Open Access

Detection of Orientia sp. DNA in rodents from Asia, West Africa and Europe

  • Jean François Cosson1Email author,
  • Maxime Galan1,
  • Emilie Bard2,
  • Maria Razzauti1,
  • Maria Bernard3,
  • Serge Morand4, 5,
  • Carine Brouat6,
  • Ambroise Dalecky6, 8,
  • Khalilou Bâ9,
  • Nathalie Charbonnel1 and
  • Muriel Vayssier-Taussat7
Parasites & Vectors20158:172

https://doi.org/10.1186/s13071-015-0784-7

Received: 24 December 2014

Accepted: 6 March 2015

Published: 21 March 2015

Abstract

Orientia bacterium is the agent of the scrub typhus, a seriously neglected life-threatening disease in Asia. Here, we report the detection of DNA of Orientia in rodents from Europe and Africa. These findings have important implications for public health. Surveillance outside Asia, where the disease is not expected by sanitary services, needs to be improved.

Keywords

Scrub typhus Zoonoses Emerging disease Rodent-borne disease Metagenomics

Findings

Orientia tsutsugamushi is the only known species belonging to the Orientia bacterial genus. The bacterium causes scrub typhus in humans. It is an obligate intracytosolic bacterium that is transmitted during feeding by larval trombiculid mites, and is hosted by rodents [1]. In Asia, approximately one million cases of scrub typhus occur annually, where it is probably one of the most underdiagnosed and underreported febrile illnesses requiring hospitalization [2], with an estimated 10% fatality rate unless treated appropriately. Formerly thought to be geographically restricted to Asia [3], Orientia was recently identified in sick patients from the Arabian Peninsula [4] and Chile [5]. Miscellaneous reports of scrub typhus-like illness have previously questioned the presence of the bacterium in the Congo [6] and Cameroon [7].

Methods

In order to generate a global picture of zoonotic bacteria that are likely to be harboured by rodents, we applied a metagenomic approach using spleen samples of 1334 rodents from France (Ardennes region), Senegal (along the Senegal River) and Thailand (northern and north-eastern provinces of Loei, Nan and Buriram). Rodents were trapped in both natural habitats and villages within rural landscapes. They were euthanized by cervical dislocation and dissected. In order to prevent cross contamination during dissection, we systematically alternated the use of two sets of dissecting instruments. After dissecting a rodent, the set used was immersed in bleach then water and let in alcohol, while we dissected another rodent with the other set [8]. Spleens were placed in RNAlater® storage solution (Sigma-Aldrich, Saint Louis, MO, USA) then stored at -20°C until further analysis. Genomic DNA was then extracted from the spleen using the DNeasy® 96 Tissue Kit (Qiagen, Germany). Spleen DNA samples were screened for the presence of bacteria using universal primers targeting the hyper variable region V4 of the 16S rRNA gene (251 bp) via Illumina MiSeq (Illumina) sequencing. The V4 region has been proven to have excellent taxonomic resolution at the genus level [9]. A multiplexing strategy enabled the identification of bacterial genera in each individual sample. We followed the method described in [10] to perform PCR amplification, indexing, pooling, multiplexing and de-multiplexing and finally taxonomic identification using the SILVA SSU Ref NR 119 database as a reference (http://www.arb-silva.de/projects/ssu-ref-nr/). In total we performed four different MiSeq runs, two with rodents from France (N = 557), one with rodents from Asia (N = 423) and one with rodents from Africa (N = 354). For each run, we systematically used negative controls (of DNA extraction and PCR) and none were positive for Orientia. Though we did not use positive controls for Orientia because the bacterium was not expected in European and African samples. We used positive controls for other bacterial genus like Leptospira, Borrelia, Bartonella and Mycoplasma, and all were found positive for the expected bacterial genera.

Results and discussion

From over a total of 1334 rodents tested, 110 were found positive for Orientia (Table 1). As expected, Orientia was detected in five sampled rodent species from Thailand: Rattus tanezumi (5 positives/67 tested), Rattus exulans (1/81), Bandicota savilei (2/26), Berylmys bowersi (1/17), and Leopoldamys edwardsi (1/10). More surprisingly, the bacterium was also detected in numerous rodents collected from both France and Senegal. In Senegal, Orientia sequences were only identified in the exotic house mouse (Mus musculus domesticus) (48 positives/207 tested), while the sympatric endemic multimammate rats (Mastomys erythroleucus) were all found to be negative (0/147), suggesting introduction of the bacterium via the exotic rodent. In France, Orientia sp. was detected in three rodent species: Myodes glareolus (44/302), Arvicola scherman (2/64) and Microtus arvalis (6/49). Positive for Orientia were trapped inside human houses in Asia and Africa, and in close proximity to human dwellings in Asia and France.
Table 1

Numbers of rodent tested and found positive for Orientia sp. for the different rodent species sampled in France, Senegal and Asia

Geographic area

Rodent species

Number tested

Number positive

France

Myodes glareolus

302

44

 

Arvicola scherman

64

2

 

Microtus arvalis

49

6

 

Microtus agrestis

7

0

 

Microtus subterraneus

4

0

 

Apodemus sylvaticus

67

0

 

Apodemus flavicolis

34

0

 

