Variation in Interleukin-4, -6, and -10 in Mastitis Milk: Associations with Infections, Pathogens, Somatic Cell Counts, and Oxidative Stress.

Poor mastitis control favors intramammary infection (IMI), which always involves CNS. This study aimed to determine the relationships of IL-4, IL-6, and IL-10 in mastitis milk with concurrent infection, bacterial pathogens, SCC, and MDA, an oxidative stress marker. All mastitis quarters from five smallholder dairy farms were sampled aseptically before morning milking and again before afternoon milking for bacteriological identification using MALDI-TOF mass spectrometry. The samples with the concomitant infection between streptococci and CNS and their pairs of another sample from the quarters were selected. In addition, samples were randomly chosen to have a controlled single infection. IL-4, IL-6, and IL-10 were measured with ELISA kits. MDA was measured using HPLC, while SCC was measured using Fossomatic™ FC. The results from a repeated measure analysis showed that IL-4 positively correlated with SCC, while IL-6 showed a negative trend. IL-4 levels were highest in CNS infections and significantly higher than in non-infected or mixed infections (p < 0.05). The IL-6 level of the mixed bacteria was highest and showed a different trend from non-infection, and the quarter was infected with streptococcal bacteria. In conclusion, from a single infection, the streptococci and CNS quarter showed varied immune responses, including trendily higher IL-6 and IL-4.

Divergent Immune Responses to Minor Bovine Mastitis-Causing Pathogens.

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.

Different cellular and molecular responses of Bovine milk phagocytes to persistent and transient strains of Streptococcus uberis causing mastitis.

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.

Exploring the potential immunomodulatory effects of gallic acid on milk phagocytes in bovine mastitis caused by Staphylococcus aureus.

Bovine mastitis caused by Staphylococcus aureus may exacerbate by resulting in significant economic losses and impacting milk quality. To date, the use of gallic acid, a phenolic compound naturally occurring in various plants, holds promise due to its potent anti-oxidant and anti-inflammatory effects in many pieces of literature, thus, making it a subject of interest in bovine innate immunity research. Here we used gallic acid to assess its potential immunomodulation on milk phagocytes in vitro challenges with mastitis-causing bacteria. Our findings indicated that cells exposed to gallic acid showed no harm to cell viability but might maintain the longevity of cells during the bacterial infection. Gallic acid-treated cells displayed reduced cell migration, phagocytosis, and bacterial killing ability, while showing an increase in ROS production, all of which are undoubtedly linked to the intracellular killing abilities of the cells. Nonetheless, the extracellular structure called neutrophil extracellular traps (NETs) was significantly released after receiving gallic acid, representing extracellular killing. We also reported that gallic acid neutralizes inflammation by regulating specific pro-inflammatory genes (IL1B, IL6, TNF) and ROS-generating genes (CYBA, LAMP1, RAC1), subsequently preventing tissue damage. Regarding apoptosis-related genes and proteins, the increased production of caspase-3 and Bcl-2 family proteins could potentially promote the longevity of cells, implicated in the mechanism of combating bacterial invasion during udder inflammation and infection. The novel role of gallic acid on milk phagocytes highlights its potential immunomodulatory properties and contributes to our understanding of its effects on bacterial-host interactions, and provides valuable molecular insights.

Exploring the distinct immunological reactions of bovine neutrophils towards major and minor pathogens responsible for mastitis.

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.

Clinical field trial of parenteral amoxicillin for the treatment of clinical and subclinical mastitis in smallholder dairy farms in the upper region of Northern Thailand.

Background and Aim: Mastitis, primarily caused by intramammary bacterial infection, is the most expensive disease in the global dairy industry due to its negative impact on milk composition and manufacturing properties. This study aimed to evaluate the efficacy of parenteral amoxicillin in the treatment of clinical and subclinical mastitis in smallholder dairy farms in Northern Thailand. Materials and Methods: A total of 51 cows with clinical and subclinical mastitis from dairy cooperatives in Lamphun and Chiang Mai provinces, Northern Thailand, were enrolled in this study. Conventional bacteriological procedures were applied to identify the causative bacteria in milk samples from these cows before and 7 days after treatment, and antibiotic susceptibility tests were conducted using the disk diffusion method for all bacteria isolated before treatment. All cows with mastitis were administered 15 mg/kg of amoxicillin (LONGAMOX®, Syva Laboratories SA, Spain) intramuscularly every other day for 3 days. Results: Environmental streptococcal bacteria (Streptococcus uberis and Streptococcus spp.) were commonly isolated from infected quarters and were highly susceptible to amoxicillin (100%). The clinical efficacy of amoxicillin treatment for clinical mastitis cases was 80.43%, and the bacteriological efficacy was 47.82%, with opportunistic staphylococcal bacteria (coagulase-negative staphylococci) and contagious streptococcal bacteria (Streptococcus agalactiae) being the most sensitive microorganisms (100%). In subclinical mastitis cases, the bacteriological efficacy of parenteral amoxicillin was 70.45%, with environmental streptococcal bacteria (S. uberis) being the most (100%) sensitive microorganisms. Conclusion: Amoxicillin is highly efficacious and can be used to treat clinical and subclinical mastitis in dairy cows, particularly mastitis caused by environmental Streptococcus spp. These findings could be used to guide treatment regimens in veterinary practice in smallholder dairy farms in Thailand.

