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).     

New molecular evidence that Teladorsagia circumcincta (Nematoda: Trichostrongylidea) is a species complex

In the polymorphic Teladorsagia circumcincta (morphs circumcincta and trifurcata), a sheep and goat line (SGL) and a goat line (GL) have been previously described on the basis of the malate dehydrogenase allozyme polymorphism (MDH-2) and of the morphology of the dorsal ray. The GL were never found alone in 1 host, so the status of species was not given to these 2 lines. To investigate further whether there are other genetic markers that will delineate them, we collected T. circumcincta worms from goat and sheep at 8 farms in Touraine (west-central France). The worms were identified individually as being SGL or GL on the basis of MDH-2 polymorphism. This distinctiveness was corroborated by sequences of the beta-tubulin isotype I gene, the second internal transcribed spacer (ITS2) of their rDNA, and the nicotinamide dehydrogenase (ND4) gene of their mDNA. The extent of the divergence in the 3 additional genetic markers was such that SGL and GL may be considered as 2 species. A third putative species was found in the SGL line based exclusively on the ND4 gene. These findings suggest that T. circumcincta is a species complex and that further investigation is required on a wider geographic scale.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Enzymes of the ornithine-glutamate-proline pathway in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

A fully functional ornithine–glutamate–proline pathway was detected in L3 and adult Haemonchus contortus and Teladorsagia circumcincta, making the parasites capable of interconversion of these amino acids. Ornithine aminotransferase (OAT) (E.C. 2.6.1.13) was a reversible pyridoxal-5-phosphate (PLP)-dependent enzyme with an optimum pH 8.5. Hydroxylamine completely inhibited OAT activity in both parasites. For all five enzymes, substrate affinity was similar for each species and life cycle stage, the notable exceptions being the nearly 10-fold lower affinity for Δ¹-pyrroline-5-carboxylate (P5C) of P5C reductase (E.C. 1.5.1.2) in adult T. circumcincta and about half for P5C for L3 H. contortus P5C dehydrogenase (E.C. 1.5.1.12). P5C synthase (E.C. 1.2.1.41) activity was similar with either NADPH or NADH as co-factor. Proline oxidase (E.C. 1.5.99.8) was a co-factor independent enzyme with an optimal pH 8.5. Despite similarities to those in the host, enzymes of this pathway may still be useful as control targets if they differ antigenically, as a supply of proline is necessary for cuticle formation.     

Population biology of the parasitic phase of Ostertagia circumcincta

Four current models for the parasitic phase of the Ostertagia circumcincta life-cycle were evaluated with respect to their ability to represent the outcome of experimental infection studies and the population biology of the parasite in the field. Neither of the two discrete-time models was able to mimic the rise and fall in parasite numbers that characterize trickle infection experiments. When the infection rate is constant, both models predict that parasite numbers will eventually reach an asymptotic equilibrium value. This happens because both models are formulated such that parasite mortality is constant when the infection rate is constant. The two continuous-time models are able to mimic trickle infection experiments because both of them represent parasite mortality as an increasing function of the infection rate and the duration of infection. However, the continuous-time models do not adequately represent the demography of the parasitic phase in the field because neither of them takes any account of the effect of variations in infection rate from host to host on the overall mean parasite death rate.     

Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD⁺ and NADP⁺, GDH activity was greater in the deaminating reaction with NADP⁺ as co-factor and more with NADH in the aminating reaction.     

Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species

The genetic structure of the root endophytes Phialocephala fortinii s.l. and Acephala applanata was analyzed in two undisturbed forests. A total of 606 strains isolated from surface-sterilized, fine roots of Picea abies and Vaccinium myrtillus were examined. Two new cryptic species of P. fortinii were recognized and host specialization of A. applanata was confirmed. This species was almost exclusively isolated from roots of P. abies. The index of association did not deviate significantly from zero within any population, suggesting that recombination occurs or had occurred. Significant gene but no genotype flow was detected among study sites for P. fortinii s.l. In contrast, several isolates of A. applanata with both identical multi-locus haplotype and identical ISSR fingerprint were found in both study sites indicating genotype flow or a recent common history.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