Rattus norvegicus

30

0

Senegal

Mus musculus

207

48

 

Mastomys erythroleucus

147

0

Asia

Bandicota indica

20

0

 

Bandicota savilei

26

2

 

Berylmys berdmorei

19

0

 

Berylmys bowersi

17

1

 

Chiropodomys gliroides

2

0

 

Hapalomys delacouri

1

0

 

Leopoldamys edwardsi

10

1

 

Leopoldamys sabanus

1

0

 

Maxomys surifer

15

0

 

Menetes berdmorei

1

0

 

Mus caroli

14

0

 

Mus cervicolor

17

0

 

Mus cookii

11

0

 

Mus fragilicauda

1

0

 

Niviventer fulvescens

17

0

 

Rattus argentiventer

1

0

 

Rattus exulans

81

1

 

Rattus losea

32

0

 

Rattus nitidus

1

0

 

Rattus tanezumi

67

5

Total

 

1334

110

In rodents, blood, spleen and other organs are routinely used for Orientia PCR detection [11], although one should notice that such assay is limited to the time window of rickettsemia, i.e. when the bacteria are infecting macrophages in peripheral blood. In our experiments we targeted the spleen because this organ is known to act primarily as a blood filter and then appears appropriate for detecting bacteria infecting blood cells. However, although there are many lines of evidence that Orientia may chronically infect humans and rodents, the persistence of the bacteria in organs, and spleen in particular, is currently poorly known [12]. Thus we cannot discard the possibility of false negatives in our assays.

Sequence analyses revealed that the Orientia sequences identified in this study were between 100 to 94.4% identical to GenBank-published Orientia sequences isolated from humans, mites and rodents in Asia. All sequences shared only 90.8 to 86.5% identity with GenBank-published Rickettsia, the closest bacterial genus to Orientia, thus consolidating our finding on the presence of Orientia in Europe and Africa. We also performed phylogenetic analyses of both the haplotypes identified in this study, and those from GenBank databases, using the neighbor-joining method [13]. Bootstrap analysis was performed on 1,000 replicates. Haplotypes from this study clustered strongly within the Orientia phylogroup and were clearly separated from the Rickettsia phylogroup. Asian haplotypes were distributed amongst Genbank haplotypes from Asia and Arabian peninsula (Chuto haplotype), African haplotypes fell with the Chuto haplotype, whereas European haplotypes clustered into a new basal phylogroup (Figure 1).
Figure 1

Phylogenetic tree based on the V4 region of the 16S rRNA gene. GenBank accession numbers are indicated. Only different haplotypes were shown. A complete list of sequences uploaded to GenBank can be provided upon request. Numbers beside branches indicate bootstrap values (>80). O: Orientia; R: Rickettsia; N: Neorickettsia; A: Anaplasma. The tree was rooted with the phylogenetically closest genus Anaplasma and Neorickettsia. Scale bar indicates evolutionary distances. Samples sequenced in the present study are marked with _*.

Conclusion

We have established the presence of Orientia DNA in spleens of rodents from Thailand, as was expected, but also in rodents collected from France and Senegal. In Asia, scrub typhus is considered as a seriously neglected life-threatening disease despite the fact that this ancient disease has been recognized within this region for many years. Our findings, together with those from other recent studies [4,5] suggest that in locales outside of Asia where the disease is not on the public health service radar, surveillance needs to be improved.

Ethical approval

Animals have been treated in accordance with the guidelines of the European Union legislation (Directive 86/609/EEC). The CBGP laboratory has received the approval (no. B 34–169–1) from the regional Head of Veterinary Service (Hérault, France), for the sampling and killing of rodents and the harvesting of their tissues. Dr Cosson has personally received the agreement “certificate d'autorisation d’expérimenter sur animaux vivants” (i.e. “certificate of authorization to experiment on live animals”) (no. C34–105) by the French administration.

Declarations

Acknowledgments

We thank Mamadou Kane, Christophe A. Diagne, Aliou Sow, Youssou Niang and Mamoudou Diallo for their help during field sampling in Senegal, Hélène Vignes for her assistance with the MiSeq sequencing, and Sylvain Piry, Alexandre Dehne-Garcia and Marie Pagès for their help with the bioinformatic analysis. This study was funded by the INRA metaprogramme PATHO-ID and by the ANR ENEMI (ANR-11-JSV7-0006). Rodents were collected in the course of studies funded by the EU grant FP7-261504 EDENext, the ADEME (APR PREST 2009) and the ANR Biodivhealthsea (ANR-11-CEPL-0002). This study was partially funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext289 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and don’t necessarily reflect the views of the European Commission. This work was also supported by the COST Action TD1303 (EurNegVec).

Authors’ Affiliations

(1)
INRA, CBGP
(2)
INRA, EpiA
(3)
INRA, GABI, Sigenae
(4)
CNRS-CIRAD, Centre d’Infectiologie Christophe Mérieux du Laos
(5)
Department of Helminthology, Faculty of Tropical Medicine, Mahidol University
(6)
Ird, CBGP
(7)
INRA, Bipar
(8)
IRD, Aix Marseille Université, LPED (UMR IRD-AMU)
(9)
Ird, CBGP, Campus ISRA/IRD de Bel Air

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Copyright

© Cosson et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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