Neutrophil activation associated with increased neutrophil acyloxyacyl hydrolase activity during inflammation in cattle.

Acyloxyacyl hydrolase (AOAH) is a lysosomal enzyme found in neutrophils and macrophages that acts to partially deacylate the lipid-A component of the endotoxin of gram-negative bacteria rendering it less toxic, yet maintaining much of its immunostimulatory potential. We have found that the activity of neutrophil AOAH per cell increased during localized inflammation. The purpose of this study was to determine the mechanism(s) responsible for these increases in neutrophil AOAH activity. Because changes in neutrophil maturity commonly are associated with inflammation, intravascular infusion of purified gram-negative bacterial lipopolysaccharide and SC injection of bovine recombinant granulocyte colony-stimulating factor was used to induce large numbers of circulating immature neutrophils. Immature neutrophils were found to have AOAH activity equal to that of mature cells; however, when neutrophils were stimulated in vitro with known activators, AOAH activity of activated cells was more than that of unstimulated cells. The increase in AOAH activity was inversely related to prestimulation activity. Increases in AOAH activity after neutrophil activation were not a result of de novo synthesis of the enzyme, because cycloheximide did not prevent activation-induced increases in activity.     

Aerobic blood culturing in cows with coliform mastitis

Aerobic culturing was performed on blood samples from 20 cows with coliform mastitis and signs of systemic illness, and on blood samples from 21 cows with mastitis caused by other agents. No bacteria other than the skin contaminant Bacillus sp were isolated from the blood.     

Studies on mammary blood flow during experimentalE. Coli endotoxin mastitis

The effect of intramammary (i.mam.) infusion ofEscherichia coli lipopolysaccharides (LPS) upon mammary blood flow (MBF) was studied in lactating goats using the electromagnetic flow technique. After i.mam. LPS administration mastitis, accompanied by a significant increase in MBF, occurred. This phenomenon showed two conspicuous peaks which occurred 11/2h and 8–10h after LPS administration. Other symptoms were: fever, increased heart rate, swelling and pain of the gland, increased chloride (MCl^?) and total cell count (TC) in milk. Since there was a marked coincidence between the increment stage of fever and the break-down of MBF peak I, studies were performed to evaluate the possible role of fever in this typical biphasis MBF pattern.

1. Mastitis was induced in one gland (homolateral) and MBF was recorded in the other (heterolateral) gland. During homolateral mastitis, clinical aspects, MCl^? and TC in the heterolateral gland were normal. For MBF, however, 3 types of responses could be distinguished. In 66% of the experiments (expts) there was a delayed MBF peak which coincided with MBF peak II of the homolateral mastitis gland. Furthermore, in 10% of these cases, a 1st MBF peak occurred simultaneously with MBF peak I of the homolateral gland. After tying off the vasoanastomoses between both udder halves, only the heterolateral MBF peak II persisted. In 33% no heterolateral peaks were present and in most cases a MBF decrease was observed between the 3rd and the 6th hours.
2. Large doses of prostaglandin E₂ (PGE₂) were infused intracerebroventricularly (i.c.v.) in normal, conscious lactating goats. In 58% of expts fever started quickly after infusion accompanied by a decrease in MBF. However, as soon as fever was established, MBF returned to normal and even rose above pre-infusion values.
3. The i.c.v. PGE₂ infusion was preceded (30 min) by i.mam. injection of LPS. In most expts fever appeared earlier than during mastitis control expts. Although symptoms of mastitis were observed, MBF peak I was lacking.
4. The antipyretic effects of 3 non-steroidal anti-inflammatory drugs (N.S.A.I.D.) on the biphasic MBF pattern during LPS mastitis were tested: Suprofen, Indomethacine and Flurbiprofen abolished fever, muscular shivers, myosis and tachycardia but had only little effect on the MBF pattern during LPS mastitis. MBF peak II was partially suppressed by these drugs, but most significantly by Flurbiprofen, which also delayed the appearance of MBF peak I. The latter is probably the consequence of its anti-inflammatory properties. None of these drugs could prevent the breakdown of MBF peak I.

It might be concluded that local mechanisms underlie the genesis of MBF peak I, but that in MBF peak II both systemic (e.g. fever) and local mechanisms are involved. The increment stage of fever may be responsible only to some extent for the breakdown of MBF peak I. Other factors than fever per se must be involved.     

