Comparison of Holstein and Jersey innate immune responses to Escherichia coli intramammary infection.

Mastitis is one of the most prevalent diseases in cattle and remains among the most costly diseases to the dairy industry. Various surveys have indicated a greater prevalence of and risk for mastitis in Holstein cows than in Jersey cows. The innate immune system comprises the immediate host defense mechanisms that respond to infection, and differences in the magnitude and rapidity of this response are known to influence susceptibility to and clearance of infectious pathogens. The reported differences in the prevalence of mastitis between Holstein and Jersey cows may suggest the occurrence of breed-dependent differences in the innate immune response to intramammary infection. The objective of the current study was to compare the acute phase and cytokine responses of Holstein and Jersey cows following intramammary infection by the bacterial pathogen Escherichia coli, a leading cause of clinical mastitis. All cows in the study were in similar stages of lactation, of the same parity, subjected to the same housing and management conditions, and experimentally infected on the same day with the same inoculum preparation. Before and after infection, the following innate immune parameters were monitored: bacterial clearance; febrile response; induction of the acute phase proteins serum amyloid A and lipopolysaccharide-binding protein; alterations in total and differential white blood cell counts; changes in milk somatic cell counts and mammary vascular permeability; and induction of the cytokines IFN-gamma, IL-1beta, IL-8, IL-12, and tumor necrosis factor-alpha. Overall innate immune responses were similar between the 2 breeds; however, temporal differences in the onset, cessation, and duration of several responses were detected. Despite these differences, intramammary clearance of E. coli was comparable between the breeds. Together, these data demonstrate a highly conserved innate immune response of Holstein and Jersey cows to E. coli intramammary infection.

Innate immune response to intramammary Mycoplasma bovis infection.

The objective of the current study was to characterize the systemic and local innate immune response of dairy cows to IMI with Mycoplasma bovis, a pathogen of growing concern to the dairy industry. Ten Holstein cows were each infused in 1 quarter with M. bovis and studied for a 10-d period. Acute phase protein synthesis, which reflects 1 parameter of the systemic response to infection, was induced within 108 h of infection, as evidenced by increased circulating concentrations of lipopolysaccharide binding protein and serum amyloid A. Transient neutropenia was observed from 84 to 168 h postinfection, whereas a constant state of lymphopenia and thrombocytopenia was observed from 84 h until the end of the study. Milk somatic cell counts initially increased within 66 h of M. bovis infusion and remained elevated, relative to control (time 0) concentrations, for the remainder of study. Increased milk concentrations of BSA, which reflect increased permeability of the mammary epithelial-endothelial barrier, were evident within 78 h of infection and were sustained from 90 h until the end of the study. Milk concentrations of several cytokines, including IFN-gamma, IL-1beta, IL-10, IL-12, tumor growth factor-alpha, and tumor necrosis factor-alpha, were elevated in response to infection over a period of several days, whereas increases in milk IL-8 were of a more limited duration. Complement activation, reflected by increased milk concentrations of complement factor 5a, was also observed over several days. Despite the indication by these observed changes that the cows mounted a prolonged inflammatory response to M. bovis intramammary infection, all quarters remained infected throughout the study with persistently high concentrations of this bacterium. Thus, a sustained inflammatory response is not sufficient to eradicate M. bovis from the mammary gland and may reflect the ongoing struggle of the host to clear this persistent pathogen.

Effect of mastitis on milk perchlorate concentrations in dairy cows.

