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Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam.

ABSTRACT: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus (Boophilus) microplus, two Amblyomma varanensis, seven Dermacentor auratus, one Dermacentor compactus and one Amblyomma sp. were submitted for MALDI-TOF MS analyses. Spectral analysis showed intra-species reproducibility and inter-species specificity and the spectra of 329 (91%) specimens were of excellent quality. The blind test of 310 spectra remaining after updating the database with 19 spectra revealed that all were correctly identified with log score values (LSV) ranging from 1.7 to 2.396 with a mean of 1.982 ± 0.142 and a median of 1.971. The DNA of several microorganisms including Anaplasma platys, Anaplasma phagocytophilum, Anaplasma marginale, Ehrlichia rustica, Babesia vogeli, Theileria sinensis, and Theileria orientalis were detected in 25 ticks. Co-infection by A. phagocytophilum and T. sinensis was found in one Rh. (B) microplus.

SUBMITTER: Huynh LN

PROVIDER: S-EPMC8500424 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam.

Huynh Ly Na LN Diarra Adama Zan AZ Pham Quang Luan QL Le-Viet Nhiem N Berenger Jean-Michel JM Ho Van Hoang VH Nguyen Xuan Quang XQ Parola Philippe P

PLoS neglected tropical diseases 20210928 9

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus (Boophilus) microplus, two Amblyomma varanensis, seven Dermacentor auratus, one Dermacentor compactus and one ...[more]

PMID: 34582467

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Project description:BackgroundTicks are obligate haematophagous ectoparasites of vertebrate hosts and transmit the widest range of pathogenic organisms of any arthropod vector. Seven tick species are known to feed on bare-nosed wombats (Vombatus ursinus), in addition to the highly prevalent Sarcoptes scabiei mite which causes fatal sarcoptic mange in most bare-nosed wombat populations. Little is known about the pathogens carried by most wombat ticks or how they may impact wombats and wombat handlers.MethodsWombat ticks were sourced from wildlife hospitals and sanctuaries across Australia and identified to species level using taxonomic keys. Genomic DNA was extracted from a subsample, and following the amplification of the bacterial 16S rRNA gene V3-V4 hypervariable region, next-generation sequencing (NGS) on the Illumina MiSeq platform was used to assess the microbial composition.ResultsA total of 447 tick specimens were collected from 47 bare-nosed wombats between January 2019 and January 2020. Five species of ticks were identified comprising wombat tick Bothriocroton auruginans (n = 420), wallaby tick Haemaphysalis bancrofti (n = 8), bush tick Haemaphysalis longicornis (n = 3), common marsupial tick Ixodes tasmani (n = 12), and Australian paralysis tick Ixodes holocyclus (n = 4). Tick infestations ranged from one to 73 ticks per wombat. The wombat tick was the most prevalent tick species comprising 94% of the total number of samples and was present on 97.9% (46/47) of wombat hosts. NGS results revealed the 16S rRNA gene diversity profile was predominantly Proteobacteria (55.1%) followed by Firmicutes (21.9%) and Actinobacteria (18.4%). A species of Coxiella sharing closest sequence identity to Coxiella burnetii (99.07%), was detected in 72% of B. auruginans and a Rickettsiella endosymbiont dominated the bacterial profile for I. tasmani.ConclusionsA new host record for H. longicornis is the bare-nosed wombat. One adult male and two engorged adult female specimens were found on an adult male wombat from Coolagolite in New South Wales, and more specimens should be collected to confirm this host record. The most prevalent tick found on bare-nosed wombats was B. auruginans, confirming previous records. Analysis of alpha-diversity showed high variability across both sample locations and instars, similar to previous studies. The detection of various Proteobacteria in this study highlights the high bacterial diversity in native Australian ticks.

Project description:BackgroundIn the last decade, an innovative approach has emerged for arthropod identification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Increasing interest in applying the original technique for arthropod identification has led to the development of a variety of procedures for sample preparation and selection of body parts, among others. However, the absence of a consensual strategy hampers direct inter-study comparisons. Moreover, these different procedures are confusing to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS is therefore a necessity, and would notably enable the sharing of reference MS databases. Here, we assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling.MethodsThree homogenization methods, two of which were manual and one automatic, were used on three distinct body parts (legs, thorax, head) of two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti, and the results evaluated. The reproducibility of MS profiles, identification rate with relevant scores and the suitability of procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood-feeding and geographical origin were evaluated using both laboratory strains and field-collected mosquitoes.ResultsRelevant score values for mosquito identification were obtained for all the three body parts assayed using MALDI-TOF MS profiling; however, the thorax and legs were the most suitable specimens, independently of homogenization method or species. Although the manual homogenization methods were associated with a high rate of identification on the three body parts, this homogenization mode is not adaptable to the processing of a large number of samples. Therefore, the automatic homogenization procedure was selected as the reference homogenization method. Blood-feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from original blood in the MS profiles of the three body parts tested from both species. Finally, a significant improvement in identification scores was obtained for field-collected specimens when MS spectra of species from the same geographical area were added to the database.ConclusionThe results of the current study establish guidelines for the selection of mosquito anatomic parts and modality of sample preparation (e.g. homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database.

Project description:Background: Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients worldwide. Delays in the identification of microorganisms often leads to a poor prognosis. The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) directly to blood culture (BC) broth can potentially identify bloodstream infections earlier, and facilitate timely management. Methods: We developed an "in-house" (IH) protocol for direct MALDI-TOF MS based identification of organisms in positive BCs. The IH protocol was initially evaluated and improved with spiked BC samples, and its performance was compared with the commercial Sepsityper™ kit using both traditional and modified cut-off values. We then studied in parallel the performance of the IH protocol and the colony MS identifications in positive clinical BC samples using only modified cut-off values. All discrepancies were investigated by "gold standard" of gene sequencing. Results: In 54 spiked BC samples, the IH method showed comparable results with Sepsityper™ after applying modified cut-off values. Specifically, accurate species and genus level identification was achieved in 88.7 and 3.9% of all the clinical monomicrobial BCs (284/301, 94.4%), respectively. The IH protocol exhibited superior performance for Gram negative bacteria than for Gram positive bacteria (92.8 vs. 82.4%). For anaerobes and yeasts, accurate species identification was achieved in 80.0 and 90.0% of the cases, respectively. For polymicrobial cultures (17/301, 5.6%), MALDI-TOF MS correctly identified a single species present in all the polymicrobial BCs under the Standard mode, while using the MIXED method, two species were correctly identified in 52.9% of the samples. Comparisons based on BC bottle type, showed that the BACTEC™ Lytic/10 Anaerobic/F culture vials performed the best. Conclusion: Our study provides a novel and effective sample preparation method for MALDI-TOF MS direct identification of pathogens from positive BC vials, with a lower cost ($1.5 vs. $ 7) albeit a slightly more laborious extracting process (an extra 15 min) compared with Sepsityper™ kit.

Dataset Information

Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam.

Publications

Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam.

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