Haemonchus contortus and Ostertagia circumcincta: fenbendazole treatment and abomasal permeability changes in sheep

Effects of fenbendazole (7.5 mg/kg of body weight infused during 90 minutes) on abomasal transmural potential difference (PD), pH, and ionic concentrations were determined in 8 lambs fitted with permanent abomasal cannulae. Four sheep were treated before inoculation and on day 28 after inoculation, with 200,000 3rd-stage Ostertagia circumcincta larvae and 4 others were similarly inoculated with 15,000 3rd-stage Haemonchus contortus larvae. In worm-free lambs, the unique effect was an increase in Na+ concentration of the abomasal contents. The O circumcincta or H contortus-inoculated lambs had an increase in PD, pH, and Na+ concentrations. Treatment further increased these concentrations, but the effects on PD occurred and ceased earlier in sheep inoculated with O circumcincta than in those inoculated with H contortus. The results indicated that part of the changes were linked to substances produced by the parasites or by the host. The increase in PD and pH was associated with a decrease in K+ concentration and indicated that these substances stimulated HCO3- transfer.     

Pepsinogen types in worm-free sheep and in sheep infected with Ostertagia circumcincta and Haemonchus contortus

Pepsinogen isolated from the plasma of Dorset wethers with abomasal burdens of Ostertagia circumcincta was found to have the same proportions of three zymogen subtypes as that isolated from the plasma of Merino wethers with abomasal burdens of Haemonchus contortus. It appears that the underlying pathophysiological response which causes increased plasma pepsinogen concentrations during abomasal parasitism may be the same irrespective of the breed of sheep or species of parasite.     

Effect of orally administered cimetidine and ranitidine on abomasal luminal pH in clinically normal milk-fed calves

OBJECTIVE: To characterize the change of pH in the abomasal lumen throughout a 24-hour period, to determine whether pH of the abomasal body differs from pH of the pyloric antrum, and to determine whether oral administration of cimetidine and ranitidine alters pH of the abomasal lumen in milk-fed calves. ANIMALS: 5 male dairy calves (4 Holsteins-Friesian, 1 Ayrshire), 5 to 15 days old. PROCEDURE: Cannulas were surgically positioned in the abomasal body and pyloric antrum of each calf. Calves received the following treatments in a randomized crossover design: milk replacer (60 ml/kg of body weight, q 12 h [untreated control calves]), milk replacer and cimetidine (50 or 100 mg/kg, q 8 h), or milk replacer and ranitidine (10 or 50 mg/kg, q 8 h). The pH of the abomasal body and pyloric antrum was measured for 24 hours, using miniature glass pH electrodes. RESULTS: Suckling of milk replacer immediately increased abomasal luminal pH from 1.4 to 6.0, followed by a gradual decrease to preprandial values by 6 hours. Preprandial and postprandial pH values were not significantly different between the abomasal body and pyloric antrum, indicating lack of pH compartmentalization in the abomasum of milk-fed calves. Administration of cimetidine and ranitidine caused a significant dose-dependent increase in mean 24-hour abomasal luminal pH. CONCLUSIONS AND CLINICAL RELEVANCE: Abomasal acid secretion in milk-fed calves is mediated in part by histamine type-2 receptors. Cimetidine and ranitidine may be efficacious in the treatment of abomasal ulcers in milk-fed calves.     

Serum and abomasal antibody response of sheep to infections with Haemonchus contortus

Serum and abomasal IgA, IgG and IgM antibody response against adult worm, L3 and egg antigens of Haemonchus contortus was monitored by the ELISA technique after one or two infections with this nematode. Following the first infection, antibody levels in serum did not change materially. After administration of a challenge dose of infective larvae, antibodies of the three immunoglobulin classes in infected animals rose slightly, but this rise appeared later than the fall in the faecal egg counts. In contrast, in abomasal mucosa, IgA anti-larval antibody levels, which did not increase materially after the primary infection, rose rapidly after a transient inhibition when sheep were challenged. A close temporal relationship was observed between the rise in local anti-worm IgA antibodies and the self-cure reaction, but antibody levels fell rapidly after worm diminution. The local antibody response was thus considered to be related to immunity of sheep to H. contortus.     