Recent surveys have identified the presence of perchlorate, a natural compound and environmental contaminant, in forages and dairy milk. The ingestion of perchlorate is of concern because of its ability to competitively inhibit iodide uptake by the thyroid and to impair synthesis of thyroid hormones. A recent study established that milk perchlorate concentrations in cattle highly correlate with perchlorate intake. However, there is evidence that up to 80% of dietary perchlorate is metabolized in clinically healthy cows, thereby restricting the available transfer of ingested perchlorate into milk. The influence of mastitis on milk perchlorate levels, where there is an increase in mammary vascular permeability and an influx of blood-derived components into milk, remains unknown. The present study examined the effect of experimentally induced mastitis on milk perchlorate levels in cows receiving normal and perchlorate-supplemented diets. Over a 12-d period, cows were ruminally infused with 1 L/d of water or water containing 8 mg of perchlorate. Five days after the initiation of ruminal infusions, experimental mastitis was induced by the intramammary infusion of 100 microg of bacterial lipopolysaccharide (LPS). Contralateral quarters infused with phosphate-buffered saline served as controls. A significant reduction in milk perchlorate concentration was observed in the LPS-challenged glands of animals ruminally infused with either water or perchlorate. In control glands, milk perchlorate concentrations remained constant throughout the study. A strong negative correlation was identified between mammary vascular permeability and milk perchlorate concentrations in LPS-infused glands. These findings, in the context of a recently published study, suggest that an active transport process is operative in the establishment of a perchlorate concentration gradient across the blood-mammary gland interface, and that increases in mammary epithelial and vascular endothelial permeability lead to a net outflow of milk perchlorate. The overall finding that mastitis results in lower milk perchlorate concentrations suggests that changes in udder health do not necessitate increased screening of milk for perchlorate.

Sequencing, chromosomal mapping, and functional characterization of bovine FLICE-like inhibitory protein (FLIP).

FLICE-like inhibitory protein (FLIP) has been shown in both humans and mice to inhibit apoptosis and NF-kappaB activation induced by pro-inflammatory mediators. The activation of NF-kappaB and the induction of apoptosis are critical events in the pathogenesis of a variety of disease states in cattle, including mastitis. Since FLIP is known to moderate these events in other species, we mapped the bovine FLIP gene, sequenced bovine FLIP cDNA, and characterized its expression in cultured primary bovine endothelial cells. Sequencing of bovine FLIP revealed approximately 83, 74, and 68% amino acid sequence identity to its porcine, human, and murine orthologs, respectively. Bovine FLIP was mapped to chromosome 2 by radiation hybrid mapping. Interestingly the region to which bovine FLIP maps contains a putative quantitative trait locus for functional herd life which is an indicator of a cow's ability to survive involuntary culling due primarily to mastitis and infertility. In addition to sequencing and mapping, the function of bovine FLIP was studied. Over-expression of bovine FLIP protected against bacterial lipopolysaccharide (LPS)- and TNF-alpha-induced apoptosis in bovine endothelial cells consistent with previous studies of human FLIP. In addition, elevated expression of bovine FLIP blocked LPS- and TNF-alpha-induced upregulation of NF-kappaB-dependent gene products as assayed by E-selectin expression. Only the full-length bovine FLIP protein could inhibit NF-kappaB activation induced by LPS, whereas the death effector domain region alone was able to inhibit TNF-alpha-induced NF-kappaB activation. Together, these data demonstrate the conservation of FLIP's ability to inhibit apoptosis and to downregulate NF-kappaB activation across species.

Increased milk levels of transforming growth factor-alpha, beta1, and beta2 during Escherichia coli-induced mastitis.

Among the gram-negative bacteria that cause mastitis, Escherichia coli are the most prevalent. The innate immune system provides initial protection against E. coli infection by detecting the presence of the foreign pathogens and by mounting an inflammatory response, the latter of which is mediated by cytokines such as IL-1beta, IL-8, and tumor necrosis factor (TNF)-alpha. Although changes in these cytokines during mastitis have been well-described, it is believed that other mediators moderate mammary gland inflammatory responses as well. The growth factors/cytokines transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are all expressed in the mammary gland and have been implicated in regulating mammary gland development. In other tissues, these growth factors/cytokines have been shown to moderate inflammation. The objective of the current study was to determine whether TGF-alpha, TGF-beta1, and TGF-beta2 milk concentrations were altered during the course of E. coli-induced mastitis. The contralateral quarters of 11 midlactating Holstein cows were challenged with either saline or 72 cfu of E. coli, and milk samples were collected. Basal milk levels of TGF-alpha, TGF-beta1, and TGF-beta2 were 98.81 +/- 22.69 pg/mL, 3.35 +/- 0.49 ng/mL, and 22.36 +/- 3.78 ng/mL, respectively. Analysis of whey samples derived from E. coli-infected quarters revealed an increase in milk levels of TGF-alpha within 16 h of challenge, and these increases persisted for an additional 56 h. Elevated TGF-beta1 and TGF-beta2 milk concentrations were detected in E. coli-infected quarters 32 h after challenge, and these elevations were sustained throughout the study. Because TGF-alpha, TGF-beta1, and TGF-beta2 have been implicated in mediating inflammatory processes, their induction during mastitis is consistent with a role for these molecules in mediating mammary gland host innate immune responses to infection.

