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.     

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

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.     

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.     

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 .     

Trends in antibacterial susceptibility of mastitis pathogens during a seven-year period

Milk samples collected from dairy cattle suspected of having mastitis were submitted to the Microbiology Laboratory of the Animal Health Diagnostic Laboratory, Michigan State University, for bacteriologic culture. A total of 2778 isolates, from the years 1994 to 2000, were isolated, identified, and subjected to in vitro antimicrobial susceptibility testing using the disk diffusion method, in accordance with National Committee on Clinical Laboratory Standards (NCCLS) standards. Isolates included in this study were Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Serratia marcesens, and Pseudomonas aeruginosa. The proportion of bacterial isolates determined to be susceptible did not change during the 7-yr period for the majority of bacterial-antibacterial interactions tested. However, analysis for linear trend in proportions determined that there were increases in the proportion of S. aureus isolates that were susceptible to ampicillin, penicillin, and erythromycin. For Strep. uberis, increases in the proportion of susceptible isolates occurred for oxacillin, sulfa-trimethoprim, gentamicin, and pirlimycin, and a decrease in the proportion of susceptible isolates occurred with penicillin. For Strep. dysgalactiae, increases in the proportion of susceptible isolates occurred with erythromycin, gentamicin, sulfa-trimethoprim, and tetracycline. For Strep. agalactiae, increases in the proportion of susceptible isolates occurred with sulfa-trimethoprim. Among E. coli isolates, there was an increase in the proportion that were susceptible to ampicillin and cephalothin. Among K pneumoniae isolates, there was an increase in the proportion that were susceptible to ceftiofur. Overall, there was no indication of increased resistance of mastitis isolates to antibacterials that are commonly used in dairy cattle.     

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.     

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.     

Management practices associated with the incidence rate of clinical mastitis.

Risk factors for the incidence rate of clinical mastitis were studied in 274 Dutch dairy herds. Variables that were associated with resistance to disease were the feeding, housing, and milking machine factors. Variables that were associated with exposure were grazing, combined housing of dry cows and heifers, and calving area hygiene. Postmilking teat disinfection in herds with a low bulk milk somatic cell count and years of practicing dry cow therapy were positively associated with the incidence rate of clinical mastitis. Herds with a low bulk milk somatic cell count and in which postmilking teat disinfection was not used had lower incidence rates of clinical mastitis than did other herds. The incidence rate of clinical mastitis caused by Escherichia coli was mostly related to housing conditions, hygiene, and machine milking. The incidence rate of clinical mastitis caused by Staphylococcus aureus was mostly related to factors associated with bulk milk somatic cell count and factors that might be due to cause and effect reversal. A strong positive correlation existed between the incidence rate of clinical mastitis caused by Streptococcus dysgalactiae and the incidence rate of clinical mastitis caused by Staph. aureus. The incidence rate of clinical mastitis caused by Streptococcus dysgalactiae was related to nutrition, milking technique, and machine milking. The incidence rate of clinical mastitis caused by Streptococcus uberis was associated with factors related to housing, nutrition, and machine milking.     

The effect of pathogen-specific clinical mastitis on the lactation curve for somatic cell count

Data from 274 Dutch herds recording clinical mastitis (CM) over an 18-mo period were used to investigate the effect of pathogen-specific CM on the lactation curve for somatic cell count (SCC). Analyzed pathogens were Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, Streptococcus uberis, other streptococci, and the culture-negative samples. The dataset contained 178,754 test-day records on SCC, recorded in 26,411 lactations of 21,525 cows of different parities. In lactations without both clinical and subclinical mastitis, SCC was high shortly after parturition, decreased to a minimum at 50 days in milk (DIM), and increased slowly toward the end of the lactation. Effects of CM on lactation curves for SCC differed among the pathogens isolated. Before a case of clinical E. coli mastitis occurred, SCC was close to the SCC of lactations without both clinical and subclinical mastitis, and after the case of CM had occurred, SCC returned rather quickly to a low level again. Similar curves were found for lactations with cases of CM associated with culture-negative samples. Before a case of clinical Staph. aureus mastitis occurred, average SCC was already high, and it remained high after the occurrence. Effects of CM associated with Strep. dysgalactiae, Strep. uberis, and other streptococci on the lactation curve for SCC were comparable. They showed a continuous increase in SCC until the case of pathogen-specific CM occurred, and afterwards SCC stayed at a higher level. Using SCC test-day records, these typical characteristics of each pathogen may be used to find more effective indicators of CM.     

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.     

A PCR-based method for the detection of Streptococcus agalactiae in milk

Bovine mastitis caused by Streptococcus agalactiae is mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiae in raw milk. The specificity of the PCR reaction is based on unique S. agalactiae DNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiae in the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1 ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.     

