone at al., Marks et al., 2017) for the presence of Borrelia spp. DNA. Because Leptospira spp. have been found in rodents in Madagascar but data on human infections were not available so far (Rahelinirina et al., 2010; Ralaiarijaona et al 2001, Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014), we included this pathogen in our investigations. All samples tested negative for Borrelia spp. and Leptospira spp. DNA. Non-specific PCR reactions, as seen with the tick samples, were not observed with human samples.
Discussion We detected Borrelia spp.-specific DNA in A. variegatum and R. microplus ticks in 23 cases. In one instance, Borrelia theileri-like sequence fragments could be identified in a R. microplus tick. In all other cases, identification failed due to poor sequence quality of the the samples. Therefore, the species identity of Borrelia detected in A. variegatum remains unclear. The screening PCR by Parola et al. has a high sensitivity and therefore provides positive results, even if infection levels are low. Such PCRs (Parola et al., 2011) lead to low yields of amplicons resulting in difficulties performing sequence analyses with the subsequent typing PCRs. High Ct-values may also be associated with non-specific reactions in real-time PCR or with the fact that Borrelia spp. diminish in the tick midgut during blood feeding. Such effects have been described for Borrelia burgdorferi in Ixodes ricinus feeding on cattle (Pacilli et al., 2014). The high Ct values even for samples with typical sigmoid real-time PCR curves suggest very low quantities of Borrelia spp. DNA in the assessed ticks. It remains unclear whether extremely low numbers of Borrelia spp. in ticks are sufficient for infections in humans. Spirochaetes in Madagascar 7
The detection of a Borrelia theileri-like sequence in Madagascar is an interesting finding. It remains unclear how this Borrelia sp. came to Madagascar and how frequently it does indeed occur. It seems possible that this Borrelia theileri-like organism came to Madagascar and other African countries with cattle from South America that were imported for cross breeding experiments. It may be that this pathogen is currently spreading. A more detailed investigation of ticks from Madagascar for the presence of borreliae seems desirable. Considering the presence of DNA of borreliae in ticks, human infections seemed possible. Human infections seemed also possible for another spirochaete, Leptospira spp., considering the presence of this pathogen in rodents in Madagascar (Rahelinirina et al., 2010; Ralaiarijaona et al 2001, Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014). We therefore investigated human blood samples of febrile Madagascan patients collected before (Boone et al 2017, Marks et al, 2017). Leptospira spp. and Borrelia spp were absent in our investigations in human blood samples in spite of the use of highly sensitive screening systems. For the Borrelia spp. PCR, no details on the sensitivity in spiked samples have been published but successful prior application with whole blood has been described (Parola et al., 2011). In the quoted study, Borrelia spp.-specific PCR outperformed traditional microscopy for the detection of relapsing fever-associated Borrelia spp. by factors 2-7 (Parola et al., 2011). The sensitivity of the Leptospira spp. PCR has been reported to be 10 bacteria/ml in whole blood from spiking experiments (Stoddard et al., 2009). Since all samples were negative, we did not have infected blood of a patient as a positive control. But we included a positive control based on plasmids as detailed above. Samples inhibition was widely absent as confirmed by a previous assessment with the same samples (Boone et al., 2017). Of note, in spite of acceptable sensitivity of the PCR used, Leptospira spp. can only be identified in the bloodstream within the 1st week after the onset of symptoms (Levett, 2001). Later stages of the disease might therefore be missed. Enrollment took place at primary health centers, therefore it was assumed that disease onset was quite recent and illnesses were not accompanied by severe complications. Details on the duration of fever are, unfortunately, unavailable, an admitted limitation of the study. Application of serological tests for leptospirosis as well as for further pathogens would have been desirable. Unfortunately, only whole blood samples were collected and stored for the study, so serum samples were not available. In future studies, serum samples should be included.
Spirochaetes in Madagascar 8
Another undeniable limitation of the study which does not allow a clear-cut association of the results is the fact that ticks and human samples were collected without spatial and timely association. Nevertheless, the complete absence of Borrelia spp. DNA in human sample material suggests that relapsing fever associated with Borrelia spp. as causative agents of FUO does not play an important role in the Madagascan population in the highlands. The situation may be different in the south of Madagascar, were we found the B. theileri-like organisms. However, our data on the absence of borreliae in human samples are in line with the literature, according to which tick-borne relapsing fever is considered as an eradicated disease on Madagascar for decades (Rodhain & Fontenille, 1989). As early as in the 1950s, transmission of tick-borne relapsing fever in Madagascar had been associated with Ornithodoros (O) moubata ticks (Colas-Belcour et al., 1952). The vanishing of Madagascan relapsing fever is supposed to be associated with a sudden decline of O. moubata populations due to unknown reasons (Rodhain & Fontenille, 1989). The present detection of low quantities of DNA of Borrelia spp. not allowing for further typing in A. variegatum ticks may be attributed to the fact that these ticks were collected while blood feeding on Zebu cattle. In contrast to O. moubata, A. variegatum and R. microplus have not been described so far as vectors for Borrelia spp. in Madagascar. African R. microplus have been incriminated as vectors of B. theileri (Smith et al., 1978; Walker et al., 2003), while for A. variegatum, there are only Borrelia-like detections from the Ivory Coast (Ehounoud et al., 2016).
The negative PCRs for Leptospira spp. in the human blood samples of the assessed Madagascan FUO patients are in line with previous observations. Sero-prevalence screening from 2001 comprising 105 Madagascans described a single instance of a low positive titer, thus a non-specific reaction cannot be excluded (Ralaiarijaona et al., 2001). In the same study, attempts to detect Leptospira-specific DNA in kidney tissue of bats, pigs and Zebu cattle failed. In contrast to these early findings (Ralaiarijaona et al., 2001), Leptospira spp. DNA was later found in bats and small mammals in Madagascar (Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014). However, human cases of leptospirosis in Madagascar were not confirmed (Desvars et al., 2013). The authors argued that the absence in humans might be due to a lack of diagnostic options in Madagascar (Desvars et al., 2013). However, the present study confirms that leptospirosis is at least not a dominating health issue in FUO patients in the Madagascan highlands. The restriction of the analysis to this regional setting is aof the study, not allowing for definite conclusions regarding the whole island. Future studies should cover a bigger number of sample collection sites and include Spirochaetes in Madagascar 9
serological investigations of human serum samples. In addition small mammals should be screened for Leptospira spp. at the respective sites of human sample collection.
Conclusion We detected Borrelia spp. DNA in Madagascan ticks and, for the first time, describe a Borrelia theileri like organism in Madagascar. The distribution of Borrelia spp. and their relevance for humans and animals is yet not clear. At least in the highlands, surrounding the capital Antananarivo, the relevance of Borrelia spp. and Leptospira spp. for humans seemslimited.
Acknowledgements Steffen Lohr and Annett Michel are gratefully acknowledged for excellent technical assistance.
Spirochaetes in Madagascar 10
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Margos, G., Vollmer, S.A., Cornet, M., Garnier, M., Fingerle, V., Wilske, B., Bormane, A., Vitorino, L., Collares-Pereira, M., Drancourt, M., Kurtenbach, K., 2009. A new Borrelia species defined by multilocus sequence analysis of housekeeping genes. Appl Environ Microbiol 75, 5410-5416.
Marks, F., von Kalckreuth, V., Aaby, P., Adu-Sarkodie, Y., El Tayeb, M.A., Ali, M., Aseffa, A., Baker, S., Biggs, H.M., Bjerregaard-Andersen, M., Breiman, R.F., Campbell, J., Cosmas, L., Crump, J.A., Espinoza, L.M., Deerin, J.F., Dekker, D.M., Fields, B.S., Gasmelseed, N., Hertz, J.T., Van Minh Hoang, N., Im, J., Jaeger, A., Jeon, H.J., Kabore, L.P., Keddy, K.H., Konings, F., Krumkamp, R., Ley, B., Løfberg, S.V., May, J., Meyer, C.G., Mintz, E.D., Montgomery, J.M., Niang, A.A., Nichols, C., Olack, B., Pak, G.D., Panzner, U., Park, J.K., Park, S.E., Rabezanahary, H., Rakotozandrindrainy, R., Raminosoa, T.M., Razafindrabe, T.J., Sampo, E., Schütt-Gerowitt, H., Sow, A.G., Sarpong, N., Seo, H.J., Sooka, A., Soura, A.B., Tall, A., Teferi, M., Thriemer, K., Warren, M.R., Yeshitela, B., Clemens, J.D., Wierzba, T.F., 2017. Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre populationbased surveillance study. Lancet Global Health 5, e310-e323.
