Analysis of somatic cell count, bacteria distribution and antimicrobial susceptibility in lactating dairy cows from small holder dairy farms in Kenya.

Metalloporphyrin-based photodynamic inactivation of multidrug-resistant environmental mastitis pathogens.

Development of a sheep model of mastitis caused by Mycoplasma bovis.

Streptococcus uberis is currently the most notableemerging mastitis pathogen in South Africa. Multilocus sequence typing (MLST) was used to investigate the sequence types (STs) of S. uberis isolated from bovine milk and their epidemiological patterns of occurrence. This retrospective, longitudinal study was conducted on a pasture-based herd of 1005 lactating cows, on which slurry-spreading had been recently introduced. Composite cow milk samples were collected quarterly during routine whole herd sampling and from clinical mastitis cases (monthly) during 2021. Streptococcus uberis isolates obtained from two routine samplings and clinical mastitis cases were stored at -80°C. In 2024, seven S. uberis isolates were added; these were from the same cows in consecutive samplings. The prevalence of S. uberis intramammary infection (IMI) was 7.44%, while 21.26% of clinical mastitis cases were caused by S. uberis. Based on conventional microbiology, 31.4% of S. uberis IMIs were recurring in consecutive samplings. A total of 42 S. uberis STs were identified from 70 isolates; 41 were novel and only 1 (ST 1613) had been previously reported in the PubMLST/GenBank database. Of the S. uberis isolates examined, 35.7% had known clonal complexes (CCs); of these, 60% were CC ST-5. Owing to the high heterogeneity, no predominant STs were observed; ST 1613 was isolated six times but did not cause clinical cases. When S. uberis was isolated from a cow more than once, only 50% of the isolates had similar STs. Where cows had multiple infections in an udder, quarters infected had different STs. In summary, this herd showed significant heterogeneity in S. uberis, with all but one ST being novel variants. Results indicate that S. uberis IMI in this herd was transient, possibly of environmental origin rather than persistent udder infections, making a point-source of infection less likely.

Antimicrobial resistance remains a major global challenge and has increased interest in plant-derived compounds as alternative or complementary therapeutic agents. This study evaluated the in vitro antibacterial activity of ethanol-extracted Piper betle L. or betel leaf (EPB), Bauhinia scandens L. stem (EBS), and Chromolaena odorata leaf (ECO) against common mastitis-associated pathogens. The three herbal samples were prepared through ethanol extraction followed by freeze-drying. Antibacterial activity was assessed using disc diffusion assays at two extract concentrations. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined through broth microdilution. Statistical analyses were performed to compare the inhibitory and bactericidal performance of the extracts. The extracts showed distinct antibacterial profiles. EPB and EBS produced the strongest inhibition zones against Staphylococcus aureus (S. aureus), while ECO displayed weaker activity. In agreement with diffusion results, EPB and EBS had the lowest MIC and MBC values for S. aureus, and both showed bactericidal MBC/MIC ratios. Activity against Escherichia coli (E. coli) was limited; measurable inhibition was observed only for EPB, and all extracts required substantially higher MIC and MBC concentrations compared with S. aureus. None of the extracts demonstrated meaningful activity against Pseudomonas aeruginosa (P. aeruginosa). Across assays, inhibition zone size showed a negative correlation with MIC values, indicating consistency between diffusion and broth-based methods. EPB and EBS demonstrated promising antibacterial activity against S. aureus, suggesting potential use as plant-based candidates for controlling Gram-positive mastitis pathogens. Their limited effects on Gram-negative bacteria (E. coli and P. aeruginosa) indicate they may be best suited for targeted rather than broad-spectrum applications. Further phytochemical characterization and in vivo studies are warranted to evaluate their therapeutic potential in livestock health management.

