Original article
Species determination and characterization of developmental stages of ticks by whole-animal matrix-assisted laser desorption/ionization mass spectrometry

https://doi.org/10.1016/j.ttbdis.2011.11.002Get rights and content

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of crude bacterial samples has been introduced as a very cost-efficient and rapid, yet highly informative tool to identify and classify bacteria. The potential of this approach to characterize whole animals, so far preferentially insects, is only evolving. Here, a simple protocol was developed to perform MALDI-MS analysis on extracts from whole ticks of 7 species and 4 developmental stages. Using commercially available software designed for the identification of bacteria, a reference database of spectra was constructed that allowed the species determination of ticks using larvae, nymphs, or adult individuals as starting material. Cluster analysis on the basis of MALDI mass spectra indicated that the primary determinant for the mass spectra was the species, followed by the developmental stages, which formed distinct clusters within the given species. With certain limitations, species identification was also possible using body parts and engorged animals. Spectra of developing Ixodes ricinus eggs showed dramatic changes with time, suggesting that, beyond its usefulness for species determination, MALDI-typing may have applications in developmental biology.

Introduction

Ticks transmit numerous infectious agents which cause severe health problems in humans and in animals (Brown et al., 2005, Estrada-Peña, 2009). In Europe, Ixodes spp. are of outstanding importance among the indigenous tick species as 90–95% of all tick bites in humans are caused by Ixodes (I.) ricinus in Western and Central Europe, and by I. persulcatus in Eastern Europe (Süss et al., 2008). I. ricinus is the vector of 95% of all tick-borne pathogens in Western and Central Europe transmitting bacterial, viral, and protozoal agents (Süss et al., 2004, Süss and Schrader, 2004). For humans, the most important I. ricinus-borne bacterial group of pathogens is the Borrelia burgdorferi sensu lato complex while the most important viral pathogen is tick-borne encephalitis virus (TBEV). In recent years, Dermacentor (D.) reticulatus, a well-known vector of Babesia spp. and Rickettsia spp., has attracted notable attention due to a significant expansion of its range in Germany (Dautel et al., 2006) and an increasing number of autochthonous Babesia canis infections in dogs (Barutzki et al., 2007). Haemaphysalis spp., Rhipicephalus spp. and even Hyalomma spp. may be encountered in rare cases in Germany (Gothe, 1968, Kampen et al., 2007, Nosek, 1971) and can be potential vectors of emerging pathogens such as Bhanja virus, Lipovnik virus, Crimean-Congo hemorrhagic fever virus, various rickettsial species and Coxiella burnetii (Süss et al., 2004, Süss and Schrader, 2004).

Classically, ticks are differentiated by morphological criteria (Sonenshine, 1991). This, however, is time consuming, requires good skills and experience in taxonomy and morphology of ticks and still may lead to misidentifications, in particular in closely related species, if nymphal or larval stages are involved or in damaged or semi-engorged specimens where morphological characteristics may be missing. To circumvent these problems, polymerase chain reaction (PCR) and other molecular biological methods have been increasingly applied in tick identification (Rumer et al., 2010). Thus, mitochondrial 12S (Beati and Keirans, 2001) and 16S rDNA sequences (Black and Piesman, 1994, Chao et al., 2009, Mangold et al., 1998) have successfully been used to differentiate closely related tick species (Moshaverinia et al., 2009, Rich et al., 1995, Zahler et al., 1995). For a targeted control of ticks and tick-associated diseases, a system of tick differentiation is highly desirable that produces correct results even in complicated cases, such as in nymphs and larvae or in damaged specimens. The system should be easy to handle, inexpensive, and allow identification of infectious agents in the same sample, e.g. by PCR analysis.

