Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections.

Parasitological and immunological parameters of experimental or naturally acquired infections with Haemonchus contortus were compared in St. Croix and Dorset lambs. In experimental infections, St. Croix lambs developed significantly greater levels of resistance to H. contortus, following primary exposure, as compared with Dorset lambs. This resistance was influenced both by age and by prior exposure to parasites. In grazing experiments on H. contortus-infected pasture, St. Croix lambs shed significantly fewer eggs as early as 5 weeks following initial exposure. Further, St. Croix lambs had more than 99% fewer worms in the abomasum at necropsy compared with age-matched Dorset lambs. Lymphoproliferative assays using peripheral blood mononuclear cells and antigen-specific serological tests demonstrated only minor differences in immune responsiveness between the two breeds despite the dramatic parasitological differences. Similarly, abomasal mucus from both breeds had elevated levels of parasite-specific antibodies and contained substances mediating larval paralysis. In contrast, St. Croix lambs which had become resistant to nematode infection had dramatically higher numbers of globule leukocytes in the abomasal mucosa compared with Dorset lambs.     

Fecundity in adult Haemonchus contortus parasites is correlated with abomasal tissue eosinophils and γδ T cells in resistant Canaria Hair Breed sheep

Canaria Hair Breed (CHB) sheep are more resistant than Canaria sheep (CS) to experimental Haemonchus contortus infection. Protective responses appear effective against the adult stage of the parasite, not as commonly reported in other breeds against the larval stages. In this study we have quantified several abomasal immune cells and correlated these with parasitological variables for each breed. A significant negative correlation between CD4+ T cell numbers and worm burden or length at 28 dpi was seen only in CS sheep. Significant negative correlations for both abomasal eosinophils and γδ/WC1+ T cells, and fecundity of the adult worms were observed only in the resistant CHB sheep breed. Tissue eosinophils and γδ/WC1+ T cells were positively correlated in CHB sheep. We suggest that the two sheep breeds have disparate immune responses following infection with the parasite and that γδ+ T cells in association with eosinophils may play a hitherto unrecognised role in modulating fecundity in H. contortus adult female parasites.     

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.     

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.     

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.     

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.     

Nematode burdens and cellular responses in the abomasal mucosa and blood of Florida Native, Rambouillet and crossbreed lambs

This experiment was carried out to compare the worm burden and cellular responses in the abomasal mucosa and blood of Florida Native and Rambouillet lambs and also in animals produced by crosses of these two breeds (generations F1 and F2). Animals were exposed to infection by gastrointestinal nematodes on three different occasions. The first infection was natural, occurring while they were suckling lambs. After weaning, they were kept indoors for 53 days and then were allowed to graze a contaminated pasture for 50 days for a second natural infection. The third infection was an artificial challenge with 6000 Haemonchus contortus infective larvae. The highest mean fecal egg counts (FEC) values were found in Rambouillet lambs followed in decreasing order by F1, F2 and Florida Native lambs. Throughout the trial, most of the high mean packed cell volumes and plasma protein levels were recorded in the F2 lambs; in contrast, most of the low values were found in the Rambouillet lambs. During the natural infection period, the highest percentages of larvae in the fecal cultures of the lambs were Haemonchus. However, high percentages of Trichostrongylus larvae were found particularly in Florida Native lambs. The mean number of blood eosinophils increased after the artificial challenge, reached a peak 21 days after infection and then declined. The highest and lowest blood eosinophil means were recorded in F2 and Florida Native lambs, respectively. The H. contortus burden was significantly higher in Rambouillet and in F1 lambs than in Florida Native and F2 lambs (p < 0.05), while no significant differences were recorded among eosinophil, mast cell and globule leucocyte counts in the abomasal mucosa (p > 0.05). The highest correlation coefficient recorded at the end of this study was between FEC and worm burden (r = 0.7). These two parameters showed a moderate negative correlation with PCV, plasma protein and mast cell counts in the abomasal mucosa. The results obtained in this study indicate that crossbreeding Florida Native and Rambouillet sheep can be a rapid way to combine and improve the characteristics of these two breeds. The parasitological results were promising, however, more studies will be necessary to verify the impact of crossbreeding in other traits.     

Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs

The effect of Haemonchus contortus infection in sheep fed with a moderate and high protein content diet was evaluated in two breeds of sheep. Forty-eight Ile de France and Santa Ines lambs were maintained indoors since birth, in worm-free conditions. The lambs were allocated after weaning in four groups of six animals per breed, which were either infected or remain uninfected and given access to either a moderately or highly metabolizable protein diet. The moderately and highly metabolizable protein diets were calculated to supply 75 and 129 g metabolizable protein per kg of dry matter (MP/kg DM), respectively. The infection consisted of a trickle infection with 300 infective larvae, three times a week, for 12 weeks. Significant differences were observed for mast cell, globule leukocyte and eosinophil counts in the abomasal mucosa of the infected groups compared to the control of both breeds (P<0.05), regardless of the diet supplied. Significantly higher IgA anti-L5 antibody was detected in the infected Santa Ines groups than in the infected Ile de France groups (P<0.05). Increased metabolizable protein supply resulted in larger body weight gain and higher packed cell volumes for both breeds (P<0.05). Both breeds showed an increased ability to withstand the pathophysiological effects of H. contortus infection when given access to the highly metabolizable protein diet. However, increased metabolizable protein supply resulted in reduced worm burdens in Santa Ines lambs but not in the Ile de France lambs (P<0.05). The present results show that the increase in protein content in growing lamb diets may benefit resistance and resilience to gastrointestinal parasites but that these benefits may vary among breeds.     

Comparative experimental Haemonchus contortus infection of two sheep breeds native to the Canary Islands

This study compares the susceptibility to Haemonchus contortus infection in two breeds of sheep endemic to the Canary Islands, the Canaria Hair Breed sheep and the Canaria sheep. Sheep were experimentally infected with 20,000 larvae of H. contortus and animals killed on days 7 and 28 post-infection. No difference between sheep breeds were detected in immature worm counts at days 7 or 28 post-infection. However, in comparison to the Canaria sheep breed, the Canaria Hair Breed sheep showed lower mean faecal egg counts, lower adult worm counts, lower number of eggs in utero and female worm stunting. Overall, these data suggest that the Canaria Hair Breed sheep has a greater resistance to H. contortus infection than Canaria sheep, and that this resistance may act at the level of the adult parasite.     

Influence of dietary protein on the pathophysiology of ovine haemonchosis in Finn Dorset and Scottish Blackface lambs given a single moderate infection

Two experiments were conducted to examine the influence of dietary protein on the pathophysiology of Haemonchus contortus in lambs of two breeds, namely Finn Dorset and Scottish Blackface. Erythrokinetic and metabolic studies using 51Cr-red cells, 59Fe-transferrin and 125I-albumin, and digestive efficiency and nitrogen balance measurements were conducted between four and six weeks after infection. Identical radioisotopic and nutritional studies were also conducted on pair-fed control lambs of both breeds. The results demonstrated that in normal control animals, there were no significant effects of diet or breed on any of the erythrokinetic, metabolic or nutritional parameters, with the possible exception of red cell survival which was considerably longer in the Finn Dorset lambs. However, in the infected animals diet and breed did influence those parameters. The severest effects were seen in the infected Finn Dorset lambs given the low protein diet. These animals had the highest level of abomasal blood loss and this was associated with low red cell volumes and high levels of both albumin catabolism and plasma iron turnover. These animals were also in negative nitrogen balance which was partly accounted for by a significantly greater urinary nitrogen loss compared with their pair-fed controls and partly due to the poorer digestibility of the crude protein fraction of the diet. In contrast, although the infected Scottish Blackface lambs had high levels of blood loss and associated pathophysiological changes, these did not differ between dietary treatments and were similar to the changes in the infected Finn Dorset lambs given the high protein diet.     

