Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a useful tool for the rapid identification of wild flea vectors preserved in alcohol

1 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a 1 useful tool for the rapid identification of wild flea vectors preserved in alcohol 2 Running title: MALDI-TOF MS identification of wild fleas 3 Antonio Zurita1, Racha Djeghar2, Rocío Callejón1, Cristina Cutillas1, Philippe Parola3, 4 Maureen Laroche3 5 1Department of Microbiology and Parasitology. Faculty of Pharmacy. University of 6 Seville. Professor García González 2, 41012 Seville, Spain. 7 2 Laboratoire d'Amélioration et Développement de la Production Végétale et Animale 8 (LADPVA), Faculté des Sciences de la nature et de la vie, Ferhat Abbas University, 9 Sétif 1, Sétif, Algérie 10 3 Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, 11 Marseille, France 12 13 * Corresponding author: Maureen Laroche. VITROME - IHU Méditerranée Infection. 14 19-21 Boulevard Jean Moulin 13005 Marseille. E- 15 mail: maureen.lar[email protected] 16 2 Abstract 17 An increasing number of papers have reported the importance of flea identification 18 since some species are considered as important vectors of several human pathogens that 19 have emerged or re-emerged recently such as Bartonella henselae or Rickettsia felis. 20 Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI- 21 TOF MS) has been evaluated in the last years for the identification of multicellular 22 organisms, including arthropods. A preliminary study corroborated by the first time the 23 usefulness of this technique for the rapid identification of fleas creating a preliminary 24 database containing the spectra of 5 species of fleas. Nevertheless, long-term flea 25 preservation in ethanol did not appear to be an adequate storage condition for specimen 26 identification by MALDI-TOF MS profiling. The goal of the present work was to assess 27 the performance of MALDI-TOF MS for the identification of seven fleas species 28 (Ctenocephalides felis, Ctenocephalides canis, Pulex irritans, Archaeopsylla erinacei, 29 Leptopsylla taschenbergi, Stenoponia tripectinata and Nosopsyllus fasciatus) collected 30 in the field and stored in ethanol for different periods of time. Our results confirmed that 31 MALDI-TOF MS can be used for identification of wild fleas stored in ethanol for 32 different periods of time. Furthermore, this technique was able to discriminate not only 33 different flea genera but also the two congeneric species C. felis and C. canis. Lastly, 34 we updated the preliminary database with the spectra of seven flea species stored in 35 ethanol. 36 Keywords: MALDI-TOF MS; flea identification; ethanol storage; Mediterranean 37 38 39 40 41 3 Introduction 42 In the last years a high number of studies have reported the importance of fleas and 43 human flea-borne infections, especially in relation to zoonotic risk (Leulmi et al., 2014). 44 Some of these flea-borne human pathogens have re-emerged recently (e.g., Bartonella 45 henselae, Rickettsia felis) suggesting that much remains to be learned about the 46 potential role of fleas as disease vectors (Eisen & Gage, 2012). We could assume that 47 the cases of flea-borne diseases reported every year in the world may be underestimated. 48 In this sense, health care professionals could not be aware of the presence of these flea- 49 borne zoonoses and thus could fail to choose the most appropriate diagnosis method and 50 treatment in each case. Furthermore, health authorities should be aware about the 51 distribution and epidemiology of flea-borne diseases in order to be ready for potential 52 outbreaks and to allocate adequate funding to the surveillance and control in these cases 53 (Bitam et al., 2010). 54 Accurate vector identification at the species level is essential in order to guide clinicians 55 regarding their patients’ care. The classification of fleas is relatively advanced, thus we 56 have a lot of knowledge about taxonomy of fleas, up to subspecies classification for 57 several families (Whiting et al., 2008). Nevertheless, it requires extensive entomology 58 knowledge of flea morphology and the availability of reference information 59 (identification keys) since they show a complex quetotaxy and morphological 60 specialization associated with ectoparasitism (Whiting et al., 2008). In the last years, 61 molecular methods have emerged for the purpose of flea identification (Whiting et al., 62 2008; Marrugal et al., 2013; Lawrence et al., 2014; Zurita et al., 2015; Zurita et al., 63 2018), therefore, different molecular markers from nuclear ribosomal DNA (rDNA) or 64 mitochondrial DNA (mtDNA) have been amplified and sequenced in order to 65 characterize some species. However, despite the several advantages of molecular 66 4 methods, no consensus sequence exists for the identification of all flea species (Zurita et 67 al., 2015). This absence of DNA sequence information for a larger number of flea 68 genera and species restricts the use of molecular biology methods for flea identification 69 (Fang et al., 2002; Feltens et al., 2010). 