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.     

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).     

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.     

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.     

Evaluation of frequent milkout for treatment of cows with experimentally induced Escherichia coil mastitis

OBJECTIVE: To evaluate the effect of frequent milkout (FMO) on the outcome of experimentally induced Escherichia coli mastitis in cows. DESIGN: Randomized complete block study. ANIMALS: 16 Holstein dairy cows. PROCEDURE: Cows were randomly assigned to 1 of 4 groups and were either not infected and not treated (NI-NT), experimentally infected with E coli and not treated (EC-NT), not infected and FMO (NI-FMO), or experimentally infected with E coli and FMO (EC-FMO). The infected quarter in cows in FMO groups was milked out every 4 hours from 16 to 36 hours and every 6 hours from 36 to 84 hours after challenge, with the aid of oxytocin administration. Somatic cell counts (SCC); times to bacterial, clinical, and systemic cures; and serum concentrations of a-lactalbumin were determined. RESULTS: Use of FMO did not appear to affect SCC. For EC-NT and EC-FMO groups, mean bacterial cure times were 203 and 159 hours, clinical cure times were 276 and 360 hours, and systemic cure times were 144 and 159 hours, respectively; these times were not significantly different. Concentrations of a-lactalbumin were significantly increased in the EC-NT group at 12 hours and in the NI-FMO group at 36 and 60 hours after challenge, compared with values of cows in other treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with results in control cows, FMO does not appear to be an efficacious treatment for experimentally induced moderate to severe E coli mastitis.     

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.     

Evaluation of systemic administration of gentamicin for treatment of coliform mastitis in cows

Recovery of cows (n = 61) with mastitis caused by gram-negative bacteria and treated systemically with an antibiotic (gentamicin) to which the bacteria were susceptible in vitro, was compared with recovery of cows (n = 13) with similar infections treated with a systemically administered antibiotic (erythromycin) to which the bacteria were resistant in vitro or with recovery of cows (n = 12) not given an antibiotic systemically. In the first part of the study, cows were selected for treatment groups by use of a diagnostic scheme designed to predict whether the mastitis was caused by gram-negative or gram-positive bacteria. In the second part of the study, all cows were treated without systemic administration of an antibiotic. Significant difference was not observed in the outcome of the disease between cows given gentamicin and cows of the other 2 treatment groups at 24 hours or at 4 weeks after treatment. At 24 hours after initial treatment, 71.9% of cows treated with gentamicin, 92.3% of those treated with erythromycin, and 45.5% not treated systemically had improved appetite. At 4 weeks after initial treatment, of the cows treated with gentamicin, 11.5% died; in 32.8%, lactation ceased in the affected mammary gland; in 21.3%, lactation was decreased in the affected gland; and 34.4% returned to normal lactation and health. Of cows treated with erythromycin, none died; in 23%, lactation ceased in the affected mammary gland; in 23%, lactation decreased in the affected gland; and 54% returned to normal lactation and health.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimal milk penicillin levels for the treatment of experimentally induced mastitis in cows.

Infection of the mammary gland (mastitis) was produced by infusing ten quarters 2/cow x 5 cows) with Staphylococcus aureus strain 305. Mastitic and normal quarters were then infused with a preparation containing two levels of penicillin G (100,000 and 200,000 IU) in 10 mL of 3% aluminum monostearate and peanut oil. Milk penicillin levels were determined prior to treatment and twice daily for eight milkings after treatment. Normal and mastitic quarters infused with 200,000 IU had higher peak levels than those infused with 100,000 IU. Milk penicillin levels were similar in mastitic and normal quarters for the first three milkings after treatment. However, residues persisted for a longer time in milk from mastitic quarters. Penicillin was not detected in milk from the untreated control quarters nor in serum samples assayed during the experiment. The in vitro penicillin G sensitivity of the udder pathogen (MIC=0.039 and MBC=0.078 IU) was well below the milk penicillin levels for the first five milkings in all cases. However, infection recurred in two of the ten quarters (one receiving 100,000 IU and one receiving 200,000 IU).     