Bovine mastitis causes substantial economic losses worldwide. Acinetobacter baumannii has emerged as a pathogen in bovine mastitis, raising considerable concerns due to its multidrug resistance and potential zoonotic transmission. However, characteristics of A. baumannii involved in bovine mastitis in China remain poorly understood, particularly with emergence of novel sequence types. From 135 Acinetobacter spp. isolates isolated in China from bovine mastitis (2021-2024), we identified a novel sequence type, ST3475. Using the known bovine strain ST3256 for comparison, we conducted hybrid whole-genome sequencing, antimicrobial susceptibility testing, and multiple phenotypic pathogenicity assays (including biofilm formation, cell adhesion/invasion, and in vivo infection models). The A. baumannii ST3475 chromosome is 3,875,774 bp with 1 plasmid (158,806 bp); it has an expanded repertoire of 70 virulence genes, including csu and bap, and 18 antimicrobial resistance genes. Furthermore, its genome contains a complement of phage elements, carbohydrate-active enzymes, and a unique carbapenemase gene bla OXA-344, indicating distinct evolution. Phenotypically, ST3475 demonstrated enhanced biofilm formation, adhesion, invasion, and cytotoxicity against bovine mammary epithelial cells. It also promoted secretion of IL-1β, IL-6, and TNF-α. In Galleria mellonella, the median lethal dose of ST3475 was 2 orders of magnitude lower than ST3256 (LD50=1,378,077 CFU/larva). In a murine mammary infection model, ST3475 caused substantial alveolar destruction and inflammatory infiltration. Phylogenetically, ST3475 belongs to clonal complex CC2 but represents a distinct, putative bovine-adapted lineage separate from major human clone CC1. Although currently susceptible to last-resort antimicrobials like carbapenems, carriage of high-risk resistance genes highlights livestock as a potential reservoir for clinically relevant resistance determinants. This study reveals ST3475 as a novel, virulent, and putative bovine-adapted lineage, underscoring the importance of One Health surveillance.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic and environmental mastitis pathogen prevalent in dairy herds worldwide. Owing to their genetic and genomic diversity, K. pneumoniae strains associated with bovine mastitis exhibit significant variation in virulence. Certain types of mastitis-causing K. pneumoniae strains exhibit enhanced pathogenicity and mammary adaptability, posing a serious threat to global public health. Bovine mastitis-causing K. pneumoniae strains can cause ultrastructural damage to bovine mammary epithelial cells (bMECs), leading to inflammatory injury, oxidative damage, apoptosis, pyroptosis, and immune evasion in bMECs. In this review, we summarize the prevalence, virulence genes, and pathogenic mechanisms of K. pneumoniae strains related to bovine mastitis. Given the increasing multidrug resistance of K. pneumoniae, we also outline the methods and mechanisms of phage therapy for K. pneumoniae infections, as well as future directions for treatment and prevention. These findings contribute to a deeper understanding of the population structure of mastitis-associated K. pneumoniae and provide valuable insights for future research on pathogenic mechanisms, vaccine development, and control strategies.

Streptococcus agalactiae, a major bovine mastitis pathogen, poses significant economic and antimicrobial resistance (AMR) challenges. This study evaluated AMR in 128 isolates from Shandong, Hebei and Inner Mongolia using the broth microdilution method. Results showed high sensitivity to most antibiotics (e.g. 100% resistance to penicillin, ceftiofur, amoxicillin, cefquinome and vancomycin) but significant resistance to tetracycline (80.7%) and daptomycin (99.3%). Inner Mongolia isolates exhibited higher resistance and mimimum inhibitory concentrations (MIC) values, reflecting regional antibiotic usage differences, guiding mastitis treatment and antibiotic stewardship in China's dairy industry.

Mastitis is one of the most economically significant diseases affecting dairy cattle worldwide, with its subclinical form causing greater production losses than the clinical form. Sphingomonas paucimobilis, a Gram-negative, yellow-pigmented, aerobic bacillus, has recently been recognized as an emerging environmental pathogen in cases of mastitis. This study aimed to isolate S. paucimobilis from milk samples of cows with mastitis in the İzmir and Aydın regions, determine its antimicrobial susceptibility, and perform genotyping. A total of 100 milk samples were collected from cows with mastitis (50 clinical and 50 subclinical cases). Conventional bacteriological methods, VITEK® Compact 2, and PCR were used for identification. Eight (8%) S. Paucimobilis isolates were obtained, including six from subclinical mastitis samples and two from clinical mastitis samples. Antimicrobial susceptibility testing by the Kirby-Bauer disk diffusion method revealed high resistance rates to amoxicillin-clavulanic acid (87.5%), ampicillin-sulbactam (100%), and cefquinome (100%), while all isolates were susceptible to cefoperazone and gentamicin. Genotyping using the (GTG)₅-PCR method showed that all isolates had distinct patterns, with genetic similarity ranging from 32% to 98%. The findings indicate that S. paucimobilis should be considered among potential environmental mastitis pathogens and highlight the need for advanced diagnostic methods and effective antimicrobial stewardship to prevent resistance development.

