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This study aimed to evaluate the inhibitory effect of lactic acid bacteria (LAB) isolated from fermented transition milk against Staphylococcus aureus from cows with mastitis adhered to teat cup rubber and stainless steel surfaces of cows. Transition milk, collected from multiparous Holstein cows, was fermented for 33 days. LAB were isolated based on their different colony morphologies on MRS agar and identified using MALDI-TOF MS analysis. The antagonistic activity was determined by the double-layer diffusion assay and the inhibition of S. aureus biofilms by LAB supernatant. Among the LAB isolates evaluated, Lacticaseibacillus paracasei, Lacticaseibacillus casei, Lactobacillus rhamnosus, Enterococcus faecalis, and Enterococcus faecium were identified. Out of the fifteen LAB isolates, nine exhibited zones of inhibition ranging from 12.53 to 25.83 mm against two strains of S. aureus. Lower concentrations of both S. aureus strains adhered to stainless steel and rubber coupons were detected after 20 min of contact with the supernatants of three selected LAB when compared to the control. These findings suggest that the metabolites produced by the selected LAB could potentially be used as an alternative method to inhibit and control antimicrobial-resistant S. aureus strains, as well as reduce their biofilm formation. [ABSTRACT FROM AUTHOR]

Nucleases and 5' nucleotidase (5'-NT) play essential roles in cell biology and are often associated with bacterial virulence. In Mycoplasma spp., which have limited metabolic capacities and rely on nutrient availability, these enzymes are of significant importance for nucleotide salvage. This study explores the potential role of 2 membrane-associated lipoproteins, the major nuclease MnuA and 5'-NT, in Mycoplasma bovis mastitis. Mutants deficient in MnuA (mnuA::Tn) and in 5'-NT (0690::Tn) were identified through genome-wide transposon mutagenesis of M. bovis PG45 type strain and their fitness and virulence were assessed both in vitro, in axenic medium, and in vivo, using murine and cow mastitis models. The mnuA::Tn mutant demonstrated reduced nuclease activity, while 0690::Tn exhibited slow log-phase growth and impaired hydrolase activity towards nucleotides as well as deoxynucleotides (dAMP and dGMP). In comparison to the parent strain, the 0690::Tn mutant displayed markedly reduced fitness, as evidenced by a significant decrease or even absence in post-challenge mycoplasma counts in murine and cow mammary tissues, respectively. Moreover, the 0690::Tn mutant failed to induce mastitis in both experimental models. Conversely, the mnuA::Tn mutant induced inflammation in murine mammary glands, characterized by neutrophil infiltration and increased expression of major inflammatory genes. In cows, the mnuA::Tn was able to cause an increase in somatic cell counts in a manner comparable to the wild type, recruit neutrophils, and induce mastitis. Collectively, these findings provide complementary insights, revealing that disruption of 5'-NT significantly attenuated M. bovis pathogenicity, whereas a MnuA-deficient mutant retained the ability to cause mastitis. Author summary: There is an increasing recognition of the crucial role that genes involved in the metabolic pathways play in bacterial pathogenicity. Although these genes are not traditionally classified as virulence factors, they may confer survival advantages and contribute to bacterial fitness and virulence. This is particularly relevant for mycoplasmas, which lack many essential biosynthesis pathways and for which nutrient availability and acquisition are critical for successful host colonization, survival, and infection. In this study, we generated Mycoplasma bovis mutants deficient in the major surface nucleases MnuA and 5'-nucleotidase (5'-NT) using random transposon mutagenesis. We evaluated these mutants in both in vitro and in vivo murine and cow models. Our findings unveiled that M. bovis 5'-NT, rather than MnuA, plays a pivotal role in pathogen survival, fitness, and virulence in bovine mastitis. The structural resemblance between M. bovis 5'-NT and other bacterial 5'-NT, which are known for their significance as virulence factors and their impact on host immune responses, underscores its potential importance. Targeting this gene could offer a promising strategy for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Mastitis, often caused by bacterial infection, is an inflammatory condition affecting the mammary glands. The condition is particularly prevalent in dairy cattle. Current treatment of bovine mastitis heavily relies on the use of antibiotics. To identify alternative solutions to antibiotic use, we evaluated the antimicrobial activity of 14 cathelicidins reported from 10 animal species. In conjunction, we assessed two bacteriocins against the bovine-mastitis causative bacterial panel, consisting of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus equi. Among the antimicrobial peptides (AMPs), cc-CATH3, ML-CATH, and PD-CATH proved to be highly active (minimum inhibitory concentration of 2–41 μg/mL, 0.2–10.3 μM) against all bacterial strains in the panel and field isolates from milk, with elevated somatic cell counts (≥ 500,000 cells/mL). Of the AMPs tested in this study, ML-CATH presented the highest level of effectiveness in controlling mastitis-associated bacterial strains while also possessing minimal cytotoxicity and functional stability against pH change and a high salt condition. The results of in silico analyses on the biochemical features of 12 helical cathelicidins revealed that the charge of AMPs appears to be a major determinant in killing Gram-negative bacteria. Furthermore, we observed a unique motif, "N(n≥3)-P(n≥1)-N(n≥3)", from the sequences of PMAP-36, cc-CATH3, ML-CATH, and PD-CATH that exhibits potent antimicrobial activity against a broad spectrum of bacteria compared to others. Our findings support the proposition that AMPs could serve as effective antimicrobial alternatives to conventional antibiotics in treating complex animal diseases caused by microbial infection, such as bovine mastitis. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus (S. aureus) mastitis results in economic losses during dairy production. Understanding the biological progression of bovine S. aureus mastitis is vital for its prevention. Lipoteichoic acid is a key virulence factor of S. aureus (aLTA), but the main biological pathways involved in its effect on bovine mammary epithetionallial cells (Mac-T) apoptosis and necrosis have not been fully explored. Folic acid (FA) has anti-inflammatory and anti-apoptotic effects. However, the role of FA in mediating the effects of aLTA on apoptosis and necrosis remains unknown. Results: We found that low concentration of aLTA inhibited apoptosis and necrosis and that high concentration promoted the apoptosis and necrosis of Mac-T. FA pretreatment alleviated high concentration of aLTA induced apoptosis. Through transcriptomic analysis, we found that nuclear receptor subfamily 4 group A (NR4A), which alters the expression of downstream genes involved in apoptosis, proliferation, and inflammation, decreased under stimulation with a low concentration of aLTA and increased under stimulation with a high concentration of aLTA. Under stimulation with a high concentration of aLTA, the expression of the NR4A subfamily could be inhibited by FA. The results showed that aLTA may affect apoptosis and necrosis through the NR4A subfamily by targeting genes involved in bacterial invasion of epithelial cells, the IL-17 signaling pathway, DNA replication, longevity regulation, the cell cycle, and tight junction pathways. We further found that the expression trends of NR4A1 and the target genes of the NR4A subfamily (PTGS2, ESPL1, MCM5, and BUB1B) in the blood of healthy cows (Healthy), subclinical mastitis cows (SCM), and SCM supplemented with FA (SCM_FA) were consistent with those observed at the cellular level in this study. Conclusions: Our study revealed that low and high concentrations of aLTA have opposite effects on apoptosis and necrosis of Mac-T and that FA can alleviate the apoptosis induced by high concentration of aLTA. Transcriptome analysis revealed that the NR4A subfamily play a role in the ability of FA to alleviate the apoptosis and necrosis induced by high concentration of aLTA. [ABSTRACT FROM AUTHOR]

Bovine mastitis remains a major problem in the global dairy cattle industry. The acute invasion of udder by pathogens induces innate immune response as the first defence mechanism in subclinical and clinical mastitis. The aim of the study was to determine inflammatory and regulatory cytokines IL-2, IL-4, TGF-β1, IL-17A, beta-defensin 3 and IL-10 and their potential changes in milk of dairy cows with subclinical and clinical mastitis, and to compare the findings with healthy animals. Milk samples from 15 holstein Friesian breed cows were used in the study. Cows were divided into three groups based on their health status (5 healthy, 5 subclinical and 5 clinical animals). All samples were tested using immunohistochemistry to evaluate IL-2, IL-4, IL-10, IL17A, TGF-β1 and β-Def 3 proteins. Expression of all proteins was detected in all milk samples. High expression of IL-2, IL-4, IL17A, TGF-β1 was detected in healthy cows' milk and in milk of cows with subclinical and clinical mastitis. However, expression of IL-10 and β-Def 3 in milk samples of healthy cows was significantly higher compared to the milk of cows with subclinical and clinical mastitis (p <.001). IL-10 and β-Def 3 can be considered as informative biomarkers in diagnosis of subclinical and clinical mastitis. [ABSTRACT FROM AUTHOR]

