Bovine Mastitis Research Articles

Development of Zinc Oxide Nanoparticle and Lemongrass Essential Oil Incorporated Nanoformulation-Based External Teat Sealant to Prevent Mastitis in Dairy Cows

Development of Zinc Oxide Nanoparticle and Lemongrass Essential Oil Incorporated Nanoformulation-Based External Teat Sealant to Prevent Mastitis in Dairy Cows

Understanding the bovine mastitis co-infections: Coexistence with Enterobacter alters S. aureus antibiotic susceptibility and virulence phenotype.

The World Health Organization recently reported an alarming evolution and spread of antibiotic resistance, a global risk factor recognized as a One Health challenge. In veterinary, the general lack of clear treatment guidelines often leads to antibiotic misuse. Bovine mastitis is responsible for major economic losses and the main cause of antibiotic administration in the dairy industry, favoring the emergence of multi-resistant phenotypes. The complexity of inter-microbial and host-pathogen interactions in the mammary gland, demonstrated by culture-independent techniques, not only complicates the prediction of antibiotic treatment outcomes but also underscores the urgent need for further research in this field. This work evaluated the interactions between S. aureus L33 and Enterobacter sp. L34 obtained from an intramammary co-infection. The behavior of the dual-species culture resembled that of the Enterobacter monoculture in all the evaluated contexts. Most of the selected S. aureus virulence factors and the antibiotic susceptibility were altered by coexisting with Enterobacter. Under the protection of Enterobacter, S. aureus was able to survive upon exposure to concentrations of cloxacillin and other antibiotics that would be bactericidal for the monoculture. This could have serious implications for bacterial clearance of mastitis originating from the underestimated co-infections. These findings highlight the importance of broadening our knowledge of how microbial interactions in intramammary infections could contribute to antibiotic treatments failures. Moreover, they open new perspectives for the design of bovine mastitis therapies that consider the ecological context in order to optimize the antibiotic usage, improve the success rates and reduce antibiotic resistance.

Wall teichoic acid glycosylation of bovine-associated Staphylococcus aureus strains.

Staphylococcus aureus (S. aureus) is one of the major causes of bovine mastitis, a disease with detrimental effects on health and wellbeing. Current control measures are costly, laborious and not always effective in eradicating S. aureus. The cell wall-linked polysaccharide wall teichoic acid (WTA) is highly immunogenic in humans and is considered as a prospective vaccine antigen based on promising pre-clinical studies in animals. WTA consist of polymerized ribitol-phosphate backbone that is modified with N-acetylglucosamine (GlcNAc) moieties in different configurations by the glycosyltransferases TarS (β-1,4-GlcNAc), TarM (α-1,4-GlcNAc) and TarP (β-1,3-GlcNAc). This study aimed to characterize the presence and genetic variation in tarS, tarM and tarP in bovine-associated S. aureus strains and how this impacts WTA-glycoprofile. Bioinformatic analyses of a whole genome sequence database consisting of 1047 S. aureus, 10 S. schweitzeri, and 6 S. argenteus strains showed that over 99% of strains contained tarS, 34 % also contained tarM, while 5 % of the strains encoded tarP in addition to tarS. The distribution of WTA-glycosyltransferase genes was similar to what has been reported for human-associated S. aureus strains. Phenotypic analysis of WTA glycosylation by flow cytometry corroborated with tarS/tarM/tarP gene presence. The WTA glycoprofile was variable between bovine-associated strains and the levels and ratios of GlcNAcylation were affected by growth conditions. Interestingly, a divergent tarM allele was present in strains of clonal complexes (CC) 49 and the mastitis-associated CC151, but its function was similar to canonical tarM. In conclusion, we demonstrated that bovine-associated S. aureus strains show similar variation in WTA GlcNAc decoration as human S. aureus strains, despite the presence of a divergent tarM allele.

Antimicrobial resistance profiles and molecular epidemiology of Klebsiella pneumoniae isolates from Scottish bovine mastitis cases.

