Excretory-secretory (ES) products of Ostertagia ostertagi, an abomasal nematode of cattle, are considered to be important for the development and survival of the parasite within the host. To gain insight in the composition of these ES products of both larval (L3, L4) and adult life stages of Ostertagia cDNA libraries of the parasite were immunoscreened with polyclonal rabbit serum raised against these ES products. This approach led to the identification of 41 proteins, amongst which are structural proteins such as actin, kinesin and vitellogenin, housekeeping proteins such as those involved in protein folding, different metabolic pathways or mitochondrial functioning and proteins associated with stress (heat shock protein) or antioxidantia (thioredoxin peroxidase). A large number of the isolated proteins were similar to hypothetical proteins of the model nematode Caenorhabditis elegans. Because somatic proteins can be non-specifically released during in vitro culturing as nematodes deteriorate, it was checked if the isolated proteins are genuinely secreted. The amino acid sequences of the translated cDNAs were investigated for signal peptides and monospecific antibodies against the isolated proteins were purified and used to develop Western blots of ES and somatic extracts. In this manner it could be proven that 15 cDNAs code for genuine secreted proteins. The identification of these ES antigens allows to select proteins with potential protective capacities, which are targets for vaccine development.

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.

The sheep scab mite, Psoroptes ovis, is a major problem in the beef cattle industry, especially in Belgian Blue (BB) cattle. This breed is naturally more predisposed to psoroptic mange but reasons for this high susceptibility remain unknown. Different immune responses could be a potential cause; thus in this study, the cutaneous immune response and in vitro cellular immune response after antigen re-stimulation were examined in naturally infested BB. Cytokine production in the skin and in circulating re-stimulated peripheral blood mononuclear cells (PBMC) demonstrated a mixed pro-inflammatory Th2/Th17 profile, with transcription of IL-4, IL-13, IL-6 and IL-17. Strong IL-17 up-regulation in the skin of BB was associated with an influx of eosinophils and other immune cells, potentially leading towards more severe symptoms. Virtually no changes in cutaneous IFN-γ transcription were detected, while there was substantial IFN-γ up-regulation in re-stimulated PBMC from infested and uninfested animals, potentially indicating a role of this pro-inflammatory cytokine in the innate immune response. In Holstein-Friesian (HF) cattle, generally more resistant to P. ovis infection, a largely similar immunologic response was observed. Differences between HF and BB were the lack of cutaneous IL-17 response in infested HF and low transcription levels of IFN-γ and high IL-10 transcription in re-stimulated PBMC from both infested and uninfested animals. Further research is needed to identify potential cell sources and biological functions for these cytokines and to fully unravel the basis of this different breed susceptibility to P. ovis.

Successful and sustainable intervention against human helminthiases depends on optimal utilisation of available control measures and development of new tools and strategies, as well as an understanding of the evolutionary implications of prolonged intervention on parasite populations and those of their hosts and vectors. This will depend largely on updated knowledge of relevant and fundamental parasite biology. There is a need, therefore, to exploit and apply new knowledge and techniques in order to make significant and novel gains in combating helminthiases and supporting the sustainability of current and successful mass drug administration (MDA) programmes. Among the fields of basic research that are likely to yield improved control tools, the Disease Reference Group on Helminth Infections (DRG4) has identified four broad areas that stand out as central to the development of the next generation of helminth control measures: 1) parasite genetics, genomics, and functional genomics; 2) parasite immunology; 3) (vertebrate) host-parasite interactions and immunopathology; and 4) (invertebrate) host-parasite interactions and transmission biology. The DRG4 was established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR). The Group was given the mandate to undertake a comprehensive review of recent advances in helminthiases research in order to identify notable gaps and highlight priority areas. This paper summarises recent advances and discusses challenges in the investigation of the fundamental biology of those helminth parasites under the DRG4 Group's remit according to the identified priorities, and presents a research and development agenda for basic parasite research and enabling technologies that will help support control and elimination efforts against human helminthiases.

The protozoan parasite Giardia is a highly prevalent intestinal pathogen with a wide host range. Data obtained in mice, cattle and humans revealed the importance of IL-17A in the development of a protective immune response against Giardia. The aim of this study was to further unravel the protective effector mechanisms triggered by IL-17A following G. muris infection in mice, by an RNA-sequencing approach. C57BL/6 WT and C57BL/6 IL-17RA KO mice were orally infected with G. muris cysts. Three weeks post infection, intestinal tissue samples were collected for RNA-sequencing, with samples from uninfected C57BL/6 WT and C57BL/6 IL-17RA KO animals serving as negative controls. Differential expression analysis showed that G. muris infection evoked the transcriptional upregulation of a wide array of genes, mainly in animals with competent IL-17RA signaling. IL-17RA signaling induced the production of various antimicrobial peptides, such as angiogenin 4 and α- and β-defensins and regulated complement activation through mannose-binding lectin 2. The expression of the receptor that regulates the secretion of IgA into the intestinal lumen, the polymeric immunoglobulin receptor, was also dependent on IL-17RA signaling. Interestingly, the transcriptome data showed for the first time the involvement of the circadian clock in the host response following Giardia infection.

