Effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows

We examined combination therapy with both lactoferrin (Lf) and antibiotics on clinical mastitis due to Staphylococcus aureus (S.aureus) on drying cows. The clinical symptoms of mastitic quarters were cured 81% of combination therapeutic quarters at 7 days post injection (dpi). Moreover, most of mammary gland secretions (MGSs) in combination therapeutic quarters were normal at 7 days after parturition. In the quarters with combination therapy, S.aureus counts, Lf concentrations and content rate of concanavalin A (Con A) low-affinity Lf decreased and were lower than in the quarters treated with Lf or antibiotics alone. The mRNA expression of tumor necrosis factor alpha (TNFalpha) of the quarters with combination therapy also decreased and was lower than that of the Lf or antibiotics treated. The mRNA expression of pro-inflammatory cytokines and chemokines in bovine mammary gland epithelial lined cells (BMEC) stimulated with Lf were lower than those of Con A low-affinity Lf stimulated BMEC. Moreover, Lf showed an inhibitory effect to the induction of pro-inflammatory cytokine mRNA expression when co-stimulated with Lf and Con A low-affinity Lf. Nuclear factor kappa B (NFkappaB) activation was also induced with Con A low-affinity Lf, and the inhibitory effects of Lf were also confirmed on BMEC co-stimulated with Lf and Con A low-affinity Lf. These results indicated that the efficacy of combination therapy with antibiotics and Lf caused antibacterial effect of antibiotics and inhibition of pro-inflammatory cytokine and chemokine production with Lf via the inhibition of NFkappaB activation.     

Small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.     

Inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.     

Lactoferrin concentrations in milk from normal and subclinical mastitic cows

The concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens. Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarter milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test. Lf concentrations (means +/- standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 +/- 0.39 and 2.70 +/- 0.39, respectively. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows. The mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05). The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01). The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods. Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score. Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis.     

Bacteriostatic activity of bovine lactoferrin in mastitic milk

The antibacterial activity of milk against a virulent strain of Escherichia coli was investigated using milk fractions from normal or inflamed glands. Mastitic whey exhibited either bactericidal or bacteriostatic activities, depending on whether bacteria were enumerated by the pour plate technique or by surface plating onto sheep blood agar. The former activity was not due to lactoferrin (Lf), which never exerted bactericidal activity, even when assayed in distilled water. Milk whey ultrafiltrate (UF) (mol. wt. less than 5000 d) was used to assay the ability of normal and mastitic milk to support the antibacterial activities of Lf against a strain of E. coli. The addition of purified Lf to UF from mastitic whey resulted in bacteriostasis, whereas Lf was without effect in UF from normal whey. It was concluded that Lf can actually slow down the growth of Lf-sensitive bacteria during mastitis, provided that plasma exudation takes place.     

Concentrations of IL-6 in Serum and Whey from Healthy and Mastitic Cows

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.     

Disposition kinetics of lactoferrin in milk after intramammary administration

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half-life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty-eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.     

Lactoperoxidase, thiocyanate, and free cystine in bovine mammary secretions in early dry period and at the start of lactation and their effect on Streptococcus agalactiae growth.

The concentrations of lactoperoxidase (LP) and thiocyanate (SCN-) in the mammary secretions of 4 dairy cows in the early dry period were similar to or higher than concentrations in the milk before drying off. The concentrations of free cystine progressively increased in the secretions beginning 3 to 5 days after the last milking; the mean concentrations were 0.66 mumoles/L before drying off and 6.66 mumoles/L after drying off. The mean concentrations of free cysteine were 0.28 mumoles/L before drying off and 1.4 mumoles/L after drying off. The secretions, when diluted in steamed milk, showed greater stimulation of Streptococcus agalactiae growth as the drying-off period progressed. This increase in stimulatory activity was attributed primarily to the increased concentrations of cystine because cystine counteracts the LP/SCN-/hydrogen peroxide inhibitory system for S agalactiae. This effect on the LP system may account for any increase in susceptibility to S agalactiae under infection during the dry period. In 3 other cows, the mammary secretions on the day of calving had lower mean concentrations of LP, SCN-, and free cystine and cysteine than those obtained 4 to 5 days before, and 7 to 8 days after calving.     

