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.     

Altered leukocyte responsiveness in dairy cows with naturally occurring chronic Staphylococcus aureus mastitis

Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.     

Lactoferrin Concentration in Milk of Bovine Clinical Mastitis

The lactoferrin (LF) concentration in the milk from dairy cows with clinical mastitis was determined to evaluate the relationship between the LF concentration (LFC) in milk and the non-specific defensive capability of the udder. The mean LFC in 368 milk samples from 319 cows with clinical mastitis was significantly higher (p<0.01) than that of normal cows. The mean LFC in milk from quarters infected with Mycoplasma bovis or Staphylococcus aureus was significantly higher (p<0.05) than that of quarters infected with coagulase-negative staphylococci (CNS). In Escherichia coli mastitis, the level of LFC in milk from cows with peracute mastitis was significantly lower (p<0.01) than that from cows with acute mastitis. In cases of mastitis due to E. coli, the mean LFC in milk from cows that needed more than 10 days to recover from the mastitis or were not cured was significantly lower (p<0.05) than that for cows which took less than 10 days to be cured. The mean LFC in milk from cows with peracute E. coli mastitis was significantly lower (p<0.05) than that for cows with mastitis associated with environmental streptococci or CNS, although these low LF levels were somewhat increased after 46 h from the occurrence of mastitis. These results suggest that the decreased levels of LF in peracute E. coli mastitis may be associated with the progress of inflammation in the early phase of mastitis.     

Somatic cell and innate immune responses in mammary glands of lactating cows to intramammary infusion of Bifidobacterium breve at pre-drying off period

Intramammary infusion of Bifidobacterium breve (B. breve)-induced somatic cell (SC) counts, chemiluminescent response (CL), lactoferrin (LF) concentrations and mastitis-causing pathogens from quarters with subclinical mastitis were measured to evaluate innate immune response of mammary glands in dairy cows at 3 to 4 weeks before drying off. SC counts in 7 quarters of 7 control cows and 5 quarters of 6 cows with mastitis increased markedly on day 1 and SC values in control cows were significantly (P<0.05) increased and returned to pre-infusion levels on day 5 after B. breve-infusion. CL values in both groups increased markedly on day 1 and then decreased after B. breve-infusion; however, CL values in cows with mastitis did not return to normal levels on day 5 and at postpartum. The CL values were highly correlated with their SC counts in milk from both groups. LF concentrations increased toward day 3 after B. breve-infusion and were higher in cows with mastitis. B. breve-infusion eliminated 16.6% (1/6) of pathogens from 6 quarters with chronic subclinical mastitis. B. breve-induced SC responses in quarters from 3 cows with mastitis showed characteristic patterns of recovery, persistent and new infections. B. breve-induced SC counts in quarters from the cows in the pre-drying off were lower (25.7–70.6%) than those of the cows in mid-lactation. The intrinsic innate immune response in cows on pre-drying off may be decreased and appears to be insufficient to eliminate pathogens from mammary gland in the pre-drying off.     

Changes in blood and milk lymphocyte sub-populations during acute and chronic phases of Staphylococcus aureus induced bovine mastitis

Staphylococcus aureus (S. aureus) often causes long-lasting chronic sub-clinical udder infections in dairy cows. To investigate if this can be due to a negative impact of S. aureus on lymphocytes important for the immune defence, alterations in proportions and expression intensity of CD4+, CD8+, WC1+, B and IL-2R+ lymphocytes was studied in blood and milk, as S. aureus mastitis developed from acute clinical to chronic sub-clinical form. Six healthy dairy cows were inoculated with S. aureus in one udder quarter per cow, and one quarter per cow acted as an uninfected control. Blood samples, and milk samples from infected and non-infected quarters were collected before infection and for five weeks after infection. All infected quarters developed acute clinical mastitis, of which five turned into chronic sub-clinical mastitis. In infected quarters, the proportions of all lymphocyte sub-sets, except WC1+ cells, differed in acute phase compared to pre-infection, while the dominant finding in the chronic phase was increased expression intensities per cell. An impact on blood lymphocytes and milk lymphocytes in non-infected quarters also occurred, mainly during the chronic phase. The most prominent finding was the increased proportion and expression of B-lymphocytes in blood, infected and non-infected quarters during chronic sub-clinical mastitis. As S. aureus can invade and survive intracellularly, a preferential stimulation of B-cells, suggesting development of a humoral response, may not be sufficient to eliminate intracellular bacteria, which could explain the persistence of the infection.     