70 In the last years MALDI-TOF MS has been used for the identification of various 71 taxonomic groups of arthropods including the Culicoides biting midges (Kaufmann et 72 al., 2011; Kaufmann et al., 2012), ticks (Karger et al., 2012; Yssouf et al., 2013a), 73 mosquitoes (Muller et al., 2013; Yssouf et al., 2013b; Raharimalala et al., 2017; 74 Mewara et al., 2018;), tsetse flies (Hoppenheit et al., 2013, 2014), sand flies (Dvorak et 75 al., 2014), fleas (Yssouf et al., 2014) and Chagas disease triatomine vectors (Laroche et 76 al., 2018). A preliminary study corroborated by the first time the usefulness of this 77 technique for the rapid identification of fleas creating a preliminary database containing 78 the spectra of 5 species of fleas (Ctenocephalides felis, Ctenocephalides canis, 79 Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata) (Yssouf et al., 80 2014). Afterwards, Nebbak et al. (2016) concluded that long-term arthropods 81 preservation in ethanol did not appear to be an adequate storage condition for specimen 82 identification by MALDI-TOF MS profiling since they observed differences at the 83 spectral profile level between the fresh specimens and those from the same species 84 stored in 70% ethanol. These differences were predominantly due to the disappearance 85 of a number of peaks in the profile of specimens conserved in 70% ethanol for more 86 than three months. Recently, (Diarra et al., 2017) proposed guidelines for the 87 automation of sample preparation, including a dealcoholisation protocol for tick 88 samples preserverd in 70% ethanol which could be used for another arthropod species. 89 The goal of the present work was to characterize for the first time seven fleas species 90 (C. felis, C. canis, Pulex irritans, A. erinacei, Leptopsylla taschenbergi, S. tripectinata 91 5 and Nosopsyllus fasciatus) stored in ethanol (70% and 96%) for different periods of 92 time by MALDI-TOF MS collected on Corse Island, East of Algeria, the French 93 Pyrenees area and the South West of Spain, using MALDI-TOF MS. Firstly, optimized 94 sample preparation protocol for fleas stored in ethanol for MALDI-TOF MS analysis 95 was established. Secondly, a MS reference spectra database was created based on 96 morphological and molecular identification of fleas. 97 Material and Methods 98 Ethical statement 99 The maintenance of laboratory colony of C. felis fleas has been approved by the 100 Institutional Animal Care and Use Committee of the Faculty of Medicine at Aix- 101 Marseille University, France. The collection of all the samples for this study in the field 102 did not involve national parks or other protected areas and endangered or protected 103 species. 104 Catching and handling procedures of animals in this study were carried out according to 105 the guidelines provided by the Institutional Animal Care of the University of Seville 106 based on RD 53/2013 of 1st January, 2013 (BOE number 34 of 8th February, 2013) of 107 the Spanish government. Rodents were trapped using Sherman traps and released into 108 its natural habitat after collecting fleas procedures. Dogs belonged to owners and public 109 dog kennels. The consent of owners and dog kennels chairman was obtained for the 110 inclusion of the dogs in the study. In Algeria, we obtained the approval of the 111 municipality of Constantine to include the dogs from the kennels. Rodents and dogs 112 were exhaustively and manually examined for fleas on the head, neck, body, sides, tail, 113 and ventral regions of each animal. No specific approval of any Institutional Animal 114 6 Care and Use Committee were needed in this study since any invasive procedure nor 115 anesthesias were required for animal handling. 