Vaccination against the brown stomach worm, Teladorsagia circumcincta, followed by parasite challenge, induces inconsistent modifications in gut microbiota composition of lambs.

BACKGROUND: Growing evidence points towards a role of gastrointestinal (GI) helminth parasites of ruminants in modifying the composition of the host gut flora, with likely repercussions on the pathophysiology of worm infection and disease, and on animal growth and productivity. However, a thorough understanding of the mechanisms governing helminth-microbiota interactions and of their impact on host health and welfare relies on reproducibility and replicability of findings. To this aim, in this study, we analysed quantitative and qualitative fluctuations in the faecal microbiota composition of lambs vaccinated against, and experimentally infected with, the parasitic GI nematode Teladorsagia circumcincta over the course of two separate trials performed over two consecutive years. METHODS: Two trials were conducted under similar experimental conditions in 2017 and 2018, respectively. In each trial, lambs were randomly assigned to one of the following experimental groups: (i) vaccinated/infected, (ii) unvaccinated/infected and (iii) unvaccinated/uninfected. Faecal samples collected from individual animals were subjected to DNA extraction followed by high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene and bioinformatics and biostatistical analyses of sequence data. RESULTS: Substantial differences in the populations of bacteria affected by immunisation against and infection by T. circumcincta were detected when comparing data from the two trials. Nevertheless, the abundance of Prevotella spp. was significantly linked to helminth infection in both trials. CONCLUSIONS: Despite the largely conflicting findings between the two trials, our data revealed that selected gut microbial populations are consistently affected by T. circumcincta infection and/or vaccination. Nevertheless, our study calls for caution when interpreting data generated from in vivo helminth-microbiome interaction studies that may be influenced by several intrinsic and extrinsic host-, parasite- and environment-related factors.

Infection with the sheep gastrointestinal nematode Teladorsagia circumcincta increases luminal pathobionts.

BACKGROUND: The multifaceted interactions between gastrointestinal (GI) helminth parasites, host gut microbiota and immune system are emerging as a key area of research within the field of host-parasite relationships. In spite of the plethora of data available on the impact that GI helminths exert on the composition of the gut microflora, whether alterations of microbial profiles are caused by direct parasite-bacteria interactions or, indirectly, by alterations of the GI environment (e.g. mucosal immunity) remains to be determined. Furthermore, no data is thus far available on the downstream roles that qualitative and quantitative changes in gut microbial composition play in the overall pathophysiology of parasite infection and disease. RESULTS: In this study, we investigated the fluctuations in microbiota composition and local immune microenvironment of sheep vaccinated against, and experimentally infected with, the 'brown stomach worm' Teladorsagia circumcincta, a parasite of worldwide socio-economic significance. We compared the faecal microbial profiles of vaccinated and subsequently infected sheep with those obtained from groups of unvaccinated/infected and unvaccinated/uninfected animals. We show that alterations of gut microbial composition are associated mainly with parasite infection, and that this involves the expansion of populations of bacteria with known pro-inflammatory properties that may contribute to the immunopathology of helminth disease. Using novel quantitative approaches for the analysis of confocal microscopy-derived images, we also show that gastric tissue infiltration of T cells is driven by parasitic infection rather than anti-helminth vaccination. CONCLUSIONS: Teladorsagia circumcincta infection leads to an expansion of potentially pro-inflammatory gut microbial species and abomasal T cells. This data paves the way for future experiments aimed to determine the contribution of the gut flora to the pathophysiology of parasitic disease, with the ultimate aim to design and develop novel treatment/control strategies focused on preventing and/or restricting bacterial-mediated inflammation upon infection by GI helminths. Video Abstract.

Characterisation of a niche-specific excretory-secretory peroxiredoxin from the parasitic nematode Teladorsagia circumcincta.

BACKGROUND: The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory-secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory-secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae). RESULTS: In this study excretory-secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory-secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells. CONCLUSIONS: In this study, we identified differences in the excretory-secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory-secretory proteins of potential importance for parasite survival and/or host immune modulation.

The rational simplification of a recombinant cocktail vaccine to control the parasitic nematode Teladorsagia circumcincta.

