Minimum inhibitory concentrations for selected antimicrobial agents against organisms isolated from the mammary glands of dairy heifers in New Zealand and Denmark.

Minimum inhibitory concentrations were determined for selected antimicrobial agents against 872 bacteria isolated from intramammary infections in heifers in New Zealand (n = 401) and Denmark (n = 471). These values were reported in micrograms per milliliters. Antimicrobial agents tested against isolates from New Zealand were penicillin, cloxacillin, cephapirin, ceftiofur, novobiocin, enrofloxacin, erythromycin, and pirlimycin. The minimum inhibitory concentrations that inhibit 90% of the strains tested for these antimicrobial agents with Staphylococcus aureus were 4.0, 0.5, 0.5, 2.0, 1.0, 0.25, 0.5, and 1.0, respectively. The minimum inhibitory concentration values that inhibit 90% of the strains tested against the Staphylococcus spp. ranged from 0.5 to 1.0 for all antimicrobics. The minimum inhibitory concentrations against streptococci were < or = 0.06, 0.5, 0.13, 0.13, 4.0, 1.0, 0.13, and < or = 0.06, respectively. Antimicrobial agents tested against isolates from Denmark included penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin plus novobiocin, erythromycin, and pirlimycin. Against S. aureus, the minimum inhibitory concentrations were 0.13, 0.5, 0.5, 0.5, 1.0, 0.25, 0.5, and 0.5, respectively. The minimum inhibitory concentrations against Staphylococcus spp. were 0.25, 0.25, 0.5, 0.5, 1.0, < or = 0.06, 0.13, 1.0, and 0.5, respectively. The minimum inhibitory concentrations against the streptococci were < or = 0.06, 0.13, 0.5, 0.5, 1.0, < or = 0.06, 0.13, 0.5, and 0.5, respectively. Minimum inhibitory concentration values for staphylococci from New Zealand and Denmark were similar to values reported for US isolates. Streptococci from New Zealand and Denmark had lower minimum inhibitory concentration values than did US isolates. Only ceftiofur and enrofloxacin were active against the Gram-negative bacilli.

Subtyping of Streptococcus dysgalactiae and Streptococcus uberis isolated from bovine mammary secretions by DNA fingerprinting.

Streptococcus dysgalactiae and Streptococcus uberis isolated from mammary secretions of cows from Tennessee and New Zealand were subtyped using polymerase chain reaction-based DNA fingerprinting. Such DNA fingerprinting using primer 8.6d (5'-GTAACGCC3') resulted in categorizing 116 S. dysgalactiae isolates into 25 different subtypes, with 17 subtypes observed in isolates from Tennessee and eight in isolates from New Zealand. All S. dysgalactiae DNA fingerprint profiles, regardless of origin, contained 700- and 330-base pair fragments. The majority of S. dysgalactiae isolates (73%) from Tennessee belonged to two subtypes. The remaining 23 isolates belonged to 15 different DNA fingerprint subtypes. Streptococcus dysgalactiae isolates from New Zealand (n = 32) were grouped into eight different subtypes; 66% belonged to two subtypes. A characteristic feature of S. dysgalactiae isolates from New Zealand was the presence of a 270-base pair DNA fragment seen infrequently in S. dysgalactiae isolates from Tennessee. When primer OPE-4 (5'-GTGACATGCC-3') was used, DNA fingerprinting differentiated S. uberis from Tennessee (n = 28) and New Zealand (n = 30) into 20 subtypes; 14 subtypes were observed in isolates from Tennessee, and six in isolates from New Zealand. All S. uberis DNA fingerprint profiles, regardless of origin, contained 1100-, 640-, and 450-base pair fragments. A characteristic feature of S. uberis isolates from New Zealand was the presence of a 300-base pair DNA fragment seen infrequently in S. uberis isolates from Tennessee. The most common subtypes of S. dysgalactiae and S. uberis from Tennessee herds were isolated in milk from lactating cows during monthly herd surveys, in milk from cows with clinical mastitis, and in mammary secretions from cows during the periparturient period, and thus were not confined to one particular stage of lactation. These data suggest that S. dysgalactiae and S. uberis from New Zealand are distinct from those isolated from the USA, and that DNA fingerprinting can be used as an epidemiological tool to differentiate streptococci and identify important sources of these mastitis pathogens on dairy farms.