ABSTRACT Mastitis in cattle is often caused by microorganism infections in the udder. The three most common pathogens are esculin‐positive streptococci (SC+), coagulase‐negative staphylococci (CNS), and Escherichia coli (E. coli). In a previous study, 10 SNPs were associated with somatic cell score and mastitis in diverse Holstein populations. We tested these SNPs for their effects on individual pathogen presence. Milk and pathogen samples of 3076 Holstein cows were collected from four farms. Samples were excluded if multiple pathogens were present at the same time. Records of the same pathogen within 14 days of each other were counted as one infection. This resulted in 1129 pathogen‐positive samples. Cases and controls were in ratios of 20:80 for SC+, 8:92 for CNS, and 11:89 for E. coli. The lasso, backward, and forward methods were used to narrow down SNPs associated with pathogen presence. The suitability of the SNPs to separate the samples into cases or controls for each pathogen was indicated using ROC curves. The Cochran‐Armitage (CAT) and the Jonckheere‐Terpstra (JTT) tests evaluated the influence of the SNPs on pathogen presence. Finally, a generalised linear mixed model (GLMM) including fixed environmental effects and a random sire effect was fitted to the binary trait of pathogen presence to test for association. In total, six out of the 10 investigated SNPs showed associations with pathogen presence based on the forward method: Two SNPs each for SC+ (rs41588957, rs41257403) and CNS (rs109934030, rs109441194), and three for E. coli (rs109934030, rs41634110, rs41636878). The CAT and GTT tests linked four SNPs (rs41588957, rs41634110, rs109441194, rs41636878) to pathogen presence, two of which were confirmed with the GLMM (rs41634110, rs109441194), with effects on CNS and E. coli. The SNPs linked to CNS and those linked to E. coli explained 13.2% and 13.8% of the variance, compared to 19% and 18.4%, respectively, of the full model with all 10 SNPs. Half of the SNP genotypes previously linked to lower SCS also decreased the probability for pathogen presence and might therefore be targets not just for lower SCS but for a better pathogen resistance.Trial Registration: Not applicable, no new data were collected for this study. [ABSTRACT FROM AUTHOR]

Streptococcus species are important pathogens implicated in bovine mastitis, causing considerable economic losses within the global dairy industry. With the development of multidrug-resistant bacteria, it is crucial to develop novel antibiotic alternatives. Here, we constructed a novel chimeric endolysin, Cly2v, which comprises the Ply2741 CHAP domain (1-155aa) and the PlyV12 CBD domain (146-314aa). Biochemical characterization analysis indicated that Cly2v exhibits a melting temperature of 50.7°C and retains stable bactericidal activity at pH = 3-10. In vitro experiments demonstrated that Cly2v exhibited more efficient bactericidal activity against Streptococcus compared to the parental endolysin Ply2741. Cly2v (25 µg/mL) can effectively inhibit and reduce biofilms formed by Streptococcus, resulting in a 68 and 44% reduction in OD590nm for S. agalactiae X2 and S. uberis 002-1 biofilms. Notably, in a mouse mastitis model, treatment with Cly2v (50 µg/gland) led to a reduction in bacterial load by 2.16 log10CFU/ml and decreased inflammatory cytokine levels in mammary tissue. To our knowledge, this represents the first application of a chimeric endolysin in the treatment of early-stage mouse mastitis induced by streptococci. Additionally, in a systemic infection model, treatment with Cly2v (400 µg/mouse) provided protection rates of up to 100 and 78% against S. agalactiae ATCC13813 infections when challenged for 1 h and 3 h, respectively. Furthermore, a significant reduction in bacterial loads was observed in the blood and organs compared to the PBS group. In summary, Cly2v possesses significant potential as an alternative antibiotic for the treatment of streptococci-induced bovine mastitis and systemic infections. [ABSTRACT FROM AUTHOR]

The wide-spreading of multidrug resistance poses a significant threat to human and animal health. Although antimicrobial peptides (AMPs) show great potential application, their instability has severely limited their clinical application. Here, self-assembled AMPs composed of multiple modules based on the principle of associating natural marine peptide N6 with ß-sheet-forming peptide were designed. It is noteworthy that one of the designed peptides, FFN could self-assemble into nanoparticles at 35.46 µM and achieve a dynamic transformation from nanoparticles to nanofibers in the presence of bacteria, resulting in a significant increase in stability in trypsin and tissues by 1.72–57.5 times compared to that of N6. Additionally, FFN exhibits a broad spectrum of antibacterial activity against multidrug-resistant (MDR) gram-positive (G+) and gram-negative (G−) bacteria with Minimum inhibitory concentrations (MICs) as low as 2 µM by membrane destruction and complemented by nanofiber capture. In vivo mouse mastitis infection model further confirmed the therapeutic potential and promising biosafety of the self-assembled peptide FFN, which can effectively alleviate mastitis caused by MDR Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and eliminate pathogenic bacteria. In conclusion, the design of peptide-based nanomaterials presents a novel approach for the delivery and clinical translation of AMPs, promoting their application in medicine and animal husbandry. [ABSTRACT FROM AUTHOR]

Nocardia cyriacigeorgica causes bovine mastitis, reduces milk quantity and quality, and is often resistant to antimicrobials. Selenomethionine (SeMet) is a form of selenium, which reduces reactive oxygen species (ROS)-mediated apoptosis and intramammary infections. However, the protective effects of SeMet on N. cyriacigeorgica-infected bovine mammary epithelial cells (bMECs) are unclear. The objective of this study was to evaluate whether SeMet mitigated N. cyriacigeorgica-induced inflammatory injury, oxidative damage and apoptosis in bMECs. Cells were cultured with or without being pretreated with 40 µM of SeMet for 12 h, then challenged with N. cyriacigeorgica (multiplicity of infection = 5:1) for 6 h. Although N. cyriacigeorgica was resistant to lincomycin, erythromycin, enrofloxacin, penicillin, amoxicillin, cephalonium, cephalexin, and ceftriaxone, 40 μM SeMet increased cell viability and inhibited lactate dehydrogenase release in infected bMECs. Furthermore, N. cyriacigeorgica significantly induced mRNA production and protein expression of TNF-α, IL-1β, IL-6, and IL-8 at 6 h. Cell membrane rupture, cristae degeneration and mitochondria swelling were evident with transmission electron microscopy. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities were down-regulated after 3, 6, or 12 h, whereas malondialdehyde (MDA) and ROS contents were significantly upregulated, with cell damage and apoptosis rapidly evident (the latter increased significantly in a time-dependent manner). In contrast, bMECs pretreated with 40 μM SeMet before infection, SOD, and GSH-px activities were upregulated (p < 0.05); MDA and ROS concentrations were downregulated (p < 0.05), and apoptosis was reduced (p < 0.05). In conclusion, 40 μM SeMet alleviated inflammation, oxidative stress and apoptosis induced by N. cyriacigeorgica in bMECs cultured in vitro. [ABSTRACT FROM AUTHOR]

Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation. [ABSTRACT FROM AUTHOR]

Background Methicillin-resistant Staphylococcus haemolyticus (MRSH) is an important pathogenic agent of bovine mastitis. Among the prominent clone lineages in dairy cows are MRSH sequence types ST3 and ST42. Little information is available on the complete characterization of SCC mec elements in MRSH. Objective In this study, two clinical isolates of MRSH ST3 and ST42 from bovine mastitis milk were selected, and their nontypable SCC mec structures were compared. Methods Two MRSH strains, MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1, were identified from bovine mastitis milk in Thailand in 2022. Minimum inhibitory concentration was used to screen for antimicrobial resistance susceptibility. Oxford Nanopore Technologies and Illumina sequencing were performed in combination to complete the genome. Their gene organization and structure of SCC mec types were analysed and compared with the whole sequences of other strains in the same sequence types. Results Both MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1 possessed the class C1 mec complex but lacked the ccr gene complex. Notably, MRSH-ST42 strain M81.1 contained a novel variant of C1 mec complex, which consisted of IS 431 - mecA -IS Sha1 - paaZ - upgQ -IS 431 , with IS 431 organized in the same orientation. Apart from class C1 mec and the heavy metal-resistant cluster, the gene composition and order of the SCC mec element varied. In ST3, variations in the SCC mec type, gene content and organization were observed. Conclusions The distinct evolution of the MRSH lineage was indicated by the various SCC mec elements. The insertion of IS Sha1 resulted in a unique variant of class C1 mec complex that demonstrated the important role of the insertion sequence in SCC mec diversification. [ABSTRACT FROM AUTHOR]

