Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

The response of immune sheep to challenge with Trichostrongylus colubriformis: enteric plasma loss and secretion of biogenic amines

Sheep were challenged with a single large dose of larvae after vaccination with irradiated Trichostrongylus colubriformis larvae. Worm counts performed on vaccinated sheep 6 or 7 days after challenge (DAC) showed that they were solidly immune and only retarded L3 larvae were recovered at this time. Enteric plasma loss (EPL) in vaccinated animals increased immediately after challenge to peak 4–6 DAC and then decreased to pre-challenge levels. In contrast, a substantial rise in EPL did not occur in unvaccinated sheep until 10 DAC. Secretion of histamine into the duodenum of immunized sheep increased significantly from 2 to 7 DAC with the highest value at 6 DAC which corresponded with a lower duodenal tissue histamine level at this time. Histamine and 5HT secretion into the small intestine of previously uninfected sheep gradually increased during 12 weeks of a trickle infection of 3000 normal T. colubriformis larvae per week. The results indicate that rejection of incoming larvae by immune sheep is accompanied by an intestinal inflammatory response involving secretion of biogenic amines and a concurrent plasma loss.     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

Development and validation of an in vitro Trichostrongylus colubriformis motility assay

Development and validation of an in vitro Trichostrongylus colubriformis motility assay. International Journal for Parasitology 17: 1441–1444. An in vitro Trichostrongylus colubriformis motility assay involving the use of a micromotility meter has been developed and validated. Four commercially available ruminant anthelmintics (albendazole, ivermectin, levamisole hydrochloride, and coumaphos) and an investigational hydrazone compound (p-toluoyl chloride phenylhydrazone) were evaluated in this assay at four concentrations each. At 100 μg ml^-1, all five treatments significantly (P 0.05) reduced the motility of ensheathed L-3 T. colubriformis larvae, thereby indicating anthelmintic activity. At this concentration, coumaphos was significantly less active than any of the other four treatments. At 10 μg ml^-1 albendazole, ivermectin, levamisole hydrochloride and the hydrazone compound were active, but coumaphos was not. At 1 μg ml^-1 albendazole, ivermectin and levamisole hydrochloride remained significantly active, but neither coumaphos nor the hydrazone compound showed significant activity. At all three of the higher concentrations (1,10 and 100 μg ml^-1), levamisole hydrochloride indicated greater activity than any of the other treatments. This difference was statistically significant at the 1 and 10 μg ml^-1 concentrations. None of the five treatments showed significant activity at the lowest concentration (0.1 μg ml^-1). The in vitro T. colubriformis motility assay proved to be sensitive, accurate, rapid, and repeatable. This assay system should be another valuable addition to the tests used to identify potential anthelmintics, monitor helminth resistance to drugs, and define the kinetics and mode of action of drugs.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

Effects of changes in temperature and saturation deficit on the survival of eggs of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae)

and 1972. Effects of changes in temperature and saturation deficit on the survival of eggs of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae) International Journal for Parasitology, 2: 439–447. In conformity with a hypothesis relating to survival of the developing T. colubriformis egg exposed to desiccation, samples of eggs initially at the early blastomere stage of development showed decreased mortality during development with increasing temperature of incubation up to 25°C, for approximately constant rates of evaporation. At 30°C there was a higher percentage mortality for fixed evaporation rate than at 20° or 25°C. It is suggested that at 30°C there may be an abrupt increase in the initial rate of water loss from the developing embryo resulting from a change in the permeability to water of the lipid layer of the egg envelope.

Fully embryonated T. colubriformis eggs were obtained by incubation at 20°C in the presence of a moderate saturation deficit during development. When such eggs were transferred to 30° and 40°C there was no mortality at the higher temperature, providing that the saturation deficit was substantially increased. A hypothesis proposed for survival at high temperature is based on analogy with water loss through the arthropod cuticle and is attributed to a decrease in permeability of the protein-chitin layer of the egg envelope under conditions of high evaporation rate, even though permeability of the lipid layer might be increased by high temperature.     

Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans.

The binding of [³H]mebendazole ([³H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [³H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [³H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [³H]MBZ-tubulin complexes and was not related to a change in the association constant of the [³H]MBZ-tubulin interaction. [³H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37°C in BZ-S T. colubriformis and 10°C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [³H]MBZ binding at 4°C. Resistance ratios derived from the amount of [³H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction ofbenzimidazole carbamates with tubulin.     

The molecular size of nematode acetylcholinesterases and their separation from nematode allergens

The molecular size of nematode acetylcholinesterases, and their separation from nematode allergens. International Journal for Parasitology 3: 735–741. Acetylcholinesterases and allergens were derived from two parasitic nematodes, Nippostrongylus brasiliensis which parasitises rats and Trichostrongylus colubriformis which infects sheep and guinea pigs.

