Immunochemical Analysis of Antigens of the Bovine Lungworm Dictyocaulus viviparus

A successful irradiated larval vaccine against the cattle lungworm, Dictyocaulus viviparus, has been available for over thirty years. At the outset of the project, however, little was known of antigens of the parasite or of the mechanism of vaccine-induced immunity. This study aimed to characterise antigens of this parasite which may be involved in protective immunity and which could eventually lead to the development of a molecularly-defined vaccine against lungworm infection. The antibody responses to surface, somatic and excretory-secretory (ES) products of larval and adult stage parasites were examined following infection or vaccination of bovine hosts and of the guinea-pig model host in which infection proceeds only to the L5 stage. No parasite proteins could be detected in ES products of infective stage larvae, despite containing substantial levels of proteinase activity. In contrast, a complex range of proteins were detected in the secretions of adult worms. Immunoprecipitation studies of radioiodinated adult ES products revealed that all of these, with the exception of two components, one of which was identified as bovine serum albumin, are antigenic to infected bovine hosts. Calves vaccinated with irradiated larvae, and therefore, not exposed to patent lungworm infection showed restricted recognition of adult ES products, thus demonstrating the stage-specific nature of D. viviparus released products. Significant heterogeneity in the specificity of the antibody response to adult ES products was observed between individual bovine and guinea-pig hosts. This individual variability was examined in the model system using inbred strains of guinea-pig and was shown to have a genetic basis, possibly being controlled by the MHC class II region. Examination of the antibody response to surface-exposed antigens of the egg, L1, L3 and adult stages of D. viviparus demonstrated both the antigenicity and stage-specificity of surface components. Immunofluorescence studies revealed significant recognition of the L3 sheath by antibody from infected and vaccinated bovine and guinea-pig hosts. In contrast, surface-exposed antigens of the adult, egg and L1 stages were recognised uniquely by calves infected with normal larvae and , therefore, exposed to patent lungworm infection. No binding of parasite specific IgG antibody was observed on the exposed surface of exsheathed L3 (i. e. the L3 cuticle) with serum from infected calves. All bovine pre-infection sera examined showed a substantial degree of IgM antibody binding to the L3 cuticle and it is proposed that this non-specific IgM antibody may block immune recognition of parasite-specific surface antigens. IgG antibody recognition of exposed L3 cuticular antigens was observed, however, with sera from hosts exposed to irradiated larvae suggesting that this immunoevasive mechanism may be overcome to some extent by vaccination. As well as differing antigenically, the L3 cuticle was found to differ biophysically from that of other stages of D. viviparus as demonstrated by its inability to bind the fluorescent lipid analogue 5-(N-octadecanoyl)aminofluorescein and to incorporate Iodine-125 into cuticular proteins. These findings may reflect changes in the surface properties of the parasite associated with host infection. Radioiodination of intact sheathed larvae identified a restricted set of proteins while a complex set of labelled proteins was observed following radioiodination of intact adult parasites. Many more adult components were labelled by the Bolton-Hunter than by the lodogen technique, probably reflecting that labelling by the latter method is more surface-restricted. There was no turnover of the major adult surface-associated antigens suggesting that surface components do not contribute to adult ES products of this parasite. Examination of the biological functions of larval and adult extracts and ES products revealed the presence of superoxide dismutase (SOD) and proteinase activities. Characterisation of the latter by pH optima, substrate specificity, inhibitor sensitivity and substrate gel electrophoresis identified multiple proteolytic activities. These enzymes may be involved in parasite invasion and survival within the host. The significant inhibition of both proteinase and SOD activities observed following incubation with immunoglobulin from immune calves may, therefore, be important in limiting parasite survival and consequently such enzymes may be of value as potential vaccine candidates. Finally, a comparison of 35S-methionine labelled polypeptides of normal and irradiated third stage larvae revealed no qualitative nor quantitative differences. It is, therefore, proposed that vaccine-induced immunity to D. viviparus may not depend on the expression of novel parasite antigens but on an enhanced immune recognition of larval stage antigens

Increased expression of a microRNA correlates with anthelmintic resistance in parasitic nematodes

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3′ UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3′ UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species

Small RNAs in parasitic nematodes - forms and functions

Small RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface

The potential for vaccines against scour worms of small ruminants

This review addresses the research landscape regarding vaccines against scour worms, particularly Trichostrongylus spp. and Teladorsagia circumcincta. The inability of past research to deliver scour-worm vaccines with reliable and reproducible efficacy has been due in part to gaps in knowledge concerning: (i) host-parasite interactions leading to development of type-2 immunity, (ii) definition of an optimal suite of parasite antigens, and (iii) rational formulation and administration to induce protective immunity against gastrointestinal nematodes (GIN) at the site of infestation. Recent ‘omics’ developments enable more systematic analyses. GIN genomes are reaching completion, facilitating “reverse vaccinology” approaches that have been used successfully for the Rhipicephalus australis vaccine for cattle tick, while methods for gene silencing and editing in GIN enable identification and validation of potential vaccine antigens. We envisage that any efficacious scour worm vaccine(s) would be adopted similarly to “Barbervax™” within integrated parasite management schemes. Vaccines would therefore effectively parallel the use of resistant animals, and reduce the frequency of drenching and pasture contamination. These aspects of integration, efficacy and operation require updated models and validation in the field. The conclusion of this review outlines an approach to facilitate an integrated research program