Arachidonic acid metabolites in milk of cows during acute coliform mastitis

Concentrations of prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TXB2) were evaluated in the milk of cows with naturally occurring (n = 3) and experimentally induced (n = 5) acute coliform mastitis. These arachidonic acid metabolites were measured by radioimmunoassay in unextracted milk. Experimental infections were induced by inoculating 600 to 1,200 colony-forming units of Escherichia coli into 1 mammary quarter per experimental cow. In the experimental cows, milk was collected before inoculation and at 12, 24, 36, 48, 60, and 72 hours after inoculation. Somatic cell concentrations, bovine serum albumin, and concentrations of PGF2 alpha and TXB2 were determined in milk collected at each sampling. Mild-to-moderate increases in milk PGF2 alpha and TXB2 concentrations were observed in cows with naturally occurring mastitis. the increases corresponded to the clinical severity and course of mastitis. In the experimental cows, increases in milk PGF2 alpha and TXB2 concentrations were observed, but the increases were not significant, using a statistical model that included factors of treatment, cows, hours after inoculation, cows-by-treatment and hours-by-treatment interactions, and random error (residual). Results of the present study indicated a large biological variability in milk arachidonic acid metabolite concentrations in cows with acute coliform mastitis, and that arachidonic acid metabolites may be important in the pathophysiologic process of acute coliform mastitis.     

Acute coliform mastitis in buffaloes (Bubalus bubalis): Clinical findings and treatment outcomes

This report was delineated to study the clinical, bacteriological and therapeutic aspects concerned with acute coliform mastitis in buffaloes. Bacteriological examination of 80 quarter milk samples obtained aseptically from 56 buffaloes with acute mastitis revealed that coliform bacteria was the most common pathogen (45 cases) followed by Staphylococcus aureus (seven cases) then Streptococcus uberis (three cases), and Streptococcus agalactiae (one case). Clinically, hotness, swelling and painful reaction with serous excretion containing clots was recorded in buffaloes with coliform mastitis. The efficacy of ceftiofur was evaluated in the treatment of buffaloes with acute coliform mastitis. Parenteral ceftiofur neither improved clinical signs nor returned milk to pre-infection production level, whereas intramammary ceftiofur and combination of intramammary with parenteral ceftiofur improved the clinical signs in 10/15 and 12/15 buffaloes, respectively. On quarter level, 3/17, 12/17 and 15/21 quarters recovered in groups received parenteral, intramammary and combination therapy, respectively. This study demonstrates that systemic ceftofur is not effective in the treatment of clinical coliform mastitis in buffaloes.     

Serum amyloid A and TNF alpha in serum and milk during experimental endotoxin mastitis

A cross-over study was conducted to investigate the effect of intramammarily infused lipopolysaccharide (LPS) on the acute phase reaction in early (EL) and in late (LL) lactation. Nine cows received intramammary injections of 100 microg of Escherichia coli 0111:B4 LPS during EL and LL. The severity of each cows systemic and local signs and change in milk appearance were recorded and scored throughout the experiment. Systemic and local signs were found to be more serious in EL cows. Tumor necrosis factor alpha (TNF alpha) was detected in milk but not in serum. Serum amyloid A (SAA) concentrations increased both in serum and in milk. The milk TNF alpha concentrations peaked at 8 h post-challenge (PC). SAA concentrations started to increase at 8 h PC, and peak concentrations were seen at 32 and 48 h PC in milk and serum, respectively. The milk TNF alpha and SAA seemed to be correlated, being on average higher in EL. Serum SAA concentration was not correlated with milk TNF alpha or SAA, nor with the severity of local or systemic signs, but was correlated with changes in milk appearance.     

Bovine mastitis therapy and why it fails

Treatment of bovine mastitis depends on the cause, the clinical manifestation and the antibiotic susceptibility of the agent. Mastitis therapy is commonly unsuccessful owing to pathological changes that occur in the udder parenchyma as a result of the inflammatory reaction to mastitogenic bacteria, pharmacokinetic properties of antimicrobial mastitis drugs, mastitogenic bacterial and related factors, and poor animal husbandry and veterinary interventions.     

Antibiotic sensitivity of bovine staphylococcal and coliform mastitis isolates over four years

Eight hundred and forty-eight strains of Staphylococcus aureus and coliforms isolated from milk samples taken from cows with clinical or subclinical mastitis were tested for their sensitivity to a range of antibiotics, comparing strains isolated in 1984, 1985, 1986 and 1987. The only increase in the proportion of resistant strains occurred with coliforms resistant to ampicillin and neomycin.     

