Use of fluorescent lectin binding to distinguish Teladorsagia circumcincta and Haemonchus contortus eggs, third-stage larvae and adult worms.

Lectin binding to carbohydrates on parasite surfaces has been investigated as a method of distinguishing adult worms, eggs and sheathed and exsheathed L3 of Teladorsagia circumcincta and Haemonchus contortus, economically important abomasal parasites in temperate climates. Both species were maintained as pure laboratory cultures of field isolates from New Zealand. Each of the four life cycle stages could be distinguished by the binding of at least one lectin: adult worms by Sambucus nigra agglutinin (SNA); eggs by peanut agglutinin (PNA), ConcavalinA and Lens culinaris agglutinin (LCA); exsheathed L3 by Griffonia simplicifolia-I lectin (GSL-I) and Lotus tetragonolobus lectin (LTL) and sheathed L3 by Aleuria aurantia lectin (AAL). The whole surface of both adult T. circumcincta and H. contortus strongly bound lectins specific for N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), mannose and fucose, but the two species could be distinguished by SNA binding only to T. circumcincta. Eggs could be distinguished by the binding of mannose-specific PNA to H. contortus and GalNAc-specific LCA and PSA to T. circumcincta eggs. GalNAc, GlcNAc and mannose lectins bound to the cuticle and over the excretory pores of a large proportion of sheathed L3 of both species, but only the H. contortus surface had exposed fucose or sialic acid complexes. The distinguishing lectin for sheathed L3 was AAL, which did not bind to T. circumcincta, but bound weakly to the head region of all fresh H. contortus and to 50-90% after 3 months storage. The cuticle of exsheathed L3 was unresponsive to all 19 lectins, and any binding was restricted to the head and tail regions. L3 exsheathed after 2-4 months storage could be distinguished by the binding of GSL-I and LTL to H. contortus but not to T. circumcincta. Lectin binding could be a useful adjunct in identifying L3, but lacked the consistency to be definitive, whereas it could be further developed as a practical method of distinguishing parasitic nematodes at other stages in the life cycle, particularly the eggs.

Neutrophil and eosinophil chemotactic factors in the excretory/secretory products of sheep abomasal nematode parasites: NCF and ECF in abomasal nematodes.

Both eosinophil chemotactic factor (ECF) and neutrophil chemotactic factor (NCF) activities were demonstrated in excretory/secretory (ES) products and homogenates of Haemonchus contortus and Teladorsagia circumcincta larvae and adult worms in a modified checkerboard assay using a micro-chemotaxis chamber. Neutrophil chemotaxis was seen in 28 of 35 experiments and eosinophil chemotaxis in 20 of 38 experiments. Chemokinetic activity for neutrophils and eosinophils (accounting for 40-50% of total cell migration) was also apparent in only three parasite products for each cell type. Significant NCF activity was present in six of seven adult worm ES products (three of four from T. circumcincta and in all three from H. contortus) and ECF activity in four of five adult ES products, whereas fewer L3 incubates, particularly of T. circumcincta, contained chemotactic activity. All parasite homogenates, with one exception for ECF, were chemotactic for both neutrophils and eosinophils. The sequential use of cellulose ultrafiltration membranes of decreasing pore size did not identify precisely the molecular weight of the NCF and ECF but indicated that the active chemicals were greater than 10 kDa and probably greater than 30 kDa.

A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida).

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.

Proteomic analysis of excretory/secretory products released by Teladorsagia circumcincta larvae early post-infection.

Teladorsagia circumcincta is an important parasitic nematode of domestic small ruminants. Drug resistance in this species is common so alternative methods of control are required. As animals develop immunity to T. circumcincta, vaccination is a valid option. Little is known about the antigens that play a role in stimulating immunity at this host/parasite interface. As responses generated between 1 and 5 dpi are known to affect development of these nematodes in their gastric niche, we focused on proteins released during the early stages of infection. To identify molecules potentially involved in immunity, we undertook a proteomics analysis of proteins released from larvae harvested at 1-, 3- and 5-days post-infection (dpi). This analysis produced peptide sequence data that was used to search information available in T. circumcincta expressed sequence tag (EST) databases and enabled identification of a number of excretory/secretory (ES) proteins. Immunoblots were performed to assess the relative molecular weight of ES antigens that were targets of local IgA responses in mucus from sheep rendered immune to infection. ELISA was performed to assess antigen-specific mucus IgA levels in individual sheep. These experiments provided preliminary evidence that the proteins identified in the larval secretome were subject to these antibody responses.

