Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Economic losses caused by mastitis and the influence of climate variation on the occurrence of the disease in a dairy cattle farm in southern Brazil.

This study evaluated the economic impacts caused by mastitis in a small dairy farm with similar characteristics and production to most dairy farms in southern Brazil and investigated if climatic variations influenced mastitis occurrence in the region. A farm with, on average, 45 lactating Holstein cattle was monitored from November 2021 to October 2022, and data on mastitis cases, bulk tank milk somatic cell count, animal treatment costs, milk production, animal disposal costs, and production losses were collected. Monthly averages of temperature, relative humidity (RH), and rainfall in the region were obtained. The greatest loss was related to the drop in milk production, resulting in 63.8% of total losses, followed by animal disposal (29.5%), milk disposal (4.6%), and treating animals with mastitis (2.0%), totaling a 10.6% reduction in the annual gross income. There were negative correlations between the clinical mastitis rate and monthly RH and between subclinical mastitis and temperature; the occurrence of subclinical mastitis and average RH were positively correlated. Our findings showed that mastitis negatively impacted the economy and that climate influenced mastitis occurrence.

Effect of 2% meloxicam injection in Holstein dairy cows on acute clinical mastitis without systemic symptoms.

This study aimed the efficacy of meloxicam (MX) in treating acute clinical mastitis (ACM) without systemic symptoms in Holstein cows by studying improvement in udder pain, changes in prostaglandin E2(PGE2) and bradykinin (BK) levels in the milk, and milk yield (MY) after healing. Forty-two cows with ACM were randomly assigned to the MX treatment group (T group; n=21) and the control group (C group; n=21). At onset of illness (day 0), the T group received a 0.5 mg/kg subcutaneous (SC) injection of MX whereas the C group received 15 mL SC of saline solution as a placebo. Udder tenderness (UT) was measured, and milk samples were collected on days 0-3. There was little change in the MY of the T group before and after healing, whereas MY in the C group was significantly lower than after healing. UT on day 3 in the T group was significantly lower than that in the C group. PGE2 levels significantly decreased from day 0 to day 3 in both groups. A significant negative correlation between PGE2 and linear score was observed on day 1 in the T group, but not in the C group. In ACM without systemic symptoms, the administration MX may be useful for restoring MY and reducing udder pain after healing.

[Selective dry cow therapy on dairy farms in Rhineland-Palatinate, Saarland and Hesse, Germany - a survey among farmers]. / Selektives antibiotisches Trockenstellen bei Milchkühen in Rheinland-Pfalz, dem Saarland und Hessen ­ eine Umfrage unter Landwirten.

OBJECTIVE: With the Regulation (EC) 6/2019, antibiotic drying off of the entire dairy herd is no longer permissible. Hence, it is necessary to establish selective antibiotic drying off (SDCT: Selective Dry Cow Therapy) in dairy herds. With the publication of the PraeRi study in 2020, systematic data for the implementation of SDCT on farms became available for several German states. For Rhineland-Palatinate, Saarland and Hesse this type of information is only available from individual projects. Therefore, the aim of this survey was to increase the knowledge concerning the implementation of SDCT in dairy farms located in these states. MATERIAL AND METHODS: An online questionnaire was sent via newsletters to farmers and was published in the regional farmers' bulletins in the described catchment area. The questionnaire inquired about the saving of antibiotics during drying off, the criteria guiding the farmer's decision (cell count from monthly dairy herd improvement data (DHI), mastitis history, microbiological examination of quarter foremilk samples, California mastitis test), use of teat sealants and the type of dry off procedure (abrupt/gradual). RESULTS: A total of 103 questionnaires were evaluated, making the response rate ~1% for Hesse, ~3% for Saarland, and ~5% for Rhineland-Palatinate based on the number of included farms. Approximately 29% of the farmers dried off one out of four cows, 20% half, 23% three out of four and 13% all cows without using antibiotics. Eighty-nine farm managers based their decision on the somatic cell counts of DHI. Additional criteria influencing the decision were the mastitis history, results of the California Mastitis Test, or a combination of both. In 76 farms cows were dried off abruptly. In 79 farms teat sealers were used. CONCLUSIONS: Application of SDCT is established in most of the farms that participated in the survey, even though the proportion varied between farms. Legal requirements are not the only reason farmers need to increasingly deal with SDCT; sustainability programs of the dairies rely on selective drying off as well. Herd veterinarians should be supportive in implementing these measures to achieve good udder health while reducing the use of antimicrobials to a necessary minimum.

