An investigation of the dynamics of intramammary infections acquired during the dry period on European dairy farms.

The dry period is acknowledged as playing a key role in mastitis epidemiology and yet surprisingly few studies have explored dry period infection dynamics in detail. The aim of this study was to investigate the dynamics of intramammary infection across a cohort of dairy herds in Europe. Five hundred and twenty-two cows were recruited from 12 farms in 6 European countries. All cows received antibiotic dry cow therapy but teat sealants were not used. All quarters of all cows were sampled for bacteriology at drying off and in the week immediately postcalving. Two ipsilateral quarters were also sampled for bacteriology in each cow 2 and 6wk after drying off. Cows were body condition scored and teats assessed for cleanliness at all sampling time points and for the presence of a keratin plug during the dry period. Other cow-level parameters such as historic somatic cell counts and milk yields before drying off were collated from farm records. Univariable and multivariable analyses were undertaken to investigate the etiology, prevalence, and dynamics of infection during the dry period and associated influential factors. In summary, environmental mastitis pathogens predominated. Although gram-positive major pathogens were typically well controlled and did not increase in prevalence across the dry period, gram-negative pathogens generally increased in prevalence. There was an increase in the number of quarters that yielded no growth across the dry period, although this was driven by minor rather than major mastitis pathogen control. Other than the presence of a gram-positive or gram-negative pathogen 6wk after drying off, the measured parameters were not influential when considering their effect on the presence of pathogens postcalving. Analysis also suggested that the early and mid dry period may be more important with respect to the timing of acquisition of infection than previously thought. We observed substantial variation in the etiology and prevalence of different pathogens on different farms with, in all cases, at least one of the 12 herds experiencing the opposite of the others with respect to increases and decreases in pathogen prevalence. Overall, this study confirms the importance of the dry period in mastitis epidemiology but highlights the importance of assessing and understanding infection dynamics on individual units. The lack of influence of the cow and quarter factors measured in this study suggests that herd and management factors may be more influential.

In vitro susceptibility of bovine mastitis pathogens to a combination of penicillin and framycetin: development of interpretive criteria for testing by broth microdilution and disk diffusion.

Dry cow therapy is an important part of mastitis control. This therapy typically consists of an antibiotic or antibiotics administered at a single dose by intramammary infusion at dry off to treat or prevent infection by prevalent mastitis pathogens. A combination dry cow therapy consisting of the active components penicillin and framycetin is currently used in several countries. Despite its use, standardized methods for the susceptibility testing of this combination against mastitis pathogens have not been established. In this study, which used Clinical and Laboratory Standards Institute methodology, preliminary interpretive criteria for the broth microdilution minimum inhibitory concentration (MIC) testing of mastitis pathogens to penicillin combined with framycetin (2:1 wt/wt) were established based on the amount of drug achieved and maintained postadministration in the udder. Based on resulting MIC distributions of recent veterinary field isolates and a subset of isolates preselected for resistance to ß-lactams or aminoglycosides and concentrations achieved postadministration, criteria for broth microdilution testing of the combination (susceptible, intermediate, resistant in micrograms per milliliter) were set as follows: Escherichia coli ≤8/4, 16/8, ≥32/16; Staphylococcus spp. ≤2/1, 4/2-8/4, >16/8; Streptococcus uberis and Streptococcus dysgalactiae <0.25/0.12, 0.5/0.25-2/1, >4/2. A disk diffusion test using disks containing 100 µg of framycetin and 10 IU of penicillin was also developed, and preliminary interpretive criteria (susceptible, intermediate, resistant in millimeters) were set based on correlation to broth MIC values and the minimization of interpretive errors between isolates tested concurrently by broth microdilution and disk diffusion as follows: E. coli ≥18, 16-17, ≤15; Staphylococcus spp. ≥21, 18-20, ≤17; Strep. uberis and Strep. dysgalactiae ≥21, 19-20, ≤18. In addition, ranges for the quality control of the testing of this combination by both broth microdilution and disk diffusion are provided. Based on these criteria and recent veterinary mastitis isolates, 96.0/96.8% of E. coli, 93.7/89.1% of Staph. aureus, 94.6/96.4% coagulase-negative staphylococci, 94.5/97.0% of Strep. uberis, and 96.7/100.0% Strep. dysgalactiae were susceptible to the combination by broth microdilution or disk diffusion, respectively. The availability of these methods will allow for the susceptibility testing of clinical isolates in the field and will also provide a way to monitor for resistance development as this combination is used going forward.

Susceptibility of coagulase-negative staphylococci to a kanamycin and cefalexin combination.

A combination of kanamycin and cefalexin was licensed in Europe in 2008 to treat bovine clinical mastitis. Preliminary broth and disk clinical breakpoints for this antibiotic combination have been proposed for Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and Escherichia coli. This study indicates that these proposed breakpoints also hold for coagulase-negative staphylococci (CNS), a group of bacteria frequently isolated in milk samples from cows with clinical mastitis. The data show that clinical bovine mastitis isolates of CNS from Europe have a high degree of susceptibility to the kanamycin/cefalexin combination, with minimal resistance to either agent alone. The use of the available kanamycin and cefalexin combination disk for testing the susceptibility of bovine mastitis isolates of Staph. aureus, Strep. uberis, Strep. dysgalactiae, and E. coli is also reliable for use in the testing of CNS, as disk results correlated with broth minimum inhibitory concentrations. The study reports, for the first time, the approved Clinical Laboratory Standards Institute quality control ranges for the kanamycin/cefalexin combination and wild-type cutoff values for major bacterial pathogens implicated in bovine mastitis.

