Short communication: Effects of supplementing diets of Holsteins with copper, zinc, and manganese on blood neutrophil function.

The effects of supplementing diets with sulfate or glycinate Cu, Zn, and Mn on blood neutrophil function were examined in 27 late-lactation Holstein cows having a mean (± standard deviation) days in milk at time of neutrophil assays of 216 ± 31 d. Cows were assigned to 9 blocks of 3 and were grouped by parity, milk production, and days in milk. Cows within each block were randomly assigned to 1 of 3 treatments: (1) control diet devoid of supplemental Cu, Zn, and Mn; (2) diet supplemented with Cu, Zn, and Mn via sulfates; and (3) diet supplemented with Cu, Zn, and Mn via glycinate form. All cows were initially fed a control total mixed ration with basal mineral concentrations of 8 mg/kg of Cu, 35 mg/kg of Zn, and 35 mg/kg of Mn for 30 d. During the treatment period, cows fed diets with mineral supplementation via sulfates or glycinate forms had target total dry matter dietary concentrations of 18 mg/kg of Cu, 60 mg/kg of Zn, and 60 mg/kg of Mn for 30 d. Control cows were fed the control diet devoid of supplemental minerals for an additional 30 d. In vitro neutrophil functions were measured after 30 d on experimental or control diets. Percentage of neutrophils phagocytizing, intracellular kill, and phagocytic index did not differ among treatments. Serum concentrations of Cu, Zn, and Mn were also not affected by dietary treatment after 30 d. Results from this study demonstrated that dietary Cu, Zn, and Mn supplemented either as sulfates or glycinate form for 30 d had no effect on either in vitro blood neutrophil function or serum concentrations of Cu, Zn, and Mn in late-lactation Holstein cows.

Effect of gradual or abrupt cessation of milking at dry off on milk yield and somatic cell score in the subsequent lactation.

The objective of this study was to assess the effect of milk cessation method (abrupt or gradual) at dry off on milk yield and somatic cell score (SCS) up to 120 d in milk during the subsequent lactation. Data from 428 cows from 8 dairy herds in Ohio were analyzed. Abrupt cessation cows kept the farm's regular milking schedule (2 or 3 times) through dry off and gradual cessation cows were milked once daily for the final week of lactation. Milk yield and SCS were collected using Dairy Herd Improvement Association test-day records. Aseptic quarter milk samples were collected approximately 1 wk before dry off, at dry off, and within 1 wk after calving for bacterial culture to determine the presence of intramammary infections. Overall, milk cessation method was not significantly associated with either milk yield or SCS in early lactation; however, interaction between the milk cessation method and herd was highly significant. Cows producing greater amounts of milk around dry off had significantly higher SCS in the following lactation. Shorter dry periods were significantly associated with decreased milk yield in the following lactation, especially among abruptly dried off cows. Additionally, as expected, several other factors, such as parity of cows and stage of lactation, were significantly associated with both outcomes. No interactions between the milk cessation method and the other explanatory variables in the final models were significant. The results of the current study suggest that higher milk yield at dry off was associated with higher SCS in the following lactation, even though milk cessation method at the end of lactation had a varying effect on test-day milk yield and SCS in different herds during the first 120 d in milk in the following lactation. The specific herd characteristics influencing this could not be identified within this study, warranting further research.

Intramammary infections and milk leakage following gradual or abrupt cessation of milking.

The objective of this study was to evaluate the effect of milking cessation method (abrupt or gradual) and daily milk yield before dry-off on milk leakage following dry-off and intramammary infections (IMI) at calving. Data from 1,086 quarters of 285 cows from 5 Ohio dairy herds were analyzed. All cows that were due to be dried off within a week were assigned to the same study group to facilitate management. Abrupt-cessation cows kept the farm's regular milking schedule through dry-off, and gradual-cessation cows were milked once daily for the final week of lactation. Aseptic technique was used to collect quarter foremilk samples at the time of enrollment (7 to 14 d before expected dry-off), the final milking before dry-off (D-O), and within 7 d of calving. Cows in the gradual-cessation group were observed for milk leakage during the period of once-daily milking. In the only herd that did not use internal teat sealants at dry-off, milk leakage after dry-off was recorded in both abrupt and gradual groups. Gradual cessation decreased milk production by 33.4% during the final week of lactation, causing milk yield at D-O to be lower for these cows compared with abrupt-cessation cows (13.2 vs. 19.8kg/d, respectively). Logistic regression models were used to model the probability of a quarter being infected at calving with any pathogen, accounting for clustering of quarters within cows and cows within herds. The final model investigating the probability of IMI at calving was stratified by parity of cows at the time of dry-off (primiparous and multiparous). Among quarters of cows that ended their first lactation, abrupt cessation of milking before dry-off and milk leakage after dry-off were associated with an increased risk of IMI at calving. Among quarters of multiparous cows, on the other hand, gradual cessation of milking before dry-off, presence of IMI at D-O, and thrice-daily milking during lactation increased the odds of IMI at calving. These results indicate that implementation of differing management practices near dry-off for different parity groups may improve mammary health within a herd.

