CNS species and antimicrobial resistance in clinical and subclinical bovine mastitis

Coagulase-negative staphylococci (CNS) are often associated with bovine mastitis. Knowledge about the relative importance of specific CNS species in different types of mastitis, and differences in antimicrobial resistance among CNS species is, however, scarce. Therefore, the aims of this study were to compare prevalence and antimicrobial susceptibility of CNS species in clinical and subclinical mastitis using material from two national surveys. Overall, Staphylococcus chromogenes and Staphylococcus epidermidis were the most common CNS species found followed by Staphylococcus simulans and Staphylococcus haemolyticus. S. epidermidis was significantly more prevalent in subclinical than in clinical mastitis, and a similar trend was observed for Staphylococcus saprophyticus, while Staphylococcus hyicus was significantly more common in clinical mastitis. The prevalence of β-lactamase producing isolates varied markedly between CNS species, and was significantly higher in S. epidermidis and S. haemolyticus (～ 40%), than in S. simulans and S. chromogenes where none or a few of the isolates produced β-lactamase. Resistance to more than one antimicrobial substance occurred in 9% and 7% of the clinical and subclinical isolates, respectively. In conclusion, the distribution of CNS species differed between clinical and subclinical mastitis indicating inter-species variation of pathogenicity and epidemiology. Overall, the prevalence of antimicrobial resistance was low, but some variation between CNS species was observed.     

Intra-species diversity and epidemiology varies among coagulase-negative Staphylococcus species causing bovine intramammary infections

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.     

Is the biofilm formation and slime producing ability of coagulase-negative staphylococci associated with the persistence and severity of intramammary infection?

Biofilm and slime formation assists bacteria in avoiding the host immune defence and antimicrobial therapy. It is suspected to affect the severity or persistence of mastitis caused by coagulase-negative staphylococci (CNS), which are a common cause of bovine mastitis. The phenotypic biofilm formation ability of 244 CNS isolates (199 isolates from bovine mastitis and 52 type and reference strains) was investigated with a tissue culture plate (TCP) assay and fluorescent in situ hybridization (FISH). Slime production of the strains was assessed using Congo red agar (CRA) plates. Additionally, genes encoding the adhesion proteins MSCRAMM (microbial surface components recognizing adhesive matrix molecules) and biofilm-associated proteins (bap) were detected. The severity of intramammary infection (IMI) in mastitis from which the isolates originated was measured with milk N-acetyl-β-D-glucosaminidase (NAGase) activity. One-third of isolates from mastitis produced biofilm when analysed with TCP or FISH. The kappa test value, measuring the agreement between two tests, differed between CNS species. Slime production was less frequent for isolates of the common mastitis species Staphylococcus chromogenes (0.2% of isolates produced slime) and Staphylococcus simulans (3.5%) compared to Staphylococcus epidermidis (40%). No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was rare in isolates originating from IMI. Only 12.7% of isolates from persistent IMI and 1.8% of isolates from spontaneously eliminated IMI produced slime. The eno gene encoding laminin-binding protein was most frequently detected among the isolates from mastitis, 75% of them having this gene. Only a few other MSCRAMM genes were detected.     

Efficacy of targeted 5-day combined parenteral and intramammary treatment of clinical mastitis caused by penicillin-susceptible or penicillin-resistant Staphylococcus aureus

Combined parenteral and intramammary treatment of mastitis caused by Staphylococcus aureus was compared to parenteral treatment only. Cows with clinical mastitis (166 mastitic quarters) caused by S. aureus treated by veterinarians of the Ambulatory Clinic of the Faculty of Veterinary Medicine during routine farm calls were included. Treatment was based on in vitro susceptibility testing of the bacterial isolate. Procaine penicillin G (86 cases due to beta-lactamase negative strains) or amoxycillin-clavulanic acid (24 cases due to beta-lactamase positive strains) was administered parenterally and intramammarily for 5 days. Efficacy of treatments was assessed 2 and 4 weeks later by physical examination, bacteriological culture, determination of CMT, somatic cell count and NAGase activity in milk. Quarters with growth of S. aureus in at least one post-treatment sample were classified as non-cured. As controls we used 41 clinical mastitis cases caused by penicillin-susceptible S. aureus isolates treated with procaine penicillin G parenterally for 5 days and 15 cases due to penicillin-resistant isolates treated with spiramycin parenterally for 5 days from the same practice area. Bacteriological cure rate after the combination treatment was 75.6% for quarters infected with penicillin-susceptible S. aureus isolates, and 29.2% for quarters infected with penicillin-resistant isolates. Cure rate for quarters treated only parenterally with procaine penicillin G was 56.1% and that for quarters treated with spiramycin 33.3%. The difference in cure rates between mastitis due to penicillin-susceptible and penicillin-resistant S. aureus was highly significant. Combined treatment was superior over systemic treatment only in the beta-lactamase negative group.     

