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PubMed2001

Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis.

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.

Animals↗
PubMed2003

Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation.

Staphylococcus aureus is one of the most common pathogens responsible for contagious mastitis in ruminants. The ability of S. aureus to form biofilm in vivo is considered to be a major virulence factor influencing its pathogenesis in mastitis. The objectives of the study were to examine in vitro slime production, biofilm formation, and the presence of the ica gene locus and icaA and icaD genes in S. aureus isolates from bovine mastitis. Thirty-two of the 35 isolates tested produced slime on Congo red agar, whereas only 24 of the isolates were found to produce biofilm in vitro. However, all the 35 isolates possessed the ica locus, icaA and icaD genes. This study indicates a high prevalence of the ica genes among S. aureus mastitis isolates, and their presence is not always associated with in vitro formation of slime or biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. aureus.

Animals↗
PubMed1997

Prevalence of capsular serotypes among Staphylococcus aureus isolates from cows with mastitis in the United States.

Development of an appropriate Staphylococcus aureus vaccine for bovine mastitis has eluded researchers for decades. The ability of S. aureus to form a protective exopolysaccharide capsule has posed a major obstacle because of the multiple serotypes and the poor immune response elicited by exopolysaccharides. This study characterized S. aureus serotypes isolated from cases of bovine mastitis obtained from veterinary diagnostic laboratories that service 44% of the dairy cattle in the United States. Major milk producing areas of the northeast, north central, Pacific coast and southwest were proportionately represented. Sub-samples of mastitic milk that contained S. aureus were frozen and sent to our laboratory for strain serotyping. The only other regional serotyping of S. aureus from bovine mastitis to date was done in France. The primary serotypes found were types 5 (51%) and 8 (18%) and 31% were non-typeable. In the current study, serotype 5 accounted for 18% of the isolates and serotype 8 for 23%. More importantly 59% of the isolates were not typeable with either type 5 or 8 antisera. These data indicate that S. aureus vaccines employing serotypes 5 and 8 would only be marginally effective in the United States. These data also suggest that development of a S. aureus vaccine for bovine mastitis should take into account regional variation in S. aureus serotypes.

Animals↗
PubMed2003

Establishing treatment protocols for clinical mastitis.

Each farm has a unique mix of mastitis pathogens and management procedures that have evolved over time. The herd veterinarian should work with the manager/owner to systematically develop treatment protocols that meet the needs and management of the farm. To establish a mastitis treatment protocol, it is necessary to develop a system to routinely identify clinical mastitis cases, develop a herd-specific severity level assessment system, manage the clinical mastitis cases based on severity level and culture result (when available), avoid antibiotic residues, and monitor the success of the system and alter the protocol as necessary.

Animals↗
PubMed2003

Investigation of mastitis problems on farms.

The production of high-quality milk is a high-priority issue for dairy farmers and milk processors. The investigation of mastitis problems on dairy farms is an area of increasing demand and is an ideal way for veterinary practitioners to increase involvement in production medicine programs. Goals for the amount of clinical and subclinical mastitis should be predefined on dairy farms, and a herd investigation initiated when needed. The use of a structured approach to mastitis problems can identify risk factors for infection, result in rapid resolution of mastitis problems, and hasten the application of appropriate preventive practices.

Animals↗
PubMed1993

Mastitis in heifers.

Udder health is typically overlooked but critically important in heifers. Mastitis pathogens of the contagious as well as environmental groups have been implicated in mastitis in heifers. Procedures considered for control of heifer mastitis include prepartum treatment in primigravid heifers, separating preweaned calves to prevent suckling, fly control, and segregation of pregnant heifers from dry cows. Further investigation is needed before general recommendations can be made relative to prepartum treatment of primigravid heifers. An improved understanding of the epidemiology and pathogenesis of heifer mastitis will aid in expanding and improving control measures.

Actinomycosis↗
PubMed1997

Udder orf infection and its role in ovine clinical mastitis caused by Pasteurella haemolytica.

During an experimental study of ovine subclinical mastitis caused by coagulase-negative staphylococci, an outbreak of contagious ecthyma occurred among ewes unvaccinated against parapox virus. The same group of ewes developed a high rate (43.7%) of clinical mastitis caused by Pasteurella haemolytica. The rate of clinical mastitis among ewes vaccinated against parapox virus was very low (3.7%) suggesting that the presence of orf in the unvaccinated ewes was contributing to the high rate of clinical mastitis. An examination of the iron, sodium, potassium and albumin concentration of milk collected from 16 unvaccinated and nine randomly selected vaccinated ewes before experimental infection with coagulase-negative staphylococci or their uninfected control mammary glands indicated significant differences in the iron (p < 0.0001) and sodium (p = 0.01) concentration. Increased iron concentration in the milk may have assisted in the development of udder infection caused by P. haemolytica as iron is easily utilised by this bacterium.

