Sequential response of milk leukocytes, albumin, immunoglobulins, monovalent ions, citrate, and lactose in cows given infusions of Escherichia coli endotoxin into the mammary gland

Changes in concentrations of both the cellular and the humoral components of milk are known to occur during mastitis. This study was conducted to determine temporal changes in the concentrations of leukocytes, albumin, immunoglobulins (Ig), monovalent ions, lactose, and citrate in milk during the initial phases of simulated mastitis. Ten cows whose udders were pathogen free and had milk leukocyte counts of less than 0.5 X 10(6)/ml were used. Two dosages of Escherichia coli endotoxin were administered to simulate various degrees of mastitis. Two quarters in each cow were infused with the endotoxin and the other 2 served as controls. Quarter milk samples were collected frequently before and after infusion. Within 2 hours after infusion of a 100-micrograms dose of endotoxin, clinical mastitis was observed in most of the infused quarters. Leukocytes, albumin, IgG1, and conductivity showed significant increases. Values before infusion and at postinfusion (PI) hour 2 were as follows: leukocytes, 0.33 and 3.65 X 10(6)/ml, respectively; albumin, 0.38 and 4.49 mg/ml; IgG1, 0.34 and 0.79 mg/ml; and conductivity, 6.0 and 6.9 mmho. Average of the peak values and their average relative time of appearance after infusion were as follows: leukocytes, 28.82 X 10(6)/ml at 16 hours; albumin, 9.37 mg/ml at 4 hours; IgG1, 1.35 mg/ml at 4 hours; and conductivity, 95.5 mmho at 10 hours. The IgG1 values tended to remain high in the presence of rapidly declining albumin concentrations, indicating the possibility of an active, rather than a passive, transfer of IgG1 from the circulation. The response to the 10-micrograms dose of endotoxin ranged from subclinical to clinically mild mastitis with lesser cellular and humoral responses.     

Measurement of phagocytosis of 32P-labeled Staphylococcus aureus by bovine leukocytes: lysostaphin digestion and inhibitory effect of cream.

A procedure to measure phagocytosis by blood and milk neutrophils was developed. One milliliter of heat-killed 32P-labeled Staphylococcus aureus ([32P]SA) (180-200 X 10(6) CFU), 1 ml of phosphate-buffered saline solution (PBSS), and 2 ml of serum, whole milk, skimmed milk, whey, or PBSS were incubated in duplicate for 60 minutes at 37 C. Isolated blood or milk nuetrophils (polymorphonuclear leukocytes (PMN), 25 X 10(6) cells/ml; 1 ml) were added and incubated at 37 C for 30 minutes. Unphagocytosed [32P]SA organisms were lysed by incubation with 5 ml of lysostaphin (10 U) at 37 C for 30 minutes, and the PMN and phagocytosed T2P]SA were removed by centrifugation. Radioactivity of the supernatant was determined in a scintillation spectrometer and was used in estimate the percentage of [32P]SA phogocytosed. With this procedure, 25 assays in duplicate could be conducted each day with an expected coefficient of variation between duplicates of 5.6%. Blood PMN phagocytosed 80, 44, 74, 72, and 11% of the [32P]SA when incubated in serum, whole milk, skimmed milk, whey, and PBSS, respectively. Mik PMN phagocytosed 78, 44, 72, 74, and 22%, respectively. The addition of cream to either skimmed milk or serum reduced phagocytosis of [32P]SA by both blood and milk PMN. The inhibitory effect of cream was verified by the microscopic observation that PMN containing large quantities of ingested fat contained fewer S aureus. Seemingly, PMN upon entering the alveoli of the mammary gland become less efficiently phagocytic for bacteria, because of the presence of milk fat globules. This phef intramammary infection by invading mastitic pathogens.     

Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis

Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli. Cows were sampled 0, 2 and 4h after infusion (p.i.). Neutrophils appeared in afferent lymph 2h p.i., and in efferent lymph and milk 4h p.i. The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4h p.i. IL-8 was also found in lymph, but at lower concentrations. The tumor necrosis factor-alpha (TNF-alpha) concentration tended to increase in afferent lymph at 2h p.i., and increased in milk at 4h p.i. The level of IL-1beta increased at 4h p.i. in milk, but was not detected in lymph. Interferon-gamma was not detected in any sample, at any time. The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1beta and TNF-alpha are not necessary for IL-8 production and release in response to endotoxin.     