Chromatographic studies indicated that the mol. wt of nematode acetylcholinesterases lies between 65,000 and 75,000. The acetylcholinesterases of both species were separated from nematode allergens whose mol. wt is in the range of 10,000–50,000.

γG binding antigens of T. colubriformis were located in fractions with a mol. wt range of 30,000–150,000. γE binding activity was confined to allergenic material with a mol. wt of less than 70,000.     

Nematodirus battus: Permeability changes,calcium binding,and phosphorylation of the eggshell during hatching

Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 ± 2.2 and 8.5 ± 1.5 μM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 ± 1.25 μM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, K_Ca′ = 1.92 μM and a second, low-affinity site, K_Ca′ = 1169.60 μM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of α-d-glucosyl-α-d-glucopyranoside and α-methyl-d-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 × 10³ and 8 × 10³ Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.     

Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri

AIMS: The objective of this study was to evaluate the effect of bile salts and cholesterol in the lipid profile of Lactobacillus reuteri CRL 1098 and to determine the relationship existing between these changes: the in vitro removal of cholesterol and the tolerance of the cells to acid and cold stress. METHODS AND RESULTS: Lactobacillus reuteri CRL 1098 was grown in the following media: MRS (deMan Rogosa Sharpe; MC, control medium), MB (MC with bile salts), MCH (MC with sterile cholesterol) and MBCH (MC with bile salts and cholesterol). Fatty acids were determined by analytical gas-liquid chromatography, and phospholipids and glycolipids by colorimetric techniques. The cells from different culture media were subjected to cold and acid stress. The MB cultures displayed a decrease in phospholipids and a low ratio of saturated : unsaturated fatty acids. The presence of the unusual C18 : 0,10-OH and C18 : 0,10-oxo fatty acids was the prominent characteristic of the bile salts growing cells. The relative increase in glycolipids and the changes in the fatty acids profiles of the MB cells would be responsible for the cholesterol remotion. The changes induced by bile salts in the lipid profile did not improve the tolerance of L. reuteri CRL 1098 to freezing and acid stress. CONCLUSIONS: The changes in lipid profiles reported in this study would play a key role in the response of Lactobacilli to environmental stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides useful information about the effect of bile salts on the cell membrane of L. reuteri, a probiotic enterolactobacillus. The steady-state response of the cells subjected to bile stress seems to be the appropriate model for evaluating the bacterial behaviour in detergent-containing gastrointestinal tracts, where the bile salts stress would presumably be continuous.     

Histamine and leukotriene concentrations in duodenal tissue and mucus of lambs selected for high and low responsiveness to vaccination and challenge with Trichostrongylus colubriformis

Lambs selectively bred for high responsiveness or low responsiveness to vaccination with irradiated Trichostrongylus colubriformis were vaccinated and challenged. Duodenal tissue histamine concentrations in both high and low responder lambs were lower at 3 days than at 28 days after challenge. At 3 days after challenge, histamine concentrations were higher in both male and female high responder lambs than in low responder lambs whereas at 28 days concentrations were increased only in high responder females. At 3 days after challenge, histamine concentrations were generally lower in mucus than in tissues, but levels were again higher in mucus from high responder groups. In duodenal tissue at 3 days after challenge, leukotriene C₄ and B₄ concentrations were similar in high and low responder animals. At the same time, concentrations of both leukotrienes were higher in mucus than in tissues, with high responder female lambs having the highest concentration. It is suggested that increased levels of histamine and leukotrienes in mucus and tissue are associated with larval rejection or exclusion.     

Bile salts of fishes collected on the Zaire River Expedition (1974-5): their chemical nature and its possible significance

Bile salts from 21 fish species caught during the Zaïre River Expedition (1974–5) have received partial or complete chemical analysis. Conclusions from the results are as follow. (1) Polypterus bile salts are biochemically advanced, but with easily detectable features regarded as relicts from ancestral stock. The bile acids include haemulcholic acid, previously found only in a marine teleost and an unidentified acid also found in a mormyrid. (2) This mormyrid has advanced bile salts, consisting almost entirely of taurine conjugates of haemulcholic acid (present in Polypterus) , and two other C₂₄ acids. (3) Three Alestes and three Distichodus species have no more than traces of primitive bile salts; their bile acids suggest a degree of activity of intestinal microorganisms during the enterohepatic circulation not previously noticed in fishes. (4) The predatory Hydrocynus has bile acids of an omnivorous rather than a carnivorous type. (5) The Barbus species have primitive bile salts closely similar to those of Cyprinidae previously examined but containing also (in two species) a newly-discovered bile alcohol sulphate: in contrast, a Varicorhinus does not have this new substance and has a chief bile salt found so far only as a minor constituent in the Cyprinidae. (6) Two Labeo species are different from all other cyprinids examined: diey both have a new major bile alcohol sulphate. (7) Four Siluriformes show evidence of enterohepatic changes that might be characteristic of some tropical freshwater fish; three have taurine-conjugated haemulcholic acid. (8) Synodontis species are remarkable in having a high proportion of unconjugated bile acid ions and this finding was confirmed with fresh material.     