Zinc concentrations in skimmed milk and whole milk samples from healthy and mastitic cows

Zinc concentrations were determined in skimmed milk and whole milk samples from healthy and mastitic cows. During spontaneous and induced mastitis, a marked decrease of Zn concentration in skimmed milk from the mastitic quarters was observed. Seemingly, there are 2 mechanisms involved in the lowering of Zn concentration in skimmed milk from mastitic quarters: a decline in Zn probably as a consequence of decreases in plasma Zn concentrations and an abrupt redistribution of Zn in the inflamed quarters.     

Outcome prediction from the first examination in clinical mastitis using ultrasonography in dairy cows

The aim of this study was to examine whether ultrasonography can be used to predict the outcome of clinical mastitis in dairy cows. Forty‐seven mastitic quarters of Holstein‐Friesian cows were examined using ultrasonography at the time of the first examination. In mastitic mammary tissue, three sonographic signs indicating tissue abnormality were found: a hyperechoic spot in the parenchyma area, structural changes to the milk duct, and non‐homogeneous parenchyma. Logistic regression was used to evaluate whether the abnormal findings in the sonographic images can be used to predict the outcome of clinical mastitis. The outcomes of clinical mastitis were defined by the return, or failure to return, to marketable milk production. The sonogram finding of non‐homogeneous parenchyma in the first examination did predict the outcome of clinical mastitis, whereas the type of systemic symptoms (severe or moderate) was not a predictor in this regression model. Therefore, ultrasound examinations of mammary glands in the first examination could be a useful method for predicting outcome of clinical mastitis. There is an economic benefit if ultrasound examination in first examination helps in the decision of whether or not to treat the mastitic cows.     

Phmacodynamics and pharmacokinetics of carprofen, a non-steroidal anti-inflammatory drug, in healthy cows and cows with Escherichia coli endotoxin-induced mastitis

The pharmacodynamics of carprofen and its pharmacokinetics in plasma and milk of healthy cows and cows with endotoxin-induced mastitis were studied after a single intravenous dose of 0.7 mg/kg body weight. Carprofen was administered to five clinically healthy cows and to the same cows 3 weeks later, 2 h after intramammary infusion of endotoxin. Mastitis developed in all endotoxin-infused quarters. The pharmacokinetic characteristics of carprofen in healthy cows were a small volume of distribution (0.09 l/kg), a relatively low systemic clearance (2.4 ml/h kg), and a long elimination half-life (30.7 h). In the mastitic cows, systemic clearance (1.4 ml/h kg) was significantly lower (P less than 0.01), and elimination half-life (43.0 h) was significantly longer (P less than 0.01) than in the normal animals. Concentrations of carprofen in milk from healthy quarters were below the limit of detection for the assay (0.022 micrograms/ml). In milk from mastitic quarters, concentrations of carprofen increased up to 0.164 micrograms/ml during the first 12 h after induction of mastitis, but were less than 0.022 micrograms/ml at 24 to 48 h. Compared with the untreated mastitic controls, carprofen treatment significantly reduced heart rate (P less than 0.01), rectal temperature (P less than 0.001), quarter swelling (P less than 0.01) and other parameters measured. Local and systemic adverse reactions to carprofen were not observed.     

Changes in subpopulations of lymphocytes in peripheral blood, and supramammary and prescapular lymph nodes of cows with mastitis and normal cows

Direct immunofluorescence (IF) and indirect IF techniques were employed to analyze the distribution of B and T lymphocyte populations in peripheral blood, and in supramammary (draining), and prescapular (non-draining) lymph nodes of cows with mastitis and normal cows. In the peripheral blood there was a significant decrease in the percent and absolute number of B lymphocytes in mastitic cows (n = 29; 17.1 +/- 10.2%; 3.4 +/- 2.7 X 10(5) cells/ml) as compared to normal cows (n = 38; 25.2 +/- 7.8%; 9.3 +/- 5.4 X 10(5) cells/ml). The percent T lymphocyte count in mastitic cows (71.2 +/- 7.1%) was slightly increased over that of normals (65.8 +/- 7.2%), although the absolute number of T lymphocytes was decreased in mastitic cows (1.49 +/- 0.91 X 10(6) cells/ml vs. 2.47 +/- 1.28 X 10(6) cells/ml). In the prescapular lymph node the percent of B lymphocytes, but not T or "null lymphocytes", decreased significantly in mastitic cows as compared to that of normals. The decrease, i.e. 32%, paralleled the 32.1% decrease found in peripheral blood B lymphocytes. In contrast, in the supramammary lymph node of mastitic cows, the percent B lymphocytes increased over that of normals (35.1 +/- 2.0% vs. 20.4 +/- 9.4%), whereas the percent T lymphocytes decreased to 54.5 +/- 2.8% compared to 70.7 +/- 3.5% in normal cows. There was no significant change in percent "null lymphocytes". The weight of prescapular lymph nodes did not change in mastitic cows when compared to that of normals. As a result, the estimated number of B lymphocytes, but not of T and "null lymphocytes", decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a Brown‐midrib corn hybrid on nutrient digestibility in wethers and on dry matter intake,performance, rumen and blood variables in dairy cows