Bovine mastitis is a complex infectious disease that develops in the mammary gland, predominantly caused by a bacterial infection of mammary tissue. Genetic variability of mastitis is well established and depends upon different quantitative trait loci (QTL) related to mastitis resistance or susceptibility. The susceptibility is often attributed to single-nucleotide polymorphisms (SNPs) in the variable cow breed genomes. Several global investigative attempts have resulted in studies mapping mastitis to the variations in the relevant genes. Reports have been attributed to dramatic genetic expression changes in Toll-Like Receptor 4 (TLR4) genes in mastitis-positive cows. However, the mechanism behind this variable genetic expression of TLR4 genes has been studied poorly. The present study aims to investigate SCM through various screening tests like somatic cell count (SCC), electric conductivity (EC), pH, and California mastitis test (CMT) in milk samples. This study also aims to investigate possible mechanisms behind this variable expression of TLR4 by comparative SNP evaluation and transcriptional factor profile mining. So that the important genetic mutations and effects thereof can be exploited in selecting specific breeds with higher mastitis resistance and milk yield. Seventy Holstein Frisian (HF) crossbred dairy cows were selected in the present study. The animals were screened based on various diagnostic tests (SCC, pH, EC, and CMT). Blood samples (5 mL) were collected for extraction of DNA followed by amplification of PPR1 and PPR2 of the promoter region and 5′UTR of the bovine TLR4 gene using specific primers. Sanger's enzymatic DNA sequencing technique sequenced the amplified PCR products. Further, the identification of SNPs was done through various bioinformatic tools used in this study. The findings of the present study revealed that CMT, EC, pH, and SCC could be used for the early detection of subclinical mastitis. In the present study, a significant increase in the EC, pH, and SCC in milk samples of animals affected with SCM was found in comparison to the healthy animals. The present study also revealed 16 SNPs falling in TLR4 promoter and 5′ untranslated region (5′UTR) sequences in mastitis-positive genotypes compared to reference genomes. The study also investigates the potential transcriptional factor program deployed in response to variable mastitis development resistance. In the present study, the allelic and genotype frequencies of all SNP variants in the three regions viz., PPR1, PPR2, and 5′UTR, were the same indicating the absence of heterozygous condition at the respective loci. The present study has wide applicability for researchers developing mastitis-resistant breeding programs and the data generated may aid in the selection of better genetic breeds. The transcription factor binding profiles can serve as concrete leads about the studies on bovine mastitis at the molecular level and may also aid global research groups working on transcription factor (TF)-based molecular pathology of mastitis. [ABSTRACT FROM AUTHOR]

Adoption of a rational management in dairy farms would improve the milk quality and farmers' income. In the current study, we aimed to describe bovine mastitis in 32 dairy herds, identify the main cow- and herd-associated risk factors, and analyze both epidemiological along with molecular characteristics of Staphylococcus aureus infecting udders. Based on Californian Mastitis Test and clinical examination, the prevalence of mastitis in cows was 52.25% (116/222), of which 6.3% was clinical mastitis and 45.94% was subclinical mastitis. Overall, 218 (24.54%) quarters suffered from mastitis, whose 29.81% (65/218) infected with S. aureus. Mastitis was lowest in mid-lactation with OR = 0.371 with 95% confidence interval (CI) of 0.141–0.976, and in cows separated from their calves (OR = 0.164, 95% CI 0.056–0.477) than suckler cows. Similar results were obtained from S. aureus related mastitis. To assess the genetic lineages of S. aureus isolates, we determined clonal complexes (CC) using DNA microarray hybridization profiles and performed spa typing. The strains were assigned to nine clonal complexes, and 19 spa types; with CC97 (44.77%), and CC22 (40.29%) were the most predominant lineages and t223 (40.29%), t7136 (10.44%), t359 (8.95%) and t267 (5.97%) were the most common spa types. A total of 88.05% (n = 59) isolates were resistant to at least one tested antibiotic while only 4.47% were multi-drug resistant strains. Higher rates of resistance were observed for penicillin (86.5%) and tetracycline (14.9%) respectively. Our results show the need for adoption of feasible mastitis program with special emphasis on sub-clinical mastitis and associated risk factors. [ABSTRACT FROM AUTHOR]

Simple Summary: Bovine mastitis is one of the most common diseases in farms and has caused huge economic losses to farmers and led to a decline in milk quality. Antibiotics are a common treatment for mastitis. However, due to the emergence of bacterial resistance, they have brought great hidden dangers to public health and safety and also endanger people's health. Therefore, the development of antibiotic alternatives in the treatment of bovine mastitis is more urgent. Studies have found that yak IL-22 has tissue repair and inflammation inhibition effects. In this study, we used the yak IL-22 gene as a potential preparation to construct a mammary gland-specific plasmid for gene therapy for bovine mastitis and evaluated the role of this therapy in the disease, laying a foundation for the possible large-scale application of this preparation in the future. Since the development of dairy farming, bovine mastitis has been a problem plaguing the whole industry, which has led to a decrease in milk production, a reduction in dairy product quality, and an increase in costs. The use of antibiotics to treat mastitis can cause a series of problems, which can bring a series of harm to the animal itself, such as the development of bacterial resistance and dramatic changes in the gut flora. However, the in vivo and in vitro antibacterial activity of yak Interleukin-22 (IL-22) and its application in mastitis caused by Staphylococcus aureus have not been reported. In this study, the mammary gland-specific expression plasmid pLF-IL22 of the yak IL-22 gene was constructed and expressed in MAC-T cells and mammary tissue of postpartum female mice. The coding region of the IL-22 gene in yaks is 573 bp, which can encode 190 amino acids, and the homology difference in the IL-22 gene in yaks is less than 30%, which indicates certain conservation. IL-22 is a hydrophilic protein with a total positive charge of four, the presence of a signal peptide, and the absence of a transmembrane domain. Sufficient expression of IL-22 effectively inhibited the high expression of inflammatory factors caused by Staphylococcus aureus, reduced the symptoms of mammary gland histopathology, and alleviated mastitis. Under the action of IL-22, the intestinal flora of mastitis mice also changed, the abundance of intestinal Bacilli, Prevotellaceae, and Alloprevotella in mice increased after treatment, and the pathogenic bacteria decreased. These findings provide new insights into the potential application of the yak IL-22 gene in the treatment of bovine mastitis in the future. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae (Strep. agalactiae) is bovine mastitis pathogen and has thus became a matter of concern to dairy farms worldwide in terms of economic loss. The aims of this study were to (a) determine virulence genes, and (b) characterize the antimicrobial resistance (AMR) profiles and AMR genes and (c) figure out the relationship between AMR phenotypes and genotypes of Strep. agalactiae isolated from dairy cows in north China. A total of 20 virulence genes and 23 AMR genes of 140 isolates collected from 12 farms in six provinces were studied. The antimicrobial susceptibility of 10 veterinary commonly used antimicrobials were tested using the broth microdilution method. Results showed that all the isolates harbored the virulence genes lacIV, gapC, and dltA. The isolates that harbored the genes lacIII, fbsA, hylB, and cfb exhibited the high prevalence (99.29%), followed by isolates that harbored lacI (98.57%), bibA (97.86%), cylE (97.14%), lacII (92.14%), cspA (52.14%), pavA (25%), bca (2.14%), and scpB (0.71%). The fbsB, lmb, spbI, bac, and ribgenes were not detected. The virulence patterns of B (fbsA_cfb_cylE_ hylB_bibA_cspA_ gapC_dltA_lacIII/IV) and C (fbsA_cfb_ bibA _ gapC_ dltA_lacIV) were dominant, accounting for 97.86% of the isolates. The following AMR genes were prevalent: pbp1A (97.14%), tet(M) (95.00%), lnu (A) (80.71%), erm (B) (75.00%), tet(O) (72.14%), blaZ (49.29%), tet(S) (29.29%), blaTEM (25.71%), erm(A) (17.14%), erm(C) (13.57%), tet (L) (10.71%), linB (2.86%), and erm(TR) (2.86%). The pbp2b, mecA1, mecC, lnu (D), erm(F/G/Q), andmef (A) geneswere not detected. Eighty percent of the isolates harbored AMR genes and were highly resistant to tetracycline, followed by macrolides (10.71%), lincosamides (9.29%) and b-lactams (4.29%). In conclusion, isolates only exhibited well correlation between tetracyclines resistance phenotype and genotype, and almost all isolates harbored intact combination of virulence genes. [ABSTRACT FROM AUTHOR]

Bovine mastitis is one of the main inflammatory diseases that can affect the udder during lactation. Somatic cell counts and sometimes microbiological tests are routinely adopted during monitoring diagnostics in dairy herds. However, subclinical mastitis is challenging to identify, reducing the possibility of early treatments. The main aim of this study was to investigate the miRNome profile of extracellular vesicles isolated from milk as potential biomarkers of subclinical mastitis. Milk samples were collected from a total of 60 dairy cows during routine monitoring tests. Small RNA sequencing technology was applied to extracellular vesicles of milk samples collected from cows classified according to the somatic cell count to identify differences in the miRNome between mastitic and healthy cows. A total of 1997 miRNAs were differentially expressed between both groups. Among them, 68 miRNAs whose FDRs were < 0.05 were mostly downregulated, with only one upregulated miRNA (i.e., miR-361). Functional analysis revealed that miR-455-3p, miR-503-3p, miR-1301-3p and miR-361-5p are involved in the regulation of several biological processes related to mastitis, including immune system-related processes. This study suggests the involvement of extracellular vesicle-derived miRNAs in the regulation of mastitis. Moreover, these findings provide evidence that miRNAs from milk extracellular vesicles can be used to identify biomarkers of mastitis. However, further studies must be conducted to validate these miRNAs, especially for subclinical diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: Bovine mastitis caused by Escherichia coli compromises animal health and inflicts substantial product losses in dairy farming. It may manifest as subclinical through severe acute disease and can be transient or persistent in nature. Little is known about bacterial factors that impact clinical outcomes or allow some strains to outcompete others in the mammary gland (MG) environment. Mastitis-associated E. coli (MAEC) may have distinctive characteristics which may contribute to the varied nature of the disease. Given their high levels of intraspecies genetic variability, virulence factors of commonly used MAEC model strains may not be relevant to all members of this group. Methods: In this study, we sequenced the genomes of 96 MAEC strains isolated from cattle with clinical mastitis (CM). We utilized clinical severity data to perform genome-wide association studies to identify accessory genes associated with strains isolated from mild or severe CM, or with high or low competitive fitness during in vivo competition assays. Genes associated with mastitis pathogens or commensal strains isolated from bovine sources were also identified. Results: A type-2 secretion system (T2SS) and a chitinase (ChiA) exported by this system were strongly associated with pathogenic isolates compared with commensal strains. Deletion of chiA from MAEC isolates decreased their adherence to cultured bovine mammary epithelial cells. Discussion: The increased fitness associated with strains possessing this gene may be due to better attachment in the MG. Overall, these results provide a much richer understanding of MAEC and suggest bacterial processes that may underlie the clinical diversity associated with mastitis and their adaptation to this unique environment. [ABSTRACT FROM AUTHOR]

