Identification of tick species and disseminate pathogen using hemolymph by MALDI-TOF MSby Amina Yssouf, Lionel Almeras, Jean-Michel Berenger, Maureen Laroche, Didier Raoult, Philippe Parola

Ticks and Tick-borne Diseases


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Accepted Manuscript

Title: Identification of tick species and disseminate pathogen using hemolymph by MALDI-TOF MS

Author: Amina Yssouf Lionel Almeras Jean-Michel Berenger

Maureen Laroche Didier Raoult Philippe Parola

PII: S1877-959X(15)00081-3


Reference: TTBDIS 481

To appear in:

Received date: 15-1-2015

Revised date: 30-3-2015

Accepted date: 28-4-2015

Please cite this article as: Yssouf, A., Almeras, L., Berenger, J.-M., Laroche,

M., Raoult, D., Parola, P.,Identification of tick species and disseminate pathogen using hemolymph by MALDI-TOF MS, Ticks and Tick-borne Diseases (2015),

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Ac ce pte d M an us cri pt 1

Identification of tick species and disseminate pathogen using hemolymph by 1


Amina Yssouf1, Lionel Almeras1, Jean-Michel Berenger1, Maureen Laroche1, Didier Raoult1, 3

Philippe Parola1*4 5 1Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales 6

Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO 7

Collaborative Center for Rickettsioses and Other Arthropod-Borne Bacterial Diseases, Faculté 8 de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France.9 10 *Address for correspondence: Philippe Parola, Unité de Recherche en Maladies Infectieuses 11 et Tropicales Emergentes (URMITE), Faculté de Médecine, 27 bd Jean Moulin, 13385 12

Marseille cedex 5, France. Phone: 33 (0) 4 91 32 43 75. Fax: 33 (0) 4 91 83 03 90. E-mail 13 address: philippe.parola@univ-amu.fr14 15

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Background. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass 16

Spectrometry (MALDI-TOF MS) is increasingly emerging tool for identification of 17 arthropods including tick vectors using whole or body part of specimens. The challenges of 18 the present study were to assess MALDI-TOF MS profiling for the both identification of tick 19 species and Rickettsia spp in infected ticks using hemolymph as protein mixture. 20

Methods. Firstly, hemolymph protein mixture from legs of 5 tick species, Rh picephalus 21 sanguineus, Rhipicephalus bursa, Dermacentor marginatus, Hyalomma marginatum rufipes 22 and Amblyomma variegatum infected by Rickettsia africae were submitted to MALDI-TOF 23

MS to assess tick species identification ability. Secondly, hemolymph MS spectra from Rh. 24 sanguineus infected or not by Rickettsia c. conorii were compared to detect protein profiles 25 changes. Finally, leg hemolymph MS spectra from new specimens of the 5 tick species were 26 tested blindly including ticks infected by R. c. conorii. Discriminating mass peaks 27 distinguishing the R. c. conorii infected and non-infected Rh sanguineus were determined.28

Results. Consistent and reproducible MS profiles were obtained into each tick species. 29

Comparison of MS spectra revealed distinct hemolymph protein profiles according to tick 30 species. MS spectra changes were observed between hemolymphs from R. c. conorii-infected 31 and non-infected Rh. sanguineus specimens, revealing 17 discriminating mass peaks. 32

Clustering analysis based on MS protein profiles highlighted that hemolymph samples were 33 grouped according to tick species. All tick hemolymph samples blindly tested against our 34 home-made arthropod MS reference database were correctly identified at the species 35 distinguishing also R. c. conorii-infected from Rickettsia-free Rh. sanguineus specimens.36

Conclusion. The present study demonstrated the use of hemolymph MS profiles for dual 37 identification of tick species and associated pathogens. This concomitant identification could 38

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Ac ce pte d M an us cri pt 3 be helpful for tick entomological diagnosis, notably for specimens removed directly on 39 patients.40

Key words: MALDI-TOF mass spectrometry; ticks; Rickettsia; entomological diagnosis. 41 42 43

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Ticks are obligate hematophagous arthropods which have parasitized every class of 44 vertebrates in almost world areas (Parola and Raoult, 2001). These ectoparasites may 45 occasionally feed on humans and transmit a variety of pathogenic agents including viruses, 46 protozoa and bacteria like Rickettsia spp (Heyman et al., 2010; Hubalek and Rudolf, 2012; 47

Parola et al., 2013). Several tick species are known as vectors of Rickettsia spp (Parola et al., 48 2013). Rhipicephalus sanguineus appear as the main tick vector and potential reservoir of 49

Rickettsia conorii conorii, the causative agent of the Mediteranean spotted fever (MSF) 50 (Parola et al., 2013). MSF is endemic in the Mediterranean area, including northern Africa 51 and southern Europe. Thus, the identification of ticks at the species level and the detection of 52

Rickettsia-associated pathogen is indispensable for vectors monitoring and control of tick-53 borne rickettsiose in these regions (Parola et al., 2013).54

Today, the reference methods for the tick species identification are generally determined 55 either by using morphological criteria or by molecular methods (Tijsse-Klasen et al., 2014), 56 and the routine method for the Rickettsia species identification in ticks remains the molecular 57 biology (Parola and Raoult 2001). The morphological identification of ticks is performed with 58 taxonomic keys for endemic species according to geographical areas (Yssouf et al., 2013a). 59

This taxonomic classification presents several limitations such as the availability of 60 identification keys, an entomological expertise, and is powerless to distinguish sibling species 61 (Karger et al., 2012). Moreover, this method could be insufficient for identification of 62 damaged or engorged specimens by the loss of morphological criteria (Parola and Raoult 63 2001). In addition, at immature tick stages, the lack of morphological criteria could prevent 64 the accurate identification. Therefore, molecular approach remains the better strategy for 65 identification of both tick species and associated Rickettsia spp (Parola and Raoult 2001).66