Mastitis in dairy cows, caused by pathogens such as Staphylococcus aureus, Escherichia coli, and Candida albicans, leads to substantial economic losses and contributes to antimicrobial resistance, emphasizing the need for natural alternatives. This study assessed the phytochemical composition, antioxidant activity, and in vitro antimicrobial effects of ethanol extracts from ten medicinal plants, including Populus balsamifera buds, Syzygium aromaticum, and Humulus lupulus, as well as two multi-component plant mixtures and commercial products against reference strains and field isolates of mastitis pathogens. Extracts exhibited total phenolic contents ranging from 2.5 to 43.0 mg GE/g, with Populus balsamifera and Syzygium aromaticum demonstrating the strongest DPPH radical scavenging activity (IC50 1.89-2.9 mg/g extract). Disc diffusion assays demonstrated broad-spectrum inhibition, particularly for Populus balsamifera (15.1-19.1 mm inhibition zones) and Humulus lupulus (9.0-18.4 mm) against key pathogens; phenolic and flavonoid contents positively correlated with antimicrobial activity (r = 0.63-0.99, p < 0.001). Multi-component mixtures provided consistent broad-spectrum effects (12.6-17.2 mm). These phenolic-rich plant extracts represent promising alternatives to reduce antibiotic use in dairy mastitis management.

Mastitis continues to be among the top 3 challenges for dairy production globally, resulting in substantial economic losses. Current pathogen-specific prevalence estimates are crucial for effective surveillance and disease control. The objective of this study was to estimate the true prevalence of herds positive for major contagious mastitis pathogens in Alberta, Canada, and to describe bulk tank SCC (BTSCC) patterns/dynamics. Using a serial cross-sectional study design, bulk tank milk (BTM) samples were collected from all active dairy producers in Alberta in December 2021 (n = 484), April (n = 486), and October 2022 (n = 484) and tested for Staphylococcus aureus, Streptococcus agalactiae and Mycoplasma bovis using a commercial multiplex qPCR kit. BTSCC data for 2021 and 2022 were obtained for each herd for each milk pick-up and production-weighted geometric mean BTSCC (PWgBTSCC) was calculated. True prevalence was estimated using Bayesian latent class models. Mixed-effect modified Poisson and linear regression models were used to assess associations among pathogen positivity, herd characteristics and PWgBTSCC. True prevalence of Staph. aureus-positive herds were 17.8, 11.0 and 13.8% in December, April and October, respectively, whereas for Strep. agalactiae, true prevalence was consistent at 0.6%. For M. bovis, true prevalence was 2.0, 4.7 and 3.6% in December, July and October, respectively. The prevalence of Staph. aureus-positive herds was higher (prevalence ratio [PR] = 1.31) among medium-sized herds (3,600-7,200 L/d milk delivered) compared with smaller herds (<3,600 L/d milk delivered). Hutterite-colony herds were more frequently Staph. aureus-positive at least once (PR = 1.23) compared with non-Hutterite herds. Non-Hutterite colony herds in central and southern regions were less frequently Staph. aureus-positive compared with the north region; however, this trend was opposite among Hutterite-colony herds. Annual PWgBTSCC for Alberta dairy herds from Nov 16, 2021 to Nov 15, 2022 was 154,000 cells/mL (median [Q1 - Q3]: 118,000 - 202,000). Herds with a medium (100,000 to 200,000 cells/mL) or high (>200,000 cells/mL) annual PWgBTSCC were more frequently Staph. aureus-positive (PR = 1.58, and 2.40, respectively) compared with < 100,000 cells/mL herds. Annual PWgBTSCC was elevated for herds testing positive for any major contagious mastitis pathogen at least once compared with consistently negative herds. Herds in the south region had an 11% lower annual PWgBTSCC compared with north-region herds. These findings provided updated prevalence estimates of major contagious mastitis pathogens and critical insights into BTSCC patterns across herd types and regions, supporting targeted within-herd diagnostic follow-up and evidence-based mastitis control strategies.

