Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei.

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Nematode burdens and immunological responses following natural challenge in Romney lambs selectively bred for low or high faecal worm egg count.

Breeding lines of Romney sheep, selected as lambs for consistently low or high faecal nematode egg count (FEC) following periods of natural challenge, have been maintained at Wallaceville for some years. In order to determine the extent to which FECs in low and high genotypes reflected their ability to resist the establishment of gastro-intestinal nematode burdens, we investigated the infection status and immune responses in 8- to 9-month-old progeny of selected rams from low and high FEC breeding lines following a period of grazing without anthelmintic treatment in autumn/early winter. In each of the 2 years of the study, outcross male progeny of the two lowest FEC (LFEC) (i.e. most 'resistant') and two highest FEC (HFEC) (i.e. most 'susceptible') rams from the divergent lines were slaughtered shortly after autumn/early winter FECs had been analysed. Post-mortem worm counts and examination of intestinal histology were then undertaken. Blood samples collected before slaughter in the second year of the study were assayed to measure serum levels of Trichostrongylus colubriformis-specific antibody and immunoglobulins (IgG1 and IgM), and numbers of circulating eosinophils. Overall, correlations between pre-slaughter FEC and total trichostrongyle burdens in the lambs proved to be very high (0.91 and 0.85, respectively, for the 2 years studied). In the first year, LFEC lambs, which were shedding only 28.6% as many strongyle eggs as their HFEC counterparts at slaughter, were found to harbour 37.6% as many adult trichostrongyle worms, while in the second year, LFEC lambs, which were shedding 16.1% as many strongyle eggs as their HFEC counterparts at slaughter, were found to harbour 33.5% as many adult trichostrongyle worms. Results, particularly in the second year, confirmed that significantly fewer worms of most of the important abomasal and small intestinal nematode species which infest lambs in New Zealand (i.e. Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Nematodirus spathiger, T. colubriformis, and Trichostrongylus vitrinus) had established in the LFEC genotypes than in their HFEC counterparts. In addition, in utero egg counts of female intestinal Trichostrongylus spp. were significantly lower in LFEC lambs than in their HFEC counterparts, indicating a reduction in fecundity of those worms which did establish. There was also some evidence of an effect of host response on the developmental composition of burdens in the case of some worm species. In relation to host responses, numbers of globule leucocytes/mucosal mast cells in the intestinal mucosa were significantly higher (P < 0.01) in LFEC lambs than in HFEC lambs in both years of the study. Numbers of connective tissue type mast cells and eosinophils in the intestinal mucosa were also significantly higher in LFEC lambs but only in the second year of the study (P < 0.01 and P < 0.05, respectively). Numbers of circulating eosinophils did not differ significantly between the genotypes. T. colubriformis-specific antibodies, IgG1 and IgM to both L3 and adult worm antigens were all significantly higher (P < 0.01 or P < 0.05) in LFEC lambs than in HFEC lambs.

The immune responsiveness of Romney sheep selected for resistance or susceptibility to gastrointestinal nematodes: lymphocyte blastogenic activity, eosinophilia and total white blood cell counts.

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)

Immunization of sheep against parasitic nematodes leads to elevated levels of globule leukocytes in the small intestine lumen.

In sheep that had been given three immunizing infections with Trichostrongylus colubriformis and Ostertagia circumcincta infective (L3) larvae, drenched after the last infection and challenged with larvae of the same species, there was a significant increase in numbers of small intestine mucosal tissue globule leukocytes (TGLs) and lumenal globule leukocytes (LuGLs) compared with sheep that had only been drenched and challenged. There was a positive correlation between the numbers of LuGLs and TGLs in the small intestine but the ratio of these two cell types was lower in non-immunized than immunized sheep. In immunized sheep positive correlations were observed between LuGLs and levels of arylsulphatase and peroxidase in the intestinal mucus and between arylsulphatase and larval migration inhibition (LMI) activity in mucus. Lumen eosinophils correlated with blood eosinophils, serum antibody against T. colubriformis correlated with peroxidase in the mucus and blood eosinophils correlated with nematode specific IgM levels in the intestinal mucus. In the abomasum, TGLs were present but not LuGLs. Sheep repeatedly infected with T. axei also had significantly more LuGLs in the small intestine than control animals. Two sheep that had a surgically prepared isolated small intestinal loop, after oral infection with T. colubriformis had TGLs and LuGLs in the intact intestine, but not in the isolated loop. Significantly more LuGLs were produced in sheep by allowing repeated T. colubriformis L3 infections to develop to adult stages compared to sheep treated with the same number of larvae, but where the infections were terminated by drenching at various intervals.

Influence of ivermectin on cellular and humoral immune responses of lambs.