Somatic extracts of Marshallagia marshalli downregulate the Th2 associated immune responses in ovalbumin-induced airway inflammation in BALB/c mice.

BACKGROUND: Recently the role of gastrointestinal nematodes in modulating the immune responses in inflammatory and immune-mediated conditions such as allergy and autoimmune diseases has been introduced. This is mainly due to the suppressive effects of somatic and excretory secretory (ES) products of nematodes on the immune responses. In this study, we evaluated the immunomodulatory potentials of somatic products of Marshallagia marshalli, a gastrointestinal nematodes of sheep, to suppress the immune-mediated responses in a murine model of allergic airway inflammation. BALB/c mice were intraperitoneally (IP) sensitized with ovalbumin (OVA)/Alum and then challenged with 1% OVA. Somatic products of M. marshalli were administered during each sensitization. The effects of somatic products on development of allergic airway inflammation were evaluated by analyzing inflammatory cells recruitment, histopathological changes, cytokines production (IL-4, IL-13, IL-10, TGF-ß) and serum antibody titers (IgG1, IgG2a). RESULTS: Somatic products of M. marshalli were able to suppress the induction of allergic airway inflammation in mice. Modulation of Th2 type responses (IL-4, IL-13, IgG1) via upregulations of IL-10 and TGF-ß production was observed after injection of somatic products of M. marshalli. In addition, inflammatory cells infiltration and pathological disorders were significantly diminished following administration of somatic products. CONCLUSIONS: Our data raised the possibility that helminths could be a potential therapeutic candidate to alleviate the inflammatory conditions in allergic asthma. According to these results, we concluded that M. marshalli may contain immune-modulatory molecules that attenuate allergic airway inflammation via induction of regulatory cytokines. Further investigations are required to identify molecules that might have potentials for development of novel therapeutic targets.

cDNA cloning of galectins from third stage larvae of the parasitic nematode Teladorsagia circumcincta.

A monoclonal antibody raised to a Teladorsagia circumcincta 31-33 kDa doublet antigen was used to immunoscreen a T. circumcincta cDNA expression library. Sheep antibodies eluted from the proteins expressed by two clones immunopositive with the monoclonal antibody specifically recognised the doublet antigen on Western blots of third stage larval extract, confirming that these clones coded for the antigen. Database searches revealed high levels of similarity with beta-galactoside-binding lectin-like proteins (Ga1BPs or galectins) from Caenorhabditis elegans and Onchocerca volvulus. By analogy with these sequences, both T. circumcincta cDNA clones contain the full-length protein coding region. The native doublet proteins could be preferentially extracted from homogenates of third stage larvae with lactose and could be affinity purified on an asialofetuin column, confirming the identity of these bands as galectins. Reverse transcriptase-polymerase chain reaction amplification using a primer based on the C. elegans Spliced Leader SL1 sequence showed that the corresponding T. circumcincta mRNAs are also trans-spliced at their 5' ends. While there are considerable nucleotide differences between the two clones, the majority are located in the non-coding regions. Within the coding region there are 87 nucleotide differences but only three of these result in amino acid substitutions.

Identification by polymerase chain reaction (PCR) of Marshallagia marshalli and Ostertagia gruehneri from Svalbard reindeer.

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.

Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta.

Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.

SDS-PAGE profiles of somatic proteins from third-stage infective larvae of Ostertagia ostertagi and Cooperia oncophora.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of somatic proteins from third-stage infective larvae of Ostertagia ostertagi and Cooperia oncophora were evaluated using laser densitometry. Several protein bands were present from both parasite preparations. A few bands from each parasite appeared to be unique. Purification of these proteins for use in serologic monitoring of cattle naturally infected with O. ostertagi and C. oncophora should allow circumvention of cross-reactivity between the two genera, which has been reported by others.

Field evaluation of a topical doramectin formulation for the chemoprophylaxis of parasitic bronchitis in calves.