Clinical characteristics and persistence of bovine mastitis caused by different species of coagulase-negative staphylococci identified with API or AFLP

The coagulase-negative staphylococcal species causing mastitis in lactating cattle were identified and possible differences in the clinical characteristics or persistence of mastitis caused by different CNS were evaluated. The effect of antimicrobial treatment was also assessed. In addition, AFLP-typing of CNS was compared with the phenotypic identification. A total of 133 clinical or subclinical quarter cases of intramammary infection caused by CNS from the practice area of the Ambulatory Clinic of the University of Helsinki were studied. Bacteriological diagnosis was based on biochemical (API) testing. Staphylococcus simulans (43.6%) followed by S. chromogenes (23.3%) were the most common CNS species isolated from the milk samples. Ninety-nine isolates were genotyped using AFLP-analysis. Only 75.0% of S. chromogenes and S. simulans isolates identified with API test were clustered with the type strains of these species. Approximately half of the mastitis cases were clinical, and in the majority clinical signs were mild. The severity and persistence of intramammary infection were unaffected by CNS species. Fifty-nine percent of the quarter cases were treated with antimicrobials, and the rest were left without treatment. Mastitis due to beta-lactamase-negative CNS was treated with penicillin G and that due to beta-lactamase-positive CNS with cloxacillin. Nineteen percent of the isolates were beta-lactamase-positive. The bacterial cure rate for quarters treated with antimicrobials was high, 85.9%, as opposed to only 45.5% for untreated quarters. Bacterial cure rates for the most common CNS species or AFLP clusters were not statistically different. Further studies on identification of CNS species are needed.     

The bovine innate immune response during experimentally-induced Pseudomonas aeruginosa mastitis

Almost half of all clinical cases of mastitis are caused by Gram-negative bacteria. Among these bacteria, intramammary infection by Pseudomonas aeruginosa remains one of the most refractory to antibiotic therapy. The ability to recognize potentially harmful pathogens whether previously encountered or not, as well as the induction of an initial pro-inflammatory response to these pathogens, are critical components of host innate immunity. Although the innate immune response to another Gram-negative mastitis-causing pathogen, Escherichia coli, has been well-characterized, little is known about the response to other Gram-negative bacteria, including P. aeruginosa. The objective of the current study was to characterize the systemic and localized bovine innate immune response to intramammary infection with P. aeruginosa. The contralateral quarters of ten mid-lactating Holstein cows were challenged with either saline or P. aeruginosa. Following the establishment of infection, milk samples were collected and assayed for changes in cytokine and growth factor concentrations, complement activation, and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), two accessory molecules involved in host recognition of Gram-negative bacteria. Initial increases in milk somatic cell counts were evident within 12h of experimental challenge and remained elevated for >or=3 weeks. Increased permeability of the mammary gland vasculature, as evidenced by elevated milk levels of BSA, was initially observed 20 h post-infection and persisted for 2 weeks. Within 32 h of challenge, increased levels of IL-8, TNF-alpha, IL-10, and IL-12 were detected, however, the elevated levels of these cytokines were not sustained for longer than a 24h period. In contrast, elevations in IL-1beta, IFN-gamma, TGF-alpha, TGF-beta1, TGF-beta2, sCD14, LBP, and activated complement factor 5 (C5a) were sustained for periods of >48 h. Systemic changes were characterized by elevated body temperature, induction of the acute phase protein synthesis of serum amyloid A and LBP, and a transient decrease in circulating neutrophils and lymphocytes. Together, these data demonstrate the capability of the mammary gland to mount a robust innate immune response to P. aeruginosa that is characterized by the induction of pro-inflammatory cytokines, complement activation, and increased levels of accessory molecules involved in Gram-negative bacterial recognition.     

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.     

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.     

Staphylococcus aureus intramammary infection elicits increased production of transforming growth factor-alpha, beta1, and beta2

In contrast to other mastitis pathogens, the host response evoked during Staphylococcus aureus intramammary infection is marked by the absence of the induction of critical cytokines, including IL-8 and TNF-alpha, which have established roles in mediating host innate immunity. The elucidation of changes in the expression of other mediators with the potential to regulate mammary inflammatory responses to S. aureus remains lacking. Transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are cytokines that regulate mammary gland development. Because these cytokines also have a demonstrated role in mediating inflammation, the objective of the current study was to determine whether S. aureus intramammary infection influences their expression. Ten cows were challenged with S. aureus and milk samples collected. Increases in milk levels of TGF-alpha were evident within 32h of infection and persisted for 16h. Increases in TGF-beta1 and TGF-beta2 levels were detected within 40h of S. aureus infection and persisted through the end of the study. Thus, in contrast to IL-8 and TNF-alpha, S. aureus elicits host production of TGF-alpha, TGF-beta1, and TGF-beta2. This finding may suggest a role for these cytokines in mediating mammary gland host innate immune responses to S. aureus.     

