Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus,Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.     

Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis

To improve diagnosis of mastitis in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of the four major bacterial causes of bovine mastitis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. The target sequence was the 16S to 23S rRNA spacer regions. The performance of the assay was examined with 117 milk samples collected from a subclinically infected herd, and the diagnostic specificities and sensitivities of the multiplex PCR were compared with conventional culture. PCR was significantly more sensitive than culture for detection of S. aureus and S. uberis, but there were no significant differences in sensitivities between PCR and culture for the detection of S. agalactiae and S. dysgalactiae. The results suggest that this multiplex PCR assay could be used as an alternative method in routine diagnosis for rapid, sensitive, and specific simultaneous detection of S. aureus, S. agalactiae, S. dysgalactiae, and S. uberis in milk samples.     

Effect of mastitis-related bacteria on total bacterial count of bulk milk supplies

Seven hundred and fifty-four bulk milk samples from a total of 9,066 tested by the Aberdeen and District Milk Marketing Board in 1984 had total bacterial counts greater than 45,000 per ml. Of the failed samples, 43.8% had more mastitis-related bacteria present than non-mastitis types. More than a quarter of the failed samples had almost pure cultures of Streptococcus agalactiae, Str. dysgalactiae, Str. uberis, Staphylococcus aureus or coagulase-negative staphylococci. No relation was found between the failed samples resulting from subclinical mastitis infections and the somatic cell count of the bulk milk. Seventy-five per cent of the financial penalties imposed on producers were attributable to mastitis bacteria in bulk milk samples .     

Nisin inhibits several gram-positive, mastitis-causing pathogens

Organisms known to cause bovine mastitis, Enterococcus faecalis ssp. liquefaciens ATCC 27959, Staphylococcus aureus ATCC 29740, Streptococcus agalactiae ATCC 27956, Streptococcus equinus ATCC 27960, Streptococcus dysgalactiae ATCC 27957, Streptococcus uberis ATCC 27958, and the neotype Staphylococcus epidermidis ATCC 14990 were examined for their susceptibility to the small peptide antibiotic, nisin. Using a disc assay, minimum inhibitory concentrations of nisin ranged from 10 to 250 micrograms/ml among the strains. Examination of the antimicrobial effect of 50 micrograms/ml nisin in milk showed nisin inhibited all gram-positive pathogens tested.     

Antimicrobial properties of the chelating agent EDTA on Streptococcal bovine mastitis isolates

To determine the efficacy of the chelating agent EDTA on microbial growth, separate cultures of two streptococcal bovine mastitis isolates, Streptococcus agalactiae and Streptococcus uberis, were exposed to known concentrations of EDTA. Bacterial cultures of 10(8) CFU/ml were exposed to concentrations of EDTA ranging from 30 to 100 mM in an in-vitro-milk environment. Multiple replications of cultures exposed to EDTA were plated during a two-hour time course. A concentration of 100 mM EDTA resulted in a 90% reduction of S. agalactiae and a 99% reduction of S. uberis. Under these experimental conditions, EDTA treatments in cultures of both isolates exhibited from 1 to 2 log reductions suggesting that EDTA is a potentially effective antimicrobial against streptococcal isolates implicated in causing bovine mastitis.     