Short communication: Environmental mastitis pathogen counts in freestalls bedded with composted and fresh recycled manure solids.

An experiment was conducted to compare bacterial counts of environmental mastitis pathogens in composted recycled manure solids bedding with those in fresh recycled manure solids. Eighteen Holstein cows were housed in 1 pen with 18 stalls. One row of 9 freestalls included mattresses and was bedded weekly with composted recycled manure solids. The second row of 9 freestalls included mattresses and was bedded weekly with fresh recycled manure solids. The back one-third of stalls toward the alleyway was covered in 25 to 50 mm of bedding. Samples were taken from the back one-third of 4 stalls for both treatments on d 0, 1, 2, and 6 of each week. After 3 wk, bedding treatments were switched between rows, making the total duration 6 wk. Mean total gram-negative bacterial counts were approximately 0.5 log10 cfu/g of dry matter lower in the composted recycled manure solids on d 0 compared with fresh recycled manure solids. Klebsiella species, coliform, and Streptococcus species counts were at least 1.0 log10 cfu/g of dry matter lower in composted compared with fresh recycled manure solids on d 0. Only gram-negative bacterial counts on d 1 were reduced in composted recycled manure solids compared with fresh recycled manure solids. Differences were not observed between treatments in gram-negative bacterial, coliform, Klebsiella species, or Streptococcus species counts on d 2 and 6. Ash content was higher in composted recycled manure solids compared with fresh recycled manure solids on d 0, 1, 2, and 6. Despite the increase in ash after composting, bacterial counts of mastitis pathogens in composted recycled manure solids were comparable with those in fresh recycled manure when used as freestall bedding.

Activity and milk compositional changes following experimentally induced Streptococcus uberis bovine mastitis.

Milk constituents and physical activity of cows experimentally infected with Streptococcus uberis mastitis were compared with those of uninfected cows. Twelve late-lactation Holsteins cows were paired based on milk production and parity. One cow in each pair was experimentally infected in the right front mammary gland with Strep. uberis. The remaining cow in each pair served as an uninfected control. Real-time analyses of milk constituents provided fat, protein, and lactose percentages at each milking. Pedometers were placed on the left front leg of all cows and activity was measured. Intramammary infections with Strep. uberis reduced milk yield in experimental cows by approximately 1.6kg/d in the first week after challenge compared with control cows. Lactose percentage in milk was reduced on d 3, 4, 5, and 6 after challenge in treatment cows compared with controls. Percentages of fat and protein in milk did not differ between infected and uninfected cows the week after infections were induced. Total steps per day were reduced and minutes resting per day were increased the week after experimental challenge in infected cows compared with control cows. The number of resting bouts did not differ between infected and uninfected cows. Changes in percentage of lactose in milk and animal activity caused by experimentally induced Strep. uberis mastitis were detected by the automated milk analyzer and pedometer systems.

Effects of various starch feeding regimens on responses of dairy cows to intramammary lipopolysaccharide infusion.