The spread of antimicrobial resistances is a global threat. This study provides a retrospective, explorative analysis of bovine mastitis samples from 2005 to 2023, regarding the antimicrobial resistance patterns of the corresponding pathogens. 41.8% of the tested samples were positive for mastitis pathogens. Regarding the occurrence of antimicrobial resistances, 18,623 tests were analysed in total, comprising the 13 most frequently identified bacterial pathogens. The highest percentage of total antimicrobial resistant samples was detected for Proteus spp. (55.2%), Bacillus spp. (44.3%) and Citrobacter freundii (40.8%), while the lowest percentage was found for Staphylococcus aureus (7.1%), Trueperella pyogenes (6.1%), and Streptococcus dysgalactiae (3.1%). Furthermore, S. uberis, S. dysgalactiae, and T. pyogenes showed an overall linear decrease of antimicrobial resistant samples from 2005 to 2023. Trends of increased resistances were also identified for individual pathogen-antimicrobial combinations. To date, long-term, large-scale studies like this have rarely been conducted, but they are of particular importance in identifying trends in the shift of antimicrobial resistances among mastitis pathogens. The results of such routine diagnostic data must be viewed with caution, as they cannot be readily extrapolated to the entire dairy population. Nevertheless, these findings emphasize the necessity of unambiguous pathogen identification, including susceptibility testing, prior to antimicrobial administration. Alternative therapy methods also become increasingly important, as well as proper farm management and hygiene to prevent infections from the beginning.

Bovine mastitis is an inflammatory disease of the mammary gland, largely caused by microbial infections and commonly managed through intramammary administration of antibiotics. However, the extensive and recurrent use of antibiotics has led to the emergence and spread of antibiotic-resistant pathogens, particularly multidrug-resistant (MDR) Staphylococcus aureus, posing significant veterinary and public health challenges. Herein, we investigated the potential of Commiphora swynnertonii resin-loaded nanoparticles as an antibiotic alternative. This study aimed to identify an effective nanocarrier platform for antimicrobial delivery of C. swynnertonii resin. To achieve this, different types of nanocarriers were explored: liposomes, alginate-based nanoparticles, chitosan-based nanoparticles (ChN), solid lipid nanoparticles (SLN), and nanostructured lipid carriers (NLC), each with or without hyaluronic acid-stearylamine conjugate (HAC). Antimicrobial activity was assessed against 13 MDR S. aureus strains isolated from mastitis cow patients. Based on minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) assays, the tested nanocarriers were ranked in ascending order of antimicrobial coverage as follows: liposomes (0% inhibitory and 0% bactericidal), ChN (15.4% inhibitory and 0% bactericidal), NLC (23% inhibitory and 7.6% bactericidal), SLN (69% inhibitory and 46.2% bactericidal), and alginate nanoparticles (100% inhibitory and 53% bactericidal). HAC-containing alginate nanoparticles achieved the strongest activity, with MIC 26-417µg/ mL and MBC 35-417µg/mL, followed by HAC-containing SLN with MIC 17-417µg/mL and MBC 278-417µg/mL. These findings highlight the potential of alginate-HAC nanoparticles as a promising platform for delivering C. swynnertonii resin constituents, offering a novel strategy to combat MDR mastitis pathogens through plant-based nanotherapeutics.