The effect of large doses of Nematodirus battus on the histology and biochemistry of the small intestine of lambs

Coop R.L., Angus K.W. and Mapes C.J. 1973. The effect of large doses of Nematodirus battus on the histology and biochemistry of the small intestine of lambs. International Journal for Parasitology3: 349–361. Large single and multiple doses of Nematodirus battus alter the structure of the small intestine, producing a low flattened, convoluted type of mucosa with extensive cellular infiltration of the lamina propria. The brush borders of the epithelial cells on the surface of the villi are narrow and the microvilli sparse and distorted.There is a reduction in alkaline phosphatase and disaccharidase activity in the mucosa of infected lambs, whereas dipeptidase activity is unaffected. The changes in enzyme activity are thought to arise from damage to mature epithelial cells on the villi.Alkaline phosphatase and disaccharidase activity in the mueosa of lambs killed 18 days after infection is correlated with the dry matter content of the faeces.     

Vaccination against the nematode Trichostrongylus colubriformis. I. Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance

Vaccination of guineapigs with homogenates of fourth larval stage and adult Trichostrongylus colubriformis and soluble products released by fourth stage larvae during in vitro maintenance, induced a high level of protection against challenge infection with the parasite.     

Comprehensive evaluation of the bactericidal activities of free bile acids in the large intestine of humans and rodents

In addition to functioning as detergents that aid digestion of dietary lipids in the intestine, some bile acids have been shown to exhibit antimicrobial activity. However, detailed information on the bactericidal activities of the diverse molecular species of bile acid in humans and rodents is largely unknown. Here, we investigated the toxicity of 14 typical human and rodent free bile acids (FBAs) by monitoring intracellular pH, membrane integrity, and viability of a human intestinal bacterium, Bifidobacterium breve Japan Collection of Microorganisms (JCM) 1192^T, upon exposure to these FBAs. Of all FBAs evaluated, deoxycholic acid (DCA) and chenodeoxycholic acid displayed the highest toxicities. Nine FBAs common to humans and rodents demonstrated that α-hydroxy-type bile acids are more toxic than their oxo-derivatives and β-hydroxy-type epimers. In five rodent-specific FBAs, β-muricholic acid and hyodeoxycholic acid showed comparable toxicities at a level close to DCA. Similar trends were observed for the membrane-damaging effects and bactericidal activities to Blautia coccoides JCM 1395^T and Bacteroides thetaiotaomicron DSM 2079^T, commonly represented in the human and rodent gut microbiota. These findings will help us to determine the fundamental properties of FBAs and better understand the role of FBAs in the regulation of gut microbiota composition.     

Protein metabolism. 6. Trichostrongylus colubriformis: skeletal protein catabolism in primary and secondary infections of the guinea pig

The urinary excretion of 3-methylhistidine (3-MH) was used as an index of muscle protein catabolism in primary and secondary infections of the guinea pig with Trichostrongylus colubriformis and in uninfected animals fed quantitatively reduced rations. Catabolism, which was depressed in all three groups, was directly related to a fall in food consumption. Possible explanations for the greater depression of catabolism in the primary infection than in the uninfected guinea pigs and its fall in the secondary infection in spite of little change in consumption are briefly discussed. It was concluded that the faster rate of whole-body protein turnover reported earlier in this series on protein metabolism in intestinal nematode infection was not partly due to a faster rate of muscle protein catabolism. It was shown that the urinary excretion of 3-MH could be validly expressed in terms of unit creatinine.     

Attempts to probe the antigens and protective immunogens of Trichostrongylus colubriformis in immunoblots with sera from infected and hyperimmune sheep and high- and low-responder guinea pigs

Comparison of antisera from sheep during primary infection and following vaccination and challenge with Trichostrongylus colubriformis, with antisera obtained following primary infection of high- and low-responder guinea pigs, failed to reveal different antigenic patterns in proteins separated from fourth stage larval extracts by two-dimensional electrophoresis and probed by the immunoblot technique.Generally, serum IgG reacted specifically with worm antigens of mol. wt greater than 94,000, whereas protection against challenge infection was elicited most effectively in the guinea pig by fractions in the 67,000–94,000 range.Most distinct separations of larval proteins by SDS-polyacrylamide gel electrophoresis were obtained by extraction of live larvae and the extracts used within 2–3 days.     

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks − 9 to − 4 relative to parturition. At the beginning of week − 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week − 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.