Endotoxin tolerance (ET) can develop in mammals that have been challenged repeatedly with sublethal amounts of lipopolysaccharide (LPS). Previous research has shown that subclinical ruminal acidosis can increase circulating concentrations of LPS. We investigated whether ET would develop in Holstein cows that were subjected to chronic subacute ruminal acidosis (SARA) or acute SARA followed by intramammary infusion of LPS. Twenty-four cows, both primiparous and multiparous, were assigned to 8 blocks of 3 cows. Cows within blocks were randomly assigned to 1 of 3 treatments: (1) control (diet DM was 24% starch and 35% NDF), (2) high starch (formulated to induce chronic milk fat depression with 29% starch and 32% NDF), and (3) acidosis (designed to cause acute bouts of milk fat depression by short-term feeding of a diet with 32% starch, some of which came from wheat grain, and 30% NDF). Cows on the control and high-starch treatments were fed their respective diets throughout the 24-d trial. The acidosis cows were fed the control diet during most of the experiment, except during two 2-d bouts (d 10 and 11 and 17 and 18 of the experiment) in which a high-starch diet was fed. Cows on the high-starch and acidosis treatments produced milk fat with an altered fatty acid profile indicative of SARA (e.g., increased concentrations of specific trans, and odd-, and branched-chain fatty acids), but only cows on the high-starch treatment had milk fat depression. Concentrations of serum amyloid A were elevated in cows on the acidosis treatment, but did not differ between control and high-starch cows. On d 20 of the experiment, all cows were given an intramammary infusion of 10 µg of LPS into 1 mammary quarter 3h after morning milking. Milk yield and DMI decreased the day of the infusion, but the response was not affected by dietary treatment. No systemic indicators of ET were observed among treatments, but evidence of an ET response at the local level of the mammary gland was observed. Cows fed the control diet had higher concentrations of serum amyloid A in milk 12 and 24h postinfusion than did cows fed the high-starch diet and higher concentrations than cows on the acidosis treatment at 12h postinfusion. Our data suggest cows that experienced varying degrees of SARA (based on altered milk fatty acid profile) and subsequent experimental endotoxin mastitis experienced a blunted inflammatory response at the level of the mammary gland, but not a systemic reduction in some inflammatory mediators.

Short communication: Bacterial counts in recycled manure solids bedding replaced daily or deep packed in freestalls.

An experiment was conducted to compare bacterial counts of mastitis pathogens in deep-packed manure solids bedding with those in manure solids bedding replaced daily from mattresses. Eighteen Holstein cows were housed in 1 pen with 18 stalls. One row of 9 stalls was equipped with mattresses topped with bedding. The back one-third of these stalls toward the alleyway was covered in 25 mm of recycled manure solids, which was removed daily for the next 6 d and replaced with bedding from the brisket board and lunge space areas of stalls. The second row of 9 stalls was bedded for 3 wk with 100 to 150 mm of deep-pack recycled manure bedding from which only fecal matter was removed daily. After 3 wk, bedding treatments were changed between rows in a switchback design. Mean total gram-negative bacterial counts did not differ between treatments throughout the experiment. Coliform and Klebsiella spp. bacterial counts were lower in daily replaced bedding compared with deep pack across the experiment and on each of d 0, 1, 2, and 6. Streptococcal counts were reduced in daily replacement stalls compared with deep-pack stalls on d 0 and greater in daily replacement stalls compared with deep-pack stalls on d 1, 2, and 6. Daily replacement of recycled manure bedding from the back one-third of the stalls appeared to be an effective approach to reducing exposure to coliforms, specifically Klebsiella, but not streptococci. However, bacterial counts in bedding from both treatments were elevated throughout the trial and resulted in considerable risk for exposure to teats and development of intramammary infections.

Short communication: antimicrobial susceptibility and frequency of resistance genes in Escherichia coli isolated from bovine mastitis.

Escherichia coli isolated from bovine milk samples submitted to the Ohio Agricultural Research and Development Center Mastitis Laboratory (Wooster) in 1985 to 1987 and in 2009 were compared for antimicrobial susceptibility and prevalence of antimicrobial resistance genes. Forty-four isolates from 1985 to 1987 and 55 isolates from 2009 were tested. Minimum inhibitory concentrations of 15 antimicrobials were determined using a commercially available broth microdilution system. Multiplex polymerase chain reaction was performed for gene detection. The percentage of isolates susceptible to trimethoprim/sulfamethoxazole, ampicillin, and kanamycin was lower in those collected in 1985 to 1987 than in isolates collected in 2009. Susceptibility did not differ between isolates from 1985 to 1987 and isolates from 2009 for the 12 other antimicrobials tested. A trimethoprim/sulfamethoxazole resistance gene was detected more frequently in isolates from 1985 to 1987 than in isolates from 2009. Gene frequencies for streptomycin resistance and tetracycline resistance were similar among 1985 to 1987 isolates and 2009 isolates. Resistance to most antimicrobials did not differ between isolates submitted to a mastitis diagnostic laboratory in 1985 to 1987 and those submitted in 2009. Changes observed indicated an increase in frequency of susceptibility in isolates to trimethoprim/sulfamethoxazole, ampicillin, and kanamycin in 2009 isolates compared with 1985 to 1987 isolates.