Bovine mastitis is one of the most economically significant diseases affecting dairy cattle, leading to substantial losses due to decreased milk yield, higher treatment costs, and lower milk quality. Iodine-based teat disinfectants are commonly used to prevent mastitis but have several limitations, such as milk residue contamination, potential skin irritation, environmental persistence, and concerns about antimicrobial resistance. Plant-derived antimicrobials have thus gained interest as potential alternatives. Clove leaf oil from Syzygium aromaticum (L.) Merr. & L.M. Perry contains eugenol and other bioactive compounds with strong antimicrobial activity. However, its use is limited by poor water solubility and physicochemical instability. This study aimed to develop and optimize a clove leaf oil nanoemulsion (CLON) using a self-nanoemulsifying drug delivery system (SNEDDS) and to evaluate its physicochemical properties and in vitro antimicrobial activity against pathogens associated with bovine mastitis. This study followed a three-phase sequential workflow. Phase I involved screening and optimizing the formulation of CLON using SNEDDS technology. Nine ratios of clove leaf oil, Polysorbate 80 (Tween 80), and Polyethylene glycol 400 (PEG 400) were initially tested, followed by optimization with an I-optimal mixture design. Phase II included physicochemical characterization and assessing the thermodynamic stability of the optimized nanoemulsion, such as measurements of transmittance, pH, emulsification time, droplet size, polydispersity index, zeta potential, and transmission electron microscopy morphology. Phase III examined the in vitro antimicrobial activity of CLON against Staphylococcus aureus, Escherichia coli, and Candida albicans through agar diffusion assays, determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and killing-time kinetics. Inhibition zone data were analyzed using analysis of variance, followed by Duncan's multiple range test. The optimized CLON formulation contained 14.29% clove leaf oil, 68.56% Tween 80, and 17.15% PEG 400. The nanoemulsion showed high optical clarity (93.36 ± 0.25%), physiological pH (7.13 ± 0.01), rapid emulsification (35.46 ± 0.08 s), nanoscale droplet size (25.19 ± 2.31 nm), low polydispersity index (0.30 ± 0.04), and strong negative zeta potential (-45.07 ± 8.15 mV), indicating excellent stability. CLON demonstrated significant concentration-dependent antimicrobial activity (p < 0.01) against all tested microorganisms. The MIC and MBC values were 12.5% and 50% (v/v), respectively, indicating bactericidal activity. In the killing-time assay, 25% CLON achieved >3 log10 colony-forming units/mL reduction within 60 min. The optimized CLON displayed favorable physicochemical properties, strong thermodynamic stability, and broad-spectrum in vitro antimicrobial activity against key bovine mastitis pathogens. These results indicate that CLON has potential as a plant-based alternative teat disinfectant to prevent mastitis. However, further in vivo studies and field trials are needed to verify its safety and practical use in dairy production systems.

Zoonotic tuberculosis (zTB), predominantly caused by Mycobacterium bovis, remains an underrecognized public health threat in many low- and middle-income countries. Although global estimates suggest that zTB accounts for approximately 1-1.5% of all human tuberculosis cases, limited molecular diagnostic capacity and underreporting likely obscure its true burden. In Southeast Asia, particularly Indonesia, increasing detection of bovine tuberculosis in dairy and beef production systems-combined with high rates of raw milk consumption, informal slaughtering practices, and weak intersectoral surveillance-may amplify the risk of human exposure. Co-infections in livestock, including mastitis and respiratory pathogens, further complicate clinical detection and may enhance bacterial shedding. This review synthesises global and national epidemiological patterns of zTB, describes major transmission pathways at the human-animal interface, and examines interactions between M. bovis infection and other livestock diseases. Critical gaps in diagnostics, surveillance integration, and food-safety regulation are identified. Strengthening One Health approaches through improved laboratory capacity, coordinated public-animal health systems, and enhanced risk mitigation along dairy and beef value chains is essential to reduce the burden of zTB in Indonesia.