116 Sample collection and morphological identification 117 Fleas belonging to C. felis and C. canis species were collected between 2011 and 2016 118 from dogs (Canis lupus familiaris) from three localities of the South West of Spain: 119 Villamanrique de la Condesa, Seville (37°14′47″N 6°18′23″W), Dos Hermanas, Seville 120 (37°17′01″N 5°55′20″W), Sanlúcar de Barrameda, Cádiz (36°46′44″N 6°21′14″W) as 121 well as Constantine, Algeria (36°21′54″N 6°36′52″E). Specimens identified as P. 122 irritans were collected from an abandoned horse stable with a flea infestation from 123 Seville, Spain (37°23′00″N 5°59′00″W) whereas, specimens identified as A. erinacei 124 were collected from hedgehogs (Erinaceus europaeus) killed on roads from Almonte, 125 Spain (37°16′00″N 6°31′00″W) and Dos Hermanas, Spain. Lastly, some samples were 126 collected from different species of rodents (see Table 1) from Corse Island (42°09′00″N 127 9°05′00″E) and Conat, France (42°36′51″N 2°21′27″E). All of flea specimens were 128 preserved in 70% and 96% ethanol at room temperature until morphological, molecular 129 and MALDI-TOF MS analyses. Some of them (C. felis, C. canis, P. irritans and A. 130 erinacei) were obtained through our own collecting efforts whereas others were 131 obtained through the assistance of colleagues (see Acknowledgements). The first ones 132 were identified by us whereas those fleas provided by our colleagues were first 133 identified by them and then morphologically corroborated by us in our laboratory. 134 Morphological identification to the species level was carried out according to different 135 publications and taxonomic keys (Marrugal et al., 2013; Jordan & Rothschild, 1912; Gil 136 Collado, 1949; Hopkins & Rothschild, 1953; Jordan, 1958; Lewis, 1967, 1993; Hastriter 137 & Tipton, 1975; Beaucournu & Launay, 1990; Giannetto et al., 1997; Beaucournu & 138 Menier, 1993, Menier & Beaucournu, 1998; Acosta & Morrone, 2013). For 139 7 morphological analysis, all specimens were examined using a Nikon microscope 140 equipped with a camera lucid system and a photomicroscope. All fleas included in this 141 study were not engorged. 142 MALDI-TOF MS analyses 143 According to Diarra et al. (2017) previously published dealcoholisation protocol of 144 arthropods for MALDI-TOF MS, each sample (flea legs and cephalothorax) was 145 introduced in a sterile Eppendorf and dried overnight at 37º C, prior to homogenization 146 in order to remove all the ethanol. Secondly, a sterile tungsten bead (Qiagen, Hilden, 147 Germany) was added to each sample plus 20 μL of a mix of 70 % (v/v) formic acid and 148 50 % (v/v) acetonitrile (Fluka, Buchs, Switzerland). The samples were then 149 homogenized using the TissueLyser device (Qiagen, Hilden, Germany) by 3 cycles of 150 30 s at a frequency of 30 Hz. The homogenized cephalothorax and legs of fleas were 151 centrifuged at 10000 r.p.m. for 30 s and 1 μL of the supernatant from each sample was 152 carefully dropped onto the MALDI-TOF target plate as previously described (Nebbak et 153 al., 2017). Each spot was then recovered with 1 μL of CHCA matrix solution composed 154 of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50 % acetonitrile 155 (v/v), 2.5 % trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water 156 (Yssouf et al., 2016) The target plate, after drying for several minutes at room 157 temperature, was introduced into the Microflex LT MALDI-TOF Mass Spectrometer 158 device (Bruker Daltonics, Germany) for analysis. The loading of the MS target plate, 159 the matrix quality, and the performance of the MALDI-TOF were performed as 160 previously described (Nebbak et al., 2017). For each target plate we used one specimen 161 of C. felis from the University Hospital Institute Méditerranée Infection of Marseille 162 laboratory rearing as a positive control and 2 μL of CHCA matrix solution as a negative 163 control. Protein mass profiles were obtained using a Microflex LT MALDI-TOF mass 164 8 spectrometer (Bruker Daltonics, Germany) using parameters previously described by 165 (Kumsa et al., 2016). The spectrum profiles obtained were visualized with Flex analysis 166 v.3.3 software and exported to ClinProTools software v.2.2 and MALDI-Biotyper v.3.0. 