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.     

Demonstration of non-inferiority of a novel combination intramammary antimicrobial in the treatment of clinical mastitis

AIM: To test the non-inferiority of a novel combination intramammary product containing penicillin and cloxacillin to a reference intramammary product containing oxytetracycline, oleandomycin, neomycin and prednisolone with regard to bacteriological cure and clinical cure.

METHODS: Clinical cases of mastitis were sourced from 30 spring-calving dairy farms in the Southland region of New Zealand. Affected quarters were infused three times at 24 hourly intervals with either the novel combination product containing 1?g penicillin and 200?mg cloxacillin, or a reference product containing 200?mg oxytetracycline, 100?mg oleandomycin, 100?mg neomycin and 5?mg prednisolone. Cows were enrolled when a farmer detected a case of clinical mastitis. Milk samples were collected for microbiological culture immediately before treatment (Day 0) and on Days 9, 16 and 23. Bacteriological cure was compared for 187 and 178 quarters treated with the reference and novel product, respectively, and clinical cure was compared for 235 and 223 quarters, respectively. Non-inferiority was assessed by calculating the difference in cure rates between the two products and constructing a 95% CI around the difference, using the variance inflation factor to account for herd level clustering. The non-inferiority margin was 20% for both bacteriological and clinical cure. Generalising estimating equation models were used to determine predictor variables.

RESULTS: The bacteriological cure percentage, adjusted to account for herd-level clustering, was 8.5 (95% CI=?1.7–21.8)% higher for quarters treated with the novel than the reference product. The adjusted clinical cure percentage was 0.3 (95% CI=?11.2–12.0)% higher for clinical quarters treated with the novel than the reference product. Bacterial species was the only covariate for bacteriological cure (p=0.003), and quarter score at enrolment (indicating udder inflammation) was the only covariate for clinical cure (p=0.032) in the multivariable models.

CONCLUSION: The novel combination product was demonstrated to be non-inferior to the reference product with regards to both bacteriological cure and clinical cure.

CLINICAL RELEVANCE: Clinicians treating mastitis now have access to this novel combination intramammary product, and demonstration of its non-inferiority compared to the existing reference product will provide options for treatment approaches. The novel product contains fewer antimicrobials; which are of a narrower spectrum of activity.     

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.     

Prevalence and herd-level risk factors for intramammary infection with coagulase-negative staphylococci in Dutch dairy herds

In this study, the prevalence of intramammary infection (IMI) with coagulase-negative staphylococci (CNS) in The Netherlands was estimated on 49 randomly selected herds with at least 40 lactating cows. In total, 4220 quarter milk samples were collected. The prevalence of CNS IMI in The Netherlands was estimated at 10.8% at quarter level and 34.4% at cow level, making it the most frequently isolated group of pathogens. Fourteen species of CNS were identified; the most frequently isolated species was Staphylococcus chromogenes (30.3%) followed by Staphylococcus epidermidis (12.9%) and Staphylococcus capitis (11.0%). Prevalence of CNS IMI was higher in heifers compared to older cows. Geometric mean quarter SCC of CNS-positive quarters was 109,000 cells/ml, which was approximately twice as high as culture-negative quarters. Quarters infected with S. chromogenes, S. capitis and Staphylococcus xylosus had a higher SCC (P<0.05) than culture-negative quarters, while quarters that were culture-positive for S. epidermidis and Staphylococcus hyicus tended to have a higher SCC than culture-negative quarters. An increased prevalence of CNS IMI was associated with the herd-level variables source of drinking water not being tap water, housing of dry cows in one group instead of multiple groups, measurement of cow SCC every month, udder health monitoring by the veterinarian, pasturing during outdoor season, percentage of stalls contaminated with milk, and BMSCC>250,000 cells/ml. Although a causal relation between these factors and prevalence of CNS is not proven and for some factors not even likely, knowledge of the associations found may be helpful when approaching CNS problems on dairy farms.     

