-
[
Parasitology,
2012]
Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins.
-
[
WormBook,
2007]
Because of their free-living life cycle alternatives, Strongyloides and related nematode parasites may represent the best models for translating C. elegans science to the study of nematode parasitism. S. stercoralis, a significant pathogen of humans, can be maintained in laboratory dogs and gerbils. Biosafety precautions necessary for work with S. stercoralis, though unfamiliar to many C. elegans researchers, are straightforward and easily accomplished. Although specialized methods are necessary for large-scale culture of the free-living stages of S. stercoralis, small-scale cultures for experimental purposes may be undertaken using minor modifications of standard C. elegans methods. Similarly, the morphological similarities between C. elegans and the free-living stages of S. stercoralis allow investigational methods such as laser cell ablation and DNA transformation by gonadal microinjection to be easily adapted from C. elegans to S. stercoralis. Comparative studies employing these methods have yielded new insights into the neuronal control of the infective process in parasites and its similarity to regulation of dauer development in C. elegans. Furthermore, we have developed a practical method for transient transformation of S. stercoralis with vector constructs having various tissue- and cell-specific expression patterns and have assembled these into a modular vector kit for distribution to the community.
-
[
Parasitol Today,
1992]
A major challenge to the development of vaccines against human lymphatic filariasis and onchocerciasis is to direct the immune response toward elimination of the early, prepathogenic larval stages and away from responses that mediate pathology. In this review, James Lok and David Abraham discuss the various animal models that have been used to investigate the pathways leading to immunity, immunological tolerance and chronic pathology in these diseases. Owing to the strict host specificities of the human-dwelling filariae, no single model serves to duplicate exactly all these aspects. Nevertheless, it has been possible to demonstrate a protective immune response invoked by and directed against incoming third-stage larvae of both lymphatic and skin-dwelling filariae. The fact that subsets of the sequelae of human filarial infection can be duplicated in animal systems should also aid in unravelling the mechanisms determining the course of infection and in ensuring that vaccine candidates do not produce an inappropriate immunopathological response. A proposed scheme for using animal models in screening candidates for a vaccine against Onchocerca volvulus is presented.
-
[
Tropenmed Parasitol,
1980]
Two species of Nearctic black fly, Simulium decorum and S. pictipes, exhibit partial susceptibility to infection with the bovine parasite, Onchocerca lienalis, when inoculated intrathoracically with microfilariae. In addition, a proportion of S. decorum females will support the development of the human parasite, O. volvulus, to the third larvae stage. Infection rates with second- or third-stage larvae seven or more days after varied among geographic strains of S. decorum, ranging from 6.5% in a strain from northern New York State to 48.7% in a strain from Georgia. The average number of larvae per infected female ranged from 1.00 to 1.78 in the three strains examined. Partial susceptibility to O. lienalis was found to persist in colonized strains of s. decorum, and cryopreserved microfilariae of this parasite retained their infectivity. Seven or more days after inoculation with microfilariae of the Guatemalan strain of O. volvulus, 16.7% of the females of S. decorum harbored second- or third-stage larvae. Development of O. lienalis and O. volvulus proceeded normally in these black flies, and moderate increases in susceptibility rate and number of infective larvae were noted in response to increased microfilarial dosages.
-
[
Curr Trop Med Rep,
2014]
Human strongyloidiasis is a threat to global health, presenting significant challenges in diagnosis and clinical management. The imperative to incorporate strongyoidiasis more fully into control programs for soil-transmitted helminths is increasingly recognized. The unique life cycles of<i>S. stercoralis</i>and congeneric species contain both free-living and parasitic generations, and transcriptomic methods have recently identified genes of potential importance to parasitism in these parasites. Proteomics recently revealed stage-specific secreted proteins that appear crucial to the host-parasite interaction. A comprehensive genome sequencing project for<i>Strongyloides</i>spp. is now nearing completion. Recent technical advances in transgenesis for<i>S. stercoralis</i>and<i>S. ratti</i>, including the first establishment of stable transgenic lines, promise to advance functional evaluations of genes expressed in conjunction with crucial life cycle events. Studies employing these methods recently bolstered the hypothesis that<i>S. stercoralis</i>uses cellular signaling pathways homologous to three that regulate dauer larval development in<i>Caenorhabditis elegans</i>to regulate morphogenesis and development of its infective third-stage larva. The free-living generation of<i>Strongyloides</i>makes classical genetics formally possible. Recent advances, such as a genetic map of<i>S. ratti</i>and a molecular genetic and karyotypic analysis of sex determination in<i>S. papillosus</i>, will greatly facilitate this approach. Advanced methods for study of chemosensation in<i>C. elegans</i>were recently applied to discover numerous host attractant molecules that mediate host finding and contact by infective third-stage larvae of<i>Strongyloides</i>spp. Finally, nucleic acid-based diagnostic methods have recently come to the fore as alternatives to parasitological and immunodiagnostic techniques.
