H.V. Simpson

Infection of sheep with adult and larval Ostertagia circumcincta: abomasal morphology.

The infection of parasite-naive sheep with approximately 15,000 adult Ostertagia circumcincta via abomasal cannulae resulted in marked changes in the structure and function of the abomasum. The functional changes, which have been characterised previously, included elevated abomasal pH and increased serum concentrations of pepsinogen and gastrin. Eight days after the transplant of adult worms, the abomasa of recipient animals were significantly heavier than those of controls (P < 0.001), the thickness of the fundic mucosa was greater (P < 0.01), there were fewer parietal cells (P < 0.01) and increases in the numbers of mitotic figures and mucus-producing cells. Mucous cell hyperplasia was also evident in the fundic mucosae of sheep receiving a trickle infection of infective, third-stage O. circumcincta larvae and was prominent within nodules associated with larval development. In non-nodular mucosa, there was hyperplasia of mucous cells and changes in the distribution of parietal cells. Decreases in the number of parietal cells at the gland base were offset by increases at a mid-gland level, probably due to chronic hypergastrinaemia, so that, overall, total parietal cell number was unaffected. Mucous cell hyperplasia and the diminution of parietal cell number are seen in a diverse range of disease states and may be mediated by host growth factors such as Transforming growth factor-alpha. Alternatively, the cellular and/or the secretory changes in response to the presence of adult worms are mediated by chemicals that are cytotoxic/inhibitory for parietal cells, and released by the parasites themselves.

Infection of sheep with adult and larval Ostertagia circumcincta: gastrin

Gastric endocrine cell populations and serum and tissue gastrin have been examined in sheep which were infected either intraruminally by tube with 150,000 Ostertagia circumcincta larvae followed by a trickle infection of 10,000 larvae thrice weekly for 8 weeks or by the transfer of 15,000 adult worms directly into the abomasum and killed 8 days later. Depletion of both antral gastrin and somatostatin was evident in both groups: tissue gastrin concentrations were reduced by 85% in the trickle infection and both G cells (gastrin-containing) and D cells (somatostatin-containing) were pale and fewer after adult worm transfer. The concurrent depletion of antral gastrin and somatostatin supports the contention that the hypergastrinaemia in parasitised sheep is largely secondary to the increase in abomasal pH. Although there was no change in the proportions of G34 and G17 in the tissues, there was an increase in the longer form of gastrin in the circulation of the larval-infected sheep, suggesting that there may be differential secretion of G17 and G34 which may be exaggerated as the rate of secretion increases. Although the fundic mucosa was thicker following trickle infection, there was no evidence of enterochromaffin-like cell hyperplasia in either infected group. It is suggested that hyper-gastrinaemia may be beneficial to the host, as it may allow the abomasum to regain the ability to acidify its contents during continued exposure to the parasites.

Excretory/secretory products of Haemonchus contortus inhibit aminopyrine accumulation by rabbit gastric glands in vitro

Abomasal parasites inhibit gastric acid secretion and reduce the number of acid secreting parietal cells either through physical contact with gastric tissue, the release of inhibitory excretory/secretory (ES) products or by initiating the host inflammatory response. To examine the role of parasite ES products, adult Haemonchus contortus were incubated in a medium based on Hanks balanced salt solution and three incubates were tested for the presence of inhibitors of acid secretion by dispersed rabbit gastric glands in vitro, using the intracellular accumulation of 14C-aminopyrine (AP) as an indirect measure of acid secretion. Acceptably sensitive rabbit preparations (80%) for testing ES products showed increased 14C-AP accumulation with either 10(-5) or 10(-4)M histamine. Sheep glands proved unsuitable as a test system as only two of six preparations responded weakly to histamine. Inhibitory activity was demonstrated in all the three parasite incubates, although responses varied quantitatively between tests, even with the same batch of incubate. In single tests, 16% ES products significantly reduced the 14C-AP ratio (P<0.05) of 10(-4)M histamine-stimulated glands (15 of 19 tests with Batch 1 and two of six with Batch 3). Even at 1 and 5%, ES products (Batch 2) were inhibitory for 10(-5)M histamine-stimulated glands: at 1% the mean reduction was 26.0% (range 7.8-54.8%, n=10), four of which were statistically significant and at 5% was 17.6% (range -15.3-53.8%, n=17), four of which were statistically significant. ES products were overall inhibitory (-12%, P<0.05), when tested on glands exposed to increasing histamine concentrations from 10(-6)M to 10(-3)M (which increased the 14C-AP ratio (P<0.001)). Responses by individual gland preparations differed (P<0.001). The active component(s) of the parasite ES products were less than 5000 molecular weight. Ammonium chloride, tested over the range of concentrations of ammonia present in worm incubates (0.2-1 mM, final concentrations in the gland incubations of 0.005-0.1 mM), had variable, but overall inhibitory effects on 10(-5)M histamine-stimulated glands (n=3). When tested with a range of histamine concentrations, 0.01 mM NH4Cl overall reduced the response by 8.6% (P<0.05)(n=4). The similarity of responses of rabbit glands to parasite ES products and to NH4Cl suggests that ammonia may be the small molecular weight ES product of adult H. contortus, which inhibited acid secretion in vitro.