Using data from five independent vaccine trials, which employed a subunit cocktail vaccine containing eight recombinant proteins to protect sheep against Teladorsagia circumcincta, a strategy was developed to simplify antigen complexity of the vaccine. A meta-analysis of data from these five trials demonstrated statistically significant reductions in cumulative faecal egg count and worm burden in vaccinated sheep when compared with those which had received adjuvant only (P = 0.009 and P < 0.0001, respectively). Relationships between antigen-specific antibody levels, antibody avidity and parasitological parameters of efficacy were analysed for each of the eight proteins in these trials. Of these, the strongest correlations between percentage reduction in cumulative faecal egg count and avidity were obtained for the vaccine antigen T. circumcincta apyrase-1 (Tci-APY-1) in relation to either total antigen-specific IgG or IgG1 in sera (P = 0.019 and P = 0.030, respectively). In addition, IgG and IgA within the serum and abomasal mucus of control (parasite challenged) lambs strongly recognised Tci-APY-1 and T. circumcincta metalloproteinase-1 (Tci-MEP-1) but only weakly bound the other six antigens, indicating Tci-APY-1 and Tci-MEP-1 are most effectively recognised by the parasite-induced antibody response. On the basis of these findings, a two-protein vaccine comprising Tci-APY-1 and Tci-MEP-1 was tested in a direct comparison with the original eight-component vaccine. A further group was immunised with Tci-MEP-1 in combination with a mutated form of Tci-APY-1 (mTci-APY-1), which had no enzymatic activity. Across the trial, the mean faecal egg count levels of the eight-antigen recipients were lower than those of the adjuvant only control group (P = 0.013) and the mean FEC of the mTci-APY-1 and Tci-MEP-1 recipients was lower, although not statistically significantly, than that of the adjuvant-only control group (P = 0.093). Mean cumulative faecal egg count levels were reduced by 43% in lambs immunised with mTci-APY-1 plus Tci-MEP-1 compared with the controls (P = 0.079).

Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations.

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the ß-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.

Niche-specific gene expression in a parasitic nematode; increased expression of immunomodulators in Teladorsagia circumcincta larvae derived from host mucosa.

Metazoan parasites have to survive in many different niches in order to complete their life-cycles. In the absence of reliable methods to manipulate parasite genomes and/or proteomes, identification of the molecules critical for parasite survival within these niches has largely depended on comparative transcriptomic and proteomic analyses of different developmental stages of the parasite; however, changes may reflect differences associated with transition between developmental stages rather than specific adaptations to a particular niche. In this study, we compared the transcriptome of two fourth-stage larval populations of the nematode parasite, Teladorsagia circumcincta, which were of the same developmental stage but differed in their location within the abomasum, being either mucosal-dwelling (MD) or lumen-dwelling (LD). Using RNAseq, we identified 57 transcripts which were significantly differentially expressed between MD and LD larvae. Of these transcripts, the majority (54/57) were up-regulated in MD larvae, one of which encoded for an ShKT-domain containing protein, Tck6, capable of modulating ovine T cell cytokine responses. Other differentially expressed transcripts included homologues of ASP-like proteins, proteases, or excretory-secretory proteins of unknown function. Our study demonstrates the utility of niche- rather than stage-specific analysis of parasite transcriptomes to identify parasite molecules of potential importance for survival within the host.

Protection of ewes against Teladorsagia circumcincta infection in the periparturient period by vaccination with recombinant antigens.

Teladorsagiosis is a major production-limiting disease in ruminants in temperate regions throughout the world and one of the key interventions in the management of the disease is the prevention of pasture contamination with Teladorsagia circumcincta eggs by ewes during the periparturient relaxation in immunity which occurs in the period around lambing. Here, we describe the immunisation of twin-bearing ewes with a T. circumcincta recombinant subunit vaccine and the impact that vaccination has on their immune responses and shedding of parasite eggs during a continuous T. circumcincta challenge period spanning late gestation and lactation. In ewes which displayed a clear periparturient relaxation in immunity, vaccination resulted in a 45% reduction in mean cumulative faecal egg count (cFEC, p=0.027) compared to control (immunised with adjuvant only) ewes. Recombinant antigen-specific IgG and IgA, which bound each of the vaccine antigens, were detected in the serum of vaccinated ewes following each immunisation and in colostrum taken from vaccinated ewes post-partum whereas low levels of antigen-specific IgG were detected in serum and colostrum from control ewes. Antigen-specific IgG and IgA levels in blood collected within 48h of birth from lambs largely reflected those in the colostrum of their ewes.

Identification and evaluation of vaccine candidate antigens from the poultry red mite (Dermanyssus gallinae).