The prevalence of mastitis in primiparous heifers in eleven Waikato dairy herds.

The objective of the study was to determine the prevalence of mastitis among primiparous heifers at calving and at drying off in 11 Waikato dairy herds during the 1993-94 dairy production season. Duplicate quarter milk samples were collected aseptically from 458 heifers within 5 days after calving for bacteriological analysis. Mastitis was diagnosed in at least one quarter in 35.6% of these heifers. Coagulase-negative staphylococci were isolated from 21.8% of the heifers. The prevalence of coagulase-negative staphylococci varied between herds from 4.3% to 44.8%. Environmental streptococci caused mastitis in 12.2% of heifers, ranging from 5.6% to 24.1% between herds. Streptococcus uberus was the pathogen identified most frequently at calving and accounted for more than 90% of the streptococcal isolates. Staphylococcus aureus and coliforms were isolated from less than 1% of samples. Clinical mastitis was observed in 8.1% of heifers at calving; environmental streptococci were isolated from 67.6% of these clinical clinical cases. Only 2.8% of heifers developed clinical mastitis during lactation and environmental streptococci were isolated from 38.5% of these cases. The prevalence of mastitis among 428 of the heifers at drying off was 64.7%; a 1.8 fold increase during lactation. Corynebactetium bovis was isolated from 43% of heifers at drying off even though it was not isolated from any heifers at calving. During the season, the prevalence of Staphylococcus aureus mastitis increased to 2.8% while mastitis caused by environmental streptococci declined to 2.8%. The prevalence of environmental mastitis pathogens decreased during lactation while contagious pathogens increased in each of the 11 herds. Ineffective post-milking teat sanitation probably contributed to the increase in mastitis caused by contagious pathogens. Specific factors were not determined that affected the variation in prevalence between herds.

Effects of freezing on the viability of nine pathogens from quarters with subclinical mastitis.

Milk samples from 45 quarters containing mastitis pathogens were collected from lactating cows to determine the viability of those pathogens after freezing. An initial bacteria count was conducted, and samples were divided into 2-ml portions and frozen. Weekly bacteria counts were conducted for 6 wk. Viability after freezing was determined on five isolates of nine bacterial species: Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Corynebacterium bovis, and Escherichia coli. Bacteria counts were converted to logarithm base 10, and analysis of variance was conducted to determine alterations in viability over the 6-wk period. Freezing of quarter milk samples for 6 wk did not affect viability of any of these pathogens.

Survey of intramammary infections in dairy heifers at breeding age and first parturition.

A survey was conducted to determine and contrast prevalence of IMI in nulligravid and primigravid dairy heifers pre- and postpartum. Contrasts were made to evaluate the risk factors of location of dairy, trimester of gestation, and season of sampling on IMI. Twenty-eight dairies in California, Louisiana, Vermont, and Washington were studied. Lacteal secretions were collected aseptically from heifers at breeding age (8 to 19 mo) from one side of the gland and again at 4 d postpartum from all quarters. Of the quarters sampled, 65.6% prepartum and 64.0% postpartum were free of IMI. The percentages of quarters with IMI from coagulase-negative staphylococci or Staphylococcus aureus IMI were 27.1 and 9% prepartum and 21.8 and 2.9% postpartum. Staphylococcus aureus IMI were most prevalent in Louisiana during the months other than summer. Location, herd, and season significantly influenced prevalence of IMI. The prevalence of IMI was greatest during the last trimester of pregnancy, ranging from 49.2% in the winter to 36.8% in the summer. The significant effects of herd location and season suggest that management variables influence prevalence of heifer IMI. Because prevalence of IMI was greatest during the last trimester of pregnancy compared with prevalence during earlier stages of pregnancy, the heifer may be most susceptible to this disease during this period of first gestation.

The visual appearance and somatic cell count of mammary secretions collected from primigravid heifers during gestation and early postpartum.

