David C. Simcock

A sequential study of the pathology associated with the infection of sheep with adult and larval Ostertagia circumcincta

Disturbances in the physiology of the abomasa of sheep infected with either adult Ostertagia circumcincta given via abomasal cannulae, or larvae (L3) given intraruminally were matched by pathological changes in tissues collected by repeated mucosal biopsy. Within 2-3 days of the transplant of adult worms, abomasal pH had increased markedly in five out of six animals, and there also had been rapid increases in serum gastrin and pepsinogen concentrations in all animals. Reductions in parietal cell number were recorded as early as 1 day after the transplant of adults and were associated with the rapid accumulation of many neutrophils and eosinophils. Mucosal hyperplasia, with increased numbers of cells closer in appearance to mucous/mucous neck cells, was a relatively late development, being most pronounced in the latter part of the infection. In sheep given larvae, changes in secretory physiology were again matched by a concurrent fall in parietal cell number and by the accumulation of inflammatory cells. Changes became maximal when most worms could be expected to be present as adults, confirming the role of adults in the natural disease. Some abnormalities were detected in biopsies collected from animals maintained free of parasites and, although milder in degree, there were similarities to those observed in parasitised tissues, there being fewer parietal cells, a modest degree of mucous cell hyperplasia and inflammatory infiltrates of predominantly neutrophils. These changes were the likely result of trauma to the tissues in the immediate vicinity of the cannula, due either to the presence of the cannula itself or to the frequent collection of biopsy material from areas close to it.

Effects of adult and larval Haemonchus contortus on abomasal secretion

Abomasal pH and serum pepsinogen and gastrin were increased in parasite-naive sheep by infection with either larval or adult H. contortus. Four sheep received 10000 larvae intraruminally and 9000 adult worms were given directly via an abomasal cannula to another 4 sheep. The latter animals were dosed orally with 0.4 mg kg-1 ivermectin 4 days after worm transfer and their recovery was monitored for a further 8 days. Whereas the presence of adult worms rapidly induced changes in the secretory activity of the abomasum, the early larval stages had minimal effects up to the 4th day post-infection. After either larval or adult infection, the initial hypergastrinaemia began at the same time as the increase in abomasal pH, but serum gastrin continued to increase after abomasal pH had reached a maximum and returned more slowly to normal values after drenching. The increase in serum pepsinogen did not precede those in the other parameters, unlike the earlier hyperpepsinogenaemia which occurs with Ostertagia infection. Three of the 8 infected sheep had no detectable serum pepsinogen increase during the parasitism while showing typical effects on abomasal pH and serum gastrin. The rapidity of the responses to the transfer of adult worms and to their removal by treatment with anthelmintic supports a role for worm excretory/secretory (ES) products which possibly are not produced by the early larval stages. The similarity of responses to H. contortus and O. circumcincta infection suggests the involvement of the same or very similar ES products.

Abomasal secretion in sheep receiving adult Ostertagia circumcincta that are prevented from contact with the mucosa.

AIMS Both adult and larval Ostertagia circumcincta infections raise abomasal pH and serum gastrin and pepsinogen concentrations, either because of physical effects of the worms or from their chemical secretions. The study was designed to examine whether adult worms require contact with the gastric mucosa to effect changes in gastric secretion. METHODS Abomasal pH and serum gastrin and pepsinogen concentrations were measured in four groups of sheep: Group A (n = 4): abomasal contents containing about 18,000 adult O. circumcincta were obtained from donor sheep, concentrated and infused into 5 microm mesh porous bags attached to surgically implanted abomasal cannulae. A second worm transplantation was carried out 1 week later to assess worm survival after 16 hours. Group B (n = 4): about 9000 adult worms, recovered after migration out of abomasal contents set in agar, were placed in small 5 microm mesh bags which were inserted through indwelling abomasal cannulae and left for 3 days. Group C (n = 2): about 3000 adult worms from the population recovered from agar were infused through abomasal cannulae which allowed. free movement in the abomasum. Group D (n = 3) was left uninfected. RESULTS Worms transplanted directly into the abomasum (Group C) caused rapid and marked effects on abomasal secretion. Adult O. circumcincta died within 16 hours of transfer into the abomasum when they were restrained within porous bags. Nevertheless, in sheep receiving 18,000 worms, abomasal pH increased soon after new feed was presented on Days 1, 2 and 4 after worm transfer; serum gastrin was elevated in three sheep from 113 hours and serum pepsinogen increased in one animal. Sheep receiving 9000 worms showed similar trends but the results were equivocal. CONCLUSIONS Adult O. circumcincta prevented from physical contact with the gastric mucosa by restraint in porous bags are able to raise abomasal pH. This study implicates parasite excretory-secretory products in mediating changes in gastric secretion caused by adult abomasal worms.