Klebsiella pneumoniae are opportunistic pathogens which can cause mastitis in dairy cattle. K. pneumoniae mastitis often has a poor cure rate and can lead to the development of chronic infection, which has an impact on both health and production. However, there are few studies which aim to fully characterize K. pneumoniae by whole-genome sequencing from bovine mastitis cases. Here, K. pneumoniae isolates associated with mastitis in dairy cattle were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing. Furthermore, whole-genome sequence data were used for phylogenetic analyses and both virulence and antimicrobial resistance (AMR) prediction, in parallel with phenotypic AMR testing. Forty-two isolates identified as K. pneumoniae were subject to whole-genome sequencing, with 31 multi-locus sequence types being observed, suggesting the source of these isolates was likely environmental. Isolates were examined for key virulence determinants encoding acquired siderophores, colibactin, and hypermucoidy. The majority of these were absent, except for ybST (encoding yersiniabactin) which was present in six isolates. Across the dataset, there were notable levels of phenotypic AMR against streptomycin (26.2%) and tetracycline (19%), and intermediate susceptibility to cephalexin (26.2%) and neomycin (21.4%). Of importance was the detection of two ESBL-producing isolates, which demonstrated multi-drug resistance to amoxicillin-clavulanic acid, streptomycin, tetracycline, cefotaxime, cephalexin, and cefquinome.

A novel broad-spectrum bacteriophage cocktail against methicillin-resistant Staphylococcus aureus: Isolation, characterization, and therapeutic potential in a mastitis mouse model.

Bovine mastitis is a considerable challenge within the dairy industry, causing significant financial losses and threatening public health. The increased occurrence of methicillin-resistant Staphylococcus aureus (MRSA) has provoked difficulties in managing bovine mastitis. Bacteriophage therapy presents a novel treatment strategy to combat MRSA infections, emerging as a possible substitute for antibiotics. This study evaluated the therapeutic potency of a novel bacteriophage cocktail against MRSA mastitis. Two new bacteriophages (vB_SauR_SW21 and vB_SauR_SW25) with potent lytic activity against MRSA were isolated and characterized. The one-step growth curve displayed a rapid latent period (20-35 min) and substantial burst size (418 and 316 PFU/ cell). In silico analyses have confirmed the absence of antimicrobial resistance or virulence factor-encoding genes within their genomes. According to the results, combining these phages augmented their host range and virulence. The phage cocktail significantly reduced bacterial burden in a BALB/c mastitis model, demonstrating efficacy comparable to antibiotic treatment. Moreover, its administration led to decreased concentrations of IL-1β and TNF-α compared to the negative control group. The bacteriophage cocktail (SW21-SW25) exhibits a promising profile for therapeutic applications and may represent a novel substitute to antibiotics for managing MRSA bovine mastitis.

Virulence of Bacteria Causing Mastitis in Dairy Cows: A Literature Review.

Bovine mastitis, a prevalent disease in dairy farms, exerts a profound negative influence on both the health and productivity of dairy cattle, leading to substantial economic losses for the dairy industry. The disease is associated with different bacterial agents, primarily Gram-positive cocci (e.g., Staphylococcus spp., Streptococcus spp.) and Gram-negative bacilli (e.g., Escherichia coli, Klebsiella pneumoniae). These pathogens induce mastitis through diverse mechanisms, intricately linked to the virulence factors they carry. Despite previous research on the virulence factors of mastitis-causing bacteria in dairy cattle, there remains a significant gap in our comprehensive understanding of these factors. To bridge these gaps, this manuscript reviews and compiles research on the virulence factors of these pathogens, focusing on their roles in mammary tissue infection, immune evasion, adherence to mammary epithelial cells, and invasion and colonization of the mammary gland. These processes are analyzed in depth to provide a comprehensive framework to promote a deeper understanding of dairy pathogenic bacteria and their pathogenic mechanisms and to provide new insights into the control of mastitis in dairy cattle.

Determining causal pathogens and inflammatory state of mastitis in dairy cows via Gram staining of precipitates in milk.