Lack of evidence for co-speciation in a parasitic nematode of grey kangaroos

Multilocus enzyme electrophoresis was used to compare specimens of the parasitic nematode Cloacina obtusa from the stomach of the eastern grey kangaroo, Macropus giganteus and the western grey kangaroo, M. fuliginosus. Allelic variation among nematodes was detected at 17 (85%) of 20 loci, but there was only a single fixed genetic difference (at the locus for isocitrate dehydrogenase, IDH) between C. obtusa from M. fuliginosus and those from M. giganteus in areas where each host occurred in allopatry. However, this fixed difference was not apparent within the zone of host sympatry. Although electrophoretic data indicate genetic divergence among allopatric populations of C. obtusa in the two host species, the magnitude of the electrophoretic difference (5%) between these populations does not refute the hypothesis that C. obtusa represents a single species. The 'usual' situation for parasitic helminths of grey kangaroos is that pairs of parasite species occur in the two host species. This situation differs for C. obtusa, where there has been a lack of speciation following a speciation event in its macropodid marsupial hosts. This finding suggests that a speciation event in the host does not necessarily lead to a speciation event for all its parasites and further highlights our lack of understanding of which processes drive speciation in parasites.     

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta

Phosphofructokinase (PFK-1) activity was examined in L₃ and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4 ± 0.2 to 1.7 ± 0.2 in L₃s and 2.0 ± 0.3 to 2.4 ± 0.4 in adults) and the K_½ for fructose 6 phosphate (from 0.35 ± 0.02 to 0.75 ± 0.05 mM in L₃s and 0.40 ± 0.03 to 0.65 ± 0.05 mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2 mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.     

Sex-dependent genetic effects on immune responses to a parasitic nematode

Background

Many disease aetiologies have sex specific effects, which have important implications for disease management. It is now becoming increasingly evident that such effects are the result of the differential expression of autosomal genes rather than sex-specific genes. Such sex-specific variation in the response to Trichuris muris, a murine parasitic nematode infection and model for the human parasitic nematode T. trichiura, has been well documented, however, the underlying genetic causes of these differences have been largely neglected. We used the BXD mouse set of recombinant inbred strains to identify sex-specific loci that contribute to immune phenotypes in T. muris infection.

Results

Response phenotypes to T. muris infection were found to be highly variable between different lines of BXD mice. A significant QTL on chromosome 5 (TM5) associated with IFN-γ production was found in male mice but not in female mice. This QTL was in the same location as a suggestive QTL for TNF-α and IL-6 production in male mice suggesting a common control of these pro-inflammatory cytokines. A second QTL was identified on chromosome 4 (TM4) affecting worm burden in both male and female cohorts. We have identified several genes as potential candidates for modifying responses to T. muris infection.

Conclusions

We have used the largest mammalian genetic model system, the BXD mouse population, to identify candidate genes with sex-specific effects in immune responses to T. muris infection. Some of these genes may be differentially expressed in male and female mice leading to the difference in immune response between the sexes reported in previous studies. Our study further highlights the importance of considering sex as an important factor in investigations of immune response at the genome-wide level, in particular the bias that can be introduced when generalizing results obtained from only one sex or a mixed sex population. Rather, analyses of interaction effects between sex and genotype should be part of future studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-193) contains supplementary material, which is available to authorized users.     

Molecular genetic analysis and ecological evidence reveals multiple cryptic species among thynnine wasp pollinators of sexually deceptive orchids

Sexually deceptive Chiloglottis orchids lure their male thynnine wasp pollinators to the flower by emitting semiochemicals that mimic the specific sex pheromone of the wasp. Sexual deception is possible because chemical rather than visual cues play the key role in wasp mate search, suggesting that cryptic wasp species may be frequent. We investigated this prospect among Neozeleboria wasp pollinators of Chiloglottis orchids, drawing on evidence from molecular phylogenetic analysis at three genes (CO1, rhodopsin and wingless), population genetic and statistical parsimony analysis at CO1, orchid associations and their semiochemicals, and geographic ranges. We found a compelling relationship between genetically defined wasp groups, orchid associations, semiochemicals and geographic range, despite a frequent lack of detectable morphological differences. Our findings reveal multiple cryptic species among orchid pollinators and indicate that chemical changes are important for wasp reproductive isolation and speciation. The diversity of Neozeleboria may have enabled, rather than constrained, pollinator-driven speciation in these orchids.     