Characterization of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae.

Gram-negative bacteria are responsible for almost one-half of the clinical cases of mastitis that occur annually. Of those gram-negative bacteria that induce mastitis, Klebsiella pneumoniae remains one of the most prevalent. Detection of infectious pathogens and the induction of a proinflammatory response are critical components of host innate immunity. The objective of the current study was to characterize several elements of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae. The inflammatory cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), 2 proteins that contribute to host recognition of gram-negative bacteria, were studied. The contralateral quarters of 7 late-lactating Holstein cows were challenged with either saline or K. pneumoniae, and milk and blood samples were collected. Initial increases in the chemoattractants C5a and IL-8, as well as TNF-alpha, were evident in infected quarters within 16 h of challenge and were temporally coincident with increases in milk somatic cells. Augmented levels of TNF-alpha and IL-8 were observed in infected quarters until >48 h postchallenge, respectively. Elevated levels of IL-12, IFN-gamma, and the antiinflammatory cytokine, IL-10, which were first detected between 12 and 20 h postinfection, persisted in infected quarters throughout the study (>96 h). Initial increases in milk LBP and sCD14 were detected 16 and 20 h, respectively, after challenge. Together, these data demonstrate that intramammary infection with K. pneumoniae elicits a host response characterized by the induction of proinflammatory cytokines and elevation of accessory molecules involved in LPS recognition.

Elevated milk soluble CD14 in bovine mammary glands challenged with Escherichia coli lipopolysaccharide.

The purpose of this study was to determine whether soluble CD14 (sCD14) in milk was affected by stage of lactation, milk somatic cell count (SCC), presence of bacteria, or lipopolysaccharide (LPS)-induced inflammation. Milk samples from 100 lactating cows (396 functional quarters) were assayed for sCD14 in milk to determine effects of stage of lactation, SCC, and intramammary infection. The concentration of sCD14 was highest in transitional milk (0 to 4 d postpartum) and in milk with high SCC (> 750,000 cells/ml). Most of the infected quarters (> 80%) were infected by coagulase-negative staphylococci and yeast. No difference was found between noninfected and infected quarters. One quarter of six healthy lactating cows was challenged with 100 microg LPS in order to study the kinetics of sCD14 during an LPS-induced inflammation. Milk samples were collected at various intervals until 72 h after injection. Rectal temperature, milk tumor necrosis factor-alpha, and interleukin-8 increased immediately after challenge. The increase in sCD14 paralleled the increase in SCC, peaked at 12 h, and started to decline after 24 h. Serum leakage, as characterized by the level of bovine serum albumin in milk, peaked at 4 h and then gradually decreased. All parameters remained at basal levels in control quarters throughout the study. In vitro experiments indicated that neutrophils released sCD14 in response to LPS in a dose-dependent manner. The results indicate that the concentration of sCD14 was significantly increased in milk after LPS challenge. The increase was not likely due to serum leakage. Instead, infiltrated neutrophils might be the main source of increased sCD14 in milk during inflammation.

Development of anti-bovine TNF-alpha mAb and ELISA for quantitating TNF-alpha in milk after intramammary injection of endotoxin.

