Sheep were immunized by multiple truncated infections with the gastrointestinal nematodes Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. Three infections with T. colubriformis of 14 days plus five booster doses of L3 stimulated highly effective protection against challenge (99%). Three infections of 14 days plus three booster doses with H. contortus also resulted in significant protection against challenge infection (87%), but the same procedure was not effective for T. circumcincta. Antibodies derived from gastrointestinal mucus of these immunized sheep were tested for their ability to reduce worm burden following injection of antibody-coated exsheathed larvae into the abomasum (H. contortus and T. circumcincta) or duodenum (T. colubriformis) of nematode-naïve sheep in a passive immunity test. The IgG fraction from the mucus of immunized sheep reduced worm burdens by 62%, 76% and 91% in three tests with T. colubriformis but was not effective for either of the abomasal dwelling nematodes H. contortus and T. circumcincta. Antibodies in immune mucus predominantly recognized two larval surface antigens on immunoblots of L3 extract, a high MW surface glycoprotein and the carbohydrate larval antigen (CarLA). Antibodies raised against purified T. colubriformis glycoprotein Tc-120 and CarLA were tested in the passive immunity model and it was found that only the antibody against CarLA resulted in a significant reduction of infection (87%). The protective anti-CarLA antibodies strongly recognized the surface of living T. colubriformis L3. Antibodies from abomasal mucus of sheep immunized by H. contortus and T. circumcincta infections reacted weakly with CarLA and the larval surface and did not reduce worm counts in a passive immunity test. The results provide further evidence that the larval surface carbohydrate antigen CarLA has potential as a mucosal immunogen for a strongylid nematode vaccine.

We examined the effects of administering recombinant bovine cytokines to non-lactating dairy cows and measured mammary gland leucocytes and the involution process. After the final milking, groups of cows were given an intramammary infusion of cytokine in two quarters. These cytokines were recombinant bovine interleukin-2 (rboIL-2) (2×105 units, n=6), recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF) (500 μg, n=4) and recombinant bovine interleukin-1β (rboIL-1β) (10 μg, n=10). Each animal also received an infusion of phosphate-buffered saline (PBS) in the other two quarters as controls. The rboIL-2 and rboGM-CSF were produced in a yeast expression system, while rboIL-1β was produced in Escherichia coli. Leucocyte numbers, bactericidal activity of leucocytes, and concentrations of citrate and lactoferrin in quarter secretion samples were monitored after infusion of cytokine or PBS. Infusion of rboIL-2 had minimal effect on leucocyte numbers and concentrations of citrate and lactoferrin. Both rboGM-CSF and rboIL-1β induced a rapid increase in the number of neutrophils and macrophages compared with control PBS quarters. Concentrations of lactoferrin in secretions were increased by rboGM-CSF and rboIL-1β compared with control PBS quarters. In addition, infusion of glands with rboIL-1β lowered the citrate[ratio ]lactoferrin molar ratio compared with PBS control quarters. The results indicate that intramammary infusion of either rboGM-CSF or rboIL-1β at cessation of milking immediately increased the number of phagocytic cells in the gland. These cytokines, in particular rboIL-1β, also increased the rate of mammary gland involution during the early dry period.

S. The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-γ (rboIFN-γ) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2i10& units) in two quarters, two animals were given rboIFN-γ (6n5i10& units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-γ in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-γ showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63n5% compared with a mean of 5n4 % for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-γ and infected quarters treated with PBS were 15n0 and 30n0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-γ to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.Bovine mastitis affects an estimated 20 % of New Zealand dairy cows and causes an enormous cost to the dairy industry from loss of milk solids, quality and yield of product. The annual loss to dairy herd owners has been estimated at $14 000\herd (Woolford, 1997). Mastitis is primarily due to the infectious animal pathogen, Staphylococcus aureus, or the environmental microorganism, Streptococcus uberis. During lactation, new infections are usually due to Staph. aureus, while new

Summary Vaccination strategies for the brushtail possum, which rely upon stimulation of mucosal immunity, are being developed for biocontrol purposes. As little is known about how to stimulate possum immune responses via a mucosal site, groups of possums were immunized intranasally with keyhole limpet haemocyanin (KLH) alone or in combination with known or novel mucosal adjuvants. Antigen-specific antibody titres in female reproductive secretions were measured by ELISA and compared with antibody titres in the serum. Antigen-induced lymphocyte proliferative responses were measured as an indicator of cell-mediated responses. Intranasal immunization with KLH alone stimulated a weak serum antibody response that was significantly increased when KLH was given with cholera toxin subunit B (CTB), recombinant possum tumour necrosis factor α (TNFα) or live Mycobacterium bovis bacillus Calmette Guerin (BCG). Antibody titres in secretions from ovarian follicles and the uterus were very low in animals administered KLH alone. Significantly higher antibody titres to KLH were present in the reproductive secretions of possums immunized with KLH plus CTB, BCG or heat-killed Mycobacterium vaccae. Antibody titres were lower in mucosal secretions than in the serum, but there was a significant correlation between the two. In addition, coadministration of live BCG with KLH produced a strong antigen-specific cell-mediated response to KLH. This study has shown that an immune response to a protein antigen can be stimulated in possums by intranasal immunization and that antigen-specific antibodies can be detected in secretions from the female reproductive tract.