Biology subjects

Martinelli, A.

Publications and source records attributed to Martinelli, A..

4 recordsLinked to original sources

bioRxiv2018

A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of ITS1 amplicons

The World Health Organization has targeted stopping the transmission of Human African Trypanosomiasis by 2030. To achieve this, better tools are urgently required to identify and monitor Trypanosome infections in human, animals, and tsetse fly vectors. This study presents a single test approach for detection and identification of Trypanosomes and their comprehensive characterization at species and sub-group level. Our method uses newly designed ITS1 PCR primers (a widely used method for detection of African Trypanosomes, amplifying the ITS1 region of ribosomal RNA genes) coupled to Illumina sequencing of the amplicon. The protocol is based on the widely used Illuminas 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. We analyzed wild tsetse flies collected from Zambia and Zimbabwe. Our results show that the traditional method for Trypanosome species detection based on band size comparisons on a gel is unable to distinguish between T. vivax and T. godfreyi accurately. Additionally, this approach shows increased sensitivity of detection at species level. Through phylogenetic analysis, we identified Trypanosomes at species and sub-group level without the need for any additional tests. Our results show T. congolense Kilifi sub-group is more closely related to T. simiae than to other T. congolense sub-groups. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any sub-groups for T. vivax and T. godfreyi, we observed distinct subgroups for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from the rest of the T. simiae Tsavo sequences suggesting that the Nannomonas group is more divergent than currently thought thus the need for a better classification criteria. This approach has the potential for refining classification of Trypanosomes and provide detailed molecular epidemiology information useful for surveillance and transmission control efforts.\n\nAuthor summaryDetection of Trypanosomes in the tsetse flies plays an important role in the control of African trypanosomiasis by providing information on circulating Trypanosome species in a given area. We have developed a method that combines multiplex PCR and next-generation sequencing for Trypanosome species detection. The method is based on the widely used bacterial metagenomic analysis protocol and uses a modular, two-step PCR process followed by sequencing of all amplicons in a single run, making sequencing of amplicons more efficient and cost-effective when dealing with large sample sizes. As part of this approach, we designed novel primers for amplifying the ITS1 region of the Trypanosome rRNA gene that is more sensitive than conventional primers. Identification of Trypanosome species is based on BLAST searches against the constantly updated NCBIs nt database, which facilitates the identification of Trypanosome subgroups. Our approach is more accurate than traditional gel-based analysis and shows how the latter is prone to misidentification. It is also sensitive and is able to discriminate between subgroups within Trypanosome species. Applied as an epidemiological tool, it has the potential to provide new, comprehensive and more accurate information on vector-pathogen-host interconnections which are key in the control and management of African trypanosomiasis.

epidemiology↗

bioRxiv2018

A major locus for ivermectin resistance in a parasitic nematode

BackgroundInfections with helminths cause an enormous disease burden in billions of animals and plants worldwide. Large scale use of anthelmintics has driven the evolution of resistance in a number of species that infect livestock and companion animals, and there are growing concerns regarding the reduced efficacy in some human-infective helminths. Understanding the mechanisms by which resistance evolves is the focus of increasing interest; robust genetic analysis of helminths is challenging, and although many candidate genes have been proposed, the genetic basis of resistance remains poorly resolved. ResultsHere, we present a genome-wide analysis of two genetic crosses between ivermectin resistant and sensitive isolates of the parasitic nematode Haemonchus contortus, an economically important gastrointestinal parasite of small ruminants and a model for anthelmintic research. Whole genome sequencing of parental populations, and key stages throughout the crosses, identified extensive genomic diversity that differentiates populations, but after backcrossing and selection, a single genomic quantitative trait locus (QTL) localised on chromosome V was revealed to be associated with ivermectin resistance. This QTL was common between the two geographically and genetically divergent resistant populations and did not include any leading candidate genes, suggestive of a previously uncharacterised mechanism and/or driver of resistance. Despite limited resolution due to low recombination in this region, population genetic analyses and novel evolutionary models supported strong selection at this Q.TL, driven by at least partial dominance of the resistant allele, and that large resistance-associated haplotype blocks were enriched in response to selection. ConclusionsWe have described the genetic architecture and mode of ivermectin selection, revealing a major genomic locus associated with ivermectin resistance, the most conclusive evidence to date in any parasitic nematode. This study highlights a novel genome-wide approach to the analysis of a genetic cross in non-model organisms with extreme genetic diversity, and the importance of a high quality reference genome in interpreting the signals of selection so identified.

genomics↗

bioRxiv2018

The dyslexia susceptibility KIAA0319 gene shows a highly specific expression pattern during zebrafish development supporting a role beyond neuronal migration.

Dyslexia is a common neurodevelopmental disorder that affects reading abilities and is predicted to be caused by a significant genetic component. Very few genetic susceptibility factors have been identified so far and amongst those, KIAA0319 is a key candidate. KIAA0319 is highly expressed during brain development but its function remains poorly understood. Initial RNA-interference studies in rats suggested a role in neuronal migration whereas subsequent work with double knock-out mouse models for both Kiaa0319 and its paralogue Kiaa0319-like reported effects in the auditory system but not in neuronal migration. To further understand the role of KIAA0319 during neurodevelopment, we carried out an expression study of the zebrafish orthologue at different embryonic stages. We report particularly high gene expression during the first few hours of development. At later stages, expression becomes localised in well-defined structures such as the eyes, the telencephalon and the notochord, supporting a role for kiaa0319 that is not restricted to brain development. Surprisingly, kiaa0319-like, which generally shows a similar expression pattern, was not expressed in the notochord suggesting a role specific to kiaa0319 in this structure. This study contributes to our understanding of KIAA0319 function during embryonic development which might involve additional roles in the visual system and in the notochord. Such a specific spatiotemporal expression pattern is likely to be under the controlled of tightly regulated sequences. Therefore, these data provide a framework to interpret the effects of the dyslexia-associated genetic variants that reside in KIAA0319 non-coding regulatory regions.

neuroscience↗

bioRxiv2017

Genomic And Transcriptomic Profiling Of Nematode Parasites Surviving After Vaccine Exposure

Some nematode species are economically important parasites of livestock, while others are important human pathogens causing some of the most important neglected tropical diseases. In both humans and animals, anthelmintic drug administration is the main control strategy, but the emergence of drug-resistant worms has stimulated the development of alternative control approaches. Among these, vaccination is considered to be a sustainable and cost effective strategy. Currently, Barbervax(R) for the ruminant strongylid Haemonchus contortus is the only registered subunit vaccine for a nematode parasite, although a vaccine for the human hookworm Necator americanus is undergoing clinical trials (HOOKVAC consortium). As both these vaccines comprise a limited number of proteins there is potential for selection of nematodes with altered sequence or expression of the vaccine antigens. Here we compared the transcriptome of H. contortus populations from sheep vaccinated with Barbervax(R) with worms from control animals. Barbervax(R) antigens are native integral membrane proteins isolated from the brush border of the intestinal cells of the adult parasite and many of them are proteases. Our findings provide no evidence for changes in expression of genes encoding Barbervax(R) antigens in the surviving parasite populations. However, surviving parasites from vaccinated animals showed increased expression of other proteases and regulators of lysosome trafficking, and displayed up-regulated lipid storage and defecation abilities that may have circumvented the vaccine effect. Implications for other potential vaccines for human and veterinary nematodes are discussed.

genomics↗