A study was conducted to evaluate the efficacy of two topical treatments with doramectin on the season-long control of lungworm and gastrointestinal infections in first grazing season (FGS) calves. At the start of the study, 20 FGS calves were randomly allocated into two treatment groups of 10 animals each. Calves in the D-group were treated with doramectin pour-on on days 0 and 56, at a dosage of 500 microg kg(-1) BW: calves in the C-group were designated as controls. A permanent pasture was divided in two blocks and these were randomly allocated to the treatment groups. Throughout the study, tracers (n = 32) were grazed on each paddock at 3-week intervals. Clinical signs of parasitic bronchitis (PB) were observed in the C-group in July and this necessitated two salvage treatments with levamisole. From day 28, post-turnout lungworm larvae were recovered from faeces of the C-calves until housing. No signs of PB were observed in the D-group during the entire grazing season. Shedding of lungworm larvae in the principals of the D-group did not occur until 112 days post-turnout. From the data obtained from the tracer calves. it appeared that larvae had overwintered on both pastures. On the C-pasture, the number of lungworms recovered from the tracer calves gradually increased to a peak in September, whereas on the D-pasture, the increase was observed only at the end of the pasture season. Both strongyle faecal egg counts and pepsinogen levels were relatively low in both groups throughout the present study. At the end of the grazing period (day 161). the principals were housed and treated with oxfendazole. During the housing period, all principal animals (D- and C-groups) and a third group of four helminth free animals (N-group) received a challenge infection with Dictyocaulus viviparus. It appeared that the different exposure to the parasite during the grazing season resulted in different establishment rates, i.e.. group C < group D < group N. The present results show that overwintering of lungworm larvae occurs in Belgium and that in such conditions, doramectin pour-on given at turnout and at 8 weeks controls PB in calves during the first grazing season.

Identical ITS-1 and ITS-2 sequences suggest Spiculopteragia asymmetrica and Spiculopteragia quadrispiculata (Nematoda: Trichostrongylidae) constitute morphologically distinct variants of a single species.

Sequences of ITS-1 and ITS-2 rDNA for adult males of Spiculopteragia asymmetrica and Spiculopteragia quadrispiculata in red deer (Cervus elaphus) were determined. They were found to be identical, suggesting that S. asymmetrica and S. quadrispiculata represent a single species and do not refute the concept of dimorphic species in the Spiculopteragia.

Isoelectric focusing of soluble proteins in the characterization of species and isolates of Nematodirus (Nematoda: Trichostrongyloidea).

Isoelectric focusing was performed on extracts from Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus, and 3 geographic isolates of Nematodirus battus. Gender-specific differences were noted within species; however, the overall protein profile of each species and isolate was distinct and reproducible and allowed unequivocal differentiation. A coefficient of similarity (Sm) for males of each species and isolate was calculated, and a dendrogram, based on evaluation of Sm by the unweighted pair-group method with arithmetic means, was produced. Although cluster analysis of the 3 isolates of N. battus indicates the North American and Weybridge isolates are similar, interpretation of the relationships and thus the history of introduction based on these data is equivocal. Isoelectric focusing is a robust method for establishing identity and has great utility in diagnostics. However, in the absence of selective histochemical staining, interpretation of identity and homology for specific bands and banding patterns is problematic, thus limiting the utility of this method for phylogenetic inference.

Proteomic analysis of Mecistocirrus digitatus and Haemonchus contortus intestinal protein extracts and subsequent efficacy testing in a vaccine trial.

BACKGROUND: Gastrointestinal nematode infections, such as Haemonchus contortus and Mecistocirrus digitatus, are ranked in the top twenty diseases affecting small-holder farmers' livestock, yet research into M. digitatus, which infects cattle and buffalo in Asia is limited. Intestine-derived native protein vaccines are effective against Haemonchus, yet the protective efficacy of intestine-derived M. digitatus proteins has yet to be determined. METHODOLOGY/PRINCIPAL FINDINGS: A simplified protein extraction protocol (A) is described and compared to an established method (B) for protein extraction from H. contortus. Proteomic analysis of the H. contortus and M. digitatus protein extracts identified putative vaccine antigens including aminopeptidases (H11), zinc metallopeptidases, glutamate dehydrogenase, and apical gut membrane polyproteins. A vaccine trial compared the ability of the M. digitatus extract and two different H. contortus extracts to protect sheep against H. contortus challenge. Both Haemonchus fractions (A and B) were highly effective, reducing cumulative Faecal Egg Counts (FEC) by 99.19% and 99.89% and total worm burdens by 87.28% and 93.64% respectively, compared to the unvaccinated controls. There was no effect on H. contortus worm burdens following vaccination with the M. digitatus extract and the 28.2% reduction in cumulative FEC was not statistically significant. However, FEC were consistently lower in the M. digitatus extract vaccinates compared to the un-vaccinated controls from 25 days post-infection. CONCLUSIONS/SIGNIFICANCE: Similar, antigenically cross-reactive proteins are found in H. contortus and M. digitatus; this is the first step towards developing a multivalent native vaccine against Haemonchus species and M. digitatus. The simplified protein extraction method could form the basis for a locally produced vaccine against H. contortus and, possibly M. digitatus, in regions where effective cold chains for vaccine distribution are limited. The application of such a vaccine in these regions would reduce the need for anthelmintic treatment and the resultant selection for anthelmintic resistant parasites.

