Characterisation and structural analysis of glyoxylate cycle enzymes of Teladorsagia circumcincta.

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min-1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01µM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.

Characterisation of a Teladorsagia circumcincta glutathione transferase.

A 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. The predicted protein consisted of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TcGST with homologues from other helminths showed that the highest identity of 53-68% with haem-binding nematode proteins designated as members of the nu class of GSTs. Substrate binding sites and conserved regions were identified and were generally conserved. The predicted 3-dimensional structures of TcGST and HcGST revealed highly open binding cavities typical of this class of GST, considered to allow greater accessibility to diverse ligands compared with other classes of GST. At 25 °C, the optimum pH for TcGST activity was pH 7, the Vmax was 1535 ± 33 nmoles.min-1. mg-1 protein and the apparent Km for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep, recognised recombinant TcGST in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins. These findings could aid in the design of novel drugs and vaccine antigens for economically important parasites of livestock.

Abomasal dysfunction and cellular and mucin changes during infection of sheep with larval or adult Teladorsagia circumcincta.

This is the first integrated study of the effects on gastric secretion, inflammation and fundic mucins after infection with L3 T. circumcincta and in the very early period following transplantation of adult worms. At 3 months-of-age, 20 Coopworth lambs were infected intraruminally with 35,000 L3; infected animals were killed on Days 5, 10, 15, 20 and 30 post-infection and 6 controls on either Day 0 or 30 post-infection. Another 15 Romney cross lambs received 10,000 adult worms at 4-5 months-of-age though surgically-implanted abomasal cannulae and were killed after 6, 12, 24 and 72 hours; uninfected controls were also killed at 72 hours. Blood was collected at regular intervals from all animals for measurement of serum gastrin and pepsinogen and abomasal fluid for pH measurement from cannulated sheep. Tissues collected at necropsy were fixed in Bouin's fluid for light microscopy, immunocytochemistry and mucin staining and in Karnovsky's fluid for electron microscopy. Nodules around glands containing developing larvae were seen on Day 5 p.i., but generalised effects on secretion occurred only after parasite emergence and within hours after transplantation of adult worms. After L3 infection, there were maximum worm burdens on Days 10-15 post-infection, together with peak tissue eosinophilia, inhibition of gastric acid secretion, hypergastrinaemia, hyperpepsinogenaemia, loss of parietal cells, enlarged gastric pits containing less mucin and increased numbers of mucous neck cells. After adult transplantation, serum pepsinogen was significantly increased after 9 hours and serum gastrin after 18 hours. Parallel changes in host tissues and the numbers of parasites in the abomasal lumen suggest that luminal parasites, but not those in the tissues, are key drivers of the pathophysiology and inflammatory response in animals exposed to parasites for the first time. These results are consistent with initiation of the host response by parasite chemicals diffusing across the surface epithelium, possibly aided by components of ES products which increased permeability. Parietal cells appear to be a key target, resulting in secondary increases in serum gastrin, pit elongation, loss of surface mucins and inhibition of chief cell maturation. Inflammation occurs in parallel, and could either cause the pathology or exacerbate the direct effects of ES products.

Molecular and biochemical characterisation of ornithine decarboxylases in the sheep abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus.

Full length cDNA encoding ornithine decarboxylases (ODC; EC 4.1.1.17) were cloned from the sheep abomasal nematode parasites Teladorsagia circumcincta (TcODC) and Haemonchus contortus (HcODC). The TcODC (1272 bp) and HcODC cDNA (1266 bp) encoded 424 and 422 amino acid proteins respectively. The predicted TcODC amino acid sequence showed 87% identity with HcODC and 65% and 64% with Caenorhabditis elegans and Caenorhabditis briggsae ODC respectively. All binding sites and active regions were completely conserved in both proteins. Soluble N-terminal His-tagged ODC proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcODC and HcODC had very similar kinetic properties: K(m) ornithine was 0.2-0.25 mM, optimum [PLP] was 0.3 mM and the pH optima were pH 8. No enzyme activity was detected when arginine was used as substrate. One millimolar difluoromethylornithine (DFMO) completely inhibited TcODC and HcODC activity, whereas 2 mM agmatine did not inhibit activity. The present study showed that ODC is a separate enzyme from arginine decarboxylase and strictly uses ornithine as substrate.

Imidazole initiates exsheathing of L3 Teladorsagia circumcincta.

The infective larvae of Teladorsagia circumcincta have a protective sheath that is lost soon after they reach the rumen of the sheep (the definitive host). Incubation in vitro with 50 mM imidazole caused more than 75% of L3 T. circumcincta to begin exsheathing within 2 hr. The initiation of exsheathing was less likely at pH 6.2 than at pH 7.8. The apparent pKa of this process was 7.08, similar to that for the conversion of imidazolium(+) to imidazole. Both the extent and the initial rate of exsheathing initiation increased with imidazole concentration (the apparent K(1/2) was about 50 mM). The initial rate of exsheathing initiation was stimulated by lactose and maltose, but not by some other carbohydrates, and by propylamine and imidazole acetic acid, but not by histidine.