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Nei, M., Kumar, S., 2000. Molecular Evolution and Phylogenetics. Oxford University Press, Inc., 1-352.
Pacilly, F.C., Benning, M.E., Jacobs, F., Leidekker, J., Sprong, H., Van Wieren, S.E., Takken, W., 2014. Blood feeding on large grazers affects the transmission of Borrelia burgdorferi sensu lato by Ixodes ricinus. Ticks Tick Borne Dis 5, 810-817.
Parola, P., Diatta, G., Socolovschi, C., Mediannikov, O., Tall, A., Bassene, H., Trape, J.F., Raoult, D., 2011. Tick-borne relapsing fever borreliosis, rural Senegal. Emerging infectious diseases 17, 883-885.
Rahelinirina, S., Léon, A., Harstskeerl, R.A., Sertour, N., Ahmed, A., Raharimanana, C., Ferquel, E., Garnier, M., Chartier, L., Duplantier, J.M., Rahalison, L., Cornet, M., 2010. First isolation and direct evidence for the existence of large small-mammal reservoirs of Leptospira sp. in Madagascar. PLoS One 5, e14111.
Ralaiarijaona, R.L., Bellenger, E., Chanteau, S., Roger, F., Pérolat, P., Rasolofo Razanamparany, V., 2001. Detection of leptospirosis reservoirs in Madagascar using the polymerase chain reaction technique. Arch Inst Pasteur Madagascar 67, 34-36.
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Spirochaetes in Madagascar 13
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Spirochaetes in Madagascar 14
Figure legends
Figure 1. Phylogenetic analysis of 16S (rrs) sequences by maximum likelihood method. In the phylogenetic tree, GenBank, reference numbers, species designation and strain name (if available) are given. The strain investigated in the present study is marked with a black circle. It forms a cluster with Borrelia spp. described from the Ivory Coast and from Brazil. The tree with the highest log likelihood (-1679.1874) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap values). A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.1000)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 33.7027% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (scale bar). The analysis involved 19 nucleotide sequences. There were a total of 881 positions in the final dataset.
Figure 2. Phylogenetic analysis by Maximum Likelihood method of flagelling (flaB) sequences. In the phylogeny, GenBank reference numbers, species designation and strain name (if available) are given. As for the 16S rRNA gene tree, in the phylogeny inferred using flaB sequences, the strain investigated in this study (Mo063b) is part of a cluster with B. theileri (as sister clade), B. lonestari and B. miyamotoi. The tree with the highest log likelihood (-2823.7008) is shown. A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.2502)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (scale bar). The analysis involved 22 nucleotide sequences. There were a total of 571 positions in the final dataset.
Figure 3. Phylogenetic analysis by Maximum Likelihood method of gyrB sequences. In the phylogeny, GenBank reference numbers, species designation and strain name (if available) are given. Strain Mo063b forms a sister clade with B. miyamotoi. This is consistent with phylogenies based on different sequences such as 16S rRNA and flaB. In case of gyrB, no sequences for B. theileri or B. lonestari were found in the GenBank database. The tree with the highest log likelihood (-4074.7247) is shown. The percentage of trees in which the Spirochaetes in Madagascar 15
associated taxa clustered together is shown next to the branches. A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.2357)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 17 nucleotide sequences. There were a total of 908 positions in the final dataset.
77 98
NR 074865.1 Borrelia duttonii Ly NR 102961.1 Borrelia crocidurae Achema
gb|GU350706.1 Borrelia hispanica Sp3 gb|CP014349.1 Borrelia hermsii HS1 Browne Mountain gb|CP005851.1 Borrelia parkeri SLO 77
NR 102958.1 Borrelia turicatae 91E135 gb|CP005745.1 Borrelia coriaceae Co53 98
gb|CP004217.1 Borrelia miyamotoi FR64b gb|CP006647.2 Borrelia miyamotoi LBgb|AY682921.1 Borrelia lonestari MO2002-V2
70
gb|KF569941.1 Borrelia theileri KAT
86
Mo063b 16S
94 90
gb|KT364342.1 Borrelia sp. TCI301 (Ivory Coast) gb|EF141021.1 Borrelia sp.
gb|CP009117.1 Borrelia valaisiana Tom4006 NR 074662.1 Borrelia afzelii HLJ01
100
NR 074854.1 Borrelia bavariensis
82
NR 102956.1 Borrelia bissettiae
75 82
gb|CP009656.1 Borrelia burgdorferi sensu stricto
0.005
Spirochaetes in Madagascar 16
gb|CP000049.1 Borrelia turicatae 91E135 98
gb|EU492387.1 Borrelia sp. IA-1
52
gb|CP005851.1 Borrelia parkeri SLO
46
gb|CP005745.1 Borrelia coriaceae Co53
26
gb|CP011060.1 Borrelia hermsii CC1
53
Candidatus Borrelia kalaharica gb|CP005830.1 Borrelia anserina BA2
75
gb|CP004217.1 Borrelia miyamotoi FR64b gb|AY850063.1 Borrelia lonestari MO2002-V1
98 97
Mo063b
100 92
gb|KF569936.1 Borrelia theileri KAT
gb|GU357612.1 Borrelia hispanica strain 33
gb|CP000993.1 Borrelia recurrentis
100
gb|CP003426.1 Borrelia crocidurae Achema 95
gb|CP000976.1 Borrelia duttonii Ly gb|JF708951.1 Borrelia microti Abyek
gb|KF422815.1 Borrelia turcica IST7 gb|AE000783.1 Borrelia burgdorferi B31 gb|CP002933.1 Borrelia afzelii
46 100
gb|CP009117.1 Borrelia valaisiana Tom4006 64
gb|CP000013.1 Borrelia bavariensis PBi 100
gb|L42885.1 Borrelia garinii Ip90
0.02
Spirochaetes in Madagascar 17
99
gb|AY934618.1 Borrelia turicatae FCB
100 37
gb|CP005851.1 Borrelia parkeri SLO gb|CP011060.1 Borrelia hermsii CC1
91
gb|CP005745.1 Borrelia coriaceae Co53 89
gb|CP005829.1 Borrelia anserina BA Mo063b
66
dbj|AB900799.1 Borrelia miyamotoi HT31
99 100
gb|CP006647.2 Borrelia miyamotoi LB-200 gb|CP000993.1 Borrelia recurrentis 100
gb|CP003426.1 Borrelia crocidurae gb|CP000976.1 Borrelia duttonii Ly
dbj|AB473537.1 Borrelia sp. Tortoise14 gb|KP067819.1 Borrelia turcica KZ11
100 85
dbj|AB529352.1 Borrelia sp.
gb|CP009117.1 Borrelia valaisiana 100
gb|CP002746.1 Borrelia bissettii DN
0.05
Spirochaetes in Madagascar 18
Table 1: Results of the typing PCRs with consecutive Sanger sequencing of samples with positive results in the Borrelia spp. screening PCR. PCRs for p41, uvrA, and pepX were only performed if sequencing of flaB, the 16S rRNA gene and gyrB led to non-conclusive results. If the more sensitive PCRs for flaB, p41, the 16S rRNA gene, uvrA, and pepX failed or led to non-conclusive results in sequencing, the less sensitive gyrB PCR was not performed.
Sample
Tick species
I.D.
Ct-value in the 16S
Results of the typing PCRs targeting the following genes:
rRNA gene screening PCR
1
Amblyomma variegatum
39
flaB (simplex)
p41 (duplex)
16S rRNA gene
uvrA
pepX
gyrB
Negative
Negative
Negative
Negative
Negative
n.a.
1
2
Amblyomma variegatum
38
Negative
Negative
Negative
Positive
Negative
n.a.
3
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
4
Amblyomma variegatum
38
Negative
Negative
Negative
Negative
Negative
n.a.
5
Amblyomma variegatum
37
negative
Negative
Negative
Negative
Negative
n.a.
6
Amblyomma variegatum
42
Negative
Negative
Negative
Negative
Negative
n.a.
7
Amblyomma variegatum
41
Negative
Negative
Negative
Negative
Negative
n.a.
8
Amblyomma variegatum
36
Negative
Negative
Negative
Negative
Negative
n.a.
9
Amblyomma variegatum
38
Negative
Negative
Negative
Positive1
Negative
n.a.
10
Amblyomma variegatum
44
Negative
Negative
Negative
Negative
Negative
n.a.
11
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
12
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
13
Amblyomma variegatum
37
Negative
Negative
Negative
Negative
Negative
n.a.
14
Rhipicephalus microplus
39
Positive1
Negative
Positive1
Negative
Negative
n.a.