Associations between pathogen-specific cases of clinical mastitis and somatic cell count patterns

Associations were estimated between pathogen-specific cases of clinical mastitis (CM) and somatic cell count (SCC) patterns based on deviations from the typical curve for SCC during lactation and compared with associations between pathogen-specific CM and lactation average SCC. Data from 274 Dutch herds recording CM over an 18-mo period were used. Pathogens found were Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, Streptococcus uberis, streptococci other than Strep. dysgalactiae and Strep. uberis, and culture-negative samples. The dataset contained 245,595 test-day records on SCC, recorded in 24,012 lactations of 19,733 cows of different parities. Pattern definitions were based on three or five consecutive test-day records. The patterns differentiated between a short or longer period of increased SCC and also between lactations with and without recovery. Logistic regression was applied to identify associations between presence of patterns and occurrence of pathogens. Occurrence of overall CM in a lactation is equally or even more accurately predicted by the presence of SCC in that lactation, than by a lactation average SCC of more than 200,000 cells/mL. Patterns can also distinguish between chances of risk for specific mastitis-causing pathogens. Clinical E. coli mastitis was significantly associated with the presence of a short peak in SCC, whereas Staph. aureus was associated with long increased SCC. Streptococcus dysgalactiae was not strongly associated with any of the defined patterns of peaks in SCC, and no single unambiguous pattern was found for Strep. uberis.     

Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.     

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.     

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.     

Within-herd prevalence thresholds for herd-level detection of mastitis pathogens using multiplex real-time PCR in bulk tank milk samples

The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis. In addition, PCR was performed on pooled herd milk samples containing milk aliquots from all lactating cows in each of the 51 herds. Only 1 out of the 51 herds was found PCR positive for Streptococcus agalactiae in bulk tank and pooled herd milk samples, and cultured quarter milk samples. Spearman's rank correlations between the cycle threshold value of bulk tank milk PCR and the apparent within-herd prevalence were calculated in regard to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis. For these pathogens, significant correlations were found. If 1 bulk tank milk sample per herd was tested, the estimated within-herd prevalence thresholds for 90% probability of detection were 27.6% for Staphylococcus aureus, 9.2% for Streptococcus dysgalactiae, and 13.8% for Streptococcus uberis on the cow level. On the quarter level, the within-herd prevalence had to be at least 32.6% for Staphylococcus aureus, 1.7% for Streptococcus dysgalactiae, and 4.3% for Streptococcus uberis to detect a herd as positive using a single bulk milk sample. The results indicate that mastitis pathogens in bulk tank milk can be identified by the applied PCR assay. Bulk tank milk examination is not a reliable tool for the identification of the named pathogens by single testing, but might be a valuable monitoring tool when used frequently with repeated testing. Furthermore, this approach could be a useful monitoring tool for detecting new pathogen occurrence in the herd.     

Evaluation of minor pathogen intramammary infection, susceptibility parameters, and somatic cell counts on the development of new intramammary infections with major mastitis pathogens

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium spp. and coagulase-negative staphylococci (CNS). The current literature comprises several studies (n=38) detailing analyses with conflicting results as to whether intramammary infections (IMI) with the minor pathogens decrease, increase, or have no effect on the risk of a quarter acquiring a new IMI (NIMI) with a major pathogen. The Canadian Bovine Mastitis Research Network has a large mastitis database derived from a 2-yr data collection on a national cohort of dairy farms, and data from this initiative were used to further investigate the effect of IMI with minor pathogens on the acquisition of new major pathogen infections (defined as a culture-positive quarter sample in a quarter that had been free of that major pathogen in previous samples in the sampling period). Longitudinal milk samplings of clinically normal udders taken over several 6-wk periods as well as samples from cows pre-dry-off and postcalving were used to this end (n=80,397 quarter milk samples). The effects of CNS and Corynebacterium spp. on the major mastitis pathogens Staph. aureus, Strep. uberis, Strep. dysgalactiae, and coliform bacteria (Escherichia coli and Klebsiella spp.) were investigated using risk ratio analyses and multilevel logistic regression models. Quarter-, cow- and herd-level susceptibility parameters were also evaluated and were able to account for the increased susceptibility that exists within herds, cows and quarters, removing it from estimates for the effects of the minor pathogens. Increased quarter-level susceptibility was associated with increased risk of major pathogen NIMI for all pathogens except the coliforms. Increased somatic cell count was consistently associated with elevated risk of new major pathogen infections, but this was assumed to be a result of low sensitivity of bacteriology to diagnose major pathogen NIMI expediently and accurately. The presence of CNS in the sample 2 samplings before the occurrence of a NIMI increased the odds of experiencing a Staph. aureus NIMI 2.0 times, making the presence of CNS a risk factor for acquiring a Staph. aureus NIMI. Even with this extensive data set, power was insufficient to make a definitive statement about the effect of minor pathogen IMI on the acquisition of major pathogen NIMI. Definitively answering questions of this nature are likely to require an extremely large data set dedicated particularly to minor pathogen presence and NIMI with major pathogens.     

Change in udder health and overmilking

Removing milker units as soon as milk flow stopped was compared to milking for a fixed time of 12 min in an 8-wk trial with 60 cows. Teats were dipped in broth culture of approximately 10(9) colony forming units per ml of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis for 4 wk. More new infections of quarters occurred in the 12-min group. In trials 2 and 3, 20 additional cows were milked with automatic milking machine detachers and 20 cows were milked with a flow control unit for 12 min each trial. New infection rates were highest in the two groups milked for 12 min. Age for lactation and cows were associated with most of the variation in the new infection rate. Cows with pointed teat ends had the highest rate of new infection. Effects of fixed-time milking on increased mammary infection and clinical mastitis were small.