Tissue distribution of cefquinome after intramammary and "systemic" administration in the isolated perfused bovine udder

Mammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. Cefquinome (88.8mg cefquinome sulphate per 8mL) was administered by the intramammary route to all quarters and/or "systemically" via the perfusion fluid at concentrations similar to those measured in plasma following intramuscular administration of 1mg cefquinome per kg body weight. Samples of the perfusate were taken over a 6-h period and from the regional lymph nodes after 6h. Using a scalpel, sections of glandular tissue - at different distances from and vertical to the teat right up to the udder base - were gathered from four quarters each per route of administration at 2, 4 and 6h. The cefquinome content of the tissue samples was analysed by high performance liquid chromatography with diode array detection and of the perfusate samples by bioassay. After intramammary administration, the concentration of cefquinome in the glandular tissue decreased exponentially with increasing distance from the teat. The addition of cefquinome to the perfusion fluid produced a mean concentration of 0.2-0.5microg/g at all glandular tissue sites. Combined intramammary and systemic treatment ensured that concentrations exceeded the MIC(90) values of the most common mastitis pathogens in all areas of the udder by 2h post-administration. There was considerable variability in the tissue concentrations of cefquinome, particularly after intramammary administration. These results suggest that for the treatment of acute mastitis a combination of both intramammary and systemic administration is likely to be advantageous in order to rapidly produce maximum cefquinome concentrations in all regions of the udder.     

Tissue distribution of oxacillin and ampicillin in the isolated perfused bovine udder

In vivo, tissue distribution of intra-mammarily administered antibiotics is mostly only assessed by sampling milk and blood. Therefore, the described study analysed whether measurement of tissue concentrations makes sense in vitro instead. Isolated bovine udders were perfused with gassed and warmed Tyrode solution. To four front and rear quarters each, 1000 mg oxacillin in 7.5 ml vehicle was administered intracisternally, completely formulated as sodium monohydrate in two lactation ointments (with or without sodium dodecylsulphate) or 80% as benzathine salt in a dry-off ointment. Over 3 h, perfusate and glandular tissue from different locations were sampled and analysed by high pressure liquid chromatography. With increasing vertical distance to the teat base, the tissue concentration of antibiotics decreased. With the lactation ointment containing sodium dodecylsulphate, lower oxacillin concentrations were reached in glandular tissue and lymph nodes compared to those without. The ointments led to a higher recovery of oxacillin in glandular tissue than in perfusate. Aluminium monostearate in the dry-off ointment caused an even poorer absorption of oxacillin into perfusate. The isolated perfused bovine udder is suitable to study the tissue distribution of antibiotics, since the results were mainly comparable with the few existing in vivo studies and show the influence of different formulations.     

The tissue distribution of antibiotics in the udder--comparison of the situation in vivo with the isolated perfused bovine udder.]

Especially for animal protection reasons, tissue concentrations of intracisternally administered antibiotics in the mammary gland hardly can be determined in the live cow. Therefore, this paper assessed the use of the isolated perfused bovine udder to study the distribution of penicillins in glandular tissue. With this intention, the in vitro results acquired with this model were compared to tissue concentrations as well as absorption data from the few in vivo studies in the literature and differences were interpreted. This approach must consider inevitable deviations of experimental materials and methods. Furthermore, in vivo the udder is included in a closed circulatory system with other metabolism and excretion feasibilities than the isolated model. Moreover, the lower flow rate in the vessels in vitro has to be taken into account with respect to absorption capacities. Nevertheless, the tissue concentrations and the distribution equilibrium across the blood-udder-barrier in both experimental concepts corresponded with each other, if the deviating conditions are considered by calculating correction factors. Advantages and disadvantages of the isolated udder are discussed critically. In conclusion, this method is a useful completion of pharmacokinetic in vivo studies that are supposed to compare intracisternally administered formulations.     