In Vitro Bacterial Competition of Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli against Coagulase-Negative Staphylococci from Bovine Mastitis Milk.

Intramammary infection (IMI) from the environment and infected quarters can cause co-infection. The objective of this study was to determine the ability of coagulase-negative staphylococci (CNS) to survive in the same environment as Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli as major pathogens. In total, 15 and 242 CNS strains were used in Experiment I and Experiment II, respectively. Both experiments were separated into three conditions: culture with CNS 24 h before (PRIOR), after (AFTER), and at the same time (EQUAL). The lack of a clear zone, regardless of size, was determined to be the key to the survival of both. The CNS species' percentages of survival against major pathogens were tested using Fisher's exact test. Differences in the percentages of survival were evident among the CNS species in all conditions. For the PRIOR condition, all CNS mostly survived when living with major strains; however, S. chromogenes could degrade S. agalactiae. Although most CNS strains were degraded in the AFTER and EQUAL conditions, some strains of S. hominis and S. simulans could resist S. aureus and S. agalactiae. In conclusion, some specific strains of CNS are able to survive in an environment with major pathogens. Research into the survival strains may indicate that the concept of novel bacteria with bacteriolytic capabilities might be possible as a novel mastitis treatment.

Comparison of Virulence Patterns Between Streptococcus uberis Causing Transient and Persistent Intramammary Infection.

The objectives of this study were determined by two experiments including Experiment 1 (EXP1) using Streptococcus uberis obtained from a weekly longitudinal study to compare virulence patterns between transient and persistent intramammary infection (IMI), and Experiment 2 (EXP2) using a stored-known-appearance PFGE strain of a contagious S. uberis to determine a change of virulence patterns after long-term transmission. For EXP1, quarter milk samples from 31 milking cows were aseptically and longitudinally collected once a week for 10 weeks. A total of 14 S. uberis isolates from quarters with 1 and >4 weeks of duration of IMI were categorized as transient and persistent IMI, respectively. For EXP2, 11 isolates of a stored-known-appearance PFGE strain of S. uberis from our previous study (1) were randomly selected, including 5 from transient IMI (1 month) and 6 from persistent IMI (>1 month). The virulence profiles of all isolates were investigated, including sua, hasAB, hasC, gapC, pauA, and CAMP factor or cfu, using PCR. The Kaplan-Meier estimates were used to calculate the duration of IMI in EXP1. Approximately 50% of field S. uberis IMI was spontaneously cured within 1 week, while 25% was not cured within 10 weeks. From EXP1, 4 virulence patterns were found in 14 isolates. The majority of patterns for transient S. uberis did not include hasAB (63.6%), the gene relating to capsule formation. Regardless of transient or persistent IMI, a high similarity of the virulence pattern within a PFGE strain was found in EXP2. Few changes of virulence pattern within a PFGE strain were found or were related to its subsequently changing to transient IMI.

Immunomodulatory Effects of Herbal Compounds Quercetin and Curcumin on Cellular and Molecular Functions of Bovine-Milk-Isolated Neutrophils toward Streptococcus agalactiae Infection.

Herbal phytochemicals featuring active ingredients including quercetin and curcumin have shown potential in treating human and animal diseases. The current study investigated their potential function in vitro for host immunomodulation associated with Streptococcus agalactiae subclinical bovine mastitis via milk-isolated neutrophils. Our results showed a positive influence on cellular migration, reactive oxygen species (ROS) generation, phagocytosis, and bacterial killing as well as neutrophil extracellular traps (NETs) release. This study also highlighted several important molecular aspects of quercetin and curcumin in milk-isolated neutrophils. Gene expression analyses by RT-PCR revealed significant changes in the expression of proinflammatory cytokines (IL1B, IL6, and TNF), ROS (CYBA), phagocytosis (LAMP1), and migration (RAC). The expression levels of apoptotic genes or proteins in either pro-apoptosis (CASP3 and FAS) or anti-apoptosis (BCL2, BCL2L1, and CFLAR) were significantly manipulated by the effects of either quercetin or curcumin. A principal component analysis (PCA) identified the superior benefit of quercetin supplementation for increasing both cellular and molecular functions in combating bacterial mastitis. Altogether, this study showed the existing and potential benefits of these test compounds; however, they should be explored further via in vivo studies.

Antibacterial Activities of Acetic Acid against Major and Minor Pathogens Isolated from Mastitis in Dairy Cows.

The present study evaluated the antimicrobial activities of acetic acid against bovine mastitis pathogens compared to lactic acid and lauric and caprylic saturated fatty acids. Eleven mastitis pathogens were isolated from sub-clinical and clinical bovine mastitis cases for the study. An initial screening of their antibacterial activities by agar well diffusion method was performed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each acid were obtained using a microdilution method; each acid was diluted from stock solution and then were diluted with culture broth to reach concentrations ranging from 4 to 0.004% w/v. The results showed acetic acid had the highest zone of inhibition against all pathogens except Escherichia coli compared with lauric and caprylic acids. The MIC and MBC were lowest for acetic acid against both Gram-positive (except Staphylococcus chromogenes from the coagulase negative staphylococci (CNS) group) and Gram-negative pathogens, intermediate for lactic and caprylic acids and greatest for lauric acid. In conclusion, acetic acid had antimicrobial activities against most mastitis pathogens compared with other acids. Further studies are needed to optimize the formulation and concentration of acetic acid for teat-dipping agent in the future.