Evaluation of propane flaming for reducing bacterial counts in sand bedding.

An experiment was conducted on a commercial dairy farm to determine the effects of propane flaming on bacterial populations of common environmental mastitis pathogens in recycled sand bedding. One row of freestalls was flamed within 12 h after recycled bedding was added to stalls and then daily for the next 6 d. Bedding in a control row of freestalls was untreated. Stalls received the same treatment for 3 consecutive weeks. After 3 wk, bedding treatments were changed between rows in a switchback design. The daily movement of the 760°C propane-fueled flame at 3.2 km/h over the surface of recycled sand bedding in stalls had a positive effect by reducing mastitis pathogen loads at different depths of bedding in a pathogen-specific manner. The greatest reduction of mastitis pathogen populations by flaming was on the surface 25 mm of recycled sand. Reductions in bacterial counts in deeper layers were less consistent. Bacterial populations in both flamed and control sand bedding were lower on the surface 25 mm compared with sand at a depth of 50 to 75 mm. The effects of subsequent flaming of sand over a week also differed among pathogens. In general, mastitis pathogens were reduced the most on the day that recycled sand was added to stalls, and flaming was less effective as sand bedding was in stalls over a 6-d period. The use of propane flaming of recycled sand was shown to have potential as a practice to control mastitis pathogen populations in bedding. The greatest advantage afforded by flaming was on the surface of bedding, with inconsistent effects deeper in the stalls. Flaming was more effective in controlling bacterial populations in fresh recycled sand than in sand after several days use

The ability of common mastitis-causing pathogens to survive an ensiling period.

A forage-based study was conducted to examine the ability of enterococci, streptococci, and gram-negative bacteria to survive a 3-wk ensiling process. Harvested grass and corn forages were each divided into 3 treatment groups consisting of 2 commercially available silage inoculants and 1 negative control group. Within 24h of harvest, a uniform amount of forage was added to each of 18 vacuum-sealable freezer bags. Inoculants 1 and 2 were applied to each of 6 bags and the remaining 6 bags were not inoculated (negative control). All bags were sealed using an industry-grade vacuum sealer to create a pillow pack. At wk 1, 2, and 3 of the ensiling process, 6 bags were opened, 2 from each of the 3 groups. Dry matter (DM), pH, and bacterial enumeration were determined on the forage before ensiling, as well as at wk 1, 2, and 3 of ensiling. The pH of grass and corn samples decreased to 5.2 and 4.1, respectively, by the end of the ensiling period. Inoculant 2 (7.3 log(10) cfu/g of DM) resulted in a greater enterococcal count in grass samples than did inoculant 1 (4.2 log(10) cfu/g of DM) at 3 wk of ensiling and both were greater than the negative control (1.2 log(10) cfu/g of DM). Counts of streptococci in grass samples at wk 3 were greater in inoculated samples (inoculant 1, 7.6 log(10) cfu/g of DM; inoculant 2, 7.0 log(10) cfu/g of DM) compared with the negative control (4.3 log(10) cfu/g of DM). Treatment differences were observed following 1 wk of ensiling in corn samples. Enterococcal counts on corn samples following the 3-wk ensiling were not significantly different between inoculant 1 (4.3 log(10) cfu/g of DM) and inoculant 2 (4.4 log(10) cfu/g of DM). However, counts in both inoculated groups were greater than in the negative control group (4.0 log(10) cfu/g of DM). No significant treatment differences were observed for streptococci counts at wk 3. Counts for gram-negative bacteria were not detectable by wk 3 of ensiling in either grass or corn samples. The addition of a silage inoculant led to greater counts of enterococci and streptococci in grass silage compared with the negative control at the end of a 3-wk ensiling period. The application of a silage inoculant led to increased enterococci counts, but not streptococci counts, in corn samples following the ensiling period. These data indicate that enterococci and streptococci are able to survive the pH decrease observed during ensiling.

