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The hypothesis that increases in the concentration of the anorectic peptide leptin may be responsible for the immune-mediated reduction in feed intake (FI) during gastrointestinal parasitism in sheep was investigated. In a 2 x 2 x 2 factorial design, the first factor was age at the start of infection (5 months old v. 17 months old). The second factor was parasite infection (no infection v. administration of eighty L3 infective Trichostrongylus colubriformis larvae/kg live weight (LW) per d three times per week for 77 d). The third factor was immunosuppressive therapy with a corticosteroid (no therapy or weekly intramuscular injection of 40 mg methylprednisolone acetate/30 kg LW). Relative to their uninfected counterparts, a 20 % reduction in FI per unit LW (FI/LW; g DM/kg LW) was observed in infected non-suppressed 5-month-old lambs from 21 to 63 d post-infection (P < 0.001) but not in comparable17-month-old ewes or in corticosteroid-treated lambs or ewes (P>0.05 for all), allowing the suggestion that the anorexia was a consequence of the developing immune response. The reduction in FI/LW in 5-month-old lambs was not associated with an increase in plasma leptin concentration. Furthermore, plasma leptin concentrations were greater in corticosteroid-treated animals (P < 0.001) and in 17-month-old animals (P < 0.001), none of which displayed an infection-induced reduction in FI/LW. Plasma leptin was positively correlated with carcass fat percentage in both 5-month-old (P = 0.016) and 17-month-old (P < 0.001) animals and did not appear to provide a direct feedback mechanism that restricted energy intake. The results do not support the hypothesis that an increase in circulating leptin is directly responsible for the immune-mediated anorexia in lambs during T. colubriformis infection.

A technique to take sequential tissue biopsy samples in multiparous, periparturient ewes from the abomasal mucosa is described, developed in parallel in Scotland and New Zealand. Samples were extracted via abomasal cannulae inserted into the wall of the abomasum and exteriorised through dorso-ventral laparotomy. Animals recovered quickly post-surgery, and tolerated the cannula and sampling without any adverse signs of pain or discomfort. The technique was deployed in two pilot studies to investigate the sequential mucosal inflammatory cell responses in well-defined parasitological models, during the periparturient relaxation of immunity in ewes infected with gastrointestinal nematodes and subjected to different feeding treatments. One experiment (Moredun Research Institute, Scotland) involved the infection of twin-bearing ewes with Teladorsagia circumcincta L3 either before, or after lambing. By feeding ewes with different levels of protein supplementation, preliminary data on the impact of nutrition on the eosinophil, mucosal mast cell and globule leucocyte responses during this period were investigated. A similar study was also performed at Lincoln University, New Zealand, to investigate these cell responses in sheep fed relatively high or low protein diets during pregnancy, and infected with a combined immunisation regime of T. circumcincta and Trichostrongylus colubriformis L3. These studies confirmed the phenomenon termed the periparturient relaxation in immunity (PPRI) where a transitory increase in faecal egg counts is observed during late pregnancy and lactation, and this effect was exacerbated during protein undernutrition. Although the number of animals was low in each experiment and the cell responses variable, the results together suggest a reduction in the number of mucosal mast cells and globule leucocyte during the PPRI when protein supply was restricted. The present paper thus describes a successful technique to monitor ovine mucosal cell populations during local immune responses in normal and pregnant sheep. It is envisaged that this technique will be a powerful adjunct to investigations into mucosal immune mechanisms and disease pathogenesis, and will be employed to confirm the influence of dietary protein on the local inflammatory cell responses during the PPRI.

Four groups of nine Saanen goat does with a naturally acquired mixed trichostrongylid infection were grazed on four paddocks. Two groups received a daily dose of Duddingtonia flagrans at the rate of 5 x 10(7) chlamydospores per animal per day for the 26-day grazing period. After a 19-day pasture resting period, 20 worm free 12-week-old tracer kids were introduced to the paddocks for 14 days prior to removal for worm burden analysis. Four groups of five does and four kids were drenched then turned out onto the paddocks and faecal egg count (FEC) monitored. The FEC between groups was comparable throughout the initial grazing period. There were significant reductions in number of Teladorsagia circumcincta (54.8%, P=0.004) and Haemonchus contortus (85.0%, P=0.02) worms recovered from tracer animals. FEC of animals subsequently grazing pasture were significantly reduced (P=0.036) with reductions of 44% observed 4 weeks post-turnout. No significant difference was observed after 6 weeks grazing. This trial has demonstrated the potential of D. flagrans to reduce larval numbers on pasture grazed by goats under New Zealand conditions.