Early detection of bovine mastitis-causing pathogens is necessary for treatment. As culturing methods are time-consuming, a more rapid detection technique is required. This study investigated the sensitivity, specificity, and detection limit of Gram staining of milk precipitates (milk Gram stain, MGS) to detect bovine mastitis-causing pathogens in milk, as well as the potential of MGS to diagnose inflammation by counting polymorphonuclear leukocytes (PMN). MGS was performed on spontaneous mastitis cases. Culture methods were also used as reference standards to calculate the sensitivity, specificity, and bacterial load in milk to determine the detection limit of MGS. PMN in the mastitic milk were counted using Gram staining. Further, somatic cell counts (SCC), interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, IL-10 and serum amyloid A (SAA) concentrations in mastitic milk were measured using cell counting and enzyme-linked immunosorbent assay. The sensitivity and specificity for all pathogens were 0.62 and 0.90, for Gram-positive were 0.67 and 0.90, and for Gram-negative were 0.50 and 1.00, respectively. The detection limits for Gram-positive and Gram-negative bacteria were 1,560 and 4,680 cfu/mL, respectively. SCC were significantly positively correlated with PMN, milk IL-6, TNF-α, and SAA concentrations, whereas PMN were significantly negatively correlated with milk IL-10 concentration. Our results suggest that MGS may be applied as a rapid method to identify causal pathogens of mastitis before culture results are determined and may also estimate inflammatory status which cannot be detected with SCC. Further clinical trials are required to elucidate whether MGS is useful in clinical veterinary settings.

Mapping Antimicrobial Resistance in Staphylococcus epidermidis Isolates from Subclinical Mastitis in Danish Dairy Cows.

Background/Objectives: Although Staphylococcus epidermidis is a key cause of subclinical mastitis in Danish dairy cows, its sensitivity to antimicrobials remains unexplored. Here, we analyzed sixty S. epidermidis isolates derived from 42 dairy cows across six conventional dairy herds in Denmark. Methods: Phenotypic resistance was measured by antimicrobial susceptibility testing and minimum inhibitory concentration (MIC) analysis, and genotypic resistance was examined through whole-genome sequencing and identification of antimicrobial resistance genes (ARGs). Correspondence between phenotypic and genotypic resistance was then evaluated by Cohen's kappa statistics. Furthermore, the presence of plasmid replicon genes and the strain diversity among the S. epidermidis isolates was investigated to associate these findings with the observed AMR patterns. Results: Results showed that 30/60 isolates (50.0%) were resistant to penicillin phenotypically, while 35/60 (58.3%) were positive for a corresponding blaZ gene (κ = 0.83, p < 0.01). A fosB gene, encoding fosfomycin resistance, was detected in all 60/60 isolates (100.0%), but fosfomycin resistance was not analyzed phenotypically. Based on MIC analysis, 3/60 isolates (5.0%) were multi-drug resistant, showing resistance towards penicillin, erythromycin, and tetracycline. However, in 11/60 genomes (18.3%), ARGs encoding resistance towards ≥3 antimicrobial classes (e.g., beta-lactams, phosphonic acid, tetracyclines, aminoglycosides, macrolides, lincosamides, and fusidane) were detected. Eleven different ARGs were detected among the 60 isolates in total. No methicillin-resistant Staphylococcus epidermidis (MRSE) were recorded. Results further showed that each herd had one primary sequence type (ST) and resistance profile associated with it, and plasmid-mediated horizontal gene transfer of ARGs was indicated This study underscores the importance of routine resistance surveillance and species-specific diagnoses to improve treatment outcomes and ensure prudent use of antimicrobials.

Regulation of LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells via TLR4-Mediated NF-κB and MAPK Signaling Pathways by Lactoferrin.