Immunobiological characterization of Graphidium strigosum experimental infection in rabbits (Oryctolagus cuniculus).

An experimental infection of rabbits with a wild isolate of the gastric nematode Graphidium strigosum was carried out. Animals (3.5 months age) were infected with 1,000 L3 administered by bucoesophagic catheter (five rabbits) or kept as uninfected control group (five animals). The infection was maintained for 3 months. Along the experimental period, some parasitological, hematological and immunological parameters were determined. Prepatent period of the infection ranged from 30 to 38 days and, at necropsy, average adult helminth counts were 430.75 +/- 126.12. No significant variations were found in packed cell volume, leukocyte, and eosinophil counts along the experimental period. Infection elicited a clear serum-specific IgG response, estimated by ELISA, during patency. Pooled sera from the patent period of the infection recognized some soluble antigens, particularly, a 67-kDa protein. Experimentally infected animals did not show cross recognition between G. strigosum, Haemonchus contortus, and Teladorsagia circumcincta. However, Western blot analysis with hyperimmune sera against H. contortus raised in rabbits and lambs showed cross reactivity between this helminth species and G. strigosum.

A preliminary proteomic survey of the in vitro excretory/secretory products of fourth-stage larval and adult Teladorsagia circumcincta.

The nature of the proteins which comprise the in vitro excretory/secretory products (ES) of the fourth-stage larva (L4) and adult Teladorsagia circumcincta are largely undefined, despite the fact that this nematode induces profound changes, in part related to parasite ES, in the cellular architecture of the glands lining the abomasal surface of infected sheep and goats. In this study, the protein components of L4 and adult ES were fractionated using 1D gel electrophoresis and the major protein bands, detected by Coomassie blue staining, excised from the gel and subjected to tryptic digest and subsequent mass spectrometric analysis. The resultant peptide mass fingerprints were used to identify 15 L4 and 13 adult ES proteins. Several proteins, such as globin and some metabolic enzymes, were present in both ES. L4 ES alone contained thioredoxin peroxidase, an enzyme that can detoxify free radicals resulting from host inflammatory responses to the parasite, a cysteine proteinase which may aid penetration of the gastric mucosa and 2 different galectins which may influence cell differentiation and morphogenesis. Adult ES contained a nucleoside diphosphate kinase homologue, an enzyme which has been linked to cellular changes and can affect liquid secretion and goblet cell degranulation.

A Cooperia punctata gene family encoding 14 kDa excretory-secretory antigens conserved for trichostrongyloid nematodes.

A polymorphic set of 14 kDa excretory-secretory (E-S) antigen-encoding cDNAs, with similarity to a previously characterized 15 kDa E-S antigen of Haemonchus contortus, was cloned from Cooperia punctata. Five cDNAs encoding predicted proteins of 70-80% identity were sequenced. Genomic analyses of individuals proved the existence of three 14 kDa E-S antigen-encoding genes, excluding that the differences reflected polymorphisms between individuals in a population. Southern blots indicated the presence of additional members of this gene family. Thus, despite the fact that heterologously expressed C. punctata 14 kDa E-S products are shown to be recognized by immune sera, potential pitfalls in the development of a recombinant vaccine are presented by this genetic diversity. Vaccine design could be further rationalized by knowledge of the function, and possible redundancy in function, of the E-S products which is presently lacking. The limitations encountered in assigning a function to the 14/15 kDa family of E-S proteins that is thus far unique to the trichostrongyloid nematodes are discussed.