Bacteriological investigations of clinical mastitis in heifers in Sweden

Bacterial analyses were carried out of 2069 udder secretions, isolated from 1481 heifers with mastitis in eight veterinary districts in Sweden. Streptococci, e.g. Streptococcus dysgalactiae and Str. uberis, dominated the bacterial flora, being isolated from 34.4 and 19.5% respectively of heifers with clinical mastitis occurring from puberty up to 14 d post partum. Bacterial species generally regarded as important pathogens in the summer mastitis complex, e.g. Actinomyces pyogenes, Stuart-Schwan coccus and strictly anaerobic bacteria such as Peptostreptococcus indolicus, Fusobacterium necrophorum and Bacteroides melaninogenicus were isolated at low frequencies (13.2, 6.3, 9.4, 3.8 and 1.3% respectively). When the cases of mastitis studied were restricted to those appearing in heifers pre partum, May 15 to October 14 (summer mastitis), these bacterial species were isolated at higher percentages (27.1, 14.4, 21.4, 13.5 and 5.2% respectively). These figures were, nevertheless, still lower than those published in reports from other countries. Whether considered up to 14 d post partum or only pre partum, there were no significant differences in the frequencies of A. pyogenes isolated at different seasons. There were geographical differences in bacterial incidence, e.g. Staphylococcus aureus was isolated significantly more often in northern regions whereas Str. dysgalactiae was more common in the south. This and other Swedish investigations support the theory that A. pyogenes and strictly anaerobic bacteria are 'secondary invaders' that depend on Str. dysgalactiae to cause a primary infection. It is stressed that the udders of all heifers should be examined daily so that cases of mastitis can be treated immediately.     

Evaluation of the Rapid Strep system for identification of gram-positive, catalase-negative cocci isolated from bovine intramammary infections

The Rapid Strep system (Analytab Products, Plainview, NY) was used to identify 199 gram-positive, catalase-negative cocci isolated from bovine intramammary infections. The system accurately identified 88.4% of isolates. The system identified 100% of 46 Streptococcus agalactiae, 100% of 48 Streptococcus dysgalactiae, 54.5% of 11 Streptococcus equinus, and 96.2% of 53 Streptococcus uberis isolates. Enterococcus spp. were identified correctly 83.3% of the time. One of 4 Streptococcus saccharolyticus strains was identified as Streptococcus bovis, the previous classification for this organism, and 8 Streptococcus equi ssp. equi strains were misidentified as Streptococcus dysgalactiae. The Rapid Strep system was determined to be an acceptable alternative to conventional methods for identification of gram-positive, catalase-negative cocci isolated from bovine intramammary infections.     

Antibiotic susceptibility patterns for environmental streptococci isolated from bovine mastitis in central California dairies

Environmental streptococci are frequently isolated from bovine mastitis in dairy cows with only limited information available on the antimicrobial susceptibility of these organisms. A total of 362 environmental streptococci isolated from cases of bovine mastitis from the central San Joaquin Valley of California over a 3-yr period were used in the study. Overall, 39.9% of the strains tested were Streptococcus uberis, 42.2% were Streptococcus dysgalactiae, and 11.1% were Enterococcus spp. The antimicrobial susceptibility for these organisms was determined for the following antimicrobial agents: penicillin, ampicillin, cephalothin, ceftiofur, penicillin + novobiocin, erythromycin, pirlimycin, tetracycline, and sulfadimethoxine. Results demonstrate substantial differences in the susceptibility patterns for the various organisms collectively referred to as the environmental streptococci. The MIC90 for penicillin was 0.06 microg/ml for 152 strains of S. dysgalactiae compared with 0.25 microg/ml for 133 strains of S. uberis. However, the Enterococcus spp. were the most resistant organisms tested. These data also indicate that the use of interpretive criteria based on human data may provide misleading results. In conclusion, these data confirm that the environmental streptococci are a diverse group of organisms comprised of several different genera and species and that identification of environmental streptococci to the species level is needed to appropriately modify control methods. Moreover, the use of the agar disk diffusion (Kirby-Bauer) susceptibility test for agents with human-based interpretive criteria is contraindicated, and these tests should only be performed with agents with mastitis specific interpretive criteria.     

