Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin

Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows. There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms. Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows. After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8. The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively. The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively. A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of sows to experimentally induced Escherichia coli mastitis

A study was conducted to compare susceptibility of sows from 2 herds to experimentally induced Escherichia coli mastitis. Four sows each from herds R and S were inoculated intramammarily at postpartum hour 8 with a strain of E coli shown previously to be capable of producing mastitis. After inoculation with E coli, sows from herd S had higher temperatures, lower WBC counts, and lower plasma protein:fibrinogen ratios than did sows from herd R. Inoculated sows from herd S lost 83% of newborn pigs due to starvation by 14 days after inoculation, whereas sows from herd R lost none. Control, noninoculated sows from both herds had normal temperatures, normal hematologic values, and minimal mortality of piglets. Levels of antibodies (complement fixing, enzyme-linked immunosorbent assay, and agglutinating) to E coli in preinoculation sera from the 2 populations of sows did not differ. Assay of lactoferrin by radial immunoassay revealed comparable concentrations in milk of sows from both herds during the first 24 hours after sows had delivered, but significantly higher values were detected in milk from sows of herd S at postpartum days 2 and 3. The basis for the marked difference in susceptibility to E coli-induced mastitis was not determined except that "susceptible" sows (herd S) were from a conventional herd and "resistant" sows (herd R) were from a specific-pathogen-free herd.     

The efficacy of bovine lactoferrin in the treatment of cows with experimentally induced Escherichia coli mastitis

The effect of bovine lactoferrin (Lf) was studied in experimental Escherichia coli mastitis, using enrofloxacin as a comparator. Mastitis was induced in six clinically healthy primiparous dairy cows by infusing 1500 colony-forming units of E. coli into a single udder quarter. The challenge was repeated into a contralateral quarter of the same cows 3 weeks later. At the first challenge, three cows were treated with 1.5 g of bovine lactoferrin intramammarily three times (12, 20 and 36 h postchallenge, PC), and the other three cows received 5 mg/kg of enrofloxacin (Baytril) parenterally (12, 36 and 60 h PC). Flunixin meglumine (2.2 mg/kg) was administered to all cows twice at 24-h intervals. During the second challenge, the treatments for the two groups were reversed. Intramammary challenge with E. coli produced clinical mastitis in all cows, but the severity of the disease varied markedly. No statistically significant differences between treatment groups were observed in clinical signs such as rectal temperature, rumen motility and general attitude. Milk somatic cell count, daily milk yield and bacterial counts in cows treated with Lf and those receiving enrofloxacin also did not differ significantly. However, a trend for a more rapid elimination of bacteria was seen in the cows treated with enrofloxacin. Milk NAGase activity also decreased significantly faster in the group treated with enrofloxacin. The concentration of lipopolysaccharide in milk compared with the number of bacteria was significantly lower in Lf than in enrofloxacin-treated cows (20 h PC).     

Acute phase response in dairy cows with experimentally induced Escherichia coli mastitis.

Six Finnish Ayrshire cows were challenged intramammarily with 1500 CFU of Escherichia coli (E. coli) into single udder quarters, and the challenge was repeated into contralateral quarters 3 weeks later. All cows received flunixine meglumine once, and 3 of them were also treated with enrofloxacin. At the 2nd challenge, treatments were changed vice versa. The development of mastitis was followed by monitoring of systemic and local clinical signs, and with serial milk and serum samples. Intramammary challenge with E. coli produced clinical mastitis in all cows, the severity of the disease varying greatly between the animals. No significant changes between the 2 treatment regimens or sequent challenges were found for any of the clinical parameters. The response of each cow followed the same pattern after both challenges; three of the cows became mildly and the other 3 either moderately or severely affected. Two severely affected cows had to be euthanized because of severe mastitis. Serum haptoglobin and amyloid-A concentrations peaked 2-3 days after bacterial challenge. Serum haptoglobin did not correlate with the severity of the disease. Serum amyloid-A rose gradually in the severely affected cows, and significant differences were found between severely versus moderately or mildly affected cows at day 4. Serum tumor necrosis factor alpha concentrations increased only in the severely affected cows. Serum cortisol response was prolonged in the severely diseased animals, and was significantly lower after the second challenge. Serum nitrite/nitrate concentration increased in the severely affected cows. This indicated excess nitric oxide production during acute E. coli mastitis. Strongly decreased milk production, and high bacterial growth in the infected quarters were best predictors for the outcome from acute E. coli mastitis.     

