Search

Preventing tick bites is a fundamental step towards reducing the impact of tick-borne protozoal, bacterial and viral diseases (TBDs) in humans and animals. The aim of this study was to evaluate the efficacy of a combination of imidacloprid 10%/permethrin 50% and of fipronil 10%/S-methoprene 12% against ticks in naturally infected dogs and to assess methodological parameters to calculate drug efficacy on tick immature stages. From July to August 2004, 45 privately owned dogs of various sexes, ages, breeds, coat length and habits were enrolled in a trial carried out in an area (radius approximately 50km) in Southern Italy. Three homogeneous groups (both for dog population and tick population) were formed: 15 dogs treated with imidacloprid 10% and permethrin 50% spot-on (group A), 15 dogs treated with fipronil 10% and methoprene 12% spot-on (group B) and 15 untreated dogs (group C). The dogs in each group were then sub-grouped according to their age and weight. Two different treatments were administered (time 0 and +28 days) to groups A and B, and the dogs were checked weekly for tick infestation until day +56 post-treatment (p.t.). Twenty-four areas distributed on the whole body surface were examined for ticks at each follow-up, while only at time 0 and at day +56 p.t., ticks were collected from the dogs and identified. For the immature stages a semi-quantitative method was adopted and the load of immature stages was evaluated and grouped into four classes up to day +56 p.t. when the mean number of immature ticks (MIT) for each infection class was evaluated. All the adult ticks collected were identified as brown dog ticks (Rhipicephalus sanguineus). Immature stages were first compared at day +28 p.t.. The efficacy of both products used in groups A and B on adult ticks was high and generally very similar. Conversely, the efficacy of imidacloprid 10% and permethrin 50% against immatures was higher than that of fipronil 10% and methoprene 12% throughout the observation period with statistically significant differences (p

Oestrosis caused by larvae of the sheep nasal botfly, Oestrus ovis (Diptera, Oestridae), is a zoonotic nasal myiasis affecting small ruminants (mainly sheep and goats) across the world, particularly in African and European Mediterranean countries. Reports of human oestrosis generally refer to people who have been closely associated with sheep and goat husbandry such as shepherds and farmers. Different clinical forms of human oestrosis are known, including external ophthalmitis, pharyngeal or laryngeal myiasis, nasal or auricular forms, with the possibility of combined clinical presentations. Reports of human infection by O. ovis have been increasing over the last few years and, recently, the first case of human oestrosis has been detected in a farmer living in the Canary Islands. Here we report the first confirmed case of human ocular myiasis by O. ovis, which has occurred in an urban environment.

