W. Cabaj

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.

Drug-abbreviated infections and development of immunity against Trichostrongylus colubriformis in sheep

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.

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)

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.

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.

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 culturedin 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.

Supravital staining of eosinophils

Live eosinophils when mixed with Acridine Orange solution and viewed microscopically using u.v. light show very intense colours of their granules (yellow, orange and red) and green nuclear staining. Their active movement, translocation of granules and degranulation can be observed in vitro. Using this method, live eosinophils can be easily differentiated and enumerated.

Immunisation of sheep by drug-abbreviated infections of Ostertagia circumcincta and Trichostrongylus colubriformis against field challenge of gastro-intestinal nematodes

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.

Immune responsiveness of Romney sheep selected for resistance or susceptibility to gastrointestinal nematodes: field studies

Long-term selection of sheep for resistance to parasite infections may be jeopardized if animals do not retain their normal ability to respond to non-parasite antigens. Therefore the antibody responses to ovalbumin (OVA) and human red blood cells (HRBC), and kinetics of peripheral blood lymphocyte phenotypes were examined in mature grazing sheep, genetically resistant or susceptible to gastrointestinal nematodes. In both lines the HRBC antibody response peaked 2 weeks after the primary injection, 1 week after the second injection and 3 weeks after the second OVA injection. The antibody titres of the resistant line sheep decreased sooner after both primary and secondary injections. The resistant line sheep had higher percentages of CD5+ and CD4+ cells than the susceptible sheep. Two injections of OVA and HRBC did not result in significant alterations in percentages of CD5+, CD4+, CD8+ and CD45R+ lymphocytes in either line. In both lines, the control groups showed a steady increase of 0.29% per week in percentages of T19+ (gamma delta) T cells which was significantly higher than in the antigen injected sheep.

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.