Bovine mastitis in Finland in 1988 and 1995--changes in prevalence and antimicrobial resistance.

Two surveys were carried out (during 1988 and 1995) to estimate the prevalence of bovine mastitis in Finland. In 1988, 17,111 quarter milk samples were obtained from 4495 cows, and in 1995 the corresponding figures were 10,410 and 2648. Antimicrobial susceptibility of mastitis pathogens was studied. Prevalence of mastitis on cow basis decreased from 47.8% in 1988 to 37.8% in 1995. Staphylococci was the largest group of pathogens isolated. The proportion of Staphylococcus aureus decreased and that of coagulase-negative staphylococci (CNS) increased. The proportion of strains resistant to at least one antibacterial drug increased with regard to S. aureus from 36.9% in 1988, to 63.6% in 1995 and with CNS from 26.6% to 49.7%. Most of the increase in antibacterial resistance was due to a higher number of beta-lactamase producing strains. Multiresistance also increased, but it was proportional to the overall increase in resistance. All the predominant mastitis streptococci were susceptible to beta-lactams tested.

Resazurin reduction as a function of respiratory burst in bovine neutrophils.

OBJECTIVE: To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay. SAMPLE POPULATION: Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows. Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide. PROCEDURE: The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate. Opsonized zymosan and phorbol myristate acetate were used as stimulants. The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions. Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments. RESULTS: Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response. The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative. Therefore, a single end-point measurement can be used for the fluorometric assay. CONCLUSIONS: The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity. The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions.

Binding of Staphylococcus aureus to milk fat globules increases resistance to penicillin-G.

The susceptibility to penicillin-G of Staphylococcus aureus strains that cause mastitis was tested in milk and in Iso-sensitest broth (ISB): The minimal inhibitory concentrations (MIC) of beta-lactamase-positive strains in milk were 10-100-fold those in ISB, whereas the MIC of beta-lactamase-negative strains in milk were some 10-fold those in ISB; beta-lactamase production was induced by milk in beta-lactamase-positive strains. Much of the increase in resistance to penicillin-G caused by milk can be attributed to milk fat globules; the increase in resistance was related to the binding capacity of the bacteria to milk fat globules as well as to capsule formation by the bacteria. It appears that the binding of the staphylococci to the fat globules and bacterial capsule formation resulted in a biofilm type of growth. In this case, the staphylococci behaved differently from the planktonic type of growth in artificial broth medium in which antibiotic susceptibility testing is usually carried out.

Collagenolytic activity and its sensitivity to doxycycline inhibition in tracheal aspirates of horses with chronic obstructive pulmonary disease.

The collagenolytic activity and its sensitivity to doxycycline inhibition in tracheal aspirates (TA) of horses with chronic obstructive pulmonary disease (COPD) was analyzed with SDS-PA gel electrophoresis (SDS-PAGE), using Type 1 collagen as the substrate. Both autoactive and total collagenase activities were significantly higher in TAs of horses with symptomatic COPD than in TAs of healthy horses. Doxycycline inhibition studies suggest that most of the TA collagenase is of the neutrophil type (MMP-8), but some is derived from other cells such as fibroblasts and monocyte/macrophages (MMP-1) and bacteria (bacterial collagenases). Drugs inhibiting collagenases in the respiratory tract might be worth a trial in the treatment of COPD in horses.

Gelatinolytic activity in tracheal aspirates of horses with chronic obstructive pulmonary disease.