Mammary secretions, obtained before and after calving, were examined for visual appearance, SCC, and bacteriology as part of a larger study determining the prevalence of IMI in 1588 primigravid heifers. Appearance of secretions was categorized into five groups: thin and watery, honey-like, serumy, milky, or thickened colostrum. Precalving secretions were further characterized as low viscosity (thin and watery, serumy, or milky) or high viscosity (honey-like and thickened colostrum). Postcalving secretions were further characterized as normal (milky, thickened colostrum) or abnormal (thin and watery, serumy, or honey-like). Infected precalving quarters (81%) had low viscosity secretions. Quarters that were uninfected precalving (75%) had high viscosity secretions. Greater than 90% of all postcalving milk samples appeared to be normal, regardless of geographic location, season, or bacterial infection status. Only 77% of the samples from quarters infected with contagious and noncontagious mastitis pathogens had normal appearance. Precalving SCC from bacteriologically negative quarters were lower than SCC from infected quarters. Similarly, postcalving SCC were lower from the bacteriologically negative quarters than from the infected quarters. Infected quarters had higher mean SCC than the uninfected quarters during both pre- and postcalving periods.

Antimicrobial susceptibility of microorganisms isolated from the mammary glands of dairy heifers.

Minimum inhibitory concentrations were determined with 1494 microorganisms isolated from the mammary glands of dairy heifers. The antimicrobial agents tested were penicillin, cloxacillin, cephapirin, ceftiofur, novobiocin, enrofloxacin, erythromycin, and pirlimycin. All minimum inhibitory concentrations were expressed as micrograms per milliliter. The isolates tested included 135 Staphylococcus aureus, 1222 Staphylococcus sp., 42 Streptococcus sp., 15 Enterococcus sp., 60 enteric species, and 20 miscellaneous organisms. The minimum inhibitory concentrations for 90% of isolates for the various antimicrobial agents with Staph. aureus were as follows: penicillin, .13; cloxacillin, .5; cephapirin, .5; ceftiofur, 1; novobiocin, .5; enrofloxacin, .5; erythromycin, .5, and pirlimycin, .5. In comparison, the minimum inhibitory concentrations for 90% of isolates for the Staphylococcus sp. were 1, 1, .5, 1, .5, .5, 1, and .5 for penicillin, cloxacillin, cephapirin, ceftiofur, novobiocin, enrofloxacin, erythromycin, and pirlimycin, respectively. The minimum inhibitory concentrations for 90% of isolates for the Streptococcus sp. were 2, 32, 2, 2, 8, 1, 64, and 32 for the respective antimicrobial agents; the minimum inhibitory concentrations for 90% of isolates were 4, 64, 32, 64, 4, 1, 4, and 4 for the enterococci. Against the Gram-negative enteric bacilli, only ceftiofur and enrofloxacin were active; minimum inhibitory concentrations for 90% of isolates were 1 microgram/ml for ceftiofur and .25 microgram/ml for enrofloxacin. Results indicated that the majority of staphylococcal strains were susceptible to the antimicrobial agents tested but that antimicrobial susceptibility varied for Streptococcus sp. Compounds currently available in intramammary infusion products demonstrated poor activity against the enteric organisms.

Effect of oxygen tension on killing of Escherichia coli by bovine polymorphonuclear neutrophil leucocytes in vitro.

A batch fermenter modified to simulate the physical conditions of an inflamed v. uninflamed mammary gland was utilized to evaluate the effect of oxygen tension on killing of Escherichia coli P4 by bovine polymorphonuclear neutrophil leucocytes. Leucocytosis was simulated in vitro 4 h after inoculation with Escherichia coli by adding bovine neutrophils isolated from peripheral blood to the culture medium. Experiments were conducted at 39 degrees C in UHT treated milk. At micro-aerophilic oxygen tension (oxygen partial pressure, 3.11 kPa), bacterial numbers declined during the first hour following addition of the neutrophils. Oxygen tension declined rapidly following PMN addition. Once oxygen was depleted, neutrophil activity was presumably diminished and Esch. coli numbers began to increase. Under anaerobic conditions (oxygen partial pressure, 0.17 kPa), no reduction in population was observed. Photomicrographs taken at the time of neutrophil addition and at subsequent time intervals demonstrated a specific association between neutrophils and the pathogen. Subsequent lysis of neutrophils associated with Esch. coli growth was seen coincident with oxygen depletion.