The application of antibiotics in the poultry and veterinary sectors is very common practice in India. Owing to the seriousness of antimicrobial resistance (AMR), the present study has illustrated the overall scenario of AMR in the poultry and veterinary sectors in India through an in-depth scoping review and key informant interview (KII). In the poultry sector, most of the studies reviewed have reported resistant bacteria isolated from chicken meat, eggs, cloacal swabs, and fecal samples, and only a few have reported the presence of resistant bacteria in and around the environment of poultry farms. The major resistant bacteria that have been reported are E. coli, Salmonella spp., S. aureus, Campylobacter jejuni, and K. pneumoniae. These bacterial isolates exhibited resistance to various antibiotics, such as azithromycin (21.43%), tetracycline (11.30-100%), chloramphenicol (4.76-100%), erythromycin (75-83.33%), ciprofloxacin (5.7-100%), gentamicin (17-100%), amikacin (4.76%), cotrimoxazole (42.2-60%), trimethoprim (89.4%), ceftriaxone (80%), and cefotaxime (14.29-70%). Like the poultry sector, different antibiotics are also used for treating clinical and subclinical bovine mastitis, which is one of the major problems plaguing the dairy sector. Several AMR bacterial strains, such as E. coli, Staphylococcus aureus, S. epidermidis, and Klebsiella pneumoniae, have been reported by many researchers and showed resistance against tetracycline (74%), oxytetracycline (47.37%), ciprofloxacin (51%), streptomycin (57.89%), cephalosporin (100%), and trimethoprim (70%). The KIIs have revealed several reasons behind these AMR scenarios, of which the growing need for the production of food animals and their products with inadequate infrastructure and a lack of proper knowledge on farm management among the farmers are the major ones. Though several government legislations and policies have been laid down, proper implementation of these policies, strict surveillance on antibiotic application in the poultry and veterinary sectors, awareness generation among farmers, and infrastructure development can help minimize the development and transmission of AMR bacteria within and from these sectors. [ABSTRACT FROM AUTHOR]

Background: The dairy sector faces formidable obstacles in the early detection of the bacteria causing subclinical mastitis and in the proactive treatment of those cases. The objective of the study was to access multiplex PCR assays (MPCR) and conventional methods for the concurrent identification of significant bacteria that cause sub-clinical mastitis and to compare these methods. 200 samples of pooled milk from cows and buffaloes in Rajasthan were gathered between 2020 and 2021. Methods: The primary bacterial pathogens in milk samples were identified using conventional methods such multiplex PCR, biochemical testing and culture. Result: From 200 combined samples of cow and buffalo milk, the traditional approach was able to extract 97 different strains. Staphylococcus aureus 54 (27%), Streptococcus spp. 30 (15%) and E. coli 13 (6.5%) were shown to be prevalent as single or mixed infections, respectively. Staphylococcus was the main pathogen that was discovered. concurrently, S. aureus, Streptococcus and E. coli. Direct detection of Staphylococcus 65 (32.5%), Streptococcus 37 (18.5%), E. coli and 16 (8%) by multiplex PCR was found in milk samples. The analysis revealed that because multiplex PCR assays have higher specificity and sensitivity than conventional procedures, they are more reliable. The multiplex PCR method employed in the current study was a simple and quick technique to identify the major pathogens and it has the potential to be a very helpful tool for determining the pathogens that cause environmental mastitis and evaluating the health of the herd. [ABSTRACT FROM AUTHOR]

Mastitis is one of the most expensive diseases of dairy animals and subclinical mastitis occupies the major proportion of mastitis. Prevalence of mastitis is highly dependent upon type of breed, climatic and housing conditions, management practices, stage of lactation, etc. and hence a study was conducted to screen the prevalence of subclinical mastitis (SCM), associated risk factors, and prevalent pathogens in small backyard and large organized dairy units in four districts in the west coast of India. A total of 240 cattle were screened for subclinical mastitis and milk samples from SCM cases were screened for the presence of major mastitis-associated pathogens. Information on managemental practices was collected and risk factors were analyzed statistically. The prevalence of clinical and subclinical mastitis was 9.93% (27/272) and 30.83% (74/240), respectively. Staphylococcus aureus, coagulase-negative Staphylococci, E coli, and Bacillus spp. were the major pathogens isolated from SCM cases. The study revealed that SCM is highly prevalent in dairy farms of coastal districts and higher in crossbred than the indigenous cattle. The absence of regular shed cleaning and the use of milking machines were significantly associated with higher SCM prevalence. Post-milking iodine teat dip was carried out by 27.5% and 88.89% of the small and large farmers and none of the farmers had adopted dry cow therapy. Around 10.0% of small farmers and 77.78% of the large farmers used milking machines. The high prevalence of SCM shows the need for active surveillance programs and the application of preventive measures in the area. [ABSTRACT FROM AUTHOR]

Mycoplasma bovis is believed to be a major cause of pneumonia, mastitis, and arthritis in cattle. Mastitis is the most prevalent production-related disease in dairy herds. The present study aimed to improve the current knowledge on the virulence factors of M. bovis-induced mastitis in dairy cows. To this end, sampling was done from farms in Chaharmahal and Bakhtiari province. The samples were monitored for growth in a specific PPLO medium in a CO2 incubator. Simultaneously, the DNA of the samples was extracted and PCR was used to detect M. bovis strains. After identifying susceptible isolates of M. bovis, P48, alpha-enolase, P81, and LppB genes were investigated in M. bovis positive PCR samples. In total, out of 204 samples, 11 isolates suspected of M. bovis were obtained (5.39%) via culture method and 21 positive samples (10.29%) were obtained through PCR. 57.14 (12/21), 66.66 (14/21), and 52.38 (11/21)% of the samples were positive concerning the presence of P48, alpha-enolase and LppB genes, respectively. P81 was not detected in any of the positive samples. The results of the present study showed that the presence of M. bovis in dairy herds in Chaharmahal and Bakhtiari could be considered among the factors that cause mastitis in cattle. alphaenolase gene was found to be more abundant than the other selected genes, which could play an important role in the future strategic measure to develop preventive measures against mycoplasmal mastitis. [ABSTRACT FROM AUTHOR]

Simple Summary: Bovine mastitis is the most common disease affecting the dairy industry and is mostly caused by intramammary infections (IMIs) due to mastitis pathogens. In this retrospective study we investigated the distribution of mastitis pathogens in all quarter milk samples (QMSs) submitted to the Bavarian Animal Health Service (TGD) in Southern Germany between 2014 and 2023. Overall, 19% of the QMSs contained mastitis pathogens and the most frequently isolated pathogens, in decreasing frequency, were non-aureus staphylococci (NAS), Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis, and Sc. dysgalactiae. However, differences were found in the distribution of the mastitis pathogens depending on the mastitis status of the quarter from which the samples originated and the time of year. The objective of this study was to investigate the distribution of mastitis pathogens in quarter milk samples (QMSs) submitted to the laboratory of the Bavarian Animal Health Service (TGD) between 2014 and 2023 in general, in relation to the clinical status of the quarters, and to analyze seasonal differences in the detection risk. Each QMS sent to the TGD during this period was analyzed and tested using the California Mastitis Test (CMT). Depending on the result, QMSs were classified as CMT-negative, subclinical, or clinical if the milk character showed abnormalities. Mastitis pathogens were detected in 19% of the QMSs. Non-aureus staphylococci (NAS) were the most common species isolated from the culture positive samples (30%), followed by Staphylococcus (S.) aureus (19%), Streptococcus (Sc.) uberis (19%), and Sc. dysgalactiae (9%). In culture-positive QMSs from CMT-negative and subclinically affected quarters, the most frequently isolated pathogens were NAS (44% and 27%, respectively), followed by S. aureus (25% and 17%, respectively) and Sc. uberis (8% and 22%, respectively). In QMSs from clinically affected quarters, the most frequently isolated pathogens were Sc. uberis (32%), S. aureus (13%), Sc. dysgalactiae (11%), and Escherichia (E.) coli (11%). The distribution of NAS and Sc. uberis increased throughout the study period, while that of S. aureus decreased. From June to October, QMSs from subclinically affected quarters increased and environmental pathogens, such as Sc. uberis, were detected more frequently. In conclusion, this study highlights the dynamic nature of the distribution of mastitis pathogens, influenced by mastitis status and seasonal factors. Environmental pathogens still play an important role, especially in clinical mastitis and seasonal dependency, with the number of positive samples continuing to increase. It is therefore essential to continue mastitis control measures and to regularly monitor the spread of mastitis pathogens in order to track trends and adapt targeted prevention measures. [ABSTRACT FROM AUTHOR]