Murine mAb reactive with recombinant bovine tumor necrosis factor-alpha (r-boTNF-alpha) were produced. An ELISA using murine mAb and rabbit polyclonal antibodies, each reactive with r-boTNF-alpha to sandwich bovine TNF-alpha was developed. Secretion of TNF-alpha in quarter milk increased 1 h after injection of 0.1 mg (four cows) or 0.5 mg (four cows) Escherichia coli lipopolysaccharide (LPS) into a mammary quarter, peaked 1 to 5 h later, and returned to control levels in 24 h. There were no differences in body temperature, SCC, TNF-alpha, and blood leukocyte responses between 0.1 and 0.5 mg of LPS. To determine effects of repeated injections of LPS into the same udder, a second injection of 0.1 mg of LPS into the same quarter (two cows) 24 h after the first injection produced a strongly attenuated TNF-alpha response. However, a normal TNF-alpha response was observed when LPS was injected into a contralateral quarter (two cows) 24 h after the first LPS injection. Leukocyte counts in blood decreased and body temperature increased substantially after each injection of LPS. Quarter milk SCC increased 200-fold 8 to 12 h after the LPS injections. It would appear that these changes were not regulated by TNF-alpha secretion because the changes were also similar after the second injection of LPS into the same mammary quarter.

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils.

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN. Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.

Apoptosis of bovine neutrophils following diapedesis through a monolayer of endothelial and mammary epithelial cells.

In a two-chamber system, isolated blood polymorphonuclear neutrophil leukocytes (PMN) were allowed to migrate (5 h, 37degrees C) in response to bovine complement component C5a across calfskin and rat-tail type I collagen-coated micropore membranes, arterial endothelial, or mammary epithelial cell monolayer on calfskin and rat-tail collagen-coated membranes, respectively. Migration through calfskin collagen-coated membranes resulted in 14.5% +/- 3.4% apoptotic PMN, which was significantly higher than 6.6% +/- 1.2% apoptotic nonmigrated C5a-treated PMN. The addition of an endothelial or epithelial cell monolayer to collagen-coated membranes prevented apoptosis of migrated PMN. After removing the membranes, nonmigrated (untreated and C5a treated) and migrated PMN were incubated for an additional 20 h. At this time point, 69.1% +/- 4.5% and 47% +/- 4.5% of PMN that have migrated through a calfskin-coated membrane and an endothelial monolayer, respectively, were apoptotic, compared with 28.2% +/- 3.0% and 21.1% +/- 4.5% apoptotic untreated and C5a-treated PMN, respectively; 46.9% +/- 4.8% of PMN that have migrated through rat-tail-coated membranes were apoptotic compared with 14.7% +/- 2.3% and 9.3% +/- 1.2% apoptotic untreated and C5a-treated PMN, respectively. Migration across rat-tail collagen-coated membranes with a monolayer of epithelial cells did not affect apoptosis of migrated PMN, even after 20 h of incubation. In conclusion, migration of PMN across collagen-coated membranes (either calfskin or rat-tail collagen) induced an apoptotic response, which was downregulated by a monolayer of endothelial cells and was negated by an epithelial cell monolayer.

Escherichia coli induces apoptosis and proliferation of mammary cells.

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1beta converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation.

Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells.

OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.

Phagocytosis and killing of Staphylococcus aureus by bovine neutrophils after priming by tumor necrosis factor-alpha and the des-arginine derivative of C5a.

