BACKGROUND: Acinetobacter lwoffii (A. lwoffii) is a Gram-negative bacteria common in the environment, and it is the normal flora in human respiratory and digestive tracts. The bacteria is a zoonotic and opportunistic pathogen that causes various infections, including nosocomial infections. The aim of this study was to identify A. lwoffii strains isolated from bovine milk with subclinical mastitis in China and get a better understanding of its antimicrobial susceptibility and resistance profile. This is the first study to analyze the drug resistance spectrum and corresponding mechanisms of A. lwoffii isolated in raw milk.
RESULTS: Four A. lwoffii strains were isolated by PCR method. Genetic evolution analysis using the neighbor-joining method showed that the four strains had a high homology with Acinetobacter lwoffii. The strains were resistant to several antibiotics and carried 17 drug-resistance genes across them. Specifically, among 23 antibiotics, the strains were completely susceptible to 6 antibiotics, including doxycycline, erythromycin, polymyxin, clindamycin, imipenem, and meropenem. In addition, the strains showed variable resistance patterns. A total of 17 resistance genes, including plasmid-mediated resistance genes, were detected across the four strains. These genes mediated resistance to 5 classes of antimicrobials, including beta-lactam, aminoglycosides, fluoroquinolones, tetracycline, sulfonamides, and chloramphenicol.
CONCLUSIONS: These findings indicated that multi-drug resistant Acinetobacter lwoffii strains exist in raw milk of bovine with subclinical mastitis. Acinetobacter lwoffii are widespread in natural environmental samples, including water, soil, bathtub, soap box, skin, pharynx, conjunctiva, saliva, gastrointestinal tract, and vaginal secretions. The strains carry resistance genes in mobile genetic elements to enhance the spread of these genes. Therefore, more attention should be paid to epidemiological surveillance and drug resistant A. lwoffii.

Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the Staphylococcus aureus isolates recovered from subclinical mastitis animals in Pothohar region of the country. A total of 278 milk samples from 17 different dairy farms around two districts of the Pothohar region, Islamabad and Rawalpindi, were collected and screened for sub clinical mastitis using California Mastitis Test. Positive milk samples were processed for isolation of Staphylococcus aureus using mannitol salt agar. The recovered isolates were analyzed for their antimicrobial susceptibility and virulence genes using disc diffusion and PCR respectively. 62.2% samples were positive for subclinical mastitis and in total 70 Staphylococcus aureus isolates were recovered. 21% of these isolates were determined to be methicillin resistant, carrying the mecA gene. S. aureus isolates recovered during the study were resistant to all first line therapeutic antibiotics and in total 52% isolates were multidrug resistant. SCCmec typing revealed MRSA SCCmec types IV and V, indicating potential community-acquired MRSA (CA-MRSA) transmission. Virulence profiling revealed high prevalence of key genes associated with adhesion, toxin production, and immune evasion, such as hla, hlb, clfA, clfB and cap5. Furthermore, the Panton-Valentine leukocidin (PVL) toxin, that is often associated with recurrent skin and soft tissue infections, was present in 5.7% of isolates. In conclusion, the increased prevalence of MRSA in bovine mastitis is highlighted by this study, which also reveals a variety of virulence factors in S. aureus and emphasizes the significance of appropriate antibiotic therapy in combating this economically burdensome disease.

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

The impact of beta-carotene on cattle fertility has been investigated in various studies; however, consensus on this issue has not been reached. In the present study, we systematically reviewed and meta-analysed 29 publications conducted between 1984 and 2022, focusing on seven fertility measures, clinical mastitis and milk yield in cows. We did not find statistically significant results in 8 out of 11 parameters (p > .05). Statistically significant results were observed for milk yield (MD: 216.25 kg in 305 days, p = .01, CI: 50.73-381.77), pregnancy at first service (OR: 1.38 CI: 1.08-1.76, p = .01) and clinical mastitis (OR: 0.59, CI: 0.44-0.80, p = .006) in favour of beta-carotene supplementation. The meta-regression revealed significant effects of \'plasma beta-carotene levels\' on \'service to per pregnancy\' and dose on \'milk yield\' (p = .04 and p = 0). In binary outcomes, \'dose × day\' and \'plasma beta-carotene concentration in the control group\' positively influenced pregnancy at first service (p = .02 and .03). In conclusion, given the positive point direction observed for some variables and insignificant results for others, there is a need for more studies. We note the very high heterogeneity of outcomes and suggest caution in interpreting results.

Biosecurity plays a crucial role in preventing the introduction of infectious diseases to a herd as well as the spread of diseases within or between animals and herds. In particular, biosecurity measures are crucial for maintaining animal health and reducing the need for the application of antibiotic substances for fighting the rising antibiotic resistance. The object of this cross-sectional study was to investigate the presence of biosecurity measures and their association with milk quality parameters, with a special focus on somatic cell count (SCC) - an indicator for udder health -, in small-scale mountain dairy farms. Therefore, for the very first time, the CLASSYFARM system was considered, which is a computer platform integrated into the Italian national veterinary portal, that processes a significant amount of data from various sources collected in the field or from other information systems (e.g. animal welfare, health status, biosecurity, antimicrobial use, slaughterhouse information). A total of 169 dairy farms were included in the study. Biosecurity measures, based on 15 questions required in the CLASSYFARM welfare assessment protocol, as well as information about husbandry systems, milking systems and pasture practices were gathered and combined with milk yield data, provided by the South Tyrolean dairy association. Farms only scored 44.00 points on average in a scoring system from 0.00 to 100.00 points that was be able to summarize 15 different biosecurity measures in one index. Our results show a clear negative correlation (-0.713) between the biosecurity index and somatic cell score (SCS) indicating that a higher level of biosecurity, which reflects the presence of biosecurity measures within a farm, is associated with lower SCC levels. Furthermore, we found significant correlations between SCS and milk production (-0.629), confirming that udder health is linked to higher milk production. Fat, protein, and the fat-to-protein ratio showed a positive correlation with SCS (0.281, 0.146, 0.106), likely to be caused by a concentration shift effect (dilution effect). Husbandry system, breed, milking system, and pasture practices seem to have an impact as well, but the main factor was the biosecurity score. This study highlights the importance of implementing biosecurity measures for ensuring animal health and thus productivity and quality in milk production, even in small-scale farms, which are characterized by limited structure availability and smaller herds compared to big dairy enterprises in the lowlands.

