A large potentiation effect of serum on the in vitro potency of tulathromycin against Mannheimia haemolytica and Pasteurella multocida.

The antimicrobial properties of tulathromycin were investigated for M. haemolytica and P. multocida. Three in vitro indices of antimicrobial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves, were established for six isolates of each organism. Each index was measured in two growth media: Mueller-Hinton broth (MHB) and calf serum. It was shown that MICs and MBCs were markedly lower in serum than in MHB. MHB:serum ratios for MIC were 47:1 (M. haemolytica) and 53:1 (P. multocida). For both serum and MHB, adjustment of pH led to greater potency at alkaline compared to acid pH. Tulathromycin MIC was influenced by size of inoculum count, being 4.0- to 7.7-fold greater for high compared to low initial counts. It was concluded that for the purpose of determining dosages for therapeutic use, pharmacodynamic data for tulathromycin should be derived in biological fluids such as serum. It is hypothesized that in vitro measurement of MIC in broth, conducted according to internationally recommended standards, may be misleading as a basis for estimating the in vivo potency of tulathromycin.

Pharmacokinetic/pharmacodynamic integration and modelling of amoxicillin for the calf pathogens Mannheimia haemolytica and Pasteurella multocida.

The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time-kill data, based on the sigmoidal Emax equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log10 reduction) and 4log10 reduction in bacterial counts. For these levels, mean AUC(0-24 h) /MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (Cmax )/MIC ratios of 13.9:1 and 25.2:1, area under concentration-time curve (AUC0-∞ )/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).

Sources of Streptococcus dysgalactiae in English and Welsh sheep flocks affected by infectious arthritis (joint ill).

In order to investigate sheep sources of Streptococcus dysgalactiae in flocks affected with joint ill, 10 sheep flocks in England and Wales with laboratory-confirmed cases of infectious arthritis (joint ill) caused by S dysgalactiae were visited during a disease outbreak while a further four flocks were visited during the lambing period in the year following an outbreak. A total of 5239 samples were collected for bacterial culture from 797 ewes and their 1314 lambs. S dysgalactiae was isolated from nine of 894 samples (1 per cent) on farms visited during an outbreak, and from 7 of 4462 samples (0.2 per cent) collected in the year following an outbreak. The 16 samples from which S dysgalactiae was isolated came from the vaginas of eight ewes, milk of one ewe, navels of four lambs, mouths of two lambs and noses of one lamb. In vitro testing of the survival of S dysgalactiae on straw, hay and in water at different temperatures was performed, and it was isolated from these substrates for up to 42, 35 and 0 days, respectively.

Pharmacokinetic-pharmacodynamic integration and modelling of florfenicol in calves.

Florfenicol was administered subcutaneously to 10 calves at a dose of 40 mg/kg. Pharmacokinetic-pharmacodynamic (PK-PD) integration and modelling of the data were undertaken using a tissue cage model, which allowed comparison of microbial growth inhibition profiles in three fluids, serum, exudate and transudate. Terminal half-lives were relatively long, so that florfenicol concentrations were well maintained in all three fluids. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined in vitro for six strains each of the calf pneumonia pathogens, Mannhemia haemolytica and Pasteurella multocida. An PK-PD integration for three serum indices provided mean values for P. multocida and M. haemolytica, respectively, of 12.6 and 10.4 for Cmax /MIC, 183 and 152 h for AUC0-24 h /MIC and 78 and 76 h for T>MIC. Average florfenicol concentrations in serum exceeded 4 × MIC and 1.5 × MIC for the periods 0-24 and 48-72 h, respectively. Ex vivo growth inhibition curves for M. haemolytica and P. multocida demonstrated a rapid (with 8 h of exposure) and marked (6 log10 reduction in bacterial count or greater) killing response, suggesting a concentration-dependent killing action. During 24-h incubation periods, inhibition of growth to a bacteriostatic level or greater was maintained in serum samples collected up to 96 h and in transudate and exudate samples harvested up to 120 h. Based on the sigmoidal Emax relationship, PK-PD modelling of the ex vivo time-kill data provided AUC0-24 h /MIC serum values for three levels of growth inhibition, bacteriostatic, bactericidal and 4 log10 decrease in bacterial count; mean values were, respectively, 8.2, 26.6 and 39.0 h for M. haemolytica and 7.6, 18.1 and 25.0 h for P. multocida. Similar values were obtained for transudate and exudate. Based on pharmacokinetic and PK-PD modelled data obtained in this study and scientific literature values for MIC distributions, Monte Carlo simulations over 100 000 trials were undertaken to predict once daily dosages of florfenicol required to provide 50% and 90% target attainment rates for three levels of growth inhibition, namely, bacteriostasis, bactericidal action and 4 log10 reduction in bacterial count.

Pharmacodynamics of florfenicol for calf pneumonia pathogens.

