Effects of yeast expressed recombinant interleukin-2 and interferon-γ on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils

Abstract

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

Effect of recombinant cytokines on leucocytes and physiological changes in bovine mammary glands during early involution

“…A low dose of IL-2 was used in the current study because an association between intramammary infusion of IL-2 into glands during the dry period and an increased incidence of abortion can occur with higher doses of 1 mg (Erskine et al 1998). The low dose of rboIL-2 given to non-lactating cattle in the current study did not enhance bactericidal activity of mammary leucocytes isolated from the secretions, which contrasted with a previous finding that a similar dose of rboIL-2, administered by the intramammary route to lactating cows, significantly increased the ability of neutrophils to kill S. aureus (Wedlock et al 2000). The bactericidal assay in the current study measured activity of both neutrophils and macrophages as significant numbers of both these cells were present in dry secretions when the assay was conducted.…”

“…Doses of rboIL-2 and rboIL-1b used in these studies were based on the results of previous trials in lactating cattle. Cattle in lactation were infused with 2r10 5 units of rboIL-2 and this dose markedly enhanced bactericidal activity of neutrophils with only a transient elevation of SCC and no effect on milk yield (Wedlock et al 2000). In a separate experiment, lactating cattle were infused with either 10 or 50 mg of rboIL-1b.…”

“…5 M-dimethylformamide (BDH Laboratory Supplies, Poole DH15 1TD, UK) containing 200 g sodium dodecyl sulphate/l (BDH), and measuring the optical density at 560 nm. The percentage killing of S. aureus was determined from a standard formazan curve relating optical density to known concentrations of bacteria as described previously (Wedlock et al 2000). The S. aureus strain was isolated from a clinical case of bovine mastitis in New Zealand.…”

“…To reduce the possibility of adverse side-effects, a lower dose of rboIL-2 was used in this study than was used in other studies (Nickerson et al 1993;Rejman et al 1995;Erskine et al 1998). The dose of rboIL-2 used in the current trial has a therapeutic effect in lactating cows and is well tolerated (Wedlock et al 2000). The effect of administering recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF), a cytokine which has not been tested in nonlactating cows, was determined.…”

Vaccination of Cattle withMycobacterium bovisCulture Filtrate Proteins and Interleukin-2 for Protection against Bovine Tuberculosis

“…Bovine IL-2 was prepared in the yeast Pichia pastoris as described previously (33) and had a specific activity of 2,500 U/g of protein. Briefly, the cDNA encoding the mature IL-2 (amino acids 21 to 155) was isolated by reverse transcription-PCR of RNA prepared from concanavalin A (ConA)-stimulated bovine lymphocytes.…”