Publication Database

Molecular and proteomic species characterisation of cyathostomins (2020)

Art

Hochschulschrift

Autor

Bredtmann, Christina (WE 13)

Quelle

Berlin: Mensch & Buch Verlag, 2020 — VI, 134 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/26362

Kontakt

Institut für Parasitologie und Tropenveterinärmedizin

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 62310
parasitologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Cyathostomins are currently considered to be the most important equine parasites, due to their ubiquitous prevalence, their ability to cause potentially fatal larval cyathostominosis and their widespread and progressing anthelmintic resistance. Multi-species infections, comprising usually more than 15 species simultaneously, impede research on the contributions of individual species to the pathogenesis as well as development of anthelmintic resistance in individual species. Research in cyathostomins e.g. on multi-species composition and species-specific pathogenicity is currently hampered by insufficient diagnostic tools. Particularly universally applicable methods, that are non-invasive and life-stage independent are needed. Molecular methods appear useful but can be resource and time-consuming, and some are limited to specific species, only. Proteomics methods, such as matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) allow species identification on the basis of matching protein patterns of organisms. In this thesis a detailed evaluation of the application of MALDI-TOF MS on cyathostomins is given. A protocol based on protein extraction with equal volumes of acetonitrile and formic acid, followed by ultrasonic homogenisation and DNA isolation after buffering with Tris-buffer was established and successfully applied. It was demonstrated that this protocol was applicable to repeatably obtain high-quality mass spectra from cyathostomins that were freshly collected during necropsy and immediately fixed in 70 % ethanol. The mass spectra of two closely related species, namely Cylicostephanus longibursatus and Cylicostephanus calicatus, were examined with a bioinformatics workflow using freely available software. Despite their close relationship, all examined specimen of both species were reliably differentiated from each other. Mass peaks were sorted according their influence on discrimination. Although a rate of identification of close to 100 % was reached, it should be noted that no single peak alone occurred only in one of the two species as it would be required to be used as biomarker for species identification. Of importance for this work is the fact that this protocol allows to retrieve protein extracts of whole worms and to perform subsequent DNA extraction for PCR. As a result, the worms that were identified morphologically, could be used for protein profiling and molecular characterisation. Two different markers, the internal transcribed spacer (ITS) 2 and cytochrome oxidase I (COI) were applied and revealed three clusters within Cylicostephanus minutus. Since two of the clusters were already described as potential cryptic species of Cys. minutus and designated as operational taxonomic units (OTU) I and II, the third was designated OTU III. A total of 22 individual worms of this species were examined and one, nine and twelve specimens could be assigned to OTU I, II, and III, respectively. For each marker and species/OTU, the sequences were compaired pairwise. The identities within the same species/OTUs were higher than between the OTUs. Identities between species and OTUs were lower for COI locus than for ITS-2 locus, indicating higher inter-species variability for COI. No identitification of the different OTUs in the proteomic analysis was possible, which could be linked to the low number of specimen available of each OTU. However, MALDI-TOF MS represents a step towards a universally applicable method for species identification of cyathostomins, provided the technical infrastructure as well as a comprehensive master spectra library is available. To set up a proteomic master spectra library, the individual species should be examined in parallel molecularly to detect cryptic species. A close examination of two additional species, Coronocyclus coronatus and Cylicostephanus calicatus has been performed in this study. Although both species are placed in different genera based on morphological descriptions, sequence identities on the ITS-2 locus were up to 100 %. This stresses the need for the employment of the COI marker that allowed, together with the ITS-2 marker, delineation of both species. Furthermore, different nuclear and mitochondrial haplotypes were identified for each species. For Cyc. calicatus, two different genospecies were identified based on two ITS-2 variants, which are associated with distinct mitochondrial haplotypes and point towards a cryptic species complex of at least two non-interbreeding species. In short, it should be summarised, that the current methods for species identification of cyathostomins are not sufficient and that the species complexity of cyathostomins is even more complicated than anticipated. To establish MALDI-TOF MS as a new method for species identification, molecular research should be performed to correctly delineate cyathostomin species that can be subsequently used for establishing a master spectra library for protein profiling.