The antimicrobial properties of florfenicol were investigated for the bovine respiratory tract pathogens, Mannheimia haemolytica and Pasteurella multocida. Three in vitro indices of efficacy and potency were determined; minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and in vitro time-kill curves for six pathogenic strains of each organism. Each was monitored in two matrices, Mueller Hinton broth (MHB) and calf serum. MBC:MIC ratios were low, 1.8 : 1 for M haemolytica in both MHB and serum and 2.4 : 1 and 2.1 : 1 for P multocida in MHB and serum, respectively. The killing action of florfenicol had the characteristics of concentration dependency against M haemolytica and codependency (on time and concentration) against P multocida. Modelling of the time-kill data after 24 hours exposure was undertaken to quantify three levels of activity for the ratio, area under concentration-time curve over 24 hours (AUC24h)/MIC; bacteriostatic action (no change in bacterial count), 3log10 reduction and 4log10 reduction in bacterial count. Mean AUC24h/MIC values for P multocida in MHB (and serum) were 22.0 (23.3) hour, 34.5 (39.9) hour and 45.8 (50.4) hour, respectively. Similar numerical values were obtained for M haemolytica. For both bacterial species, interstrain variability was low; coefficients of variation ( per cent) in serum for 3log10 and 4log10 reductions in count were, respectively, 14.3 and 24.1 for P multocida and 7.8 and 11.4 for M haemolytica. These data form a rational basis for dosage selection for treatment of calf pneumonia caused by M haemolytica or P multocida.

Survival of Actinobacillus pleuropneumoniae outside the pig.

Transmission of Actinobacillus pleuropneumoniae is primarily thought to be via direct transfer of mucus from pig to pig. For transfer between farms, the organism may need to persist in the wet or dried state to be carried on an inanimate surface. The survival of A. pleuropneumoniae was investigated under controlled laboratory conditions. In aqueous suspension, survival was improved by the presence of NaCl and mucin; it was prolonged at lower temperature. In dry state, it survived best on a hydrophobic surface either under desiccated conditions or saturated humidity. Detectable viability was maintained for 3-4 days. When frozen, A. pleuropneumoniae survived for more than 17 weeks at -20 °C, but the viability declined to 0.01% during that time. Survival at -70 °C was effective for long term storage. Results obtained from this investigation would be applicable for sampling method, transport techniques, epidemiological study, and biosecurity implementation.

Pharmacokinetic and pharmacodynamic integration and modelling of marbofloxacin in calves for Mannheimia haemolytica and Pasteurella multocida.

The pharmacokinetics (PK) and pharmacodynamics (PD) of marbofloxacin were established in calves for six strains of each of the pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. The distribution of marbofloxacin into inflamed (exudate) and non-inflamed (transudate) tissue cage fluids allowed comparison with the serum concentration-time profile. To establish the PD profile, minimum inhibitory concentration (MIC) was determined in Mueller-Hinton broth (MHB) and calf serum. Moderately higher MICs were obtained for serum compared to MHB. An initial integration of PK-PD data established C(max)/MIC ratios of 45.0 and AUC(24h)/MIC values of 174.7 h, based on serum MICs, for both bacterial species. Using bacterial time-kill curves, generated ex vivo for serum marbofloxacin concentrations, PK-PD modelling established three levels of growth inhibition: AUC(24 h)/MIC ratios for no reduction, 3 log(10) and 4 log(10) reductions in bacterial count from the initial inoculum count were 41.9, 59.5 and 68.0 h for M. haemolytica and 48.6, 64.9 and 74.8 h for P. multocida, on average respectively. Inter-strain variability for 3 log(10) and 4 log(10) reductions in bacterial count was smaller for P. multocida than for M. haemolytica. In conjunction with literature data on MIC(90) values, the present results allowed prediction of dosages for efficacy for each organism for the three levels of growth inhibition.

Porcine circovirus type 2 infection before and during an outbreak of postweaning multisystemic wasting syndrome on a pig farm in the UK.

The presence of porcine circovirus type 2 (PCV-2) and other pathogens before and during an outbreak of postweaning multisystemic wasting syndrome (PWMS) in pigs is evaluated in this study. At the time of the outbreak on a large commercial pig farm in the UK, serum samples and data were collected in two independent on-going research projects, one in weaned pigs and the other in sows. Serum samples of growing pigs and sows were PCV-2-antibody and PCR positive before and during the PMWS outbreak. Upon sequencing, PCV-2 isolates collected before the outbreak were identified as PCV-2a, and isolates collected during the outbreak were identified as PCV-2b, suggesting a shift of PCV-2 genotypes present on the farm. Pigs in the weaner study were from sows originating from different breeders and an association of sow origin and PCV-2 serostatus in offspring was found. Further, pigs had higher odds to be PCV-2 antigen positive if the sow was PCV-2 antibody positive around farrowing, the sow was of higher parity, and were less likely to test antigen positive if the sow was sourced from a particular breeder. The findings of this study highlight the potential role of the immune status of the sow on the occurrence of PMWS.