Animals↗
PubMed1981

The relationship between the levels of free fatty acids, lipoprotein lipase, carboxylesterase, N-acetyl-beta-D-glucosaminidase, somatic cell count and other mastitis indices in bovine milk.

A large-scale survey of milks from healthy and mastitic bovine quarters was undertaken to establish the influence of mastitic infection on milk lipase activity and free fatty acid (FFA) level. Mastitic milks tended to have higher FFA levels, but lower lipoprotein lipase activities compared with milk from healthy quarters. These effects became significant at relatively severe levels of infection. The elevated FFA was attributable to higher FFA levels on secretion and to greater lipolysis during storage. Levels of carboxylesterase activity increased with severity of mastitis and showed high positive correlation with mastitis indices. Marked increases in carboxylesterase, N-acetyl-beta-D-glucosaminidase and phospholipase occurred following the induction of mastitis by intramammary infusion of Escherichia coli endotoxin, in parallel with changes in somatic cell count and other mastitis indices. Relativity little change in lipoprotein lipase activity was observed.

Acetylglucosaminidase↗
PubMed1984

Milk trypsin-inhibitor capacity as an indicator of bovine mastitis--a novel principle which can be automated.

As mastitis is associated with leakage of small molecular weight plasma proteins, such as alpha 1-antitrypsin, into milk, this antitrypsin can be used as an indicator of mastitis. A colorimetric procedure was developed for large scale monitoring of milk antitrypsin activity, using microtitration plates and the Multiskan system. The effect of stage of lactation and age of the cow on the antitrypsin concentration and its interrelationship with other mastitis indicators (bovine serum albumin (BSA), somatic cell count) was analysed by computer programs on 1029 cows. Milk antitrypsin activity was high after parturition owing to colostral inhibitors. After the first month of lactation the assay measures only blood-derived antitrypsin and is a good indicator for detecting an increased permeability between blood and milk due to mastitis. Increasing lactation number only slightly affected the antitrypsin and BSA concentrations whereas somatic cell content was markedly affected.

Animals↗
PubMed1993

Classification of newly calved cows into moderate and severe responders to experimentally induced Escherichia coli mastitis.

In the present study newly calved cows were tentatively classified as moderate and severe responders to experimentally induced Escherichia coli mastitis based upon the reactive oxygen species (ROS)-generating capacity of their blood neutrophils before infection. The groups differed in blood and milk composition prior to infection. This initial classification was supported by the corresponding variation in clinical symptoms and in the changes in milk production and composition measured during mastitis. Responses of newly calved cows to Esch. coli challenge varied from mild to severe symptoms of inflammation in infected glands and differed in the intensity of systemic disturbances and general illness. Losses in milk yield and compositional changes were most pronounced in inflamed glands and in severe responders. In inflamed glands milk yield and composition did not return to preinfection level in either moderate or severe responders. The yields of lactose, alpha-lactalbumin, casein and fat followed the same pattern as milk yield. It is concluded that the severe and long lasting systemic disturbances observed in severe responders can be ascribed to absorption of endotoxin from infected glands into circulation, indicating the important role of endotoxin in the pathology of coliform mastitis in periparturient cows. Evaluation of the ROS-generating capacity of blood neutrophils and blood and milk composition before infection might help to predict the cow's sensitivity to Esch. coli mastitis.

Animals↗
PubMed1988

Consequences of diagnostic errors in mastitis therapy trials.

The effect of errors that occur in the diagnosis of intramammary infectious mastitis on the precision of experiments measuring the efficacy of mastitis therapy has been investigated. Diagnostic errors within the range found by experienced workers can create large biases in the apparent cure rate of therapy particularly at cure rates of less than 0.5. Using confirmed methods of diagnosis rather than single samples and reducing the probabilities of false positive and false negative diagnoses to 0.01 and 0.05 respectively, the biases in the apparent cure rates are reduced to acceptable levels. A method is given for calculating the rates of occurrence of false positive and false negative diagnoses from the results of trials using confirmed diagnoses. These errors cannot be calculated from therapy trial data when diagnosis is based on single milk samples. Because the bias in the measurements of the cure rate is greatest at the lowest levels of elimination, estimates of spontaneous recovery in untreated quarters have the greatest error. For this reason experiments incorporating an untreated control group of infected quarters usually reduce the precision of the therapy trials. An experiment in which the efficacy of a test product is measured relative to a reference product has advantages. It minimizes the difficulties arising from scale of measurement, diagnostic errors and herd differences in response rate, and makes possible comparisons between trials. Further investigations are required on the importance of spontaneous recovery, particularly for studies of Escherichia coli therapy and dry period therapy. The results of this investigation have relevance to all types of mastitis investigation that measure the change in mastitis status of udder quarters, i.e. new infection rates.

