Pharmacological composition based on bacteriocinnisin in experiments in vitro and in vivo.

Background: Antibiotic resistance is a global health problem related to the transmission of bacteria and genes between humans and animals. The development of new drugs with antimicrobial activity research is an urgent task of modern science. Aim: The article presents data of in vitro and in vivo experiments on new pharmaceutical composition based on nisin. Methods: The antimicrobial activity was studied on the mastitis pathogens. To identify microorganisms the Matrix-Assisted Lazer Desorption/Ionization Time-of-Flight (MALDI-TOF) (mass spectrometry) method was performed using. To determine sensitivity, the serial dilution method and the diffusion method were used. On laboratory animals, biochemical, hematological, and histological research methods were used. Female nonlinear white laboratory rats were used, which were divided into one control group and three experimental ones. Results: "Duration" factor was statistically significant for the following indicators: hemoglobin, hematocrit, leukocytes, lymphocytes, erythrocyte sedimentation rate, and eosinophils. The "Dose" factor did not show significance for any indicator, which means that the effect was similar regardless of the dose chosen. When analyzing the biochemical indicators, significant differences were found in the "Duration" and "Dose" factors, in the direction of a decrease in the indicators of total protein, globulins, urea, and an increase in the concentration of alkaline phosphatase. When conducting histological studies in the first experimental group, it was established that there were no changes in the structural and functional units of the organs. In animals of the second experimental group, the presence of reversible pathological processes of a compensatory nature was noted. More profound changes in the structure of the studied organs were recorded in the third experimental group. Conclusion: An in vitro study on cell cultures showed that the pharmacological composition has high antimicrobial activity against isolates from the mammary gland secretion of cows with mastitis. An in vivo study on laboratory animals showed that the developed composition belongs to the IV class of substances "low-hazard substances". Histological examination made it possible to select the safest dose of the pharmacological composition of no more than 500 mg/kg.

1,25 dihydroxyvitamin D3-mediated effects on bovine innate immunity and on biofilm-forming Staphylococcus spp. isolated from cattle with mastitis.

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host's immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0-200 nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72 h. Concentrations of 0-100 mM for 24 h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24 h decreased the internalization of S. aureus V329 into MAC-T cells (0-100 nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10 nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200 nM of the metabolite for 24 h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0-200 nM for 12 and 24 h, respectively). In contrast, the conditioned media (0-100 nM for 24 h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host's endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol's potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.

Pathogen-specific patterns of milking traits in automatic milking systems.

Early detection of IMI can improve animal health and welfare in dairy herds. The implementation of sensors and automatic milking systems (AMS) in dairy production inherently increases the amount of available data and hence also the potential for new approaches to mastitis management. To use the full potential of data from AMS and auxiliary sensors, a better understanding of physiological and pathological changes in milking traits associated with different udder pathogens may be imperative. This observational study aimed to investigate pathogen-specific patterns in milking traits recorded in AMS. The milking traits included; online SCC (OCC), electrical conductivity (EC), milk yield (MY), and average milk flow rate (AMF). Data were collected for a study period of 2 years and included 101,492 milkings from 237 lactations in 169 cows from one farm. Measurements of OCC were recorded at cow level and data on EC, MY, and AMF were obtained at quarter level. In addition to the data obtained from the AMS, altogether 5,756 quarter milk samples were collected. Milk samples were obtained monthly for bacteriological culturing. We included findings of 13 known mastitis pathogens to study pathogen-specific patterns in milking traits. These patterns were compared with those in a baseline group consisting of cows that did not have any positive milk culture results throughout the lactation period. Patterns of the milking traits are described for all positive samples both across 305 DIM, and in the 15-d period before a positive bacteriological sample. The association between a positive sample and the milking traits [ln(OCC); EC-IQR, the ratio between the quarter with the highest and the quarter with the lowest level of EC; and MY] for the 15 d before the detection of a pathogen was assessed using mixed effects linear regression models. All pathogens were associated with alterations in the level and variability of ln(OCC) relative to lactations with no positive bacteriological samples. A positive sample for Staphylococcus aureus was associated with increased values for MY during the 15 d before a positive diagnosis. It is biologically plausible to interpret changes in OCC and EC-IQR as consequences of an IMI, while higher MY in bacteriologically-positive cows is most likely linked to the increased risk of infection in high-yielding cows. In this study, the most notable changes in the traits (OCC and EC-IQR) were observed for Staph. aureus and Streptococcus dysgalactiae, followed by Streptococcus simulans, Streptococcus uberis, and Lactococcus lactis. Even if we did not detect significant associations between positive bacteriology and EC-IQR, visual assessment and descriptive statistics indicated that there might be differences suggesting that it could be an informative trait for detecting infection when combined with OCC and possibly other relevant traits using machine learning algorithms.

Inferring Causalities of Environmental and Genetic Factors for Differential Somatic Cell Count and Mastitis Pathogens in Dairy Cows Using Structural Equation Modelling.

The aim of this study was to establish and evaluate a structural equation model to infer causal relationships among environmental and genetic factors on udder health. For this purpose, 537 Holstein Friesian cows were genotyped, and milk samples were analyzed for novel traits including differential somatic cell counts and specific mastitis pathogens. In the structural model, four latent variables (intramammary infection (IMI), production, time and genetics) were defined, which were explained using manifest measurable variables. The measurable variables included udder pathogens and somatic differential cell counts, milk composition, as well as significant SNP markers from previous genome-wide associations for major and minor pathogens. The housing system effect (i.e., compost-bedded pack barns versus cubicle barns) indicated a small influence on IMI with a path coefficient of -0.05. However, housing system significantly affected production (0.37), with ongoing causal effects on IMI (0.17). Thus, indirect associations between housing and udder health could be inferred via structural equation modeling. Furthermore, genotype by environment interactions on IMI can be represented, i.e., the detection of specific latent variables such as significant SNP markers only for specific housing systems. For the latent variable genetics, especially one SNP is of primary interest. This SNP is located in the EVA1A gene, which plays a fundamental role in the MAPK1 signaling pathway. Other identified genes (e.g., CTNNA3 and CHL1) support results from previous studies, and this gene also contributes to mechanisms of the MAPK1 signaling pathway.