Kill rate of mastitis pathogens by a combination of cefalexin and kanamycin.

AIM: To assess the bacterial killing rate produced by a combination of cefalexin and kanamycin at two different concentration ratios. METHODS AND RESULTS: Time-kill kinetics of cefalexin and kanamycin, individually and in combination, were determined against one strain each of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. The combination was tested using two fixed ratios (cefalexin : kanamycin ratios of 1·25 : 1 and 1 : 2·3) and two concentrations of each ratio. Time-kill curves produced with either ratio were quite similar. Against most bacterial species, higher concentrations produced faster kill. In all cases, the combination of cefalexin and kanamycin showed faster and greater kill at lower antibiotic concentrations than those observed with either drug alone. CONCLUSIONS: The combination of cefalexin and kanamycin results in a fast initial killing of major mastitis pathogens at both concentration ratios. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of cefalexin and kanamycin achieved rapid bacterial kill at concentrations and ratios that can be achieved in vivo following intramammary infusion of a mastitis treatment.

Evaluating the in vitro susceptibility of bovine mastitis pathogens to a combination of kanamycin and cefalexin: Recommendations for a disk diffusion test.

Cows suffering from bovine mastitis have markedly reduced milk production because of inflammation within the udder subsequent to infection and damage from bacterial toxins. Antibiotic treatment is commonly used as a preventative and therapeutic measure for bovine mastitis. The most common pathogens include Staphylococcus aureus, various streptococci (Streptococcus dysgalactiae, Streptococcus uberis), and coliforms (Escherichia coli), which can be contracted from other infected cows or from the environment. A combination of kanamycin and cefalexin (1:1.5 wt/wt) is currently used therapeutically in Europe for the treatment of bovine mastitis, although standardized methods for the in vitro determination of the susceptibility of target pathogens have not been developed. This study evaluates the appropriate broth microdilution testing criteria for kanamycin and cefalexin administered in combination and reports the development of a disk diffusion test. At a ratio of kanamycin:cefalexin relevant to that observed in milk postadministration (10:1 wt/wt), the minimum inhibitory concentrations were determined against 307 isolates of target mastitis pathogens (staphylococci, streptococci, and E. coli). Based on achievable concentrations in milk and the resulting distribution of minimum inhibitory concentrations, preliminary broth breakpoints for kanamycin/cefalexin (10:1 fixed ratio) of or=32/3.2 microg/mL resistant were applied to evaluated staphylococci, streptococci, and E. coli. Parallel testing by disk diffusion and resulting error-rate bounded analysis using a combined disk concentration of 30 microg of kanamycin and 15 microg of cefalexin resulted in the establishment of preliminary disk interpretive breakpoints of >or=20 mm susceptible, 18 to 19 mm intermediate, and

Systemic treatment of subclinical mastitis in lactating cows with penethamate hydriodide.

A randomized controlled field trial was performed to evaluate the efficacy of a 3-d treatment regimen with i.m. penethamate hydriodide compared with no treatment in lactating cows with subclinical mastitis. To be included, a cow had to have 2 somatic cell counts (SCC) 300,000 cells/mL at the last 3 monthly controls, 1 or more quarters with SCC >250,000 cells/mL, and the same bacterial species isolated in 2 consecutive samples 2 to 4 d apart. A total of 151 quarters from 92 cows were monitored for 2 mo following treatment. Quarter milk samples were examined for bacteriological cure (BC) and SCC at 14, 28, and 60 d after treatment. Bacteriological cure was defined as not having the same bacterial species isolated from the quarter milk samples taken at 14 and 28 d posttreatment as in the samples taken before treatment. Systemic treatment with penethamate resulted in BC in 59.5% of quarters and 52.2% of cows, compared with 16.7 and 10.9% in the untreated cows. Somatic cell count decreased significantly in the penethamate-treated cows, steadily in the case of BC and transiently when the infections persisted. This study confirms that systemic treatment of subclinical mastitis with penethamate is effective and that BC of infected quarters has a sustained positive effect on milk SCC during the 2 mo following treatment.

Comparative efficacy of local and systemic antibiotic treatment in lactating cows with clinical mastitis.

The intramuscular administration of penethamate hydriodide over 3 consecutive days and the intramammary administration of an ampicillin/cloxacillin combination were compared in lactating cows suffering from infectious clinical mastitis in one quarter, through an open, randomized, controlled multicenter field trial. Clinical examinations were carried out on d 1 (immediately before treatment), 3, 8, 17, and 22. Milk samples were taken from affected quarters for bacteriological analysis on d 1, 17, and 22, and from all quarters for somatic cell count (SCC) determination on d 1, 8, 17, and 22. There was no significant difference in bacteriological and clinical cure rates between the 2 treatment groups. The systemic treatment with penethamate resulted more frequently in a reduction of the milk SCC below the threshold of 250,000 cells/mL. This also occurred in the adjacent quarters not affected by clinical mastitis but with an SCC above 250,000 cells/mL before treatment. These findings suggest that the parenteral treatment with penethamate provides collateral cure on the quarters of the cows affected by subclinical mastitis. The number of quarters per cow affected by clinical or subclinical mastitis should be considered when selecting an antibiotic treatment by the local or systemic route.