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.

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.

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.

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.

Association between postpartum pyrexia and uterine bacterial infection in dairy cattle.

The temperature of 90 dairy cattle was recorded for the first 10 days after parturition and the animals were categorised as either normal (< 39.7 degreesC) or pyrexic. Swabs were collected from the uterine lumen seven, 14, 21 and 28 days after parturition for aerobic and anaerobic culture; bacteria were identified and their growth was scored semiquantitatively. Blood samples were collected three times a week for the estimation of the concentrations of acute phase proteins. The cows' temperatures were often above the accepted normal range, but it was not a good indicator of the number of bacteria in the uterus. However, pyrexia was correlated with the presence of specific uterine pathogens (P < 0.05) and in particular with Prevotella species (P < 0.01). The pyrexic animals had a higher plasma concentration of the acute phase protein (alpha1-acid glycoprotein (P < 0.05). Although pyrexia is an indicator of postpartum inflammation, additional clinical signs are necessary to identify uterine bacterial infection.

Effect of intrauterine administration of oestradiol on postpartum uterine bacterial infection in cattle.

After parturition fewer first dominant follicles are selected in the ovary ipsilateral to the previously gravid uterine horn in cattle. However, the presence of a large oestradiol-secreting follicle in the ipsilateral ovary is a predictor of fertility, possibly due to a localised effect of oestradiol which increases the rate of elimination of the ubiquitous uterine bacterial contamination that occurs after calving. The present study tested the hypothesis that oestradiol reduces uterine bacterial contamination when administered into the uterine lumen around the expected time for selection of the first postpartum dominant follicle. Animals were infused with saline (n=15) or 10mg oestradiol benzoate (n=15) into the previously gravid uterine horn on Days 7 and 10 postpartum. Peripheral coccygeal blood samples were collected daily and oestradiol concentrations measured by radioimmunoassay (RIA). Uterine lumen swabs were collected 7, 14 and 21 days postpartum for aerobic and anaerobic culture, bacteria were identified and growth scored semi-quantitatively. Plasma oestradiol concentrations were higher for treated animals between Days 7 and 14 (1.4+/-0.1 versus 2.0+/-0.2 pg/ml, P<0.05). Control animals had a similar bacterial growth score on Days 7 and 14, with a lower value on Day 21 (5.7+/-1.0 and 6.1+/-0.7 versus 0.3+/-0.1, P<0.05). However, treated animals had a surprising higher bacterial load on Day 14, than on Days 7 or 21 (7.1+/-0.9 versus 4.0+/-0.6 or 3.6+/-0.6, P<0.05). The increased score was attributable to more pathogens associated with endometritis on Day 14 than Day 7 (5.1+/-1.0 versus 2.5+/-0.5, P<0.05), in particular Prevotella melaninogenicus (1.5+/-0.5 versus 0.7+/-0.2, P<0.05) and Fusobacterium necrophorum (1.5+/-0.4 versus 0.3+/-0.2, P<0.05). In conclusion, administration of oestradiol into the uterine lumen surprisingly increased uterine pathogenic anaerobic bacterial contamination. Thus, it is unlikely that increased fertility associated with a first dominant follicle in the ipsilateral ovary is a consequence of the elimination of bacterial contamination by ovarian oestradiol.

Identification of equine cecal bacteria producing amines in an in vitro model of carbohydrate overload.

Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO(4); 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.

Effect of postpartum manual examination of the vagina on uterine bacterial contamination in cows.

Twenty-one days after they had calved, the vaginas of 34 cows were examined with a clean gloved hand, and 26 cows were left unexamined. Swabs were collected from the lumen of the uterine body of the cows on the same day and seven days later; bacteria were identified by aerobic and anaerobic culture, and bacterial growth was scored semi-quantitatively. On the same days, blood samples were collected and the concentrations of acute phase proteins were measured, and the diameters of the cows' uterine horns were measured by transrectal ultrasonography. The vaginal examinations did not result in uterine bacterial contamination or an acute phase protein response, and they did not affect the diameters of the uterine horns.

Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle.

First postpartum dominant follicles are preferentially selected in the ovary contralateral to the previously gravid uterine horn. The aim of the present study was to test the hypothesis that uterine bacterial contamination alters the location of ovarian follicle emergence and selection, and inhibits follicle growth and function. Swabs were collected from the uterine body lumen of cattle on days 7, 14, 21 and 28 after parturition. Bacteria were identified by aerobic and anaerobic culture; bacterial growth was scored semiquantitatively and animals were categorized into standard or high bacterial contamination categories on the basis of the number of colonies detected. Follicular growth and function were monitored by daily transrectal ultrasonography, and estimation of plasma FSH, oestradiol and progesterone concentrations. There was no effect of bacterial contamination on plasma FSH concentration profiles or emergence of the ovarian follicle wave. When uterine bacterial growth scores were high on day 7 or day 21 after parturition, fewer first (1/20 versus 15/50; P < 0.05) or second (1/11 versus 13/32; P < 0.05) dominant follicles were selected in the ipsilateral compared with the contralateral ovary, respectively. The diameter of the first dominant follicle was smaller in animals with a high day 7 bacterial score (P < 0.001), dominant follicle growth was slower (P < 0.05) and oestradiol secretion was decreased (P < 0.05). The present study provides evidence for an effect of the uterus on the ovary after parturition, whereby uterine bacteria have a contemporaneous localized effect on ovarian follicle selection and subsequent growth and function, but not on initial emergence.

Actinobacillus pleuropneumoniae serotype 1 carrying the defined aroA mutation is fully avirulent in the pig.

The aroA gene from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 was isolated and sequenced. The gene complemented the aroA mutation in Escherichia coli AB2829. A kanamycin resistance cassette was inserted into the aroA gene and the mutant gene was reintroduced into A. pleuropneumoniae by allelic replacement. Intratracheal infection of susceptible pigs with A. pleuropneumoniae aroA caused no signs of respiratory disease or lung lesions in any of the animals at a dose 10(4) times the dose reliably known to induce acute pleuropneumonia; all animals infected with the unaltered control strain developed acute disease. The aroA mutant was rapidly eliminated from the lungs and tonsil of infected animals. The mutant may represent a safely attenuated strain for use in live bacterial vaccination or the delivery of antigen by the intranasal route. However, the residence time of the mutant in the respiratory tract of the pig may be too short for it to be useful in generating a protective immune response.