The gelatinolytic activity in tracheal aspirates (TA) of horses with chronic obstructive pulmonary disease (COPD) was analyzed using SDS-PAGE-gelatin-gel electrophoresis (zymography) and compared to TAs from healthy controls. The 110-90 kD MMP-9 type gelatinase was high in symptomatic disease phases (permanent disease 0.46 +/- 0.15, p < 0.001; or intermittent disease 0.47 +/- 0.12, p < 0.001) compared to healthy controls (0.10 +/- 0.07). Similarly, the overall gelatinolytic activity, the activity in high-mw gelatinolytic bands (210-190 and 150 kD) and in proteolytically processed fragments in the 50-40 kD range were high, whereas the 75-65 kD MMP-2 was not altered. These findings suggest that MMP-9 type gelatinases, originating possibly from neutrophils or macrophages, and products thereof have a role in the pathogenesis of equine respiratory diseases, whereas MMP-2 type gelatinases represent house-keeping proteinases involved with normal tissue remodelling. The gelatinolytic activity in TAs correlated with the beta-glucuronidase activity, which indicates that they are simultaneously elevated in the respiratory secretions of horses suffering from COPD and might both be of same origin, or have a causal relationship.

Giving selenium supplements to dairy cows strengthens the inflammatory response to intramammary infection and induces a growth-suppressing effect on mastitis pathogens in whey.

Supplementing the feed of selenium-deficient diary cows with selenium (Se)-yeast or selenite at a level of 0.2 p.p.m. induced self-cure of subclinical mastitis; the prevalence of quarters harbouring subclinical mastitis (bacteriological criteria) decreased to about one half during the 8 week supplementation period. Three phenomena became apparent to explain the beneficial effect of selenium on mastitis: 1. The recruitment of phagocytes to the infected milk compartment of the udder was improved due to Se-supplementation; the correlation between infection and the respective inflammatory response, as indicated by the somatic cell count (SCC) and the N-acetyl-beta-D-glucosaminidase activity (NAGase) of milk, was poor in selenium-deficient cows. Selenium supplementation significantly improved the correlation; 2. Selenium supplementation induced an unspecified antibacterial activity in milk lactoserum (whey), restricting in vitro growth of the mastitis pathogens Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis, bacterial growth rates in whey samples became inversely correlated with the respective blood glutathione peroxidase activities (GSH-Px); and 3. Selenium supplementation had an effect on redox activities and sulfhydryl activities in whey. These changes were correlated with in vitro bacterial growth rates.

Disposition of enrofloxacin (Baytril) into the udder after intravenous and intra-arterial injections into dairy cows.

Enrofloxacin (Baytril) was injected into arteries supplying the udder of dairy cows. The idea was to avoid primary distribution, metabolism and elimination and thus to deliver the drug to the target organ at higher concentration. Enrofloxacin injected into the abdominal aorta or the external iliac artery resulted in high initial enrofloxacin retention by the udder and high milk concentrations. Injection of enrofloxacin into the abdominal aorta resulted in 2.2 times higher milk peak concentration of the drug than intravenous injection into the jugular vein. Injection of the drug into one of the two external iliac arteries allowed drug concentrations of milk from the udder halves to be compared: when enrofloxacin was injected into the right external iliac artery, the peak milk enrofloxacin concentration from the right udder half was 4.8 times that of the left udder half. The bulk of enrofloxacin was absorbed from the milk compartment of the udder before the next regular milking 6.5 h later. By this time, the metabolite ciprofloxacin had accumulated in milk. Pharmacokinetic values for enrofloxacin and its metabolite ciprofloxacin are given separately for serum and milk whey following three intravascular dosing routes.

Inhibition of the protease activity in tracheobronchial aspirates of horses with chronic obstructive pulmonary disease.

OBJECTIVES: To clarify the role of proteolytic enzymes in the pathogenesis of chronic obstructive pulmonary disease (COPD) in horses, and to investigate new possibilities for treatment of this disease by interfering in the proteolytic process. DESIGN: Effect of antiproteolytic activity of selected protease inhibitors on tracheal aspirates was studied in vitro, and the inhibition profiles were compared with those of purified proteases. SAMPLE POPULATION: Respiratory tract secretions with antiproteolytic activity from 9 horses with COPD. PROCEDURE: Caseinolytic agar-diffusion assay. RESULTS: The protease-inhibition profile of tracheal aspirates differed from horse to horse. The profiles did not resemble that of any of the pure proteases. Acetylcysteine, pentamidine, and diminazene were most effective in inhibiting proteolytic activity in tracheal aspirates in vitro. CONCLUSIONS: A mixed type of proteolytic activity is present in the respiratory tract secretions of horses with COPD. CLINICAL RELEVANCE: Acetylcysteine, pentamidine, and diminazene seem to have potential to be used in vivo to protect the lungs of horses with COPD from proteolytic damage.

