Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins

Abstract

Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI-TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex-specific profiles within both morphospecies could not be reliably discriminated using MALDI-TOF MS. In conclusion, MALDI-TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.

Genetic variability, cryptic species and phylogenetic relationship of six cyathostomin species based on mitochondrial and nuclear sequences

“…minutus OTU I was considered a valid and independent genospecies after several PCR and molecular cloning repetitions. These findings support the evidence for the presence of a cryptic species complex, as previously reported 15 , 25 . Regarding the two specimens that switched between nuclear and mitochondrial clusters it was unfortunately impossible to exclude contamination of PCRs due to depletion of the DNA samples.…”

“…Regarding incomplete lineage sorting, use of additional marker sequences is highly recommended. In the context of the present project, it was also aimed to evaluate proteomic (MALDI-TOF MS) data for cyathostomin species 25 and this limited the number of DNA sequences that could reliably amplified per specimen. Therefore, it was decided to focus on the two markers that showed the most robust amplification and the best chance to obtain both, phylogenetic signal and barcoding sequences.…”

“…After DNA extraction of individual cyathostomin specimens with the NucleoSpin Tissue XS Kit (Macherey–Nagel, Düren, Germany), PCRs targeting the internal transcribed spacer 2 (ITS-2) 40 and a partial cytochrome c oxidase I (COI) 26 were conducted using a high-fidelity DNA polymerase 25 . PCR products were cloned into the pSC-Bamp/kan vector using the StrataClone Blunt PCR Cloning Kit (Agilent Technologies, Waldbronn, Germany), and one clone with insert per PCR fragment of each individual worm was sequenced by LGC Genomics (Berlin).…”

“…To further complicate the situation, the validity of some cyathostomin species is questionable, and others might represent cryptic species complexes 15 , 16 . To overcome the limitation of morphological identification, diverse techniques have been applied to study cyathostomins, such as serological 17 – 19 and common molecular methods 20 – 24 as well as, more recently, proteomic approaches 25 . This study applies molecular methods to assess the utility of two different genes—the nuclear second internal transcribed spacer (ITS-2) and the mitochondrial partial cytochrome c oxidase subunit I (COI)—as barcode regions and the phylogenetic relationships of six common species of cyathostomins ( Cor.…”

MALDI-TOF mass spectrometry as a diagnostic tool in human and veterinary helminthology: a systematic review

“…Coronocyclus coronatus , C. labiatus and C. labratus ) and found distinct protein spectra among adult helminths of different species [66]. These findings were recently confirmed and substantiated by another study on the application of MALDI-TOF for differentiation of cyathostomins, which was published in April 2019 [147].…”