Experimental challenge of bovine mammary glands with Enterococcus faecium during early and late lactation.

Mammary glands of early and late lactation cows were challenged with Enterococcus faecium of bovine origin to determine in vivo pathogenicity and milk somatic cell count (SCC) responses. A total of 20 early lactation and 18 late lactation mammary glands were challenged. Two isolates highly adaptive and 2 isolates poorly adaptive for in vitro growth in mammary secretion were used as challenge strains of bacteria. Challenged quarters of early lactation cows were more susceptible to intramammary infection caused by E. faecium than those of late lactation cows. Intramammary challenge with isolates poorly adaptive for in vitro growth in mammary secretions resulted in 94.7% of quarters infected compared with 36.8% of the quarters infused with the isolates highly adaptive for in vitro growth in mammary secretions. Milk from quarters infused with the isolates poorly adaptive for in vitro growth had higher SCC and bacterial counts compared with quarters challenged with the isolates highly adaptive for in vitro growth. A stage of lactation effect within treatment groups was measured when milk SCC were compared between early and late lactation cows. Milk SCC in uninfused (negative control) quarters were lower in early lactation cows compared with late lactation cows. Conversely, in quarters infused with isolates poorly adaptive for in vitro growth, SCC were higher in early lactation cows compared with late lactation cows on d 2, 3, 4, 15, 16, and 17 postchallenge. In quarters infused with isolates highly adaptive for in vitro growth, SCC response did not differ between early and late lactation cows. In vitro growth of E. faecium in mammary secretion was inversely related to in vivo pathogenicity in the mammary glands of early and late lactation cows.

Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae.

AIMS: Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes. METHODS AND RESULTS: Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X. szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X. budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite((R)) XAD 1180, and eluted with methanol : 1 n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX. CONCLUSIONS: Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P. nicotianae.

Relative bioavailability of all-rac and RRR vitamin E based on neutrophil function and total alpha-tocopherol and isomer concentrations in periparturient dairy cows and their calves.

The objective of this experiment was to determine whether source of supplemental alpha-tocopherol fed to periparturient dairy cows affects neutrophil function and vitamin E status of the cow and the neonatal calf. Starting 14 d before anticipated calving and continuing until 14 d post-parturition, cows were fed diets with no supplemental vitamin E or with 2,500 IU/d of vitamin E from all-rac alpha-tocopheryl acetate or RRR alpha-tocopheryl acetate. All-rac alpha-tocopherol contains equimolar amounts of all 8 stereoisomers, whereas the RRR contains only the RRR isomer. Concentrations of alpha-tocopherol in cow plasma, colostrum, milk, and blood neutrophils were greatest for the RRR treatment, intermediate for all-rac, and lowest for cows fed no supplemental vitamin E. The concentration of alpha-tocopherol in plasma of newborn calves was very low and not affected by treatment but after 6 feedings of their dam's colostrum or milk, concentrations in calf plasma followed the same treatment pattern as cow plasma. The number of bacteria phagocytized was greater by neutrophils from cows fed all-rac vitamin E than for the other 2 treatments, which resulted in a greater number of bacteria being killed. For cows fed all-rac vitamin E, the RRR isomer comprised about 20% of the alpha-tocopherol consumed but approximately 60% of the alpha-tocopherol in plasma and milk. This enrichment was caused mostly by an almost complete discrimination against the 2S isomers. Because all-rac alpha-tocopherol is 50% 2S isomers, these data suggest that 1 g of all-rac tocopheryl acetate is equivalent to 0.5 g of RRR tocopheryl acetate.

Factors associated with the risk of antibiotic residues and intramammary pathogen presence in milk from heifers administered prepartum intramammary antibiotic therapy.

