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

doi:10.1016/j.ttbdis.2015.04.013

Ticks and Tick-borne Diseases

Volume 6, Issue 5, July 2015, Pages 579-586

https://doi.org/10.1016/j.ttbdis.2015.04.013 Get rights and content

Abstract

Background

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is increasingly emerging tool for identification of arthropods including tick vectors using whole or body part of specimens. The challenges of the present study were to assess MALDI-TOF MS profiling for the both identification of tick species and Rickettsia spp. in infected ticks using hemolymph as protein mixture.

Methods

Firstly, hemolymph protein mixture from legs of 5 tick species, Rhipicephalus sanguineus, Rhipicephalus bursa, Dermacentor marginatus, Hyalomma marginatum rufipes and Amblyomma variegatum infected by Rickettsia africae were submitted to MALDI-TOF MS to assess tick species identification ability. Secondly, hemolymph MS spectra from Rh. sanguineus infected or not by Rickettsia c. conorii were compared to detect protein profiles changes. Finally, leg hemolymph MS spectra from new specimens of the 5 tick species were tested blindly including ticks infected by R. c. conorii. Discriminating mass peaks distinguishing the R. c. conorii infected and non-infected Rh sanguineus were determined.

Results

Consistent and reproducible MS profiles were obtained into each tick species. Comparison of MS spectra revealed distinct hemolymph protein profiles according to tick species. MS spectra changes were observed between hemolymphs from R. c. conorii-infected and non-infected Rh. sanguineus specimens, revealing 17 discriminating mass peaks. Clustering analysis based on MS protein profiles highlighted that hemolymph samples were grouped according to tick species. All tick hemolymph samples blindly tested against our home-made arthropod MS reference database were correctly identified at the species distinguishing also R. c. conorii-infected from Rickettsia-free Rh. sanguineus specimens.

Conclusion

The present study demonstrated the use of hemolymph MS profiles for dual identification of tick species and associated pathogens. This concomitant identification could be helpful for tick entomological diagnosis, notably for specimens removed directly on patients.

Introduction

Ticks are obligate hematophagous arthropods which have parasitized every class of vertebrates in almost world areas (Parola and Raoult, 2001). These ectoparasites may occasionally feed on humans and transmit a variety of pathogenic agents including viruses, protozoa and bacteria like Rickettsia spp. (Heyman et al., 2010, Hubalek and Rudolf, 2012, Parola et al., 2013). Several tick species are known as vectors of Rickettsia spp. (Parola et al., 2013). Rhipicephalus sanguineus appear as the main tick vector and potential reservoir of Rickettsia conorii conorii, the causative agent of the Mediterranean spotted fever (MSF) (Parola et al., 2013). MSF is endemic in the Mediterranean area, including northern Africa and southern Europe. Thus, the identification of ticks at the species level and the detection of Rickettsia-associated pathogen is indispensable for vectors monitoring and control of tick-borne rickettsiose in these regions (Parola et al., 2013).

Today, the reference methods for the tick species identification are generally determined either by using morphological criteria or by molecular methods (Tijsse-Klasen et al., 2014), and the routine method for the Rickettsia species identification in ticks remains the molecular biology (Parola and Raoult, 2001). The morphological identification of ticks is performed with taxonomic keys for endemic species according to geographical areas (Yssouf et al., 2013a). This taxonomic classification presents several limitations such as the availability of identification keys, an entomological expertise, and is powerless to distinguish sibling species (Karger et al., 2012). Moreover, this method could be insufficient for identification of damaged or engorged specimens by the loss of morphological criteria (Parola and Raoult, 2001). In addition, at immature tick stages, the lack of morphological criteria could prevent the accurate identification. Therefore, molecular approach remains the better strategy for identification of both tick species and associated Rickettsia spp. (Parola and Raoult, 2001). Nevertheless, DNA-based methods entail maximum time-consuming, are cost expensive for the consumables and requires information on gene sequences.

This last decade, a proteomic approach, the Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technology was tested for identification and classification of some arthropod groups like Drosophilia (Feltens et al., 2010). More recently, MALDI-TOF MS was successfully employed for identification of hematophagous arthropods like Culicoides biting midges (Kaufmann et al., 2012), mosquitoes (Müller et al., 2013, Yssouf et al., 2013b, Yssouf et al., 2014a), tsetse flies (Hoppenheit et al., 2013), sand flies (Dvorak et al., 2014) and fleas (Yssouf et al., 2014b). This innovative proteomic strategy was presented as an alternative tool to discriminate tick species using whole specimens or body parts (Karger et al., 2012) such as legs only (Yssouf et al., 2013a). A recent pioneering study underlined that the submission of tick leg protein extracts to MALDI-TOF MS may allow one-step identification of tick species and tick-borne bacterial pathogens, including recurrent-fever Borrelia infecting Ornithodoros ticks (Fotso Fotso et al., 2014). More recently, MALDI-TOF MS was successfully applied for distinction of the presence of R. c. conorii or R. slovaca pathogens in Rhipicephalus sanguineus and Dermacentor marginatus using tick legs (Yssouf et al., 2015).