15
Amblyomma variegatum
40
Negative
Negative
Negative
Negative
Negative
n.a.
16
Amblyomma variegatum
37
Negative
Negative
Positive1
Negative
Negative
n.a.
1
17
Amblyomma variegatum
36
Negative
Negative
Negative
Positive
Negative
n.a.
18
Amblyomma variegatum
41
Negative
Negative
Negative
Negative
Negative
n.a.
19
Amblyomma variegatum
36
Negative
Negative
Negative
Negative
Negative
n.a.
20
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
21
Amblyomma variegatum
38
Negative
Negative
Positive2
Negative
Negative
n.a.
1
22
Amblyomma variegatum
43
Negative
Negative
Positive
Negative
Negative
n.a.
23
Rhipicephalus microplus
22
Positive3
n.a.
Positive3
n.a.
n.a.
Positive3
Spirochaetes in Madagascar 19
1
Sequence analysis failed due to poor sequence quality. 2Sequence analysis suggested B. afzelii (99% identity (1 bp-mismatch), 100% query coverage), but only after 150 bp with poor sequence quality
were removed. 3Sequence analyses in figures 1-3 and tables 2-4. n.a. = not applicable.
Spirochaetes in Madagascar 20
Table 2: Estimates of Evolutionary Divergence between 16SrRNA Sequences. The analysis involved 19 nucleotide sequences. There were a total of 881 positions in the final dataset.
Mo063b 16SrRNA EF141021.1 Borrelia sp. Brazil
0,000
KT364342.1 Borrelia sp. Ivory Coast
0,000
0,000
KF569941.1 Borrelia theileri
0,003
0,003
0,003
AY682921.1 Borrelia lonestari
0,008
0,008
0,008
0,007
NR_102958.1 Borrelia turicatae
0,014
0,014
0,014
0,013
0,013
CP004217.1 Borrelia miyamotoi
0,013
0,013
0,013
0,011
0,014
0,015
CP006647.2 Borrelia miyamotoi
0,015
0,015
0,015
0,014
0,016
0,015
0,002
CP005851.1 Borreliaparkeri
0,015
0,015
0,015
0,014
0,014
0,001
0,016
0,016
CP005745.1 Borrelia coriaceae
0,016
0,016
0,016
0,015
0,015
0,009
0,017
0,017
0,010
CP014349.1 Borrelia hermsii
0,016
0,016
0,016
0,015
0,017
0,007
0,015
0,015
0,008
0,011
GU350706.1 Borrelia hispanica
0,015
0,015
0,015
0,014
0,014
0,006
0,016
0,016
0,007
0,010
0,008
NR_074865.1 Borrelia_duttonii
0,016
0,016
0,016
0,015
0,015
0,007
0,017
0,017
0,008
0,011
0,009
0,001
NR_102961.1 Borrelia crocidurae
0,016
0,016
0,016
0,015
0,015
0,007
0,017
0,017
0,008
0,011
0,009
0,001
0,000
CP009117.1 Borrelia valaisiana
0,034
0,034
0,034
0,033
0,035
0,029
0,035
0,038
0,028
0,034
0,031
0,033
0,034
0,034
NR_074854.1 Borrelia bavariensis
0,035
0,035
0,035
0,035
0,038
0,034
0,040
0,042
0,033
0,039
0,036
0,035
0,036
0,036
0,009
NR_102956.1 Borrelia bissetti
0,037
0,037
0,037
0,036
0,039
0,033
0,039
0,041
0,031
0,037
0,035
0,035
0,036
0,036
0,008
0,003
NR_074662.1 Borrelia afzelii
0,037
0,037
0,037
0,036
0,039
0,033
0,036
0,039
0,031
0,038
0,035
0,034
0,035
0,035
0,008
0,008
0,006
CP009656.1 Borrelia burgdorferi
0,037
0,037
0,037
0,036
0,039
0,033
0,039
0,041
0,031
0,037
0,035
0,035
0,036
0,036
0,009
0,005
0,001
0,007
Spirochaetes in Madagascar 21
Table 3: Estimates of Evolutionary Divergence between flaB Sequences. The analysis involved 22 nucleotide sequences. There were a total of 601 positions in the final dataset. Mo063b flaB KF569936.1 Borrelia theileri
0,013
AY850063.1 Borrelia lonestari
0,043
0,040
CP004217.1 Borrelia miyamotoi
0,081
0,085
0,089
CP005830.1 Borrelia anserina
0,099
0,095
0,118
0,085
KT970516.1 Candidatus B. kalaharica
0,110
0,102
0,114
0,094
0,059
GU357612.1 Borrelia hispanica
0,114
0,114
0,134
0,110
0,095
0,089
CP005745.1 Borrelia coriaceae
0,118
0,114
0,118
0,102
0,079
0,079
0,102
CP000049.1 Borrelia turicatae
0,120
0,116
0,120
0,087
0,066
0,063
0,102
0,072
CP003426.1 Borrelia crocidurae
0,120
0,116
0,140
0,122
0,107
0,100
0,025
0,122
0,110
EU492387.1 Borrelia
0,120
0,116
0,120
0,092
0,068
0,065
0,104
0,074
0,010
0,105
CP000993.1 Borrelia recurrentis
0,122
0,122
0,138
0,120
0,105
0,102
0,022
0,116
0,112
0,013
0,106
CP000976.1 Borrelia duttonii
0,122
0,122
0,138
0,120
0,105
0,102
0,019
0,112
0,112
0,013
0,106
0,003
JF708951.1 Borrelia microti
0,124
0,124
0,136
0,122
0,103
0,104
0,020
0,114
0,114
0,015
0,108
0,005
0,002
CP011060.1 Borrelia hermsii
0,124
0,120
0,130
0,095
0,071
0,072
0,115
0,085
0,070
0,118
0,068
0,121
0,120
0,123
CP005851.1 Borrelia parkeri
0,128
0,120
0,124
0,092
0,072
0,065
0,100
0,066
0,012
0,112
0,015
0,110
0,110
0,112
0,070
KF422815.1 Borrelia turcica
0,160
0,156
0,173
0,141
0,124
0,126
0,127
0,139
0,127
0,134
0,123
0,142
0,142
0,144
0,134
0,130
AE000783.1 Borrelia burgdorferi
0,176
0,178
0,198
0,182
0,159
0,157
0,167
0,178
0,159
0,176
0,161
0,182
0,180
0,182
0,172
0,165
0,157
CP002933.1 Borrelia afzelii
0,174
0,172
0,196
0,178
0,145
0,145
0,167
0,167
0,157
0,174
0,159
0,184
0,184
0,186
0,159
0,163
0,153
0,063
CP009117.1 Borrelia valaisiana
0,176
0,178
0,198
0,176
0,159
0,153
0,161
0,167
0,155
0,172
0,153
0,178
0,178
0,180
0,166
0,161
0,145
0,059
0,043
CP000013.1 Borrelia bavariensis
0,186
0,189
0,211
0,197
0,168
0,165
0,178
0,178
0,170
0,185
0,172
0,191
0,191
0,193
0,187
0,172
0,166
0,071
0,067
0,059
L42885.1 Borrelia garinii
0,184
0,186
0,209
0,191
0,151
0,155
0,176
0,171
0,159
0,178
0,161
0,189
0,189
0,191
0,166
0,165
0,153
0,069
0,052
0,048
0,017
Spirochaetes in Madagascar 22
Table 4: Estimates of Evolutionary Divergence between gyrB Sequences. The analysis involved 19 nucleotide sequences. There were a total of 908 positions in the final dataset. Mo063b gyrB AB900799.1 Borrelia miyamotoi
0,081
CP006647.2 Borrelia miyamotoi
0,087
0,008
CP011060.1 Borrelia hermsii
0,110
0,101
0,109
AY934616.1 Borrelia turicatae
0,112
0,110
0,118
0,069
CP005851.1 Borrelia parkeri
0,112
0,108
0,116
0,068
0,013
AY934618.1 Borrelia turicatae
0,114
0,112
0,120
0,069
0,002
0,013
CP005745.1 Borrelia coriaceae
0,126
0,114
0,122
0,080
0,078
0,078
0,078
CP005829.1 Borrelia anserina
0,134
0,118
0,122
0,106
0,100
0,098
0,102
0,114
CP003426.1 Borrelia crocidurae
0,141
0,136
0,139
0,119
0,130
0,131
0,130
0,134
0,146
CP000976.1 Borrelia duttonii
0,143
0,137
0,140
0,121
0,131
0,132
0,131
0,136
0,147
0,001
CP000993.1 Borrelia recurrentis
0,145
0,140
0,143
0,123
0,134
0,135
0,134
0,138
0,151
0,006
0,004
KP067819.1 Borrelia turcica
0,155
0,134
0,136
0,139
0,141
0,141
0,141
0,140
0,172
0,145
0,146
0,150
AB529352.1 Borrelia sp.