Use of predictive modeling to evaluate the manipulation of milking frequency, temperature, and oxygen tension on growth of Escherichia coli in an artificial intramammary environment.

A method was developed to evaluate frequent milking as a means of controlling intramammary infection. An artificial intramammary environment was used to determine growth responses of Escherichia coli (P4) to natural changes in the mammary gland resulting from bacterial invasion. Physical conditions manipulated in this model were growth medium, temperature, and oxygen tension. Mathematical modeling was then incorporated to generate predictions concerning growth dynamics of the organism when milking frequency was changed. To test accuracy of the model, initial predictions were derived from bacterial growth data in which E coli was incubated in tryptose soy broth for 12 hours at 37 C and PO2 equal to 23.3 mm of Hg. These predictions matched closely with experimental data in which 12-, 4-, and 2-hour milking intervals were simulated in the artificial intramammary environment. The mathematical model was then used to characterize growth rate data from in vitro experiments in ultra-high temperature-treated milk and in vivo experimental infection data generated with E coli (P4). Predictions generated from this model suggested that increasing milking frequency to 4 or 6 times daily controls growth of E coli for a prolonged period and that 12 times daily milking may lead to elimination of the bacterium.

Effects of temperature and oxygen tension on growth of Escherichia coli in milk.

An in vitro system was developed to mimic conditions within normal and mastitic mammary glands. The system consisted of a modified batch fermenter that allowed for manipulation of temperature, oxygen tension, and pH. Experiments in tryptose-soy broth and UHT-treated milk were conducted to evaluate growth characteristics of Escherichia coli P4 as physical conditions were manipulated. The effect of bacterial growth on oxygen tension and pH were also evaluated. Growth of E. coli was inhibited as temperature was increased from 37 to 41 degrees C and as oxygen tension was decreased from microaerophilic to anaerobic levels. At bacterial populations > 6 log10 cfu/ml, microaerophilic cultures became anaerobic. The pH followed a similar trend; however, after a significant decrease in pH, mean bacterial populations were 7.1 log10 cfu/ml in tryptosesoy broth and 8.2 log10 cfu/ml in UHT-treated milk. This dynamic model demonstrated potential use in evaluation of growth characteristics of mammary gland pathogens in the lactating mammary gland.

Evaluation of a 1% iodophor postmilking teat sanitizer.

A natural exposure field trial a with positive control was conducted to evaluate bacteriological efficacy and teat conditioning qualities of an experimental postmilking teat dip. An experimental 1% iodine postmilking teat sanitizer with a 10% emollient system was compared with a 1% iodine plus 10% glycerin teat sanitizer. Efficacy of the two sanitizers was equivalent for all new IMI, major pathogens, and environmental pathogens. The products were not equivalent for efficacy against coliforms and coagulase-negative staphylococci. Fewer coliform IMI were diagnosed in the control group than in the treatment group. Differences were determined for efficacy against coagulase-negative staphylococci in favor of the treatment product. The products were equivalent for all clinical mastitis, including previously existing IMI that became clinical. The products were not equivalent for all or new clinical IMI with major pathogens, all environmental pathogens, or coliforms. Fewer infections were diagnosed in the control group than in the treatment group. Teat end and teat skin conditions improved with the use of the triple emollient, postmilking teat sanitizer under the winter conditions experienced during this field trial.

Winter evaluation of a postmilking powdered teat dip.

A powdered teat dip designed for winter usage was evaluated for bacteriological efficacy and teat conditioning qualities. A positive control, natural exposure field trial was conducted for 3 mo on 509 lactating cows. Two sets of cows, primiparous and multiparous, were used. The trial compared efficacy of a powdered teat dip with a teat dip of 1% iodine plus 10% glycerin. Bacteriological efficacy among primiparous cows was equivalent for all major mastitis pathogens, environmental pathogens, and streptococci other than Streptococcus agalactiae. Efficacy was not equivalent against coagulase-negative staphylococci and all mastitis pathogens. Results suggested that the positive control product was more efficacious. Among multiparous cows, efficacy was equivalent against environmental mastitis pathogens and bacteriologically negative, clinical mastitis. The products were not equivalent against Staphylococcus aureus, coagulase-negative staphylococci, or all major mastitis pathogens, once again suggesting that the positive control product was more efficacious. Data indicated that germicidal activity of the powdered dip was not sufficient to reduce the incidence of new IMI caused by contagious or minor pathogens normally associated with teat skin. Application of a powdered postmilking teat dip during 3 winter mo in Idaho resulted in improved teat end condition among primiparous and multiparous dairy cows. Teat skin condition improved among primiparous but not among multiparous cows.

