Bovine milk-derived cells express transcriptome markers of pluripotency and secrete bioactive factors with regenerative and antimicrobial activity.

The bovine mammary stem/progenitor cell secretome stimulates regeneration in vitro and contains proteins associated with antimicrobial defense. This has led to the exploration of the secretome as a biologic treatment for mastitis, a costly inflammation of the udder commonly caused by bacteria. This study reports on a population of bovine mammary stem/progenitor cells isolated non-invasively from milk (MiDCs). MiDCs were characterized by immunophenotyping, mammosphere formation assays, and single cell RNA sequencing. They displayed epithelial morphology, exhibited markers of mammary stem/progenitor cells, and formed mammospheres, like mammary gland tissue-isolated stem/progenitor cells. Single cell RNA sequencing revealed two sub-populations of MiDCs: epithelial cells and macrophages. Functionally, the MiDC secretome increased fibroblast migration, promoted angiogenesis of endothelial cells, and inhibited the growth of mastitis-associated bacteria, including antibiotic-resistant strains, in vitro. These qualities of MiDCs render them a source of stem cells and stem cell products that may be used to treat diseases affecting the dairy industry, including mastitis.

Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-ß-1,4-mannanase.

ß-Mannanases from the glycoside hydrolase 26 (GH26) family are retaining hydrolases that are active on complex heteromannans and whose genes are abundant in rumen metagenomes and metatranscriptomes. These enzymes can exhibit distinct modes of substrate recognition and are often fused to carbohydrate-binding modules (CBMs), resulting in a molecular puzzle of mechanisms governing substrate preference and mode of action that has not yet been pieced together. In this study, we recovered a novel GH26 enzyme with a CBM35 module linked to its N terminus (CrMan26) from a cattle rumen metatranscriptome. CrMan26 exhibited a preference for galactomannan as substrate and the crystal structure of the full-length protein at 1.85 Å resolution revealed a unique orientation of the ancillary domain relative to the catalytic interface, strategically positioning a surface aromatic cluster of the ancillary domain as an extension of the substrate-binding cleft, contributing to galactomannan preference. Moreover, systematic investigation of nonconserved residues in the catalytic interface unveiled that residues Tyr195 (-3 subsite) and Trp234 (-5 subsite) from distal negative subsites have a key role in galactomannan preference. These results indicate a novel and complex mechanism for substrate recognition involving spatially remote motifs, distal negative subsites from the catalytic domain, and a surface-associated aromatic cluster from the ancillary domain. These findings expand our molecular understanding of the mechanisms of substrate binding and recognition in the GH26 family and shed light on how some CBMs and their respective orientation can contribute to substrate preference.

Effects of supplement type and narasin inclusion on supplement intake by Bos indicus beef bulls grazing a warm-season forage.

This study aimed to evaluate the effects of supplement type and narasin inclusion on the frequency and supplement intake of grazing Bos indicus beef bulls. Four hundred animals were ranked by initial BW (383 ± 35 kg) and allocated into one of four paddocks of Brachiaria brizantha cv. Marandú (100 animals/paddock). Paddocks were randomly assigned to receive either a mineral salt (MIN) or a protein-energetic supplement (PREN) containing or not narasin (N) for a 90-d period. An individual electronic data capture system with 11 feed bunks was used to individually measure supplement intake and meal frequency in each paddock. The evaluations and analysis of individual intake, frequency of visits to the feeder, and intake per visit (I/V) were performed every 15 d and classified as periods (PR1 through PR6). All data were analyzed as a 2 × 2 factorial design with the PROC MIXED procedure of SAS. A supplement type × N × PR interaction was observed (P < 0.0001) for daily supplement intake. No differences were observed between MIN, whereas PREN had a greater (P ≤ 0.03) supplement intake on PR1 and PR3, but a reduced supplement intake on PR6 compared with PREN + N (P = 0.02). Moreover, no supplement type × N interaction (P = 0.47) or N (P = 0.44) effects were observed for daily supplement intake in the present study. A supplement type × N × PR interaction was detected (P < 0.0001) for the frequency of visits in the feeders. Throughout the experimental period, animals from the MIN + N had a greater (P ≤ 0.02) frequency of visits compared with MIN cohorts. A supplement effect was detected for I/V (P = 0.02), whereas neither a narasin effect (P = 0.74) nor interactions (P ≥ 0.16) were observed. Animals offered PREN had a greater I/V when compared with MIN cohorts (145 vs. 846 g/d for MIN and PREN, respectively; SEM = 16.1). When these data are reported as percentage of days visiting the feeder within each PR, MIN and MIN + N animals visited the feeder for 25.8% and 35.9% of the days, respectively. Conversely, no differences were observed (P = 0.65) in the overall mean visits per PR between PREN and PREN + N (12.8 vs. 12.3 d for PREN and PREN + N, respectively; SEM = 0.195). As percentage of days visiting the feeder, PREN and PREN + N visited the feeder for 85.1% and 81.9% of the days, respectively. In summary, narasin inclusion did not reduce supplement intake, regardless of supplement type, but increased the frequency of visits to the feeder for the MIN treatment.

Minimum inhibitory concentrations of cephalosporin compounds and their active metabolites for selected mastitis pathogens.