0,157
0,135
0,138
0,140
0,149
0,149
0,149
0,143
0,173
0,144
0,146
0,150
0,017
AB473537.1 Borrelia sp.
0,161
0,139
0,140
0,150
0,156
0,159
0,156
0,150
0,177
0,144
0,146
0,150
0,040
0,042
CP009117.1 Borrelia valaisiana
0,204
0,210
0,205
0,211
0,212
0,206
0,210
0,202
0,232
0,209
0,208
0,209
0,207
0,205
0,218
CP002746.1 Borrelia bissettiae
0,208
0,222
0,217
0,218
0,214
0,212
0,213
0,211
0,244
0,210
0,209
0,210
0,218
0,219
0,219
0,063
CP002933.1 Borrelia afzelii
0,216
0,214
0,209
0,221
0,213
0,208
0,211
0,206
0,241
0,218
0,219
0,221
0,211
0,215
0,225
0,052
0,064
CP000013.1 Borrelia bavariensis
0,214
0,233
0,229
0,223
0,219
0,215
0,219
0,219
0,248
0,215
0,214
0,215
0,227
0,225
0,235
0,061
0,063
0,059
Spirochaetes in Madagascar 23
S0001-706X(17)30720-9 https://doi.org/10.1016/j.actatropica.2017.10.002 ACTROP 4463
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
17-6-2017 25-9-2017 2-10-2017
Please cite this article as: Hagen, Ralf Matthias, Frickmann, Hagen, Ehlers, Julian, Kruger, ¨ Andreas, Margos, Gabriele, Hizo-Teufel, Cecilia, Fingerle, Volker, Rakotozandrindrainy, Raphael, Kalckreuth, Vera von, Im, Justin, Pak, Gi Deok, Jeon, Hyon Jin, Rakotondrainiarivelo, Jean Philibert, Heriniaina, Jean No¨el, Razafindrabe, Tsiry, Konings, Frank, May, Jurgen, ¨ Hogan, Benedikt, Ganzhorn, J¨org, Panzner, Ursula, Schwarz, Norbert Georg, Dekker, Denise, Marks, Florian, Poppert, Sven, Presence of Borrelia spp.DNA in ticks, but absence of Borrelia spp.and of Leptospira spp.DNA in blood of fever patients in Madagascar.Acta Tropica https://doi.org/10.1016/j.actatropica.2017.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar
Short title: Spirochaetes in Madagascar
Ralf Matthias Hagen1,2,#, Hagen Frickmann2,3,#, Julian Ehlers4, Andreas Krüger2, Gabriele Margos5, Cecilia Hizo-Teufel5, Volker Fingerle5, Raphael Rakotozandrindrainy6, Vera von Kalckreuth7, Justin Im7, Gi Deok Pak7, Hyon Jin Jeon7, Jean Philibert Rakotondrainiarivelo6, Jean Noël Heriniaina6, Tsiry Razafindrabe6, Frank Konings7, Jürgen May8, Benedikt Hogan8, Jörg Ganzhorn4, Ursula Panzner7, Norbert Georg Schwarz8, Denise Dekker8, Florian Marks7,°, Sven Poppert9,°,*
1
Department
of
Preventive
Medicine,
Bundeswehr
Medical
Academy
Munich,
Neuherbergstraße 11, 80937 Munich, Germany; 2Department of Tropical Medicine at the Bernhard Nocht Institute, Bundeswehr Hospital Hamburg, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; 3Institute for Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, 18057 Rostock, Germany; 4Biological Faculty, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany;
5
National Reference
Center for Borrelia, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), Branch Oberschleißheim, Veterinärstraße 2, 85764 Oberschleißheim, Germany; 6
Department of Microbiology and Parasitology, University of Antananarivo, BP 566,
Antananarivo
101,
Madagascar;
7
International
Vaccine
Institute,
1
Gwanak-ro,
8
Nakseongdae-dong, Gwanak-gu, Seoul, Republic Korea; Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; 9University Medical Center, University Hospital Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany
#
Both authors contributed equally to this work.
°Both authors also contributed equally to this work. Spirochaetes in Madagascar 1
*Corresponding author: Sven Poppert, M.D., 9University Medical Center, University Hospital Hamburg-, Martini-Straße 52, 20251 Hamburg, Germany, Email: [email protected], phone number: 0049-176-80106141
Conflicts of interest: None.
Highlights
Borrelia spp. DNA was detected in Amblyomma (A.) variegatum ticks and Rhipicephalus (R.) microplus ticks in Madagascar.
DNA of a Borrelia (B.) theileri-related borreliae was detected in R. microplus for the first time in Madagascar.
Only low amounts of Borrelia spp. DNA were detected in the assessed ticks, indicating low numbers of organisms and indicating an at the most limited risk of transmission to humans.
Borrelia spp. DNA was absent in the blood of fever patients from the highlands in Madagascar.
Leptospira (L.) spp. DNA was absent in the blood of fever patients from the highlands in Madagascar.
Abstract The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flab sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1,009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target Spirochaetes in Madagascar 2
DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar.
Key words: Borrelia spp.; Leptospira spp.; tick-borne relapsing fever; leptospirosis; fever of unknown origin; Madagascar, tick
Introduction Data on the prevalence of the spirochaetes Borrelia spp. and Leptospira spp. in Madagascar and their health-related relevance on the human population are scarce. Previous reports suggested a complete absence of leptospirosis and tick-borne relapsing fever in humans in Madagascar (Rodhain & Fontenille, 1989, Desvars et al., 2013). However, most recently, we have identified Rickettsia spp. in Madagascan ticks (Keller et al., 2016) and Brucella spp. in human blood samples of Madagascan fever patients (Boone et al., 2017), although little was known on the local occurrence of these pathogens before. Concerning Leptospira, small mammals that can serve as reservoirs are present in Madagascar and rodents have been shown to be infected with Leptospira (Rahelinirina et al., 2010; Ralaiarijaona et al 2001, Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014). Data on humans are extremely scarce (Desvars et al., 2013). Concerning Borrelia, suitable vectors like Ornithodoros moubata ticks are present in Madagascar (Colas-Belcour et al., 1952, Rodhain & Fontenille, 1989). There is no reliable information on the frequency of tick bites in humans in Madagascar. However, tick bites in humans are reportedly frequently, especially in farmers (personal correspondence of the authors). Recent data on the frequency of relapsing fever in humans in Madagascar are lacking (Rodhain & Fontenille, 1989). In the here described investigation, we assessed tick samples that were collected previously from Zebu cattle in Madagascar for another study (Keller et al., 2016) for the presence of DNA of Borrelia spp. and identified borrelia DNA. In a next step, we investigated human blood samples, that were collected in the scope of a study on fever of unknown origin (FUO) (Boone et al., 2017, Marks et al., 2017) for the presence of DNA of the spirochaetes Borrelia spp. and Leptospira spp..
