This study investigated the presence, distribution, and antimicrobial resistance profiles of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in a dairy herd located in Northern Italy. The feces of clinically healthy calves, their mothers, and the cows treated for mastitis, as well as water, environmental samples, and waste milk were collected and subjected to bacteriological culture on CHROMagar^TM ESBL plates. A questionnaire was administered to identify risk factors. The isolates were identified as E. coli by MALDI-TOF MS and subjected to the double-disk synergy test (DDST) and minimal inhibitory concentration (MIC) assay. As a result, ESBL E. coli was isolated from the feces of 28 of 37 (75.67%) calves, the feces of 2 of 3 (66.67%) treated cows, 8 of 14 (57.15%) environmental samples, and waste milk. All ESBL isolates showed multiple resistances and were categorized as multidrug-resistant (MDR). Several risk factors for ESBL E. coli selection and diffusion were identified, including lack of routine cleaning of calf feeding and housing equipment, administration of waste milk to male calves, and blanket dry cow therapy. In conclusion, this study highlighted the presence of MDR, ESBL E. coli in the feces of most dairy calves, and their association with different sample sources. Accordingly, adding to the prudent use of antibiotics, the adoption of adequate farm hygiene and biosecurity measures might also help prevent the spread and transmission of ESBL E. coli within the herd.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR. Moderate numbers (103 CFU ml−1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment. The use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.

S. aureus is the most frequently isolated mastitis pathogen in southern Chile. Hygiene during milking time and DCT have not been successful to control this pathogen. Vaccination has been suggested as a useful tool to combat mastitis and several vaccines have been developed and evaluated worldwide. This study presents the results of a pilot trial to evaluate the effects of a new vaccine produced in Chile against S. aureus mastitis on udder health, humoral immune response, and milk production after an experimental intramammary challenge with a heterologous S. aureus strain. Four 2 mL doses of the vaccine were administered intramuscularly to five pregnant heifers free of IMI infections, using another five heifers as controls. Ten days after last immunisation two mammary quarters of all heifers were challenged with a pathogenic strain of S. aureus. Heifers were monitored for clinical signs, bacterial count, SCC, differential leukocyte count in blood, S. aureus antibody, and milk yield during 14 days after challenge. No systemic effects were observed in any of the challenged heifers, and severe clinical cases were only observed in the control group. The SCCs were always higher in heifers of the control group. The challenged quarters of the control group yielded the highest counts of S. aureus, but no significant difference was observed between vaccinated and non-vaccinated groups. Serum antibody titres were twice as high in the vaccinated group compared to the control group, and average milk yield reduction was lower in the vaccinated group. In conclusion, this vaccine was able to stimulate the humoral immune response of vaccinated heifers which could have a beneficial effect against new IMI by S. aureus and help to combat this mastitis pathogen.

Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM.

Waste milk (WM), a byproduct of dairy production, is often used as a cost-effective feed for calves, but it can contain pathogens and antimicrobial residues, which pose health risks. This study examined the microbiological quality and the presence of antimicrobial residues in WM from 36 dairy farms in southern Chile. In a cross-sectional study, WM samples were collected, and farm management data were gathered through a questionnaire. The samples were analyzed for total bacterial load, coliforms, staphylococci, streptococci/streptococci-like organisms (SSLOs), Salmonella spp., Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium bovis, Mycoplasma spp., Prototheca spp., methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum beta-lactamase (ESBL)-producing E. coli. Antimicrobial residues were detected using a commercial test. A high average bacterial load (7.63 × 10⁶ CFU/mL) and significant levels of staphylococci, SSLOs, and coliform were found. In addition, S. aureus (33.3%), MAP (11.1%), and Salmonella spp. (2.8%) were detected. ESBL-E. coli was found on five farms, with bla_CTX-M being the predominant gene. Antimicrobial residues, mainly beta-lactams, were present in 55.6% of samples. These results show that WM in this region frequently contains pathogens, antimicrobial residues, and resistant bacteria. Current farm practices, such as feeding untreated WM to calves, may contribute to the spread of antimicrobial resistance and compromise calf health.