Interdependence and distribution of subclinical mastitis and intramammary infection among udder quarters in dairy cattle

The objective of the present study was to determine the distribution of subclincial mastitis and intramammary infection (IMI) across udder quarters and to quantify, if any, the level of interdependence between quarters. Subclinical mastitis was defined as an udder quarter somatic cell count of > or =250,000. Intramammary infection was defined as the presence of pathogens in an udder quarter. The data used in the analyses consisted 6577 test-day records from 2034 lactations with observations on subclinical mastitis and 8428 test-day records from 2575 lactations with observations on IMI; each test-day record consisted data on all four udder quarters. Records were from animals on three research herds in southern Ireland. Observed distributions of IMI were compared to expected distributions, estimated using a binomial probability distribution, assuming independence among quarters. Generally, the observed frequencies deviated significantly from the expected frequencies, indicating some level of interdependence between quarters. Intraclass correlations were estimated from a mixed model including cow and test-day record nested within cow as random effects; fixed effects in the model included herd, year of calving, month of calving, parity, days in milk at sampling and interactions. Intraclass correlations within test-day record ranged from 0.14 to 0.50 for subclinical mastitis and from 0.02 to 0.40 for IMI. Thus, substantial interdependence between quarters exists which should be accounted for when analysing data from experiments across udder quarters. Failure to do so may result in incorrect inferences from statistical tests.     

Disposition kinetics of lactoferrin in milk after intramammary administration

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half-life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty-eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.     

Antimicrobial susceptibility of Prototheca zopfii isolated from bovine intramammary infections

In vitro antimicrobial susceptibility tests were carried out on 48 strains of Prototheca zopfii, an achlorophyllous algae causing refractory mastitis in dairy cows; 27 antimicrobials were evaluated. All strains were susceptible to both myxin and nystatin. In addition, 22 strains were susceptible to amphotericin B, 21 to polymyxin B, and 18 to gentamicin. Only 1 strain was susceptible to kanamycin. All strains were resistant to ampicillin, bacitracin, carbenicillin, cephalothin, chloramphenicol, clotrimazole, cloxacillin, erythromycin, flucytosine, ketoconazole, lincomycin, miconazole, neomycin, nitrofurazone, novobiocin, oleandomycin, penicillin, rifampin, streptomycin, tetracycline, and vancomycin.     

Plasminogen activation in the bovine udder

Plasmin is a serin protease with a broad substrate specificity which might cause disintegration of basal membranes, epithelium and surrounding matrix. Plasmin might also elicit degradation of tissue (Mullins & Rohrlich 1983).     

Determination of cloxacillin residues in dairy cows after intramammary administration

The aim of this study was to perform a comparative analysis of the characteristics of cloxacillin (CLO) (MRL of withdrawal in bovine milk is 30 ng/g) after a single intramammary (IMM) dose in the dry period (DP) and lactation (LP), and to establish a high‐performance liquid chromatography (HPLC) analytical method for CLO detection in milk. The research was conducted on a group of 10 cows in DP and 10 in LP. A single dose of 600 mg of CLO was administrated by the IMM route for a single quarter in DP and 500 mg for a single quarter in LP. CLO concentration was analyzed by HPLC. CLO was monitored at a wavelength of 206 nm. Pharmacokinetic calculations were performed using Phoenix^® WinNonlin^® 6.4 software. The calibration curve was linear over the range of 13.03–28 019.00 ng/g with the coefficient of determination R² > 0.999. CLO withdrawal in both the LP and DP group had a biphasic nature. The total CLO elimination in the DP and LP group was reached after 36 and 6.5 days, respectively. A quantitative and confirmatory method for the determination of CLO in fresh milk has been established. We have confirmed that the withdrawal of CLO in the DP group is not a linear process and has a stepwise character.     

Immune responses to intramammary infusion with soluble (ovalbumin) and particulate (S uberis) antigens in the preparturient bovine udder

Pregnant non-lactating cows were immunised by intramammary infusion with killed Streptococcus uberis into one quarter and ovalbumin into another, at one week (group 2) or one week and two weeks (group 1) before the expected date of parturition. A small IgG1 and IgG2 antibody response to ovalbumin was detected in the serum of these cows. There was also a small increase in IgG1 and IgA serum antibody activity to S uberis. In whey the response was restricted to IgA with activity to S uberis. The IgA antibody response to S uberis in group 1 was significantly greater in the quarter immunised with bacteria than that of the control quarters for up to two months after calving. In contrast, the serum IgA response was short or absent in a number of animals.     