Depression of polymorphonuclear leukocyte function associated with experimentally induced Escherichia coli mastitis in sows

In a study of susceptibilities of sows from 2 herds to experimentally induced Escherichia coli mastitis, a marked difference was seen. The "susceptible" sows were from a conventional herd and "resistant" sows were from a specific-pathogen-free herd. The purpose of the study was to determine whether deficient neutrophil function was associated with increased susceptibility to E coli-induced mastitis. Four in vitro procedures were used to evaluate polymorphonuclear leukocyte (PMN) function: (i) random migration under agarose, (ii) ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus, (iii) quantitative nitroblue tetrazolium reduction, and (iv) iodination. After parturition and intramammary inoculation with E coli, sows from the susceptible herd were neutropenic and the neutrophils which were present in the peripheral blood had reduced function. Specifically, there were depressed random migration under agarose, S aureus ingestion, and iodination when compared with PMN function in resistant sows. These data indicate that susceptibility to E coli mastitis was associated with deficiencies in PMN numbers and function. Potential causes of the neutrophil dysfunction are discussed and include possible systemic hormonal aberrations or the presence of an inapparent viral or bacterial infection.     

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.     

Changes in thermal nociceptive responses in dairy cows following experimentally induced Escherichia coli mastitis

Background

Mastitis is a high incidence disease in dairy cows. The acute stage is considered painful and inflammation can lead to hyperalgesia and thereby contribute to decreased welfare. The aim of this study was to examine changes in nociceptive responses toward cutaneous nociceptive laser stimulation (NLS) in dairy cows with experimentally induced Escherichia coli mastitis, and correlate behavioral changes in nociceptive responses to clinical and paraclinical variables.

Methods

Seven Danish Holstein-Friesian cows were kept in tie-stalls, where the E. coli associated mastitis was induced and laser stimulations were conducted. Measurements of rectal temperature, somatic cell counts, white blood cell counts and E. coli counts were conducted. Furthermore, scores were given for anorexia, local udder inflammation and milk appearance to quantify the local and systemic disease response. In order to quantify the nociceptive threshold, behavioral responses toward cutaneous NLS applied to six skin areas at the tarsus/metatarsus and udder hind quarters were registered at evening milking on day 0 (control) and days 1, 2, 3, 6 and 10 after experimental induction of mastitis.

Results

All clinical and paraclinical variables were affected by the induced mastitis. All cows were clinically ill on days 1 and 2. The cows responded behaviorally toward the NLS. For hind leg stimulation, the proportion of cows responding by stepping was higher on day 0 than days 3 and 6, and the frequency of leg movements after laser stimulation tended to decrease on day 1 compared to the other days. After udder stimulation, the proportion of cows responding by stepping was higher on day 1 than on all other days of testing. Significant correlations between the clinical and paraclinical variables of disease and the behavioral responses toward nociceptive stimulation were found.

Conclusions

Changes in behavioral responses coincide with peaks in local and systemic signs of E. coli mastitis. During the acute stage of E. coli mastitis nociceptive thermal stimulation on hind leg and mammary glands results in decreased behavioral responses toward nociceptive stimulation, which might be interpreted as hypoalgesia.     

Identification of a bovine mastitis Escherichia coli subset

Eleven Escherichia coli isolates from clinical bovine mastitis cases (mastitic strains) and 11 from the cowshed environment (environmental strains) were compared, to determine if the former were a subset of the latter. The mastitic and environmental strains could not be distinguished according to O antigen and antibiotic sensitivity. All mastitic isolates showed significantly (P<0.0001) faster growth in milk and faster lactose fermentation than most (approximately 64%) environmental strains, but growth rates in nutrient broth did not differ. The rates of lactose fermentation and growth in milk were positively correlated. Adhesion and phagocytosis of mastitic strains by bovine PMN were significantly (P<0.0001) lower than those of environmental strains, and correlated negatively with growth in milk and lactose fermentation. The average percentages of killing by bovine leukocytes in the two sources were not statistically different. All mastitic strains were serum sensitive, whereas most ( approximately 72%) environmental ones were resistant. Finally, pulse-field gel electrophoresis revealed two main pulse type clusters, sharing a similarity coefficient of 79%. Cluster 1 comprised only environmental strains, whereas cluster 2 comprised mostly mastitic strains and only three environmental ones. Four mastitic strains shared a similarity coefficient of less than 74% with the other strains and were not included in the clusters. Our results suggest that clinical bovine mastitis E. coli isolates may form a subset of the general environmental E. coli population; they seem better able to multiply in the udder medium and to evade the host cellular innate immune response, and are genetically distinct from most environmental strains.     