Materials and methods Four hundred and three ticks were included in the study. Sixteen ticks (A. variegatum, 6 males; R. microplus, 10 females) were collected in 2013 from 8 free-ranging Zebu cattle in the region Atsimo-Andrefana while working on a project on turtle ticks (Ehlers et al., 2016) and screened for borrelia as a proof-of-principle approach. In addition, 387 ticks were Spirochaetes in Madagascar 3
collected in 2012 at slaughter-houses located in Antananarivo, the capital of Madagascar, from 83 Zebu cattle from the regions of Bongolava, Haute Masiatra, Itasy, Menabe, Mampikony, and Vakinkaratra, Madagascar (Keller et al., 2016). Identification was performed according to the guides and keys of Voltzit & Keirans and Walker et al. (Voltzit & Keirans, 2003; Walker et al., 2003). Ticks species included Amblyomma variegatum (n=249, 81 females, 150 males, and 18 nymphs) and Rhipicephalus microplus (n=138, 111 females, 27 males). DNA from ticks was extracted as previously described using QIAamp DNA® Mini Kits (Qiagen) (Keller et al 2016). All nucleic acid extractions of ticks were screened for Borrelia spp.-specific DNA as described (Parola et al., 2011). Samples with positive results for Borrelia spp. were further characterized by PCR and Sanger sequencing of the target genes flaB (P41), uvrA, pepX, gyrB and a longer fragment of the 16S rRNA gene as described previously (Barbour et al, 1996; Schwan et al., 2005; Margos et al., 2009, Margos et al., 2015; Assous et al., 2016; Venczel et al., 2016). PCRs for p41, uvrA, and pepX were only performed if sequencing of flaB, the 16S rRNA gene and gyrB led to inconclusive results. In case the PCRs for flaB, p41, the 16S rRNA gene, uvrA, and pepX failed or led to inconclusive sequencing results, the less sensitive gyrB PCR was not performed (Table 1). Sequencing was conducted by Eurofins Genomics (Ebersberg, Germany). Sequence alignment, genetic distance analyses and construction of phylogenetic trees was done in MEGA5 (Kimura, 1980; Tamura et al., 2011). Basic local alignment search tool (BLAST) searches in GenBank were conducted. Genetic distance analyses were based on the Kimura 2-parameter model (Kimura, 1980). The evolutionary history was inferred using the Maximum Likelihood method based on the General Time Reversible model (Nei & Kumar, 2000). Initial trees for the heuristic search were obtained by applying Neighbour-Joining and BioNJ algorithms to a matrix of pair-wise distances estimated using the Maximum Composite Likelihood (MCL) approach, followed by selecting the topology with superior log likelihood value. To calculate node support values, 1,000 bootstrap repeats were chosen. A discrete Gamma distribution was used to model evolutionary rate differences among sites [+G]. The rate variation model allowed for some sites to be evolutionarily invariable [+I]. The trees were drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+noncoding for flaB and gyrB sequences. All positions containing gaps and missing data were eliminated. Further information is provided in the figure legends. Human EDTA-blood samples were collected between 09/2011 and 06/2013 from Madagascan patients with FUO (≥38.5°C) (von Kalckreuth et al., 2016; Boone et al 2017, Spirochaetes in Madagascar 4
Marks et al, 2017). Collection took place at paediatric wards as well as in inpatient or outpatient departments of the basic health centers Centre de Santé de Base (CSB) II Imerintsiatosika, Itasy Region, CSB II Fenoarivo and CSB II Isotry, Antananarivo, and of the clinic Centre Hospitalier Universitaire (CHU) Tsaralalana, Antananarivo, and included into the study. The median age of included patients was 21 years (IQR 11, 34). The age-distribution analysis indicated 48 (4.8%) of patients between 0 and 1 year of age, 73 (7.2%) patients between 2 and 4 years of age, 220 (21.8%) patients between 5 and 14 years of age, and 668 (66.2%) patients older than 14 years. Written informed consent was obtained from the patients or the next-of-kin in case of minors. All samples were stored at -20°C. We included all available samples of patients with body temperature above 38.5°C, that contained more than 1 ml. 1,009 of 1251 samples could be included, each sample representing one patient. 242 samples were not used, because of insufficient sample volume. The same samples were in parallel used for another study on brucellosis, Q fever and melioidosis (Boone et al 2017). In 174 (17.2%) cases, the corresponding patients had received an antibiotic drug before acquisition of the sample. Patients of all ages presenting with current fever or self-reported history of fever within the past 72 hours were eligible for enrolment. Blood was sampled from all patients meeting inclusion criteria and enrolled in the study. All health facilities recruiting patients represented primary level health services, therefore patients enrolled were assumed to present with early onset disease. Unfortunately no serum samples were collected for this study. Nucleic acid was extracted from 1 ml EDTA blood per patient sample using FlexiGene DNA® kits (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Following DNA extraction of human blood samples, real-time screening PCRs for Leptospira spp. and Borrelia spp. were performed as previously described (Stoddard et al., 2009; Parola et al., 2011). Positive controls were based on designed plasmids with a pEX-A128 vector backbone (eurofins Genomics, Ebersberg, Germany) and PCR-specific inserts. For the Leptospira spp.-specific screening PCR, a 254-base-pair insert of the lipL32 gene of L. interrogans serovar Icterohaemorrhagiae (Genbank: GU183106.1) was chosen (sequence: 5’TCATACGAACTCCCATTTCAGCGATTACGGCAGGAATCCAAACATAGAGATAGTATGCTT TTTTGTTTCCATCGACTAAACCGTCCGGCGCTTGTCCTGGCTTTACGTATCCGTAATAGT TGATCACAGATCCGTAGGGAAGTAACGTTTTTACGGTTTCGTTTGTCCCTGGGATTGTGT CCTCGCTCAGAACAAAAGAGCTTTTTAGGCTTGGCAGACCACCGAAAGCACCACAAGC GGTAATGCTTGCAAAG-3’). For the Borrelia spp.-specific PCR, a 148-base-pair fragment of the 16S rRNA gene of Borrelia burgdorferi (Genbank: L39081.1) was used (sequence: 5’AGCCTTTAAAGCTTCGCTTGTAGATGAGTCTGCGTCTTATTAGTTAGTTGGTAGGGTAAA Spirochaetes in Madagascar 5
TGCCTACCAAGGCGATGATAAGTAACCGGCCTGAGAGGGTGAACGGTCACACTGGAAC TGAGATACGGTCCAGACTCCTACGGGAGGC-3’). The plasmids were adjusted to cycle threshold (Ct) values of about 30 by serial dilutions. Ethical clearance was obtained from the Malagasy Ethical Committee; for patients recruited at the CSB-II in Imerintsiatosika and CSB-II Isotry ethical clearance was additionally obtained from the Institutional Review Board of the International Vaccine Institute.
Results In a very first step, we investigated 16 ticks (A. variegatum, 6 males; R. microplus, 10 females) that were collected in 2013 from 8 free-ranging Zebu cattle in the region AtsimoAndrefana while working on a project on turtle ticks (Ehlers et al., 2016). This was done as a proof-of-principle approach. Unexpectedly, one R. microplus tick turned out to be positive for borreliae. In a next step, we subsequently assessed 387 further ticks that were collected for a study on rickettsiae in a slaughter house in the capital Antananarivo (Keller at al 2016) (Table 1) and performed additional PCRs and sequencing of the positive samples for species identification. A total of 23 out of 403 ticks were positive for Borrelia spp. The positive ticks comprised 21 A. variegatum (21/255; 8.2%, 18 males, 2 females, 1 nymph) and 2 R. microplus (2/148; 1.4%, 2 females). Only real-time PCR curves with characteristic sigmoid shapes in the screening PCR (Parola et al., 2011) were considered positive. The mean cycle-threshold (Ct) value was 38.2 (± standard deviation (SD) 4.1). If questionable PCR curves were taken into account, the number of positive results would rise to 57 out of 255 A. variegatum ticks (22.4%, 56 adults (8 females, 48 males), 1 nymph) and 5 out of 148 R. microplus ticks (3.4%, all female). The Ct-values would be similar with 37.9 ± 2.9 (mean value ± standard deviation). PCRs of 23 ticks showed no atypical curves and were considered truly positive for Borrelia spp. DNA and were thus further analyzed (Table 1). One of these was the above mentioned tick from Southern Madagascar, all other positive ticks were from the slaughterhouse of Antananarivo. Species identification of Borrelia spp. by typing PCRs with subsequent Sanger sequencing failed for 22 out of 23 samples as shown in Table 1. Although 8 out of 110 typing PCRs were positive in these 22 ticks, low sequence quality did not allow further analyses. DNA detected in the R. microplus tick collected from a free-ranging Zebu cattle in the southwest of the island gave suitable sequencing results for 16S rRNA (GenBank accession KY066479), flaB (KY070335) and gyrB (KY070336). BLAST searches using the 16S rRNA PCR fragments revealed two hits with 100% identity with non-specified Borrelia spp. detected in R. microplus from Cote d'Ivoire (GenBank accession KT364342.1) and Brazil (EF141021.1), respectively Spirochaetes in Madagascar 6
(Yparraguirre et al., 2007; Ehounoud et al., 2016). Both strains also showed the highest identity when the flaB fragment was assessed (KT364345.1, EF141022.1), but were not considered for phylogenetic analysis due to short sequence length (query coverage 38% and 61%, respectively). Phylogenetic and genetic distance analyses were performed using the 16S rRNA, flaB and gyrB fragments in MEGA5 (Kimura, 1980). These analyses showed that - when available - Borrelia theileri formed a sister clade to the strain investigated here (Fig. 1, 2). For gyrB, no sequences for B. theileri and B. lonestari were found in GenBank and the strain clustered next to B. miyamotoi (Fig 3). However, the result is consistent with the phylogenies for 16S rRNA and flaB as B. miyamotoi fell into the clade that contained strain Mo063b and B. theileri. Genetic distance values obtained for the 16S rRNA fragment, for flaB and gyrB confirmed the close relatedness of Mo063B with B. theileri (Tables 2-4). Since B. theileri has the potential to infect humans and vectors of relapsing fever-related borreliae are present in Madagascar, we assessed 1,009 human blood samples which were collected for a study on FUO (Boone at al., Marks et al., 2017) for the presence of Borrelia spp. DNA. Because Leptospira spp. have been found in rodents in Madagascar but data on human infections were not available so far (Rahelinirina et al., 2010; Ralaiarijaona et al 2001, Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014), we included this pathogen in our investigations. All samples tested negative for Borrelia spp. and Leptospira spp. DNA. Non-specific PCR reactions, as seen with the tick samples, were not observed with human samples.
