Effects of extracellular Ca2+ on phagocytosis and intracellular Ca2+ concentrations in polymorphonuclear leukocytes of postpartum dairy cows

The aim of this study was to determine the effects of extracellular Ca(2+) concentration ([Ca(2+)](e)) on phagocytosis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine polymorphonuclear leukocytes (PMNs). The experiments were performed by using blood samples from parturient paretic and clinically normal parturient cows and manipulating the [Ca(2+)](e) in vitro. Phagocytosis by PMNs (with and without stimulation with phorbol myristate acetate and inhibition with cytochalasin B) and resting [Ca(2+)](i) were significantly lower in parturient paretic cows. Repletion of Ca(2+) in the extracellular media for the samples from these animals increased phagocytosis and resting [Ca(2+)](i). In the blood of clinically normal parturient cows, decreasing the [Ca(2+)](e) decreased phagocytosis and resting [Ca(2+)](i) in PMNs, but increasing the [Ca(2+)](e) did not affect phagocytosis. These results suggest that the hypocalcemic condition of parturient paretic cows in vivo causes decreased phagocytosis and resting [Ca(2+)](i) in PMNs, which may partly contribute to greater susceptibility to infection.     

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.     

Intramammary application of ozone therapy to acute clinical mastitis in dairy cows

The infusion of ozone into the inflamed quarter of cows with clinical mastitis was performed and the efficacy of ozone therapy was evaluated. Ozone was infused into the inflamed quarter via a teat canal using ozone gas generating equipment. Nineteen Holstein cows with acute clinical mastitis were divided into two groups: 15 cows treated with ozone therapy, and 4 cows treated with antibiotic therapy. Systemic and local clinical signs, California Mastitis Test scores, the mastitis causing pathogens, electronic conductivity of milk, and somatic cell counts in milk from ozone- and antibiotic-treated quarters, were compared between the groups. Sixty percent (9/15) of cows with acute clinical mastitis treated with ozone therapy, did not require any antibiotics for recovery. This newly developed ozone therapy method was proven to be effective, safe, and cost effective, and carries no risk of drug residues in milk.     

Altered leukocyte responsiveness in dairy cows with naturally occurring chronic Staphylococcus aureus mastitis

Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.     

Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows

The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows. Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows. Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01). A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters. The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters. The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01). At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively). The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve. In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis. In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters. The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12. The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival. In conclusion, our results suggest that a combination of local and systemic action of E. coli endotoxin is involved in the priming of milk PMN during mastitis.     

Glycogen in Leukocytes from Bovine Blood and Milk

Glycogen content was determined quantitatively by the Anthrone reagent method in leukocytes obtained from blood and milk of five cows. Distribution of glycogen in leukocytes was studied by microscopic examination of slides stained by Periodic acid-Schiff (PAS) reaction. Blood glucose concentrations were investigated in these animals by standard procedures. In two of five cows both blood glucose levels and blood leukocyte glycogen levels on the same day were determined for six consecutive days. One hundred and two blood leukocyte samples from five cows had a mean glycogen content of 1.32 ± 0.04 (S.E.) mg/10⁹ WBC, and 6.11 ± 0.17 (S.E.) mg/10⁹ PMNs. Leukocyte preparations from 80 samples of milk comprising 97 to 98% PMNs contained 3.81 ± 0.18 (S.E.) mg glycogen/10⁹ milk leukocytes. In PAS preparations of blood and milk leukocytes glycogen was found almost exclusively in PMNs. Glycogen granules, present frequently in PMNs and occasionally in monocytes and large lymphocytes from blood, were not observed in those from milk. The glycogen level in milk leukocytes was significantly lower (P = <0.01) than that of the blood PMNs in every cow, and the overall mean difference between levels for milk leukocytes and blood PMNs was highly significant (P = <0.001). Mean blood glucose concentration in the five cows was 44.46 ± 0.66 (S.E.) mg%. There was no significant relationship between blood glucose and blood leukocyte glycogen levels in the five corresponding cows; nor between blood glucose and blood PMN glycogen levels on the same day in either of two cows investigated. Leukocyte preparations from milk samples obtained on the second day following intramammary infusion of endotoxin consistently contained markedly less glycogen than the leukocyte preparations from first day post-infusion samples.

These tended to level off and became intermediate between first and second day levels. It is postulated that the poor phagocytic competence of leukocytes from bovine mammary glands compared to their counterparts in blood observed by various workers may be due partially to low energy reserves in these cells.

