Valine partitioning and kinetics between the gastrointestinal tract and hind limbs in lambs with an adult Trichostrongylus colubriformis burden.

Intestinal parasitic infection increases the demand for AA because of increased protein synthesis in the intestine and increased luminal losses of AA, and these increased demands may be supported by increased mobilization of AA from the skeletal muscles. Two experiments were conducted to determine the effects of parasitic infection on valine kinetics within the gastrointestinal tract and hind limbs of lambs fed fresh forages. On d 1, lambs were given 6,000 stage-3 Trichostrongylus colubriformis larvae per day for 6 d (n = 6) or kept as parasite-free controls (n = 6) and fed fresh lucerne (Medicago sativa; Exp. 1) or fresh sulla (Hedysarum coronarium; Exp. 2). On d 48, valine kinetics within the mesenteric- (MDV) and portal-drained viscera (PDV) and hind limbs were obtained by carrying out concurrent infusions of para-amminohippuric acid into the mesenteric vein and indocyanin green into the abdominal aorta (for blood flow), and [3,4-(3)H]valine into the jugular vein and [1-(13)C]valine into the abomasum for 8 h (for kinetics). During the infusions, blood was collected from the mesenteric and portal veins and from the mesenteric artery and vena cava, and plasma was harvested. After the 8-h infusion, lambs were euthanized, ileal digesta were collected, and tissues were sampled from the intestine and muscle (biceps femoris). Tissues, digesta, and plasma were analyzed for valine concentration, specific radioactivity, and isotopic enrichment. In both experiments, intestinal worm burdens on d 48 were greater in parasitized lambs (P = 0.0001 and 0.003). In Exp. 1, parasitic infection increased (P = 0.03) the total valine irreversible loss rate (ILR) in the MDV and PDV. In Exp. 2, luminal ILR of valine in the MDV was reduced (P = 0.01); however, ILR of valine in the PDV was unaffected. Despite these changes within the MDV and PDV, parasitic infection did not affect the ILR of valine within the hind limbs, and valine transport rates were largely unchanged. We suggest that the increased mobilization of AA from the hind limbs that might have occurred in the early phase of inflammation was no longer required when the parasitic infection was established. The MDV and PDV data may indicate that the non-MDV parts of the PDV play an important role in this adaptation, which warrants further study.

Serpin expression in the parasitic stages of Trichostrongylus vitrinus, an ovine intestinal nematode.

Members of the genus Trichostrongylus, such as T. vitrinus, being endemic in Northern Europe, are among the principal causative nematodes which contribute to parasitic gastro-enteritis in sheep world-wide, inhabiting the proximal small intestine and causing damage to the mucosa. This results in impaired nutrient absorption as well as a pronounced inflammatory response with cellular infiltration of the mucosa, including a pronounced mast cell response. These mast cells release serine proteinases that enhance the passage of effector cells and macromolecules across epithelial boundaries and into direct contact with the invading parasite. The adult and larval stages of T. vitrinus release a number of serine proteinases in vitro that may contribute to tissue invasion and nutrient acquisition in vivo. This study describes the molecular cloning and characterization of a serine proteinase inhibitor (serpin) that is present in extracts of all the parasitic stages, becoming more abundant as the life-cycle progresses. The serpin is present in the in vitro excretory/secretory products (ES) of 4th-stage larval and adult parasites, being more abundant in the former. The serpin was expressed in E. coli and the recombinant protein was a potent inhibitor of several host serine proteinases including mast cell proteinases. The serpin may regulate the activity of the parasite serine proteinases or it may modulate the host immune response to the parasite by inhibiting the activity of serine proteinases released from host inflammatory cells.

Microarray analysis of selection lines from outbred populations to identify genes involved with nematode parasite resistance in sheep.