Elimination kinetics of ceftiofur hydrochloride after intramammary administration in lactating dairy cows

OBJECTIVE: To determine the elimination kinetics of ceftiofur hydrochloride in milk after intramammary administration in lactating dairy cows. DESIGN: Prospective study. ANIMALS: 5 lactating dairy cows. PROCEDURE: After collection of baseline milk samples, 300 mg (6 mL) of ceftiofur was infused into the left front and right rear mammary gland quarters of each cow. Approximately 12 hours later, an additional 300 mg of ceftiofur was administered into the same mammary gland quarters after milking. Milk samples were collected from each mammary gland quarter every 12 hours for 10 days. Concentrations of ceftiofur and its metabolites in each milk sample were determined to assess the rate of ceftiofur elimination. RESULTS: Although there were considerable variations among mammary gland quarters and individual cows, ceftiofur concentrations in milk from all treated mammary gland quarters were less than the tolerance (0.1 microg/mL) set by the FDA by 168 hours (7 days) after the last intramammary administration of ceftiofur. No drug concentrations were detected in milk samples beyond this period. Ceftiofur was not detected in any milk samples from nontreated mammary gland quarters throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Ceftiofur administered by the intramammary route as an extra-label treatment for mastitis in dairy cows reaches concentrations in milk greater than the tolerance set by the FDA. Results indicated that milk from treated mammary gland quarters should be discarded for a minimum of 7 days after intramammary administration of ceftiofur. Elimination of ceftiofur may be correlated with milk production, and cows producing smaller volumes of milk may have prolonged withdrawal times.     

Comparison of four test procedures to identify Staphylococcus aureus isolated from bovine intramammary infections

A comparison was made between conventional tube coagulase, macrocupsular coagulase, latex agglutination, and miniaturized biochemical test systems for identification of Staphylococcus aureus of bovine origin. A total of 303 gram-positive, catalase-positive cocci of bovine origin were tested. Agreement between each pair of 4-hour tube coagulase, macrocupsular coagulase, latex agglutination, and miniaturized biochemical test results within isolates was greater than 95.0. Seventeen (5.6%) isolates were test negative for 4-hour tube coagulase, but test positive for 24-hour tube coagulase. Thirteen (76.5%) of these isolates were identified as S hyicus, 3 were S aureus, and 1 was not identified.     

Florfenicol pharmacokinetics in lactating cows after intravenous, intramuscular and intramammary administration

Pharmacokinetics of florfenicol 30% injectable solution was determined in lactating cows after intravenous, intramammary and intramuscular administration. Serum concentration-time data generated in the present study were analysed by non-compartmental methods based on statistical moment theory. Florfenicol half-life was 176 min, mean residence time 129 min, volume of distribution at steady-state 0.35 L/kg, and total body clearance 2.7 mL/min·kg after intravenous administration at 20 mg/kg. The absorption after intramuscular administration appeared slow and the kinetic parameters and the serum concentration vs. time curve were characteristic of absorption rate-dependent elimination. The absorption after intramammary administration of florfenicol at 20 mg/kg was good (53.9%) and resulted in serum concentrations with apparent clinical significance. The intramammary administration resulted in serum florfenicol concentrations that were significantly higher than the respective serum concentrations following Intravenous administration 4 h after administration and thereafter. Florfenicol absorption was faster from the mammary gland than from the muscle. The maximum serum concentrations ( C _max) were 6.9 μg/mL at 360 min after intramammary administration and 2.3 μg/mL at 180 min after intramuscular administration. The bioavailability of florfenicol was 54% and 38% after intramammary and intramuscular administration, respectively. The C _max in milk was 5.4 μg/mL at 180 min after intravenous and 1.6 μg/mL at 600 min after intramuscular administration.     