Heifers (n=136) from 5 herds were treated with a commercially available beta-lactam intramammary (IMM) antibiotic preparation containing cephapirin sodium at 10-21 d prior to anticipated parturition to evaluate the risk of antibiotic residues occurring in milk postpartum and to determine factors associated with antibiotic residues and IMM pathogen presence in milk postpartum. Mammary secretions collected from quarters before antibiotic administration and during weeks 1, 2 and 3 postpartum were analyzed for mastitis pathogens. Composite milk was collected at milkings 3, 6 and 10 postpartum and analyzed for beta-lactam residues using a microbial inhibition antibiotic residue screening test. Antibiotic residues were confirmed with beta-lactamase treatment and re-tested for residues. Residues were detected in 28.0, 8.82 and 3.68% of milk samples obtained at the third, sixth, and tenth milking postpartum, respectively. Increases in interval between prepartum antibiotic therapy and parturition and an increase in the postpartum interval to sampling were associated with a decrease in risk of antibiotic residues. The presence of antibiotic residues in milk at the third milking was associated with a reduced risk for IMM pathogen prevalence in the first 21 d postpartum. Lower somatic cell counts, an increase in mean milk yield over 200 days in milk and a reduction in IMM pathogen prevalence were associated with the presence of an antibiotic in milk postpartum. Screening milk for antibiotic residues in milk postpartum following prepartum antibiotic therapy in heifers is recommended to reduce the risk for antibiotic residue contamination of milk.

Genomic typing of enterococci isolated from bovine mammary glands and environmental sources.

Enterococcal isolates (n = 102) from various sources of bovine origin on 1 farm were characterized using pulsed field gel electrophoresis analysis of SmaI restriction patterns. Isolates originated from feed samples (n = 6), bedding samples (n = 15), and bovine quarter-milk samples (n = 81). Isolates collected from milk samples included those from high-somatic cell count cows (n = 42), postpartum milk samples (n = 16), and clinical mastitis samples (n = 23). Species evaluated included Enterococcus faecium (n = 68), Enterococcus casseliflavus (n = 29), and Enterococcus faecalis (n = 5). A total of 20 clusters representing 44 isolates were detected when a similarity cut-off level of 75% was applied to interpret the pulsed field gel electrophoresis results. Fifteen of the clusters contained only isolates from milk samples. Four clusters contained isolates from bedding and milk samples. One cluster contained only isolates from feed samples. Clusters comprised of a single species represented 17 of the 20 total clusters. These results suggest enterococci from bovine origin were genetically diverse, whereas a limited number of isolates from various sources appeared to cluster together.

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria.

AIMS: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram-positive and Gram-negative bacteria, and were tested against mastitis isolates from dairy cows. METHODS AND RESULTS: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell-free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4-methyl-2-oxovaleric acid sodium salt. CONCLUSIONS: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.

In vitro growth of enterococci of bovine origin in bovine mammary secretions from various stages of lactation.

In vitro growth responses of Enterococcus faecium and Enterococcus faecalis were tested in cell-free, fat-free bovine mammary secretions. Mammary secretions were collected during the dry period, and during early, late, and extended lactation. Sixty-three enterococcal isolates from aseptically collected bovine quarter milk samples and bedding samples from a commercial dairy herd were tested. Isolates from bovine quarter milk samples originated from mammary glands with clinical mastitis, cows with composite somatic cell score >4, postpartum milk samples, or from routine milk samples submitted to a mastitis diagnostic laboratory. Source of enterococcal isolates and the species significantly contribute to the ability of organisms to multiply in mammary secretions from various stages of lactation. Isolates collected from milk samples of the commercial herd and isolates from milk submitted to a mastitis diagnostic lab did not display enhanced growth in mammary secretions compared with isolates from bedding. Growth responses of E. faecalis were greater than those for E. faecium in secretions collected during the dry period, late lactation, and extended lactation. Bacterial growth did not differ between enterococcal species in mammary secretion collected from cows in early lactation. Differences in bacterial growth between E. faecalis and E. faecium in mammary secretions may indicate differences between species in susceptibility of mammary glands during the lactation cycle.

Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows.