Characterization of Staphylococcus simulans strains isolated from cases of bovine mastitis

This study was conducted to characterize Staphylococcus simulans isolated from cases of bovine mastitis. A total of 134 isolates of S. simulans selected from 80 quarters from 61 cows or heifers in 37 different herds were characterized by EcoRI ribotyping. From 22 quarters two to seven consecutive isolates taken at weekly intervals were selected. Furthermore, three isolates from clinical infections in humans and two reference strains were included. A total of 16 different ribotypes were found, however, two types predominated. In most herds more than one type was found. From the 22 different quarters, where 76 paired or multiple isolates were at disposal, the same ribotype was constantly found in the same quarter. This study showed that S. simulans causing bovine mastitis could be divided into relatively large number of different types, but that two types predominated. More than one type could be found in the same herd and within different quarters of the same cow, but ribotyping confirmed that S. simulans could be the cause of persistent and stable infections.     

Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders.

The species of 203 strains of coagulase-negative staphylococci (CNS), isolated from bovine udder quarters was determined; all were tested for hydrophobicity and encapsulation, attributes that may relate to virulence. Twelve species were identified, of which Staphylococcus simulans, (34.5%), S. chromogenes (16.7%), S. epidermidis (13.8%) and S. xylosus (8.9%) were the most frequent. The majority of strains possessed a hydrophilic cell surface. However, strains from two species (S. chromogenes and S. epidermidis) were more hydrophobic than the others. Only five strains were encapsulated (S. xylosus, 3; S. saprophyticus, 1; and S. sciuri, 1). Judging from the low frequencies of hydrophobic and encapsulated strains, and comparing with strains isolated from clinical cases, it is suggested that these properties are not major virulence determinants of CNS.     

Coagulase-negative staphylococci-emerging mastitis pathogens

Coagulase-negative staphylococci (CNS) have become the most common bovine mastitis isolate in many countries and could therefore be described as emerging mastitis pathogens. The prevalence of CNS mastitis is higher in primiparous cows than in older cows. CNS are not as pathogenic as the other principal mastitis pathogens and infection mostly remains subclinical. However, CNS can cause persistent infections, which result in increased milk somatic cell count (SCC) and decreased milk quality. CNS infection can damage udder tissue and lead to decreased milk production. Staphylococcus simulans and Staphylococcus chromogenes are currently the predominant CNS species in bovine mastitis. S. chromogenes is the major CNS species affecting nulliparous and primiparous cows whereas S. simulans has been isolated more frequently from older cows. Multiparous cows generally become infected with CNS during later lactation whereas primiparous cows develop infection before or shortly after calving. CNS mastitis is not a therapeutic problem as cure rates after antimicrobial treatment are usually high. Based on current knowledge, it is difficult to determine whether CNS species behave as contagious or environmental pathogens. Control measures against contagious mastitis pathogens, such as post-milking teat disinfection, reduce CNS infections in the herd. Phenotypic methods for identification of CNS are not sufficiently reliable, and molecular methods may soon replace them. Knowledge of the CNS species involved in bovine mastitis is limited. The dairy industry would benefit from more research on the epidemiology of CNS mastitis and more reliable methods for species identification.     

A multilocation clinical trial in lactating dairy cows affected with clinical mastitis to compare the efficacy of treatment with intramammary infusions of a lincomycin/neomycin combination with an ampicillin/cloxacillin combination