Intramammary preparation of enrofloxacin hydrochloride-dihydrate for bovine mastitis (biofilm-forming Staphylococcus aureus).

BACKGROUND: Chronic bovine mastitis is linked to biofilm-producing Staphylococcus aureus (bp-Sa) or Staphylococcus coagulase-negative (bp-Scn). OBJECTIVES: Bp-Sa and bp-Scn were treated with intramammary preparations of either enrofloxacin HCl·2H2O-dimethyl-sulfoxide-chitosan (enro-C/DMSO/chitosan) or enro-C alone. Their potential to inhibit and degrade biofilm formation in vitro was also assessed. METHODS: Milk samples were obtained from the affected quarters in a herd. Phenotypical and genotypical identifications as biofilm-producing Staphylococcus species were carried out. Enro-C/DMSO/chitosan and enro-C alone were assessed to determine their in vitro efficacy in interfering with biofilm formation and their bactericidal effects. A prolonged eight-day treatment with a twice-daily intramammary insertion of 10 mL of enro-C/DMSO/chitosan or enro-C alone was set to evaluate the clinical and bacteriological cures on day 10 in 15 cows per group and the biofilm-inhibiting ability. RESULTS: Fifty-seven percent of the isolates were identified as Staphylococcus spp., of which 50% were bp-Sa, 46% bp-Scn, and 4% Staphylococcus pseudintermedius. One hundred percent of the S. aureus isolated and 77% of Staphylococcus coagulase-negative were biofilm producers. In both groups, the icaA and icaD biofilm-producing genes were identified. The experimental preparation could inhibit biofilm formation, degrade mature biofilms, and have well-defined microbicidal effects on planktonic and biofilm bacteria. The respective clinical and bacteriological cure rates were 100% and 80% for enro-C/DMSO/chitosan and 41.7% and 25% for enro-C alone. CONCLUSIONS: Enro-C/DMSO/chitosan eliminates bp-Sa and bp-Scn from cases of chronic bovine mastitis.

Forsythiaside A attenuates mastitis via PINK1/Parkin-mediated mitophagy.

BACKGROUND: Bovine mastitis is the most common animal production disease in the global dairy industry, which affects the health of dairy cows. When bovine mastitis occurs, the mitochondrial metabolism of breast tissue increases, and the relationship between inflammation and mitophagy has become a hot topic for many scholars. The abuse of antibiotics leads to the increase of resistance to bovine mastitis. FTA is one of the main effective components of Forsythia suspensa, which has anti-inflammatory, anti-infection, anti-oxidation and anti-virus pharmacological effects, and has broad application prospects in the prevention and treatment of bovine mastitis. However, the relationship between the anti-inflammatory effects of FTA and mitophagy is still unclear. PURPOSE: This study mainly explores the anti-inflammatory effect of FTA in bovine mastitis and the relationship between mitophagy. METHODS: MAC-T cells and wild-type mice were used to simulate the in vitro and in vivo response of mastitis. After the pretreatment with FTA, CsA inhibitors and siPINK1 were used to interfere with mitophagy, and the mitochondrial function impairment and the expression of inflammatory factors were detected. RESULTS: It was found that pre-treatment with FTA significantly reduced LPS induced inflammatory response and mitochondrial damage, while promoting the expression of mitophagy related factors. However, after inhibiting mitophagy, the anti-inflammatory effect of FTA was inhibited. CONCLUSION: This study is the first to suggest the relationship between the anti-inflammatory effect of FTA and mitophagy. PINK1/Parkin-mediated mitophagy is one of the ways that FTA protects MAC-T cells from LPS-induced inflammatory damage.

Endolysin NC5 improves early cloxacillin treatment in a mouse model of Streptococcus uberis mastitis.

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 µg) combined with either a low (23.5 µg) or high (235.0 µg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog10; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. KEY POINTS: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.

Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae.

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45-55%) compared to the Norwegian isolates (10-13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Fighting antibiotic resistance in the local management of bovine mastitis.