Hypergastrinaemia, abomasal bacterial population densities and pH in sheep infected with Ostertagia circumcincta

Serum gastrin and pepsinogen concentrations, food intake, abomasal pH and abomasal aerotolerant and anaerobic bacterial populations were measured in sheep infected with Ostertagia circumcincta to search for links between hypergastrinaemia, food intake and changes in the abomasal environment. Abomasal pH and serum gastrin and pepsinogen concentrations were elevated in each of five sheep infected via abomasal cannulae with 150000 exsheathed larval stage three, followed 11 days later by 100000 sheathed larvae given intraruminally. Unparasitised abomasa contained aerotolerant bacterial population densities of between 10(3) and 10(6) cells ml(-1) and these did not change significantly following parasitism. In contrast, anaerobic bacterial population densities increased markedly by about 10(4)-fold following parasitism. Anaerobic numbers changed rapidly when abomasal pH increased from 2.5 to 3.5. At pH 4 and above, anaerobic bacterial numbers approached levels expected in rumen contents but parameters other than pH did not relate to bacterial numbers. Brief periods when serum gastrin was lower than expected, coinciding with raised abomasal pH, were not explicable by increased bacterial numbers. Food intake, which decreased for a variable period from around Day 5 p.i., correlated poorly with serum gastrin concentration, suggesting hypergastrinaemia is not the sole cause of anorexia in parasitised animals. The survival of substantial numbers of rumen bacteria in the abomasum at only slightly raised pH may significantly lower the bacterial protein available to the sheep.

Reduced Ostertagia circumcincta burdens in milk-fed lambs

Abstract Aims: To compare the susceptibility to parasitism by Ostertagia circumcincta of lambs fed entirely with bovine milk or weaned on to solid feed at 3 weeks of age. In addition, the effect of a single daily feed of milk on worm burdens was assessed. Methods: Eight lambs were assigned to each of the 3 diets: milk (M), milk plus solid feed (cereal-based pellets and lucerne chaff) (MS), or solid feed only (S). Those to be fed solid feed were converted from complete milk feeding to the designated diet during their third week of life. From 3 weeks of age, all lambs were infected with 1000 O. circumcincta larvae twice weekly for 6 weeks; 4 lambs from each diet group were given normal sheathed L3 and another 4 were infected with exsheathed larvae. Faecal egg counts (FEC) and serum gastrin and pepsinogen concentrations were monitored from Day 17 after first infection, and worm burdens and abomasal pH and morphology were determined at necropsy. Results: Total worm burdens and FEC were significantly lower in the M than MS and S groups, whereas there was no significant difference between those receiving sheathed and exsheathed larvae. The milk-fed lambs had a smaller reticulo-rumen and omasum and a more acidic abomasal pH. Serum gastrin and pepsinogen were increased in all groups, irrespective of diet or type of larvae used for infection. Conclusions: The cause of the lower worm establishment in lambs fed only milk was probably not failure to exsheath in the immature gastro-intestinal tract, as there were similar worm burdens in lambs whether sheathed or exsheathed larvae were administered. The lower pH of the abomasal contents of the preruminant lambs may have been a factor, as the parasites have previously been shown to die more rapidly in vitro at low pH. Alternatively, the milk itself had adverse effects on the parasites, but was ineffective when combined with solid feed. There was no benefit from feeding a milk plus solid diet over a solid diet.

Effect of Ostertagia circumcincta excretory/secretory products on gastrin release in vitro

It has been suggested that parasite excretory/secretory (ES) products may be capable of direct stimulation of gastrin secretion and of contributing to the hypergastrinaemia typical of abomasal parasitism. Ostertagia circumcincta ES products were tested on an ovine antral mucosal preparation which had been developed for a pharmacological study of gastrin secretion in the sheep. Its responsiveness to chemical stimulation was established by stimulation with amino acids and amines: tryptophan (0.1-5 mM) and phenylalanine (10-100 mM) stimulated gastrin release (151-160 and 117-129%, respectively), whereas glycine (0.1-100 mM) was without effect; ammonium sulphate, but not sodium sulphate, stimulated gastrin release in concentrations from 1mM (122%) to 50mM (148%). ES products were prepared by incubation of exsheathed third-stage larvae (L3) or parasites recovered on Day 8 p.i. (L4), Day 12 p.i. (10% L4, 90% immature adults), Day 21 p.i. (5% L4, 30% immature adults, 65% adults), Day 22 p.i. (20% immature adults, 80% adults), Day 30 p.i. (adults) and Day 35 p.i. (adults), or a mixed-age parasite population. Worms were recovered from agar and incubated in either distilled water or Hanks balanced salt solution (HBSS) adjusted to pH 2.5, 3.5, 4.5, 5.0 or 7.4. HBSS pH 7.4 was also prepared with antibiotics, without glucose, and with antibiotics but without glucose. Survival of Day 21 and 35 worms and exsheathed L3 in water or in a series of HBSS adjusted to pH 2.5, 3.5, 4.5, 5.0 or 7.4 was assessed from the percentage of motile parasites. L3 slowly became immotile over several days except in HBSS pH 2.5, in which survival was reduced, whereas adult worms did not tolerate incubation at 37 degrees C in water or HBSS at pH 2.5, retained motility for about 2 days at pH 3.5, but survived well at pH 4.5 and above. Incubates prepared from all stages of O. circumcincta, both in media favourable and unfavourable for parasite survival, failed to stimulate consistently the secretion of gastrin by tissue from both parasite-naive and previously exposed sheep, whereas a considerable number of incubates were significantly inhibitory. The inhibitor may not be produced by the nematodes, but by contaminating abomasal or environmental microflora, as inhibitory activity was predominantly generated by prolonged incubation, it was less potent when glucose was omitted and was not present in media containing antibiotics. This study did not find evidence for a gastrin stimulant in O. circumcincta ES products, but did demonstrate the acid intolerance of adult worms and suggests that abomasal microbes may be capable of modulating the secretory activity of the host digestive tract.

Gastrin secretion by ovine antral mucosa in vitro

The effect on gastrin and somatostatin release in sheep of stimulatory and inhibitory peptides and pharmacological agents was investigated using an in vitro preparation of ovine antral mucosa. Carbachol stimulated gastrin release in a dose-dependent manner but had no effect on somatostatin release. As atropine blocked the effect of carbachol, cholinergic agonists appear to stimulate gastrin secretion directly through muscarinic receptors on the G-cell and not by inhibition of somatostatin secretion. Both vasoactive-intestinal peptide (VIP) and gastric-inhibitory peptide (GIP) increased somatostatin release but did not inhibit basal gastrin secretion, although VIP was effective in reducing the gastrin response to Gastrin-releasing peptide (GRP). Porcine and human GRP were stimulatory to gastrin secretion in high doses but bombesin was without effect. The relative insensitivity to GRP (not of ovine origin) previously reported from intact sheep may be caused either by a high basal release of somatostatin or by the ovine GRP receptor or peptide differing from those of other mammalian species.

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.

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.

Shall we dance? Music as a port of entrance to maternal-infant intersubjectivity in a context of postnatal depression.

The present study introduces the concept of a mother-infant group therapy that makes use of music, singing, and moving to establish maternal-infant intersubjectivity. It was conducted in a residential mother-baby unit for mothers with postnatal depression and their infants over a 5-week period. Maternal-infant intersubjectivity of four dyads in relation to the group dynamics were microanalyzed for Sessions 1 and 5. We made within-session (i.e., beginning-middle-end) and between-session (Session 1 vs. Session 5) comparisons for the number of intersubjectivity moments (ISMs), total time of intersubjectivity (IST), and the mean duration of ISMs on a dyadic (i.e., own mother/infant involved) and a nondyadic level (i.e., own mother/infant not involved). In addition, three ISM levels (degree of group contribution) were distinguished. The results indicated a significant increase of ISMs/IST from Session 1 to Session 5. Within-session analyses showed that ISMs/IST significantly decreased through Session 1 and remained stable throughout Session 5. Intersubjectivity occurred mainly on ISM Level 1 during Session 1 and on ISM Level 3 during Session 5, suggesting increased dyadic autonomy and self-efficacy. The results are discussed in relation to the musical characteristics of mother-infant dyads, music improvisation techniques, group processes, and intersubjective development.