Lactoferrin (LF), a member of the transferrin family, is widely present in mammalian milk and other secretions, exhibiting anti-inflammatory, antibacterial, and anti-infective properties. Although the biological functions of LF have been extensively studied, there are few reports on its effects and molecular mechanisms concerning bovine mastitis caused by bacterial infection. This study used bovine mammary epithelial cells (BMECs) cultured in vitro as the research model. An inflammatory injury model was established by stimulating BMECs with LPS to investigate whether LF at different concentrations (10, 50, 100, and 200 μg·mL-1) could inhibit the inflammatory response before and after the onset of inflammation. The expression of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α at both the gene and protein levels was detected using RT-qPCR and ELISA. Western blotting was employed to evaluate the phosphorylation levels in the inflammatory signaling pathways MAPK/P38/ERK and NF-κB/P65, while RT-qPCR was used to examine the impact on TLR4 receptor gene expression. The results display that pretreatment with LF prior to LPS-induced inflammation in BMECs reduced the expression of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α at both the gene and protein levels (p < 0.05). LF also inhibited the phosphorylation of NF-κB/P65 and MAPK/P38/ERK signaling pathways and downregulated TLR4 receptor gene expression (p < 0.05). However, when LF was added after the onset of LPS-induced inflammation, inflammatory cytokine expression and phosphorylation levels in the NF-κB/P65 and MAPK/P38/ERK pathways remained elevated, along with high expression of the TLR4 receptor gene (p < 0.05). These findings show that LF can antagonize LPS-induced inflammatory responses in BMECs and reduce cytokine expression, exhibiting anti-inflammatory effects when administered before inflammation. Conversely, when LF is added post-inflammation, it appears to enhance cytokine expression, potentially promoting the recruitment of more cells or factors to resolve inflammation rapidly. Both effects are mediated through the TLR4 receptor and the NF-κB/P65 and MAPK/P38/ERK signaling pathways.

Prevalent and Drug-Resistant Phenotypes and Genotypes of Escherichia coli Isolated from Healthy Cow's Milk of Large-Scale Dairy Farms in China.

Escherichia coli is a common cause of mastitis in dairy cows, which results in large economic losses to the livestock industry. The aim of this study was to investigate the prevalence of E. coli in raw milk in China, assess antimicrobial drug susceptibility, and identify key antibiotic resistance genes carried by the isolates. In total, 350 raw milk samples were collected from large-scale farms in 16 provinces and cities in six regions of China to assess the resistance of E. coli isolates to 14 antimicrobial drugs. Among the isolates, nine resistance genes were detected. Of 81 E. coli isolates (23.1%) from 350 raw milk samples, 27 (33.3%) were multidrug resistant. Antimicrobial susceptibility testing showed that the 81 E. coli isolates were resistant to 13 (92.9%) of the 14 antibiotics, but not meropenem. The resistance gene blaTEM was highly distributed among the 27 multidrug-resistant isolates with a detection rate of 92.6%. All isolates carried at least one resistance gene, and 19 patterns of resistance gene combinations with different numbers of genes were identified. The most common gene combinations were the one-gene pattern blaTEM and the three-gene pattern blaTEM-blaPSE-blaOXA. The isolation rate of E. coli in raw milk and the identified resistance genes provide a theoretical basis for the rational use of antibiotics by clinical veterinarians.

Retrospective Analysis of Penicillin G Minimum Inhibitory Concentration for Gram-Positive Isolates of Non-Severe Clinical Mastitis.

Background: Despite penicillin having a longstanding reputation as being scientifically approved for the treatment of bovine mastitis, its market share and practical application rate seem rather low. While in some countries, cases of mild and moderate mastitis are treated almost completely with simple penicillin, in other countries, penicillin is rarely used as a mono-substance in udder tubes. Methods: Based on minimal inhibitory concentration (MIC) studies of 1489 isolates of Gram-positive microorganisms isolated from bovine mastitis cases, the extent to which penicillin preparations can fulfil their role as first-line treatment and in how many cases insufficient efficacy must be assumed was assessed in comparison with more recent studies on the achievable levels of active substances in milk. Results: Of the isolates, 76% had an MIC of ≤0.125 µg/mL and 95% of the isolates had an MIC of ≤1 µg/mL. Conclusions: The data show that in Northern Germany, it can be assumed that penicillin is a good choice in most cases of mastitis caused by Gram-positive mastitis pathogens, at least from the perspective of antibiotic resistance.

Protective effect of curcumin-loaded zeolitic imidazolate framework-8-based pH-responsive drug delivery system against Staphylococcus aureus infection.