A study was conducted to compare the efficacy in lactating dairy cows of intramammary infusions in quarters affected with clinical mastitis between a formulation containing 330 mg lincomycin and 100 mg neomycin in a 10-mL aqueous solution (LINCOCIN FORTE S, Pharmacia & Upjohn) and a formulation containing 75 mg ampicillin and 200 mg cloxacillin in an oil suspension (AMPICLOX, Pfizer Animal Health). This study was designed as a multicentre clinical trial involving investigators in France, Germany and Belgium and carried out according to the European Commission guidelines on Good Clinical Practices. Cows in the herds were monitored for clinical mastitis. When evidence of clinical mastitis was detected in a single quarter, a pretherapy milk sample was collected from the affected quarter. After milk sampling, the cow was assigned to one of the two treatment groups at random and treated with an intramammary infusion of one syringe of either LINCOCIN FORTE S or AMPICLOX for three successive milkings in the mastitic quarter. At 4-5, 13-15 and 20-22 days after first infusion, the veterinarian returned to the farm to conduct a clinical examination and collect milk samples from the affected quarter. Milk samples were cultured for the presence of mastitis organisms and somatic cell count (SCC) was measured. Following a 10-month study period, 256 cases were enrolled in the study. A total of 232 and 189 cases were analysed for clinical cure and for clinical-plus-bacteriological cure, respectively. The proportions of cases cured clinically and cured clinically-plus-bacteriologically were compared between the two treatment groups. Somatic cell count differences between treatment groups were also tested. The clinical cure rate for LINCOCIN FORTE S (62.5%) was significantly better than for AMPICLOX (51.8%) (P = 0.035). The clinical-plus-bacteriological cure rate was also significantly better for LINCOCIN FORTE S (38.1%) than for AMPICLOX (21.7%) (P = 0.005). Among bacteriologically cured cases, the SCC declined in both treatment groups but the SCC was significantly higher for the AMPICLOX group than for the LINCOCIN FORTE S group (P = 0.036). In conclusion, clinical cure rate, clinical-plus-bacteriological cure rate, and SCC level were significantly better with LINCOCIN FORTE S than for AMPICLOX.     

Antimicrobial resistance and genotypic characterization of coagulase-negative staphylococci over the dry period

Coagulase-negative staphylococci (CNS) have become the most frequently isolated organisms from bovine intramammary infections in recent years. While antimicrobial resistance (AR) is not considered a major problem among mastitis pathogens, concerns over emerging AR in general are increasing worldwide. Little information exists about the association between AR and one of the most common mastitis control measures, antibiotic dry cow therapy. The primary objective of the current study was to determine the prevalence of AR in CNS isolated before and after antimicrobial dry cow therapy. An additional objective was to genotypically characterize selected CNS isolates using pulsed-field gel electrophoresis (PFGE), to assess diversity and persistence of organisms over the dry period. Resistance against 10 antimicrobials was determined using a broth microdilution method and compared between CNS isolates collected at dry-off and at calving and from cows treated or not treated with intramammary antimicrobial products at dry-off (752 cows in total). Results suggested that increasing age of a cow and dry cow treatment when combined with high milk somatic cell count at dry-off and positive clinical mastitis history, were associated with increased AR to most beta-lactam antimicrobials and sulfadimethoxine. PFGE results suggested considerable diversity among the tested isolates as well as some clusters within cows and herds. PFGE would be useful in distinguishing between potentially persisting infections and cures and reinfections with different CNS strains.     

Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis

ABSTRACT: Coagulase-negative staphylococci (CNS) are in several countries the most common bacteria isolated in subclinical mastitis. To investigate the innate immune response of cows to infections with two common mastitis-causing CNS species, Staphylococcus epidermidis and Staphylococcus simulans, experimental intramammary infection was induced in eight cows using a crossover design. The milk somatic cell count (SCC), N-acetyl-β-D-glucosaminidase (NAGase) activity, milk amyloid A (MAA), serum amyloid A (SAA) and proinflammatory cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor α (TNF-α) were determined at several time points before and after challenge. All cows became infected and showed mild to moderate clinical signs of mastitis. The spontaneous elimination rate of the 16 infections was 31.3%, with no difference between species. Infections triggered a local cytokine response in the experimental udder quarters, but cytokines were not detected in the uninfected control quarters or in systemic circulation. The innate local immune response for S. simulans was slightly stronger, with significantly higher concentrations of IL-1β and IL-8. The IL-8 response could be divided into early, delayed, or combined types of response. The CNS species or persistency of infection was not associated with the type of IL-8 response. No significant differences were seen between spontaneously eliminated or persistent infections.     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

Prevalence and persistence of coagulase-negative Staphylococcus species in three dairy research herds