The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery

<p>Background: The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.</p> <p>Results: Here we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates.</p> <p>Conclusions: The H. contortus genome and transcriptome provides an essential platform for postgenomic research in this and other important strongylid parasites. </p&gt

A novel technique for retrospective genetic analysis of the response to vaccination or infection using cell-free DNA from archived sheep serum and plasma

Genetic variation is associated with differences in disease resistance and susceptibility among individuals within a population. To date, molecular genetic analyses of host responses have relied on extraction of genomic DNA from whole blood or tissue samples. However, such samples are not routinely collected during large-scale field studies. We demonstrate that cell-free genomic DNA (cfDNA) may be extracted and amplified from archived plasma samples, allowing retrospective analysis of host genetic diversity. This technique was also applicable to archived serum samples up to 35 years old and to different ruminant species. As proof of concept, we used this cfDNA approach to genotype the major histocompatibility complex (MHC) class II DRB1 locus of 224 Merino sheep which had participated in field trials of a commercial Haemonchus contortus vaccine, Barbervax®, in Australia. This identified a total of 51 different DRB1 alleles and their relative frequencies. This is the first study to examine host MHC diversity using DNA extracted from archived plasma samples, an approach that may be applied to retrospective analyses of genetic diversity and responses to vaccination or infection across different species and populations

The parasitic nematode Haemonchus contortus lacks molybdenum cofactor synthesis, leading to sulphite sensitivity and lethality in vitro

Sulphite oxidase has an essential role in detoxifying environmental and endogenously generated sulphite into sulphate and requires the molybdenum cofactor (Moco) to function. Until recently it was believed that the synthesis pathway for Moco was so important for survival that it was conserved in all multicellular animals. Here we report the use of comparative genomics to identify the absence of the first enzyme involved in Moco synthesis in Haemonchus contortus, a highly pathogenic and economically important helminth of livestock that, similar to many parasitic nematode species, has proved difficult to maintain in vitro. We show that Moco deficiency in Haemonchus leads to a high sensitivity to environmental sulphite and limits the ability to maintain the early parasitic larval stages in vitro. Analogous losses in Moco synthesis in other recently sequenced nematode species are also identified. These findings may lead to improved culture methods for parasitic nematodes and to novel approaches for their control

Organoids in parasitology: a game-changer for studying host-nematode interactions

Abstract not currently available

A novel member of the let-7 microRNA family is associated with developmental transitions in filarial nematode parasites

Background: Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event. Results: microRNAs differentially expressed during the early post-infective stages of Brugia pahangi L3 were identified by microarray analysis. One of these, bpa-miR-5364, was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade III nematodes, and is a novel member of the let-7 family, which are known to have key developmental functions in the free-living nematode Caenorhabditis elegans. Predicted mRNA targets of bpa-miR-5364 were identified using bioinformatics and comparative genomics approaches that relied on the conservation of miR-5364 binding sites in the orthologous mRNAs of other filarial nematodes. Finally, we confirmed the interaction between bpa-miR-5364 and three of its predicted targets using a dual luciferase assay. Conclusions: These data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. This study is the first to identify parasitic nematode mRNAs that are verified targets of specific microRNAs and demonstrates that post-transcriptional control of gene expression via stage-specific expression of microRNAs may be important in the success of filarial infection

Differences in immune responses to Haemonchus contortus infection in the susceptible Ile de France and the resistant Santa Ines sheep under different anthelmintic treatments regimens

Understanding the immunological basis of resistance to gastrointestinal nematode infections in livestock is important in order to develop novel methods of parasite control such as vaccination or genetic selection for parasite resistance. The present study aimed to investigate differences in immune response between parasite resistant Santa Ines and susceptible Ile de France sheep breeds to natural Haemonchus contortus infection. Parasitological parameters, humoral immunity, local and circulating cellular immune responses were evaluated in 19 Santa Ines and 19 Ile de France lambs undergoing different anthelmintic treatments regimens: suppressive treatments (SUP) or targeted selective treatments (TST) over a 5-month grazing period. Santa Ines lambs had significantly lower Haemonchus faecal egg count and worm burden compared to Ile de France regardless of treatment regime. In addition, circulating blood eosinophils count and parasite-specific IgG levels were significantly higher and more rapidly induced in Santa Ines lambs. Abomasal immune responses were generally greater in the resistant breed, which had significantly higher levels of parasite-specific IgA in mucus, and elevated number of globule leukocytes and CD3+ T cells within the abomasal mucosal. Furthermore, numbers of POU2F3+ epithelial cells, a tuft-cell specific transcription factor, were also elevated in the Santa Ines breed, suggesting that this breed is better able to initiate T-helper type 2 immune responses within the abomasum. In conclusion, the differential immunological responses detailed here are relevant to understanding resistance to gastrointestinal nematodes in other host breeds, as well as to resistance breeding as a sustainable control approach for parasitic infections