Apoptosis of bovine neutrophils during mastitis experimentally induced with Escherichia coli or endotoxin

OBJECTIVE: To determine whether apoptosis of neutrophils was accelerated during mastits experimentally induced by use of Escherichia coli or E coli endotoxin and whether differences were apparent in the response to E coli or endotoxin. ANIMALS: 11 healthy lactating Holstein cows. PROCEDURE: Blood samples were collected from cows at various intervals after intramammary inoculation with E coli or endotoxin. Percentage of apoptotic neutrophils detected after in vitro incubation for 3 hours was determined. Fluorescein isothiocyanate-labeled annexin-V in combination with propidium iodide was used to distinguish apoptosis and necrosis of neutrophils. Total and differential circulating leukocyte counts and rectal temperature were determined at the time of collection of blood samples. Milk yield and milk somatic cell counts were determined at the time of milking. RESULTS: Inoculation of endotoxin did not accelerate in vitro induction of neutrophil apoptosis. However, inoculation of E coli increased the percentage of apoptotic neutrophils. At 18 hours after inoculation, 20% of the neutrophils were apoptotic, compared with 5% before inoculation. Milk somatic cell count and rectal temperature increased, milk production and total leukocyte count decreased, and percentage of immature neutrophils increased after inoculation with E coli or endotoxin. However, kinetics of the responses were more rapid, more severe, and of shorter duration during endotoxin-induced mastitis. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro induction of apoptosis of neutrophils was accelerated only during E coli-induced mastitis and not during endotoxin-induced mastitis. Endotoxin inoculation as a model for studying coliform mastitis in dairy cows should be viewed with caution.     

Epigenetic mechanisms contribute to enhanced expression of immune response genes in the liver of cows after experimentally induced Escherichia coli mastitis

Endotoxins, such as lipopolysaccharide (LPS), are released during infection with Gram-negative bacteria, which can result in excessive activation of toll-like receptor (TLR) signalling. The aim of the present study was to investigate whether epigenetic mechanisms are involved in controlling the onset and progression of the systemic inflammatory response. Using chromatin accessibility by real-time (CHART) PCR to assess livers from cows with experimentally induced Escherichia coli mastitis, this study demonstrated that the chromatin at the site of the promoters of the genes encoding TLR2, TLR4, lipopolysaccharide binding protein (LBP) and haptoglobin (HP) was opened up 24 h after infection, accompanied by enhanced mRNA expression by these genes. Such modulation did not occur in the same samples for the αS1-casein promoter, which served as a negative control. Demethylation of the TLR4 promoter accompanied opening up of chromatin. These data suggest that modulation of epigenetic factors might offer a novel approach to treating adverse systemic reactions elicited in cows with 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.     

Progressive pathology of severe Escherichia coli mastitis in dairy cows

An investigation was made using light and electron microscopy of the progressive pathological changes in nine experimental and two natural cases of severe Escherichia coli mastitis in dairy cows. The duration of infection varied from 18 hours to 13 days. Epithelial lesions were not found in glands which had been infected for more than 24 hours. However, the epithelia of the sinuses and large ducts became hyperplastic after 60 hours of infection and by six days hyperplasia was extensive on the crests of folds. The leucocyte response in the lumina of the glands and subepithelial tissue showed a progressive change from an acute neutrophil reaction to a chronic mononuclear cell infiltration within the first 36 hours of infection. The only changes affecting the secretory tissue occurred after six days of infection and were typical of mammary gland involution which was probably a direct consequence of anorexia.     

Flunixin meglumine and flurbiprofen in cows with experimental Escherichia coli mastitis

The effect of flunixin meglumine and flurbiprofen on the course of experimental Escherichia coli mastitis was examined. Nine cows (within one month post partum) were inoculated intramammarily with 20 x 10(5) viable E coli in both rear quarters. Three cows remained untreated (controls); three cows received three injections of flunixin meglumine and three cows received flurbiprofen as two intravenous infusions. Flunixin meglumine and flurbiprofen were initially given before clinical signs were observed. Treatment was repeated if the cows' temperature increased by more than 1 degree C. In the untreated cows, rectal temperature and heart rate increased from three hours after infection, and rumen motility (both frequency and amplitude) decreased from four hours after infection. Treatment with flunixin meglumine or flurbiprofen almost completely abolished the febrile response during the first nine hours after infection, and the decrease in rumen motility was less pronounced in the treated animals. These results suggest that the decrease in rumen motility during E coli mastitis is at least partly due to a mechanism involving prostaglandin.     

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

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

Influence of liver fat on experimental Escherichia coli mastitis in periparturient cows

Eleven cows with a wide range of liver fat (5.7 to 51.4 per cent) at seven days post partum were experimentally infected in a single quarter with a capsular Escherichia coli at 10 days post partum. The results suggested that a fatty liver in itself does not influence the severity of mastitis. All animals had clinical mastitis 10 hours after infection but no animals became severely ill and no treatment was given. Four out of five animals in the group with less than 20.2 per cent liver fat had bacteria in their milk at 10 hours after infection but these bacteria were eliminated by 12 hours. The six animals in the group with more than 28.3 per cent fat in their liver retained viable bacteria in the udder for much longer; with two animals bacteria were shed and abnormal milk was secreted for up to four months despite antibiotic therapy.     