Mastitis, generally caused by pathogenic microorganisms, is a serious disease in dairy farming. Staphylococcus aureus (S. aureus) is one of the main pathogens that induces mastitis in dairy cows. It evades the innate and adaptive immune responses of dairy cows, causing recessive transmission and harming the health of the mammary glands. Antibiotics remain the primary treatment; however, their excessive use can lead to antimicrobial resistance. Therefore, it is necessary to develop new strategies to replace antibiotic therapies. The zeolitic imidazolate framework (ZIF-8) is a metal-organic skeleton material with applications in biology and drug delivery. This study aimed to construct a novel nanodrug delivery system for S. aureus infection by combining ZIF-8 with curcumin (ZIF-8@CCM), which exhibits antibacterial and anti-inflammatory properties. Bovine mammary epithelial cells (BMECs) and mice were used to evaluate the therapeutic efficacy and biotoxicity of the system, and to explore the protective mechanism of ZIF-8@CCM. The results showed that ZIF-8@CCM exhibited high drug loading capacity, stability, and pH responsiveness. Both in vitro and in vivo experiments revealed that ZIF-8@CCM effectively released encapsulated curcumin in response to the acidic microenvironment induced by bacterial infection, which in turn enhanced the bactericidal efficacy. It not only prevents biofilm formation, but also mitigates the toxic side effects associated with drug treatments, showing excellent bioavailability and biocompatibility. Furthermore, ZIF-8@CCM also attenuated S. aureus-induced inflammatory through suppressing the activation of TLR2-NF-κB pathway. Consequently, ZIF-8@CCM is an effective targeted antibacterial and anti-inflammatory drug, showing promise as a novel therapeutic agent for the clinical management of S. aureus-induced mastitis in dairy cows.

Microbiota do leite de vacas com mastite clínica: uma abordagem etiológica

ABSTRACT: Bovine mastitis is the most impacting disease of dairy industry, and it is characterized by a complexity of causal agents, which have revealed a geographical variation among regions and countries. The mastitis-related pathogens have been traditionally classified as contagious or environmental, based on habits of the microorganisms and transmission routes. Besides, the severity of mammary infections has been associated with the virulence of the pathogens, and immune and nutritional status of the hosts. Considering this scenario, we investigated the etiological pattern, clinical severity scores, and days in milk (DIM) data in 4,273 clinical cases of bovine mastitis among ten large-dairy farms located in Brazil. Streptococcus dysgalactiae (283/4,273 = 6.6%), Escherichia coli (190/4,273 = 4.4%), Prototheca spp. (112/4,273 = 2.6%), and Streptococcus uberis (95/4,273 = 2.2%) were the predominant pathogens isolated. Among 4,273 clinical cases, clinical severity score was available in 43.8% (1,871/4,273) animals. From these, 69.8% (1,306/1,871), 27.3% (510/1,871), and 2.9% (55/1,871) were scored as mild, moderate, and severe, respectively. Pathogens were observed mainly in samples obtained during the first 100 days in milk, and clinical severity scored mainly as mild. Our results contributed to the etiological identification, clinical severity scoring, and milking aspects of mastitic cows in dairy farms with a history of clinical mammary infections.

Prevalence, Antibiotic Resistance Patterns, and Virulence Factors of Staphylococcus aureus Isolates Associated with Bovine Mastitis in Northern Bangladesh

Prevalence, Antibiotic Resistance Patterns, and Virulence Factors of Staphylococcus aureus Isolates Associated with Bovine Mastitis in Northern Bangladesh

Effect of double disinfectant application during premilking teat disinfection on teat cleanliness, bacterial count, and mastitis in pasture-grazed dairy cows

Effect of double disinfectant application during premilking teat disinfection on teat cleanliness, bacterial count, and mastitis in pasture-grazed dairy cows

Decoding epigenetic markers: implications of traits and genes through DNA methylation in resilience and susceptibility to mastitis in dairy cows