Evaluation of two fluorogenic assays for identification of Streptococcus species isolated from bovine mammary glands

Liquid and agar assays that utilized 4-methylumbelliferyl-conjugated beta-D-glucuronide, beta-D-galactoside, or N-acetyl-beta-D-glucosaminide, and agglutination by Dolichos biflorus lectin were evaluated for identification of Streptococcus species isolated from bovine mammary glands. A greater number of Streptococcus uberis isolates were negative for N-acetyl-beta-D-glucosaminidase by the liquid assay compared with the agar assay. Enzyme profiles for Streptococcus dysgalactiae were similar by both assays. Streptococcus dysgalactiae was the only species that agglutinated when mixed with lectin from D. biflorus. Most Streptococcus agalactiae isolates were positive for beta-D-glucuronidase and beta-D-galactosidase by both assays. Two Streptococcus equinus strains had negative enzyme profiles by the liquid assay; however, both strains had enzyme profiles consistent for S. equinus by the agar method. Incorporation of 4-methylumbelliferyl-conjugated substrates into trypticase soy agar did not appear to alter agar characteristics and eliminated aliquoting substrates and inoculating tubes. More than one enzyme profile was produced per Streptococcus species or serogroup by both methods. However, some profiles were similar between species, which hindered accurate identification of Streptococcus species.     

Efficacy of chlorhexidine as a postmilking teat disinfectant for the prevention of bovine mastitis during lactation

A natural exposure trial was conducted for 12 mo in a herd of 150 lactating Jersey cows to determine efficacy of a .35% chlorhexidine teat dip containing a glycerine emollient for the prevention of bovine intramammary infections. Right teats of cows were dipped in the experimental teat dip after milking machine removal and left teats were not dipped. The herd was free of Streptococcus agalactiae and had a low prevalence of Staphylococcus aureus. Most new major pathogen intramammary infections resulted from Streptococcus species, primarily Streptococcus uberis and Streptococcus dysgalactiae. New infections by Streptococcus species were significantly lower in teats dipped in chlorhexidine than in undipped teats. Overall efficacy of the chlorhexidine teat dip against major mastitis pathogens was 50%. The experimental teat dip also reduced coagulase-negative Staphylococcus species infections 49.0% and Corynebacterium bovis infections 65.2%. Overall efficacy against minor mastitis pathogens was 54.0%. No irritation or chapping of teats dipped in the experimental teat dip was observed.     

Relationship between the level of N-acetyl-beta-D-glucosaminidase (NAGase) in bovine milk and the presence of mastitis pathogens

Changes in the level of the tissue damage marker enzyme, N-acetyl-beta-D-glucosaminidase (NAGase) in quarter fore milks were found to be related to the presence and types of pathogenic bacteria present and to somatic cell counts (SCC). Minor pathogens (coagulase-negative staphylococci, Corynebacterium bovis) elicited a mild SCC increase (from a mean of 243 X 10(3)/ml in healthy quarters to 504 X 10(3)/ml in infected quarters) with marginal tissue damage (mean NAGase activity increased from 21 in healthy quarters to 28 in infected quarters). Major pathogens (i.e. Staphylococcus aureus, Streptococcus agalactiae, Str. dysgalactiae and Str. uberis) caused more severe tissue damage (mean NAGase of 48) and SCC increases (mean, 2803 X 10(3)/ml). The NAGase test could also be used effectively on composite milk samples where regular monthly NAGase analysis was able to identify correctly 74% of animals having infected quarters. The possibility of combining SCC and NAGase data in order to give a more definite diagnosis of bovine mastitis is discussed.     

Minimum inhibitory concentrations and disk diffusion zone diameter for selected antibiotics against streptococci isolated from bovine intramammary infections

The antimicrobial susceptibility to 11 antibiotics was determined of 358 streptococcal isolates representing six species was determined. All isolates were from milk samples obtained from clinical or subclinical cases of bovine mastitis. Agar disk diffusion zone diameters and minimum inhibitory concentrations were obtained for all isolates. Of the antibiotics tested that are available for mastitis therapy, cephalothin, penicillin, ampicillin, novobiocin, and erythromycin demonstrated acceptable activity against nonenterococcal streptococci, but only penicillin and ampicillin demonstrated moderate activity against Enterococcus faecalis. Enterococcus faecalis demonstrated substantial resistance to cephalothin, erythromycin, gentamicin, streptomycin, and tetracycline, while the nonenterococcal streptococci demonstrated substantial resistance to gentamicin, streptomycin, and tetracycline. Streptococcus uberis demonstrated greater antibiotic resistance than did Streptococcus dysgalactiae, while Aerococcus viridans demonstrated the greatest overall resistance of the nonenterococcal species.     