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.     

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.     

Immune response during the onset of coliform mastitis in dairy cows vaccinated with STARTVAC®

The immune response during the onset of coliform mastitis in vaccinated cows was investigated by measuring lactoferrin (LF), interleukin-8 ( IL-8), and interleukin-1β (IL-1β) concentrations and somatic cell counts in 28 milk samples at the onset of acute coliform mastitis (ACM) and 73 milk samples at the onset of peracute coliform mastitis (PCM). Vaccinated ACM, unvaccinated ACM, and vaccinated PCM showed significantly higher values for LF and IL-1β levels than unvaccinated PCM (p < .01). The IL-8 concentration was lower in vaccinated PCM than in unvaccinated PCM (p < .05). There was no significant difference in somatic cell counts for each parameter. There were no significant differences in the parameters between vaccinated and unvaccinated ACM cows, or vaccinated ACM and PCM cows. From the above results, it is suggested that mastitis vaccination improved the early immune response, particularly at the onset of PCM, and played a large role in host defense against the initial infection.     

Incidence of mastitis in beef cows after intramuscular administration of oxytetracycline

There is limited information on the value of antibiotic therapy for mastitis in beef cows. Effects of antibiotic treatment at weaning and the subsequent calving on calf weaning weight, milk somatic cell counts, milk components, and intramammary infection were studied in beef cows. Additionally, effects of number of infected mammary quarters, number of dry mammary quarters, type of intramammary pathogen, and parity on response variables were determined. Cows (n = 192) were randomly assigned to treatments in a 2 x 2 factorial arrangement; factors were time of treatment (weaning and after calving) and treatment (vehicle and vehicle plus antibiotic). Oxytetracycline (LA-200) or vehicle was administered intramuscularly following collection of quarter milk samples at weaning and calving. Percentage of infected cows and quarters averaged 43.4 and 16.4%, respectively, at calving and increased (P < 0.05) to 53.7 and 29.7% at weaning. Calves from cows with one or two dry quarters weighed 12.7 kg less (P < 0.05) at 90 d after calving and 18.7% less (P < 0.05) at 212 d after calving than calves from cows with no dry quarters. Calves from cows with three or four infected quarters weighed 17.5 kg less (P < 0.05) at 90 d and 25.5 kg less (P < 0.05) at weaning than calves from cows with two or fewer infected quarters. Infections by Staphylococcus aureus and coagulase-negative staphylococci were the most common and accounted for 67 and 78% of the infections. Percentages of infected cows and quarters, infections caused by S. aureus, and dry quarters increased (P < 0.05) with parity. No differences were found among antibiotic treatments for any of the response variables studied. Intramuscular oxytetracycline was not effective in the control of mastitis in beef cows under the conditions of the study.     

Evaluation by enzyme-linked immunosorbent assay of local and systemic production of milk immunoglobulins to surface exopolysaccharide antigen in cows with staphylococcal mastitis

An enzyme-linked immunosorbent assay (ELISA) was developed to evaluate milk immunoglobulin levels to surface exopolysaccharide antigen of Staphylococcus aureus in cows with staphylococcal mastitis. Quarter milk samples were obtained from 24 lactating dairy cows on two occasions, one month apart. Cows were classified as S. aureus-positive (S. aureus cultured from at least one quarter on both sample dates) or S. aureus-negative. Individual quarter samples were tested for IgA (representing local synthesis) and IgG1 (primarily of systemic origin) specific for staphylococcal surface exopolysaccharide antigen. No significant differences were found for specific IgA or IgG1 between S. aureus-positive and S. aureus-negative cows, nor between infected and non-infected quarters of S. aureus-positive cows. The data indicate that, in cows with staphylococcal mastitis, milk immunoglobulins specific for exopolysaccharide antigen are not significantly increased by either the systemic or the local immune response.     

Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens.     

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.     