Discussion We detected Borrelia spp.-specific DNA in A. variegatum and R. microplus ticks in 23 cases. In one instance, Borrelia theileri-like sequence fragments could be identified in a R. microplus tick. In all other cases, identification failed due to poor sequence quality of the the samples. Therefore, the species identity of Borrelia detected in A. variegatum remains unclear. The screening PCR by Parola et al. has a high sensitivity and therefore provides positive results, even if infection levels are low. Such PCRs (Parola et al., 2011) lead to low yields of amplicons resulting in difficulties performing sequence analyses with the subsequent typing PCRs. High Ct-values may also be associated with non-specific reactions in real-time PCR or with the fact that Borrelia spp. diminish in the tick midgut during blood feeding. Such effects have been described for Borrelia burgdorferi in Ixodes ricinus feeding on cattle (Pacilli et al., 2014). The high Ct values even for samples with typical sigmoid real-time PCR curves suggest very low quantities of Borrelia spp. DNA in the assessed ticks. It remains unclear whether extremely low numbers of Borrelia spp. in ticks are sufficient for infections in humans. Spirochaetes in Madagascar 7
The detection of a Borrelia theileri-like sequence in Madagascar is an interesting finding. It remains unclear how this Borrelia sp. came to Madagascar and how frequently it does indeed occur. It seems possible that this Borrelia theileri-like organism came to Madagascar and other African countries with cattle from South America that were imported for cross breeding experiments. It may be that this pathogen is currently spreading. A more detailed investigation of ticks from Madagascar for the presence of borreliae seems desirable. Considering the presence of DNA of borreliae in ticks, human infections seemed possible. Human infections seemed also possible for another spirochaete, Leptospira spp., considering the presence of this pathogen in rodents in Madagascar (Rahelinirina et al., 2010; Ralaiarijaona et al 2001, Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014). We therefore investigated human blood samples of febrile Madagascan patients collected before (Boone et al 2017, Marks et al, 2017). Leptospira spp. and Borrelia spp were absent in our investigations in human blood samples in spite of the use of highly sensitive screening systems. For the Borrelia spp. PCR, no details on the sensitivity in spiked samples have been published but successful prior application with whole blood has been described (Parola et al., 2011). In the quoted study, Borrelia spp.-specific PCR outperformed traditional microscopy for the detection of relapsing fever-associated Borrelia spp. by factors 2-7 (Parola et al., 2011). The sensitivity of the Leptospira spp. PCR has been reported to be 10 bacteria/ml in whole blood from spiking experiments (Stoddard et al., 2009). Since all samples were negative, we did not have infected blood of a patient as a positive control. But we included a positive control based on plasmids as detailed above. Samples inhibition was widely absent as confirmed by a previous assessment with the same samples (Boone et al., 2017). Of note, in spite of acceptable sensitivity of the PCR used, Leptospira spp. can only be identified in the bloodstream within the 1st week after the onset of symptoms (Levett, 2001). Later stages of the disease might therefore be missed. Enrollment took place at primary health centers, therefore it was assumed that disease onset was quite recent and illnesses were not accompanied by severe complications. Details on the duration of fever are, unfortunately, unavailable, an admitted limitation of the study. Application of serological tests for leptospirosis as well as for further pathogens would have been desirable. Unfortunately, only whole blood samples were collected and stored for the study, so serum samples were not available. In future studies, serum samples should be included.
Spirochaetes in Madagascar 8
Another undeniable limitation of the study which does not allow a clear-cut association of the results is the fact that ticks and human samples were collected without spatial and timely association. Nevertheless, the complete absence of Borrelia spp. DNA in human sample material suggests that relapsing fever associated with Borrelia spp. as causative agents of FUO does not play an important role in the Madagascan population in the highlands. The situation may be different in the south of Madagascar, were we found the B. theileri-like organisms. However, our data on the absence of borreliae in human samples are in line with the literature, according to which tick-borne relapsing fever is considered as an eradicated disease on Madagascar for decades (Rodhain & Fontenille, 1989). As early as in the 1950s, transmission of tick-borne relapsing fever in Madagascar had been associated with Ornithodoros (O) moubata ticks (Colas-Belcour et al., 1952). The vanishing of Madagascan relapsing fever is supposed to be associated with a sudden decline of O. moubata populations due to unknown reasons (Rodhain & Fontenille, 1989). The present detection of low quantities of DNA of Borrelia spp. not allowing for further typing in A. variegatum ticks may be attributed to the fact that these ticks were collected while blood feeding on Zebu cattle. In contrast to O. moubata, A. variegatum and R. microplus have not been described so far as vectors for Borrelia spp. in Madagascar. African R. microplus have been incriminated as vectors of B. theileri (Smith et al., 1978; Walker et al., 2003), while for A. variegatum, there are only Borrelia-like detections from the Ivory Coast (Ehounoud et al., 2016).
The negative PCRs for Leptospira spp. in the human blood samples of the assessed Madagascan FUO patients are in line with previous observations. Sero-prevalence screening from 2001 comprising 105 Madagascans described a single instance of a low positive titer, thus a non-specific reaction cannot be excluded (Ralaiarijaona et al., 2001). In the same study, attempts to detect Leptospira-specific DNA in kidney tissue of bats, pigs and Zebu cattle failed. In contrast to these early findings (Ralaiarijaona et al., 2001), Leptospira spp. DNA was later found in bats and small mammals in Madagascar (Lagadec et al., 2012; Desvars et al., 2013; Dietrich et al., 2014). However, human cases of leptospirosis in Madagascar were not confirmed (Desvars et al., 2013). The authors argued that the absence in humans might be due to a lack of diagnostic options in Madagascar (Desvars et al., 2013). However, the present study confirms that leptospirosis is at least not a dominating health issue in FUO patients in the Madagascan highlands. The restriction of the analysis to this regional setting is aof the study, not allowing for definite conclusions regarding the whole island. Future studies should cover a bigger number of sample collection sites and include Spirochaetes in Madagascar 9
serological investigations of human serum samples. In addition small mammals should be screened for Leptospira spp. at the respective sites of human sample collection.
Conclusion We detected Borrelia spp. DNA in Madagascan ticks and, for the first time, describe a Borrelia theileri like organism in Madagascar. The distribution of Borrelia spp. and their relevance for humans and animals is yet not clear. At least in the highlands, surrounding the capital Antananarivo, the relevance of Borrelia spp. and Leptospira spp. for humans seemslimited.
Acknowledgements Steffen Lohr and Annett Michel are gratefully acknowledged for excellent technical assistance.
Spirochaetes in Madagascar 10
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Keller, C., Krüger, A., Schwarz, N.G., Rakotozandrindrainy, R., Rakotondrainiarivelo, J.P., Razafindrabe, T., Derschum, H., Silaghi, C., Pothmann, D., Veit, A., Hogan, B., May, J., Girmann, M., Kramme, S., Fleischer, B., Poppert, S., 2016. High detection rate of Rickettsia africae in Amblyomma variegatum but low prevalence of anti-rickettsial antibodies in healthy pregnant women in Madagascar. Ticks Tick Borne Dis 7: 60-65
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Lagadec, E., Gomard, Y., Guernier, V., Dietrich, M., Pascalis, H., Temmam, S., Ramasindrazana, B., Goodman, S.M., Tortosa, P., Dellagi, K., 2012. Pathogenic Leptospira spp. in bats, Madagascar and Union of the Comoros. Emerg Infect Dis 18, 1696-1698.
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Ralaiarijaona, R.L., Bellenger, E., Chanteau, S., Roger, F., Pérolat, P., Rasolofo Razanamparany, V., 2001. Detection of leptospirosis reservoirs in Madagascar using the polymerase chain reaction technique. Arch Inst Pasteur Madagascar 67, 34-36.