The effect of oxfendazole terminated infections with Haemonchus contortus on the development of immunity in sheep

The relative contribution of third (L3), fourth (L4) or adult stages of Haemonchus contortus to the development of immunity was evaluated in three groups of sheep subjected to infections terminated by oxfendazole treatments at the L3, L4 or adult stage. A control group did not receive immunising infections. All the groups were challenged with 5000 L3, to evaluate the protection provided by the different protocols. All sheep were necropsied at the end of the experiment to count the abomasal worm burdens. A marked reduction in egg counts after challenge infection was only observed in sheep in which the infection was terminated in the adult stage (Group 4). A significant reduction in worm burden was also observed in Group 4. The immunising infections and/or the challenge infection resulted in moderately elevated IgG antibody levels against L3, L4 and adult somatic antigens in all the groups. In contrast, a strong IgG response against H. contortus excretory/secretory (ES) antigens was observed in the groups in which the immunising infection was terminated in the L4 and the adult stage. An elevated lymphocyte proliferation response against Haemonchus ES antigens was found only in the group that had their immunising infection terminated at the adult stage. The combined data suggest that exposure to and elicited immunological responses to ES antigens are important for the development of immunity against H. contortus.     

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.     

Uptake of albendazole and albendazole sulphoxide by Haemonchus contortus and Fasciola hepatica in sheep

The pattern of in vivo uptake of albendazole (ABZ) and its major metabolite, ABZ-sulphoxide (ABZSO), by Haemonchus contortus and Fasciola hepatica recovered from ABZ-treated sheep, was investigated. Concentration profiles of both compounds were simultaneously measured in target tissues/fluids from the same infected sheep. In addition, the proportion of the (+) and (-) ABZSO enantiomers was determined in plasma, bile and F. hepatica recovered from treated sheep. Sheep naturally infected with H. contortus were intraruminally (i.r.) treated with ABZ (micronized suspension, 7. 5mg/kg) and the plasma concentrations of ABZSO and ABZ-sulphone (ABZSO(2)) determined in addition to the concentration of ABZ and ABZSO in H. contortus, abomasal mucosa and fluid content samples. In addition, F. hepatica artificially infected sheep were treated i.r. with the same ABZ suspension (7.5mg/kg), and samples of blood, bile, liver tissue and adult flukes were collected and analysed by HPLC to determine the concentrations of ABZ and both enantiomers of ABZSO. ABZSO and ABZSO(2) were the analytes recovered in plasma with ABZ and ABZSO present in H. contortus. ABZ was the analyte recovered at the highest concentration in H. contortus and abomasal mucosa, whereas higher concentrations of ABZSO were measured in abomasal fluid content. Only low concentrations of ABZ were detected in F. hepatica and bile, but markedly higher concentrations of ABZ were measured in liver tissue. ABZSO was the main molecule recovered in F. hepatica, plasma and bile samples collected from ABZ-treated sheep. The (+) enantiomer of ABZSO was recovered at a higher proportion in plasma (75%), bile (78%) and F. hepatica (74%) after ABZ administration to infected sheep.     

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.     

Towards the eradication of Haemonchus contortus from sheep flocks in Sweden

Previous studies have shown that between-year transmission of Haemonchus contortus in Swedish sheep flocks is almost entirely as over-wintered populations within housed animals, and not on pasture. This suggests that eradication of this parasite is a realistic possibility. Thus, two sheep farms with a history of H. contortus infection on the Swedish island of Oland were selected for study. During the winter housing period of 2003/2004 all ruminants (sheep and cattle) on both farms were treated with ivermectin. Monitoring by faecal egg counts and infective larval differentials of ewes and lambs for the subsequent two grazing seasons, together with total abomasal worm counts of 10 lambs from each farm at the end of the first grazing year, showed that this objective was achieved.     