Somatic cell count detection is the daily work of dairy farms to monitor the health of cows. The feasibility of applying near-infrared spectroscopy to somatic cell count detection was researched in this paper. Milk samples with different somatic cell counts were collected and preprocessing methods were studied. Variable selection algorithm based on hybrid strategy and modelling method based on ensemble learning were explored for somatic cell count detection. Detection model was used to diagnose subclinical mastitis and the results showed that near-infrared spectroscopy could be a tool to realize rapid detection of somatic cell count in milk. [ABSTRACT FROM AUTHOR]

Importance: Identifying bovine mastitis agents using molecular methods to reveal their phylogenetic relationships and antimicrobial resistance profiles is essential for developing up-to-date databases in mastitis cases that cause severe economic losses. Objective: This study examined bacterial mastitis agents in cows with clinical and subclinical mastitis observed in various dairy cattle farms to reveal their phylogenetic relationships and antibiotic resistance properties. Methods: Sixty-two clinical and subclinical bovine mastitis milk samples were collected from 15 dairy farms. The polymerase chain reaction (PCR) was used to amplify the 16S rRNA gene regions of the bacteria. The 16S rRNA gene sequences obtained from sequencing include the V4-V6 regions. The strains were compared using a similarity analysis method that produced phylogenetic trees using the Molecular Evolutionary Genetics Analysis 11 program. Antibiotic susceptibilities were determined using the Kirby-Bauer disk diffusion method. Results: Sixty-three bacteria were isolated and identified in this study. The most isolated bacteria from all mastitis cases were Staphylococcus spp. (30.2%), Escherichia coli (25.4%), Streptococcus spp. (14.3%), and Aerococcus spp. (7.9%), respectively. The phylogenetic trees were drawn from the 16S rRNA sequences. Some of these bacteria showed resistance to different types of antibiotics at varying rates. Conclusions and Relevance: The bacteria isolated in this study originated from environmental sources. Regular cleaning of barns and proper hygiene practices are essential. Regular screenings for mastitis should be conducted in herds instead of the random or empirical use of antibiotics. [ABSTRACT FROM AUTHOR]

Bovine clinical mastitis is an economically important disease in dairy industry worldwide resulting in reduction of milk yield and quality. Among mycotic mastitis, Candida spp. are commonly occurring opportunistic mycosis in immunocompromised animals. The microorganism's causing mastitis has high zoonotic potential and has been linked with rapid growth and introduction of antimicrobial resistance between animals and humans. The present study was conducted to isolate and identify the common pathogenic Candida spp. from bovine mastitis cases in India. The isolates were phenotypically characterized by culturing on Sabouraud's dextrose agar, Hichrome Candida differential agar and germ tube production test. Antibiogram was also performed to determine their antifungal activities. The phenotypically positive isolates were confirmed by polymerase chain reaction (PCR) and genetically analyzed by targeting 18S-ITS1-5.8S-ITS2-28S region specific for Candida spp. and identified the yeast at the species level. The antibiogram showed the isolates were highly sensitive with ketoconazole, clotrimazole and miconazole. The PCR assay identified C. lusitaniae and C. tropicalis based on the two distinctive amplicon sizes (592bp and 737bp) respectively. Also, the sequence analysis and phylogeny confirmed C. lusitaniae in six sequences and C. tropicalis in one sequence. It is worth noting that in this study, the species identification was consistent among PCR and genetic analysis. Therefore, the PCR based identification system of the fungal species performed in this study could be an efficient and time saving tool for early diagnosis of clinical mastitis in milch animal, which allows prompt control and application of speedy effective treatment. [ABSTRACT FROM AUTHOR]

Abstract Mastitis, often caused by bacterial infection, is an inflammatory condition affecting the mammary glands. The condition is particularly prevalent in dairy cattle. Current treatment of bovine mastitis heavily relies on the use of antibiotics. To identify alternative solutions to antibiotic use, we evaluated the antimicrobial activity of 14 cathelicidins reported from 10 animal species. In conjunction, we assessed two bacteriocins against the bovine-mastitis causative bacterial panel, consisting of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus equi. Among the antimicrobial peptides (AMPs), cc-CATH3, ML-CATH, and PD-CATH proved to be highly active (minimum inhibitory concentration of 2–41 μg/mL, 0.2–10.3 μM) against all bacterial strains in the panel and field isolates from milk, with elevated somatic cell counts (≥ 500,000 cells/mL). Of the AMPs tested in this study, ML-CATH presented the highest level of effectiveness in controlling mastitis-associated bacterial strains while also possessing minimal cytotoxicity and functional stability against pH change and a high salt condition. The results of in silico analyses on the biochemical features of 12 helical cathelicidins revealed that the charge of AMPs appears to be a major determinant in killing Gram-negative bacteria. Furthermore, we observed a unique motif, “N(n≥3)-P(n≥1)-N(n≥3)”, from the sequences of PMAP-36, cc-CATH3, ML-CATH, and PD-CATH that exhibits potent antimicrobial activity against a broad spectrum of bacteria compared to others. Our findings support the proposition that AMPs could serve as effective antimicrobial alternatives to conventional antibiotics in treating complex animal diseases caused by microbial infection, such as bovine mastitis.

Abstract The wide-spreading of multidrug resistance poses a significant threat to human and animal health. Although antimicrobial peptides (AMPs) show great potential application, their instability has severely limited their clinical application. Here, self-assembled AMPs composed of multiple modules based on the principle of associating natural marine peptide N6 with ß-sheet-forming peptide were designed. It is noteworthy that one of the designed peptides, FFN could self-assemble into nanoparticles at 35.46 µM and achieve a dynamic transformation from nanoparticles to nanofibers in the presence of bacteria, resulting in a significant increase in stability in trypsin and tissues by 1.72–57.5 times compared to that of N6. Additionally, FFN exhibits a broad spectrum of antibacterial activity against multidrug-resistant (MDR) gram-positive (G+) and gram-negative (G−) bacteria with Minimum inhibitory concentrations (MICs) as low as 2 µM by membrane destruction and complemented by nanofiber capture. In vivo mouse mastitis infection model further confirmed the therapeutic potential and promising biosafety of the self-assembled peptide FFN, which can effectively alleviate mastitis caused by MDR Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and eliminate pathogenic bacteria. In conclusion, the design of peptide-based nanomaterials presents a novel approach for the delivery and clinical translation of AMPs, promoting their application in medicine and animal husbandry. Graphical Abstract

Abstract Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation. Graphical Abstract

Early detection of Mycoplasmal mastitis is greatly hampered by late seroconversion, slow growth of Mycoplasma organisms, intermittent shedding, and the high cost of diagnostic tests. To improve future diagnostic development, examining the available techniques is necessary. Accordingly, the present study systematically reviewed M. bovis diagnostic studies published between January 2000 and April 2023 utilizing the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol. The protocol registration was performed according to the Open Science Framework (osf.io/ug79h), and the electronic search was conducted in the World Catalog, Mendeley, ProQuest, ScienceDirect, Semantic Scholar, PubMed, Google Scholar, Prime Scholar, and PubMed Central databases using a Boolean operator and inclusion and exclusion criteria. Of the 1194 pieces of literature retrieved, 67 studies were included. Four broad categories of up to 16 diagnostic approaches were reported: microbial culture, serological, DNA-based, and mass spectrometry. Overall, DNA-based techniques were the most published (48.0%), with recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) as the most promising user-friendly, equipment-free techniques. On the other hand, mass spectrometry was reported as the least utilized (2.9%) given the high equipment cost. Though costly and laboratory-allied, DNA-based techniques, particularly PCRs, were reported as the most rapid and specific approach.