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Spirochaetes in Madagascar 14
Figure legends
Figure 1. Phylogenetic analysis of 16S (rrs) sequences by maximum likelihood method. In the phylogenetic tree, GenBank, reference numbers, species designation and strain name (if available) are given. The strain investigated in the present study is marked with a black circle. It forms a cluster with Borrelia spp. described from the Ivory Coast and from Brazil. The tree with the highest log likelihood (-1679.1874) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap values). A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.1000)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 33.7027% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (scale bar). The analysis involved 19 nucleotide sequences. There were a total of 881 positions in the final dataset.
Figure 2. Phylogenetic analysis by Maximum Likelihood method of flagelling (flaB) sequences. In the phylogeny, GenBank reference numbers, species designation and strain name (if available) are given. As for the 16S rRNA gene tree, in the phylogeny inferred using flaB sequences, the strain investigated in this study (Mo063b) is part of a cluster with B. theileri (as sister clade), B. lonestari and B. miyamotoi. The tree with the highest log likelihood (-2823.7008) is shown. A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.2502)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (scale bar). The analysis involved 22 nucleotide sequences. There were a total of 571 positions in the final dataset.
Figure 3. Phylogenetic analysis by Maximum Likelihood method of gyrB sequences. In the phylogeny, GenBank reference numbers, species designation and strain name (if available) are given. Strain Mo063b forms a sister clade with B. miyamotoi. This is consistent with phylogenies based on different sequences such as 16S rRNA and flaB. In case of gyrB, no sequences for B. theileri or B. lonestari were found in the GenBank database. The tree with the highest log likelihood (-4074.7247) is shown. The percentage of trees in which the Spirochaetes in Madagascar 15
associated taxa clustered together is shown next to the branches. A discrete gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.2357)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 17 nucleotide sequences. There were a total of 908 positions in the final dataset.
77 98
NR 074865.1 Borrelia duttonii Ly NR 102961.1 Borrelia crocidurae Achema
gb|GU350706.1 Borrelia hispanica Sp3 gb|CP014349.1 Borrelia hermsii HS1 Browne Mountain gb|CP005851.1 Borrelia parkeri SLO 77
NR 102958.1 Borrelia turicatae 91E135 gb|CP005745.1 Borrelia coriaceae Co53 98
gb|CP004217.1 Borrelia miyamotoi FR64b gb|CP006647.2 Borrelia miyamotoi LBgb|AY682921.1 Borrelia lonestari MO2002-V2
70
gb|KF569941.1 Borrelia theileri KAT
86
Mo063b 16S
94 90
gb|KT364342.1 Borrelia sp. TCI301 (Ivory Coast) gb|EF141021.1 Borrelia sp.
gb|CP009117.1 Borrelia valaisiana Tom4006 NR 074662.1 Borrelia afzelii HLJ01
100
NR 074854.1 Borrelia bavariensis
82
NR 102956.1 Borrelia bissettiae
75 82
gb|CP009656.1 Borrelia burgdorferi sensu stricto
0.005
Spirochaetes in Madagascar 16
gb|CP000049.1 Borrelia turicatae 91E135 98
gb|EU492387.1 Borrelia sp. IA-1
52
gb|CP005851.1 Borrelia parkeri SLO
46
gb|CP005745.1 Borrelia coriaceae Co53
26
gb|CP011060.1 Borrelia hermsii CC1
53
Candidatus Borrelia kalaharica gb|CP005830.1 Borrelia anserina BA2
75
gb|CP004217.1 Borrelia miyamotoi FR64b gb|AY850063.1 Borrelia lonestari MO2002-V1
98 97
Mo063b
100 92
gb|KF569936.1 Borrelia theileri KAT
gb|GU357612.1 Borrelia hispanica strain 33
gb|CP000993.1 Borrelia recurrentis
100
gb|CP003426.1 Borrelia crocidurae Achema 95
gb|CP000976.1 Borrelia duttonii Ly gb|JF708951.1 Borrelia microti Abyek
gb|KF422815.1 Borrelia turcica IST7 gb|AE000783.1 Borrelia burgdorferi B31 gb|CP002933.1 Borrelia afzelii
46 100
gb|CP009117.1 Borrelia valaisiana Tom4006 64
gb|CP000013.1 Borrelia bavariensis PBi 100
gb|L42885.1 Borrelia garinii Ip90
0.02
Spirochaetes in Madagascar 17
99
gb|AY934618.1 Borrelia turicatae FCB
100 37
gb|CP005851.1 Borrelia parkeri SLO gb|CP011060.1 Borrelia hermsii CC1
91
gb|CP005745.1 Borrelia coriaceae Co53 89
gb|CP005829.1 Borrelia anserina BA Mo063b
66
dbj|AB900799.1 Borrelia miyamotoi HT31
99 100
gb|CP006647.2 Borrelia miyamotoi LB-200 gb|CP000993.1 Borrelia recurrentis 100
gb|CP003426.1 Borrelia crocidurae gb|CP000976.1 Borrelia duttonii Ly
dbj|AB473537.1 Borrelia sp. Tortoise14 gb|KP067819.1 Borrelia turcica KZ11
100 85
dbj|AB529352.1 Borrelia sp.
gb|CP009117.1 Borrelia valaisiana 100
gb|CP002746.1 Borrelia bissettii DN
0.05
Spirochaetes in Madagascar 18
Table 1: Results of the typing PCRs with consecutive Sanger sequencing of samples with positive results in the Borrelia spp. screening PCR. PCRs for p41, uvrA, and pepX were only performed if sequencing of flaB, the 16S rRNA gene and gyrB led to non-conclusive results. If the more sensitive PCRs for flaB, p41, the 16S rRNA gene, uvrA, and pepX failed or led to non-conclusive results in sequencing, the less sensitive gyrB PCR was not performed.
Sample
Tick species
I.D.
Ct-value in the 16S
Results of the typing PCRs targeting the following genes:
rRNA gene screening PCR
1
Amblyomma variegatum
39
flaB (simplex)
p41 (duplex)
16S rRNA gene
uvrA
pepX
gyrB
Negative
Negative
Negative
Negative
Negative
n.a.
1
2
Amblyomma variegatum
38
Negative
Negative
Negative
Positive
Negative
n.a.
3
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
4
Amblyomma variegatum
38
Negative
Negative
Negative
Negative
Negative
n.a.
5
Amblyomma variegatum
37
negative
Negative
Negative
Negative
Negative
n.a.
6
Amblyomma variegatum
42
Negative
Negative
Negative
Negative
Negative
n.a.
7
Amblyomma variegatum
41
Negative
Negative
Negative
Negative
Negative
n.a.
8
Amblyomma variegatum
36
Negative
Negative
Negative
Negative
Negative
n.a.
9
Amblyomma variegatum
38
Negative
Negative
Negative
Positive1
Negative
n.a.
10
Amblyomma variegatum
44
Negative
Negative
Negative
Negative
Negative
n.a.
11
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
12
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
13
Amblyomma variegatum
37
Negative
Negative
Negative
Negative
Negative
n.a.
14
Rhipicephalus microplus
39
Positive1
Negative
Positive1
Negative
Negative
n.a.
15
Amblyomma variegatum
40
Negative
Negative
Negative
Negative
Negative
n.a.
16
Amblyomma variegatum
37
Negative
Negative
Positive1
Negative
Negative
n.a.
1
17
Amblyomma variegatum
36
Negative
Negative
Negative
Positive
Negative
n.a.
18
Amblyomma variegatum
41
Negative
Negative
Negative
Negative
Negative
n.a.
19
Amblyomma variegatum
36
Negative
Negative
Negative
Negative
Negative
n.a.
20
Amblyomma variegatum
39
Negative
Negative
Negative
Negative
Negative
n.a.
21
Amblyomma variegatum
38
Negative
Negative
Positive2
Negative
Negative
n.a.
1
22
Amblyomma variegatum
43
Negative
Negative
Positive
Negative
Negative
n.a.
23
Rhipicephalus microplus
22
Positive3
n.a.
Positive3
n.a.
n.a.
Positive3
Spirochaetes in Madagascar 19
1
Sequence analysis failed due to poor sequence quality. 2Sequence analysis suggested B. afzelii (99% identity (1 bp-mismatch), 100% query coverage), but only after 150 bp with poor sequence quality
were removed. 3Sequence analyses in figures 1-3 and tables 2-4. n.a. = not applicable.
Spirochaetes in Madagascar 20
Table 2: Estimates of Evolutionary Divergence between 16SrRNA Sequences. The analysis involved 19 nucleotide sequences. There were a total of 881 positions in the final dataset.