     

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degrees C to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P < 0.01) in early lactating cows (15.1%, n = 9) compared with midlactating cows (5.3%, n = 10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n = 7) and midlactating cows (34.0%, n = 8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n = 7) was significantly higher than that in midlactating cows (14.2%, n = 8) (P < 0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis.     

Anti-inflammatory effects of intramammary infusions of glycyrrhizin in lactating cows with mastitis caused by coagulase-negative staphylococci

OBJECTIVE: To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS). ANIMALS: 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis. PROCEDURE: Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL. RESULTS: In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.     

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.     

Decreased neutrophil bactericidal activity during phagocytosis of a slime-producing Staphylococcus aureus strain

Phagocytosis and intracellular killing by bovine polymorphonuclear leukocytes (PMN) are important host defence mechanisms against mastitis caused by Staphlylococcus aureus. We compared the phagocytosis and overall killing of a non slime-producing (NSP) S. aureus and its slime-producing (SP) variant by blood PMN, using an in vitro bacteriological assay. Seven clinically healthy Holstein-Friesian dairy cows in mid-lactation stage were used for this purpose. The percentages of overall killing for the NSP and SP variant were 34+/-3% and 21+/-4% (P < 0.05) and the corresponding percentages of phagocytosis were 40+/-4% and 31+/-4%, respectively. A significant positive correlation (r = 0.79; P < 0.001) was found between phagocytosis and overall killing. These results suggest that the presence of slime was responsible for a decreased phagocytic ingestion and overall killing.     

Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes

Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.     

Modulation of the inflammatory reaction and neutrophil defense of the bovine lactating mammary gland by growth hormone.

This review is focused on the possible interactions of prolactin and somatotrope hormone in the modulation of inflammation of the mammary gland. Several different models are examined: Escherichia coli, Streptococcus uberis, and endotoxin mastitis. Subsequently, the release of growth hormone and insulin-like growth factor during fever and mastitis, the immunophysiological effects of GH on E. coli mastitis, S. uberis and endotoxin mastitis, the galactopoietic action of rBST on healthy and mastitis cows as well as the immunologic effects of GH on leukocytes in healthy and diseased cows are discussed. It can be concluded that the underlying regulation of the neuro-endocrine network is fundamental in the normal function of the immune system.     

In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate

OBJECTIVE: To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS). SAMPLE POPULATION: Peripheral blood PMNs obtained from 12 healthy Beagles. PROCEDURES: The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry. RESULTS: The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA-induced generation of reactive oxygen species.     

Effect of mastitis on macro-minerals of bovine milk and blood serum in Sudan

Milk and blood serum from clinically mastitis infected, subclinically mastitis infected and healthy Friesian cows (15 samples from each of 3 groups) were evaluated for macrominerals (sodium, potassium, calcium, magnesium and phosphorus). The milk from cows infected with subclinical mastitis revealed a significant decrease in potassium (P < 0.001) and a significant increase in sodium and phosphorus content (P < 0.01). Similarly, the milk from cows with the clinical form of the disease showed a significant increase in sodium (P < 0.001) and a significant decrease in potassium, magnesium (P < 0.001) and calcium (P < 0.01). Comparison of healthy cow's milk with that from cows with subclinical mastitis revealed a highly significant increase in sodium (P < 0.001). Comparison of healthy cow's milk with that of clinically mastitic milk showed a highly significant decrease in levels of calcium, magnesium (P < 0.001) and potassium (P < 0.01). However, sodium increased highly significantly (P < 0.001). Comparison of macro-minerals in milk from cows with subclinical and clinical mastitis revealed a significant decrease in potassium contents (P < 0.05) compared with that of healthy cows. Potassium levels were found to decrease significantly (P < 0.05) in subclinically infected cow's blood serum. However, calcium and phosphorus showed a significant decrease (P < 0.01) in blood serum samples from the clinically infected cows.     

Expression of CD3 and CD11b antigens on blood and mammary gland leukocytes and bacterial survival in milk of cows with experimentally induced Staphylococcus aureus mastitis.