Coagulase-negative Staphylococcus species (CNS) were isolated from 11.3% (1407 of 12,412) of mammary quarter milk samples obtained from cows in three dairy research herds in 2005. Approximately 27% (383/1407) of CNS was identified to the species level. The species distribution among those CNS identified from all herds was Staphylococcus chromogenes (48%), Staphylococcus hyicus (26%), Staphylococcus epidermidis (10%), Staphylococcus simulans (7%), Staphylococcus warneri (2%), Staphylococcus hominis (2%), Staphylococcus saprophyticus (1%), Staphylococcus xylosus (1%), Staphylococcus haemolyticus (<1%), Staphylococcus sciuri (<1%), and Staphylococcus intermedius (<1%). Staphylococcuschromogenes was the predominant CNS isolated from all three herds; however, differences were seen in the prevalence of other CNS species. A total of 158 CNS (S. chromogenesn=66, S. hyicusn=38, S. epidermidisn=37, S. simulans n=10, and S. warneri n=7) were analyzed by pulsed-field gel electrophoresis (PFGE). The majority (33/41) of CNS isolated from the same mammary quarter on more than one occasion had the same PFGE pattern indicating persistence of the same infection over time. When all PFGE patterns for each CNS were analyzed, no common pulsotype was seen among the three herds indicating that CNS are quite diverse. Composite milk somatic cell count (SCC) data were obtained +/-14d of when CNS were isolated. Average milk SCC (5.32 log(10)/ml) for cows in which CNS was the only bacteria isolated was significantly higher than the average milk SCC (4.90 log(10)/ml) from cows with quarter milk samples that were bacteriologically negative.     

Antimicrobial susceptibility of coagulase-negative Staphylococcus species isolated from bovine milk

Coagulase-negative Staphylococcus (CNS) isolates (n=168) obtained from milk from heifers and dairy cows were screened for minimum inhibitory concentration (MIC) to antimicrobials used commonly for mastitis therapy. Of the 10 CNS species included in the study, the predominant species were Staphylococcus chromogenes (n=61), Staphylococcus epidermidis (n=37), Staphylococcus hyicus (n=37), and Staphylococcus simulans (n=16). The majority of CNS was susceptible to ampicillin, oxacillin, cephalothin, and ceftiofur. Erythromycin and pirlimycin were also very effective in vitro inhibitors of CNS. The only exception was observed with S. epidermidis. Of 37 S. epidermidis evaluated, 13 (35%) exhibited efflux-based resistance to erythromycin (> or =16 microg/ml) encoded by msrA and one isolate carried ermC encoding ribosomal methylase-based resistance to both erythromycin (> or =64 microg/ml) and pirlimycin (> or =64 microg/ml). A total of 17 S. epidermidis, 11 S. chromogenes, and one S. hyicus exhibited phenotypic resistance to ampicillin (> or =0.5 microg/ml). Constitutive beta-lactamase production was observed in all ampicillin resistant isolates except 4 S. epidermidis that exhibited inducible beta-lactamase production. Induced beta-lactamase production was also observed in 13 S. epidermidis that were phenotypically susceptible to the entire MIC panel. All isolates that produced beta-lactamase either constitutively or by induction carried blaZ. S. epidermidis (n=12, 32%) that were resistant to methicillin (oxacillin > or =0.5 microg/ml) carried low affinity penicillin-binding protein encoded by mecA. Most multi-drug resistant (MDR) S. epidermidis (> or =2 resistance genes) were resistant to ampicillin, erythromycin and methicillin. All except one MDR S. epidermidis had icaAB, which encodes for polysaccharide intercellular adhesion. Based on pulsed field gel electrophoresis, MDR S. epidermidis were closely related genotypically, and were isolated from different cows on the same farm suggesting clonal dissemination. Bovine S. epidermidis share antimicrobial resistance patterns and virulence determinants of strains observed in human infections. Studying CNS at the species level can provide valuable information about species-specific differences that can be vital data for effective mastitis therapy and management.     

Effect of teat dipping with a germicide barrier teat dip in late gestation on intramammary infection and clinical mastitis during the first 5 days post-partum in primiparous cows

The effect of teat dipping with a barrier teat dip prior to parturition on intramammary infection (IMI) and clinical mastitis during the first 5 days post-partum was investigated in a split udder trial in 149 Holstein-Frisian heifers. Their left front and right hind quarters were dipped three times weekly (i.e. Monday, Wednesday and Friday) with a barrier teat dip containing 0.1% polyvidon iodine from day 260 of gestation until parturition. The opposite quarters (right front and left hind quarter) served as untreated control. Bacteria were isolated from 52.2% of quarter milk samples collected immediately after parturition prior to first machine milking. Staphylococcus aureus and coagulase-negative staphylococci (CNS) were predominantly found in the samples (29.2 and 35.6% of the positive samples, respectively). At parturition 6.7% of the heifers showed signs of clinical mastitis and another 27.5% developed signs of clinical mastitis during the first five days of lactation. No significant differences were found between treated and control quarters regarding IMI and incidence of clinical mastitis. Teat dipping prior to parturition in primigravid dairy heifers did not improve udder health in this trial.     