In Thailand and Laos alone, approximately 10 million people are infected with the liver fluke Opisthorchis viverrini [1]. Chronic infection with this parasite is considered the leading cause of cholangiocarcinoma (CCA, or bile-duct cancer) in large areas of Southeast Asia [2]. In these regions, CCA caused by O. viverrini is typically diagnosed 30–40 years after infection, with death occurring within 3–6 months post diagnosis [3]. O. viverrini is characterised by a three-host life cycle, with prosobranch snails of the genus Bithynia and cyprinid fishes acting as first and second intermediate hosts, respectively, while piscivorous mammals, including dogs, cats, and humans, act as definitive hosts [2]. Over the last two decades, much attention has been paid to studies on the epidemiology, developmental biology, and diagnosis of O. viverrini [4], while recent biotechnological advances are contributing large-scale explorations of the fundamental molecular biology of this liver fluke, with a view toward identifying key molecules essential for its development, reproduction, and survival, as well as dissecting the molecular pathways leading to the development of CCA [5]–[8]. These advances provide a solid foundation for the development of novel strategies to fight this devastating disease. However, long-term control of O. viverrini–induced cancer strictly relies on the development of integrated approaches, targeting the parasite as well as its intermediate hosts. Recently, an article in PLOS Neglected Tropical Diseases by Adema and colleagues [9] served to highlight the substantial gap in knowledge of aspects of the fundamental molecular biology of molluscs harbouring parasites, as well as the extraordinary opportunities that modern research toolkits, including microarray platforms, RNA interference, and high-throughput sequencing, offer for investigations of snail-parasite interactions [9]. Indeed, despite the massive expansion in the demand for and access to low-cost, high-throughput sequencing, large-scale genomic analyses of snails are limited to the draft genome sequence of the pulmonate snail intermediate host of Schistosoma blood flukes (Biomphalaria glabrata Genome Initiative at http://biology.unm.edu/biomphalaria-genome/index.html). Until now, there has been no genomic or transcriptomic information available for the prosobranch snail intermediate hosts of carcinogenic liver flukes, such as O. viverrini and Clonorchis sinensis. In order to provide the research community with a solid resource for molecular studies of these organisms, we generated the first reference transcriptome of Bithynia siamensis goniomphalos (Gastropoda, Bithyniidae), the intermediate host of O. viverrini in areas of northeast Thailand, Lao PDR, Cambodia, and South Vietnam, where the incidence of CCA is highest [cf. 10] (cf. Figure 1). A cDNA library from adult snails [11] was constructed, sequenced using RNA-seq (Illumina), and annotated using an established bioinformatic workflow [12]. Briefly, snails were collected from a natural body of water in Muang District, Khon Kaen Province, northeast Thailand (Figure 1); the taxonomic identity of the specimens was confirmed based on characteristic morphological features of the shells [13]; RNA was extracted from whole adult, parasite-free B. siamensis goniomphalos (n = 5), reverse-transcribed to cDNA, adaptor-ligated, and paired-end sequenced on a Genome Analyzer II (Illumina). Almost 50 million high-quality (Phred score >28) reads were generated; the assembly, produced using the Trinity software [14], yielded 167,029 contigs >200 bp in length, with a GC content of 44.4% (Table 1). Using sequence homology–based searches [12], approximately 40% of the assembled contigs could be annotated (cf. Table 1). A total of 32,026 contigs could be annotated with Gene Ontology terms (via Blast2GO; [15]), according to the categories “biological process,” “cellular component,” and “molecular function.” Approximately 77,000 non-overlapping protein sequences (of which ∼15,000 were full-length) could be inferred from the transcriptome of B. siamensis goniomphalos via BLASTx alignments with protein sequences available in public databases. Of the B. siamensis goniomphalos transcripts encoding proteins that could be mapped to orthologues in the Clusters of Orthologous Groups (COGs) database [16] (Table 1), the largest set was assigned to “general function prediction only” (16%), followed by “translation, ribosomal structure and biogenesis” (10%), “replication, recombination and repair” (7%), and “transcription” (7%) (Table 1). Approximately 54,000 protein-coding transcripts had orthologues in one of the 29 known biological pathways in the KEGG database (Table 1), including “regulation of actin cytoskeleton” (4%), “focal adhesion” (4%), and “spliceosome” (3.9%) (Table 1). Among the major protein classes included in the B. siamensis goniomphalos transcriptome were peptidases (n = 1,944; 4%), kinases (n = 4,757; 9%), phosphatases (n = 2,151; 4%), GTPases (n = 2,533; 5%), receptors (n = 6,243; 11%), transcription factors (n = 1,478; 3%), and channels and transporters (n = 1,287; 2%). Annotation information linked to each transcript characterised in the present study, including top BLASTx hit, GO classification, KEGG pathway mapping, and COG orthologues, is available from Table S1. In the absence of a reference genome for Bithynia spp., the annotation of the sequence data analysed herein was based on comparison with data available in public databases. The relatively small proportion of B. siamensis goniomphalos annotated protein sequences (∼60%) is likely to reflect the paucity of genomic sequence information available for prosobranch molluscan species in these databases [17]; supported by the availability of this dataset, as well as of the draft genome sequence of B. glabrata, future sequencing efforts will provide the depth of coverage required for the determination of the genome of Bithynia spp., which, in turn, will pave the way for comparative genomic studies of prosobranch and pulmonate snails harbouring parasite infections. This knowledge will be pivotal to improve our understanding of the biology of snail-borne parasites, and their “choice” of distinct snail species as their intermediate hosts. Figure 1 Distribution and prevalence of Opisthorchis viverrini, cholangiocarcinoma, and Bithynia spp. snails in Thailand. Table 1 Summary of the RNA-seq data for Bythinia siamensis goniomphalos prior to and following assembly, and bioinformatics annotation and analyses. Both raw and assembled sequence data generated in the present study are freely available from the Sequence Read Archive (SRA) and the Transcriptome Shotgun Assembly Sequence Database at NCBI (http://www.ncbi.nlm.nih.gov) under accession numbers SRR768418 and GAGS0000000, respectively. To our knowledge, this sequence data represents the first large-scale transcriptomic resource for a prosobranch mollusc intermediate host of a human platyhelminth and represents a major contribution to future fundamental explorations of the developmental biology of O. viverrini in Bithynia spp. snails, as well as the molecular interactions occurring at the snail-parasite interface. We suggest that the extensive sequence data generated in this study will be of great value in future studies aimed at exploring the changes in gene transcription occurring in Bithynia spp. upon infection with O. viverrini eggs and at different time points following infection. Besides yielding a general picture of the modified biology of trematode-infected snails, this data will set a basis for the identification of key genes, gene products, biological pathways, and/or bacterial and viral symbionts [cf. 9] involved in the cascade of molecular events leading to the development of the parasite through the stages of miracidium, sporocyst, redia, and cercaria. In turn, this advance will ultimately result in the development of novel targeted strategies to control snail-borne NTDs. New molecular technologies have a tremendous potential to aid our efforts in controlling snail-borne diseases. However, despite the unanimous acknowledgment that an increased understanding of the fundamental molecular biology of parasite-harbouring snails will provide us with a range of new tools to aid current efforts aimed at controlling snail-borne infections, the progressive but steady decline of malacology expertise and funding for snail vector–related research [cf. 9] poses a serious obstacle to the application of such technologies to snail-oriented NTD control. With this first step toward the establishment of a reference database for genetic research of prosobranch snail vectors of parasitic helminths, we hope to stimulate integrated, interdisciplinary research across malacology, helminthology, genomics, and bioinformatics, in the bid to fight snail-borne infections.