Genomic fingerprints of Staphylococcus aureus of bovine origin by polymerase chain reaction-based DNA fingerprinting.

Staphylococcus aureus (n = 75) isolated from mammary secretions of cows with subclinical and clinical mastitis from several geographic locations in the USA were examined using polymerase chain reaction-based DNA fingerprinting. DNA fingerprints were produced using a synthetic oligonucleotide primer (5'GTAACGCC3') to produce a distinct spectrum of amplified DNA fragments facilitating a high degree of resolution for differentiating S. aureus strains. PCR-based DNA fingerprinting grouped the 75 S. aureus isolates into 19 distinct profiles. The technique differentiated closely related strains within and between geographic locations. Findings suggest that certain types are found across geographic regions suggesting a common clonal type. Within herd data suggest heterogeneity among subclinical and clinical isolates of S. aureus strains. Compared to existing typing methods, PCR-based DNA fingerprinting is easy to perform and interpret. Use of PCR-based DNA fingerprinting may allow for a more detailed investigation of the epidemiology of S. aureus mastitis in dairy cows.

Evolution of udder hygiene. Premilking teat sanitation.

Compared with post-milking teat dipping, predipping is in its infancy. Few controlled studies have been published on premilking teat disinfection/sanitation. Experimental challenge procedures resulted in consistent reductions for predipping compared with no udder preparation and conventional udder preparation, indicating a potential for effectiveness. Challenge studies that included the treatments of predip only and postdip only indicated that interactions during the milking process require elucidation. Positive benefits have been observed for predipping in field trials among some herds, but wide variation has been observed between herds in all field trial evaluations. Parity, DIM, and season of the year had major interactions on efficacy of predipping in Pankey et al's 1987 report; other trials have been of shorter duration and precluded analysis of these interactions. Interactions among these variables influenced incidence of mastitis by environmental pathogens. The length of time after milking before teats are contaminated probably is a major influence on predip efficacy. Predipping has reduced incidence of new IMIs and new cases of clinical mastitis. Unfortunately, certain factors negated these positive effects. These factors need to be defined. Producers should monitor effects of predipping to determine whether the investment in product and time has an economic return under the conditions of their dairy.

Efficacy evaluations on five chlorhexidine teat dip formulations.

Three developmental postmilking teat dip formulations containing chlorhexidine digluconate were evaluated against Staphylococcus aureus and Streptococcus agalactiae in sequential experimental exposure trials. Two additional commercial chlorhexidine digluconate teat dip products were evaluated in natural exposure trials. Under conditions of experimental challenge, the developmental formulations were efficacious against Staph. aureus but did not significantly reduce incidence of new IMI by Strep. agalactiae. None of the three formulations of a conventional germicide used as teat sanitizers effectively reduced incidence of new Strep. agalactiae IMI under experimental challenge conditions. In the natural exposure trials with negative controls, a .35% chlorhexidine teat sanitizer had efficacy of 88.7% against Staph. aureus and 51.4% against Strep. agalactiae. The .5% chlorhexidine product reduced Staph. aureus and Strep. agalactiae IMI by 86 and 56%, respectively.

Evaluation of 57 teat sanitizers using excised cow teats.