The abomasal histology of worm-free sheep given primary and challenge infections of Haemonchus contortus

The histological changes in the abomasal mucosa of pairs of worm-free ewes and lambs were compared at various stages after infection and reinfection with 350 Haemonchus contortus L3 per kg bodyweight. In terms of worm burdens the ewes were relatively resistant to reinfection compared with the lambs and the histological changes in the abomasum at most stages after infection were more marked in the adult animals. These included increases in the numbers of mast cells, globule leucocytes, eosinophils and IgA plasma cells in the mucosa.     

Evidence for a parasite-mediated inhibition of abomasal acid secretion in sheep infected with Ostertagia leptospicularis

The acid secretory capacity of the abomasal mucosa was studied in sheep experimentally infected with Ostertagia leptospicularis. The acidity of the abomasal contents, permanently recorded by a pH probe located inside the abomasum, decreased markedly to mean levels between pH 5 and 6. Subcutaneous administration of histamine or carbachol successfully stimulated acid secretion (pH 3.4). The results indicate that the abomasal mucosa harboured a population of functional parietal cells which were also identified immunohistochemically (H(+)/K(+)-ATPase). Ultrastructural investigation before stimulation revealed that the majority of these cells was in a resting state. Despite high serum gastrin levels, the acid secretion was blocked either at the level of the parietal cell or the enterochromaffin-like cell by an unknown factor, possibly mediated by the parasites. This is the first report of a parietal cell dysfunction associated with a nematode infection in the abomasum. It is suggested that the parasites induce changes in their environment which favour their survival and/or increase their reproduction.     

Stimulated pepsinogen secretion from dispersed abomasal glands exposed to Ostertagia species secretion

Carbachol and secretions from Ostertagia species parasites significantly (P less than 0.001) stimulated isolated preparations of dispersed gastric glands from bovine and ovine abomasal mucosa to secrete pepsinogen. Atropine reduced the response to both secretagogues. Live adult and larval stages of Ostertagia ostertagi and O circumcincta and homogenates of these parasites did not significantly (P greater than 0.05) increase pepsinogen production from bovine or ovine gland preparations.     

Resistance to macrocyclic lactone anthelmintics by Haemonchus contortus and Ostertagia circumcincta in sheep in New Zealand

AIM: To establish the efficacy of oral formulations of ivermectin and moxidectin against naturally acquired abomasal nematode infections on a North Island sheep farm. METHODS: Two controlled slaughter trials were undertaken. In the first, 30 sheep on pasture were randomly allocated on the basis of faecal egg count to 1 of 3 groups, comprising an untreated control group and 2 treatment groups. One treatment group was given a single oral dose of ivermectin and the other a single oral dose of moxidectin, both at the manufacturer's recommended dose rates of 0.2 mg/kg liveweight. Six days after treatment, all animals were slaughtered and their abomasa recovered for worm counting. The second trial, which involved 47 animals, was essentially the same as the first except that, as well as involving the slaughter of 30 sheep from all 3 groups, 6 days after treatment, it also included a further 8 untreated control animals and 9 moxidectin treated animals which were slaughtered 27 days after treatment. RESULTS: At 6 days after treatment, moxidectin was highly effective against all 3 of the abomasal nematodes present. While ivermectin was similarly effective against Trichostrongylus axei 6 days after treatment, it was not effective against either Ostertagia circumcinta or Haemonchus contortus, against which average efficacies of only 63.6% and 61.6%, respectively, were recorded. At 27 days after treatment, moxidectin, was also highly effective against T. axei (97.3% reduction) but not against either H. contortus (71.4% reduction) or O. circumcinta (61.0% reduction). CONCLUSIONS: These results provide the first record of macrocyclic lactone resistance in H. contortus in sheep or in any other host in New Zealand, and the first case where such resistance has been exhibited in more than one parasite species at a time. Although the therapeutic efficacy of moxidectin was high against these resistant H. contortus and O. circumcincta strains, resistance to moxidectin was indicated by its diminished prophylactic activity against them. It is suggested that this reduction in the prophylactic activity of moxidectin is also likely to reduce its apparent current high therapeutic efficacy. CLINICAL RELEVANCE: As well as providing further evidence that it can no longer be automatically assumed that macrocylic lactone anthelmintics will be effective on sheep farms in this country, these findings also present a warning that increasingly complex parasite control options may have to be faced in the future.     