One such potential system for species determination could be intact cell mass spectrometry (ICMS) which was originally introduced for the identification and classification of bacteria (Heller et al., 1987, Platt et al., 1988), but has been expanded to other microorganisms like fungi (Marinach-Patrice et al., 2009, Marklein et al., 2009) and protozoans (Villegas et al., 2006) and also to tissue cultures (Karger et al., 2010). Also, characterization of whole insects (Campbell, 2005, Feltens et al., 2010, Kaufmann et al., 2011, Perera et al., 2005) has been reported. Commercial software packages and databases for the identification of microorganisms by MALDI-TOF mass spectrometry are available (MALDI Biotyper™ by Bruker Daltonics, AXIMA@SARAMIS™ by Schimadzu & AnagnosTec). Characteristic features of ICMS, also referred to as MALDI-typing, are the minimalistic sample preparation procedures followed by comparison of sample spectra with reference spectra from authentic samples. Although it is generally assumed that most of the analyzed molecules are proteins, no sequence data are required. The similarity between 2 spectra is assessed by statistical means and can be used for different purposes. In our study, we have used commercial software which has been designed for the identification of bacteria. It calculates a similarity score from the number of masses that are present in the sample and in reference spectra of authentic samples within certain mass tolerance frames and from the reliability with which these masses appear in technical replicates of the reference. The software suggests empirical cut-off values that define species or genus borders. Also, clustering algorithms can be applied to mathematical distances between 2 spectra in order to indicate relations between the organisms. For tissue cultures from a broad variety of animals, it has been shown that spectrum-based cluster analysis can give a fair approximation of phylogenetic relations between organisms (Karger et al., 2010). This study was initiated by a monitoring program in which ticks were collected at different sites in Germany with the aim to determine the influence of climatic and microclimatic changes on tick activity and to screen ticks for disease agents. For some specimens, particularly those collected from hosts such as small mammals and birds, species identification turned out to be problematic as most of the ticks were engorged nymphs or larvae and more or less damaged. Therefore, a quick, simple, inexpensive, and reliable identification procedure would have been helpful for the documentation of the collected samples, preferentially one that is compatible with the extraction of RNA and DNA for the following PCR-based screening for tick-borne pathogens. Hence, the potential of MALDI-typing of whole-animal homogenates for this purpose was explored. Among other issues, the focus was set on the questions if a reliable species determination is possible, if field samples collected from different biotopes or animals are a proper starting material, and if engorged ticks or damaged samples can be analyzed.

Section snippets

Ticks

A collection of reference spectra was constructed for I. canisuga, I. hexagonus, I. persulcatus, I. ricinus, I. scapularis, Dermacentor reticulatus, and Rhipicephalus sanguineus. Table 1 gives an overview. Whenever available, laboratory-bred ticks were preferred. However, to provide a representation in the reference database for the variability of the population, some field-collected samples, set in italics in Table 1, were also included.

Processed adult D. reticulatus were field-collected in

Identification of tick species by whole-animal mass spectrometry and construction of a reference database

Exemplary spectra of I. ricinus developmental stages in the full mass range are given in Fig. 1A. Concerning the resolution of peaks and their reproducibility, the quality of spectra from whole ticks was comparable to that obtainable from bacterial samples like the extract of E. coli that has been used for calibration. Apart from a number of species-specific masses that clearly distinguished different tick species (data not shown), characteristic mass signatures were also found for the

Discussion

Whole-animal mass spectrometry as described here allows a reliable determination of the tick species included in this study. The scores obtained with Biotyper software were similar to those of bacterial samples so that this software can be used for the purpose without further adjustments. As scores over 2.00 were achieved for the correct classification of almost all samples, this value can be considered a reasonable cut-off for defining the species of an unknown sample. Thus, whole-animal mass

Acknowledgements

We thank Katja Bauer, Angela Dramburg, Eva-Maria Franke, and Elisabeth Hasse for excellent technical assistance, Prof. Dr. Eberhard Schein (Berlin, in memoriam), Elisabeth Meyer-Kayser (Bad Langensalza), and Dr. Joachim Müller (Magdeburg) for providing samples.

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