Haemonchus contortus and Trichostrongylus colubriformis in pen-trials with Javanese thin tail sheep and Kacang cross Etawah goats

Weight gain costs due to infection were higher in sheep than goats, 28 and 17.5%, respectively, for Trichostrongylus colubriformis and 48.7 and 32.2%, respectively, for Haemonchus contortus. The extent of bodyweight cost attributed to anorexia in sheep infected with H. contortus was higher (13.5 g/day) than in sheep infected with T. colubriformis (2.3 g/day). On the other hand, bodyweight cost due to the other pathogenic effects in sheep infected with T. colubriformis were higher (35.6 g/day) compared to sheep infected with H. contortus (10.9 g/day). A strong relationship between faecal egg count and worm count (r=0.79, P=0.006) was shown only in sheep infected with T. colubriformis. About half of the infected sheep and goats had low or zero faecal egg counts throughout the study. In about 40% the egg count rose initially but became low by weeks 10-16, whereas in about 10% counts increased progressively throughout the period of observation and these animals also had the highest numbers of worms at slaughter. Packed cell volume was reduced in sheep and goats infected with H. contortus but serum protein and haemoglobin levels were unaffected. Sheep infected with T. colubriformis had a higher level of eosinophilia after 8 weeks (18.4%) than sheep infected with H. contortus (11.4%), whereas this pattern was reversed in goats and levels were also lower (4.1 and 8.9%, respectively). There was no apparent relationship between eosinophilia and resistance to infection with H. contortus or T. colubriformis.     

Influence of dietary protein on parasite establishment and pathogenesis in Finn Dorset and Scottish Blackface lambs given a single moderate infection of Haemonchus contortus

An experiment was conducted to examine the influence of dietary protein on parasite establishment and pathogenesis of Haemonchus contortus in lambs of two breeds. Finn Dorset and Scottish Blackface. The lambs were introduced to high protein (170 g crude protein (CP) kg-1 dry matter (DM) or low protein (88 g CP kg-1 DM) diets at three months old and infected one month later with 125 larvae (kg bodyweight)-1. Blood and faecal samples were collected for analysis and bodyweights recorded weekly. The lambs were killed 20 weeks after infection. A second experiment was conducted in Blackface lambs solely to determine the influence on parasite establishment of the high and low protein diets following infection with 125 larvae kg-1. These lambs were killed five or 14 weeks after infection. Results showed that in Scottish Blackface lambs dietary protein did not significantly influence the establishment of H contortus. However, in Finn Dorset lambs dietary protein may have influenced parasite establishment since lambs on the low protein diet had a higher faecal egg output four weeks after infection and more severe clinical signs than infected lambs of the same breed on a high protein diet.     

Rumen function and digestion of Merino sheep divergently selected for genetic difference in resistance to Haemonchus contortus

The aim of this experiment was to investigate the effect of Haemonchus contortus infection on rumen function and digestion of Merino sheep from lines divergently selected for genetic resistance to H. contortus. Rumen function and whole-tract digestibility were determined in 29 Merino weaner wethers from the CSIRO Haemonchus selection flock, increased resistance to Haemonchus (IRH), decreased resistance to Haemonchus (DRH) and random bred control (C) selection lines. Wethers were fed a restricted low quality roughage diet (5.9 MJ ME/kg DM, 29 g MP/kg DM) to maintain weight and were either uninfected (NIL) or infected with H. contortus (INF). Measurements were taken at week 7 of infection, while animals were housed in metabolic crates. Animals were then euthanased to determine abomasal worm counts. IRH had significantly lower worm egg count than DRH and C lines however, adult worm and larval counts, though lower in IRH animals, did not differ significantly. DM and OM digestibility and in sacco degradability of IRH and DRH selection lines were greater than C animals. Rumen function of animals from the IRH line was altered in response to H. contortus infection with an increase in fluid outflow and turnover rate and a decrease in propionic acid concentration. These changes may be a component of a greater host resistance and begin to explain the failure of IRH animals to translate lower worm egg count into greater bodyweight gain.