Neutrophil function and the severity and incidence of mastitis in dairy cows is related to the intake of many antioxidant nutrients. Because vitamin C is the major water-soluble antioxidant in mammals, we examined the effect of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccahride (LPS) in periparturient dairy cows. At 2 wk before anticipated calving, Holstein cows were fed diets that provided 0 (16 cows) or 30 (15 cows) g/d of supplemental vitamin C (phosphorylated ascorbic acid). Treatments continued until 7 d after cows received an infusion of 10 microg of LPS into one quarter of the mammary gland (on average, this occurred 32 d postcalving). Supplementation of vitamin C increased plasma concentrations of vitamin C at calving, but no differences were observed in samples taken 24 h postinfusion. Concentrations of vitamin C in milk (24 h postinfusion) and in neutrophils (calving and 24 h postinfusion) were not affected by treatment, but vitamin C concentrations in neutrophils isolated from milk were about 3 times greater than concentrations in blood neutrophils. The LPS infusion did not alter concentrations of vitamin C in plasma or milk, suggesting that the LPS model did not produce the same effects as a bacterial infection of the mammary gland with respect to antioxidant effects. Supplemental vitamin C had no effect on neutrophil phagocytosis or bacterial kill. Dietary vitamin C reduced the milk somatic cell count but did not affect the febrile response or milk production following LPS infusion.

Bacterial counts in organic materials used as free-stall bedding following treatment with a commercial conditioner.

An acidic conditioner was tested in recycled manure and sawdust used as free-stall bedding to determine the effect on bacterial counts of common environmental mastitis pathogens. Free stalls were bedded with 10 kg of either kiln-dried sawdust or recycled manure per stall. All bedding was removed and fresh bedding was applied every 7 d. Approximately 1 kg of commercial bedding conditioner containing 93% sodium hydrosulfate (wt/wt) was spread evenly over bedding in the back one-third of each treated stall. Control bedding received no treatment. Cows were bedded on the same material for 3 consecutive weeks. After 3 wk on a bedding treatment, cows remained in the same pens and bedding treatments were changed between rows within pens in a switchback design. The addition of bedding conditioner to sawdust reduced gram-negative bacterial, coliform, Klebsiella spp., and streptococcal counts immediately after application and 1 d after application. Bacterial counts did not differ between treated and untreated sawdust on d 2 and 6 after the conditioner treatment. Each bacterial count measured was reduced in recycled manure immediately after application. Gram-negative bacterial and streptococcal counts were reduced in treated recycled manure compared with untreated recycled manure on d 1 after conditioner was applied. Bacterial counts did not differ between treated and untreated recycled manure bedding on d 2 and 6 after application in free stalls. The antibacterial activity of the conditioner was related to the pH of the bedding and was diminished by d 2 after application in both bedding types.

Effects of prepartum intramammary antibiotic therapy on udder health, milk production, and reproductive performance in dairy heifers.

Preparturient heifers (n = 561) from 9 herds in 6 US states and 1 Canadian province were enrolled in a study to test the hypothesis that prepartum intramammary therapy would cure existing intramammary infections (IMI) and lead to increased milk production, reduced linear somatic cell count (LSCC), and improved reproductive performance. Mammary secretions were collected 10 to 21 d before expected calving from each quarter. Heifers were then assigned by identification number to receive intramammary therapy consisting of infusion of one tube per mammary quarter of a lactating cow commercial antibiotic preparation containing cephapirin or to a nontreated control group. Overall, 34.1% of mammary quarters were infected with a mastitis pathogen before parturition and 63.4% of heifers had at least one mammary quarter infected. The coagulase-negative staphylococci (CNS) caused the majority (74.8%) of prepartum IMI. Coagulase-positive staphylococci, environmental streptococci, and coliforms accounted for 24.5% of prepartum infections. Treatment had a significant effect on the cure rate of infected mammary quarters. Mammary quarters that were infected prepartum and treated with antibiotics had a 59.5% efficacy of cure rate and the percentage reduction in heifers with IMI was 51.9. Control quarters had a spontaneous cure rate of 31.7%. Treatment did not significantly affect milk production or LSCC in the first 200 d of lactation; however, there was a significant treatment by herd interaction for milk production. Quarters cured of either CNS or major pathogens had a lower LSCC in the first 200 d of lactation. No significant effect on services per conception or days open between treatment and control groups was observed. This trial demonstrated that prepartum intramammary antibiotic therapy did reduce the number of heifer IMI postpartum. Milk production, LSCC, and reproductive performance during the first 200 d of the first lactation were not significantly affected by treatment. Given these results, use of prepartum intramammary antibiotic therapy in heifers as a universal strategy to increase milk production in first-lactation dairy cows may not be warranted.