Field trials on the prophylaxis of intramammary infections in pregnant heifers

The study was carried out in 5 farms on 174 pregnant heifers. Clinical examination of the udder and bacteriological tests of quarter secretion were performed between the 8th and 3rd week before parturition, and then the animals were divided into a control group (64 heifers) and 3 experimental groups and immediately treated. A group of 32 experimental heifers was injected once with antioxidants (Vitamin A--600,000 i.u.; Vitamin D3--200,000 i.u.; Vitamin E--1.5 mg/kg b.w., Selenium--0.022 mg/kg b.w., i.m.). The next group (26 heads) was intramammary infused with antibiotic DC product (cloxacillin). Heifers from last experimental group (52) were injected with lysosyme dimer in a single dose of 0.02 mg/kg b.w. Clinical and bacteriological examinations were made during the first week after calving. The presence of bacteria was found in secretion of 22.6-38.9% udder quarters in 56.2-71.2% of pregnant heifers. The number of infected quarters (cows) did not change distinctly in the first week after calving except the lysozyme dimer group, where a decrease by 30% was noted. The percentage of quarters with elevated somatic cell count was higher in antibiotic DC group and closely similar in the other groups. None of examined methods showed an acceptable prophylactic effect. Clinical mastitis cases during first week after parturition were mostly caused by Escherichia coli, Staph. chromogenes, Staph. simulans, Staph. aureus, Staph. hyicus, Str. uberis, Str. acidominimus and Enterococcus faecalis.     

A comparative efficacy trial between cefuroxime and cloxacillin as intramammary treatments for clinical mastitis in lactating cows on commercial dairy farms

AIMS: To assess the efficacy of a commercial intramammary preparation containing cefuroxime as a treatment for clinical mastitis in lactating dairy cows. METHODS: Clinical mastitis cases (n=440) were identified during early to mid lactation on 36 seasonally-calving commercial dairy herds in south-western Victoria, Australia, that ranged in size from 140 to 550 cows. A milk sample for bacterial culture was collected from each affected quarter prior to treatment. Cases were treated using a commercial intramammary antibiotic product containing either 250 mg cefuroxime or 200 mg cloxacillin, according to the manufacturer's recommendations, and the recommended milk with-holding period was observed. The assessment of clinical cure of each case was made by the farm owner/manager at the end of the milk with-holding period. Post-treatment milk samples were taken from clinically cured quarters at each of three consecutive milkings, commencing 7 days after the end of the milk with-holding period and submitted for bacterial culture. RESULTS: Pathogenic bacteria were isolated from 252/416 (60.6%) pre-treatment milk samples submitted from eligible cases. Streptococcus uberis was the most frequent isolate, being cultured from 31.7% of cases. Staphylococcus aureus was isolated from 18.3% of pre-treatment samples, and Esherichia coli from 7.0%. The clinical cure rate for all eligible cases was 81.7%. There was no significant difference in clinical cure rates between cases treated with cefuroxime (186/225=82.7%) and cases treated with cloxacillin (154/191=80.6%). The trial had 80% power to detect a significant difference if the actual cure rates differed by at least 12.6%. There was a superior clinical response rate (p=0.04) for mastitis cases from which E. coli was isolated that were treated with cefuroxime (18/19=95%) compared with cloxacillin 6/10=60%), but case numbers were low (n=29). The overall bacteriological cure rate for the trial was 70% (69/98 cases assessed). There was no significant difference (p=0.27) in bacteriological cure rate between cases treated with cefuroxime (42/56=75%) and cases treated with cloxacillin (27/42=64%). CONCLUSIONS: This trial demonstrated that cefuroxime was an effective intramammary treatment for clinical mastitis and had similar clinical and bacteriological cure rates to cloxacillin, an accepted industry-standard product. Activity against Gram-negative and Gram-positive bacteria was demonstrated for cefuroxime which, along with the economic benefits of less discarded milk compared with other antibiotic preparations, indicated that cefuroxime is likely to be an appropriate intramammary antibiotic for the treatment of clinical mastitis in commercial dairy herds during early to mid lactation in Victoria, Australia.