Reconstitution of mastitic milk by adding blood plasma and leukocytes into low cell count milk.

Milk from inflamed quarters is high in somatic cells, proteolytic activity and lysosomal enzyme activity. Addition of homologous blood plasma and leukocytes into low cell count milk did not increase the plasmin activity, total proteolytic activity or N-acetyl-beta-D-glucosaminidase (NAGase) as anticipated, even if the reconstituted milk samples were stimulated by endotoxin or opsonized zymosan. Addition of urokinase activated the latent proteolytic system in plasma-supplemented milk. The reactive sulfhydryls in milk samples with different treatments decreased significantly after incubation, indicating the presence of an oxidative mechanism in milk (eg, sulfhydryl oxidase). The high background resazurin reduction of fresh milk might be due, in part, to the cellular activity of somatic cells. This study indicates that a more complex system including inflammatory mediators and complex cellular interactions is required for expression of plasmin-activator activity and for full activation of the proteolytic and lysosomal enzymes in mastitic milk.

Quantitative determination of bovine serum haptoglobin in experimentally induced Escherichia coli mastitis.

A high performance liquid chromatography (HPLC) method was developed, validated and used to analyse haptoglobin concentrations in serum samples taken from eight cows which had been challenged twice intramammarily with Escherichia coli. The results of the HPLC were compared with those from a photometric assay. The kinetics of the haptoglobin response were analysed with pharmacokinetic computer software. In contrast with the photometric assay, the HPLC was sufficiently sensitive to detect normal background levels of bovine serum haptoglobin. The serum haptoglobin concentrations of healthy cows ranged from 22 to 47 mg litre-1. As the concentration of haptoglobin increased, the results of the two methods correlated well (r = 0.96). A 52-fold increase in serum haptoglobin was detected after the first challenge with E coli. The mean pharmacokinetic parameters of the response after the first challenge were: lag phase 12 hours, t2/1increase 20 hours, tmax 72 hours, t1/2decrease 46 hours, and mean residence time 112 hours. The second challenge three weeks later resulted in a significantly lower haptoglobin response, the area under curve being 35 per cent of that after the first challenge. The clinical signs and inflammatory changes in the milk did not differ significantly between the challenges.

A fluorometric beta-glucuronidase assay for analysis of bacterial growth in milk.

The growth of common mastitis-causing bacteria in milk was followed by a fluorometric technique based on the release of fluorescent 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl-beta-D-glucuronide by the beta-glucuronidase of bacterial or milk origin. Three of four Escherichia coli strains, all four strains of Streptococcus uberis (4/4) and Streptococcus agalactiae (4/4) produced beta-glucuronidase. Four Staphylococcus aureus strains (4/4) and one E. coli strain, though unable to produce the enzyme, activated the milk beta-glucuronidase most probably by lowering the pH of bacterial cultures in milk for optimum activity of the indigenous enzyme. The beta-glucuronidase of milk, Str. uberis and Str. agalactiae origin had similar optimum pH ranges (5.3-6.6) while E. coli beta-glucuronidase was more active at neutral or slightly alkaline pH (6.8-7.7). The increase of beta-glucuronidase activity in milk cultures of E. coli, Str. uberis, Str. agalactiae and S. aureus seemed to parallel the increase of colony forming units and were dependent on the inoculum size. The time to reach a predetermined enzyme threshold in E. coli-milk cultures showed excellent linear relationship with the inoculum size.

Comparisons of selenite and selenium yeast feed supplements on Se-incorporation, mastitis and leucocyte function in Se-deficient dairy cows.