Effects of Teladorsagia (Ostertagia) circumcincta infection on lambs selected for high fleece weight.

The physiological processes leading to the expression of the resilient phenotype, which allow animals to maintain a relatively higher production level during infection, have been investigated in lambs from a closed flock selected for 40 generations for high fleece weight (HFW), but with higher FEC and worm burdens than their unselected control (C) flock run in parallel. After recovery from surgery to implant abomasal cannulae, eight parasite-naive lambs from each flock were infected intraruminally at 4.5 months-of-age with 50,000 Teladorsagia circumcincta L3. Blood, abomasal fluid and faecal samples were collected daily for measurement of serum gastrin and pepsinogen concentrations, blood eosinophils, abomasal pH and FEC. Four lambs from each flock were euthanased on Day 8 post-infection and the other four on Day 28 post-infection. At necropsy, abomasal contents and tissues were collected for worm counts, abomasal lymph nodes and fundic tissue for cytokine gene expression and fundic tissue for histopathology. Expression of resilience appeared to be age-dependent as there were no significant differences in either FEC or worm burden between lambs from the two flocks, unlike older HFW lambs in a previous study. Abomasal secretion did not differ between flocks. Histopathological changes were typical of parasitism: inflammatory cells, mainly eosinophils and lymphocytes, were numerous in nodular areas and there were fewer TGF-alpha positive parietal cells, many of which were vacuolated. By Day 28 p.i., globule leucocytes were present. Mucosal thickness was significantly greater on Day 8 than Day 28 p.i. (p=0.000) and in C than HFW lambs. There were fewer parietal cells on Day 28 than on Day 8 p.i. (p=0.003) for pooled data. Circulating eosinophil counts increased moderately in both groups, significantly less in the HFW lambs. Fewer tissue and blood eosinophils in the HFW than C group on Day 8 p.i. were consistent with cytokine gene expression patterns, particularly lower IL-5 levels. Worm count decreased by 90% by Day 28 p.i., along with declining tissue eosinophil counts and IL-13 gene expression and increasing IL-10 and IL-4 gene expression. Food intake was depressed less in the HFW lambs, suggesting that maintenance of appetite could be an important aspect of the physiological basis for resilience. Although the resilient phenotype was not apparent at the younger age, lesser effects on food intake, differences in ALN cytokine profiles and lower blood and tissue eosinophil numbers in the HFW lambs may lead to the expression of resilience when older.

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta.

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30h and adult worms for 4-6h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30h. An initial external ammonia concentration of 600 microM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.

Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta.

Sarcosine (N-methylglycine) is an intermediate in glycine degradation and can also be synthesised from glycine in mammals. Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta differed from that of mammals in that creatinase activity was present and sarcosine was demethylated only by sarcosine oxidase (SOX) and not by sarcosine dehydrogenase (SDH). The mean SOX activity was 30 nmolmin(-1)mg(-1) protein in homogenates of L3 and adult worms of both parasites and the apparent Km for sarcosine was 1.1 mM. Addition of 2 mM Cd(2+) inhibited activity by 30%. There was no SDH activity with either NAD(+) or NADP(+) as co-factor. Mean creatinase activity in L3 T. circumcincta and adult worms of both species was 31±6 nmolmin(-1)mg(-1) protein, but was undetectable in L3 H. contortus. Activity was inhibited by up to 70% by Cu(2+), Fe(2+), Fe(3+) and Zn(2+). Possessing creatinase would allow host creatine to be incorporated into amino acids by the parasites.

Effects of weaning and infection with Teladorsagia circumcincta on mucin carbohydrate profiles of early weaned lambs