Gastrointestinal nematodes infect sheep grazing contaminated pastures. Traditionally, these have been controlled with anthelmintic drenching. The selection of animals resistant to nematodes is an alternative to complete reliance on drugs, but the genetic basis of host resistance is poorly understood. Using a 10,204 bovine cDNA microarray, we have examined differences in gene expression between genetically resistant and susceptible lambs previously field challenged with larval nematodes. Northern blot analysis for a selection of genes validated the data obtained from the microarrays. The results identified over one hundred genes that were differentially expressed based on conservative criteria. The microarray results were further analyzed to identify promoter motifs common to the differentially expressed genes. Motifs identified in upregulated gene promoters were primarily restricted to those promoters; however, motifs identified in downregulated gene promoters were also found in the promoters of upregulated genes but not in the promoters of genes whose expression was unaltered. Protein Annotators' Assistant was used for lexical analysis of the differentially expressed genes, and Gene Ontology was used to look for metabolic and cell signaling pathways associated with parasite resistance. Two pathways represented by genes differentially expressed in resistant animals were those involved with the development of an acquired immune response and those related to the structure of the intestine smooth muscle. Genes involved in these processes appear from our analysis to be key genetic determinants of parasite resistance.

Effects of the excretory/secretory products of Trichostrongylus colubriformis on the growth of different cell lines.

The effects of the excretory/secretory (ES) products of the parasitic nematode Trichostrongylus colubriformis were examined on the proliferation of seven cell lines derived from a digestive or non-digestive origin. The excretory/secretory products of T. colubriformis were incorporated in the culture medium of the different cell lines and cell proliferation was measured by means of the 5-bromo-2'-deoxy-uridine (Brdu) assay. An increase in cell numbers was found with the three epithelial intestinal cells (RIC, IEC-6, IRD-98) and with epithelial kidney cells (MDCK). In contrast, an inhibition in the proliferation of epithelial ovarian cells (CHO) and fibroblasts (3T3) was observed with the addition of the excretory/secretory products and no effect was detected on the cell growth of hepatocytes (HepG2). These data are discussed with respect to the tissue specificity of the existing mitogenic effect of the worms on the intestinal crypt cells during parasitism.

Superoxide dismutase and total antioxidant status of larvae and adults of Trichostrongylus colubriformis, Haemonchus contortus and Ostertagia circumcincta.

Superoxide dismutase (SOD), a cytosolic enzyme that is specific for scavenging superoxide radicals, is involved in protective mechanism(s) in tissue injury following oxidative processes and phagocytosis. The presence of SOD activity in larval and adult Trichostrongylus colubriformis, Haemonchus contortus and Ostertagia circumcincta was examined using a xanthine-xanthine oxidase assay and by polyacrylamide gel electrophoresis (PAGE) and non-denaturing sodium dodecyl sulfate (SDS)-PAGE followed by specific enzyme staining. Total antioxidant status was determined using the Randox Laboratories kit. The infective larval stages (L3) of the three species contained 8-10 times more activity than the corresponding adults. SOD activity from adult parasites was sensitive to KCN and SDS and may therefore belong to a Cu/Zn and Mn class of enzymes. SOD from the larvae was sensitive only to KCN, suggesting that it may belong to a Cu/Zn class of enzymes. Insignificant interspecies variation was observed when SOD isozyme profiles of larvae were compared. PAGE showed at least five bands of SOD activity with molecular weights of between 18 and 205 kDa. Examination of total antioxidant status showed that non-enzymatic antioxidant potential was also present, but only in the infective larvae. The level of antioxidants in the three genera of larvae studied was similar and amounted to about 0.33-1.07 microM/mg of protein.

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

Mineral content of the free-living nematode Turbatrix aceti and ruminant parasite Trichostrongylus colubriformis.

1. The parasitic nematode Trichostrongylus colubriformis and free-living nematode Turbatrix aceti were examined for element content. 2. T. colubriformis contained significantly less phosphorus, potassium, manganese and boron than T. aceti. 3. T. colubriformis contained significantly more nitrogen and copper than T. aceti. 4. Comparative study of the chemical composition of parasitic vs free-living nematodes may facilitate in vitro cultivation of the parasitic forms.

A routine diagnostic assay for the detection of benzimidazole resistance in parasitic nematodes using tritiated benzimidazole carbamates.

Resistance to benzimidazoles (BZs) in parasitic nematodes has recently been shown to be due to a reduction in the ability of BZs to bind the structural protein, tubulin, in resistant isolates. Based on these observations the development and standardisation of a routine diagnostic assay has been undertaken by measuring the binding of tritiated mebendazole to crude supernatants of L3 larvae. The assay is rapid, requiring less than 2 h, and is robust, highly reproducible and sensitive to minor changes in the resistance status of parasite populations. Investigation of the routine application and validity of this assay has been documented using 24 isolates of known resistance status from the species Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta: In all cases the observed binding and calculated susceptibility factors were in accordance with their respective resistance status.