T-helper cells from naive to committed

An endotoxin-induced mastitis model was used to study permeability changes associated with increased milk matrix metalloproteinase (MMP) activity in early inflammation. One quarter of two cows was inoculated with endotoxin (Escherichia coli 055:B5). Blood, milk, and whey were collected before and repeatedly after inoculation for 48 h. The profile and amounts of gelatinolytic MMPs were determined by zymography; gelatinase A (72 kD MMP-2) and gelatinase B (92 kD MMP-9) were identified by Western immunoblotting. Bovine serum albumin (BSA) and trypsin inhibitor capacity (TIC) were used as markers of capillary permeability with parallel examination of neutrophil penetration from blood to milk. Five clinical E. coli mastitis milk samples and five milk samples from cows with healthy udders were analyzed to detect whether increased levels of gelatinolytic MMP-2 and MMP-9 have a role in naturally occurring mastitis with endotoxin involvement. Milk MMP levels increased 2h after the endotoxin challenge. Both MMP-2 and MMP-9 were involved in this early proteolytic event. These increased MMP levels are associated with increased capillary permeability, evidenced first by the penetration of small molecular weight proteins, such as BSA and TIC. Later, 6-12h post endotoxin inoculation, neutrophilic leucocytes also entered the site, as they require larger tissue damage in basal membrane and interstitial tissue structures than BSA and TIC to extravasate. In naturally occurring disease, increased MMP-2 and MMP-9 levels were detected in milk. Thus, gelatinases, especially MMP-2, are involved in the early degradation of the blood-milk barrier, which precedes the penetration of blood-derived cellular components into milk in endotoxin-induced mastitis. In the future, measuring MMP in milk/whey might be a useful tool for diagnosing early mastitis.     

Efficacy of flunixin meglumine for the treatment of endotoxin-induced bovine mastitis

The clinical effect of flunixin meglumine administration was determined in cows with acute mastitis induced by intramammary administration of endotoxin. In 12 lactating cows, 10 micrograms of Escherichia coli 026:B6 endotoxin were administered via a teat cannula into the teat cistern of single randomly selected rear quarters. Cows were challenge exposed as pairs. One cow in each pair was administered parenteral flunixin meglumine (6 cows) and 1 cow per pair was administered saline solution (6 cows). Multiple doses (7) of 1.1 mg of flunixin meglumine/kg of body weight or saline solution were administered at 8-hour intervals beginning 2 hours after endotoxin. Cow and quarter clinical signs as well as milk somatic cell concentrations, bovine serum albumin, electrical conductivity, and milk production were determined before and for 14 days after endotoxin inoculation. Intramammary endotoxin produced signs characteristic of acute coliform mastitis. Quarter and systemic abnormalities occurred and milk production was reduced by approximately 50% at 12 hours after endotoxin. Flunixin meglumine therapy significantly (P less than or equal to 0.05) reduced rectal temperatures and quarter signs of inflammation and improved clinically graded depression when compared with these signs in saline solution-treated controls. Milk production and laboratory indicators of inflammation in milk were not significantly (P greater than 0.05) different for flunixin meglumine vs saline solution controls. The clinical response observed was consistent with the antipyretic, analgesic, and anti-inflammatory properties of flunixin meglumine.     

共轭亚油酸对奶牛乳脂肪球粒径及分布的影响

旨在探索共轭亚油酸（CLA）对奶牛乳脂肪球粒径（MFG）及分布的影响。本试验选取24头体况相近的泌乳中期荷斯坦奶牛（体重（583±34.6）kg,产奶量（27.2±2.4）kg·d^-1）,分为4组,每组6个重复,每个重复1头牛。对照组（C组）饲喂基础日粮,低剂量组（L组）饲喂基础日粮+150 g·d^-1 CLA,中剂量组（M组）饲喂基础日粮+300 g·d^-1 CLA,高剂量（H组）饲喂基础日粮+400 g·d^-1 CLA。连续饲喂5 d,测量奶牛采食量、产奶量和乳成分,采用Mastersizer 3000激光粒度仪测量乳脂肪球颗粒平均直径以及比例分布。结果发现,饲喂CLA对奶牛采食量、产奶量、乳蛋白和乳糖含量没有显著影响（P>0.05）,CLA处理极显著降低了乳脂肪的含量（P<0.01）。与对照组相比,CLA处理组牛乳中脂肪球粒径D_[3,2]和D_[4,3]显著减少（P<0.05）,并且随CLA添加剂量的增加,脂肪球分布比例呈现小脂肪球增多而大脂肪球减少的变化趋势。综上,在本试验条件下,饲粮CLA水平能够改变奶牛乳脂肪球粒径大小和比例,为阐明反式脂肪酸CLA造成奶牛低脂乳的内在机制提供基础。     