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. [ABSTRACT FROM AUTHOR]

The mastitis caused by Klebsiella pneumoniae (K. pneumoniae) is increasing in the dairy cows. To investigate the epidemic of K. pneumoniae of China, 131 strains were isolated from 495 clinical mastitis milk samples (26.5%) from 14 provinces in China. Among the isolates, K57 was the dominant serotype (45.0%) and 19 (14.5%) isolates were identified as hypervirulent K. pneumoniae (hvKP). The mrkA, entB, wabG and fimH genes were prevalent virulence genes while rmpA, magA, and ycf were not found in K. pneumoniae. Furthermore, K. pneumoniae had serious antibiotic resistance and multiple β-lactamase genes, including blaTEM, blaSHV, blaNDM, blaCTX-M, blaDHA, and blaKPC. Biofilm was an important factor in bacterial resistance and persistent infection, and 77.1% isolates could form biofilm. Although acylated homoserine lactone (AHL, a Gram-negative bacterial quorum sensing signal molecule) was not confirmed among the K. pneumoniae isolates, exogenous AHLs could reduce the biofilm formation ability of the K. pneumoniae strains. Three new ST types (ST6781, ST6782, and ST6783) were first identified in this study. The MLST phylogenetic tree showed the distribution of mastitis associated K. pneumoniae strains had no regular pattern, which confirmed high genomic diversity of mastitis associated K. pneumoniae. In conclusion, the high rate of isolation and serious antibiotic resistance of K. pneumonia were found in this study and indicated a potential threat to public health from the food chain. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the activation pathway(s) triggered by Minthostachys verticillata essential oil (EO) in bovine mammary epithelial cells (MAC-T) challenged with a strain of bovine Staphylococcus aureus. MAC-T cells were stimulated with EO, S. aureus or pre-treated with EO and then challenged with S. aureus. Cytokine's release was measured by ELISA. The mRNA for TLR2, TLR4, NOD2, MyD88 and NFκB was quantified by RT-qPCR. S. aureus adherence and internalization was also evaluated. MAC-T cells stimulated with S. aureus synthesized high levels of IL-1ß and IL-6 were kept up to 48 h, while IL-4 levels were not altered. Cells pre-treated with EO for 2 and 6 h and then challenged with S. aureus showed a significant increase of IL-1β and IL-6. However, in these cells, a decrease in IL-1ß and IL-6 levels and an increase of IL-4 values was observed from 24 h. No significant increase in the expression levels of TLR2 or NOD2 was detected in all stimulated cells. However, the expression of TLR4, MyD88 and NFκB was increased in cells stimulated with S. aureus at 2 and 6 h as well as in cells pre-treated with EO between 2 and 6 h and then challenged with S. aureus. The NFκB expression levels was similar to control at 24 h in all stimulated cells, although pro-inflammatory cytokine levels and TLR4 and MyD88 expression levels remained high in cells stimulated with S. aureus. This results suggested the activation of other pathways independent of MyD88 by the pathogen that involucrated the activation of others transcription factors. Pre-treatment with EO during 2, 6 and 24 h did not affect S. aureus adherence but decreased its internalization. In conclusion, pre-treatment with EO increased the IL-1β and IL-6 synthesis during the first hours post-challenged with S. aureus up-regulating TLR4/MyD88/NFκB pathway. Furthermore, EO increased the IL-4 levels from 6 to 24 h down-regulating the NFκB and possibly other transcription factors activated by the pathogen, which decreased its internalization into MAC-T cells. [ABSTRACT FROM AUTHOR]

Group B Streptococcus sequence type 103 is known primarily as a bovine mastitis pathogen. In Brazil, it has circulated in cattle and humans since the 1990s. It lacks scpB and, in humans, was found only among carriage isolates. Bovine–human interspecies transmission may have contributed to its evolution and spread.

Background and Aim: Bovine mastitis is an inflammation of the mammary gland of dairy cattle that causes economic losses due to poor quantity and quality of milk. The extensive or incorrect use of antibiotics has increased in the veterinary field, leading to the emergence of antibiotic-resistant pathogens worldwide. This study aimed to investigate bovine mastitis bacterial pathogens in Sakon Nakhon, Thailand. Materials and Methods: A total of 35 dairy farms were screened for clinical and subclinical mastitis using the California Mastitis Test and clinical examination. Polymerase chain reaction was used to characterize bacterial species-induced mastitis (380 isolates) in cattle and antimicrobial resistance genes, and disk diffusion and broth microdilution were used to characterize antimicrobial susceptibility. Results: The prevalence of Staphylococcus epidermidis (38.10%; 32/84)-induced mastitis in cattle was considerably high, followed by Streptococcus agalactiae (33.33%), Streptococcus uberis (25%), Klebsiella pneumoniae (8.33%), and Staphylococcus aureus (4.76%). In this study, Staphylococcus spp. isolates demonstrated 100% susceptibility to cefoxitin, and no antibiotic-resistance genes were identified. Tetracycline (TET) and macrolide-resistant genes of Streptococcus spp. revealed that tetM was predominant in 55.63% (79/142), followed by tetS + erm(B) (16.90%). Antibiotic susceptibility tests revealed the following resistance profiles to bacterial species: TET (85.92%), clindamycin (29.58%), erythromycin (15.49%), levofloxacin (14.08%), and penicillin (0%). Gram-negative bacterial isolates (K. pneumoniae [8.33%], Klebsiella variicola [2.38%], Klebsiella quasipneumoniae [1.19%], and Escherichia coli [1.19%]) were recovered and still susceptible to meropenem (100%), ceftazidime (97.06%), ceftriaxone (79.41%), and ciprofloxacin (79.41%). Conclusion: This result suggested that mastitis pathogens in this area were susceptible to most antimicrobials, with the exception of streptococci against TET. In this study, limited data were available including one from small-holder dairy farms and study only dairy farms in Sakon Nakhon, Thailand. So, more farms should be included in the future studies.

Bovine mastitis is a worldwide disease affecting dairy cattle and causes major economic losses in the dairy industry. Recently, the emergence of microbial resistance to the current antibiotics complicates the treatment protocol which necessitates antibiotic stewardship and further research to find new active compounds. Recently, phytobiotics have gained interest in being used as an alternative to antibiotics in the poultry industry as an antibiotic stewardship intervention. This study evaluated the in vitro antibacterial activity of 16 flavonoids against bovine mastitis pathogens. Two flavones: 2-(4-methoxyphenyl)chromen-4-one (1) and 2-(3-hydroxyphenyl)chromen-4-one (4) showed inhibition of the growth of Klebsiella oxytoca with MIC values range (25–50 µg mL− 1) followed by a structure-activity relationship (SAR) study indicating that the presence of a hydroxyl group at C-3' or methoxy at C-4' increases the activity against Klebsiella oxytoca while the presence of hydroxyl group at C-7 decreases the activity. Furthermore, a structure-based drug development approach was applied using several in silico tools to understand the interactions of active flavones at the active site of the DNA gyrase protein. Compound (4) showed a higher docking score than quercetin (standard) which is known to have antibacterial activity by inhibiting the DNA gyrase. In addition, the structure-based pharmacophores of compound (4) and quercetin showed similar pharmacophoric features and interactions with DNA gyrase. Based on our findings, compounds (1) and (4) are promising for further study as potential anti-microbial phytochemicals that can have a role in controlling bovine mastitis as well as to investigate their mechanism of action further. [ABSTRACT FROM AUTHOR]

Background: Bovine mastitis results in significant economic losses for the dairy industry globally due to milk production losses and decreased herd efficiency. This research aimed to isolate, select, and characterize indigenous lactobacilli with probiotic properties. A total of 40 lactobacilli were isolated from healthy milk samples of cattle and identified at the species level through 16S rDNA sequencing. All isolates were initially screened for antimicrobial activity, and selected isolates underwent in vitro assessment of probiotic properties. Results: Among the lactobacilli isolates, varying levels of activity (9 to 19 mm) against cattle mastitogens; Stapylococcus aureus (Staph. aureus), Escherichia coli (E. coli) and Streptococcus dysgalactiae (Strep. dysgalactiae) were observed in the well diffusion assay. These isolates demonstrated auto-aggregation (ranging from 14.29 ± 0.96% to 62.11 ± 1.09%) and co-aggregate (ranging from 9.21 ± 0.14% to 55.74 ± 0.74%) with mastitogens after 2 h. Lactobacillus (Lb.) plantarum CM49 showed sensitivity to most antibiotics tested and exhibited strong inhibitory effects, with mean log10 reductions of 3.46 for Staph. aureus, 2.82 for E. coli, and 1.45 for Strep. dysgalactiae in co-culture experiments. Furthermore, Lb. plantarum CM49 significantly decreased the adhesion rate of mastitogens on the bovine mammary cell line and mouse model, demonstrating its potential effectiveness in preventing mastitis. Conclusion: It is concluded that Lb. plantarum CM49 has remarkable probiotic potential with activity against cattle mastitogens in the laboratory and cell culture and competitively excludes mastitogens from bovine mammary cells and ameliorates Staph. aureus-induced mastitis in mice. [ABSTRACT FROM AUTHOR]

Background: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. Results: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus (2.7%), E. durans (1.2%), E. galinarum (0.8%). Conclusions: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented. [ABSTRACT FROM AUTHOR]

