A longitudinal observational study was carried out to explore transmission dynamics and duration of infection of Streptococcus uberis. Quarter milk samples were collected aseptically for bacterial culture from all lactating cows once a month over a 10-mo period. Molecular typing of S. uberis mastitis was performed using pulsed-field gel electrophoresis (PFGE). Molecular typing was used to determine episodes of S. uberis intramammary infection (IMI). Comparisons of spontaneous cure among PFGE types were performed using Fisher's exact chi-squared tests. Differences of duration among PFGE types and between periods of lactation were tested with Kaplan-Meier survival curves and Cox's proportional hazard model. Among a total of 851 quarter samples, 145 milk samples were detected with S. uberis presence. Based on results of PFGE, 66 episodes of S. uberis IMI were determined. From the 8 main PFGE types (A-H), PFGE type D, E, F1, F2, G, and H had only one episode indicating no evidence for transmission, subsequently defined as environmental S. uberis strains. In contrast, PFGE types A1, A2, B, C1, and C2 had at least 2 infection episodes caused by the same strain in different quarters or cows, indicating that these strains would be able to transmit to other quarters or cows. These strains were defined as contagious strains. The majority of IMI were attributable to PFGE type A1 (55%), B (17%), and A2 (11%). Spontaneous cures were observed in 35 IMI episodes. Of these 35 IMI cures, 91.4% were in IMI with duration of infection of 1 mo, n = 25, and 2 mo, n = 6. The remaining 8.6% was in IMI with duration of infection >2 mo, n = 4. Based on results from Cox's proportional hazard model, environmental S. uberis episodes were likely to have spontaneous cure with shorter duration compared with contagious S. uberis with PFGE type B (hazard ratio = 8.4). Quarters infected with S. uberis strain PFGE type A in early lactation were more likely to persist compared with those infected in late lactation (hazard ratio = 7.57). In conclusion, the majority of S. uberis IMI in this herd were transient and showed spontaneous cure. In addition to environmental S. uberis IMI, at least 3 types of contagious IMI S. uberis can be defined as (1) short duration of IMI and likely to have spontaneous cure, (2) long duration and unlikely to have spontaneous cure, and (3) wide range of duration of IMI either transient or persistent where spontaneous cure may occur depending on host defense capacity.

Traditionally, non-aureus staphylococci and mammaliicocci (NASM) were not considered significant players in bovine mastitis. This study investigated the involvement of NASM (Staphylococcus hominis and Staphylococcus chromogenes) and lactic acid bacteria (LAB) strains (Weissella paramesenteroides) through bovine neutrophil responses. Bovine neutrophils displayed minimal apoptosis upon NASM and LAB challenge. Neutrophils expressed high TLR2 after challenge, but TLR6 expression varied and remained low in NASM pathogen recognition. Bovine neutrophils effectively engulfed and killed LAB, but their activity was significantly impaired against NASM. This was evident in S. chromogenes, where reduced TLR6 recognition and a weakened phagocytic response likely contributed to a lower bactericidal effect. Regardless of the bacteria encountered, intracellular ROS production remained high. S. chromogenes-challenged neutrophils displayed upregulation in genes for pathogen recognition (TLRs), ROS production, and both pro- and anti-apoptotic pathways. This response mirrored that of Weissella. except for CASP9 and BCL2, suggesting these bacteria have divergent roles in triggering cell death. Our findings suggest that S. chromogenes manipulates bovine neutrophil defenses through coordinated changes in functional responses and gene expression, while LAB strains have a weaker influence on apoptosis.

Bovine mastitis is primarily caused by a group of bacteria known as Staphylococcus and Streptococcus. However, additional types of bacteria, such as bovine non-aureus staphylococci and mammaliicocci (NASM) as well as lactic acid bacteria (LAB), are considered minor pathogens and have less impact on cows. Modulating bovine neutrophil activities and gene expressions in response to bacterial stimuli prompted the cells to execute effector functions to combat udder infections. Although neutrophils can manage major mastitis-causing bacteria, this strategy has not been tested against minor pathogens, i.e. NASM, Weissella spp. Our main objective was to investigate how neutrophils interacted with major and minor pathogens during in vitro bacterial stimulation. The results reveal that neutrophils performed offensive duties regardless of the type of bacteria encountered. Neutrophils generated high levels of reactive oxygen species, efficiently phagocytosed both types of bacteria, and facilitated extracellular killing by releasing NET structures against all bacteria. In addition, neutrophils migrated preferentially towards the majors rather than the minors, although myeloperoxidase (MPO) degranulation did not differ substantially across bacteria. Furthermore, the killing capacity of neutrophils was not dependent on any particular bacterium. The correlation of effector functions is intimately linked to the up-regulation of genes associated with the above functions, except for IL6, which was down-regulated. Furthermore, neutrophil apoptosis can be modulated by altering apoptosis-associated genes in response to harmful stimuli. These findings provide valuable information on how neutrophils react to major and minor mastitis-causing bacteria. However, future research should explore the interplay between minor pathogens and the host’s responses.