Host-pathogen gene expression profiles during infection of primary bovine mammary epithelial cells with Escherichia coli strains associated with acute or persistent bovine mastitis

Escherichia coli intramammary infection (IMI) is often acute with local and systemic clinical manifestations that clear within 7 days. However, if not diagnosed early and treated, E. coli IMI could result in generalized systemic reaction and death. Persistent E. coli IMI is characterized by mild clinical manifestations followed by acute episodes of clinical mastitis during lactation. Factors responsible for pathogenesis of E. coli IMI and variation in clinical manifestations are not known. There are studies indicating that the outcome of E. coli IMI is mainly determined by cow factors. However, recent research demonstrated that virulence attributes of E. coli strains have significant impact on the outcome of E. coli IMI. The aims of this study were; (a) to compare gene expression profiles of PBMEC cocultured with strains of E. coli associated with acute or persistent IMI and; (b) to identify genes of E. coli induced during bacterial interaction with PBMEC. Utilizing cDNA we analyzed gene expression patterns of PBMEC cocultured with strains of E. coli using non-treated PBMEC as negative control. We evaluated also expression patterns of virulence associated genes of E. coli after co-culture with PBMEC using qRT-PCR. Our results showed that infection by both strains induced increased expression of pro-inflammatory cytokines, chemokines and innate immune response and apoptosis related genes. Our qRT-PCR results showed significant up-regulation of ler, eae, flic and iutA genes mainly in the strains of E. coli associated with persistent IMI. The pathogenesis and clinical severity of E. coli IMI may be determined by combined effects of host-pathogen factors.     

Controlling acute Escherichia coli mastitis during the periparturient period with recombinant bovine interferon gamma

The efficacy of recombinant bovine interferon (rBoIFN)-gamma against experimentally induced Escherichia coli mastitis during the periparturient period was investigated. Dairy cows intramammarily treated with rBoIFN-gamma 24 h before the E. coli challenge had fewer infected quarters, lower clinical scores, and infections of shorter duration when compared to placebo-treated animals. All rBoIFN-gamma treated cows survived the experimental E. coli challenge. However, placebo treated cows had a 42% mortality rate attributed to coliform mastitis within 3 days of the challenge. Results from this study suggest that intramammary infusion of rBoIFN-gamma can prevent the rapid, unrestricted growth of E. coli within the mammary gland and inhibit the subsequent development of an unlimited inflammatory response under experimental conditions. It is likely that controlling severe local inflammatory reactions may also decrease the pathological alterations to mammary parenchymal tissue that often accompanies acute coliform mastitis during the periparturient period. The potential for prophylactic treatment of perinatal dairy cows with rBoIFN-gamma to regulate the rate, severity, and duration of naturally occurring coliform mastitis during periods of heightened susceptibility is discussed.     

Intramammary administration of gentamicin as treatment for experimentally induced Escherichia coli mastitis in cows.

In 8 Holstein cows, 50 colony-forming units (CFU) of Escherichia coli was administered into 1 mammary gland. Infections were established in all inoculated glands. In 4 of the 8 cows, 500 mg of gentamicin sulfate was administered by intramammary infusion 14 hours after inoculation; the other 4 cows were untreated controls. Infusions of gentamicin also were given after each of the 3 successive milkings after the initial infusion, so that a total dose of 2 g of gentamicin was given to each of the treated cows. During the 33-hour treatment period and for the first milking after the last infusion of gentamicin, the treated cows had a mean gentamicin concentration of greater than or equal to 31.0 micrograms/ml in milk samples that were collected from inoculated quarters immediately before each milking. Concentrations of 0.34 and 0.69 micrograms of gentamicin/ml were detected in milk from 2 cows at 8 days after inoculation with E coli. Mean serum concentrations of gentamicin were greater than or equal to 0.37 micrograms/ml throughout the treatment period and the first 12 hours after the last infusion, with a mean peak concentration of 0.96 micrograms/ml at 24.4 hours. The range of peak concentration of gentamicin detected in urine from all treated cows was 42 to 74.4 micrograms/ml. Peak concentration of E coli in milk in the treated cows (6.08 +/- 1.02 log10 CFU/ml) did not significantly (P greater than 0.05) differ from that of the control cows (5.26 +/- 1.00 log10 CFU/ml). Similarly, mean duration of infection in the treated cows (54 hours) did not differ significantly from that of the control cows (48 hours).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequestration of N-acetyl-beta-D-glucosaminidase in somatic cells during experimental bovine mastitis induced by endotoxin, Staphylococcus aureus or Streptococcus agalactiae