In view of the extensive use of anthelmintics in sheep and the fact that their activity may in part depend upon the immune system, we were interested to determine if ivermectin had any influence on aspects of the sheep immune response. Ten parasite-free 6-month-old lambs were drenched with ivermectin and 1 day later were given intravenously human erythrocytes and subcutaneously ovalbumin. Ten other lambs with injected antigens were not drenched and served as controls. Both groups were bled at intervals for cells and serum. The procedure was repeated on day 28. Lymphocytes from the drenched lambs, cultured in vitro in RPMI plus 50% autologous serum collected up to 7 and 14 days after the first and second antigen injections respectively, had decreased blastogenic activity compared with lymphocytes from control lambs. Similar results were obtained with lymphocytes cultured in RPMI 1640 supplemented with 50% autologous serum plus concanavalin A (Con A) or phytohaemagglutinin (PHA). When washed, lymphocytes were cultured in RPMI 1640 supplemented with 5% foetal calf serum (FCS) or 5% FCS plus Con A or PHA, decreased blastogenesis was observed but blastogenesis depression was not as marked as that observed with autologous serum. Similar antibody responses were seen for the drenched and control groups in response to the two injections of both antigens except that after the second injection, there was a significant reduction in antibody response to ovalbumin in the ivermectin-treated lambs. There were no differences in serum complement or serum nitric oxide levels between the two groups at any stage, but insulin-like growth factor-1 levels were significantly reduced in serum of the ivermectin-treated group, 4 days after each drench. Growth hormone levels were consistently significantly higher 22 days after both drenchings. There was no difference in mean body weight increase between the groups during the experiment.

Levamisole and its influence on the immune response of lambs.

Ten parasite-free lambs were drenched with 8 mg/kg of levamisole on days 0 and 28 and were injected with human erythrocytes and ovalbumin one day after each drench. Ten other antigen-injected lambs were not drenched with anthelmintic as controls. Lymphocytes from the control and drenched lambs were cultured in vitro with RPMI 1640 plus 5% fetal calf serum (FCS), with 50% autologous serum only, with concanavalin A (Con A) or with phytohaemagglutinin (PHA). Decreased blastogenesis was observed in cells from the drenched lambs cultured in the presence or absence of mitogen and was most obvious when 50% autologous serum was used, particularly with PHA, and when lymphocytes were collected 3 and 7 days after the first and 3 days after the second antigen injection. There were no significant changes in antibody titres between the groups. Decreased serum complement activity was seen 3 days after the second antigen injection in the drenched lambs. Although there was a significant reduction in the serum insulin-like growth factor I levels 4 days after each levamisole drench, the drenched lambs gained significantly more weight than the non-drenched control lambs.

Fenbendazole and its effect on the immune system of the sheep.

Ten parasite-free 6-month-old lambs were drenched on days 0 and 28 with fenbendazole and 1 day after each drench were injected with human erythrocytes and ovalbumin. Ten other lambs injected with the antigens were not drenched with anthelmintic and served as controls. Lymphocytes from the fenbendazole-drenched lambs collected 3 days after the first antigen injections and cultured in vitro in RPM1 1640 plus 5% foetal calf serum, and lymphocytes collected at 3 and 7 days and cultured in RPM1 plus 50% autologous serum, had decreased blastogenic activity compared with lymphocytes from control lambs. Similarly, decreased blastogenesis was observed with lymphocytes collected 7 days after the second antigen injections from drenched lambs and cultured in 50% autologous serum containing concanavalin A. In contrast, increased blastogenesis was seen with lymphocytes collected 14 days after the second antigen injections from the drenched lambs and cultured in 50% autologous serum containing phytohaemagglutinin. Similar antibody responses were seen for the drenched and control lambs in response to the injections of both antigens except that, after the second injection, there was a significant reduction in antibody response to human erythrocytes in the fenbendazole-treated lambs. Decreased serum complement levels were seen particularly 3 and 7 days after the second antigen injections in drenched lambs. These serum samples had increased conglutinin activity. At the end of the experiment, the fenbendazole-drenched lambs were significantly heavier than the control lambs. However, this did not appear to be related to any effects of fenbendazole on levels of growth promoting hormones.

Sheep small intestinal mast cells/globule leucocytes: ability to divide.