Animals↗
PubMed1992

Trials of a novel mastitis sensor on experimentally infected cows.

Numerous attempts to develop in-line systems for the measurement of the electrical conductivity of milk as an aid to the detection of mastitis have been described. The major technical problem of fouling of electrodes exposed to milk has been overcome in the sensor tested here by use of an inductive system of measurement. The practical application of this system in the milking parlour is reported. The novel mastitis sensor has been shown to detect incipient mastitis before there are visible signs of abnormal milk. Conductivity measurements have the advantage over other diagnostic procedures in that the information is available immediately without additional effort. Normalized conductivity and milk yield variation results indicate the potential for developing a practical, automatic system for mastitis detection.

Animals↗
PubMed1992

Isolation and characterization of alpha 2-macroglobulin from mastitis milk.

Whey samples were screened for the presence of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). From an enzymic test, alpha 2M levels in normal whey varied in the range 0.49-0.84% of the serum level, whereas in mastitis whey the activity was markedly increased, reaching values between 0.91 and 138.5% (median 7.2%) of standard serum level. In mastitis milk samples but not in normal milk alpha 2M was also detected by double immunodiffusion and Western blotting. The proteinase inhibitor was purified from a mastitis milk sample with high alpha 2M activity (138.5% of serum level). In SDS-PAGE, native-PAGE and in double immunodiffusion analysis the inhibitor appeared indistinguishable from plasma-derived alpha 2M. The alpha 2M preparation from mastitis whey migrated essentially as native alpha 2M, representing the 'slow' form of the molecule. Treatment with trypsin transformed the alpha 2M preparation to the electrophoretic 'fast' form, but treatment with methylamine had only a minor effect. The receptor recognition sites were not exposed on the isolated alpha 2M molecule but could be readily exposed by treatment of the proteinase inhibitor with trypsin, which further proved that the isolated alpha 2M was in the entire native, functionally active state.

Animals↗
PubMed2003

Haptoglobin and serum amyloid A in milk and serum during acute and chronic experimentally induced Staphylococcus aureus mastitis.

Local and systemic changes in the acute phase proteins, haptoglobin and serum amyloid A (SAA), were studied in six dairy cows during the acute and chronic phases of experimentally induced Staphylococcus aureus mastitis. Haptoglobin and SAA were measured in serum, and in milk from infected and healthy control udder quarters within each cow. Concentrations of haptoglobin and SAA increased rapidly in both serum and milk during the acute phase of mastitis and followed a similar pattern. Significantly raised milk concentrations of SAA were also found during chronic subclinical mastitis. Serum concentrations of SAA also tended to be higher during the chronic phase than pre-infection. Increases in milk haptoglobin and SAA were specific for the infected udder quarters. In conclusion, measurement of SAA in milk samples could be a useful tool in diagnosing mastitis.

Acute Disease↗
PubMed2005

Use of partial budgeting to determine the economic benefits of antibiotic treatment of chronic subclinical mastitis caused by Streptococcus uberis or Streptococcus dysgalactiae.

The economic effect of lactational antibiotic treatment of chronic subclinical intramammary infections due to Streptococcus uberis or Streptococcus dysgalactiae was explored by means of partial budgeting. Effects at cow level and herd level were modelled, including prevention of clinical mastitis episodes and the prevention of transmission of infections. Input variables for our deterministic model were derived from literature or based on 2002/2003 dairy prices and farming conditions in The Netherlands. Sensitivity analysis was used to examine the effect of uncertainty around input variables or changes in price estimates. On farms where pathogen transmission was prevented through proper udder health management, 3-d antibiotic treatment during lactation resulted in an average net profit of euro+11.62 over no treatment while 8-d antibiotic treatment had an average negative net result of euro-21.83. Sensitivity analysis showed that profitability depends on the probability of treatment-induced cure, pathogen transmission rates, culling rate, retention pay-off, and costs of antibiotic treatment. Three-day antibiotic treatment of chronic subclinical streptococcal mastitis is economically profitable over a range of input values for cure probabilities, transmission rates and losses due to culling. In contrast, 8-d lactational treatment is only profitable for very valuable animals, on farms where the risk of pathogen transmission is high and/or when the farmer is likely to cull a high percentage of cows with subclinical mastitis. Because bacterial flora, cow characteristics and management differ widely between farms, the economic outcome of lactational treatment of chronic subclinical streptococcal mastitis may be highly farm-dependent.