The dairy cows at the Estonian Agricultural University appeared to have an extremely low selenium status. The selenium level was 5.6 micrograms/l in whole blood and 3.2 micrograms/l in milk, on average. The blood glutathione peroxidase was consequently extremely low. The effects of organic selenium (selenized yeast) and sodium selenite were compared in a feeding experiment on 100 dairy cows. Selenium incorporation, udder health and the in vitro function of blood neutrophils were monitored. Supplementation of the feed either with 0.2 ppm organic selenium or sodium selenite for 8 weeks, increased the blood selenium level (geometric mean) within this period from the back-ground level (about 5.6 micrograms/l) to 167 (Se-yeast) and to 91 micrograms/l (selenite). The respective change in whole blood glutathione peroxidase (GSH-PX) was from 0.22 to 3.0 (Se-yeast) and to 2.3 (selenite) microKat/g Hb. Blood GSH-PX continued to increase up to 10 weeks after the supplementation was stopped. The bioavailability of yeast selenium was superior to selenite: the relative bioavailability (selenite = 1) of yeast selenium was 1.4 if blood GSH-PX, 1.9 if blood selenium, and 2.7 if milk selenium was used as the response criterion. Selenium-supplementation showed a positive effect on udder health. The percentage of quarters harbouring mastitis pathogens dropped from 22.9 to 13.0 in the Se-yeast group and from 18.4 to 7.4 in the selenite group during the supplementation period. The effect of selenium on mastitis was also reflected as a decrease in the output of milk somatic cells and N-acetyl-beta-D-glucosaminidase (NAGase). The time-luminescence profile of zymosan-induced activity of blood neutrophils became skewed to the left in Se-supplemented cows.

Inhibition of the proteinase activity in mastitic milk.

The antiproteolytic activity of selected proteinase inhibitors was studied in mastitic bovine milk and urokinase-activated normal milk using a caseolytic agar diffusion assay. The inhibition profiles of mastitic milk and urokinase-activated milk were compared with those of purified proteinases. The proteinase inhibition profile of mastitic milk did not resemble that of any of the pure proteinases, indicating a mixed type of proteinase system in mastitic milk. The trypanocidals diminazene (equivalent to Berenil) and pentamidine (equivalent to Lomidine), together with aprotinin (Trasylol), showed most promise when considering possible applications in mastitis to break up the proteolytic cascade within the inflamed udder.

D-dimer improves the prognostic value of combined clinical and laboratory data in equine gastrointestinal colic.

The discriminating ability of 15 parameters alone or in combinations, including results from analysis of plasma endotoxin, the Nycomed plasma D-Dimer test and phospholipase A2, were analyzed to predict morbidity and mortality in equine gastrointestinal colic. Endotoxaemia was a characteristic feature of the colic horses. The problem of adequately predicting nonsurvivors among colic horses required several parameters to be included in the logistic model: if the "classical parameters", (heart rate, respiratory rate, PCV, anion gap) were included in the model, addition of plasma D-dimer, phospholipase A2, and Cl- significantly improved the predictive value of the logistic model. Increasing heart rate and D-dimer together with decreasing chloride was a risk factor for nonsurvival. The sensitivity of this three-parameter logistic model to predict nonsurvival was 78% and specificity 77%. The Nycomed D-Dimer test is recommended as a horse-site test to predict disseminated intravascular coagulation and nonsurvival in equine colic.

Antibacterial susceptibility of bovine-mastitis pathogens tested directly in milk from infected quarters.

Antibacterial susceptibilities of bovine-mastitis pathogens were analysed directly in 57 mastitic milk samples without inoculation with exogenous organisms. Aseptically collected milk was mixed with serial dilutions of antibacterials and the growth was observed using 2,3,5-triphenyltetrazolium chloride (TTC) reduction the following day. The results were compared with those obtained by using calibrated bacterial inocula in turbidimetric minimum-inhibitory-concentration (MIC) determination in broth cultures, and in TTC-broth culture-test and TTC-normal milk-test. The results of different methods all correlated positively when the entire data was used. However, taking the direct test in mastitic milk as the 'true' result, the total discrepancies varied from 34.7% to 48.8%. Antibacterial activities of the trimethoprim-sulphadoxine combination, and of spiramycin and ampicillin, decreased significantly when nutrient broth was replaced by milk as the test medium. The efficacy of trimethoprim-sulphadoxine as an antibacterial agent was also dependent on the source of milk.