The number of mucosal mast cells/globule leukocytes (MMC/GLs) increase in the intestinal mucosa in response to nematode parasite infections but it is not known if this accumulation is due to in situ cell division, derivation from elsewhere or some combination of both. To determine if MMC/GLs can divide, cells were obtained from immunized Romney sheep and cultured in vitro in RPMI 1640. For cultures supplemented with 10, 20 or 30% foetal lamb serum (FLS) or foetal calf serum (FCS) and without concanavalin A (Con A), cell division had ceased by day 2, but with Con A (3 micrograms ml-1) cell division continued to day 9. Better growth of cells was obtained with the higher concentrations of serum. However the use of 30% or 50% autologous serum with Con A lead to cell death but 10% serum permitted limited growth. The detrimental effect of autologous serum could be overcome by increasing the Con A concentration. It was established that an alpha-macroglobulin present in autologous serum can bind Con A. This macroglobulin appears to have a higher avidity for Con A than does the receptor(s) on the surface of MMC/GLs. Our data seem to indicate that a direct interaction of Con A with the cell surface or a receptor(s) is responsible for MMC/GLs division.

A technique for the isolation and purification of viable mucosal mast cells/globule leukocytes from the small intestine of parasitised sheep.

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.

Oxfendazole treatment of non-parasitized lambs and its effect on the immune system.

Ten parasite-free lambs were drenched with oxfendazole on days 0 and 28 and, one day after each drench, were injected with human erythrocytes and ovalbumin. Ten other antigen-injected lambs were not drenched (controls). Lymphocytes collected 3 days after each antigen injection and cultured in RPMI 1640 plus 5% fetal calf serum (FCS) and lymphocytes collected 3 days after the first and 3 and 7 days after the second antigen injection and cultured in 50% autologous serum had decreased blastogenic activity compared with control lymphocytes. After the second drench, decreased blastogenesis was seen with lymphocytes collected on days 3 and 7 and cultured in 5% FCS and concanavalin A (Con A) and on day 3 when cultured in 5% FCS and phytohaemagglutinin (PHA). Decreased blastogenesis was also seen with lymphocytes collected 7 and 29 days after the second injection of antigen and cultured in 50% autologous serum plus Con A and on days 3, 7 and 29 when cultured in 50% autologous serum and PHA. Significantly depressed antibody responses to both antigens were seen after the second drench. The serum complement level was depressed 3 days after the second injection of antigen. Serum nitric oxide levels were significantly depressed 3 and 21 days after the first and 7 and 21 days after the second injection of antigen. There were no differences in levels of growth-promoting hormones but the drenched lambs gained significantly more weight than the controls.

Globule leukocytes in the lumen of the small intestine and the resistance status of sheep infected with parasitic nematodes.

The presence of globule leukocytes in the lumen (LuGLs) of the small intestine was studied in Romney sheep reared parasite free and then experimentally infected or immunized with Trichostrongylus colubriformis infective larvae, or naturally infected when gazed on pasture. It was discovered that high numbers of LuGLs were associated with both parasitic infection and the sheep protective immune response. A high positive correlation was observed between LuGLs and tissue globule leukocytes. The highest correlation (r = 0.92) observed was between larval migration inhibition (LMI) and numbers of LuGLs. This was the only correlation between LMI and other parameters studied. Significant positive correlations were also observed between LuGLs and IgG1 and eosinophils. There was a negative correlation between LuGLs and the number of parasites in the intestine and parasite egg production. Progeny of genetically resistant and susceptible sires had significantly different abilities to produce LuGLs.

Plasma and organ counts of 3H in normal or immune mice and sheep injected with 3H-epsilon-dinitrophenyl-lysine.

Mice and sheep with circulating antibody to dinitrophenol (Dnp) retained injected 3H-epsilon-Dnp-lysine in their circulation for three and seven days respectively. In normal mice and sheep the 1 h post injection counts of 3H/g of kidney were higher than those per ml of plasma. In immune mice, plasma 3H counts were always higher than kidney counts, whereas at 1 h in immune sheep, plasma and kidney counts were similar. Liver, spleen and lung counts were also made in some experiments. Increased 3h counts could be demonstrated in mice treated passively with sheep anti-Dnp serum and then given 3h-epsilon-Dnp-lysine. The fast gamma-globulin of sheep antiserum is mainly responsible for retention of hapten in mouse plasma.

Antibody-hapten interactions in vivo in mice and sheep.

From 5 min to 5 h after an intravenous injection of one of the haptens, elipson-dinitrophenyl-lysine, 2,4-dinitrophenol (DNP), or procaine, mice that were actively immunised against these haptens held more of the hapten in their plasma than did normal mice. Over the same time interval, mice that had been passively immunised with sheep anti-procaine antisera and then treated with procaine held more procaine in their plasma than did mice treated with normal sheep serum. When procaine or DNP was administered orally or intraperitoneally to sheep with circulating antibody to the hapten, the antibody titre was usually reduced 1 h after dosing but returned to the pre-dosing titre by 24 h. Experiments indicated that the reduction in antibody titre was due to in vivo neutralisation of antibody by the hapten.