-
[
Parasite Immunol,
2008]
Ease of experimental gene transfer into viral and prokaryotic pathogens has made transgenesis a powerful tool for investigating the interactions of these pathogens with the host immune system. Recent advances have made this approach feasible for more complex protozoan parasites. By contrast, the lack of a system for heritable transgenesis in parasitic nematodes has hampered progress toward understanding the development of nematode-specific cellular responses. Recently, however, significant strides towards such a system have been made in several parasitic nematodes, and the possible applications of these in immunological research should now be contemplated. In addition, methods for targeted cell ablation have been successfully adapted from Caenorhabditis elegans methodology and applied to studies of neurobiology and behaviour in Strongyloides stercoralis. Together, these new technical developments offer exciting new tools to interrogate multiple aspects of the host-parasite interaction following nematode infection.
-
[
Parasitology,
2016]
Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.
-
[
Curr Clin Microbiol Rep,
2016]
Signaling or communication between host and parasite may occur over relatively long ranges to enable host finding and acquisition by infective parasitic nematode larvae. Innate behaviors in infective larvae transmitted from the soil that enhance the likelihood of host contact, such as negative geotaxis and hypermotility, are likely mediated by mechanoreception and neuromuscular signaling. Host cues such as vibration of the substratum, elevated temperature, exhaled CO2, and other volatile odorants are perceived by mechanosensory and chemosensory neurons of the amphidial complex. Beyond this, the molecular systems that transduce these external cues within the worm are unknown at this time. Overall, the signal transduction mechanisms that regulate switching between dauer and continuous reproductive development in Caenorhabditis elegans, and doubtless other free-living nematodes, have provided a useful framework for testing hypotheses about how the morphogenesis and development of infective parasitic nematode larvae and the lifespan of adult parasites are regulated. In C. elegans, four major signal transduction pathways, G protein-coupled receptor signaling, insulin/insulin-like growth factor signaling, TGF-like signaling and steroid-nuclear hormone receptor signaling govern the switch between dauer and continuous development and regulate adult lifespan. Parasitic nematodes appear to have conserved the functions of G-protein-coupled signaling, insulin-like signaling and steroid-nuclear hormone receptor signaling to regulate larval development before and during the infective process. By contrast, TGF-like signaling appears to have been adapted for some other function, perhaps modulation of the host immune response. Of the three signal transduction pathways that appear to regulate development in parasitic nematodes, steroid-nuclear hormone signaling is the most straightforward to manipulate with administered small molecules and may form the basis of new chemotherapeutic strategies. Signaling between parasites and their hosts' immune systems also occurs and serves to modulate these responses to allow chronic infection and down regulate acute inflammatory responses. Knowledge of the precise nature of this signaling may form the basis of immunological interventions to protect against parasitism or related lesions and to alleviate inflammatory diseases of various etiologies.
-
[
Bio Protoc,
2014]
Splinkerette PCR (spPCR) is a newly developed and efficient method to ascertain and characterize genomic insertion sites of transgenes. The method described in this protocol was successfully applied to confirm piggyBac transposon-mediated integration of transgenes into chromosomes of the parasitic nematode Strongyloides ratti. This work is described in detail in Shao et al. (2012) and presented here in a simplified diagram (Figure 1). Using this method, chromosomal loci of integration were determined based on target site and 5'- and 3' flanking sequences. Therefore, spPCR can be a useful method to confirm integrative transgenesis in functional genomic studies of parasitic nematodes. Potter and Luo (2010) contains a protocol for use of spPCR to detect and map piggyBac transposon-mediated chromosomal integrations in Drosophila, and was the source of our method for Strongyloides. The splinkerette- and piggyBac-specific oligos described in that reference could be used without modification in Strongyloides. For interested readers, a general review of the biology of parasitic nematodes in the genus Strongyloides may be found in Viney and Lok (2007), and a methods-based article on S. stercoralis as an experimental model, with information on transgenesis, may be found in Lok (2007).
-
[
Curr Trop Med Rep,
2019]
Purpose of Review: spp. and related parasitic nematodes. Recent Findings: in a new murine model of human strongyloidiasis. Summary: demonstrated the potential of this pathway as a novel chemotherapeutic target in parasitic nematodes.