B-mode ultrasonography of the bovine udder and teat

The udders and teats of cows were examined by brightness mode (B-mode) ultrasonography. A 5-MHz linear array transducer and a 5-MHz or 10-MHz mechanical sector transducer were used in the examinations. In lactating animals, the teat sinus, gland sinus, and lactiferous ducts were imaged easily. The scans also allowed visualization of the layers of the teat wall. The annular fold and the folding of the mucosa at the junction of the teat sinus and the papillary duct were seen best with the 5-MHz and 10-MHz mechanical sector transducers. In one cow lacking milk flow from one quarter, a mass at the junction of the papillary duct and the teat sinus was observed ultrasonographically. Surgical removal of the mass resulted in a return of milk flow from that quarter.     

Studies on the modulation of leucocyte subpopulations and immunoglobulins following intramammary infusion of beta 1,3-glucan into the bovine udder during the dry period

Inflammatory and immunological reactions after intramammary infusion of beta 1,3-glucan were studied during the steady dry period and involution phase of the bovine udder. The effects of a single intramammary infusion of two different doses (100 and 200 mg) of beta 1,3-glucan were evaluated during the steady dry period. In a second study, the effects of beta 1,3-glucan at drying off were studied by using two treatment regimens; a single infusion at drying off, compared with two infusions of the compound, at drying off and again 2 weeks later. Total and differential leucocyte counts were measured in both blood and udder secretions. Additionally, the expression of receptors for CD14 and MHC class II on leucocytes, and the expression of receptors for CD4, CD8, WC1, IL2R and B-cells on lymphocytes was measured in mammary secretions by flow cytometric analyses. The concentrations of immunoglobulins in udder secretions were measured by radial immunodiffusion. The results showed that a single intramammary infusion of beta 1,3-glucan during the steady dry period causes transient enhancement of some aspects of the inflammatory and immune responses. The increases in somatic cell counts, numbers of monocytes/macrophages, and in proportions of CD14+ and MHC class II+ leucocytes in udder secretions were dose-dependent. Infusion of beta 1,3-glucan also slightly increased the proportion of CD4+ lymphocytes and the concentrations of IgG1 and IgG2 in dry secretions. Infusion of beta 1,3-glucan at drying off seemed to accelerate the involution process through an increase in somatic cells, particularly in the numbers of macrophages, in mammary secretions. The numbers of lymphocytes and polymorphonuclear leucocytes, the proportions of IL2R+ lymphocytes, the proportions of CD14+ or MHC class II+ leucocytes and the concentrations of IgG1 and IgG2 also increased in comparison with untreated controls. Moreover, a second infusion of beta 1,3-glucan tended to prolong this response, indicating that this might be an effective means of enhancing the mammary defence against udder infections closer to calving. In conclusion, the results indicate the intramammary infusion of beta 1,3-glucan could be used to enhance the defence mechanisms of the bovine udder against infections, especially during early involution.     

Persistence of staphylococcal species and genotypes in the bovine udder

Staphylococci are a major cause of intramammary infections (IMI) in ruminants. The main aim of this study was to investigate staphylococcal IMI in dairy cattle with emphasis on persistence and distribution of staphylococcal species and genotypes. With a sampling interval of 4-8 weeks, over a year, 4030 samples from 206 cows in 4 herds were collected. Coagulase-negative staphylococci (CNS) and Staphylococcus aureus were detected in 13.2% and 4.2% of the samples, respectively. Selected CNS isolates from quarter milk samples were identified to species level using sodA sequencing. Staphylococcus chromogenes (32%) and Staphylococcus simulans (25%) predominated. The proportion of S. chromogenes was greater in primiparous (52%) than in multiparous cows (12%), while the opposite was the case for Staphylococcus epidermidis (6% and 21%, respectively). Isolates from possibly persistent IMI were selected for pulsed-field gel electrophoresis (PFGE). Six staphylococcal species were found to cause persistent IMI; S. aureus, S. chromogenes, S. simulans, S. epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri. It was shown that several pulsotypes (PTs) within each species were associated with persistent infections, but only a few were spread and caused persistent IMI in multiple cows within a herd. Of special interest was the observation that only one, or a few, strains of each species caused persistent IMI in multiple cows within a same herd. This indicates strain differences with respect to transmissibility and pathogenicity.     