Bovine mastitis is a widespread infectious disease with a significant economic burden, accounting for 80 % of the antibiotic usage in dairy animals. In recent years, extensive research has focused on using biomimetic approaches such as probiotics, bacteriocins, bacteriophages, or phytochemicals as potential alternatives to antibiotics. The local administration of therapeutic molecules through the intramammary route is one of the most commonly strategies to manage bovine mastitis. This review highlights the most important findings in this field and discusses their local application in mastitis therapy. In contrast to antibiotics, the proposed alternatives are not limited to promote bacterial death but consider other factors associated to the host microenvironments. To this end, the proposed biomimetic strategies can modulate different stages of infection by modifying the local microbiota, preventing oxidative stress, reducing bacterial adhesion to epithelial cells, modulating the immune response, or mediating the inflammatory process. Numerous in vitro studies support the antimicrobial, antibiofilm or antioxidant properties of these alternatives. However, in vivo studies incorporating these components within pharmaceutical formulations with potential clinical application are limited. The development of secure, stable, and effective drug delivery systems based on the proposed options is necessary to achieve real alternatives to antibiotics in the clinic.

Antimicrobial activities of Agave fructans against multi-resistant and biofilm-producing Staphylococcus aureus isolated from bovine mastitis.

Bovine mastitis is an emerging disease that causes large economic losses. Staphylococcus aureus its main etiological agent, is multi-resistant to antimicrobials and produces biofilm. The objective of this study was to investigate the effect of Agave fructans (AF), a type of prebiotic, on multi-resistant and biofilm-forming isolates of S. aureus. Ten isolates of S. aureus from bovine subclinical mastitis previously characterized as highly resistant to antimicrobials and biofilm formers were used in this study. The growth kinetics of S. aureus in the presence of AF was evaluated by the Baranyi and Roberts microbial growth model using the DMFit program. The antibacterial activity of AF against S. aureus was studied by the well-diffusion method and the effect on biofilm formation by the crystal violet method. All assays were performed in triplicate for each isolate and an ANOVA with Tukey's post hoc was performed considering p < 0.05 as significant. The AF showed a decrease in maximum growth rate (µmax) and OD max levels (Ymax) in all isolates with all concentrations. Also, zones of inhibition were observed due to the effect of all AF concentrations in all isolates in a dose-dependent manner. Interestingly, S. aureus biofilm formation was inhibited by all AF concentrations assessed in this study. More investigations are required to elucidate the mechanisms of action of AF on S. aureus as well as in vivo studies to evaluate its therapeutic efficacy for bovine mastitis.

Impact of a Novel Homeopathic Complex Medicine on the Management of Multiple Antibiotic-Resistant Bovine Mastitis: An Open-Label, Non-Randomized, Placebo-Controlled Trial.

BACKGROUND: Bovine mastitis is characterized by an inflammatory process in the mammary gland and represents one of the main diseases affecting a dairy herd. Management of mastitis is most commonly via antibiotics, but the rising incidence of multiple antibiotic resistance (MAR) means that additional options are needed. Homeopathic products can be administered in dairy farming for a range of clinical reasons and may be preferential due to the absence of residues. OBJECTIVES: The aim of this study was to assess the potential of a novel homeopathic complex medicine in managing bovine mastitis. METHODS: Twenty-four lactating Holstein cows with mastitis were divided into two groups: the homeopathic complex group received a homeopathic complex daily for 60 days at a dose of 20 g/d; the placebo group received the calcium carbonate vehicle without homeopathic medicines at the same dose and repetition. The main outcome measure was somatic cell count (SCC; cells/mL), with additional outcome measures including milk production (kg/d), milk constituents (percentage of protein, fat, lactose and total milk solids), and serum levels of cortisol, glucose, ammonia and lactic acid. All outcomes were measured at the beginning of the study and after 30 and 60 days. Milk samples were also collected from all animals at the beginning of the study, confirming a high (>0.2) MAR index for isolated bacterial cultures. RESULTS: Assessment of SCC showed a statistically significant difference favoring the homeopathic complex versus placebo group at day 60. A reduction in serum cortisol levels and an increase in fat, lactose and total milk solids in animals treated with the homeopathic complex at day 60 were also seen. Other outcome measures did not show statistically significant inter-group differences. CONCLUSION: The results of this non-randomized, open-label, placebo-controlled trial suggest the potential for a novel homeopathic complex medicine in management of multiple antibiotic-resistant bovine mastitis, thus offering dairy farmers an additional option to antibiotics and making dairy products safer for consumer health and milk production more sustainable.

Adoption and decision factors regarding selective treatment of clinical mastitis on Canadian dairy farms.