OBJECTIVES: To evaluate effects of proinflammatory mediators on phagocytosis and killing of Staphylococcus aureus, the oxidative burst (OB), and expression of receptors for opsonins by bovine neutrophils. SAMPLE POPULATION: Neutrophils from 10 cattle. PROCEDURE: Neutrophils were primed with recombinant bovine tumor necrosis factor-alpha (TNF-alpha) or the des-arginine derivative of bovine C5a (C5a(desArg)) and mixed with S aureus. Phagocytosis and OB were measured by use of flow cytometry. Rate of phagocytosis and intracellular killing were evaluated. Expression of receptors for immunoglobulins and the C3bi fragment of complement were estimated by use of flow cytometry. RESULTS: Priming of neutrophils by TNF-alpha improved phagocytosis of S aureus with a concentration-dependent effect. Phagocytosis of preopsonized washed bacteria was increased by activation of neutrophils with C5a(desArg). Phagocytosis was optimal when neutrophils primed with TNF-alpha were activated with C5a(desArg). The OB of phagocytizing neutrophils was highest when TNF-alpha and C5a(desArg) were used in combination. Bactericidal activity of neutrophils was stimulated by priming with TNF-alpha or C5a(desArg). Binding of bovine IgM or IgG2 to bovine neutrophils was not stimulated byTNF-alpha, C5a(desArg), or both, and aggregated IgG1 did not bind to neutrophils regardless of their activation state. Both TNF-alpha and C5a(desArg) increased expression of beta2 integrins (CD18), with the highest expression when they were used in combination. CONCLUSIONS AND CLINICAL RELEVANCE: The mediators TNF-alpha and C5a(desArg) stimulated phagocytic killing by neutrophils and potentiated each other when used at suboptimal concentrations. Bovine neutrophils have enhanced bactericidal activities at inflammatory sites when TNF-alpha, C5a(desArg), or both are produced locally.

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin.

OBJECTIVE: To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils. ANIMALS: 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources. PROCEDURE: Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils. RESULTS: MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes. CONCLUSION: MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells.

Evaluation of selected antibiotic residue screening tests for milk from individual goats.

Because somatic cell counts (SCC) of caprine milk are higher than SCC of bovine milk, the performance of antibiotic residue tests for screening bovine milk was investigated for caprine milk. Eighty-five does that were free of antibiotic usage for at least 30 d and that were free of clinical mastitis were sampled at three milkings during a 37-d period. At each sampling, foremilk was collected for bacteriological analysis, and composite bucket milk samples were collected for antibiotic testing and SCC. Day of lactation, parity, 305-d mature equivalent milk yield, and SCC averaged 221 d (57 to 577 d), 2.3 lactations (one to nine lactations), 1160 kg (623 to 1750 kg), and 2.2 x 10(6)/ml (0.3 to 30.7 x 10(6)/ml), respectively. The mean Dairy Herd Improvement Association test day milk yield for the month of sample collection was 3 kg (1.4 to 6.4 kg). Intramammary infections were present in 54% of the goats and in 36% of the udder halves. Assays included positive (5 and 10 ppb of penicillin-G and 50 ppb of ceftiofur) and negative controls that had been prepared in caprine milk and controls supplied by the manufacturers. One false-negative outcome and one false-positive outcome were recorded. For one sampling day, a positive linear relationship existed between SCC and the results of one test, and a quadratic relationship existed between SCC and the results of another test. The antibiotic residue screening tests for milk from individual goats adequately identified milk that was free of antibiotic. These tests are therefore recommended for use with caprine milk.

Sensitization of the bovine mammary gland to Escherichia coli endotoxin.

The effect of repeated infusions of Escherichia coli endotoxin on the acute phase response in the bovine mammary gland was assessed through the concentrations of tumor necrosis factor alpha (TNF-alpha) in milk. Four clinically normal lactating cows received two intramammary infusions of E coli endotoxin (33 micrograms) 24 h apart in the same mammary quarter. Along with the second infusion, the cows received one dose of endotoxin in the contralateral quarter. Milk was collected at varying intervals before and after infusion and TNF-alpha concentrations were determined by ELISA. Following the first infusion at 0 h, the mean concentrations of TNF-alpha augmented from undetectable concentrations to a maximum of 0.4 ng/mL at 4 h and declined to below 0.04 ng/mL at 24 h, the time of the second infusion. In the quarters challenged twice, the increase in TNF-alpha concentrations was abrupt, culminating at 11.7 ng/mL 6 h later (at 30 h). The increases in TNF-alpha concentrations were similar in the contralateral quarters infused once. TNF-alpha concentrations in the control, uninfused quarters of infused cows remained undetectable (< 0.04 ng/mL). Despite the low TNF-alpha response following the first infusion, mean somatic cell counts increased markedly, being only slightly lower than after the second infusion (10(7)/mL and 5 x 10(7)/mL at 8 h and 32 h, respectively) in the quarters challenged twice. After the first infusion, none of the cows developed fever, but following the second infusion, rectal temperature increased markedly, culminating 6 h after the second infusion. These results show that an infusion in one quarter of an amount of endotoxin sufficient to induce a pronounced cell recruitment but insufficient to induce a marked TNF-alpha secretion following the first infusion sensitized not only that quarter but also the contralateral one to a second infusion with the endotoxin. It is thus possible that sensitization of the whole udder follows a first contact with a moderate dose of endotoxin in one quarter.