Stochastic bio-economic model of bovine intramammary infection

Although the dynamics of transmission play an important role in the occurrence of intramammary infection (IMI), they have not been considered in previous models used to estimate the cost of IMI. The bio-economic model described includes within-herd dynamics of pathogen-specific IMI. The model simulated Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli IMI stochastically and estimated the cost of these IMI in a herd of 100 dairy cows in a situation where a quota is applied to milk production. A Reed–Frost model was used for S. aureus, S. uberis, and S. dysgalactiae IMI and a Greenwood model for E. coli IMI. Economic analysis was conducted per pathogen for clinical and subclinical IMI. The parameters used in the model were based on the literature and were deemed credible and valid. Median annual incidence of clinical and subclinical IMI for all pathogens varied considerably. This variation was greatest for S. aureus IMI. The annual incidence of IMI in a herd of 100 dairy cows caused by S. aureus varied between 0 and 88 cases, with a median of 5 cases and the 5th and 95th percentiles of 0 to 36 for clinical IMI, and a median of 7 cases with the 5th and 95th percentiles of 0 to 52 for subclinical IMI. In consequence, the average total annual net costs also varied widely for S. aureus IMI. Clinical IMI costs were € 1375, with the 5th and 95th percentiles of 0 to 4716 and subclinical IMI costs were € 1219, with the 5th and 95th percentiles of 0 to 4030. The average annual net cost due to the 4 simulated pathogens combined was € 4896 and varied from € 915 to € 11,287 in a herd of 100 dairy cows. The bio-economic model developed for this study will be utilized as a tool to investigate the economic impact of management of pathogen-specific IMI.     

Detection of classical and newly described staphylococcal superantigen genes in coagulase-negative staphylococci isolated from bovine intramammary infections

The coagulase-negative staphylococci (CNS) are the most prevalent mastitis pathogen group yet their virulence characteristics have not been well described. We investigated the presence of 19 classical and newly described staphylococcal superantigen (SAg) genes in CNS isolates from bovine intramammary infections (IMI). A total of 263 CNS representing 11 different Staphylococcus spp. were examined, and 31.2% (n = 82) of CNS isolates had one or more SAg genes; there were 21 different SAg gene combinations. The most prevalent combination of SAg genes (seb, seln and selq; n = 45) was found in S. chromogenes, S. xylosus, S. haemolyticus, S. sciuri subsp. carnaticus, S. simulans and S. succinus. The genes for SAgs appear to be widely distributed amongst CNS isolated from bovine IMI.     

Minimum inhibitory concentrations of 20 antimicrobial agents against Staphylococcus aureus isolated from bovine intramammary infections in Japan

Minimum inhibitory concentrations (MICs) of 20 antimicrobial agents were determined against 51 isolates of Staphylococcus aureus from bovine intramammary infections. Fourteen (27.4%) isolates were resistant to benzylpenicillin, but none of the isolates was resistant to cloxacillin, nafcillin, or cephems. Among aminoglycosides, gentamicin was the most active, with an MIC50 of 0.2 microg/ml, followed by kanamycin, with an MIC50 of 0.78 microg/ml. Five isolates (9.8%) were resistant to dihydrostreptomycin, three isolates (5.9%) to kanamycin and two isolates (3.9%) to gentamicin. Resistance to erythromycin was observed in two isolates (3.9%). Tylosin was less active than erythromycin, with MIC50s of 1.56 microg/ml versus 0.39 microg/ml, but none of the isolates was resistant to this antibiotic. Oxytetracycline MICs were situated in the range of 0.39-1.56 microg/ml for 48 susceptible isolates. Although 19 (37.3%) isolates were resistant to one or more antimicrobial agents, a single resistance pattern was most frequent: benzylpenicillin (12 isolates), dihydrostreptomycin (two isolates) and kanamycin (one isolate). There were no isolates resistant to antimicrobial agents such as methicillin, lincomycin, clindamycin, chloramphenicol, florfenicol and virginiamycin, which have not been approved for use in cattle husbandry in Japan.