Induction of Escherichia coli mastitis in cows fed selenium-deficient or selenium-supplemented diets

Ten Holstein heifers were fed a selenium-deficient (SeD) diet (0.04 mg of Se/kg on a total ration dry-matter basis) 3 months before calving and throughout their first lactation. A selenium-supplemented (SeS) diet (2 mg of Se/head/d) was fed to a group of 10 heifers. In about the 14th week of lactation, the cows were challenge-exposed to Escherichia coli by administering 15 to 40 colony-forming units (CFU) into 1 mammary gland. Selenium concentration (microgram/ml) in blood around the time of challenge exposure was 0.033 +/- 0.002 (mean +/- SEM) in SeD and 0.132 +/- 0.006 in SeS cows. Infections were established in all challenge-exposed quarters. The frequency of quarter atrophy and agalactia, and reduction in whole-udder milk yield in the first 4 days after challenge exposure, were greater (P less than 0.05) in the SeD cows. Log10 peak bacterial concentrations in milk were higher (P less than 0.05) in SeD (7.63 +/- 0.34 CFU/ml) than in SeS cows (5.57 +/- 0.66 CFU/ml). Mean log bacterial concentration was significantly higher (P less than 0.05) from 12 to 20 hours after challenge exposure in SeD than in SeS cows. Duration of infection was significantly greater (P less than 0.05) in SeD (162.0 +/- 12.0) than in SeS cows (114.4 +/- 18.0 hours). Milk somatic cell counts increased significantly more slowly (P less than 0.05) in SeD than in SeS cows from 8 to 16 hours after challenge exposure. Ratios of milk somatic cells to bacteria in milk were significantly lower (P less than 0.05) in SeD than in SeS cows at 12 and 16 hours after challenge exposure.     

Virulence factors of Escherichia coli isolated from cows with acute mastitis

A total of 184 Escherichia coli isolates recovered from cows with acute mastitis were examined for recognized pathogenic mechanisms and virulence factors commonly found in pathogenic groups of E coli. A modification of the Eng procedure (for detecting complement deficiencies in serum) was used to test for resistance to different animal sera. The Sereny test (for invasiveness), infant mouse test (for heat-stable enterotoxin), and Y-1 adrenal tumor cell assay (for heat-labile enterotoxin) were used. Hemagglutination tests, using rabbit, sheep, and guinea pig RBC, were done with and without added mannose. All of the 184 isolates were serum resistant in all tested sera. None of the isolates was invasive. Only 1 isolate was positive for heat-stable enterotoxin and 2 cultures were positive for heat-labile enterotoxin. Multiple patterns of hemagglutination were observed. The majority of the isolates exhibited both mannose-sensitive and mannose-resistant hemagglutinins with guinea pig and rabbit RBC. A few strains were positive only in mannose-sensitive or mannose-resistant hemagglutination tests. A few strains were negative in all hemagglutination tests. Based on our results, E. coli from cows with acute mastitis lack the virulence factors commonly observed in other E coli groups associated with disease. Serum resistance was the only characteristic that could be related to virulence.     

Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine.

A prospective cohort study was undertaken in two commercial California dairies. The treatment group, 246 cows, received three doses of a whole cell bacterin of J5 Escherichia coli (mutant of E. coli O111:B4) plus Freund's incomplete adjuvant vaccine (two in the dry period and one after calving) while 240 unvaccinated cows served as controls. Thirty-five cases of clinical coliform mastitis were diagnosed, six in vaccinated cows and 29 in unvaccinated cows. Bacteria isolated from the clinical cases included 15 E. coli five Klebsiella pneumoniae, three K. oxytoca, three K. ozaenae, five Enterobacter aerogenes, three Serratia marcescens and one Serratia spp. Four control cows were culled, three of them because of chronic coliform mastitis and one because of postcoliform infection agalactia. Incidence rate of clinical gram-negative mastitis was 2.57% in vaccinated cows and 12.77% in unvaccinated cows. The estimated risk ratio, the measure of risk of having clinical gram-negative mastitis for vaccinated cows to unvaccinated cows, was 0.20 (p less than 0.005), indicating a strong relationship between vaccination and lack of clinical gram-negative mastitis. The results of this trial indicate that the administration of the E. coli J5 vaccine is protective against natural challenge to gram-negative bacteria, and reduces the incidence of clinical gram-negative mastitis in dairy cows during the first three months of lactation.