Proteinases are known to be capable of prolonging the survival of endoparasites in a host. We were therefore interested in knowing whether immunization of lambs against a proteasome (multisubunit proteinases) preparation obtained from Trichostrongylus colubriformis infective third-stage larvae (L3) would have any effect on the immune response to a single challenge infection with the same organism. A total of 21 penned lambs aged 8 months were divided into 3 equal groups. Group 1 was immunized on three occasions with increasing amounts of a proteasome-enriched fraction obtained from infective L3. Group 2 was given a similar amount of protein from the initial supernatant of homogenized larvae. Group 3 (controls) received adjuvant plus saline solution only. All groups were challenged with 60,000 infective T. colubriformis larvae at 28 days after the last immunization. Significant protection was obtained only when the initial supernatant extract was used to immunize lambs. The proteasome preparation seemed to have immunosuppressive effects through the stimulation of nonspecific IgE production. Significantly lower levels of specific IgE were observed in lambs immunized with the proteasome-enriched fraction, and levels of specific IgG antibodies were increased. We suggest that proteasome fractions of T. colubriformis may serve as useful preparations for the study of mechanisms of IgE production in parasitized sheep.

Twenty, 2-yr-old angora goats kept on nematode larvae-contaminated pasture since birth were divided into 2 equal groups. Goats from group 1 were immunized by drug-abbreviated infection, a procedure that gave high protection against field challenge in 12-moold sheep. Group 1 was orally dosed 3 times with increasing numbers of Trichostrongylus colubriformis and Teladorsagia circumcincta infective larvae. Each time, the infection was abbreviated with Oxfendazole (OXF) 15 days after dosing. Group 2 received only OXF. After the third dose of OXF, the goats were grazed together on the same pasture and fecal egg counts determined. No protection in immunized goats was achieved. In fact, immunized goats produced significantly more nematode eggs than the nonimmunized group.

A very high level of protection was achieved against homologous (up to 97%) and heterologous (up to 87%) infections in 12-month-old Romney sheep immunised with oxfendazole-abbreviated infections of Ostertagia circumcincta and Trichostrongylus colubriformis. No significant protection occurred following ivermectin-abbreviated infections. None of the immunised sheep showed an increase in antibody level against excretory-secretory antigen of T. colubriformis infective larvae. The immunisation procedures did not cause a decrease in wool production, or liveweight gains compared with non-immunised controls.

Two divergent lines of Romney sheep have been selected on the basis of differences in faecal egg counts (FEC) to natural poly-generic parasite challenge in New Zealand. However, it is not known if the expression of resistance or susceptibility extends to parasitic nematodes that were not a major part of the selection challenge. To examine this, the immune response to infection with Trichostrongylus axei was examined in these sheep lines. Changes in the proportions of CD5+, CD4+, CD8+, T19+ and CD45R+ lymphocytes and parasite specific antibody titres in peripheral blood were monitored each week in six resistant and six susceptible line lambs that were maintained indoors in pens during the course of 14 weekly infections with 10,000 T. axei larvae. No difference in FEC was observed. Similarly, no significant difference in T. axei specific antibody titre between sheep lines was seen although antibody titre increased steadily from Week 4. Significant increases in the proportions of CD5+, CD4+, CD8+ and T19+ cells occurred in both resistant and susceptible line lambs during Weeks 1-4 of infection. Following peak levels, proportions of CD5+, CD4+ and CD8+ cells fell with the rate of decline of CD5+ and CD4+ cells significantly greater in the resistant line lambs. Proportions of CD45R+ cells showed significant changes with time that were the inverse of those of CD5+, CD4+ and CD8+ cells. Susceptible line lambs showed higher proportions of CD5+ and lower proportions of CD45R+ cells than resistant lambs before infection with T. axei. Overall, during infection, these differences were maintained while CD4+ and T19+ levels were higher in the susceptible line lambs.