Bovine mastitis caused by infectious pathogens, mainly Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), constitutes a major destructive challenge for the dairy industry and public health. Berberine chloride (BER) and Cyperus rotundus possess a broad spectrum of anti-inflammatory, antioxidant, antibacterial, and antiproliferative activities; however, their bioavailability is low. This research aimed first to prepare an ethanolic extract of Cyperus rotundus rhizomes (CRE) followed by screening its phytochemical contents, then synthesis of BER and CRE loaded chitosan nanoparticles (NPs) (BER/CH-NPs and CRE/CH-NPs), afterward, the analysis of their loading efficiency in addition to the morphological and physicochemical characterization of the formulated NPs employing Scanning Electron Microscopy (SEM), Zeta Potential (ZP), Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) assessments compared to their crude forms to evaluate the enhancement of bioavailability and stability. Isolation of bacterial strains from the milk of mastitic cows, used for induction of mammary gland (MG) inflammation in female albino rats, and a preliminary investigation of the prophylactic oral doses of the prepared NPs against S. aureus-induced mastitis in female rats. The minimal inhibitory concentration (MIC) of BER/CH-NPs and CRE/CH-NPs is 1 mg/kg b.w. BER/CH-NPs and CRE/CH-NPs alone or in combination show significant (P ≤ 0.05) DPPH radical scavenging activity (69.2, 88.5, and 98.2%, respectively) in vitro. Oral administration of BER/CH-NPs and CRE/CH-NPs to mastitis rats significantly (P ≤ 0.05) attenuated TNF-α (22.1, 28.6 pg/ml), IL-6 (33.4, 42.9 pg/ml), IL-18 (21.7, 34.7 pg/ml), IL-4 (432.9, 421.6 pg/ml), and MPO (87.1, 89.3 pg/ml) compared to mastitis group alongside the improvement of MG histopathological findings without any side effect on renal and hepatic functions. Despite promising results with BER and CRE nanoparticles, the study is limited by small-scale trials, a focus on acute administration, and partially explored nanoparticle-biological interactions, with no economic or scalability assessments. Future research should address these limitations by expanding trial scopes, exploring interactions further, extending study durations, and assessing economic and practical scalability. Field trials and regulatory compliance are also necessary to ensure practical application and safety in the dairy industry. In conclusion, the in vitro and in vivo results proved the antioxidant and anti-inflammatory efficacy of BER/CH-NPs and CRE/CH-NPs in low doses with minimal damage to the liver and kidney functions, supposing their promising uses in mastitis treatment. [ABSTRACT FROM AUTHOR]

Background: Several diagnostic environments in Uganda lack real-time, robust and high-throughput technologies for comprehensive typing of microbes, which is a setback to infectious disease surveillance. This study combined various wet laboratory diagnostics to understand the epidemiology of pathogenic staphylococci isolated from animals in Uganda and the implications for global health security priorities. Methods: A retrospective study was conducted employing records and pathogenic staphylococci (from animals) archived at the Central Diagnostic Laboratory (CDL), Makerere University, Uganda, between January 2012 and December 2019. The bacteria were speciated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tested for virulence factors [beta lactamases, lecithinase, deoxyribonuclease (DNase), haemolysins] and resistance to ten antimicrobials of clinical and veterinary relevance. Tetracycline and methicillin resistance genes were also tested. Results: The prevalent diseases were mastitis in cattle and skin infections in dogs. Of the 111 staphylococci tested by MALDI-TOF MS, 79 (71.2%) were Staphylococcus aureus, 27 (24.3%) were Staphylococcus pseudintermedius and 5 (4.5%) were Staphylococcus schleiferi. All these strains expressed haemolysins. The prevalence of strains with lecithinase, penicillinase, cephalosporinase and DNase was 35.9% (14/39), 89.7% (35/39), 0.0% (0/39) and 87.2% (34/39), respectively. Staphylococci were primarily resistant to early penicillins (over 80%), tetracycline (57.7%), and chloramphenicol (46.2%). Minimal resistance was noted with cloxacillin (0.0%), ciprofloxacin (9.6%), and cefoxitin (3.8%). The prevalence of multidrug resistance (MDR) was 78.8% for general staphylococci, 82.2% for S. aureus, 73.1% for S. pseudintermedius, and 60.0% for S. schleiferi. Multidrug resistant staphylococci were significantly more prevalent in the cattle isolates than in the dog isolates (P < 0.05). The prevalence of methicillin-resistant staphylococci (MRS) tested by resistance to cefoxitin and mecA carriage was 3.8%. These four strains were all isolated from dog skin infections. The tetK gene was the most predominant (35.4%), followed by tetM (25.0%). Conclusion: In resource-constrained settings, the approach of integrated diagnostics promises sustainable disease surveillance and the addressing of current capacity gaps. The emergence of MRS (zoonotic bacteria) in companion animals creates a likelihood of reduced treatment options for related human infections, a threat to global health. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus , particularly multi-drug resistant strains, presents significant challenges in dairy farming due to its role in causing bovine mastitis, which leads to substantial economic losses and limited treatment options. Seeking alternative therapies, we investigated the potential of a topical formulation derived from the medicinal herb Salvia officinalis to combat S. aureus growth and biofilms associated with bovine mastitis. Through systematic extraction in different solvents and fractionation by column chromatography, we isolated and identified three key multicyclic terpenoids—ferruginol, sugiol, and sclareol—exhibiting significant antimicrobial activity. The formulation effectively inhibited biofilm formation, with minimum inhibitory concentration (MIC) values ranging from 0.09 to 0.74 mg ml−1 against clinical S. aureus strains, comparable to or lower than those of the pure compounds. Moreover, it displayed robust anti-adhesive properties, reducing biofilm formation by 20%–79% at subinhibitory concentrations. Furthermore, the formulation successfully disrupted pre-existing biofilms, achieving reductions ranging from 30% to 82%. Cytotoxicity assays confirmed the safety of the formulation on mammary epithelial cells, with cell viability maintained at 100% at MIC. Our findings underscore the therapeutic potential of Sa. officinalis -derived compounds in managing bovine mastitis caused by S. aureus , emphasizing their antimicrobial efficacy and safety profile. [ABSTRACT FROM AUTHOR]

Mastitis is a significant inflammatory condition of the mammary gland in dairy cows. It is caused by bacterial infections and leads to substantial economic losses worldwide. The disease can be either clinical or sub-clinical and presents challenges such as reduced milk yield, increased treatment costs, and the need to cull affected cows. The pathogenic mechanisms of mastitis involve the activation of Toll-like receptors (TLRs), specifically TLR2 and TLR4. These receptors play crucial roles in recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses through the NF-κB signaling pathway. Recent in vitro studies have emphasized the importance of the TLR2/TLR4/NF-κB signaling pathway in the development of mastitis, suggesting its potential as a therapeutic target. This review summarizes recent research on the role of the TLR2/TLR4/NF-κB signaling pathway in mastitis. It focuses on how the activation of TLRs leads to the production of proinflammatory cytokines, which, in turn, exacerbate the inflammatory response by activating the NF-κB signaling pathway in mammary gland tissues. Additionally, the review discusses various bioactive compounds and probiotics that have been identified as potential therapeutic agents for preventing and treating mastitis by targeting TLR2/TLR4/NF-κB signaling pathway. Overall, this review highlights the significance of targeting the TLR2/TLR4/NF-κB signaling pathway to develop effective therapeutic strategies against mastitis, which can enhance dairy cow health and reduce economic losses in the dairy industry. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Bovine mastitis is a prevalent infectious disease in dairy herds worldwide, resulting in substantial economic losses. Staphylococcus aureus is a major cause of mastitis in animals, and its antibiotic resistance poses challenges for treatment. Recently, renewed interest has focused on the development of alternative methods to antibiotic therapy, including bacteriophages (phages), for controlling bacterial infections. In this study, 2 lytic phages, vB_SauM_JDYN (JDYN) and vB_SauM_JDF86 (JDF86), were isolated from the cattle sewage effluent samples collected from dairy farms in Shanghai. The 2 phages have a broad bactericidal spectrum against Staphylococcus of various origins. Genomic and morphological analyses revealed that the 2 phages belonged to the Myoviridae family. Moreover, JDYN and JDF86 remained stable under a wide temperature and pH range and were almost unaffected in chloroform. In this study, we prepared a phage cocktail (PHC-1) which consisted of a 1:1:1 ratio of JDYN, JDF86, and SLPW (a previously characterized phage). We found that PHC-1 showed the strongest bacteriolytic effect and the lowest frequency of emergence of bacteriophage insensitive mutants compared with monophages. Bovine mammary epithelial cells and lactating mice mastitis models were used to evaluate the effectiveness of PHC-1 in vitro and in vivo, respectively. The results demonstrated that PHC-1 treatment significantly reduced bacterial load, alleviated inflammatory response, and improved mastitis pathology. Altogether, these results suggest that PHC-1 has the potential to treat S. aureus -induced bovine mastitis and that phage cocktails can combat antibiotic-resistant S. aureus infections. [ABSTRACT FROM AUTHOR]