Mo063b 16SrRNA EF141021.1 Borrelia sp. Brazil
0,000
KT364342.1 Borrelia sp. Ivory Coast
0,000
0,000
KF569941.1 Borrelia theileri
0,003
0,003
0,003
AY682921.1 Borrelia lonestari
0,008
0,008
0,008
0,007
NR_102958.1 Borrelia turicatae
0,014
0,014
0,014
0,013
0,013
CP004217.1 Borrelia miyamotoi
0,013
0,013
0,013
0,011
0,014
0,015
CP006647.2 Borrelia miyamotoi
0,015
0,015
0,015
0,014
0,016
0,015
0,002
CP005851.1 Borreliaparkeri
0,015
0,015
0,015
0,014
0,014
0,001
0,016
0,016
CP005745.1 Borrelia coriaceae
0,016
0,016
0,016
0,015
0,015
0,009
0,017
0,017
0,010
CP014349.1 Borrelia hermsii
0,016
0,016
0,016
0,015
0,017
0,007
0,015
0,015
0,008
0,011
GU350706.1 Borrelia hispanica
0,015
0,015
0,015
0,014
0,014
0,006
0,016
0,016
0,007
0,010
0,008
NR_074865.1 Borrelia_duttonii
0,016
0,016
0,016
0,015
0,015
0,007
0,017
0,017
0,008
0,011
0,009
0,001
NR_102961.1 Borrelia crocidurae
0,016
0,016
0,016
0,015
0,015
0,007
0,017
0,017
0,008
0,011
0,009
0,001
0,000
CP009117.1 Borrelia valaisiana
0,034
0,034
0,034
0,033
0,035
0,029
0,035
0,038
0,028
0,034
0,031
0,033
0,034
0,034
NR_074854.1 Borrelia bavariensis
0,035
0,035
0,035
0,035
0,038
0,034
0,040
0,042
0,033
0,039
0,036
0,035
0,036
0,036
0,009
NR_102956.1 Borrelia bissetti
0,037
0,037
0,037
0,036
0,039
0,033
0,039
0,041
0,031
0,037
0,035
0,035
0,036
0,036
0,008
0,003
NR_074662.1 Borrelia afzelii
0,037
0,037
0,037
0,036
0,039
0,033
0,036
0,039
0,031
0,038
0,035
0,034
0,035
0,035
0,008
0,008
0,006
CP009656.1 Borrelia burgdorferi
0,037
0,037
0,037
0,036
0,039
0,033
0,039
0,041
0,031
0,037
0,035
0,035
0,036
0,036
0,009
0,005
0,001
0,007
Spirochaetes in Madagascar 21
Table 3: Estimates of Evolutionary Divergence between flaB Sequences. The analysis involved 22 nucleotide sequences. There were a total of 601 positions in the final dataset. Mo063b flaB KF569936.1 Borrelia theileri
0,013
AY850063.1 Borrelia lonestari
0,043
0,040
CP004217.1 Borrelia miyamotoi
0,081
0,085
0,089
CP005830.1 Borrelia anserina
0,099
0,095
0,118
0,085
KT970516.1 Candidatus B. kalaharica
0,110
0,102
0,114
0,094
0,059
GU357612.1 Borrelia hispanica
0,114
0,114
0,134
0,110
0,095
0,089
CP005745.1 Borrelia coriaceae
0,118
0,114
0,118
0,102
0,079
0,079
0,102
CP000049.1 Borrelia turicatae
0,120
0,116
0,120
0,087
0,066
0,063
0,102
0,072
CP003426.1 Borrelia crocidurae
0,120
0,116
0,140
0,122
0,107
0,100
0,025
0,122
0,110
EU492387.1 Borrelia
0,120
0,116
0,120
0,092
0,068
0,065
0,104
0,074
0,010
0,105
CP000993.1 Borrelia recurrentis
0,122
0,122
0,138
0,120
0,105
0,102
0,022
0,116
0,112
0,013
0,106
CP000976.1 Borrelia duttonii
0,122
0,122
0,138
0,120
0,105
0,102
0,019
0,112
0,112
0,013
0,106
0,003
JF708951.1 Borrelia microti
0,124
0,124
0,136
0,122
0,103
0,104
0,020
0,114
0,114
0,015
0,108
0,005
0,002
CP011060.1 Borrelia hermsii
0,124
0,120
0,130
0,095
0,071
0,072
0,115
0,085
0,070
0,118
0,068
0,121
0,120
0,123
CP005851.1 Borrelia parkeri
0,128
0,120
0,124
0,092
0,072
0,065
0,100
0,066
0,012
0,112
0,015
0,110
0,110
0,112
0,070
KF422815.1 Borrelia turcica
0,160
0,156
0,173
0,141
0,124
0,126
0,127
0,139
0,127
0,134
0,123
0,142
0,142
0,144
0,134
0,130
AE000783.1 Borrelia burgdorferi
0,176
0,178
0,198
0,182
0,159
0,157
0,167
0,178
0,159
0,176
0,161
0,182
0,180
0,182
0,172
0,165
0,157
CP002933.1 Borrelia afzelii
0,174
0,172
0,196
0,178
0,145
0,145
0,167
0,167
0,157
0,174
0,159
0,184
0,184
0,186
0,159
0,163
0,153
0,063
CP009117.1 Borrelia valaisiana
0,176
0,178
0,198
0,176
0,159
0,153
0,161
0,167
0,155
0,172
0,153
0,178
0,178
0,180
0,166
0,161
0,145
0,059
0,043
CP000013.1 Borrelia bavariensis
0,186
0,189
0,211
0,197
0,168
0,165
0,178
0,178
0,170
0,185
0,172
0,191
0,191
0,193
0,187
0,172
0,166
0,071
0,067
0,059
L42885.1 Borrelia garinii
0,184
0,186
0,209
0,191
0,151
0,155
0,176
0,171
0,159
0,178
0,161
0,189
0,189
0,191
0,166
0,165
0,153
0,069
0,052
0,048
0,017
Spirochaetes in Madagascar 22
Table 4: Estimates of Evolutionary Divergence between gyrB Sequences. The analysis involved 19 nucleotide sequences. There were a total of 908 positions in the final dataset. Mo063b gyrB AB900799.1 Borrelia miyamotoi
0,081
CP006647.2 Borrelia miyamotoi
0,087
0,008
CP011060.1 Borrelia hermsii
0,110
0,101
0,109
AY934616.1 Borrelia turicatae
0,112
0,110
0,118
0,069
CP005851.1 Borrelia parkeri
0,112
0,108
0,116
0,068
0,013
AY934618.1 Borrelia turicatae
0,114
0,112
0,120
0,069
0,002
0,013
CP005745.1 Borrelia coriaceae
0,126
0,114
0,122
0,080
0,078
0,078
0,078
CP005829.1 Borrelia anserina
0,134
0,118
0,122
0,106
0,100
0,098
0,102
0,114
CP003426.1 Borrelia crocidurae
0,141
0,136
0,139
0,119
0,130
0,131
0,130
0,134
0,146
CP000976.1 Borrelia duttonii
0,143
0,137
0,140
0,121
0,131
0,132
0,131
0,136
0,147
0,001
CP000993.1 Borrelia recurrentis
0,145
0,140
0,143
0,123
0,134
0,135
0,134
0,138
0,151
0,006
0,004
KP067819.1 Borrelia turcica
0,155
0,134
0,136
0,139
0,141
0,141
0,141
0,140
0,172
0,145
0,146
0,150
AB529352.1 Borrelia sp.
0,157
0,135
0,138
0,140
0,149
0,149
0,149
0,143
0,173
0,144
0,146
0,150
0,017
AB473537.1 Borrelia sp.
0,161
0,139
0,140
0,150
0,156
0,159
0,156
0,150
0,177
0,144
0,146
0,150
0,040
0,042
CP009117.1 Borrelia valaisiana
0,204
0,210
0,205
0,211
0,212
0,206
0,210
0,202
0,232
0,209
0,208
0,209
0,207
0,205
0,218
CP002746.1 Borrelia bissettiae
0,208
0,222
0,217
0,218
0,214
0,212
0,213
0,211
0,244
0,210
0,209
0,210
0,218
0,219
0,219
0,063
CP002933.1 Borrelia afzelii
0,216
0,214
0,209
0,221
0,213
0,208
0,211
0,206
0,241
0,218
0,219
0,221
0,211
0,215
0,225
0,052
0,064
CP000013.1 Borrelia bavariensis
0,214
0,233
0,229
0,223
0,219
0,215
0,219
0,219
0,248
0,215
0,214
0,215
0,227
0,225
0,235
0,061
0,063
0,059
Spirochaetes in Madagascar 23