A pilot study to determine the production and health benefits of milking visibly lame cows twice daily compared with three times daily

A randomized clinical trial was conducted on lame cows to study the effect of milking frequency on milk production, lameness prevalence, and body condition score (BCS). At the beginning of the study, the entire herd of lactating Holstein dairy cows was visual locomotion scored (VLS) by 2 trained veterinarians. Lame cows (VLS > 2) were eligible for the study. The initial study population consisted of 270 cows randomly allocated to the three-times-daily milking frequency group (MFG) and 230 cows randomly allocated to the twice-daily MFG. Milking frequencies did not significantly affect average milk production. Cows in the twice-daily MFG had a significant increase in BCS, however, compared with cows in the three-times-daily MFG (P-value < 0.001). In addition, the probability of lameness in cows in the three-times-daily MFG was 36% higher than for cows in the twice-daily milking routine (P-value = 0.006).     

共轭亚油酸诱导的奶牛低脂乳症中脂肪球膜甘油磷脂的组成差异分析

旨在分析共轭亚油酸(CLA)诱导奶牛低脂乳症对牛乳脂肪球膜甘油磷脂含量的影响.选取体况相近、泌乳中期的10头荷斯坦奶牛进行自身前后对照设计,试验期12 d,第1天至第6天每头牛饲喂基础日粮+CLA 400 g·d-1,第7天至第12天恢复为饲喂基础日粮,每天记录产奶量及采食量,分析乳成分及乳脂肪球(MFG)粒径参数,对...     

Sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood

A study was conducted to examine sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood, including variation attributable to isolation of neutrophils from blood, variation between duplicate determinations of percentage phagocytosis, and the variation in the ability of neutrophils isolated from blood (over repeated collections from the jugular vein) to phagocytose. For the phagocytosis assay, jugular venous blood from each of 4 cows was divided into 2 equal portions. The neutrophils were isolated by lysis of red blood cells with 0.2% sodium chloride. The neutrophils (2 x 10(7)) were incubated in duplicate with 32P-labeled Staphylococcus aureus ([32P]SA; 2 x 10(8)) in skimmed milk samples (2.5% final concentration) prepared from 4 cows. This process was repeated thrice on neutrophils isolated from 4 cows at 2-week intervals. The proportions of variation in percentage of 32P-labeled S aureus phagocytosed between duplicate neutrophil isolations and between duplicate assay determinations were 0 and 1%. Differences among skimmed milk sources and among runs, using blood neutrophils taken at different times from the same donor cow, accounted for 62 and 36% of the total variation. The results indicated that variation arising from blood neutrophil isolation introduced into a phagocytosis assay within a single-day trial is of no concern. The large variation among skimmed milk sample sources indicated differences among cows in the ability of their milk to support phagocytosis. The variation in neutrophil isolations over time for any cow was considered too large to allow for evaluation of physiologic and environmental effects on phagocytosis of neutrophils isolated from blood.     

Effects of the perfusion of beta-, beta2-, or beta3-adrenergic agonists or epinephrine on in situ adipose tissue lipolysis measured by microdialysis in underfed ewes

The effects of isoproterenol (ISO, a non-selective beta-agonist), terbutaline (TER, a selective beta2-agonist), CL316243 (CL, a selective beta3-agonist), and epinephrine (EPI, beta- and alpha2-agonist) on in situ lipolytic response of s.c. adipose tissue were investigated in vivo, using a microdialysis method to measure glycerol release, in 12 adult nonlactating and ovariectomized, underfed Lacaune ewes. All the adrenergic compounds were perfused for 120 min at 10(-6), 10(-5), and 10(-4) M. They had no lipolytic effect at 10(-6) M. Isoproterenol and EPI at 10(-5) and 10(-4) M enhanced, in the same way, maximal response and area under the concentration curve (AUC) of dialysate glycerol, thus suggesting that involvement of alpha2-adrenoceptors in the control of in situ lipolysis is of minor importance in underfed ewes. Terbutaline had only a slight lipolytic effect at 10(-5) M. This low effect could be due to a lower affinity of TER than of ISO for the beta2-adrenoceptors. The beta3-agonist, CL, had no lipolytic effect whatever the concentration perfused. Further studies are needed to prove the putative presence of beta3-adrenoceptors and their possible role in the ovine adipose tissue.     