The aim of the present trials was to determine the effect of an experimental Brown‐midrib (Bm) corn hybrid in relation to a commercial corn hybrid (Con) on digestibility in wethers and on dry matter intake (DMI), milk yield and milk composition in dairy cows. Digestibility of crude fibre (CF), neutral detergent fibre (NDFom) and acid detergent fibre (ADFom) were higher for Bm (CF Con: 57.8%; Bm: 67.2%; NDFom Con: 56.8%; Bm: 64.8%; ADFom Con: 52.0%; Bm: 63.9%), but concentration of net energy for lactation did not differ (Con: 6.4 MJ/kg DM; Bm: 6.3 MJ/kg DM). A total of 64 lactating German Holstein cows were assigned to one of the two dietary treatments Con or Bm according to milk yield, lactation number, days in milk and live weight. In Trial 1, cows were fed a total mixed ration consisting of 50% corn silage (Con or Bm) and 50% concentrate on dry matter (DM) basis. In Trial 2, the same animals were fed the respective silage for ad libitum intake and 5.3 kg of concentrate DM per animal per day. In Trial 1, DMI and milk‐fat content were decreased significantly for the Bm‐treatment (DMI Con: 22.5 kg/day; Bm: 21.5 kg/day; milk fat Con: 3.8%; Bm: 3.3%). In Trial 2, milk yield and fat‐corrected milk (FCM) were increased significantly, whereas milk‐fat% was decreased significantly (milk yield Con: 25.8 kg/day; Bm: 29.4 kg/day; FCM Con: 27.2 kg/day; Bm: 29.6 kg/day; fat Con: 4.4%; Bm: 4.0%). Diets did not influence ruminal pH or temperature. Diets, furthermore, did not influence rumination in either trial. Additional research on digestibility and rumen fermentation should, however, be carried out using dairy cows at respective intake levels as trials with wethers cannot be transferred to high‐yielding ad libitum fed cows.     

Subclinical,chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.     

Assessment of strategy in selective dry cow therapy for mastitis control.

Seven hundred and three cows were treated at drying off, using three main types of therapy: control, long-acting intramammaria and short-acting intramammaria. Selective dry cow therapy in infected quarters was used. Of the cows with one or two infected quarters one to six weeks before drying off, 57.7 per cent had one or more new infected quarters at drying off. Thus selective dry cow therapy on quarter basis determined from the results of samples taken one to six weeks before drying off would give "inadequate" therapy (i.e. new infection in non-treated quarters at drying off) for more than 50 per cent of the cows. For those cows given "adequate" therapy (no new infection at drying off) long-acting therapy, short-acting and control yielded 53.6%, 49.3% and 30.7% healthy cows respectively two to five weeks post partum. In cows with "inadequate" therapy the treatment groups yielded 42.9%, 52.9% and 29.7% healthy cows respectively. Our conclusion is that if long-acting intramammaria are to be used, all the quarters of infected cows should be treated, whereas if short-acting preparations are to be used, treatment can be restricted to infected quarters only.     

Interferon-gamma and tumor necrosis factor-alpha levels in sera and whey of cattle with naturally occurring coliform mastitis

Concentrations of interferon-gamma (lFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-gamma and TNF-alpha-specific ELISA. In serum and whey samples from healthy cows. IFN-gamma was almost undetectable and TNF-alpha was detected at low levels. At the onset of illness, INF-gamma in sera and whey and TNF-alpha in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-gamma and TNF-alpha in whey from mastitic cattle decreased significantly as the cows recovered.     