Ticks possess a circulatory system containing a circulating fluid, termed hemolymph. The hemolymph assay has long been used in live ticks to detect bacteria, particularly the spotted fever group Rickettsia (Burgdorfer, 1970). The distal portion of a leg is amputated, and the hemolymph that appears at the site can be smeared onto a microscope slide, stained by Gimenez and Giemsa staining or immuno-detection methods, and examined for the presence of bacteria (Parola and Raoult, 2001).

Thus, the aim of the present study was to investigate the use of MALDI-TOF MS to simultaneously identify the tick species and the presence of associated pathogens by using protein mixture of hemolymphs. The hemolymph MS protein profiles from 5 tick species were compared to determine the reproducibility and uniqueness of their respective MS protein profiles, taking into account their Rickettsia infectious status. Subsequently, the hemolymph MS spectra from the 5 tick species were added to our home-made arthropod reference MS spectra database, which was then used to identify new tick specimens by blind tests.

Section snippets

Tick samples used for hemolymph extraction

Five adult tick species including Rhipicephalus sanguineus, Rhipicephalus bursa, Dermacentor marginatus, Hyalomma marginatum rufipes and Amblyomma variegatum infected by Rickettsia africae, laboratory-reared at the URMITE (Socolovschi et al., 2009), were used in the present study. All these tick species were collected in the field and laboratory breed. For detail about origins and collection time, see Table 1. The D. marginatus specimens were breaded in environmental incubator at 19 °C with a

Molecular detection of R. c. conorii and R. africae in ticks

Detection of R. c. conorii and R. africae infections from Rh. sanguineus and A. variegatum specimens, respectively, was performed by qPCR (Ogata et al., 2000, Yssouf et al., 2014c). All laboratory reared Rh. sanguineus specimens (7/7) predicted to be R. c. conorii-infected were confirmed by molecular biology. Additionally, the absence of R. c. conorii was confirmed in all (7/7) laboratory-reared Rh. sanguineus ticks predicted not to be infected. About ticks from A. variegatum species, R. africae

Discussion

Recent studies evidenced that MALDI-TOF MS is now a relevant, rapid and at low cost alternative method for identification of some arthropod groups, including several tick species (Yssouf et al., 2013a, Müller et al., 2013). For tick, it was demonstrated that MS spectra from leg protein extracts enabled to identify accurately specimens at the species level (Yssouf et al., 2013b). More recently, the proof-of-concept to identify simultaneously tick species and borne-pathogen by applying this

Conclusions

The present study demonstrated the possibility to use tick hemolymph protein mixture for species identification by MALDI-TOF MS. After this proof-of-concept, the detection of intra-cellular pathogenic agents within tick hemolymph by MALDI-TOF MS was demonstrated achievable. This rapid, accurate, simple and economic strategy allows tick species identification and Rickettsia spp. detection, exempting entomological knowledge. Nevertheless, the main limitation of this strategy is the need for

Competing interests

The authors declare that they have no competing interests

Authors’ contribution

YA, PP, AL conceived and designed the experiments. YA, BJ, LM performed the experiments. YA, AL, PP analyzed the data. LM contributed reagents/materials/analysis tools. YA, AL, PP wrote the paper. RD contributed to the paper redaction.

Acknowledgements

We thank Jérome Terras (URMITE-IRD198, Marseille, France) and Christophe Flaudrops (URMITE-IRD198, Marseille, France) for their technical contribution in this study.

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Original articleIdentification of tick species and disseminate pathogen using hemolymph by MALDI-TOF MS

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Tick samples used for hemolymph extraction

Molecular detection of R. c. conorii and R. africae in ticks

Discussion

Conclusions

Competing interests

Authors’ contribution

Acknowledgements

Int. J. Parasitol.

Ticks Tick Borne Dis.

Ticks Tick Borne Dis.

Clin. Microbiol. Infect.

Comp. Immunol. Microbiol. Infect. Dis.

Hemolymph test. A technique for detection of rickettsiae in ticks

Am. J. Trop. Med. Hyg.

Accurate identification of Culicidae at aquatic developmental stages by MALDI-TOF MS profiling

Parasites Vectors

Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Parasites Vectors

Discrimination of different species from the genus Drosophila by intact protein profiling using matrix-assisted laser desorption ionization mass spectrometry

BMC Evol. Biol.

MALDI-TOF mass spectrometry detection of relapsing fever Borrelia crocidurae in Ornithodoros sonrai ticks

PLoS Negl. Trop. Dis.