An aqueous extract of the haematophagous poultry ectoparasite, Dermanyssus gallinae, was subfractionated using anion exchange chromatography. Six of these subfractions were used to immunise hens and the blood from these hens was fed, in vitro, to poultry red mites. Mite mortality following these feeds was indicative of protective antigens in two of the subfractions, with the risks of mites dying being 3.1 and 3.7 times higher than in the control group (P<0.001). A combination of two-dimensional immunoblotting and immunoaffinity chromatography, using IgY from hens immunised with these subfractions, was used in concert with proteomic analyses to identify the strongest immunogenic proteins in each of these subfractions. Ten of the immunoreactive proteins were selected for assessment as vaccine candidates using the following criteria: intensity of immune recognition; likelihood of exposure of the antigen to the antibodies in a blood meal; proposed function and known vaccine potential of orthologous molecules. Recombinant versions of each of these 10 proteins were produced in Escherichia coli and were used to immunise hens. Subsequent in vitro feeding of mites on blood from these birds indicated that immunisation with Deg-SRP-1 (serpin), Deg-VIT-1 (vitellogenin), Deg-HGP-1 (hemelipoglycoprotein) or Deg-PUF-1 (a protein of unknown function) resulted in significantly increased risk of mite death (1.7-2.8times higher than in mites fed blood from control hens immunised with adjuvant only, P<0.001). The potential for using these antigens in a recombinant vaccine is discussed.

The emergence of resistance to the benzimidazole anthlemintics in parasitic nematodes of livestock is characterised by multiple independent hard and soft selective sweeps.

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the "genetic signature" of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.

Suppression of ovine lymphocyte activation by Teladorsagia circumcincta larval excretory-secretory products.

Teladorsagia circumcincta is an important pathogenic nematode of sheep. It has been demonstrated previously that stimulation of murine T lymphocytes with excretory-secretory (ES) products derived from fourth stage larvae of T. circumcincta (Tci-L4-ES) results in de novo expression of Foxp3, a transcription factor intimately involved in regulatory T cell function. In the current study, Foxp3⁺ T cell responses in the abomasum and the effects of Tci-L4-ES on ovine peripheral blood mononuclear cells (PBMC) following T. circumcincta infection were investigated. T. circumcincta infection resulted in a significant increase in numbers of abomasal Foxp3⁺ T cells, but not an increase in the proportion of T cells expressing Foxp3. Unlike in mice, Tci-L4-ES was incapable of inducing T cell Foxp3 expression but instead suppressed mitogen-induced and antigen-specific activation and proliferation of ovine PBMC in vitro. This effect was heat labile, suggesting that it is mediated by protein(s). Suppression was associated with up-regulation of interleukin-10 (IL-10) mRNA, and specific monoclonal antibody neutralisation of IL-10 resulted in a 50% reduction in suppression, indicating involvement of the IL-10 signaling pathway. Suppression was significantly reduced in PBMC isolated from T. circumcincta infected vs. helminth-naïve lambs, and this reduction in suppression was associated with an increase in Tci-L4-ES antigen-specific T cells within the PBMC. In conclusion, we have identified a mechanism by which T. circumcincta may modulate the host adaptive immune response, potentially assisting survival of the parasite within the host. However, the impact of Tci-L4-ES-mediated lymphocyte suppression during T. circumcincta infection remains to be determined.

Successful immunization against a parasitic nematode by vaccination with recombinant proteins.

Infection of humans and livestock with parasitic nematodes can have devastating effects on health and production, affecting food security in both developed and developing regions. Despite decades of research, the development of recombinant sub-unit vaccines against these pathogens has been largely unsuccessful. We have developed a strategy to identify protective antigens from Teladorsagia circumcincta, the major pathogen causing parasitic gastroenteritis in small ruminants in temperate regions, by studying IgA responses directed at proteins specific to post-infective larvae. Antigens were also selected on the basis of their potential immunomodulatory role at the host/parasite interface. Recombinant versions of eight molecules identified by immunoproteomics, homology with vaccine candidates in other nematodes and/or with potential immunoregulatory activities, were therefore administered to sheep in a single vaccine formulation. The vaccine was administered three times with Quil A adjuvant and the animals subsequently subjected to a repeated challenge infection designed to mimic field conditions. Levels of protection in the vaccinates were compared to those obtained in sheep administered with Quil A alone. The trial was performed on two occasions. In both trials, vaccinates had significantly lower mean fecal worm egg counts (FWECs) over the sampling period, with a mean reduction in egg output of 70% (Trial 1) and 58% (Trial 2). During the period of peak worm egg shedding, vaccinates shed 92% and 73% fewer eggs than did controls in Trials 1 and 2, respectively. At post mortem, vaccinates had 75% (Trial 1) and 56% (Trial 2) lower adult nematode burdens than the controls. These levels of protection are the highest observed in any system using a nematode recombinant sub-unit vaccine in the definitive ruminant host and indicate that control of parasitic helminths via vaccination with recombinant subunit vaccine cocktails is indeed an alternative option in the face of multi-drug resistance.

A comparative study of the effects of four treatment regimes on ivermectin efficacy, body weight and pasture contamination in lambs naturally infected with gastrointestinal nematodes in Scotland.