Continuous cultures of macrophages derived from the 8-day epiblast of the pig.

Secondary macrophage cell cultures were generated from the primary culture of epiblasts of 8-d-old pig blastocysts. The epiblast-derived macrophagelike (EDM) cells have a morphology and ameboid behavior that is typical of tissue histocytes. The cells reacted positively with monoclonal antibodies specific for pig granulocyte-macrophage lineage cells, and were not reactive with monoclonal antibodies specific for pig B and T lymphocytes. Marked phagocytic behavior and the formation of phagosomes were demonstrated following incubation with FITC-labeled bacteria. The EDM cells stained positively for nonspecific acid esterase that was not inhibited by sodium fluoride. DiI-acetylated-LDL was rapidly taken up by the cells. Transmission electron microscopy of the EDM cells showed phagolysosomes, numerous cytoplasmic vacuoles, large, lobed nuclei, and numerous pseudopods or filopodia at the cell surface. Strong reactivity of the cells with anti-CD14 monoclonal antibody was observed. Further, cytotoxic activity was produced from the EDM cells after exposure to lipopolysaccharide in a concentration and time-dependent manner. The cultures could be maintained and expanded for several months on STO co-culture. Their derivation from the epiblast of the pig demonstrates the possibility of obtaining hemopoietic cell cultures from the preimplantation blastocysts of all mammals.

Cell culture system for studying bovine neutrophil diapedesis.

Neutrophils are the major defense against bacterial infection in the bovine mammary gland. Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli. This study describes the development of a system of cell culture that can be used to study neutrophil diapedesis through secretory and ductal mammary epithelial barriers. The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ductal epithelial cells layered on a porous membrane. Confluence was determined by electrical resistance and trypan blue diffusion. Neutrophil diapedesis occurred from the basal to the apical surface of the monolayers. Purified complement C5a, fetal bovine serum that had been activated by zymosan, and fetal bovine serum that had been activated by Escherichia coli induced neutrophil diapedesis. Neutrophil diapedesis was greater across ductal cell monolayers. Blood neutrophils from five cows differed in their ability to migrate through the multilayered culture system in response to C5a. Monoclonal antibodies to C5a blocked diapedesis induced by purified C5a but had no effect on diapedesis induced by fetal bovine serum that had been activated by zymosan or by fetal bovine serum that had been activated by E. coli endotoxin, indicating that factors other than C5a were chemotactic for neutrophils. Monomeric IgG2, immune complexes, and E. coli endotoxin did not induce neutrophil diapedesis.

Intramammary defense against infections induced by Escherichia coli in cows.