Experimental mastitis was induced in cows by intramammary infusion of endotoxin, Staphylococcus aureus or Streptococcus agalactiae. The inflammatory response was monitored by somatic cell counting and N-acetyl-beta-D-glucosaminidase (NAGase). NAGase activity was analysed in fresh milk samples in parallel with samples treated by a cycle of freezing and thawing combined with detergent treatment to release the cell-bound NAGase. Before the udder reacted by inflammation, the total NAGase activity consisted of free extracellular activity. Later on when the inflammation was established, much of the milk NAGase remained sequestered intracellularly. S agalactiae was linked with a high degree of cellular NAGase sequestration indicating a blockage of the lysosomal release function from the phagocytes. S aureus delayed the inflammatory response.     

Efficacy and pharmacokinetics of enrofloxacin and flunixin meglumine for treatment of cows with experimentally induced Escherichia coli mastitis

The efficacy of flunixin alone and together with enrofloxacin in treatment of experimental Escherichia coli mastitis was compared using six cows. The cross-over study design was used. Pharmacokinetics of flunixin and enrofloxacin were also studied in these diseased cows. The response of each cow was similar after the first and second challenge and the individual reaction seemed to explain the severity of clinical signs. The most important predictive factor for outcome of E. coli mastitis was a heavy drop in milk yield. Treatment with enrofloxacin and flunixin enhanced elimination of bacteria, but the difference from those receiving flunixin alone was not significant. Two cows, which had received no antimicrobial treatment (Group 1), were killed on day 4 postchallenge. One cow was killed after the first and the other after the second challenge. Cows receiving combination therapy produced 0.9 L more milk per day during the study period than cows which had only received flunixin (P < 0.05). Based on our findings, antimicrobial treatment might be beneficial in the treatment of high-yielding cows in early lactation. The absorption of enrofloxacin was delayed after subcutaneous administration, the mean apparent elimination half-life being about 23 h, whereas after i.v. administration elimination t(1/2) was only 1.5 h. The majority of the antimicrobial activity in milk originated from the active metabolite, ciprofloxacin, which could be measured throughout the 120-h follow-up period after the last subcutaneous administration. No differences were present in the pharmacokinetic parameters of flunixin between treatment groups: mean elimination half-life was 5.7-6.2 h, volume of distribution 0.43-0.49 L/kg and clearance 0.13-0.14 L h/kg. No flunixin or merely traces were detected in milk: one of the three cows had a concentration of 0.019 mg/L 8 h after administration.     

Neutrophil apoptosis during experimentally induced Staphylococcus aureus mastitis

The objective of this study was to determine whether neutrophil apoptosis and their consequent elimination by macrophages from the mammary gland is modulated by an infection caused by Staphylococcus aureus (S. aureus). The study was performed on twenty mammary glands of 5 virgin heifers. A buffered physiological solution (PBS) was administered as a means of control into the mammary glands of the heifers and after 168 h, the glands were inoculated with S. aureus. The samples of cell populations were obtained by lavages of the mammary glands in 4 intervals (24, 48, 72 and 168 h) after the experimental infection. Flow cytometry was used for determination of Annexin-V positivity and propidium iodide (PI) negativity of neutrophils. Light microscopy was used for determination of neutrophil karyopyknosis. Cytochemistry was used for the detection of myeloperoxidase-positive (MPO+) macrophages. Instillation of S. aureus resulted in an intramammary infection which persisted during the following experimental period. The total number of both Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils peaked at 24 h after both of PBS and S. aureus administration. The highest percentages of Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils were detected 48 and 168 h after PBS and S. aureus administration, respectively. The total number of MPO+ macrophages was the highest 24 h and 48 h after PBS and S. aureus administration, respectively; the percentage of MPO+ macrophages was the highest at 72 h in both cases. The dynamics of resolution of mastitis caused by S. aureus was very similar to the resolution of inflammatory response of the mammary gland after PBS administration. Mechanisms of cell pathogen elimination as well as inflammation resolution were very intensively involved; nevertheless, the mammary gland infection persisted. An early inclusion of the mechanisms of an acute inflammatory resolution thus paradoxically led to chronic infection.     

Adherent and invasive Escherichia coli are associated with persistent bovine mastitis

Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.     

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

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