Intramammary infections and risk factors for clinical mastitis in herds with low somatic cell counts in bulk milk

Ten herds with low somatic cell counts in bulk milk had an incidence of clinical mastitis of only 2.2 per 100 cows whereas 10 other herds with similarly low cell counts had an incidence of 53.6 per 100 cows. The major pathogens in the herds with a high incidence were Escherichia coli, Streptococcus uberis, Staphylococcus aureus and the coagulase-negative staphylococci. The percentage of uninfected quarters in the herds with a high incidence of clinical mastitis was 21.4 per cent compared with 12.2 per cent in the herds with a low incidence of clinical mastitis. The prevalence of coagulase-negative staphylococci, Corynebacterium bovis and Micrococcus species was higher in the herds with a low incidence of clinical mastitis. There was a significant linear relationship between the percentage of uninfected quarters and the incidence of clinical mastitis in the herds with a high incidence of clinical mastitis. In herds with a low incidence of clinical mastitis significantly less teat disinfection after milking was practised. The results suggest that infections with minor pathogens tend to protect cows against mastitis, and that teat disinfection after milking may increase the percentage of uninfected quarters and lead to an increased risk of clinical mastitis in herds with low somatic cell counts in bulk milk.     

Intramammary Infusion of β1,3‐glucan for Prevention and Treatment of Staphylococcus aureus Mastitis

Udder health problems associated with Staphylococcus aureus infections in dairy cows are difficult to control and antibiotics have limited effects. Lately, more interest has been directed towards ways to stimulate the innate immune mechanisms of the animal for better prevention and treatment of mastitis. The objectives of this study were to investigate if intramammary infusion at drying off with the immune modulator β1,3‐glucan can make the udder more resistant to experimental intra mammary S. aureus infection at this time, and to study if intramammary infusion of β1,3‐glucan into lactating udder quarters with chronic subclinical S. aureus infection can stimulate the clearing of the infection. Another aim was to evaluate the effect of β1,3‐glucan on the expression of major histocompatibility complex (MHC class II) on mammary leucocytes, measured by flow cytometry, during these circumstances. The results indicated a slight, but not statistically significant, positive effect of β1,3‐glucan at drying off on the clinical and anti‐bacterial response to S. aureus infection, but no therapeutic effect of β1,3‐glucan treatment of udder quarters with chronic subclinical S. aureus mastitis. However, the proportion of MHCII+ milk lymphocytes tended to increase after glucan infusion in those udder quarters indicating a stimulation of the antigen presenting ability. To further evaluate a possible preventive effect of β1,3‐glucan infusion at drying off more studies are needed involving a larger number of animals.     

Sensitivity to various antibiotics of coagulase-negative staphylococci isolated from samples of milk from Dutch dairy cattle

During recent years the prevalence of coagulase-negative staphylococci in milk samples from Dutch dairy cows has increased. In 1999 16.2% of the bacteria isolated from milk collected from cows with subclinical mastitis were coagulase-negative staphylococci. In 2004 this proportion was 42.2%. The proportion of coagulase-negative staphylococci of the bacteria isolated from milk samples from cows with clinical mastitis was 7.3% in 1999 and 14.1% in 2004. In this study, the susceptibility of 108 coagulase-negative staphylococci to oxacillin, cefquinome, streptomycin, neomycin, penicillin, and the combination of nafcillin, penicillin, and streptomycin was tested. The isolates were cultured from milk collected from cows with mastitis and typed using the Api-Staph system. Eight species were identified. Staphylococcus chromogenes was the predominant species (41.7%), followed by Staphylococcus xylosus (15.7%) and Staphylococcus simulans (10.2%). With the agar dilution method all strains proved to be sensitive to cefquinome and 90% to oxacillin. Three isolates (2.8%) were mecA-positive. Despite the agar dilution results, these three isolates should be considered resistant to all beta-lactam antibiotics (penicillins, penicillins combined with a beta-lactamase inhibitor and all generations of cephalosporins). In the agar diffusion test, all isolates proved to be sensitive to the combination of nafcillin-penicillin-streptomycin, 99% were sensitive to neomycin and 1% intermediate sensitive, and 95% were sensitive to streptomycin, 4% resistant, and 1% intermediate sensitive. The coagulase-negative staphylococci were highly resistant to penicillin (37.4%), although the level of resistance varied between species, from 0% for Staphylococcus simulans to 100% for Staphylococcus saprophyticus. Because coagulase-negative staphylococci are resistant to several antibiotics, sensitivity testing is important for targeted treatment of mastitis.