Bovine mastitis is a leading cause of economic loss in dairy farming and is increasingly complicated by antimicrobial resistance (AMR), posing challenges to treatment and public health. This study aimed to investigate the prevalence, bacterial etiology, and AMR patterns of mastitis pathogens in dairy herds from the Banat region of Romania. A retrospective analysis was conducted on 420 dairy cows from five localities. Mastitis diagnosis involved clinical examination, indirect tests (California Mastitis Test (CMT), R-Mastitest), and bacteriological culture. Antimicrobial susceptibility was assessed using the VITEK® 2 system. Out of 420 cows, 120 (28.6%) were diagnosed with mastitis. The predominant pathogens were Staphylococcus aureus (33.3%) and Streptococcus agalactiae (22.5%). Most infections were monomicrobial (70%) and affected a single under quarter (77.5%). Beta-lactam resistance was widespread among both Gram-positive and Gram-negative isolates, particularly against penicillin and ampicillin. Multidrug-resistant (MDR) strains were identified in 33.3% of all isolates, with 100% of Gram-negative isolates exhibiting MDR profiles. The high prevalence of S. aureus and S. agalactiae, along with widespread beta-lactam resistance and frequent MDR phenotypes, highlights the urgent need for routine AMR surveillance and targeted antimicrobial therapy in bovine mastitis control programs.

Recurrent bovine mastitis is a global concern that causes substantial economic losses and is exacerbated by pathogen internalization into mammary epithelial cells, and the emergence of antimicrobial resistance. These challenges necessitate the development of alternative antimicrobial strategies with multimodal activity. In this study, the naturally occurring molecule octanoic acid (OA) was evaluated for its antimicrobial efficacy and multitargeted mode of action against mastitis-associated pathogens. OA exhibited rapid bactericidal activity within 1h and significantly reduced bacterial pathogenicity by attenuating toxin activity and inhibiting pathogen adhesion and internalization into epithelial cells. Transcriptomic analysis of Staphylococcus aureus revealed extensive OA-induced transcriptional alterations across multiple functional categories, including virulence regulation, stress response, metabolism, DNA replication and repair, membrane-associated functions, and transport systems, suggesting a broad cellular response to OA exposure. OA treatment also upregulated endogenous antimicrobial peptide (AMP) gene expression in MAC-T cells and did not induce detectable resistance even after 30 serial passages. Membrane perturbation was supported by molecular dynamics simulations and validated experimentally using DiBAC assays. In vivo toxicity assessment using Galleria mellonella demonstrated no observable toxicity up to 1000mM OA. In addition, quantum chemical, physicochemical, and ADME/Tox analyses provided predictive insights into the chemical stability, drug-likeness, and safety profile of OA. Collectively, these findings suggest that OA exerts a multifaceted antimicrobial effect and represents a promising candidate for the development of next-generation antimicrobials targeting recurrent and resistant infections. KEY POINTS: • Octanoic acid (OA) rapidly kills mastitis pathogens via multimodal mechanisms. • OA prevents adhesion and internalization and mitigates toxicity in vitro and in silico. • OA alters mRNA expression profiles, revealing key antimicrobial pathways.

Bovine mastitis is a major bioeconomic and animal health challenge in dairy systems and is traditionally managed with intensive antibiotic therapy, contributing to antimicrobial resistance (AMR). This study explored the therapeutic potential of essential oils (EOs) from two native species of the Valdivian temperate rainforest, Laureliopsis philippiana (Tepa; LP_EO) and Drimys winteri (Canelo; DW_EO), against priority mastitis pathogens. Gas Chromatography-Mass Spectrometry (GC-MS) was used to characterize EO composition, and in vitro antibacterial and antifungal activities were evaluated against clinical isolates of Staphylococcus aureus, Streptococcus uberis and the azole-resistant yeast Pichia kudriavzevii by disk diffusion and broth microdilution assays. Both EOs were dominated by monoterpenes; LP_EO was richer in oxygenated monoterpenes (eucalyptol, terpinen-4-ol), whereas DW_EO showed a pinene-rich profile (β-pinene, α-pinene). DW_EO produced significantly larger inhibition zones than LP_EO against S. aureus and P. kudriavzevii and exhibited lower MIC50/MIC90 values for S. aureus, S. uberis and P. kudriavzevii. Notably, DW_EO showed a higher inhibitory activity against P. kudriavzevii with a MIC90 of 4 mg/mL. These findings support DW_EO as a high-potential dual-action phytotherapeutic candidate for developing formulations and complementary tools within sustainable bovine udder health and antimicrobial stewardship frameworks.