Sympathoadrenal and immune system activation during the periparturient period and their association with bovine coliform mastitis. A review

Increased incidence of clinical mastitis in high-yielding cows during early lactation has been attributed to a depressed functional capacity of the immune system. Sympathoadrenal factors are known to play an important role in modulating the host susceptibility and resistance to infectious diseases. Of primary importance in combating acute intramammary infections are polymorphonuclear leukocytes (PMN), as they represent one of the early lines of immunological defense. The release of stress hormones at parturition and during the first weeks of lactation has been proposed to partly contribute to the impaired function of PMN. Here, we summarize the current understanding of the stress-induced peripheral effectors, i.e. the limbs of the sympathetic system and the hypothalamic-pituitary-adrenal axis, on PMN function around parturition and during coliform mastitis. The questions as to whether and how stress induced secretion of glucocorticoids and catecholamines might affect the lactating dairy cow's udder health will be addressed.     

Relationship between serologic recognition of Escherichia coli 0111:B4 (J5) and clinical coliform mastitis in cattle

Serum IgG1 ELISA titers recognizing gram-negative core antigens (Escherichia coli [J5]) were studied at a large dairy in central California. Population mean log10 titer was 2.7357 (equivalent to 1:544) with a SE of 0.03843. Titers increased with increased lactation number (unstandardized regression coefficient = 0.06733). Changes in lactation number accounted for only 6.77% of titer variation. Titers less than 1:240 were associated with 5.33 times the risk of clinical coliform mastitis. Also, older cattle were at greater risk to develop clinical coliform mastitis. These factors apparently affect incidence in a nonlinear fashion, with greatly increased risk associated with titers less than 1:240 and with fourth or greater lactations.     

The proteomic advantage: label-free quantification of proteins expressed in bovine milk during experimentally induced coliform mastitis

Coliform mastitis remains a primary focus of dairy cattle disease research due in part to the lack of efficacious treatment options for the deleterious side effects of exposure to LPS, including profound intra-mammary inflammation. To facilitate new veterinary drug approvals, reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis. Most attempts to characterize the host response to LPS, however, have been accomplished using ELISAs. Because a relatively limited number of bovine-specific antibodies are commercially available, reliance on antibodies can be very limiting for biomarker discovery. Conversely, proteomic approaches boast the capability to analyze an unlimited number of protein targets in a single experiment, independent of antibody availability. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), a widely used proteomic strategy for the identification of proteins in complex mixtures, has gained popularity as a means to characterize proteins in various bovine milk fractions, both under normal physiological conditions as well as during clinical mastitis. The biological complexity of bovine milk has, however, precluded the complete annotation of the bovine milk proteome. Conventional approaches to reducing sample complexity, including fractionation and the removal of high abundance proteins, has improved proteome coverage, but the dynamic range of proteins present, and abundance of a relatively small number of proteins, continues to hinder comparative proteomic analyses of bovine milk. Nonetheless, advances in both liquid chromatography and mass spectrometry instrumentation, including nano-flow liquid chromatography (nano-LC), nano-spray ionization, and faster scanning speeds and ionization efficiency of mass spectrometers, have improved analyses of complex samples. In the current paper, we review the proteomic approaches used to conduct comparative analyses of milk from healthy cows and cows with clinical mastitis, as well as proteins related to the host response that have been identified in mastitic milk. Additionally, we present data that suggests the potential utility of LC-MS/MS label-free quantification as an alternative to costly labeling strategies for the relative quantification of individual proteins in complex mixtures. Temporal expression patterns generated using spectral counts, an LC-MS/MS label-free quantification strategy, corresponded well with ELISA data for acute phase proteins with commercially available antibodies. Combined, the capability to identify low abundance proteins, and the potential to generate temporal expression profiles, indicate the advantages of using proteomics as a screening tool in biomarker discovery analyses to assess biologically relevant proteins modulated during disease, including previously uncharacterized targets.     

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.     

Phagocytosis of serum-resistant and serum-sensitive coliform bacteria (Klebsiella) by bovine neutrophils from blood and mastitic milk

Phagocytic activity of neutrophil leukocytes from bovine blood and mastitic milk was determined for 2 strains of Klebsiella, 1 resistant and the other sensitive to serum bactericidal activity. Both strains were easily phagocytized in the presence of an opsonic agent, but milk neutrophils seemed to be less efficient than blood neutrophils in this respect. Phagocytosis was maximal after incubation for 60 minutes at 37 C and decreased markedly with reduction in incubation temperature. The opsonic activity of mastitic milk was considerably higher than that of normal milk and approached that of fresh bovine serum. Precolostral calf serum was deficient in opsonic activity and anti-bovine leukocyte serum was antiphagocytic.