Physiological characteristics of Streptococcus dysgalactiae and Streptococcus uberis and the effect of the lactoperoxidase complex on their growth in a chemically-defined medium and milk

Aerobic or anaerobic degradation of glucose by Streptococcus dysgalactiae and Streptococcus uberis yielded products qualitatively similar to those observed previously for Streptococcus agalactiae. There were, however, quantitative differences. Though acetoin was formed during aerobic growth of Streptococcus uberis, there was none with Streptococcus dysgalactiae. Differences between Streptococcus dysgalactiae and Streptococcus uberis in their aerobic metabolism of glucose was in lower oxygen consumption (.5 mol/mol of glucose), greater conversion of glucose to lactic acid, and lower molar growth yields with Streptococcus uberis. Cell suspensions of Streptococcus uberis had strong peroxidase activity, and no hydrogen peroxide accumulated during the respiration on glucose. With Streptococcus dysgalactiae, there was more oxygen consumed during growth (1.5 mol/mol of glucose used), greater conversion of glucose to acetic and formic acids and carbon dioxide, and a cell yield of about 6 g of dry cells more per mole of glucose than with Streptococcus uberis. This increase in molar growth yield with Streptococcus dysgalactiae over Streptococcus uberis could be nearly all accounted for by differences in the amount of substrate level adenosine triphosphate generated. Cell suspensions oxidizing glucose accumulated hydrogen peroxide and showed no peroxidase activity. Streptococcus dysgalactiae showed the same growth relationships in three milk media as Streptococcus agalactiae, although growth and acid formation values were much lower. Growth inhibition by the lactoperoxidase complex was reversed with cystine. Acid formation by Streptococcus uberis was decreased by the lactoperoxidase complex and increased by the addition of cystine; however, neither appeared to affect the growth of the organism.     

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

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

Growth of gram-positive mastogenic bacteria in normal, simulated bulk tank, and mastitic milk held at simulated fluctuating temperatures of farm bulk tank

Growth of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus bovis, and Streptococcus uberis was studied in normal milk, simulated bulk tank milk, and aseptic mastitic milk held at simulated fluctuating temperatures of farm bulk tank for 48 h. With the exception of S. bovis, growth rates of the other five bacteria were similar in both normal and simulated bulk tank milk. Mastitic milk inhibited growth of all bacteria studied. A 24-h adjustment period occurred before most of the bacteria started growing. The mastitis level in a dairy herd may be monitored by cultures of bulk tank milk samples and by calculations as discussed in this study.     

Evaluation of experimental teat dip containing sodium chlorite and lactic acid by excised teat assay

An experimental teat dip containing sodium chlorite and lactic acid, diluted in water, was evaluated by excised teat protocol. The teat dip was tested against 21 microorganisms. Included were: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Numerous strains were tested for strain differences. Environmental bacteria were included because of their increasing importance as a cause of bovine mastitis. All excised teats were dipped in a bacterial suspension containing about 1 X 10(8) cfu/ml. Negative control teats were not dipped in a germicidal compound. Positive controls were dipped in 1% iodophor. Effectiveness of the experimental teat dip was expressed as the percent reduction in mean log of bacteria recovered from dipped teats as compared to numbers recovered from control teats. The sodium chlorite - lactic acid dip caused a greater percent log reduction than iodophor for 14 of 21 strains tested. However, differences were generally slight. The experimental teat dip appeared effective against Gram-negative bacteria. Some differences in percent log reduction were observed between strains of the same species. Lowest effectiveness and greatest strain variation were observed with Staphylococcus aureus for both dips tested.     