BackgroundSCP/TAPS proteins of parasitic helminths have been proposed to play key roles in fundamental biological processes linked to the invasion of and establishment in their mammalian host animals, such as the transition from free-living to parasitic stages and the modulation of host immune responses. Despite the evidence that SCP/TAPS proteins of parasitic nematodes are involved in host-parasite interactions, there is a paucity of information on this protein family for parasitic trematodes of socio-economic importance.Methodology/principal findingsWe conducted the first large-scale study of SCP/TAPS proteins of a range of parasitic trematodes of both human and veterinary importance (including the liver flukes Clonorchis sinensis, Opisthorchis viverrini, Fasciola hepatica and F. gigantica as well as the blood flukes Schistosoma mansoni, S. japonicum and S. haematobium). We mined all current transcriptomic and/or genomic sequence datasets from public databases, predicted secondary structures of full-length protein sequences, undertook systematic phylogenetic analyses and investigated the differential transcription of SCP/TAPS genes in O. viverrini and F. hepatica, with an emphasis on those that are up-regulated in the developmental stages infecting the mammalian host.ConclusionsThis work, which sheds new light on SCP/TAPS proteins, guides future structural and functional explorations of key SCP/TAPS molecules associated with diseases caused by flatworms. Future fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new approaches for the control of parasitic diseases.

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

Background The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention.

Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism., Author Summary In the present study, we constructed a cDNA library enriched for molecules of the infective third-stage larva (L3) of Ascaris suum, the common roundworm of pigs. Using the method of suppressive-subtractive hybridization (SSH), we explored transcription of a subset of molecules by microarray analysis and conducted bioinformatic analyses to characterize these molecules, map them to biochemical pathways, and predict genetic interactions based on comparisons with Caenorhabditis elegans and/or other organisms. The results provide interesting insights into early molecular processes in A. suum. Approximately 60% of the L3-enriched molecules discovered had homologues in C. elegans. Probabilistic analyses suggested that a complex genetic network regulates or controls larval growth and development in A. suum L3s, some of which might be involved in or regulate the switch from the free-living to the parasitic stage. Functional studies of these molecules to elucidate developmental processes in Ascaris could assist in identifying new targets for intervention.