Association af changes in the bacterial ecology of bovine mastitis with changes in the use of milking machines and antibacterial drugs.

The results of mastitis bacteriology made by the National Veterinary and Food Research Institute in Finland during the past 50 years (1.15 million samples) are viewed in relation to simultaneous changes in dairy cow management. Although intensive preventive measures have been applied for decades, the prevalence of bovine mastitis has not decreased. Instead, pathogenic bacteria are becoming progressively less susceptible to the available therapy. In part this must be due to the emergence of antibiotic resistant strains of bacteria, but it seems that the bacterial spectrum has also changed. The incidence of Streptococcus agalactiae (group B streptococci) has decreased, while in contrast, the incidence of staphylococci, initially Staphylococcus aureus and later coagulase-negative staphylococci, has increased. Results suggest that external pressure, like changes in animal husbandry, including antimicrobial treatments and introduction of modern milking machines, act as selective forces on the bacterial species which cause bovine mastitis.

Relevance of sensitivity testings (MIC) of S. aureus to predict the antibacterial action in milk.

Bacterial susceptibility testings were carried out in parallel Iso-sensitest broth (ISB) and bovine milk cultures using 16 antibacterials and 4 sensitive strains of mastitic isolates of Staphylococcus aureus. Bacterial activities were analyzed by continuous turbidity monitoring (broth cultures), continuous fluorometric monitoring of the resazurin-reducing redox activity, and by analyzing the triphenyltetrazolium (TTC)-reducing capacity at the end of the incubation period. To obtain an equipotent bacteria-suppressing activity, milk cultures required in general several times more antibiotic than the respective ISB cultures. Antibacterial activities of sulfadoxine-trimethoprim, vancomycin, novobiocin, macrolides, aminoglycosides and oxytetracycline were most effectively suppressed by milk. Aminoglycosides suffered additionally from reduction of oxygen in the incubation environment. The beta-lactams (penicillin G, oxacillin, cephalothin, ceftiofur, ampicillin, ampicillin-clavulanic acid), gentamicin and enrofloxacin showed extremely variable sensitivity results depending on the S. aureus/milk combination.

A comparative study of intravenous and rectal administration of propofol in piglets.

To compare the effectiveness of propofol given intravenously and rectally, ten piglets received propofol intravenously. On the next day, the same piglets and five other piglets were given suppositories containing propofol. Serial blood samples were collected for the analysis of propofol plasma concentration. The time course of the total plasma drug concentration was fitted into a bi-exponential function using a least square fitting regression computer programme. The volume of distribution was 2.5-2.8 l.kg-1, mean elimination half-life, 23.9 min and mean clearance 0.08 l.kg-1.min-1. The mean bioavailability by the rectal route was low. In contrast to the intravenously administered propofol, none of the piglets slept when given propofol rectally, reflecting the extremely low plasma propofol concentration. In veterinary medicine, propofol would seem to be clinically valuable for inducing intravenous anaesthesia, but would be ineffective when given rectally. The findings indicate that with the dosage forms used here, propofol would be clinically ineffective if given rectally to human infants and children.

Automation of the resazurin reduction test using fluorometry of microtitration trays.

Microtitration plate technique and -fluorometry was applied to automate the resazurin reduction test for monitoring bacterial numbers in broth cultures and milk. The effect of resazurin and resorufin concentration, bacterial species, growth medium, pH, and redox potential on the fluorescence response was studied. The timing of the appearance of maximum fluorescence was directly related to the logarithm of the number of colony-forming units (log CFU). Fresh milk and heat-treated milk contain interfering redox systems. The technique based on microtitration plate fluorometry, when fully automated, seems to provide a high-capacity system for analyzing bacterial numbers in foodstuffs and other media.