As clinical mastitis (CM) treatments are responsible for a large portion of antimicrobial use on dairy farms, many selective CM treatment protocols have been developed and evaluated against a blanket treatment approach of CM cases. Selective treatment protocols use outcomes of diagnostic tests to exclude CM cases from antimicrobial treatment when they are unlikely to benefit. To tailor interventions to increase uptake of selective treatment strategies, a comprehension of current on-farm treatment practices and factors affecting treatment decisions is vital. Two questionnaires were conducted among 142 farms across 5 provinces participating in the Canadian Dairy Network for Antimicrobial Stewardship and Resistance in this cross-sectional study. Self-reported adoption of selective CM treatments by dairy farmers was 64%, with median of 82% of cows treated in those herds using selective treatment. Using logistic regression models, the odds to implement a selective CM treatment protocol increased with a decreasing average cow somatic cell count. No other associations were identified between use of a selective CM treatment protocol and farm characteristics (herd size, CM incidence, province, milking system, and housing system). Three subsets of farmers making cow-level CM treatment decisions were identified using a cluster analysis approach: those who based decisions almost exclusively on severity of clinical signs, those who used various udder health indicators, and farmers who also incorporated more general cow information such as production, age, and genetics. When somatic cell count was considered, the median threshold used for treating was >300,000 cells/mL at the last Dairy Herd Improvement test. Various thresholds were present among those considering CM case history. Veterinary laboratories were most frequently used for bacteriological testing. Test results were used to start, change, and stop treatments. Regardless of protocol, reasons for antimicrobial treatment withheld included cow being on a cull list, having a chronic intramammary infection, or being at end of lactation (i.e., close to dry off). If clinical signs persisted after treatment, farmers indicated that they would ask veterinarians for advice, stop treatment, or continue with the same or different antibiotics. Results of this study can be used to design interventions targeting judicious mastitis-related antimicrobial use, and aid discussions between veterinarians and dairy producers regarding CM-related antimicrobial use.

Intramammary calcitriol treatment of mastitis alters profile of milk somatic cells and indicators of redox activity in milk.

The objective of this experiment was to determine the effect of intramammary calcitriol treatment on indicators of inflammation during an intramammary bacterial infection. Lactating Holstein cows were challenged with intramammary Streptococcus uberis. At the onset of mild or moderate mastitis, cows were randomly assigned to receive 10 µg of intramammary calcitriol (CAL, n = 7) or placebo control (CON; n = 6) after every milking for 5 days. Data were analyzed by ANOVA with mixed models using the MIXED procedure of SAS with significance declared at P ≤ 0.05. Milk somatic cells, mastitis severity scores, rectal temperatures, and milk bacterial counts did not differ between treatments. Calcitriol decreased the percentage of CD11b+CD14- cells in milk compared with CON (CON = 81 vs. CAL = 61 ± 5%). Antioxidant potential and concentrations of 15-F2t- isoprostanes in milk of infected quarters also were lower in CAL compared with CON. Transcripts for the 25-hydroxyvitamin D 24-hydroxylase and inducible nitric oxide synthase were greater in milk somatic cells of CAL compared with CON, but those for ß-defensin 7, metallothionein 1 A and 2 A, thioredoxin and thioredoxin reductase did not differ between treatments. Although clinical signs of severity did not differ, CAL influenced the composition of milk somatic cells and redox activity in milk of infected quarters.

Using biologically synthesized TiO2 nanoparticles as potential remedy against multiple drug resistant Staphylococcus aureus of bovine mastitis.

Presently, there is considerable emphasis on biological synthesis of nanoparticles containing bioactive reducing compounds with an aim to mitigate the harmful effects of pollutants. The approach under study is simple and ideal for the production of durable antimicrobial nanomaterials by novel single-step green synthesis of TiO2 metal oxide nanostructures using ginger and garlic crude aqueous extracts with bactericidal and catalytic activity. A variety of experimental techniques were used to characterize the synthesized nanomaterials. As demonstrated using x-ray diffraction and ultra-violet visible spectroscopy, the produced nanoparticles exhibited high absorption at 318 nm with size varying between 23.38 nm for ginger and 58.64 nm for garlic in biologically-reduced TiO2. At increasing concentrations (500, 1000 µg/50 µl), nanoparticles reduced with garlic exhibited enhanced bactericidal efficacy against multiple drug-resistant S. aureus and effectively decomposed toxic methylene blue (MB) dye. In conclusion, biologically-reduced TiO2 nanoparticles may prove an effective tool in the fight against microbial illnesses and drug resistance.

