Ticks are obligate hematophagous parasites of the order Acari. These arthropods can feed on every known class of vertebrate and can bite people (1
). Ticks are currently the second leading vector of human infectious diseases and can carry bacterial (1
), viral (1a
), and protozoan pathogens (2
). However, only in 1982, with the identification of Borrelia burgdorferi
as the etiological agent of Lyme disease, was the major effect of ticks on public health recognized, leading to an increased awareness of tick-borne diseases (3
). Since then, more than 15 tick-borne rickettsioses have emerged throughout the world (4
The removal of a tick from the human body is a common situation, and patients may visit a physician with an attached or removed tick. Certain tick species are well-known vectors of human diseases, such that identifying the species, which will alert the physician to the diseases that may have been transmitted, is clinically helpful if such information is obtained quickly (1
). Indeed, recent studies confirm that the use of doxycycline prophylaxis following an Ixodes
tick bite is useful for the prevention of Lyme disease (4
). Similar postexposure regimens could also prevent tick-borne relapsing fever in areas of endemicity (5
). However, for prophylactic treatment to be effective, it must be delivered shortly after potentially infectious ticks are removed from patients (6
Ticks species can be morphologically identified using taxonomic keys for endemic species in several geographic regions (1
). However, morphological identification can be difficult because it requires some entomological expertise, and it is difficult to identify a specimen that is damaged or at an immature stage of its life cycle (1
). Molecular methods, such as the sequencing of the mitochondrial 12S (7
), 18S (8
), and 16S ribosomal DNAs (rDNAs) (7
), mitochondrial cytochrome oxidase subunit 1 (COX1), and nuclear internal transcribed spacer 2 (ITS2), have been developed to identify arthropods, including ticks (9
). However, there is currently no PCR assay that can distinguish tick species, and ideal PCR primer pairs that can amplify the relevant gene fragments are not available.
In addition to the technical and logistical drawbacks of PCR assays, this approach is further limited by the availability of gene sequences in GenBank. Protein profiling by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is now increasingly common for the routine identification of microorganisms in clinical microbiology (10
). This revolutionary, reliable, and cost-effective technique is simpler and faster than conventional phenotypic and molecular methods for the identification of human pathogens (10
The MALDI-TOF MS approach was first applied to arthropods for the differentiation of Drosophila
). It was found that protein extracts obtained from whole specimens generated reproducible spectra (11
). Species-specific protein profiles have also been be used to differentiate three species of aphids (insects that feed on plants) (12
). In 2011, a blind test in which 111 wild specimens were compared to the database profiles of Culicoides
species showed that MALDI-TOF MS can differentiate species of Culicoides
biting midges collected in the field (13
). More recently, a MALDI-TOF MS study of seven ticks reported that whole ticks or body parts, excluding the legs, generate spectra that are sufficient for species identification (14
The objective of the present study was to investigate the use of MALDI-TOF MS for the rapid differentiation of tick species using only their legs. Our goals were to establish a reference database, to evaluate the MALDI-TOF MS-based identification system in a blind test, and to evaluate this new identification tool using ticks removed from patients.
Although other factors should be considered, the identification of ticks that have bitten or been removed from patients is the first step in assessing the risk of infection (1
). In this study, all ticks removed from patients were potential vectors of pathogens that depend on the involved tick species for propagation. In Europe, D. marginatus
is one of the primary vectors of R. slovaca
and R. raoultii
, two spotted fever group rickettsiae responsible for tick-borne lymphadenopathy (TIBOLA), also called Dermacentor
-borne necrosis erythema and lymphadenopathy (DEBONEL) or SENLAT, which is defined as scalp eschar and neck lymphadenopathy after a tick bite (21a
). I. ricinus
is a major vector of several bacterial agents, including the causative agent of Lyme borreliosis, Borrelia garinii
), B. miyamotoi
, which causes relapsing fever (21b
), A. phagocytophilum
, the causative agent of human granulocytic anaplasmosis (22
), and Rickettsia helvetica
, an emerging pathogen (4
, the brown dog tick, is a primary vector of Rickettsia conorii
, the causative agent of life-threatening Mediterranean spotted fever (23
), Rickettsia rickettsii
is the vector of Rocky Mountain spotted fever in southern regions of the United States (23a
), and R. massiliae
is an emerging pathogen (23b
). Rhipicephalus bursa
is a known vector of several cattle parasites as well as a putative vector of Crimean-Congo hemorrhagic fever in certain regions of the world, such as Turkey (24
). The potential for these ticks to transmit disease agents was illustrated in this study by the detection of several pathogens in the ticks that had bitten patients. This finding highlights the clinical need for the species identification of ticks.