Factors contributing to the contamination of peripheral intravenous catheters in dogs and cats.

The aim of this study was to identify factors that contribute significantly to the bacterial contamination of peripheral intravenous catheters in dogs and cats. Between January and June 2005, intravenous catheters were removed from 84 dogs and 15 cats at the Queen Mother Hospital for Animals, Royal Veterinary College. None of the factors under consideration was significantly associated with bacterial contamination, but 42.9 per cent of the animals with clinical signs consistent with a peripheral catheter-related infection, 34.8 per cent of the animals in which blood had been collected from the catheter immediately after its insertion, and 21.1 per cent of the animals in which a T-connector rather than a Y-connector had been used had contaminated cannulae, compared with 19.0 per cent, 19.7 per cent and 8.3 per cent, respectively, of the animals that did not have signs of such an infection, from which blood was not taken immediately, and that had a Y-connector rather than a T-connector. Binary logistic regression showed that the animals with clinical signs of a catheter-related infection were 10 times more likely to have a contaminated catheter (odds ratio [OR] 10.9, 95 per cent confidence interval [CI] 0.89 to 134) and the animals fitted with Y-connectors rather than T-connectors were 10 times less likely to have a contaminated catheter (OR 0.10, 95 per cent CI 0.008 to 1.25).

PCR specific for Actinobacillus pleuropneumoniae serotype 3.

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.

Multiplex PCR that can distinguish between immunologically cross- reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains.

We describe a highly sensitive and specific multiplex PCR, based on capsular loci and the species specific apxIV gene, that unequivocally differentiates serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains that are cross-reactive in conventional immunological tests.

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo.

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow.

In cattle, the first postpartum dominant follicle grows slower and produces less oestradiol in animals with high numbers of bacteria contaminating the uterine lumen. However, only bacteria that are uterine pathogens are correlated with severe clinical disease and an increased inflammatory response. It is unknown whether the effect on the ovary in relation to uterine bacterial contamination is associated with the presence of recognised uterine pathogens. Therefore, the present study examined the relationship between pathogenic bacteria in the postpartum uterine lumen, follicle growth and function and the formation of a competent corpus luteum. In addition, peripheral plasma concentrations of immune mediators were quantified. Swabs were collected from the uterine lumen of cattle on day 7 postpartum. Bacteria were cultured and identified and bacterial growth was scored semi-quantitatively. Animals were categorized into high or low recognized uterine pathogen contamination groups based on the number of colonies. Ovarian structures were monitored by daily transrectal ultrasonography and blood samples were collected. In animals with high numbers of uterine pathogens on day 7 postpartum, the diameter of the first postpartum dominant follicle was smaller and plasma oestradiol concentrations were lower. In addition, these animals had smaller corpora lutea, which produced less progesterone. Furthermore, animals with a high day 7 uterine pathogen growth density had higher peripheral concentrations of acute phase proteins. Thus, contamination of the uterus with recognized uterine pathogens is associated with ovarian dysfunction during the postpartum period. Furthermore, infection results in an increase in the production of inflammatory mediators.

Causative organisms and somatic cell counts in subclinical intramammary infections in milking goats in the UK.

The bacterial causes of subclinical mastitis were determined in samples of milk taken from one half of the udders of 159 goats in three different herds. The mean prevalence of subclinical infection was 33 percent, with prevalences of 26 percent, 39 percent and 42 percent in the three herds. Staphylococcus aureus was isolated from seven (13 percent) of the 53 infected halves, coagulase-negative staphylococci accounted for 47 percent, Corynebacterium species for 31 percent and alpha-haemolytic streptococci for 6 percent of the infected samples. The mean somatic cell count of the uninfected milk samples was 428,000 cells/ml, and 93 percent of uninfected samples had counts less than 1,000,000 cells/ml; the mean cell count of the infected samples was 2,785,000 cells/ml.

Minimum inhibitory concentrations of some antimicrobial drugs against bacteria causing uterine infections in cattle.

The minimum inhibitory concentrations (MICs) of oxytetracycline, cephapirin, cephapirin/mecillinam, cefquinome, ceftiofur and enrofloxacin, candidate antibiotics for the principal bacteria associated with uterine infections: Escherichia coli, Arcanobacterium pyogenes and the anaerobic bacteria Fusobacterium necrophorum and Prevotella melaninogenicus, were determined by the agar dilution method. The bacteria were isolated from animals with clinical metritis and/or endometritis. For E coli, cefquinome and enrofloxacin had the lowest MIC90 and MIC50 values (< 0.06 microg/ml), and oxytetracycline and cephapirin had the highest values. For A pyogenes, oxytetracycline had the highest MIC50 value (16 microg/ml), but all the cephalosporins had values below 0.06 microg/ml. For the anaerobic bacteria, enrofloxacin and oxytetracycline had the highest MIC50 values but all the cephalosporins had values of 0.06 microg/ml or below.