The scientific community identified non stool-based biomarkers as the way forward to support soil-transmitted helminth (STH; Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus) and schistosome (S. mansoni and S. haematobium) deworming programs. This support is needed in making the decision of whether or not to stop preventive chemotherapy intervention efforts and to ultimately transition towards a post-intervention surveillance phase. We applied a two-step micro-array approach to identify antigenic linear epitopes in the STH and S. mansoni proteomes. In a first experiment, we identified antigenic peptides by applying sera from 24 STH and/or S. mansoni infected Ethiopian children on a high-density peptide microarray containing 3.3 million peptides derived from the complete STH and S. mansoni proteomes. A second array experiment with 170,185 peptides that were recognized in the first array was designed to identify non-specific antibody reactivity by applying sera from 24 healthy individuals from Belgium (a non-endemic country). From this array testing cascade, several peptides were identified for STH but none of them appeared to be unique for one species. We therefore concluded that for STH, none of the peptides revealed to be sufficiently sensitive or species specific. For S. mansoni, some promising peptides were identified prompting future investigation. Based on these results, it is unlikely that linear epitopes would be highly useful in detecting species-specific antibody responses to STH in endemic communities. For S. mansoni, one particular peptide of the micro-exon gene 12 (MEG-12) protein deserves further research. In addition, this study emphasizes the need of well-characterized biobanks for biomarker discovery, particularly when the integration of multiple disease programs is envisioned.

Conventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA).

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity.

Ascaris is a soil-transmitted nematode that causes ascariasis, a neglected tropical disease affecting predominantly children and adolescents in the tropics and subtropics. Approximately 0.8 billion people are affected worldwide, equating to 0.86 million disability-adjusted life-years (DALYs). Exploring the molecular biology of Ascaris is important to gain a better understanding of the host-parasite interactions and disease processes, and supports the development of novel interventions. Although advances have been made in the genomics, transcriptomics and proteomics of Ascaris, its lipidome has received very limited attention. Lipidomics is an important sub-discipline of systems biology, focused on exploring lipids profiles in tissues and cells, and elucidating their biological and metabolic roles. Here, we characterised the lipidomes of key developmental stages and organ systems of Ascaris of porcine origin via high throughput LC-MS/MS. In total, > 500 lipid species belonging to 18 lipid classes within three lipid categories were identified and quantified-in precise molar amounts in relation to the dry weight of worm material-in different developmental stages/sexes and organ systems. The results showed substantial differences in the composition and abundance of lipids with key roles in cellular processes and functions (e.g. energy storage regulation and membrane structure) among distinct stages and among organ systems, likely reflecting differing demands for lipids, depending on stage of growth and development as well as the need to adapt to constantly changing environments within and outside of the host animal. This work provides the first step toward understanding the biology of lipids in Ascaris, with possibilities to work toward designing new interventions against ascariasis.

Anthelmintic resistance among GINs in cattle is a worldwide issue. Identifying the early signs of anthelmintic resistance (AR) is necessary to sustainably manage bovine parasitic infections. This study aimed to evaluate the resistance status of bovine parasitic nematodes against FBZ on a farm with a known history of broad-spectrum anthelmintic usage in Ecuador. FBZ efficacy was analyzed using a fecal egg count reduction test (FECR test) and β-tubulin 1 mutation identification in Cooperia spp., the dominant nematode parasite identified before and after treatment. According to the FECR test, the nematode population was susceptible to FBZ. After amplifying and cloning the β-tubulin 1 of Cooperia spp., an F200Y mutation was found in 43% of the pooled larva coproculture after treatment. This study reports, for the first time, the presence of F200Y resistance-conferring mutation in Cooperia spp. in Ecuador. Although the nematode population was phenotypically susceptible to FBZ, the presence of F200Y suggests the existence of resistance in the early stages. Our findings highlight the need to implement alternative control strategies for parasitic infections besides broad-spectrum anthelmintics.

Digital Dermatitis (DD) is a common disease of dairy cows, the pathogenesis of which is still not clear. This study examined some host responses associated with the typical lesions, in an attempt to further elucidate the pathogenesis of the disease. Twenty four samples representing the 5 different clinical stages of DD (M0-M4) were collected from slaughtered cattle for histopathological and immunological analyses.

Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections.

