Anti-inflammatory therapy in acute endotoxin-induced bovine mastitis

Acute mastitis was induced in lactating cows by intramammary challenge with 10 g of Escherichia coli lipopolysaccharide. The cows were monitored clinically prior to and for 96 hours after challenge. Milk production, complete blood counts, serum enzyme activities and milk indicators of inflammation were evaluated.Endotoxin challenge was in 7 groups of 3 cows each. Within the groups, cows were randomly assigned to 3 intravenous treatments: saline controls, steroid (one dose of dexamethasone at 0.44 mg/kg) and non-steroidal agent (two doses of flunixin meglumine at 1.1 mg/kg, 8 h apart).Anti-inflammatory therapy reduced rectal and mammary gland surface temperatures. Milk production was significantly reduced (p<0.05) in cows treated with dexamethasone. Although dexamethasone treatment produced significant increases (p<0.05) in blood leukocytes and segmented neutrophils, milk somatic cell concentrations were not significantly altered. Flunixin meglumine did not alter milk production or blood or milk leukocytes.     

Immunity to Escherichia coli in pigs: Antibody Secretion by the Mammary Gland after Intramammary or Intramuscular Vaccination with an E. coli Vaccine

Indirect hemagglutinating antibody titres in individual gland samples of colostrum and milk from 13 sows were measured. Five of the sows were vaccinated via a mammary gland and five by the intramuscular route with a live formalinised Escherichia coli vaccine and three remained as non-vaccinated controls.

Antibody titres were higher in colostral and milk whey from the vaccinated sows than from non-vaccinated groups. The inoculated gland in the group of sows given vaccine by the intramammary route secreted milk containing markedly more antibodies to the vaccine E. coli strain than did the non-vaccinated glands. Milk from the vaccinated gland did not contain higher titres to heterologous E. coli O antigens than milk from non-vaccinated glands. Serum titres were the same or higher than the titres in colostrum from non-vaccinated glands.

     

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.     

Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection.

Daily injections of an anti-inflammatory milk-derived factor (MDF) into mice increased resistance to Staphylococcus aureus challenge, and reduced leukocyte infiltration. Intraperitoneal injection of MDF into lactating mice prior to S. aureus intramammary challenge resulted in greater milk secretory activity and less inflammation compared with untreated controls, but had little effect on the number of S. aureus recovered from mammary tissue. Infusion of MDF directly into mouse mammary glands prior to challenge reduced S. aureus recovered after challenge. Incubation of bovine mammary macrophages in medium supplemented with MDF enhanced phagocytosis of opsonized S. aureus. In addition, infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer infections (five of ten) than in control quarters (seven of nine). Repeated daily injections of 5 mg MDF into S. aureus-infected quarters increased the percent of mammary neutrophils and decreased the recovery of S. aureus, but did not eliminate infections. Intravenous injection of 8 g MDF into cows resulted in pronounced leukopenia while the accompanying effect on mammary leukocytes was less marked but followed a similar course. Results suggest that the use of MDF in mice enhanced resistance to experimental infection and was beneficial in maintaining mammary secretory activity and reducing inflammation after bacterial challenge. In the cow, MDF promoted phagocytosis in vitro and was effective against challenge when infused intramammarily.     

Evaluation of the Microbiological Status of Milk and Various Structures in Mammary Glands from Naturally Infected Dairy Cows

A knowledge of the microbiological status of milk and of the different structures in the mammary glands has great importance in elucidating the pathogenesis of mammary gland infections. The objective of this study was to evaluate the microbiological status of various structures in the mammary glands from naturally infected dairy cows following slaughter. A total of 94 samples of milk, 184 samples of mammary parenchyma, 168 samples of gland cisterns, and 168 samples of teat cisterns were collected for microbiological examination. Microorganisms were detected in 59.9% of all samples, 67.0% of the milk samples, 70.1% of the mammary parenchymas, 55.9% of the gland cisterns and 48.8% of the teat cistern samples. When all samples were considered, coagulase-negative Staphylococcus were the most prevalent (35.7%) followed by coagulase-positive Staphylococcus (12.2%), Corynebacterium bovis (2.4%), Prototheca sp. (1.9%), and Streptococcus dysgalactiae (1.5%). There was a significantly higher occurrence of microorganisms in the milk and mammary parenchyma compared to the gland cisterns and teat cisterns. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46⁺/CD3⁻) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis.     

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.     

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.     

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS‐induced mastitis in dairy cows

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long‐term manipulated glucose and insulin concentrations in combination with a LPS‐induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT‐qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha‐lactalbumin, insulin‐induced gene 1, κ‐casein and acetyl‐CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS‐induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.     

CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats

Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 μg kg⁻¹ body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 × 10⁶ colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 × 10⁶ CFU ml⁻¹ E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpG-ODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats.     