Differences in mucin glycosylation in milk-fed and early weaned lambs may influence susceptibility to parasitism, particularly the greater cellular content and higher sulphation of mucins in young and unweaned lambs. Weaning also reduced the percentage of Gal (p<0.05) in fundic mucin and galactosamine (GalN) (p<0.01) in duodenal mucin, but had no noticeable effect on fucosylation or sialylation. Four experimental groups of lambs were studied (n=3): (1) 3 days old; (2) 9 weeks old milk-fed; (3) 9 weeks old weaned at 3 weeks-of-age on to lucerne chaff and cereal-based pellets (4) 9 weeks old weaned and infected with 1000 Teladorsagia circumcincta L3 twice weekly for 5 weeks. Fundic and duodenal mucin monosaccharides were analysed chemically and fundic, antral and duodenal tissues were stained with lectins, periodic acid Schiff (PAS), Alcian blue/PAS (AB/PAS) and high iron diamine. Age-related maturation of mucin glycosylation was prominent in young lambs: reduced total fundic mucins and increasing fucosylation and decreasing sialylation and sulphation of all mucins, as well as changes in the types of linkages of Fuc and sialic acids. By 9 weeks-of-age, there were no longer sialylated mucins in fundic surface mucus cells, only neutral mucins, while in Brunners glands, there was reduced sialylation and large amounts of neutral mucins. In the neonates, both fundic and duodenal tissues contained only small amounts of mucins terminating with alpha-1,2-linked Fuc, which became the principal linkage in 9 weeks old lambs. Duodenal mucins in 3 days old lambs contained both alpha-2,6- and alpha-2,3-linked sialic acids, whereas the alpha-2,3 linkage was not present in older lambs. Parasitism increased the percentage of galactose, but reduced total and neutral fundic mucins, as well as sulphation and sialylation. There was both decreased sialylation and sulphation in duodenal mucins. Although no change in fucosylation was apparent from chemical analysis, infection reduced lectin staining for alpha-1,2-linked fucose in antral and duodenal tissues and alpha-1,6- and alpha-1,3-linked fucose in the duodenum. These changes in fundic and duodenal mucins were similar to those previously seen on Day 28 p.i. after a single infection of 4-9 months old sheep with T. circumcincta larvae.

Milk and milk components reduce the motility of Ostertagia circumcincta larvae in vitro

Abstract AIM: To examine the effects in vitro of bovine milk and milk products and soymilk on the motility of sheathed and ex- sheathed L3 Ostertagia circumcincta (also known as Teladorsagia circumcincta) as a measure of larval viability and infectivity. METHODS: L3 were exsheathed in 0.2% sodium hypochlorite, resuspended in Hank’s Balanced Salt Solution (HBSS) pH 7.4 and incubated with test solutions at 37°C for up to 48 h. The motility of 50 larvae from each incubate was assessed at selected times using a McMaster slide. Larvae were considered immotile only if straight and not moving. Fresh bovine milk, homogenised milk (3.3% fat), low-fat milk (0.2% fat) and lamb milk replacer were diluted with HBSS pH 7.4 to concentrations from 1.6–100%, and incubated with exsheathed L3 for 1, 24 or 48 h. Bovine whey protein was tested in concentrations of 5–15% at pH 2.5–6.5, casein at 5 or 7.5%, and skim milk powder from 5–15% at pH 5.5 or 6.5, all for 2, 4 or 24 h. Soymilk was tested in concentrations of 1.6–100% for 1, 2, 24 or 48 h. HBSS was used as the control solution. Sheathed L3 were incubated in HBSS pH 7.4, 50% homogenised milk in HBSS, or 50% soymilk in HBSS. Each solution was incubated for 1, 2, 24 or 48 h. RESULTS: The motility of exsheathed L3 was reduced by fresh bovine milk, homogenised milk, low-fat milk, lamb milk replacer, whey, casein and skim milk solutions, but not by soymilk. The mean percentage (and SE) immotile at 48 h were: fresh milk 38% (SE 20); homogenised milk 65% (SE 7); low-fat milk 57% (SE 5); lamb milk replacer 43% (SE 7); and soymilk 7% (SE 0.5). Larval immotility increased in whey protein solutions from 5–15%, from pH 2.5–6.5 and from 2 to 24 h (all p<0.001); in skim milk from 5–15% (p<0.001), and was greater at pH 6.5 than at pH 5.5 (p<0.001); in casein from 5–7.5% (p<0.001), but was no different at pH 5.5 and 6.5. The motility of sheathed L3 was reduced at 24 h (p=0.009) and 48 h (p<0.001) by 50% homogenised milk, but not by 50% soymilk or HBSS. CONCLUSIONS: Bovine milk proteins, or components associated with the proteins, reduced the motility of both sheathed and exsheathed L3 O. circumcincta. Soymilk had no effect on nematode motility. Lower larval motility may reduce worm establishment and be a contributing factor to the smaller burdens of gastrointestinal nematodes in milk-fed animals compared with animals after weaning.

Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta.

Full length cDNA encoding arginine kinases (AK) were cloned from Teladorsagia circumcincta (TcAK) and Haemonchus contortus (HcAK). The TcAK and HcAK cDNA (1080 bp) encoded 360 amino acid proteins. The predicted amino acid sequence showed 99% similarity with each other and 94% with a Caenorhabditis elegans AK. Soluble N-terminal His-tagged AK proteins were expressed in Escherichia coli strain BL21, purified and characterised. All binding sites were completely conserved in both proteins. The recombinant TcAK and HcAK had very similar kinetic properties: K(m) arginine was 0.35 mM, K(m) ATP was 0.8-0.9 mM and the pH optima were pH 7.5. Arginine analogues strongly inhibited recombinant enzyme activities (up to 80%), whilst other amino acids decreased activities by a maximum of 20%. TcAK and HcAK are potential vaccine candidates because of the strong antigenicity of invertebrate phosphagens and kinases and presence in metabolically active parts of the worm.

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 Mn(2+) 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.