Simple Summary: Antimicrobial therapy is the most frequently used medical intervention for bovine mastitis in the dairy industry. In this study, we aimed to monitor the extent of the antimicrobial resistance problem in Staphylococcus aureus. The multiple antibiotic resistance was calculated for bovine and human strains, using phenotypic and genotypic methods. It is a cause for concern that values were higher than acceptable limits for almost all antibiotics currently used in the dairy industry. Genotypic analysis was used for assessing the degree of penetrance of the resistance genes in Staphylococcus. Given that penetrance in S. aureus was strongly positively correlated with the multi-antibiotic resistance index (MARI), it may be possible to use the same limit value for management decision purposes. In fact, values of penetrance over 0.20, along with MARI values over 0.40, raise questions over the validity of current antimicrobial treatment programs/strategies, which the study suggests ought to be changed. Antimicrobial therapy is the most frequently used medical intervention for bovine mastitis in the dairy industry. This study aims to monitor the extent of the antimicrobial resistance (AMR) problem in Staphylococcus aureus in the dairy industry in Western Romania. Twenty farms were selected by random sampling in a transverse epidemiological study conducted across four counties in Western Romania and divided into livestock units. This study assessed the association between the resistance genes to phenotypic expression of resistance and susceptibility. Isolates of S. aureus were identified and q-PCR reactions were used to detect antibiotic resistance genes. One hundred and fifty bovine and 20 human samples were positive for S. aureus. Twenty five percent of bovine isolates (30/120) and none(0/30) of the human isolates were methicillin-resistant S. aureus (MRSA). All isolates were susceptible to fosfomycin, ciprofloxacin, netilmicin, and resistant to ampicillin and penicillin. S. aureus isolates regarded as phenotypically resistant (R) were influenced by the origin of the samples (human versus bovine, χ2 = 36.510, p = 0.013), whether they were methicillin-resistant S. aureus (χ2 = 108.891, p < 0.000), the county (χ2 = 103.282, p < 0.000) and farm of isolation (χ2 = 740.841, p < 0.000), but not by the size of the farm (χ2 = 65.036, p = 0.306). The multiple antibiotic resistance index was calculated for each sample as the number regarded as phenotypically resistant (R)/total antibiotics tested (MARI = 0.590 ± 0.023) was significantly higher (p < 0.000) inmethicillin-resistant S. aureus (0.898 ± 0.019) than non-methicillin-resistant S. aureus (0.524 ± 0.024) isolates. For the antibiotics tested, the total penetrance (P%) of the resistance genes was 59%, 83% for blaZ, 56% for cfr, 50% for erm(B), 53% for erm(C), 57% for mecA and 32% for tet(K). Penetrance can be used as a parameter for guidance towards a more accurate targeting of chemotherapy. P% in S. aureus was strongly positively correlated with the multiple antibiotic resistance index (r = +0.878, p < 0.000) with the potential to use the same limit value as an antibiotic management decision criterion. Considering cow mastitis, the penetrance value combined with the multiple antibiotic resistance index suggests that penetrance could serve as a useful parameter for more precise targeting of chemotherapy for S. aureus. [ABSTRACT FROM AUTHOR]

Simple Summary: This review outlines the use of metabolomics to detect biomarkers for bovine mastitis, offering insights into disease prediction and early diagnosis. It details the use of various biological samples for metabolomic analysis, including milk, blood, urine, rumen fluid, feces, and mammary tissue. Bovine mastitis is an inflammatory disease of the mammary glands, and its pathogenesis and diagnosis are complicated. Through qualitative and quantitative analysis of small-molecule metabolites, the metabolomics technique plays an important role in finding biomarkers and studying the metabolic mechanism of bovine mastitis. Therefore, this paper reviews the predictive and diagnostic biomarkers of bovine mastitis that have been identified using metabolomics techniques and that are present in samples such as milk, blood, urine, rumen fluid, feces, and mammary tissue. In addition, the metabolic pathways of mastitis-related biomarkers in milk and blood were analyzed; it was found that the tricarboxylic acid (TCA) cycle was the most significant (FDR = 0.0015767) pathway in milk fluid, and glyoxylate and dicarboxylate metabolism was the most significant (FDR = 0.0081994) pathway in blood. The purpose of this review is to provide useful information for the prediction and early diagnosis of bovine mastitis. [ABSTRACT FROM AUTHOR]

Innovative approaches in nanotechnology provide a potentially promising alternative to untreatable cases of mastitis caused by genus Prototheca spp. algae infections. Drying of the teats of the affected animals or culling are typically the outcomes of mastitis in dairy cattle caused by these pathogens. A major issue in both veterinary medicine and animal breeding is the Prototheca species' widespread resistance to the current methods of managing infections and the available drugs, including antibiotics. Commercial antifungal preparations are also ineffective. Nanotechnology, an emerging discipline, has the potential to create an effective alternative treatment for protothecal mastitis. The aim of the paper is to combine the literature data on the use of nanotechnology in the control of mastitis, taking into account data on combating mastitis caused by Prototheca spp. infections. The databases employed were PubMed, Google Scholar, and Scopus, focusing on literature from the last 20 years to ensure relevance and currency. Studies conducted in vitro have demonstrated that nanomaterials have significant biocidal activity against mastitis infections of different etiologies. Analyzed research papers show that (NPs), such as AgNPs, CuNPs, AuNPs, etc., may not negatively impact various cell lines and may be effective agents in reducing the pathogens' viability. However, it is also critical to assess the risks involved in using nanomaterials. [ABSTRACT FROM AUTHOR]

Group B Streptococcus sequence type 103 is known primarily as a bovine mastitis pathogen. In Brazil, it has circulated in cattle and humans since the 1990s. It lacks scpB and, in humans, was found only among carriage isolates. Bovine-human interspecies transmission may have contributed to its evolution and spread. [ABSTRACT FROM AUTHOR]

Mycoplasma bovis (M. bovis) is a significant bovine pathogen associated with various diseases, including bovine bronchopneumonia and mastitis resulting in substantial economic losses within the livestock industry. However, the development of effective control measures for M. bovis is hindered by a limited understanding of its virulence factors and pathogenesis. Nucleomodulins are newly identified secreted proteins of bacteria that internalize the host nuclei to regulate host cell gene expression and serve as critical virulence factors. Although recent reports have initiated exploration of mycoplasma nucleomodulins, the efficiency of conventional techniques for identification is very limited. Therefore, this study aimed to establish high-throughput methods to identify novel nucleomodulins of M. bovis. Using a direct biotinylation (DB) approach, a total of 289 proteins were identified including 66 high abundant proteins. In parallel, the use of proximity-based biotinylation (PBB), identified 28 proteins. Finally, seven nucleomodulins were verified to be nuclear by transfecting the bovine macrophage cell line BoMac with the plasmids encoding EGFP-fused proteins and observed with Opera Phenix, including the known nucleomodulin MbovP475 and six novel nucleomodulins. The novel nucleomodulins were four ribosomal proteins (MbovP599, MbovP678, MbovP710, and MbovP712), one transposase (MbovP790), and one conserved hypothetical protein (MbovP513). Among them, one unique nucleomodulin MbovP475 was identified with DB, two unique nucleomodulins (MbovP513 and MbovP710) with PBB, and four nucleomodulins by both. Overall, these findings established a foundation for further research on M. bovis nucleomodulin-host interactions for identification of new virulence factors. [ABSTRACT FROM AUTHOR]

Background: Coagulase-negative Staphylococcus species are an emerging cause of intramammary infection, posing a significant economic and public health threat. The aim of this study was to assess the occurrence of coagulase-negative Staphylococcus species in bovine milk and dairy farms in Northwestern Ethiopia and to provide information about their antibiotic susceptibility and virulence gene profiles. Methods: The cross-sectional study was conducted from February to August 2022. Coagulase-negative Staphylococcus species were isolated from 290 milk samples. Species isolation and identification were performed by plate culturing and biochemical tests and the antimicrobial susceptibility pattern of each isolate was determined by the Kirby-Bauer disc diffusion test. The single-plex PCR was used to detect the presence of virulent genes. The STATA software version 16 was used for data analysis. The prevalence, proportion of antimicrobial resistance and the number of virulent genes detected from coagulase-negative Staphylococcus species were analyzed using descriptive statistics. Results: Coagulase-negative Staphylococcus species were isolated in 28.6%, (95% CI: 23.5–34.2) of the samples. Of these, the S. epidermidis, S. sciuri, S. warneri, S. haemolyticus, S. simulans, S. chromogens, S. cohnii, and S. captis species were isolated at the rates of 11, 5.2, 3.4, 3.1, 3.1, 1, 1, and 0.7% respectively. All the isolates showed a high percentage (100%) of resistance to Amoxicillin, Ampicillin, and Cefotetan and 37.5% of resistance to Oxacillin. The majority (54.2%) of coagulase-negative isolates also showed multidrug resistance. Coagulase-negative Staphylococcus species carried the icaD, pvl, mecA, hlb, sec, and hla virulent genes at the rates of 26.5%, 22.1%, 21.7%, 9.6%, 9.6% and 8.4% respectively. Conclusion: The present study revealed that the majority of the isolates (54.2%) were found multidrug-resistant and carriage of one or more virulent and enterotoxin genes responsible for intramammary and food poisoning infections. Thus, urgent disease control and prevention measures are warranted to reduce the deleterious impact of coagulase-negative species. To the best of our knowledge, this is the first study in Ethiopia to detect coagulase-negative Staphylococcus species with their associated virulent and food poisoning genes from bovine milk. [ABSTRACT FROM AUTHOR]

Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5–10) and a wide temperature range (4–42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 μg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection. [ABSTRACT FROM AUTHOR]