167 (Bruker Daltonics, Germany) for analysis and data processing (smoothing, baseline 168 subtraction and peak picking). MSPs of each species were created as previously 169 described (Nebbak et al., 2017). Only the high quality and reproducible spectra were 170 loaded into the MALDI Biotyper v.3.0 to create a spectral database with 2–6 specimens 171 for each species as previously described (Diarra et al., 2017). To upgrade the database, 172 MSP reference spectra were created using spectra from at least two specimens per 173 species by the automated function of the MALDI-Biotyper software v3.0. (Bruker 174 Daltonics). MS spectra were created with an unbiased algorithm using information on 175 the peak position, intensity and frequency. This database was formerly composed of a 176 total of five flea species (C. felis, C. canis, A. erinacei, X. cheopis and S. tripectinata) 177 plus others arthropods species described in (Boucheikhchoukh et al., 2017). The spectra 178 files are available on request and transferable to any Bruker MALDI-TOF device. 179 For the blind test analysis, 148 good quality spectra from the seven species of fleas 180 tested were selected to query the upgraded database. Since the protein extracted from 181 each specimen was spotted on the MALDI-TOF target plate in quadruplicate, each 182 specimen was associated with four spectra. The results of the database queries are 183 presented as log score values (LSVs) for each spectrum, corresponding to a matched 184 degree of signal intensities of mass spectra of the query and the reference spectra. LSVs 185 range from 0 to 3. LSVs allow good evaluation of the reproducibility between a queried 186 spectrum and a reference spectrum as it is the result of thorough comparison of peaks 187 position and intensity between those two spectra. A LSV was obtained for each 188 9 spectrum of the samples tested blindly. For each specimen, the spectrum with the 189 highest LSVs was selected for identification (Laroche et al., 2018). 190 On the other hand, in order to visualize MS profiles similarity and distances, 191 hierarchical clustering of the mass spectra of 3-4 specimens of each tested species was 192 performed using the dendrogram function of MALDI Biotyper, version 3.0. 193 Dendrograms are based on the results of Composite Correlation Index (CCI) matrix. 194 CCIs are calculated by dividing spectra into intervals and comparing these intervals 195 across a dataset. The composition of correlations of all intervals provides the CCI, 196 which is used as a parameter that defines the distance between spectra. A CCI match 197 value of 1 represents complete correlation, whereas a CCI match value of 0 represents 198 an absence of correlation (Laroche et al., 2018). 199 Molecular identification 200 For molecular purposes the abdomens of each specimen were cut off and transferred to 201 a 1.5 mL tube containing 180 μL of G2 lysis buffer (Qiagen, Hilden, Germany), and 20 202 μL of proteinase K (Qiagen, Hilden, Germany), and incubated at 56º C overnight. DNA 203 extraction was performed with an EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany) 204 according to manufacturer recommendations. Flea DNAs were then eluted in 100 μL of 205 Tris EDTA buffer using the DNA extracting EZ1 Advanced XL Robot (Qiagen, Hilden, 206 Germany). The DNA was either immediately used or stored at −20° C until molecular 207 analysis. The DNA extracting EZI Advanced XL Robot was disinfected after each batch 208 of extraction as per the manufacturer’s recommendations, to avoid cross-contamination. 209 The nuclear marker ITS1 of two to six samples for each flea species was amplified by 210 standard PCR using a Bio-Rad Thermocycler (Bio-Rad Laboratories, Hercules, 211 California, USA). For this purpose, we used the DNA of those specimens added to the 212 16 misidentified on the basis of their morphological characters included in taxonomic keys. 359 Particularly the classification of the subspecies of C. felis in the Afrotropical zone (C. 360 felis strongylus) has often been misidentified as C. canis based on the sole criterion of 361 the cephalic profile; and likewise, C. orientis in Asia (Beaucournu & Kock, 1990). 