The snail Bithynia siamensis goniomphalos acts as the first intermediate host for the human liver fluke Opisthorchis viverrini , the major cause of cholangiocarcinoma (CCA) in Northeast Thailand. The undisputed link between CCA and O. viverrini infection has precipitated efforts to understand the molecular basis of host–parasite interactions with a view to ultimately developing new control strategies to combat this carcinogenic infection. To date most effort has focused on the interactions between the parasite and its human host, and little is known about the molecular relationships between the liver fluke and its snail intermediate host. In the present study we analyse the protein expression changes in different tissues of B. siamensis goniomphalos induced by infection with larval O. viverrini using iTRAQ labelling technology. We show that O. viverrini infection downregulates the expression of oxidoreductases and catalytic enzymes, while stress-related and motor proteins are upregulated. The present work could serve as a basis for future studies on the proteins implicated in the susceptibility/resistance of B. siamensis goniomphalos to O. viverrini , as well as studies on other pulmonate snail intermediate hosts of various parasitic flukes that infect humans. Biological significance Despite the importance and high prevalence of opisthorchiasis in some regions of Southeast Asia and the direct relationship between infection by Opisthorchis viverrini and the incidence of cholangiocarcinoma, little is known of the modifications induced by this parasite in its snail intermediate hosts. This time-course study provides the first in-depth quantitative proteomic analysis of experimentally infected Bithynia siamensis goniomphalos . We show how motor and stress-related proteins are upregulated in infected snails, while O. viverrini infection downregulates the expression of oxidoreductases and catalytic enzymes. This work serves as a basis for the development of new strategies, focused on the invertebrate intermediate hosts, to control parasite transmission.

Thelazia callipaeda (Spirurida, Thelaziidae) infects the eyes of humans and rabbits as well of domestic and wild carnivores (i.e., dogs, cats, and foxes). The first three cases of infection by T. callipaeda eyeworms in wolves (Canis lupus) are here described with up to 96 nematodes collected from a single animal. The host competence of wolf was demonstrated by the retrieval of adult worms in the eyes of all examined animals. All nematodes collected were evaluated by morphologic and molecular techniques. Sequence analysis of the partial mitochondrial cytochrome c oxidase subunit 1 gene (cox1) confirmed that only one haplotype of T. callipaeda was present. The competence of wolves as a definitive host for T. callipaeda is discussed in view of the relevance of wild fauna in maintaining and spreading eyeworm infection in humans and domestic animals.

Background Tissue inhibitors of metalloproteases (TIMPs) are a multifunctional family of proteins that orchestrate extracellular matrix turnover, tissue remodelling and other cellular processes. In parasitic helminths, such as hookworms, TIMPs have been proposed to play key roles in the host-parasite interplay, including invasion of and establishment in the vertebrate animal hosts. Currently, knowledge of helminth TIMPs is limited to a small number of studies on canine hookworms, whereas no information is available on the occurrence of TIMPs in other parasitic helminths causing neglected diseases. Methods In the present study, we conducted a large-scale investigation of TIMP proteins of a range of neglected human parasites including the hookworm Necator americanus, the roundworm Ascaris suum, the liver flukes Clonorchis sinensis and Opisthorchis viverrini, as well as the schistosome blood flukes. This entailed mining available transcriptomic and/or genomic sequence datasets for the presence of homologues of known TIMPs, predicting secondary structures of defined protein sequences, systematic phylogenetic analyses and assessment of differential expression of genes encoding putative TIMPs in the developmental stages of A. suum, N. americanus and Schistosoma haematobium which infect the mammalian hosts. Results A total of 15 protein sequences with high homology to known eukaryotic TIMPs were predicted from the complement of sequence data available for parasitic helminths and subjected to in-depth bioinformatic analyses. Conclusions Supported by the availability of gene manipulation technologies such as RNA interference and/or transgenesis, this work provides a basis for future functional explorations of helminth TIMPs and, in particular, of their role/s in fundamental biological pathways linked to long-term establishment in the vertebrate hosts, with a view towards the development of novel approaches for the control of neglected helminthiases.