OBJECTIVES: To differentiate early (1 to 8 days) from late (9 to 14 days) inflammatory phases and assess relationships between leukocyte phenotype and bacterial recovery in cows with Staphylococcus aureus-induced mastitis. ANIMALS: 10 first-lactation Holstein cows. PROCEDURE: Blood and milk samples were collected from 4 or 6 cows before and after intramammary infusion of sterile broth or S. aureus, respectively. Flow cytometric expression of CD3 and CD11b antigens on blood and milk leukocytes, leukocyte differential counts, bacterial counts in milk, and somatic cell counts were determined longitudinally. RESULTS: Density of CD3 molecules decreased on blood lymphocytes and increased on milk lymphocytes after infusion of bacteria. Density of CD11b molecules on lymphocytes and phagocytes and percentage of CD11b+ lymphocytes in milk increased significantly after infusion; maximum values were achieved during the early inflammatory phase. Density of CD3 and CD11b molecules on milk lymphocytes and macrophages, respectively, 1 day after inoculation were negatively correlated with bacterial recovery on day 1 and days 9 to 14, respectively. Density of CD11b molecules on milk macrophages and the ratios of phagocyte to lymphocyte percentages and polymorphonuclear cell to macrophage percentages in milk differentiated the early from the late inflammatory phase. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of bovine mammary gland macrophages and T cells in response to intramammary infusion of S. aureus was associated with an inability to culture this bacterium from milk. Identification of specific inflammatory phases of S. aureus-induced mastitis in cows may allow for the design of more efficacious treatment and control programs.     

Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were higher in the serum and milk of the cows with mastitis than in the cows in the other two groups. Four of the cows with extramammary inflammatory conditions had serum amyloid A concentrations in serum above 100 microg/ml, but negligible concentrations in milk, indicating that a pathogen must be present in the mammary gland for serum amyloid A to accumulate in milk. The acute phase protein concentrations in milk increased significantly with increasing somatic cell count, suggesting that they may be indicators of the severity of an infection.     

Concentrations of corticosteroids, leukocytes, and immunoglobulins in blood and milk after administration of ACTH to lactating dairy cattle: effects on phagocytosis of Staphylococcus aureus by polymorphonuclear leukocytes

A study was conducted to determine relationships among plasma and milk corticosteroids, milk immunoglobulins, and phagocytosis of Staphylococcus aureus by polymorphonuclear leukocytes (PMN) isolated from milk of cows given injections of 0 (saline solution only), 100, and 200 IU of ACTH. Also determined were the effects of ACTH on mobilization of PMN into milk from the mammary gland irritated by infusion of 100 ml of saline solution containing 0.1% oyster glycogen. Cows (n = 10) were injected 6 times within a 48-hour period with 0, 100, or 200 IU of ACTH. Immediately before cows were given the 5th injection, 2 mammary quarters were infused with the saline-glycogen solution. Five hours after the 6th injection, milk from infused quarters was collected, and PMN were isolated, washed, and resuspended in autologous skimmed milk collected 5 hours after the 4th injection and before the udder was infused. Isolated PMN were incubated with S aureus and percentage of phagocytosis was determined. Concentrations of total corticosteroids in plasma and milk increased after cows were given injections of 100 and 200 IU of ACTH. The concentrations of IgA, IgG1, IgG2, IgM, and bovine serum albumin in milk after 4 injections of 0, 100, and 200 IU of ACTH were similar to preinjection (base line) concentrations. Injections of 100 and 200 IU of ACTH significantly increased the concentrations of total circulating leukocytes. Concentrations of leukocytes in milk tended to increased with increasing doses of ACTH, but the differences were not significant. Injection of 100 and 200 IU of ACTH reduced phagocytosis of S aureus by PMN. After 60 minutes of incubation, phagocytosis averaged 57% and 54%, respectively, for the ACTH treatments and 70% for controls (saline only). Results indicate that injections of ACTH that result in physiologic and pharmacologic plasma concentrations of corticosteroids inhibit phagocytosis. Impairment of phagocytosis appeared to be a direct effect of corticosteroid concentration o PMN and was not due to reduced concentrations of immunolobulins. These data indicate that reduced phagocytosis by PMN could be important in increased susceptibility to disease during stress in lactating dairy cows.     

Immunobiology of bovine mammary gland: apoptosis of somatic cells in milk during naturally occurring mastitis

Mastitis remains a major cause of economic losses in dairy herds. It is believed, that the enhancement of natural defense mechanisms in mammary gland may be helpful in the treatment of bovine mastitis. Our study was designed to assess the apoptosis of leukocytes isolated from bovine milk during subclinical staphylococcal mastitis. Milk samples were collected from cows naturally infected with one pathogen--Staphylococcus aureus and from animals with mastitis caused by several pathogens, including S. aureus. It has been determined that the rate of apoptosis was lower in mastitic milk, as compared with control samples, although varied significantly between groups. High percentage of apoptotic cells was detected in milk with high counts of pathogenic bacteria. In all groups the rate of apoptosis was dependent on the bacterial load, although various correlations were identified. Thus, it is postulated, that the rate of apoptosis of somatic cells in mastitic milk is related to the etiology of infection and is determined by the bacterial load.     

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.     

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.