BACKGROUND: Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows\' udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available.
RESULTS: Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens.
CONCLUSIONS: The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable - especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment.

Staphylococcus haemolyticus is a cause of bovine mastitis, leading to inflammation in the mammary gland. This bacterial infection adversely affects animal health, reducing milk quality and yield. Its emergence has been widely reported, representing a significant economic loss for dairy farms. Interestingly, S. haemolyticus exhibits higher levels of antimicrobial resistance than other coagulase-negative Staphylococci. In this study, we synthesized silver/silver chloride nanoparticles (Ag/AgCl-NPs) using Solanum lasiocarpum root extract and evaluated their antibacterial and antibiofilm activities against S. haemolyticus. The formation of the Ag/AgCl-NPs was confirmed using UV-visible spectroscopy, which revealed maximum absorption at 419 nm. X-ray diffraction (XRD) analysis demonstrated the crystalline nature of the Ag/AgCl-NPs, exhibiting a face-centered cubic lattice. Fourier transform infrared (FT-IR) spectroscopy elucidated the functional groups potentially involved in the Ag/AgCl-NPs synthesis. Transmission electron microscopy (TEM) analysis revealed that the average particle size of the Ag/AgCl-NPs was 10 nm. Antimicrobial activity results indicated that the minimum inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) of the Ag/AgCl-NPs treatment were 7.82-15.63 μg/mL towards S. haemolyticus. Morphological changes in bacterial cells treated with the Ag/AgCl-NPs were observed under scanning electron microscopy (SEM). The Ag/AgCl-NPs reduced both the biomass of biofilm formation and preformed biofilm by approximately 20.24-94.66 % and 13.67-88.48 %. Bacterial viability within biofilm formation and preformed biofilm was reduced by approximately 21.56-77.54 % and 18.9-71.48 %, respectively. This study provides evidence of the potential of the synthesized Ag/AgCl-NPs as an antibacterial and antibiofilm agent against S. haemolyticus.

Bacillus licheniformis is a gram-positive, endospore-forming, saprophytic and facultative anaerobe that is resistant to heat and environmental conditions. This study was the first to isolate and confirm B. licheniformis as a cause of bovine mastitis in Iran. In the summer of 2020, 105 samples of mastitic milk were collected from dairy farms around Tehran and sent to the microbiology laboratory of the Faculty of Veterinary Medicine at the University of Tehran. The bacterial pathogens were identified using selective and differential culture media and confirmed by PCR to contain the toxin synthetase genes licA, licB and licC in mastitic isolates of B. licheniformis. Resistance patterns to 19 antibiotics were determined for two isolates of B. licheniformis. Staphylococcus aureus and Escherichia coli were identified as the most important organisms in the samples. B. licheniformis was isolated from the two samples containing all three genes. Both isolates were resistant to streptomycin, trimethoprim-sulfamethoxazole, cefixime, ampicillin, bacitracin, clindamycin, and gentamicin. B. licheniformis was reported for the first time in Iran as a cause of bovine mastitis with clinical symptoms. The first isolation of toxin-producing strains of B. licheniformis from mastitic cows in Iran raises concerns about the safety of dairy products. In principle, selected strains with toxigenic potential should not be used as feed additives and animal feed. However, whole genome sequencing is proposed to search for genes coding for toxins.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of \"phage tail fiber.\" In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

Retrospective evaluation of udder recovery following treatment of the inflamed quarter with acoustic pulse technology (APT) of cows with subclinical mastitis was done on 4 Israeli commercial dairy farms. Here, we evaluated the APT treatment as a tool to manage subclinical mastitis and its economic consequences in commercial farms. Recovery of the infected glands following APT treatment was compared to the customary no-treatment (NT) for cows with subclinical mastitis. Over 2 years, 467 cows with subclinical mastitis were identified. Subclinical mastitis was defined by elevated somatic cell count (SCC; >1 × 106 cells/mL) in the monthly test-day milk sample; 222 cows were treated with APT and 245 cows were not treated and served as control. Differences between treatment groups in culling, milk quality, milk yield and bacterial elimination were analyzed. After treatment, cure from bacteria was calculated only for cows with pre-isolated bacteria. The percentage of sampled cows determined as cured (no bacterial finding) in the NT group was 32.7% (35/107) (30.9% Gram negative; 32.4% Gram positive) and in the APT-treated group, 83.9% (42/55) (89.4% Gram negative; 80.6% Gram positive). Culling rate due to mastitis was significantly lower (>90%) in the APT-treated vs. NT group. Recovery was 66.0% in the APT group compared to 11.5% in the NT group at 90 d post-treatment. Average milk volume per cow in the APT-treated group was 16.1% higher compared to NT cows. Based on the study, savings incurred by using APT to treat only subclinical cows per 100-cow herd can total $15,106/y, or $309 per treated subclinically infected cow.