OBJECTIVE: To compare the minimum inhibitory concentration (MIC) of cephapirin and ceftiofur with MICs of their active metabolites (desacetylcephapirin and desfuroylceftiofur) for selected mastitis pathogens. SAMPLE: 488 mastitis pathogen isolates from clinically and subclinically affected cows in commercial dairy herds in Wisconsin. PROCEDURES: Agar dilution was used to determine MICs for Staphylococcus aureus (n = 98), coagulase-negative staphylococci (99), Streptococcus dysgalactiae (97), Streptococcus uberis (96), and Escherichia coli (98). RESULTS: All S aureus isolates were susceptible to cephapirin and ceftiofur. Most coagulase-negative staphylococci were susceptible to cephapirin and ceftiofur. For E coli, 50 (51.0%; cephapirin) and 93 (94.95%; ceftiofur) isolates were susceptible to the parent compounds, but 88 (89.8%) were not inhibited at the maximum concentration of desacetylcephapirin. All S dysgalactiae isolates were susceptible to ceftiofur and cephapirin, and consistent MICs were obtained for all compounds. Most S uberis isolates were susceptible to cephapirin and ceftiofur. Of 98 S aureus isolates classified as susceptible to ceftiofur, 42 (42.9%) and 51 (52%) were categorized as intermediate or resistant to desfuroylceftiofur, respectively. For 99 coagulase-negative staphylococci classified as susceptible to ceftiofur, 45 (45.5%) and 17 (17.2%) isolates were categorized as intermediate or resistant to desfuroylceftiofur, respectively. For all staphylococci and streptococci, 100% agreement in cross-classified susceptibility outcomes was detected between cephapirin and desacetylcephapirin. No E coli isolates were classified as susceptible to desacetylcephapirin. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in inhibition between parent compounds and their active metabolites may be responsible for some of the variation between clinical outcomes and results of in vitro susceptibility tests.

Enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus recovered from milk of cows with subclinical mastitis.

OBJECTIVE: To evaluate enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus in milk obtained from cows with subclinical mastitis. SAMPLE: Milk samples obtained from 350 cows (1,354 mammary glands) on 11 Wisconsin dairy farms. PROCEDURES: Of 252 S aureus isolates obtained from 146 cows, 83 isolates (from 66 cows with subclinical mastitis) were compared genotypically by use of pulsed-field gel electrophoresis and via PCR identification of toxic shock syndrome toxin 1 (TSST-1) and classical S aureus enterotoxin genes (sea, seb, sec, sed, and see). RESULTS: Among the 83 S aureus isolates, ≥ 1 enterotoxin genes were identified in 8 (9.6%). Enterotoxin gene distribution was as follows: TSST-1, 7 isolates (8.4%); sec, 5 isolates (6.0%); and sed, 2 isolates (2.4%). Enterotoxin genes sea, seb, and see were not identified. Twelve pulsotypes and 5 subtypes were identified among the 83 isolates; 5 of the 12 pulsotypes were represented by only 1 isolate. In cows of 1 herd, only a single S aureus pulsotype was detected; in cows on most other farms, a variety of pulsotypes were identified. One pulsotype was recovered from 4 farms (n = 23 cows) and another from 5 other farms (16). Isolates with an enterotoxin gene were represented by 6 pulsotypes. CONCLUSIONS AND CLINICAL RELEVANCE: S aureus classical enterotoxins and TSST-1 were rarely recovered from milk samples obtained from cows with subclinical mastitis in Wisconsin. Diverse pulsotypes of S aureus were detected within and among farms, indicating that different strains of S aureus cause subclinical mastitis in dairy cows.

Relationship between results of in vitro susceptibility tests and outcomes following treatment with pirlimycin hydrochloride in cows with subclinical mastitis associated with gram-positive pathogens.

OBJECTIVE: To determine the association between results of in vitro antimicrobial susceptibility tests and outcomes in cows that received intramammary treatment with pirlimycin hydrochloride for subclinical mastitis associated with gram-positive pathogens. DESIGN: Case-control study. ANIMALS: 132 dairy cows (178 mammary glands with subclinical mastitis caused by 194 pathogen isolates). PROCEDURES: Cows with positive results for a California mastitis test (CMT) were assigned to receive 50 mg of pirlimycin via intramammary administration into each CMT-positive mammary gland every 24 hours for 2 consecutive days or no treatment. Duplicate milk samples were collected before treatment and approximately 21 days later. Target pathogens included coagulase-negative Staphylococcus spp (n = 118 isolates), Streptococcus spp (28), Staphylococcus aureus (7), and other gram-positive cocci (30). Antimicrobial susceptibilities were determined via broth microdilution. RESULTS: Overall treatment success rate was 66% (128/194) for both groups. In vitro resistance to pirlimycin ranged from 0% (0/7 isolates of S aureus) to 50% (13/26 isolates of other gram-positive cocci). For the treated group, 62 of 94 (66%) target pathogens were classified as treatment successes and 32 (34%) were classified as failures. Similarly for the control group, 66 of 100 (66%) target pathogens were classified as treatment successes, whereas 34 (34%) were classified as failures. CONCLUSIONS AND CLINICAL RELEVANCE: Many target pathogens from cows with subclinical mastitis were eliminated without treatment, and treatment with pirlimycin did not improve the treatment success rate. Results of in vitro antimicrobial susceptibility tests were not useful as predictors of treatment success following intramammary treatment with pirlimycin.