Refugia-based drenching regimes have been widely recommended to slow development of anthelmintic resistance but there are few comparisons between different treatment approaches in the UK. The impact of four ivermectin treatment regimes on drug efficacy, lamb body weight and nematode contamination during a 154 day grazing season were evaluated in a consecutive five year field study. Regimes were whole-flock treatment every 4 weeks (NST), targeted selective treatment (TST) based on individual performance, strategic whole-flock treatments at pre-determined times (SPT) or whole-flock treatment when clinical signs were apparent (MT). Mean numbers of ivermectin drenches administered per season were 4.0, 1.8, 2.0 and 1.4 for NST, TST, SPT and MT groups, respectively. The mean anthelmintic efficacy (AE) for each treatment group was based on faecal egg count reduction post-treatment employing a bootstrap sampling based algorithm. Mean AE was 95-98% for all groups in 2006 and mean AE (95% confidence limits) for NST declined to 62% (55%, 68%) in 2010. In comparison, AE for TST, SPT and MT in 2010 were 86% (81%, 92%), 86% (83%, 90%) and 83% (78%, 88%), respectively. Body weight in TST and SPT was similar to NST in all years (p > 0.05), however MT lambs were lighter than NST in 2006-2008 (p â©½ 0.04). Tracer lamb worm burdens was lowest in NST but was not significantly different between other groups. Overall, both the TST and SPT regimes appeared to maintain animal performance and conserve anthelmintic efficacy compared with a neo-suppressive anthelmintic treatment regime.

A survey of the trichostrongylid nematode species present on UK sheep farms and associated anthelmintic control practices.

A survey of sheep farms from across the UK was conducted to establish information on farming practices, the trichostrongylid nematode species present and anthelmintic usage. Questionnaires and faecal samples were returned from 118 farms. First stage larvae (L(1)) were cultured from faecal samples and used for PCR analysis to determine the presence/absence of selected trichostrongylid species. Teladorsagia circumcincta was the only species present on 100% of farms. Haemonchus contortus was found on ∼50% of farms and was widespread throughout the UK. The most common Trichostrongylus spp. was T. vitrinus, found on 95% of farms. Determining the anthelmintic dose rate based on the weight of the heaviest animal in the flock to avoid under dosing was carried out on 58% of farms and was associated with a significantly lower mean epg (p<0.001) in lambs. However, the weight of animals was only estimated (as opposed to animals weighed) on 32% of farms. Macrocyclic lactones (ML) were the most commonly used anthelmintic class for ewes, whilst benzimidazoles (BZ) were the most widely used in lambs. Twenty-two of the surveyed farms had confirmed anthelmintic resistance, of these, 18 had BZ resistance, one had levamisole (LEV) resistance and 3 had resistance to both BZ and LEV. No farms in this survey reported resistance to ML. Location had a significant effect on the incidence of anthelmintic resistance on the farms in this survey (p=0.002). There was evidence of a lower risk of anthelmintic resistance occurring on farms from Scotland compared to those in England (p(f)=0.047) and Wales (p(f)=0.012). Farm type, flock type and open or closed status did not have any significant effect on the incidence of anthelmintic resistance when all other factors were taken into consideration.

Oral dosing with papaya latex is an effective anthelmintic treatment for sheep infected with Haemonchus contortus.

BACKGROUND: The cysteine proteinases in papaya latex have been shown to have potent anthelmintic properties in monogastric hosts such as rodents, pigs and humans, but this has not been demonstrated in ruminants. METHODS: In two experiments, sheep were infected concurrently with 5,000 infective larvae of Haemonchus contortus and 10,000 infective larvae of Trichostrongylus colubriformis and were then treated with the supernatant from a suspension of papaya latex from day 28 to day 32 post-infection. Faecal egg counts were monitored from a week before treatment until the end of the experiment and worm burdens were assessed on day 35 post-infection. RESULTS: We found that the soluble fraction of papaya latex had a potent in vivo effect on the abomasal nematode H. contortus, but not on the small intestinal nematode T. colubriformis. This effect was dose-dependent and at tolerated levels of gavage with papaya latex (117 µmol of active papaya latex supernatant for 4 days), the H. contortus worm burdens were reduced by 98%. Repeated treatment, daily for 4 days, was more effective than a single dose, but efficacy was not enhanced by concurrent treatment with the antacid cimetidine. CONCLUSIONS: Our results provide support for the idea that cysteine proteinases derived from papaya latex may be developed into novel anthelmintics for the treatment of lumenal stages of gastro-intestinal nematode infections in sheep, particularly those parasitizing the abomasum.