Interleukin-1β infusion in bovine mammary glands prior to challenge with <Emphasis Type="Italic">Streptococcus uberis</Emphasis> reduces bacterial growth but causes sterile mastitis

Dairy cows are especially vulnerable to intramammary infection by the bacterial pathogen Streptococcus uberis in the dry period. Use of immunotherapeutic agents at drying off could increase cellular defences in the gland and prevent establishment of new S. uberis infections. This study investigated the potential of infusing recombinant bovine interleukin-1 beta (rbIL-1beta) in the mammary glands as a prophylactic agent against subsequent intramammary challenge with S. uberis in the early dry period. Immediately after the last milking at commencement of the dry period, one cow from each of 10 monozygous twinsets was infused with 10 microg of rbIL-1beta in two quarters and the other twin was infused with the carrier agent, sterile phosphate buffered saline. Twenty-four hours later, the quarters were infused with 10(3) colony-forming units (CFU) of S. uberis. Bacteriology, somatic cell count (SCC), concentrations of specific cytokines and antibody responses were monitored in mammary gland secretions and sera for the next 21 days. Infusion of rbIL-1beta into mammary glands at commencement of the dry period was associated with less new S. uberis intramammary infections, as determined by the number of quarters with bacterial growth. However, high SCC in quarters following infusion of rbIL-1beta masked the full beneficial effect of this procedure.     

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid‐lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control‐fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat‐inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme‐linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post‐partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up‐regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk‐extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non‐invasive milk‐extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.     

Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO.     

Intracellular fate of strains of <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from dairy cows with acute or chronic mastitis

Research on mastitis in dairy cows caused by Escherichia coli has reported the emergence of strains capable of inducing chronic mastitis and that these strains adhered to and internalized into bovine mammary epithelial cells better than strains of E. coli isolated from acute mastitis. To understand mechanisms and strategies used by chronic E. coli strains to survive intracellularly internalization studies using bovine mammary epithelial cells treated with inhibitors of caveolae-mediated endocytosis (CME) and receptor-mediated endocytosis (RME), double immunofluorescence labeling confocal laser and fluorescence microscopy were conducted. Internalization studies showed that strains chronic E. coli strains persisted intracellularly longer than acute E. coli strains. Treatment of bovine mammary epithelial cells CME or RME inhibitors resulted in lower numbers of intracellular E. coli strains associated with chronic or acute mastitis than untreated controls. In addition, when selective CME inhibitors were used significantly fewer chronic E. coli were detected intracellularly than acute E. coli or untreated controls. Confocal laser microscopy showed that chronic E. coli strains colocalized preferentially with caveolae whereas acute strains did so with early endosomes, an early step of RME. These results suggest that strains of E. coli associated with chronic mastitis exploit lipid rafts/CME to internalize into and move through mammary epithelial cells. By exploiting this endocytosis pathway, chronic E. coli strains avoid bactericidal mechanisms such as endosome acidification and endosome-lysosome fusion, thus allowing intracellular survival. Data from this study helps to explain how these strains are capable of causing chronic E. coli mastitis.     

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.     

Dynamics of leukocytes and cytokines during experimentally induced Streptococcus uberis mastitis

Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.     

The elimination of serum-resistant Escherichia coli from experimentally infected single mammary glands of healthy cows.

Neutrophils isolated from mammary glands stimulated with a staphylococcal culture filtrate efficiently killed serum-resistant strains of Escherichia coli. This study was extended and it was shown that an infusion of wide ranging numbers (5 X 10(1) to 5 X 10(6)) of the same strains of E coli into a single mammary gland resulted in bacterial growth, which was eliminated following neutrophil infiltration. This elimination occurred before the appearance of any clinical signs. Once bacterial kill had started in the gland, it continued in the milk after withdrawal from the gland. These results offer an explanation of why causative microbial agents cannot be isolated from some cases of clinical mastitis.     

Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma

Effects of recombinant bovine interferon (rBoIFN) gamma on mammary gland neutrophil activity during the periparturient period were studied. Bovine mammary gland neutrophils were isolated and incubated in mammary gland secretions obtained from Holstein-Friesian cattle during the last 2 weeks of gestation. Cell functions were evaluated following treatment with 10 U, 100 U, and 1000 U of rBoIFN-gamma. Bacterial phagocytosis, bactericidal activity and chemiluminescence were significantly lower for neutrophils incubated in mammary gland secretions when compared with control neutrophils incubated in Hank's balanced salt solution. Treatment of mammary neutrophils with rBoIFN-gamma reversed the suppressive effects of mammary secretions resulting in higher chemiluminescent activity and significantly more bacterial phagocytosis and bactericidal activity when compared with untreated controls. Results from these preliminary in vitro data suggest that rBoIFN-gamma therapy may modulate mammary gland neutrophil functions in vivo and possibly facilitate the rapid clearance of mastitis-causing pathogens mammary glands during the periparturient period.