Serial passage in resistant sheep drives the infectivity and fitness of Teladorsagia circumcincta in susceptible lambs: Experimental evidence

Gastrointestinal nematodes (GIN) of small ruminants have adapted their life history strategies to thrive in diverse and fluctuating environments. Environments which alter their expression of life traits may also drive changes in the infection or transmission dynamics, particularly if transferred to a foreign setting. This study aimed to explore how repeated exposure to a resistant sheep host environment would alter the life history traits and infection dynamics of Teladorsagia circumcincta when consequently infected in susceptible lambs. Following just three generations of passage in resistant sheep, T. circumcincta significantly increased their infectivity and fitness in susceptible lambs compared to a control population. This is the first evidence to indicate the resistant host environment can drive such rapid changes in the expression of GIN life traits, with potentially undesirable epidemiological outcomes.     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

First evidence of cryptic species diversity and significant population structure in a widespread freshwater nematode morphospecies (Tobrilus gracilis)

Free‐living nematodes are ubiquitous and highly abundant in terrestrial and aquatic environments, where they sustain ecosystem functioning by mineralization processes and nutrient cycling. Nevertheless, very little is known about their true diversity and intraspecific population structure. Recent molecular studies on marine nematodes indicated cryptic diversity and strong genetic differentiation of distinct populations, but for freshwater nematode species, analogous studies are lacking. Here, we present the first extensive molecular study exploring cryptic species diversity and genetic population structure of a widespread freshwater nematode morphospecies, Tobrilus gracilis, from nine postglacially formed European lakes. Taxonomic species status of individuals, analysed for fragments of the mitochondrial COI gene and for the large (LSU) and small (SSU) ribosomal subunits, were determined by morphological characteristics. Mitochondrial and nuclear markers strongly supported the existence of three distinct genetic lineages (Tg I–III) within Tobrilus gracilis, suggesting that this morphospecies indeed represents a complex of highly differentiated biological species. High genetic diversity was also observed at the population level. Across the nine lakes, 19 mitochondrial, and seven (LSU) and four (SSU) nuclear haplotypes were determined. A phylogeographical analysis revealed remarkable genetic differentiation even among neighbouring lake populations for one cryptic lineage. Priority and persistent founder effects are possible explanations for the observed population structure in the postglacially colonized lakes, but ask for future studies providing direct estimates of freshwater nematode dispersal rates. Our study suggests therefore that overall diversity of limnetic nematodes has been so far drastically underestimated and challenges the assumed ubiquitous distribution of other, single freshwater nematode morphospecies.     

Untangling the mechanisms of cryptic species coexistence in a nematode community through individual-based modelling

Cryptic species are morphologically identical but genetically distinct, and are prominent across numerous phyla. The coexistence of such closely related species on local scales would seem to run counter to traditional coexistence and competition theory; it has been hypothesized as a consequence of differences in their resource use or tolerances to environmental conditions. We developed an individual-based model of a community of three cryptic Litoditis marina (nematode) species, to understand how individual-level interspecific and intraspecific interactions might explain the coexistence of these closely related species. The model incorporates individuals' reproduction, competition, dispersal and resource use. Data characterizing the cryptic species (growth rates, dispersal ability, competitive interactions and responses to changing environmental conditions) were obtained from laboratory experiments involving both mono- and multispecific nematode cultures, and are used to parameterize the model. Simulation studies are used to investigate which individual-level mechanisms of dispersal and interaction lead to the characteristic population-level patterns observed experimentally. Our results highlight the key role of intraspecific competition in mediating dispersal and therefore co-occurrence of the cryptic species. The differences in dispersal also influence the response of the cryptic species to competition, a combination of factors that provides an explanation for their co-occurrence. These results provide insights into how changes in individual-level processes can be amplified to affect population-level co-occurrence.