Streptococcus uberis is one of the most frequently isolated bacterial species from mastitis cows and presents significant control challenges due to its high genetic diversity. This study aimed to determine genetic variations of Strep. uberis causing mastitis across 3 high-prevalence farms (farm A, B, and C). Isolates were obtained from different longitudinal studies and were used to study relationships between virulence factors (VF), antimicrobial resistance (AMR), and strain diversity. Data on farm management and the mastitis control policy were collected during longitudinal collection of milk samples and revealed different policies among the 3 farms. Among all available Strep. uberis isolates, strains were selected based on the characteristics of infection episodes, choosing a maximum of 2 isolates per episode. Selected isolates were confirmed using MALDI-TOF and subsequently used for whole-genome sequencing (WGS). All VF and AMR genes were categorized into the core genes, as present in all isolates; the accessory gene, as present in more than 50% of all isolates; and the unique genes for the genes that were present in less than 50% of all isolates. Accessory genes and unique genes were used to determine the relationships between VF and AMR using the Fisher exact chi-squared tests. The WGS results from a total of 138 Strep. uberis isolates, obtained from 92 episodes, revealed 7 distinct phylogroups (I to VII) and 32 gene patterns. Farm B, the only farm with long-time dairy experience and managed solely by experienced dairy farmers, exhibited the highest genetic diversity. In contrast, the other farms, dominated by persistent Strep. uberis IMI, showed fewer dominant patterns and lower diversity. A lower AMR prevalence in farm C (10.7%, 6/56) was associated with lower antibiotic use, as antimicrobial usage must be authorized by a local veterinarian, but the farmers of farms A and B designed their own use. In addition, both phylogenetic and farm factors revealed a significant association between virulence and AMR . From all identified 35 VF genes and 16 AMR genes, the core pattern included 21 core genes and 8 accessory genes from the VF genes, whereas the unique genes contained 6 VF and all 16 AMR genes. The results revealed that an increase in the number of AMR genes was associated with greater virulence diversity. Significant correlations were observed with overall VF gene presence or absence (r = 0.437), as well as with the absence of VF genes from the core pattern (R = 0.523). No AMR genes were detected in patterns without VF variations from the core pattern. Furthermore, a significant correlation was demonstrated between AMR genes and specific VF genes, notably with the presence of cfu and the absence of hasA. Thus, these findings highlight the complex relationship between virulence factors and resistance genes, which are potentially influenced by factors such as farm management practices and bacterial traits. This underscores the need for further studies that should be specific to each mastitis scenario, enabling the development of effective control strategies to reduce the impact of Strep. uberis mastitis.

Background and Aim: Bovine mastitis, an inflammatory condition of the mammary gland, is a critical economic issue in the dairy industry. Antimicrobial resistance (AMR) to mastitis-causing pathogens poses a significant threat to dairy operations in Thailand and Cambodia. This study investigates the AMR of mastitis pathogens in Thailand and Cambodia. It focuses on detecting methicillin-resistant Staphylococcus aureus, vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant enterococci by identifying the presence of mecA, vanA, and vanB genes in bacterial isolates. Materials and Methods: A total of 65 bacterial isolates (55 S. aureus from Thailand and 10 Enterococcus faecium from Thailand and Cambodia) were analyzed. Disk diffusion tests were conducted for antibiotic susceptibility, and polymerase chain reaction was employed to detect AMR genes. Results: S. aureus isolates showed resistance to penicillin (21.8%), tetracycline (9.1%), and gentamycin (7.3%). Three isolates were identified as multidrug-resistant (MDR), resistant to tetracycline, gentamycin, and penicillin. E. faecium isolates exhibited high resistance to tetracycline (100%) and penicillin (90%), with 60% classified as MDR. Phenotypic analysis identified VRSA in 11% of S. aureus isolates. However, mecA, vanA, and vanB genes were not detected in any isolate. Conclusion: Mastitis pathogens in this study pose significant AMR challenges, especially with MDR S. aureus and E. faecium, and phenotypically VRSA without the vanA gene. The findings highlight the need for judicious antibiotic use in dairy farms and further studies with broader sampling. Keywords: antimicrobial resistance, dairy farms, mastitis, methicillin-resistant Staphylococcus aureus, multidrug resistance, vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus.

Streptococcus uberis is frequently isolated from milk collected from dairy cows with mastitis. According to the host’s immunity, bacterial virulence, and their interaction, infection with some strains can induce persistent subclinical inflammation, while infection with others induces severe inflammation and transient mastitis. This study compared the inflammatory response of milk-isolated white blood cells (mWBCs) to persistent and transient S. uberis strains. Quarter milk samples were collected aseptically for bacterial culture from all lactating cows once a week over a 10-week period. A transient and noncapsular strain with a 1-week intramammary infection duration was selected from this herd, while a persistent and capsular S. uberis strain with an intramammary infection longer than 2 months from our previous study was selected based on an identical pulse field gel electrophoresis pattern during the IMI episode. Cellular and molecular responses of mWBCs were tested, and the data were analyzed using repeated analysis of variance. The results showed a higher response in migration, reactive oxygen species generation, and bacterial killing when cells were stimulated with transient S. uberis. In contrast, the persistent strain led to increased neutrophil extracellular trap release. This study also highlighted several important molecular aspects of mWBCs. Gene expression analyses by real-time RT-PCR revealed a significant elevation in the expression of Toll-like receptors (TLR-1, TLR-2, TLR-6) and proinflammatory cytokines (tumor necrosis factor-alpha or TNF-α) with the transient strain. Additionally, Streptococcus uberis capsule formation might contribute to the capability of these strains to induce different immune responses. Altogether, these results focus on the immune function of activated mWBCs which demonstrate that a transient strain can elicit a stronger local immune response and, subsequently, lead to rapid recovery from mastitis.