Our results suggest that the MALDI-TOF MS spectra of protein extracts from tick legs are a suitable tool for identifying ticks. The results obtained using this method corroborated those from morphological and molecular identification methods.
Overall, 63% of the laboratory tick specimens were identified by MALDI-TOF MS with scores of >2, which are considered to be reliable scores for the identification of bacterial species (15
Ticks collected in the field or removed from patients were reliably identified with lower score values, but each specimen's spectrum matched a reference spectrum. Only one tick was not identified by MALDI-TOF MS, R. bursa (identification confirmed by 12S sequencing). This tick was not identified because the corresponding spectrum was not present in our database. The species not represented in our database, which were tested as controls (fleas, mosquitoes, bugs, bees, and beetles), had low scores, less than 1.1, and none of their spectra matched the reference spectra in the database.
The establishment of a cutoff score for accurate identification would be ideal. When ticks removed from patients were tested, most (76%) were identified with scores of >1.7, and all were identified with scores of >1.3. However, it is not known if a score of 1.3 can be used as a definitive cutoff in other regions. The ticks used to construct the database are representative of the tick species in France and correspond to the species that have been removed from French patients, including returned travelers, and sent to our reference centers in the past 15 years (P. Parola, unpublished observations). In addition, the vectors included in this study are not closely related, and it must be confirmed that this MALDI-TOF MS method can differentiate closely related species, e.g., Ixodes species. However, when D. marginatus specimens removed from patients were tested against the database, which contained reference spectra for this species and for the closely related species D. reticulatus, the identification of the specimens was unequivocal.
Using the MSP dendrogram function of MALDI Biotyper, version 3.0, all but one tick (R. bursa
) clustered according to their genera, species, and strain. A similar discrepancy was observed in a preliminary study using the entire body of the tick (14
). Therefore, although MALDI-TOF MS has opened new doors for the phylogenetic study of arthropods and bacteria, additional data are still needed, and detailed studies should be conducted.
Contrary to a recent report (14
), we found that the use of tick legs appears to be an effective means to identify tick vectors if a reference database is available. The remainder of the body can be reserved for other purposes, such as the detection of pathogens, as performed in this study. However, various storage conditions (10
) and chemical extraction methods (26
) appear to influence the results of the MALDI-OF MS analysis of arthropod specimens. To expand our database, we will test a large number of tick species. Some species are available in collections. However, it seems that long-term storage in 70% ethanol reduces the reproducibility of the MALDI-TOF MS spectra (13
). It will also be useful to evaluate the use of MALDI-TOF MS for the identification of frozen specimens.
In conclusion, we showed in this study that MALDI-TOF MS is an efficient approach for the rapid identification of tick vectors. This method was used for the first time to identify ticks removed from patients. The results were obtained rapidly relative to the time required for molecular methods, and the completion of this assay does not require any specific entomological expertise. One damaged tick removed from a patient was successfully identified using MALDI-TOF MS as I. ricinus
, a vector of Lyme disease. The knowledge of the tick species can be used to inform the clinician's decision as to whether to prescribe prophylactic doxycycline treatment. At the very least, the identity of the tick species can tell physicians which specific clinical signs they should look for in their patients. The rapid identification of ticks, and most likely of other arthropod vectors, is now possible in any laboratory with a MALDI-TOF MS system. In our unit, results are now available for clinicians in less than 1 h, with no requirement for entomological expertise. Our database contains reference spectra for ticks removed from humans in our area. This database can be shared and used directly by any clinical microbiology laboratory equipped with a MALDI Biotyper system. Because MALDI-TOF studies with bacteria and yeast have demonstrated significant variation in the protein profile based on geographical region (27
), it will be interesting to test ticks of the same species from a variety of geographical regions to determine if this technique can be used as a regional or global tool. We will continue to add new reference spectra to our database to test the ability of this MS method to discriminate closely related species and to define a definitive cutoff score for species identification. Finally, it will be informative to determine whether MALDI-TOF MS can be used to identify not only tick vectors but also the microorganisms with which the ticks are infected.