Antimicrobial efficacy of Thymus vulgaris extract against some Staphylococcus species isolated from subclinical mastitis in cattle in Basrah province, Iraq.

Background: Thymus vulgaris extracts can play a significant role as alternatives for antimicrobial agents against bovine staphylococcus mastitis. Aim: This research's goal was to evaluate the antibacterial properties of an extract from T. vulgaris as an alternative to antibiotics for bovine Staphylococcus mastitis. In addition, it is important to know the effect of the extraction methods (hot alcoholic, cold alcoholic, and hot water extract) on their effectiveness. Methods: Two hundred ten cow milk samples from different areas of Basrah province had been suffering subclinical mastitis reported by using the California mastitis test (CMT). Staphylococcus species were identified by conventional microbiological technique, GP24 Kit, and nuc gene. Antimicrobial activity of various concentrations of T. vulgaris extracted (75, 50, 25) mg/ml with different methods of extraction (hot alcoholic, cold alcoholic, and hot water extract). Results: Out of 210 samples, 99 (47.1%) were positive for the CMT, and the identification rate of Staphylococci spp. by conventional microbiological technique and GP24 kit was 78 (78.8%). Out of 78 isolates of Staphylococcus spp. 48 (61.5%) were identified as Staphylococcus aureus, by using both molecular techniques using PCR and miniaturized Kit GP24 and employing the miniature GP24, the remaining 30 (38.5%) were determined to be different species of Staphylococcus. Antibacterial activity of various concentrations of T. vulgaris extracted (75, 50, 25) mg/ml with different methods of extraction revealed that hot alcoholic extract (100%) was more effective than cold alcoholic extract (66.7%), whereas there is no effect on the bacteria species with the hot water extract. Conclusion: Thymus vulgaris extracts can play a significant role as alternatives for antimicrobial agents against bovine staphylococcus mastitis.

Effects of Encapsulation of Caesalpinia sappan L. with Cyclodextrins for Bovine Mastitis.

The main components of Caesalpinia sappan L. (CS) are brazilin and brazilein, which show high potential in pharmacologic applications. However, these have been drastically limited by the poor water solubility and stability. The present study investigates the formation of inclusion complexes F1, F2, and F3 between CS and ß-cyclodextrin (ßCD), hydroxypropyl-ß-cyclodextrin (HPßCD), and methyl-ß-cyclodextrin (MßCD), respectively. These complexes were characterized by Fourier transform infrared spectroscopy (FT-IR). The results showed that the highest encapsulation efficiency and loading capacity of CS extract were 44.24% and 9.67%, respectively. The solubility and stability of CS extract were significantly increased through complexation in phase solubility and stability studies. The complexes F1-F3 showed mainly significant antibacterial activities on gram-positive bacteria pathogens causing mastitis. Moreover, the expression levels of COX-2 and iNOS were significantly decreased in LPS-induced inflammatory cells at concentrations of 50 and 100 µg/mL. In addition, treatment of complex F3 (CS/MßCD) in bovine endothelial cells remarkably increased the chemokine gene expression of CXCL3 and CXCL8, which were responsible for immune cell recruitment (9.92 to 11.17 and 8.23 to 9.51-fold relative to that of the LPS-treated group, respectively). This study provides a complete characterization of inclusion complexes between CS extract and ßCD, HPßCD, and MßCD for the first time, highlighting the impact of complex formation on the pharmacologic activities of bovine mastitis.

Risk factors of high somatic cell count and differential somatic cells in early lactation associated with selective dry cow therapy.