Persistence of detectable residues of penicillin and cloxacillin in normal and mastitic quarters following intramammary infusion

The excretion rates of sodium penicillin and sodium cloxacillin from treated and untreated quarters of normal and mastitic cows were studied. Penicillin was detected in normal and infected quarters for 72 hours after treatment. Cloxacillin was detected in normal and infected quarters for 64 and 48 hours, respectively, after treatment. Differences in the excretion rates of both antibiotics from normal and infected treated quarters were not significant. Penicillin was detected in the untreated quarters of both normal and mastitic cows but cloxacillin was only detected in the untreated quarters of mastitic cows.     

Elimination kinetics of chlorhexidine in milk following intramammary infusion to stop lactation in mastitic mammary gland quarters of cows

OBJECTIVE: To evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in cows. DESIGN: Prospective study. ANIMALS: 6 cows. PROCEDURE: The study was performed in 2 phases. Three cows were studied in each phase. All cows were treated with chlorhexidine suspension by infusion into a mastitic mammary gland quarter after 2 milkings 24 hours apart. Foremilk samples (100 mL) were collected from treated and untreated (controls) mammary gland quarters of each cow. Chlorhexidine was extracted from raw milk, and residue concentrations were quantified by use of high-performance liquid chromatography. Foremilk samples from days 2, 5, and 8 were analyzed in phase I, and samples from time 0 and days 3, 7, 14, 21, 28, 35, and 42 were analyzed in phase II. RESULTS: In phases I and II, there was no quantifiable transference of chlorhexidine to milk in untreated mammary gland quarters. Measurable chlorhexidine residues were found in milk from treated mammary gland quarters of 2 cows throughout the 42-day sample period in phase II. Estimated mean elimination half-life for chlorhexidine in milk was 11.5 days. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the long elimination half-life of chlorhexidine in milk from treated mammary gland quarters, the lack of human dietary exposure data to suggest a food tolerance for chlorhexidine in food products, and the Food and Drug Administration's published zero tolerance for chlorhexidine in uncooked edible calf tissues, we do not recommend extralabel use of chlorhexidine suspension as a treatment to stop lactation in mastitic mammary gland quarters of cows.     

Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk

To elucidate the occurrence of heat-stable toxin-producing strains among mastitic Bacillus isolates, 100 milk samples of mastitic cows from different parts of Finland were screened. Bacillus was identified as the major organism in 23 samples. Toxinogenic Bacillus isolates identified by sperm cell motility inhibition assay were isolated from six samples. Four isolates belonged to the species Bacillus pumilus and two to Bacillus licheniformis. The toxic substances were heat-stable and soluble to methanol thus being of non-protein nature. The methanol extracted substances disrupted the sperm cell plasma membrane permeability barrier at exposure concentrations of 1-15 microg ml(-1) (B. pumilus) or 20-30 microg ml(-1) (B. licheniformis). The toxic properties of the two mastitic B. licheniformis strains were similar to those of B. licheniformis strains known to produce the lipopeptide lichenysin A and the synthetase genes lchAA, lchAB and lchAC for lichenysin were found in the mastitic strains by PCR. Toxin synthetase genes for the syntheses of lichenysin or surfactin were searched but not found in the toxic B. pumilus strains. The ribopatterns of the mastitic B. pumilus and B. licheniformis isolates were similar to those of the toxinogenic strains described earlier from food poisoning incidents and contaminated indoor air. B. licheniformis and B. pumilus survive pasteurization and other heat treatments as spores. Toxin-producing strains of these species in the dairy production chain may thus be of food safety concern.     

ELISA for bovine interleukin-1 receptor antagonist and its application to mastitic sera and whey

A sandwich ELISA for the bovine IL-1 receptor antagonist (bIL-1ra) was developed using recombinant (r) bIL-1ra produced by Escherichia coli, anti-rbIL-1ra rabbit IgG, its biotinylated one and avidin-peroxidase. This ELISA system enabled detection of rbIL-1ra at a concentration of more than 2 ng/ml. This ELISA was applied to quantitation of bIL-1ra in sera and whey of mastitic and healthy cows. The results indicate that although IL-1ra levels in healthy and mastitic sera and whey were comparable, serum IL-1ra/IL-1beta ratio of euthanized cows was significantly lower than that of the recovered.