Aspects of the local immune response to nematode challenge were investigated in vivo in isolated loops of the upper small intestine of mature sheep that were immunised by repeated infections with Trichostrongylus colubriformis infective larvae (L3). Groups of 3 sheep were challenged either through the loop (Group 1) or orally (Group 2) with T. colubriformis L3, the third group served as unchallenged controls (Group 3). Nematode specific antibody levels, mast cell proteinase levels (SMCP) and larval migration inhibition (LMI) activity were determined in loop secretions for 4 weeks after challenge. The intestinal loops remained functional throughout the experiment. Groups 1 and 2 were re-challenged 2 weeks after the first challenge, and all 3 Groups were slaughtered 2 weeks later. Histopathological examination showed elevated numbers of globule leukocytes (GL) in both the nematode-challenged loop and unchallenged small intestine of Group 1 and small intestine of Group 2 indicating that nematode infections induce the local appearance of large numbers of GL. Oral, but not loop challenge caused increased antibody levels in loop secretions when compared to unchallenged controls. Only loop-challenged sheep showed a peak in loop fluid SMCP levels 10-13 days after the first challenge which coincided with a peak in numbers of mucosal GL. The isolated loops of all 3 groups showed highly elevated numbers of eosinophils when compared to the intact small intestine. Loop fluid of all 3 groups showed a high level of LMI activity reflecting the high level of nematode-resistance induced by the immunisation procedures. Sheep in Groups 1 and 2 were both able to expel challenge infections, and when compared to Group 3, showed higher blastogenic activity of unstimulated cells derived from a mesenteric lymph node in the region of the challenged part of the intestine. The present experiment showed that surgically constructed intestinal loops provided a model system by which the substantial changes associated with the local intestinal immune response to challenge with T. colubriformis could be investigated.

A protective immune response without liveweight loss can be induced in sheep against T. colubriformis but results depend on the anthelmintic used and duration of immunizing infections. More than 90% protection was achieved in sheep immunized by three 15- or 7-day oxfendazole abbreviated infections or three 21-day nonabbreviated infections. Only 41% protection was induced by 3-day oxfendazole abbreviated infections. Significantly higher worm burden and faecal egg counts were present after challenge in sheep immunized by 7-day levamizole abbreviated infections compared to 7-day oxfendazole abbreviated infection. Liveweight gains of sheep immunized by 15- and 7-day abbreviated infections were not significantly different than non infected controls. Liveweight loss seemed to be associated with high activity of mucus peroxidase and high numbers of eosinophils in the intestinal lumen. High parasite numbers seemed to be associated with low activity of alkaline phosphatase in mucus. Mucus peroxidase, arylsulphatase, larval migration inhibition of mucus, mucus or serum antibody against L3 excretory/secretory antigen or somatic L3, L4 and adult antigen were not associated with protection.

Blastogenic activity, eosinophil and total white blood cell counts (TWBC) were examined over a period of 14 weeks in Romney lambs, genetically resistant or susceptible to gastrointestinal nematodes. The lambs were infected with 5000 infective Trichostrongylus colubriformis larvae twice weekly. Compared to preinfection levels, the blastogenic activity of unstimulated lymphocytes in lambs of both lines peaked at week 3, and was significantly higher in resistant than in susceptible lambs. These changes may have been due to in vivo polyclonal activation. Lymphocytes from susceptible sheep responded more strongly to Con A, PHA and PWM than cells from resistant sheep. Counts per minute (c.p.m) for Con A- and PHA-stimulated lymphocytes increased in both lines of sheep from week 2 to week 7 and then returned to initial levels. An increase in c.p.m. in PWM-stimulated cell cultures was observed from weeks 3 to 5 in both groups. The blastogenic activity for LPS-stimulated cultures was significantly higher for resistant than susceptible sheep at weeks 3 and 4. No significant correlations between the decline in faecal egg counts (FEC) and the blastogenic activity was observed. Eosinophil counts in peripheral blood began to increase one week earlier in resistant than in susceptible sheep. No significant correlation between FEC and eosinophil counts was observed in resistant lambs, whereas in susceptible lambs a significant correlation was found between FEC and eosinophil counts at some sampling times. TWBC in resistant lambs steadily increased with infections whereas susceptible lambs showed a decrease until week 5 and then steadily increased. There was no significant correlation between the decline in FEC and TWBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Romney sheep, 1-2 years old, immunized by at least three anthelmintic abbreviated infections of 80-100,000 Trichostrongylus colubriformis larvae usually produced high numbers of intestinal mucosal mast cells/globule leukocytes (MMC/GLs). In isolating these cells, the importance of maintaining the intestine at 37 degrees C, removal of mucus with dithiothreitol, enzymatic dispersion and careful in vitro handling procedures for maximising cell viability are emphasised. The MMC/GLs were separated from most contaminant cells by using a Percoll discontinuous gradient. MMC/GLs collected at the 60/100% Percoll interface were passed through a complement coated nylon wood column to remove the contaminating eosinophils. Viable MMC/GLs were able to grow in vitro in the presence of Concanavalin A and survive in culture for up to 30 days. The MMC/GLs were readily identified by ultraviolet light microscopy after staining with auramine O.