Derivatives of epinephrine, norepinephrine, octopamine and histamine formed by homogenates of Trichostrongylus colubriformis, a nematode parasite of ruminants.

1. Radiolabeled epinephrine, norepinephrine, octopamine and histamine were introduced into homogenates of mixed sexes of the nematode Trichostrongylus colubriformis and the labeled derivatives formed during incubation were identified by HPLC. Vanillylmandelic acid (VMA), 4-hydroxy-3-methoxyphenyl glycol (HMPG) and metanephrine were formed from epinephrine. 2. VMA, HMPG and normetanephrine were produced from norepinephrine. 3. Octopamine was converted into norepinephrine, synephrine and epinephrine. 4. Imidazole-4-acetic acid, 1,4-methylimidazole acetic acid and acetylhistamine were formed from histamine. 5. Inhibitors of monoamine oxidase, catechol-O-methyltransferase and phenylethanolamine-N-methyltransferase were tested for their effects on the formation of certain of these derivatives, and the results suggested that these enzymes were active in the homogenate.

Alteration of Trichostrongylus colubriformis egg permeability by Bacillus thuringiensis israelensis toxin.

A toxin from the bacterium Bacillus thuringiensis israelensis is lethal to nematode eggs. Exposure of eggs of the ruminant nematode Trichostrongylus colubriformis to the toxin significantly increased the eggs' permeability to radiolabeled phenylalanine within 2 hr. Calcium chloride inhibited the toxin-induced change in egg permeability. Iodine staining of eggs that were exposed to the microbial toxin revealed that egg permeability was altered within 5 min and was dependent on the dose of toxin. Addition of 34 mM sucrose, 17 mM sodium chloride, or 17 mM potassium chloride to the eggs' medium increased the toxin's lethality. Exopeptidase activity in eggs of T. colubriformis was reduced significantly after exposure to the B. t. israelensis toxin. Tetrodotoxin, tetraethylammonium chloride, ouabain, 4-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid (SITS), 4,4'-diisothiocyano-2,2'disulfonic acid stilbene (DIDS), valinomycin, and sodium vanadate, which affect membrane transport, had no significant effect on the activity of B. t. israelensis toxin for eggs. Likewise, a series of nucleotides and their derivatives had no effect on the toxin's activity. Ovicidal activity of the microbial toxin was increased by 4-aminopyridine (4.4 X), but was decreased by furosemide (97 X), nigericin (263 X), or monensin (125 X). Microscopic measurement of T. colubriformis eggs after treatment with the microbial toxin revealed no significant size change.

Sex steroid content and metabolism in Trichostrongylus colubriformis (Nematoda).

Testosterone, progesterone and cholesterol were found in mixed sexes of the nematode Trichostrongylus colubriformis from goats, according to thin-layer, gas-liquid and high-performance liquid chromatography. The structure of these steroids was confirmed by proton nuclear magnetic resonance and mass spectroscopy. Melting points of the worms' steroids were similar to authentic standards of the steroids. Estradiol was not detected in worms from either goat sex. Cholesterol was about 0.08% of the worms' dry weight in helminths from either sex of host. Testosterone was 0.02% of the dry weight when worms were taken from male goats, but only 0.005% from female goats. Progesterone was not detected in worms from male goats, but was 0.005% of the dry weight of helminths from female hosts. Incubation of a worm preparation with tritiated steroids showed that progesterone was converted to 17-alpha-hydroxyprogesterone, based on retention during radioactive thin-layer and gas-liquid chromatography, and co-crystallization. Testosterone, cholesterol and 17-beta-estradiol were not metabolized.

Tubulin and benzimidazole-resistance in Trichostrongylus colubriformis (Nematoda).

Benzimidazole treatment produced greater effects on microtubule-dependent acetylcholinesterase secretion, the presence of microtubules in intestinal cells, and colchicine binding in susceptible compared with benzimidazole-resistant Trichostrongylus colubriformis. In addition, the binding of benzimidazoles was markedly reduced in preparations from the latter strain, indicating that the mechanism of resistance to benzimidazoles in this nematode involves a reduced affinity of tubulin for benzimidazoles.