Quantitative analysis of Staphylococcus aureus in skimmed milk powder by real-time PCR

A large-scale outbreak of food poisoning caused by consumption of skimmed milk powder contaminated with staphylococcal enterotoxin A (SEA) occurred in Japan. No viable Staphylococcus aureus was detected in the skimmed milk powder, however, sea and nuc genes of S. aureus were detected in it by PCR. The number of S. aureus in skimmed milk powder was estimated by quantitative real-time PCR.     

Tumor necrosis factor-alpha and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and of NO(x) (sum of nitrite and nitrate as indicators of endogenous nitric oxide production) in milk and blood plasma were measured in three mastitis models in dairy cows in early lactation. Escherichia coli P4:O37 bacteria or endotoxin O111:B4 were administered into both left quarters of 12 and 6 cows, respectively. Six of the E. coli-infected cows were treated with a bactericidal antibiotic (Enrofloxacin; Bayer AG, Leverkusen, Germany) i.v. at 10 hr and subcutaneously (sc) at 30 hr after infection. NO(x) concentrations transiently increased maximally 10- to 11-fold in milk of E. coli-infected quarters with or without antibiotic treatment at 24 hr and after endotoxin administration. NO(x) concentrations did not change in milk of unchallenged quarters and in blood plasma. Increases of NO(x) were proceeded by a transient (96- to 149-fold) rise of milk TNF-alpha concentrations, which in endotoxin-administered quarters was maximal at 6 hr and in infected quarters without or with Enrofloxacin treatment at 10 and 14 hr. In blood plasma TNF-alpha concentrations only moderately increased to peaks in endotoxin-administered cows at 6 hr and in E. coli-infected cows at 14 hr postchallenge. In one severely sick, nontreated E. coli-infected cow milk, TNF-alpha response at 14 hr was excessive and followed by a spectacular rise of NO(x) concentration in milk between 48 and 72 hr. In conclusion, a possible clinical relevance of nitric oxide production associated with a rise of intramammary and systemic TNF-alpha during acute mastitis by E. coli infection and endotoxin in lactating dairy cows is indicated, but could not be inhibited by antibiotic treatment.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

Endotoxin-induced bovine mastitis: immunoglobulins, phagocytosis, and effect of flunixin meglumine

Milk whey immunoglobulins (Ig) and phagocytosis of staphylococci by milk polymorphonuclear neutrophilic leukocytes (PMN) were measured in 12 cows (allotted to 6 pairs) during acute bovine mastitis induced by intramammary inoculation of endotoxin. Six of these cows (or 1 in each pair) were treated with flunixin meglumine and were compared with the others (given only saline solution). The endotoxin inoculation comprised 10 micrograms of Escherichia coli O26:B6 lipopolysaccharide injected into one of the rear quarters (mammae). Flunixin meglumine was administered parenterally at a dosage of 1.1 mg/kg every 8 hours (total of 7 doses) beginning at 2 hours after endotoxin was injected. Milk samples were obtained, and whey samples were prepared from each quarter of each cow 3 times before inoculation and at 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours after endotoxin was inoculated. Significant increases (P less than 0.05) in milk whey IgG1, IgG2, IgM, and IgA concentrations were observed in whey samples from endotoxin-inoculated quarters. Greatest relative increase was seen for IgG2. Increased whey Ig concentrations were not observed in quarters which were not inoculated with endotoxin. Concentrations of whey IgG1 and IgM in endotoxin-inoculated quarters were significantly (P less than 0.05) decreased in flunixin meglumine-treated cows, compared with those in saline solution-treated cows. Significant increases in phagocytosis of staphylococci by milk PMN were observed in whey samples from endotoxin-inoculated quarters. Significant differences in PMN phagocytosis were not found in whey samples from cows given flunixin meglumine when compared with whey samples from cows given saline solution.     