ABSTRACT Cattle farming faces challenges linked to intensive exploitation and climate change, requiring the reinforcement of animal resilience in response to these dynamic environments. Currently, genetic selection is used to enhance resilience by identifying animals resistant to specific diseases; however, certain diseases, such as mastitis, pose difficulties in genetic prediction. This study introduced the utilization of enzymatic methyl sequencing (EM-seq) of the blood genomic DNA from twelve dairy cows to identify DNA methylation biomarkers, with the aim of predicting resilience and susceptibility to mastitis. The analysis uncovered significant differences between cows resilient and susceptible to mastitis, with 196,275 differentially methylated cytosines (DMCs) and 1,227 Differentially Methylated Regions (DMRs). Key genes associated with the immune response and morphological traits, including ENOPH1, MYL10 and KIR2DL5A, were identified by our analysis. Quantitative trait loci (QTL) were also highlighted and the body weight trait was the most targeted by DMCs and DMRs. Based on our results, the risk of developing mastitis can potentially be estimated with as few as fifty methylation biomarkers, paving the way for early animal selection. This research sets the stage for improved animal health management and economic yields within the framework of agricultural sustainability through early selection based on the epigenetic status of animals.

Conventional and molecular identification of methicillin-resistance Staphylococcus aureus (MRSA) associated with human skin lesions and bovine mastitis

Background. The objective of this study was to identify the prevalence of Staphylococcus aureus (S. aureus) taken from human skin lesions and bovine mastitis samples as well as determine the rate of occurrence of methicillin-resistance S. aureus (MRSA) and to analysis of the genetic connections between isolated bacteria with local and global strains. Methods. A total of 350 specimens were collected, including 150 specimens from human with different skin lesions and 200 specimens from cattle with mastitis. Results. Cultivation methods and biochemical evaluation demonstrated the presence of 86 (57.3%) and 98 (49%) S. aureus isolates in human and animal samples, respectively. The resistance pattern showed that each individual S. aureus isolate shown resistance to penicillin. (100%) followed by erythromycin (86% and 95.5%), Clindamycin (69.7% and 90.8%), Oxacillin (63.9% and 82.6%), Levofloxacin (50% and 55.1%), Tetracyclin (19.7% and 12.2%), Vancomycin (15.1% and 9.1%), Teicoplanin (8.1% and 20.4%) and Gentamicin (9.3% and 5.1%) for human and animals, respectively. The mecA gene was detected in all human Oxacillin resistant isolates (100%), while it was detected only in 73 (82.6) phenotypically Oxacillin resistant isolates. PCR partial gene sequencing that targeted housekeeping gene loci; arcC, aroE, glpF, gmk, pta, tpi, and yqi was done. The sequences were analyzed using multilocus sequence typing (MLST). These isolates carried the above-mentioned genes as follows; 2 (6.7%), 2 (6.7%), 2 (6.7%), 2 (6.7%), 6 (6.7%), 3 (10%), and 2 (6.7%) human isolates, and 10 (13.3%), 14 (18.7%), 8 (10.7%), 6 (8%), 10 (13.3%), 3 (4%), and 2 (2.7%) for milk isolates, respectively, and the MLST via a phylogenetic evaluation revealed close-nucleotide similarities between local and NCBI-based human and cow sequences. Conclusion. Ultimately, the findings of the current investigation suggest that S. aureus is commonly identified in human skin lesions and milk samples and a lot of these isolates the bacteria included significant resistance genes, identifying a possible origin of the spread of resistance to human strains. Furthermore, the obtained information about the genetic similarities between these isolates highlights the animals as an important source for transporting the virulent pathogens to human which leads to health risks to public health.

Fifteen-Year Retrospective Study: Prevalence of Bacterial Isolates in Mastitis-Suspected Milk Samples Submitted to Central Veterinary Laboratory, Nepal

Bovine mastitis, a prevalent mammary gland inflammation, significantly impacts dairy production worldwide. This study investigated the changing landscape of mastitis-causing bacteria in Nepal. A retrospective analysis of secondary data was conducted, utilizing technical bulletins published by the Central Veterinary Laboratory (CVL) in Kathmandu over a fifteen-year period (2003/2004 to 2017/2018). The analysis focused on 3,445 bovine milk samples screened for mastitis using the California Mastitis Test (CMT).The analysis revealed Staphylococcus spp. as the most prevalent bacterial isolate (2,948 positive samples), followed by E. coli and Streptococcus spp. This finding suggests a potential shift in the dominant mastitis pathogens in Nepal, with Staphylococcus spp. potentially surpassing E. coli compared to previous reports. These results highlight the importance of implementing hygiene practices during milking to reduce the spread of contagious pathogens like Staphylococcus spp. Additionally, maintaining clean milking environments can help minimize E. coli infections. Further research is warranted to understand the factors driving this potential shift in bacterial prevalence and develop targeted prevention and control strategies for mastitis in Nepal.