Fifty-seven teat dip formulations were tested for germicidal activity with an excised teat assay. Streptococcus agalactiae (ATCC 27956), Staphylococcus aureus (ATCC 29740), and Escherichia coli+ (a wild strain isolated from an Ohio cow 2956RR during the dry period) were used. Log reductions ranged from 5.6480 to 1.8330 for Strep. agalactiae compared with undipped controls. Staphylococcus aureus were reduced from 4.8972 to 1.0102. Escherichia coli were reduced from 5.8677 to 1.1450. The percentages of the products tested that had log reductions < 3 against Strep. agalactiae, Staph. aureus, and E. coli were 24, 30, and 61. Four teat dip formulations contained 4% sodium hypochlorite; all were effective against Strep. agalactiae, Staph. aureus, and E. coli. The three formulations containing glutaraldehyde were effective against the three mastitis pathogens. Formulations containing 1 and .5% iodine were not consistently effective against Strep. agalactiae, Staph. aureus, or E. coli. Teat sanitizers containing .5% chlorhexidine also showed inconsistency. Variations in formulation for products containing iodine or chlorhexidine affected germicidal activity.

Increased susceptibility to intramammary infection following removal of teat canal keratin.

Influence of teat canal keratin on susceptibility to intramammary infection was investigated in lactating Jersey cows. In each of two replicate trials, keratin was removed from the left teats of 20 cows immediately before milking. Immediately after milking, all teats were exposed to bacterial challenge by immersion in a suspension of Streptococcus agalactiae (5 x 10(7) cfu/ml). Bacterial challenge was repeated after the next four milkings. Foremilk samples were obtained for 8 d after keratin removal to determine infection status. A mammary quarter was classified as infected based solely upon the bacteriological criteria outlined by the National Mastitis Council. The rate of infection in quarters from which keratin was removed was greater than that in control quarters. Infection rates were 26.3% for keratin-removed quarters and 8.3% for control quarters in trial 1 and 13.5 and 0%, respectively, in trial 2. When more stringent criteria (recovery of greater than 100 cfu of S. agalactiae/ml in three or more successive milk samples and a SCC of greater than 10(6)) were used to identify a subset of infections that were clearly intramammary, infection rates were 9.3% for keratin-removed quarters and 1.4% for control quarters. Thus, partial removal of keratin from the teat canal compromised the ability of the teat to prevent passage of bacterial pathogens from the external environment into the mammary gland.

The influence of intensively managed rotational grazing, traditional continuous grazing, and confinement housing on bulk tank milk quality and udder health.

Monthly bulk tank milk samples and veterinary records were analyzed for 1 yr on 15 Vermont dairy farms. Data were evaluated using ANOVA to compare effects of grazing management systems on milk quality and udder health. Systems evaluated were intensively managed rotational grazing, traditional continuous grazing, and confinement housing. Bulk tank samples were evaluated for standard plate count, bacterial type counts on tryptose-blood-esculin agar, and SCC. Veterinary records were evaluated for incidence of clinical mastitis, udder edema, and teat injuries. Within- and between-treatment group analyses were conducted by season, herd size, and udder sanitation systems. Mean standard plate counts were lower in rotationally grazed herds than counts of confined herds during the grazing season. Similarly, rotationally grazed herds with fewer than 60 cows had lower standard plate counts than confined herds of similar size. Mean bulk tank counts of streptococci other than Streptococcus agalactiae during the grazing season differed among treatments. The lowest counts occurred in rotationally grazed herds. Among herd using predip products recognized as efficacious, fewer streptococci other than S. agalactiae were isolated from bulk tank milk of rotationally grazed herds than confined herds. Rotationally grazed herds using postdips recognized as efficacious had lower SCC than those using unrecognized postdips. No udder health differences were observed among grazing treatments.

Modification of the Excised Teat Germicidal Activity Assay.

The Excised Teat Assay is an in vitro germicidal activity assay for evaluation of bovine teat sanitizers. The rinse procedure of this assay was modified and compared to the standard method. The rinse modification used an ultrasonic cleaner to vibrate the quencher solution to remove bacteria from excised teats after challenge. In alternating trials, bacteria were rinsed from teats by the standard procedure or by the ultrasonic procedure. A 1% iodine based teat sanitizer, a chlorhexidine based teat sanitizer, and a negative control were used in the study. Staphylococcus aureus (ATCC 29740) was used as the challenge organism. No significant differences were obtained by analysis of variance when comparing procedures or in comparing procedures within treatments when an experienced technician conducted the procedure. When an inexperienced technician conducted the procedure, significant differences were obtained between procedures and within treatments when comparing procedures. The modified rinse procedure provided more consistent results than the standard rinse procedure when used by an inexperienced technician.