Endotoxin levels in milk and plasma of mastitis-affected cows measured with a chromogenic limulus test

A chromogenic limulus test ("Toxicolor") was applied to cow's milk and plasma after treatment with perchloric acid to remove interfering factors. The endotoxin levels in normal cow's milk and plasma were all less than 10 pg ml-1. In acute mastitis, the milk endotoxin level averaged (1.1 +/- 0.7) X 10(3) pg ml-1 in the cases where Gram-negative bacteria were isolated, while the plasma endotoxin concentration was normal. The endotoxin levels in the quarters infected with Gram-positive bacteria were all normal, both in milk and plasma. In gangrenous mastitis due to Gram-negative bacteria, the endotoxin concentration was very high in both milk [(9.3 +/- 5.3) X 10(6) pg ml-1] and plasma (85.2 +/- 68.2 pg ml-1). In similar cases due to Gram-positive bacteria, endotoxin levels were all normal, both in milk and plasma, resembling the acute mastitis due to Gram-positive bacteria. The test was considered suitable for the diagnosis of mastitis due to Gram-negative organisms and the levels of endotoxin detected would aid in assessing the prognosis.     

Effects of two anti-inflammatory drugs on physiologic variables and milk production in cows with endotoxin-induced mastitis

OBJECTIVE: To determine the effects of 2 anti-inflammatory drugs in lactating Holstein cows with endotoxin-induced mastitis. ANIMALS: 30 multiparous Holstein cows that had been lactating for 30 to 60 days. PROCEDURE: Bacterial culture of milk samples and physical examinations established that study cows were in good health and free of mastitis. Mastitis was induced in 1 front mammary gland by intramammary administration of purified bacterial endotoxin. Cows were allocated into 1 of 3 treatment groups: untreated endotoxic mastitis (n = 9), endotoxic mastitis plus flunixin meglumine (9), and endotoxic mastitis plus isoflupredone acetate (10). Heart rate, rectal temperature, mammary surface area, and rumen motility were recorded hourly for 14 hours following endotoxin administration. Flunixin meglumine or isoflupredone acetate was administered after mammary swelling and rectal temperature > or = 40 degrees C had developed. Milk production was evaluated from 5 days before to 10 days after induction of mastitis. RESULTS: Neither drug ameliorated loss of milk production or swelling of the affected mammary gland. Both drugs reduced mean heart rate during the 14 hours following endotoxin administration, compared with untreated control cows. Cows treated with flunixin meglumine had increased rumen motility and decreased rectal temperature during the same period, compared with all other cows. CONCLUSIONS AND CLINICAL RELEVANCE: Neither drug enhanced recovery of milk production following endotoxin-induced mastitis. Flunixin meglumine decreased rectal temperature, whereas isoflupredone did not; however, it has not been established that reduction of fever is beneficial to cows with naturally occurring mastitis.     

Effect of citrus pulp silage feeding on concentration of beta‐cryptoxanthin in plasma and milk of dairy cows

Citrus pulp is known to contain a functional molecule of beta‐cryptoxanthin which is one of the carotenoids showing anti‐oxidative capacity. Influences of citrus pulp silage feeding to dairy cows on beta‐cryptoxanthin concentration in plasma, other blood properties and milking performances were investigated. Four Holstein cows were fed total mixed ration (TMR) containing citrus pulp silage 20% dry matter (DM) for 2 weeks with free access to the TMR. Dry mater intake, milk production and milk components 2 weeks later were not altered compared with those of the control group without citrus pulp silage. Activities of aspartate aminotransferase, alanin aminotransferase and gamma‐glutamyltranspeptidase in plasma were not affected by feeding of citrus pulp silage. Concentrations of protein, albumin, sulfhydryl residue, ascorbic acid, thio‐barbituric acid reactive substance and urea nitrogen in plasma were also not altered by citrus pulp silage feeding. Concentration of beta‐cryptoxanthin in plasma was increased approximately 20‐fold compared with the control group (P < 0.05). Content of beta‐cryptxanthin in pooled milk fat fraction was also increased approximately three times compared with that of the control group. Feeding of TMR containing citrus pulp silage 15% DM for 30 days to eight dairy cows also increased plasma beta‐cryptoxanthin concentration 30‐fold compared with that before feeding.     

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.