This research paper addresses the hypothesis that the in vivo criterion of bovine somatic cell count (SCC)<200,000 cells/ml milk as a diagnostic marker for healthy mammary tissue is not suitableto be adopted to milk samples taken post slaughter. To study immune mechanisms associated with intramammary infections, we developed a mammary explant model. As SCC is routinely applied to differentiate between healthy and inflamed mammary tissue, donor cows were selected based on their milk SCC obtained in vivo. Furthermore, milk cell differentiation for early mastitis detection via flow cytometry allows identification of leucocyte subpopulations and complements SCC. To replace in vivo examination and allow for post mortem selection of donor cows, this explorative study aimed to investigate how slaughter influences the reliability of SCC and differential milk cell count (DMCC) and to assess their validity as diagnostic markers for udder health in bovine milk samples obtained post slaughter. Therefore, quarter milk samples from cows were obtained in vivo and post mortem and analysed to determine SCC and DMCC and identify major mastitis pathogens. The logarithmized numbers of SCC, non-viable cells, viable cells, lymphoid cells, polymorphonuclear (PMN) and large cells per ml milk were compared using linear mixed-effects models in milk samples obtained from cows in vivo and post mortem. The number of lymphoid cells, PMN and large cells was significantly higher in milk samples obtained post mortem than in vivo, with PMN being the most prominent cell population. Higher milk SCC values measured post mortem might be explained by migration of leucocytes into the periphery during slaughter. This should be considered when modelling intramammary infection in vitro using udder tissue. Reflecting these findings, it is not feasible to endorse SCC as a reliable marker for post mortem selection of donor cows with healthy mammary tissue for in vitro models.

Gram-positive (GP) and gram-negative (GN) bacteria are the most common pathogens responsible for mastitis infections. Gram-negative infections typically elicit a more intense inflammatory response than GP infections, potentially influencing both cow behavior and milking performance before the onset of clinical mastitis (CM). Automatic milking systems (AMS) can detect early signs of CM through indicators such as deviations in milk yield, electrical conductivity, milk color, and milking behavior. However, the relationship between specific pathogen types and changes in milking behavior has not been widely explored. The objective of this study was to investigate whether milking behavior and performance during the week preceding CM diagnosis are associated with the type of pathogen involved (i.e., GN, GP, mixed infection [MX], and no-growth [NG]), and how this association varies across different lactation stages. The study considered data from 49,899 milking events in 3,467 lactations from September 2023 to June 2024 in a commercial dairy in Colorado, United States. Cows were grouped in a freestall barn system with 62 automatic milking units and were allowed to visit the milking station at intervals based on their lactation stage. To facilitate comparisons among pathogen categories, the lactations were divided into 3 stages based on the heathy control group's average milk peak DIM: prepeak (1-72 DIM), postpeak (73-144 DIM), and late lactation (145 DIM to dry-off). Depending on the pathogen isolated from milk, CM cases were classified into 4 categories: GN, GP, MX, and NG. The analyses were conducted separately for each stage of lactation, and a healthy control group (HLT) was identified as a reference for the comparisons using the average DIM of each period (prepeak: 36 DIM, postpeak: 103 DIM, and late lactation: 243 DIM). Multivariate mixed linear and logistic regression models for repeated measures were fitted to calculate estimated marginal means and probabilities, respectively, including pathogen category as the main predictor and using parity and milk yield as covariates. To assess the effect of time before CM diagnosis on the dynamics of the variables in the study, data were analyzed at 3 time points: -7, -4, and 0 d relative to the CM diagnosis. A total of 431 quarter milk samples were submitted for bacteriological analysis from cows affected by CM, resulting in 50.0%, 23.4%, 16.2%, and 10.4% GN, GP, NG, and MX, respectively. Overall, cows with CM caused by GN and GP pathogens showed significant alterations in milking behavior and performance, which in some cases were evident 7 d before diagnosis. This study provides novel insights into how specific mastitis-causing pathogen groups affect cow milking behavior and performance in AMS.