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.

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.

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.

The kinetic properties of the glutamate dehydrogenase of Teladorsagia circumcincta and their significance for the lifestyle of the parasite.

Like other nematodes, both L(3) and adult Teladosagia circumcincta secrete or excrete NH(3)/NH(4)(+), but the reactions involved in the production are unclear. Glutamate dehydrogenase is a significant source NH(3)/NH(4)(+) in some species, but previous reports indicate that the enzyme is absent from L(3)Haemonchus contortus. We show that glutamate dehydrogenase was active in both L(3) and adult T. circumcincta. The apparent K(m)s of the L(3) enzyme differed from those of the adult enzyme, the most significant of these being the increase in the K(m) for NH(4)(+) from 18mM in L(3) to 49mM in adults. The apparent V(max) of the oxidative deamination reaction was greater than that of the reductive reaction in L(3), but this was reversed in adults. The activity of the oxidative reaction of the L(3) enzyme was not affected by adenine nucleotides, but that of the reductive reaction was stimulated significantly by either ADP or ATP. The L(3) enzyme was more active with NAD(+) than it was with NADP(+), although the activities supported by NADH and NADPH were similar at saturating concentrations. While the activity of the oxidative reaction was sufficient to account for the NH(3)/NH(4)(+) efflux we have previously reported, the reductive amination reaction was likely to be more active.

Thermotolerance of L3 Ostertagia (Teladorsagia) circumcincta and some of its enzymes.

Parasitic nematodes of ruminants can be expected to experience temperatures in excess of 40 degrees C in faeces on pasture and, perhaps, in the host. L3 Ostertagia (Teladorsagia) circumcincta survived for at least 90 min at 45 degrees C in vitro in water, but the larvae were inactivated rapidly by only slightly higher temperatures. The glycolytic enzymes hexokinase and pyruvate kinase were inactivated in a similar temperature range, whereas malate dehydrogenase maintained its activity at temperatures in excess of 50 degrees C. These data imply that the loss of glycolytic activity might explain the loss of larval motility at temperatures between 45 degrees C and 50 degrees C.

Effects of excretory/secretory products of Haemonchus contortus on cell vacuolation.

Excretory/secretory (ES) products of the gastric nematode, Haemonchus contortus, have been implicated in the inhibition of gastric acid secretion which follows infection. Parietal cell vacuolation has been observed in abomasal sections from parasitised sheep, and ES prepared in vitro has been reported to cause vacuolation and to increase neutral red (NR) uptake in epithelial cell cultures. We have used the latter approach to examine, at the cellular level, the effects of ES prepared from L3 and adult nematodes. Both NR uptake and cellular vacuolation were increased following exposure to larval or adult ES products. ES preparations from adult worms induced more extensive vacuolation then those from L3 worms and, as with VacA treatment, adherent cells remained viable despite high levels of vacuolation. Whereas VacA-induced vacuolation resulted in NR uptake predominantly localised in vacuoles, this appeared not to be the case with ES-induced vacuolation, suggesting that different mechanisms might be involved. Both ES and VacA exposure was associated with an increased rate of cell detachment.

Excretory/secretory products of sheep abomasal nematode parasites cause vacuolation and increased neutral red uptake by HeLa cells.

Excretory/secretory (ES) products of Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus have been implicated in the inhibition of gastric acid secretion and vacuolation, and the loss of parietal cells associated with abomasal parasitism. Vacuolation of epithelial (HeLa) cells caused by adult O. circumcincta or L3 O. circumcincta or H. contortus ES products have been examined by differential interference contrast microscopy and by the neutral red uptake assay. ES products caused visible vacuolation of HeLa cells, and this effect was enhanced by 8 mM NH4Cl. Some parasite ES products caused a marked detachment of cells from the coverslip. At lower concentrations of ES products, neutral red uptake was usually increased above the control, but at higher concentrations of ES products, uptake was often decreased, probably because of cell detachment. Although generally consistent with direct observations of HeLa cell vacuolation by parasite chemicals, neutral red uptake was not a satisfactory quantitative assay.

The failure of Haemonchus contortus excretory/secretory products to stimulate gastrin secretion in vitro.

Excretory/secretory (ES) products collected from exsheathed L3 or parasitic stages of Haemonchus contortus were tested in vitro for gastrin stimulatory properties using an ovine abomasal antral mucosal preparation. In addition, the motility of exsheathed L3 and parasites recovered on weeks 2, 6 and 8 post-infection was studied in water, saline, saline with glucose or ovine blood and in HBSS pH 2.5-7.4. Parasitic stages became immotile rapidly in water and HBSS pH 2.5, more slowly in HBSS pH 3.5, but nearly 100% remained motile for 48 h when blood was included in the medium. Exsheathed L3 motility was reduced only by water and HBSS pH 2.5, and then only in the second week of incubation. Gastrin secretion was not consistently increased by any of the parasite ES products tested in vitro, therefore, they probably do not stimulate the G cell directly to produce the hypergastrinaemia seen in parasitised sheep.