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days.Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.     

Synergistic influence of Ostertagia ostertagi and Trichostrongylus axei on Ostertagia ostertagi larval inhibition and abomasal lesions in cattle

Parasite-free 4-month-old calves were inoculated with Ostertagia ostertagi and/or Trichostrongylus axei followed 6 weeks later by increasing doses of O ostertagi for 8 weeks. Clinical signs of parasitism, fecal egg counts, and plasma pepsinogen concentrations were monitored, and gross lesions and parasite burdens were determined postmortem. Clinical signs of parasitism were not observed and weight gains were not affected in experimentally infected calves. In calves infected with O ostertagi, mean plasma pepsinogen concentrations were greater than for control calves and were diagnostically significant 4 weeks after inoculation and during the last 4 weeks of serial inoculations with O ostertagi. In calves that were given O ostertagi and T axei, abomasal pH was significantly increased, and abomasal lesions were more pronounced than in control calves or in calves inoculated with only O ostertagi or T axei. Abomasal lymph nodes were enlarged in all parasitized calves; other lymph nodes in the calves inoculated with both O ostertagi and T axei were usually smaller than in calves inoculated with only O ostertagi or T axei. Numbers of O ostertagi-inhibited larvae were small in all inoculated calves, but the percentage inhibition was significantly greater in calves inoculated with both O ostertagi and T axei. The percentage inhibition was 3.53% for the O ostertagi-inoculated calves and 7.07% for calves inoculated with both O ostertagi and T axei. These percentages indicated a synergistic effect of concurrent abomasal parasitism, whereas a synergistic effect on T axei worm burden was not observed. The low percentage of larval inhibition indicated that factors other than host resistance are involved in naturally occurring pretype II ostertagiosis.     

Effect of parasitism with Ostertagia circumcincta on pharmacokinetics of fenbendazole in sheep

Plasma and abomasal fluid concentrations of fenbendazole and its two major metabolites in sheep experimentally infected with Ostertagia circumcincta were compared with those in the same sheep when non-parasitised. Bio-availability of the drug was reduced in the parasitised state. There was also a reduction in the proportion of drug present in the form of metabolites in parasitised as compared with non-parasitised animals.     

Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens

Primary and secondary serum antibody responses to Haemonchus contortus were studied in Castellana sheep. Ten-month-old sheep were infected (200 L3/kg live weight (lw)) and challenged (400 L3/kg lw) or uninfected and equally challenged with H. contortus. Primary infections induced a partially protective response upon challenge, characterized by higher serum protein levels, longer prepatent periods, lower fecal egg counts, and significant reduction in the establishment rate of the parasite and abomasal adult and L4 worm burdens. The resistant status of the infected and challenged sheep was not clearly related either to the serum specific antibody levels (IgG: IgG1, IgG2; IgM; IgA) estimated by ELISA or to immunodetection patterns in the Western blots.     

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.     

Identification of Ostertagia ostertagi specific cells in bovine abomasal lymph nodes

To investigate the contribution of different bovine cell subpopulations in the development of in vitro induced responses by Ostertagia ostertagi third larval antigen extract (L3), bovine abomasal lymph node cell suspensions were depleted of specific cell populations. The depleted cell suspensions were subsequently assayed for their proliferative responses to O. ostertagi L3 antigen extract. Proliferative responses to O. ostertagi L3 antigen extract were restricted to a CD2+ CD4- CD8- cell population and MHC II+ cells different from B-cells were of major importance. Depletion of CD4, CD8, CD4CD8, IgM or CD21 positive cells did not decrease proliferation to L3 antigen extract. Depletion of gammadelta T-cells, which also comprise a subpopulation of CD2+ CD4- CD8- cells, reduced proliferation to L3 antigen extract only in one animal. The results suggest that either gammadelta T-cells could be involved in the proliferation or that another as yet unidentified population is important for proliferation. The precise role of these populations during infection with O. ostertagi and the mechanism by which these cells may influence the host immune response are important issues that remain to be elucidated.     

Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep

This study was carried out to evaluate the relationship of abomasal inflammatory cells and parasite-specific immunoglobulin A (IgA) in mucus, with the resistance to Haemonchus contortus infection in three breeds of sheep naturally infected with gastrointestinal nematodes. The breeds were the native Santa Ines sheep, and the European Suffolk and Ile de France breeds. Mast cells, eosinophils and globule leucocytes were enumerated in abomasal mucosa. Eosinophils within the sub-mucosa also were counted separately. Histamine concentration was estimated in abomasal tissue samples. Enzyme-linked immunosorbent assay was carried out in mucus samples to determine the level of IgA anti-H. contortus third and fifth instar. There were no significant differences among group means of these variables (P>0.05). The correlation coefficients between fecal egg counts (FEC)xmast cells (r=-0.490; P<0.05) and FECxeosinophils in sub-mucosa (r=-0.714; P<0.01) was significant in the Santa Ines sheep. In the Ile de France group, the correlation coefficients between globule leucocytesxFEC (r=-0.879; P<0.001) and histaminexworm burden (r=-0.833; P<0.01) were also significant. In the Santa Ines and Ile de France sheep, correlation coefficients between IgA anti-L3xworm burden and IgA anti-L3xFEC were negative. In general, inflammatory cells and IgA-parasite-specific in abomasum were inversely associated with H. contortus worm burden and FEC indicating that they may impair parasite development or fecundity in the three breeds of sheep. However, similar mean values of inflammatory cells and IgA were found in the resistant (Santa Ines) and in the susceptible (Suffolk and Ile de France) breeds of sheep. The enumeration of cells by histological assessment does not provide information on their functional activity, which may be different among breeds. Thus, the effect of breed on the functional activity of these and other inflammatory cells is an important area for further study.     

Effect of sodium cromoglycate on the response to Ostertagia circumcincta in adult sheep

Serum pepsinogen concentrations rose more rapidly and to higher levels in adult sheep infected with Ostertagia circumcincta and treated orally with the mast cell stabilising agent sodium cromoglycate than they did in adult sheep infected with the parasite which remained untreated. Sodium cromoglycate did not affect the serum pepsinogen concentrations or abomasal pH in uninfected sheep.     

Effect of Ostertagia ostertagi secretions and various putative secretagogues and inhibitors on aminopyrine accumulation in dispersed bovine abomasal gland cells

Aminopyrine accumulation in suspensions of isolated gastric glands was used to determine the effect of Ostertagia ostertagi secretions and putative secretagogues and inhibitors on abomasal parietal cells. Parasite secretions did not affect acid production nor did histamine. Dibutyryl cyclic adenosine monophosphate (cAMP) and pentagastrin significantly increased aminopyrine accumulation by gastric glands and cimetidine, omeprazole and thiocyanate significantly decreased aminopyrine accumulation confirming their roles as stimulators and inhibitors of gastric acid production, respectively.     

Persistence of dead Ostertagia ostertagi in the abomasal mucosa following anthelmintic treatment

Anthelmintic trails, conducted with albendazole, fenbendazole and ivermectin for efficacy against gastrointestinal nematodes, principally inhibited early fourth larval stages of Ostertagia ostertagi in naturally infected cattle. Cattle wee slaughtered seven to 20 days after treatment. O ostertagi was the predominant abomasal nematode recovered with occasional small numbers of Haemonchus species and Trichostrongylus axei. Control calves uniformly had very large O ostertagi infections, primarily early fourth stage larvae. Viable surviving worms and variable numbers of dead and degenerate worms were recovered in abomasal contents and washings. These O ostertagi larvae and adults were characterised by adherent debris or proteinaceous material, degenerated cuticles and distortion of internal structures. This study demonstrated the necessity for proper timing of slaughter for anthelmintic trial evaluation to allow clearance of dead nematodes, specifically O ostertagi larvae which are sequestered in the abomasal glandular tissue. Nematode collection within seven to 12 days after treatment will include dead and degenerate larval nematodes. The peripheral coating of larvae was suggestive of the Splendore-Hoeppli effect which has been associated with immunological responsiveness. The antigenic stimulus for this material and the lymphocyte and eosinophil infiltration was suspected to be early fourth stage O ostertagi larvae within the mucosa but was not identified definitively.