The routine use of intramammary antimicrobial products in all dairy cows at the beginning of the dry period is no longer allowed in European Union (EU) countries due to the new Regulation (EU) 2019/6 to reduce antimicrobial resistance. This study investigated the application of a selective dry cow therapy scheme and the risk factors of high individual milk somatic cell count (SCC) and individual neutrophil count in early lactation, as a response to the application of a selective dry cow therapy (SDCT) protocol. The study was carried out on three commercial farms, and a total of 243 lactating cows were monitored at the end of lactation and at the beginning of the next one, 91 of which were dried off without the use of antimicrobials (NoT) based on milk SCC, differential somatic cell count (DSCC), and the response of Vetscan DC-Q milk analyser, using a secret algorithm. The remaining 152 cows received antimicrobials (T). After calving, similar means were observed between the two treatment groups for SCC (4.8 vs 4.9 log10 cells/ml for T and NoT, respectively, P = 0.5) and total milk leucocyte count (TLC) (5 vs 5.1 log10 cells/ml for T and NoT, respectively, P = 0.7) in milk. However, the use of antimicrobials led to a lower DSCC (58 vs 64% for T and NoT, respectively, P = 0.01) and lower percentage of neutrophils (59 vs 64% for T and NoT, respectively, P = 0.05), although the levels of DSCC and percentage of neutrophils in cows dried off without antimicrobials remained lower than the risk threshold suggested by the international literature. A logistic regression was computed after the application of selective dry cow therapy to identify risk factors of high milk SCC (≥100 000 cells/mL) at the beginning of lactation. Increased milk SCC after calving was related to high SCC at the end of lactation and abandonment of antimicrobial therapy at dry-off. Moreover, the length of the dry period, milk protein content, and flank cleanliness in the last test day before dry-off were other factors in the logistic regression. Neutrophil counts at the beginning of the next lactation were affected by the same factors that influenced SCC, together with milk production, TLC, and macrophages on the last test day. The results obtained in these studied farms showed that selective dry cow therapy may be applied without adversely affecting the next lactation.

Assessment of antibiotic use patterns in bovine mastitis treatment in the dairy sector in Serbia.

The dairy sector is essential for the world's food systems, playing a vital role in agricultural production. Yet, mastitis, an inflammation of the mammary gland, affects a considerable portion of the dairy cow population annually, leading to reduced productivity and sustainability. Insight into therapeutic protocols is essential for a better understanding of the situation on farms with the aim of developing new or harmonizing existing protocols in the mastitis treatment. This study conducted on dairy farm in Serbia aimed to explore the most commonly used antibiotics during bovine mastitis treatment and their association with daily milk yield. Data from 100 lactating cows, including therapeutic protocols, lactation numbers, mastitis episodes, milk yield, and antibiotic usage, were obtained from a computerized database and statistically analysed. The results demonstrated a high prevalence of narrow-spectrum antibiotics usage, with the majority of cows treated with combination therapy. The route of antibiotic application did not significantly impact daily milk yield, while treatment duration had no discernible effect. Nonetheless, specific antibiotics were associated with milk yield variations, suggesting the need for careful antibiotic selection and management in mastitis therapy. These findings emphasize the importance of prudent antibiotic use to safeguard animal health and long-term milk production sustainability.

Development of engineered endolysins with in vitro intracellular activity against streptococcal bovine mastitis-causing pathogens.

Bacteriophage-derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic-resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high-throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram-positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time-kill assays in phosphate-buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta-lactam penicillin used to intramammarily treat bovine Gram-positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add-on in the treatment of bovine streptococcal mastitis.

Antimicrobial resistance of Streptococcus uberis isolated from bovine mastitis: Systematic review and meta-analysis.

Streptococcus uberis is one of the most common pathogens associated with bovine mastitis, commonly treated with antimicrobials (AM), favoring the appearance of antimicrobial resistance (AMR). The objective of this work was to determine the proportion of phenotypic AMR among S. uberis isolated worldwide from bovine intramammary infections between the years 1983-2022, and to assess the variables associated by means of a systematic review and metanalysis. Sixty articles were eligible for quantitative review. Ninety-four independent studies were obtained. The antimicrobials evaluated in more S. uberis strains were penicillin (21,987 strains), oxacillin (21,727 strains), erythromycin (20,013 strains), and ampicillin (19,354 strains). Most of the studies included in this meta-analysis were from Europe (44), followed by America (25), Africa (10), Asia (10), and Oceania (5). Among the included articles, 22 were published from 1983 to 2006, 23 from 2007 to 2012, 25 from 2013 to 2015, and the remaining 24 after 2016. Penicillin, erythromycin, and tetracycline were the antimicrobials with >25 studies. Therefore, the following analyses were performed only for these antimicrobials, presenting a high heterogeneity index (I2). The variability observed for penicillin and tetracycline was only explained, partially, by continent of origin. The variability observed for erythromycin was not explained by any of the potential explanatory variables included in this study. The S. uberis proportion of resistance to antimicrobials is highly variable and probably influenced by many factors other than those studied in this meta-analysis, where it was not possible to inform a unique average proportion of resistance.