362 Recently, Linardi & Santos (2012) and Marrugal et al. (2013) concluded that the shape 363 of the head and the chaetotaxy of the tibia and metepisternite showed significant intra- 364 specific variations and, therefore, these two characteristics should be cautiously used for 365 interspecific diagnosis between C. felis and C. canis. Based on these results, they argued 366 that some others morphological criteria (relative size of the first and second genal spines 367 or male and female genitalia) must be considered in order to discriminate between these 368 two species (Fig. 3). Moreover, although both species are recognized vectors of R. felis 369 and several Bartonella spp. pathogens (Lawrence et al., 2015), numerous authors 370 reported much lower prevalence of these bacteria in C. canis (Horta et al., 2006; Kumsa 371 et al., 2014; Nasereddin et al., 2014). These studies confirm the need for an 372 unambiguous identification of these two species. As illustrated on the dendrogram 373 created with MS spectra of the seven included flea species, we could suggest the 374 usefulness of this technique to discriminate different flea families (Pulicidae, 375 Ceratophyllidae and Stenoponiidae) but also closely related species such as the two 376 congeneric species C. felis and C. canis. Nevertheless, although results are encouraging 377 for the distinction of closely related species, this robustness would be confirmed with 378 more specimens, particularly C. orientis, the major Ctenocephalides species in South 379 East Asia, closely related to C. felis and C. canis (Linardi & Santos, 2012). 380 MALDI-TOF MS has been developed during the last years as an important tool not only 381 for the taxonomic identification but also for the phylogenetic classification of 382 microorganisms (Freiwald & Sauer, 2009). In our study, dendrogram results are in 383 17 concordance with former phylogenetic studies which reported a close phylogenetic 384 relation between Ctenocephalides and Archaeopsylla genera. Thus, in these studies, 385 specimens belong to both genera clustered together composing the Archaeopsyllini 386 subfamily within Pulicidae family (Zhu et al., 2015; Zurita et al., 2017). However, we 387 agree with Yssouf et al. (2013b) who argued that the MALDI-Biotyper software cannot 388 yet be considered to be a reliable tool for studying the phylogeny of arthropods. The 389 number of flea is still too limited in our study to hypothesize that MS spectra could be 390 used as a taxonomic tool as it is already the case for bacteria. Therefore, although 391 MALDI-TOF MS has opened new doors for phylogenetic studies in this phylum, 392 additional data are still needed, and detailed studies should be carried out, especially 393 within Siphonaptera order where the MS data are still limited. 394 In conclusion, our results confirm that MALDI-TOF MS could be used for 395 identification of fleas preserved in alcohol. It requires the creation of a specific database 396 with specimens stored in ethanol since spectra from fresh specimens differ from those 397 obtained from ethanol-preserved specimens. Accordingly, we updated the preliminary 398 database with the spectra of seven flea species stored in ethanol (C. felis, C.canis, P. 399 irritans, A. erinacei, L. taschenbergi, N. fasciatus and S. tripectinata). This protocol 400 was developed and validated on seven flea species. Our reference spectra are available 401 on request for laboratories equipped with Bruker MALDI-TOF MS devices to upgrade 402 their own databases. The rapid identification of wild flea species by MALDI-TOF MS 403 in only one step would constitute a relevant discover in epidemiology and entomology 404 field since this technique shows some advantages compared with classic and molecular 405 methods such as the low consumable costs, the minimal time required for sample 406 preparation or the rapid availability of the results. For this reason, this study should 407 encourage other scientific teams about the necessity of setting up an updated MALDI- 408 18 TOF MS database. This tool is also important for clinicians as this study illustrates that 409 wild fleas collected from a patient or at his house could be preserved in alcohol 410 allowing a fast and reliable subsequent identification, which is critical information for a 411 medical doctor. 412 Conclusion 413 The present study confirms that the drawbacks of ethanol preservation for MALDI-TOF 414 MS identification of arthropods can be circumvented with the appropriate sample 415 preparation. This approach is fast, with low running costs and validated on field 416 samples. It can be used by operators with no entomological training, and opens 417 numerous perspectives such as high throughput vector monitoring and identification of 418 fleas collected from patients. 419 Acknowledgments 420 The present work was supported by a FEMS (Federation of European Microbiological 421 Societies) Research Grant and a grant of the V Plan Propio de Investigación of the 422 University of Seville, Spain. 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