Intestinal helminths are potent regulators of their host's immune system and can ameliorate inflammatory diseases such as allergic asthma. In the present study we have assessed whether this anti-inflammatory activity was purely intrinsic to helminths, or whether it also involved crosstalk with the local microbiota. We report that chronic infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb) altered the intestinal habitat, allowing increased short chain fatty acid (SCFA) production. Transfer of the Hpb-modified microbiota alone was sufficient to mediate protection against allergic asthma. The helminth-induced anti-inflammatory cytokine secretion and regulatory T cell suppressor activity that mediated the protection required the G protein-coupled receptor (GPR)-41. A similar alteration in the metabolic potential of intestinal bacterial communities was observed with diverse parasitic and host species, suggesting that this represents an evolutionary conserved mechanism of host-microbe-helminth interactions.

The aim of this study was to explore the mechanisms of resistance against invading Ascaris suum larvae in pigs. Pigs received a low dose of 100 A. suum eggs daily for 14 weeks. This resulted in a >99% reduction in the number of larvae that could migrate through the host after a challenge infection of 5000 A. suum eggs, compared to naïve pigs. Histological analysis at the site of parasite entry, i.e. the caecum, identified eosinophilia, mastocytosis and goblet cell hyperplasia. Increased local transcription levels of genes for IL5, IL13, eosinophil peroxidase and eotaxin further supported the observed eosinophil influx. Further analysis showed that eosinophils degranulated in vitro in response to contact with infective Ascaris larvae in the presence of serum from both immune and naïve animals. This effect was diminished with heat-inactivated serum, indicating a complement dependent mechanism. Furthermore, eosinophils were efficient in killing the larvae in vitro when incubated together with serum from immune animals, suggesting that A. suum specific antibodies are required for efficient elimination of the larvae. Together, these results indicate an important role for eosinophils in the intestinal defense against invading A. suum larvae.

Resistance against macrocyclic lactones such as ivermectin is widespread among parasitic gastrointestinal nematodes of small ruminants and is rapidly increasing in cattle parasites. ABC transporters of the subfamily B, the so-called P-glycoproteins (Pgps) have been frequently implicated in ivermectin resistance and are a major cause of multi-drug resistance in protozoa and helminths. The Pgp inhibitor verapamil (VPL) dramatically enhanced susceptibility of the cattle parasitic nematode Cooperia oncophora to ivermectin in vitro as measured in a larval developmental assay and a larval migration inhibition assay using third stage larvae. Moreover, VPL completely restored susceptibility to ivermectin in a resistant isolate resulting in virtually identical dose-response curves of susceptible and resistant isolates in the presence of VPL. Further characterisation of the molecular mechanisms resulting in Pgp-mediated ivermectin resistance is still hampered by the lack of molecular and biochemical information for Pgps of parasitic nematodes. Using PCR with degenerate primers, fragments of four different C. oncophora Pgps could be amplified and the Conpgp-2, previously implicated in macrocyclic lactone resistance in Haemonchus contortus, and Conpgp-3 full-length cDNAs were obtained by RACE PCR. The pgp sequences presented here contribute important data required to systematically screen resistant C. oncophora isolates for up- or down-regulation of Pgps and for the detection of single nucleotide polymorphisms in Pgps to detect selection of specific Pgp alleles by anthelmintics as early as possible.

The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential.

The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs.

Ascariasis, caused by Ascaris suum, is an important soil-transmitted parasitic disease of pigs worldwide. It leads to significant economic losses in the pork industry, as a consequence of low feed conversion efficiency in pigs and liver condemnation at slaughter. Despite ascariasis still being widespread on pig farms in many developing and the industrialised countries, there are surprisingly limited data on porcine ascariasis in China, where nearly half of the world's total pork is produced.

Psoroptic mange is a common disease of livestock, caused by Psoroptes ovis. Compared to Holstein-Friesian (HF) cattle, the Belgian Blue (BB) cattle breed is highly susceptible to the infestation. However, the mechanism for this difference is still unclear. To determine the factors responsible for this breed susceptibility, the immune response to P. ovis was studied in experimentally infested BB and HF cattle, using clinical signs, histology, immunohistochemical profiling and gene expression analysis of skin biopsies. The mite numbers and lesion area of BB cattle were greater than in HF during the whole study period. Significant influxes of eosinophils in the epidermis and dermis were detected in comparison with the pre-infestation samples in both breeds, with significantly higher eosinophils in BB at 6 weeks post infestation (wpi). Mast cell numbers were unaffected at all stages of infestation in HF, but were significantly elevated relative to pre-infestation in BB cattle at 2 and 6 wpi. The more pronounced cutaneous eosinophilia and higher IL-4 levels at 6 wpi in BB cattle suggest that a Th2-type immune response is underlying the higher susceptibility of the BB breed. In naturally infested BB cattle, development of the psoroptic mange lesions and eosinophils and CD3+ T cell areas were severely depressed after anti-inflammatory treatment with dexamethasone. Together, these results suggest that a stronger Th2-type immune response to P. ovis causes the skin lesions in psoroptic mange in BB cattle and that local anti-inflammatory treatment could potentially be an alternative to control the pathology caused by this parasite.

Hookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored.