I. Scott

Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis

After reports of the apparent failure of monepantel to reduce the egg counts of goats on a farm in the lower North Island of New Zealand, faecal egg count reduction tests were conducted in goats and lambs resident on the property, and a confirmatory, slaughter study was conducted using 12 sheep, sourced elsewhere, that were grazed on the farm for approximately 5 weeks. In the egg count reduction test in goats, 8 animals were given monepantel at 3.9 mg/kg (just over 1.5× the sheep dose rate of 2.5mg/kg), whilst four received 7.7 mg/kg (just over 3× the sheep dose). In the egg count reduction test in sheep, 15 lambs were treated with 3.0mg/kg of monepantel. For the confirmatory study, the sheep were housed indoors for 2 weeks before half were treated with 2.9 mg/kg monepantel and the animals were killed for worm counts 9 days later. There was no evidence of efficacy in either egg count reduction test, or in the goats, the two dose rates used appeared equally ineffective. Likewise, there were no significant reductions in egg counts or worm burdens in the slaughter study. Monepantel was ineffective against at least two gastrointestinal nematode species, Teladorsagia circumcincta and Trichostrongylus colubriformis. These findings represent the first report from the field of resistance having developed to the anthelmintic monepantel with severe resistance developing in more than one species after being administered on 17 separate occasions to different stock classes and in less than 2 years of the product first being used on the farm in question.

Rumpshaker mouse: A new X-linked mutation affecting myelination: Evidence for a defect in PLP expression

SummaryThis report describes a new X-linked mutation in mice, named rumpshaker (rsh) which is associated with hypomyelination of the central nervous system. Myelination commences appropriately but the majority of sheaths fail to develop normally. Oligodendrocytes are increased in number and have prominent Golgi apparatus, rough endoplasmic reticulum and free ribosomes. Occasional cisternae of rough endoplasmic reticulum are distended. Some dense lamellar inclusions occur in Oligodendrocytes but overall, degenerative changes and cell death are uncommon. Immunostaining demonstrates a major defect in expression of PLP/DM-20. Using site-specific antisera directed at different portions of the PLP/DM-20 molecule, the major defect appears to be with PLP where virtually no myelin sheaths are positive. Antiserum against the C-terminal common to PLP and DM-20 shows reduced but definite myelin staining. Genetic analysis indicates a locus at or close to the PLP/jimpy (jp) locus. The rsh mutation, however, differs fromjp in that affected mice have normal longevity, can breed, produce substantially more myelin and have increased numbers of Oligodendrocytes.

A key mechanism of pathogenesis in sheep infected with the nematode Teladorsagia circumcincta

Abstract Infection of sheep with the abomasal nematode Teladorsagia circumcincta can cause a relative protein deficiency and reduce growth rate in growing lambs. A key event appears to be the destruction of junctions between epithelial cells. If the infection is heavy or prolonged, this leads to increased mucus production, hyperplasia, decreased acid production, gastrinemia, inappetance and pepsinogenemia. The severity of the infection depends upon the extent of concurrent infection, the nutritional status of the host and genetically controlled variation in the ability to mount protective immune responses.

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.

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.

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.

The distribution of pepsinogen within the abomasa of cattle and sheep infected with Ostertagia spp. and sheep infected with Haemonchus contortus

The effect of nematode infections on the production of pepsinogen by ruminants was investigated immunohistochemically and biochemically. Abomasal tissues were collected from parasite-naive cattle and sheep, from sheep infected with predominantly Ostertagia circumcincta, sheep infected experimentally with Haemonchus contortus and cattle infected with Ostertagia ostertagi. Pepsinogen was also assayed biochemically in homogenates of fundic mucosae from sheep infected with predominantly O. circumcincta. Infection with Ostertagia spp. parasites was associated mainly with nodular hyperplasia, resulting in increased numbers of cells that produce both pepsinogen and mucus. Measured biochemically, nodules contained more pepsinogen than adjacent more normal mucosa (p < 0.05), and this effect was largely attributable to the greater mass of nodules. Infection of sheep with H. contortus was associated with generalised hyperplasia, characterised by increased numbers of mucopeptic cells and in at least one animal with reductions in parietal cell numbers. At the same time, the zymogen granule content of chief cells was reduced. Similar changes were occasionally seen in sheep infected predominantly with O. circumcincta. Generalised hyperplasia is likely to be indicative of the presence of ambulatory parasitic stages as opposed to those confined to nodules. The potential for the enhanced production of pepsinogen by increased numbers of cells with a joint mucous cell and zymogenic cell phenotype may offset decreases in the numbers of chief cells or reductions in chief cell activity.

Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1–3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

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.

Sub-optimal efficacy of ivermectin against Parascaris equorum in foals on three Thoroughbred stud farms in the Manawatu region of New Zealand

Abstract AIM: To ascertain whether resistance was present in Parascaris equorum to the macrocyclic lactone anthelmintic ivermectin, using faecal egg count reduction tests. METHODS: Thirty-nine foals aged between 11 and 28 weeks on three Thoroughbred stud farms (Farms A; n=20, B; n=5 and C; n=14) were treated with ivermectin (Day 0) and faecal egg counts (FEC) were monitored before and for 21 (Farms A and B) or 14 (Farm C) days after treatment. On Farms A and B, the foals were treated with a macrocyclic lactone/benzimidazole/praziquantel combination on Day 21 and FEC assessed on Day 35. The three farms were all in the Manawatu region in the southern half of New Zealands North Island. RESULTS: Of the 39 foals, 15 were not shedding P. equorum eggs on the day they were treated with ivermectin, but all 15 did so post-treatment. The FEC on Farms A and B showed no evidence of a reduction at any time between Day 7 and 21; egg output increased steadily over this period. Following combination treatment FEC were reduced by 94% on farm A and 100% on Farm B. On Farm C, there was a 69% reduction in P. equorum FEC on Day 14. Twelve of the 14 foals on Farm C were shedding strongylid eggs on Day 0 and on Day 14 FEC were reduced by 84%. CONCLUSIONS: Without comparison to untreated control animals, these results do not allow a reliable estimation of the exact level of efficacy of ivermectin against P. equorum, but they nevertheless show that, within the farms studied, ivermectin no longer achieved a complete kill of egg-laying adults and that foals treated with this drug may continue to shed considerable numbers of eggs after treatment. The results also indicate that ivermectin had sub-optimal efficacy against strongylid nematodes on one of the farms.