OBJECTIVE: To examine Escherichia coli lipopolysaccharide (LPS) effects on expression of CD14 and CD18 cell surface receptors and lectin/carbohydrate-mediated nonopsonic phagocytosis of E coli. DESIGN: Cell isolation, monoclonal antibody, phagocytosis, and flow cytometric studies. ANIMALS: 4 clinically normal lactating Holstein cows for studies on CD14 and CD18, and 2 for phagocytosis studies. PROCEDURE: Binding of CD14 and CD18 monoclonal antibodies to blood and milk neutrophils and mononuclear leukocytes was studied by flow cytometry before and after intramammary injection of LPS, and nonopsonic phagocytosis of E coli by blood neutrophils was determined. Presence of intracellular CD14 was determined after in vitro incubation of neutrophils in skimmed milk and after fixation and permeabilization of freshly isolated neutrophils. RESULTS: Before LPS injection, percentages of blood neutrophils and large mononuclear (LMO) cells expressing CD14 averaged 3 and 63% and 68 and 35% for mammary neutrophils and LMO cells, respectively. After LPS injection, CD14 was only detected on blood and mammary LMO cells (61 and 25%); receptor expression increased by 1.8- and threefold, respectively. In vitro incubation of neutrophils in skimmed milk increased the percentage of neutrophils expressing CD14. The number of blood neutrophils staining positive for CD14 increased after permeabilization of the plasma membrane, which was blocked by unlabeled anti-CD14 monoclonal antibodies. Before LPS, percentages of blood neutrophils and LMO cells expressing CD18 averaged 93 and 95% and was 88 and 55% for mammary neutrophils and LMO cells, respectively. After LPS, percentages of mammary neutrophils and LMO cells expressing CD18 increased to 100 and 95%, respectively. Expression of CD18 was 2.6-fold higher for mammary neutrophils before injection of LPS, compared with blood neutrophils, either before or after LPS. In absence of opsonins, neutrophils with adherent and phagocytosed E coli averaged 83 and 14%. CONCLUSIONS: LPS modulated expression of CD14 and CD18 and lectin-carbohydrate interactions mediated nonopsonic phagocytosis of E coli. An intracellular pool of CD14 exists in bovine neutrophils and is capable of translocating to the cell surface. CLINICAL RELEVANCE: Development of methods to maximize expression of CD14 receptors on mammary neutrophils involved in production of tumor necrosis factor-alpha, and nonopsonic phagocytosis could result in reducing prevalence of mastitis in dairy cows.

Reactivity of purified complement component 3b with bovine neutrophils and modulation of complement receptor 1.

OBJECTIVE: To study binding of purified complement component C3b to bovine blood and mammary neutrophils (PMN) after various treatments and determine their ability to modulate receptor numbers. DESIGN: Cell isolation, activation, and flow cytometric studies. ANIMALS: Healthy lactating Holstein cattle. PROCEDURE: Complement component C3b (18,300 kd) was isolated from bovine serum by column chromatography, and flow cytometric assays using fluorescein isothiocyanate-labeled C3b were developed to evaluate binding to PMN complement receptor 1. Multiple substances were tested to determine their overall effect on C3b binding to PMN. Blood and milk PMN were isolated by differential centrifugation and exposed to optimal concentrations of recombinant human C5a, formyl-methyl leucyl phenylalanine, recombinant bovine interferon-gamma, variable concentrations of phorbol myristate acetate (0.01 to 100 ng), calcium ionophore A23187, serum-opsonized zymosan, zymosan-activated serum (ZAS), zymosan-activated plasma (ZAP), and hydrocortisone acetate (25 and 70 ng). Additionally, mammary and blood PMN were preincubated in skim milk and whey. RESULTS: Variable concentrations of phorbol myristate acetate caused a dose-dependent increase in percentage of PMN binding C3b, and increased the amount of C3b bound per cell. Significant increases were observed after PMN treatment with calcium ionophore, serum opsonized zymosan, ZAS, and ZAP; conversely, incubation of PMN with hydrocortisone acetate resulted in reduced overall binding of C3b. Mammary PMN consistently bound more C3b, which was attributed to their activation during migration into the mammary gland. Binding of C3b was inhibited by skim milk. Activation of blood PMN with PMA, ZAS, and ZAP elicited larger responses than those observed for mammary PMN. CONCLUSIONS: Modulation of complement receptors on bovine PMN is possible. Additionally, significant difference between the level of binding of C3b to blood and milk PMN, with milk PMN having higher binding, may be attributable to migration of PMN into the mammary gland, causing increased receptor expression. CLINICAL RELEVANCE: Contribution to a greater understanding of the role of complement in bovine immunologic systems, leading to testing for in vivo enhancement of bovine immune responses to invading pathogens.