Protein degradation in bovine milk caused by Streptococcus agalactiae

Streptococcus (Str.) agalactiae is a contagious mastitis bacterium, often associated with cases of subclinical mastitis. Different mastitis bacteria have been evaluated previously from a diagnostic point of view, but there is a lack of knowledge concerning their effect on milk composition. Protein composition is important in achieving optimal yield and texture when milk is processed to fermented products, such as cheese and yoghurt, and is thus of great economic value. The aim of this in vitro study was to evaluate protein degradation mainly caused by exogenous proteases originating from naturally occurring Str. agalactiae. The samples were incubated at 37°C to imitate degradation caused by the bacteria in the udder. Protein degradation caused by different strains of Str. agalactiae was also investigated. Protein degradation was observed to occur when Str. agalactiae was added to milk, but there were variations between strains of the bacteria. Caseins, the most economically important proteins in milk, were degraded up to 75% in milk inoculated with Str. agalactiae in relation to sterile ultra-high temperature (UHT) milk, used as control milk. The major whey proteins, α-lactalbumin and β-lactoglobulin, were degraded up to 21% in relation to the sterile control milk. These results suggest that different mastitis bacteria but also different strains of mastitis bacteria should be evaluated from a milk quality perspective to gain knowledge about their ability to degrade the economically important proteins in milk.     

A multi-valved milking machine cluster to control intramammary infection in dairy cows

A milking machine claw incorporating valves to prevent movement of milk between teats during milking, and its contribution to the prevention of udder infection under experimental and field conditions, is described. Under experimental conditions a suspension of Streptococcus agalactiae and Str. dysgalactiae was introduced into the milking cluster during milking; 11 of 40 quarters became infected using a conventional claw piece whereas none of 40 quarters milked with the multi-valve claw developed intramammary infection. In a 12-month experiment in ten commercial herds, each split into two equal susceptibility groups, the multi-valved cluster reduced the incidence of new infection with coliforms and Str. uberis by 17% (P less than 0.1). The incidence of all new infections and new clinical cases was 14 and 25% lower compared to conventional clusters but these reductions were not significant.     

An inhibitor typing scheme for Streptococcus uberis

A typing scheme was used to test 15 strains of Streptococcus uberis according to their production of (P-type) and sensitivity to (S-type) bacteriocin-like inhibitory substances. Twelve of the strains were inhibitor producers and nine different P-types were detected. All of the strains were typable according to inhibitor sensitivity, ten different S-types being distinguished. Both the P-type and S-type designations of the strains were reproducible on repeated testing. By combination of P-typing and S-typing, highly discriminatory inhibitor 'fingerprints' of the strains could be obtained. This scheme would appear to have considerable potential for typing isolates of Str. uberis as an aid to investigations into the epidemiology of Str. uberis mastitis in dairy cattle.     

Antibacterial effect of caprylic acid and monocaprylin on major bacterial mastitis pathogens

Bovine mastitis is the most significant economic drain on the worldwide dairy industry. Concerns regarding poor cure rates, emergence of bacterial resistance, and residues in milk necessitate development of alternative therapeutic approaches to antibiotics for treatment of mastitis. A variety of free fatty acids and their monoglycerides have been reported to exert antimicrobial activity against a wide range of microorganisms. The objective of our study was to examine the efficacy of caprylic acid, a short-chain fatty acid, and its monoglyceride, monocaprylin, to inactivate common mastitis pathogens, including Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Milk samples containing 50 mM or 100 mM caprylic acid, and 25 mM or 50 mM monocaprylin were inoculated separately with a 3-isolate mixture of each of the 5 pathogens, and incubated at 39°C. Populations of surviving bacteria were determined at 0 min, 1 min, 6 h, 12 h, and 24 h of incubation. Both caprylic acid and monocaprylin reduced all 5 pathogens by >5.0 log cfu/mL after 6 h of incubation. Among the bacterial species tested, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis were most sensitive, and E. coli was most tolerant to caprylic acid and monocaprylin. Results of this study indicate that caprylic acid and monocaprylin should be evaluated as alternatives or adjuncts to antibiotics as intra-mammary infusion to treat bovine mastitis.