AVALIAÇÃO DA OZONIOTERAPIA COMO TRATAMENTO ALTERNATIVO PARA MASTITE SUBCLÍNICA BOVINA

Mastitis is one of the main diseases affecting dairy cattle worldwide, characterized by inflammation of the mammary gland. This condition causes significant economic losses, including the disposal of contaminated milk, withdrawal periods after the use of antibiotics, reduced milk quality, as well as additional costs for treatments, veterinary assistance, and early culling of affected animals. The disease can present in two forms: clinical and subclinical. Infection is often treated with antibiotics, but this approach has been questioned due to the residuals left in the milk, the risks to consumers, and the product loss during the withdrawal period. Due to these factors, alternative treatments have been studied. Among them, ozone therapy has stood out for its antimicrobial (fungicidal, virucidal, and bactericidal), anti-inflammatory, and immunomodulatory properties. The objective of this study is to evaluate ozone therapy as an alternative treatment for bovine subclinical mastitis. This study was approved by the Ethics Committee for the Use of Animals at the Universidade Iguaçu – UNIG, with protocol PEBIO/UNIG Nº 014/2023. The research was conducted at the farm school Cacaria, at the UNIG campus in Nova Iguaçu. Three female cattle were diagnosed with subclinical mastitis after performing the California Mastitis Test (CMT) and the strip cup test, and were treated with ozone therapy. A 50% improvement was observed in the animals, with worsening in three teats, while the other three showed no changes after the use of ozonized saline, direct ozone gas application to the affected teats, and "bagging." Although the treated cases initially showed a positive therapeutic response, the treatments were not conducted continuously, which compromised long-term effectiveness. It is assumed that this contributed to the difficulty in achieving full recovery of the animals. This scenario reinforces the importance of a personalized therapeutic approach, considering the severity of the infection, the individual response of each animal, and the management conditions of the farm.

A Disintegrin and Metalloproteinase 17 Disrupts Bovine Macrophage MER Proto-Oncogene Tyrosine Kinase Integrity to Impede Apoptotic Cell Clearance and Promote Inflammation in Clinical Mastitis.

In clinical mastitis of dairy cows, the abnormal accumulation of apoptotic cells (ACs) and subsequent secondary necrosis and inflammation pose significant concerns, with macrophage-mediated efferocytosis, crucial for ACs clearance, remaining unexplored in this context. In nonruminants, MER proto-oncogene tyrosine kinase (MERTK) receptors are essential for efferocytosis and A Disintegrin and Metalloproteinase 17 (ADAM17) is thought to play a role in regulating MERTK integrity. This study aimed to delineate the in situ role of efferocytosis in clinical mastitis, with a particular focus on the interaction between MERTK and ADAM17 in bovine macrophages. In mastitic mammary tissue, a significant accumulation of ACs was observed, along with active macrophage efferocytosis. Western blotting analysis revealed elevated expressions of MERTK and ADAM17, and immunofluorescence confirmed that MERTK is predominantly localized within CD163+ macrophages. Additionally, elevated levels of soluble MERTK (sol-MER) in serum indicated impaired integrity and functionality of MERTK. In vitro experiments with the bovine macrophage cell line Bo-mac cells selectively phagocytosed apoptotic MAC-T cells, a process associated with increased MERTK phosphorylation and an anti-inflammatory phenotype. The activation of ADAM17 by Phorbol 12-myristate 13-acetate (PMA) induced sol-Mer release and impaired efferocytosis, with these effects reversed by the ADAM17 inhibitor TAPI-1. Bo-mac efferocytosis was influenced by the presence and activation of MERTK. Silencing MERTK interrupted efferocytosis, a phenomenon also observed with the inhibition of MERTK phosphorylation by UNC2025, which concurrently suppressed anti-inflammatory cytokine production. These findings suggest that targeting the MERTK-ADAM17 axis could enhance inflammatory resolution, providing a novel therapeutic strategy for dairy cattle health management.