Animals↗
PubMed2006

L-lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase activities in bovine milk as indicators of non-specific mastitis.

Systematic factors affecting the activities of L-lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) and somatic cell count (SCC), the association between the activities of LDH and NAGase and SCC with respect to udder health status, and the ability of LDH and NAGase to classify cows in udder health categories for early detection of mastitis were studied. A dataset of records from 74 Danish Holstein, 76 Danish Red and 47 Jersey cows on one research farm was used. Cows were grouped into healthy and clinically mastitic. A healthy cow was defined as having no veterinary treatment and SCC<100,000 cells/ml. A clinically infected cow was one receiving veterinary treatment after showing clinical signs of mastitis and SCC >800,000 cells/ml. Breed, month of production, and days in milk significantly influenced (P<0.001) LDH activity, NAGase activity and SCC in both healthy and clinically mastitic cows. In healthy cows, LDH activity, NAGase activity and SCC started at a high level immediately after calving and decreased to low levels approximately 30-40 d post partum. All the three parameters increased due to clinical mastitis. NAGase activity had numerically higher variation in healthy cows than in clinically mastitic cows (CV=56.2% v. CV=53.5%). The relationship between LDH activity and SCC was stronger in milk from clinically mastitic than from healthy cows (r=0.76 v. r=0.48 and r=0.67 v. r=0.44 for correlation of observed values and residuals, respectively). LDH activity had higher sensitivity than NAGase activity (73-95% v. 35-77%) while specificities were in a similar range (92-99%). Further, sensitivities for LDH activity were more robust to changes in the threshold value than those for NAGase activity. Opportunities for automated, in-line real-time mastitis detection are discussed.

Acetylglucosaminidase↗
PubMed2004

Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak.

Biopsy by fine needle aspiration together with microbiological examination and scanning electron microscopy were evaluated in diagnosis of clinical bovine mastitis in a Prototheca zopfii outbreak. Fine needle aspiration was performed in 21 mammary quarters from ten Holstein cows presenting clinical mastitis caused by P. zopfii. The algae were previously identified in the microbiological examination of milk collected from these cows. Material aspirated from these 21 mammary glands was submitted to cytological staining (Gram, Giemsa and/or Shor staining). Fine needle aspiration enabled cytological identification of the algae in these 21 mammary glands, from which P. zopfii was isolated in the milk. Simultaneously, five mammary fragments collected by fine needle aspiration from these 21 mammary glands presenting clinical mastitis were also submitted to microbiological examination. P. zopfii was also isolated from these five fragments. Scanning electron microscopy technique also identified three of these five P zopfii strains isolated from mammary fragments collected by cytological aspiration. These results suggest that fine needle aspiration may be an alternative method for the diagnosis of clinical mastitis.

Animals↗
PubMed2000

Characterisation of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway.

Eighty-six Staphylococcus aureus isolates from cases of bovine mastitis were characterised biochemically and with respect to serotype, multilocus enzyme electrophoresis genotypes, antibiotic sensitivity, and production of enterotoxins A through D (SEA-D) and toxic shock syndrome toxin-1 (TSST-1). The samples were obtained from 81 different cows from 79 Norwegian dairy herds in 10 different counties in southern Norway. There was an equal representation of isolates from cases of acute, chronic and subclinical mastitis. Multilocus enzyme electrophoresis using 13 genetic loci showed that 69 of 86 isolates had the same electrophoretic type. This common electrophoretic type comprised isolates that differed in the expression of other phenotypical characteristics studied. Fifty-eight percent of the isolates produced one or more enterotoxins, predominantly a combination of SEC and TSST-1. Capsular serotyping revealed that 95% of the isolates belonged to serotype 8. No correlation was found between the factors studied and the clinical classification of mastitis. It appears that the majority of S. aureus isolates recovered from cases of bovine mastitis in Norway are genetically closely related and express common phenotypical characteristics.

Acute Disease↗