Effect of dry cow therapy on antimicrobial resistance of mastitis pathogens post-calving.

The aim of this study was to evaluate the effect of dry cow therapy (DCT) on the antimicrobial resistance (AMR) profile of mastitis pathogens post-calving. A repository of isolates based on a DCT trial was utilized for the current study. A stratified random survey sample of cows from the trial were identified within the strata of season, herd, and trial treatment resulting in 382 cows. All isolates from the 382 cows were selected for the current study, which identified 566 isolates from milk samples collected at dry off (S1), post-calving (S2), and at the first clinical mastitis event up to 150 days in milk (S3). The AMR profiles were determined using broth microdilution method. Less than 10% of the coagulase-negative Staphylococcus species (CNS) isolates (n = 421) were resistant to tetracycline, ceftiofur, penicillin/novobiocin or erythromycin, while higher proportions of resistance to sulfadimethoxine (72%) and penicillin (28%) were observed. All Staphylococcus aureus (S. aureus) isolates (n = 4) were susceptible to all tested AMD except sulfadimethoxine, to which all isolates were resistant. Similarly, all Streptococcus spp. (n = 37) were susceptible to penicillin, penicillin/novobiocin, and ampicillin while resistant to tetracycline (17%). All coliforms (n = 21) were susceptible to ceftiofur, but resistance was recorded for sulfadimethoxine (70%), cephalothin (56%), and tetracycline (43%). The increased resistance percent from S1 to S2 was observed in CNS isolates from AMD-treated cows, with the highest increase recorded for penicillin (12.2%). Parametric survival interval regression models were used to explore the association between antimicrobial drug (AMD) therapy at dry off and the AMR phenotype post-calving. The accelerated failure-time metric was adopted to minimum inhibitory concentration measurements to permit interpretation of model exponentiated coefficients. Models for cows with CNS isolated at both S1 and S2 showed increased resistance against cephalothin, oxacillin, and ceftiofur in cows that received DCT from the same drug class, or a class with a shared resistance mechanism. In contrast, resistance of CNS isolates to tetracycline were associated with any AMD therapy at dry off. Resistance of CNS isolates to Penicillin decreased in CNS isolates in cows that received any AMD therapy at dry off compared to those that didn't. The study provided evidence that dry-cow IMM AMD was associated with AMR post-calving.

Estimation of the performance of two real-time polymerase chain reaction assays for detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in pooled milk samples in a field study.

The early detection of major mastitis pathogens is crucial for the udder health management of dairy herds. Testing of pooled milk samples, either individual test-day cow samples (TDCS) or aseptically collected pre-milk quarter samples (PMQS) may provide an easy to use and cost-effective group level screening tool. Therefore, the aim of this study was (1) to evaluate the sensitivity (Se) and specificity (Sp) of 2 commercial multiplex real-time PCR test kits applied to pooled milk samples using a Bayesian latent class analysis and (2) to estimate the probability of detection in relation to the pool size and the number of cows positively tested by bacteriological culture (BC) within a pool. Pools of 10, 20 and 50 cows were assembled from 1,912 test-day samples and 7,336 PMQS collected from a total of 2,045 cows from 2 commercial dairy farms. Two commercial quantitative real-time PCR kits were applied to detect Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in the pooled samples, and a BC was applied to PMQS yielding a cumulative pool result. A pool was considered BC-positive if it contained at least one BC-positive PMQS. Pathogens were more frequently detected in the PMQS pools than in the TDCS pools. Pools of 10 cows showed the highest probability of detection irrespective of sample type or type of PCR kit compared with larger pool sizes. Estimation with a Bayesian latent class analysis resulted in a median Se in PMQS pools of 10 cows for Staph. aureus of 63.3% for PCR kit I, 78.1% for PCR kit II, and 95.5% for BC; the Sp values were 97.0%, 97.6%, and 89.1%, respectively. The estimated median Se for Strep. species for PCR kits ranged between 77.5 and 85.6% and for BC between 73.7% and 79.2%; the median Sp values ranged between 93.6 and 99.2% for PCR kits, and between 96.9% and 97.4% for BC. In addition, the probability of detection increased with an increasing number of BC-positive cows per pool. To achieve a probability of detection of 90%, the estimated number of positive cows in PMQS pools of 10 cows for kit I was 4.1 for Staph. aureus, 1.5 for Strep. agalactiae, and 1.3 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 6.9, 1.9, and 2.0 positive cows were required, respectively. For Kit II and PMQS pools, the number of positive cows required was 2.8 for Staph. aureus, 1.4 for Strep. agalactiae, and 1.2 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 5.3, 1.8, and 2.0 positive cows were required, respectively. In conclusion, the type of samples used for pooling, the pool size and the number of infected cows per pool determine the probability of detecting an infection with major mastitis pathogens within a pool by PCR testing.

Seasonal, physiological and bacteriological risk factors for subclinical mastitis in dairy cows maintained under different farming conditions.

Subclinical mastitis (SCM) is a major health problem of dairy animals in India and across the globe. An identification of potential risk factors of SCM can help for efficient udder health management in dairy animals. In this study, apparently healthy cows (HF crossbred: n = 45; Deoni: n = 43) were screened for SCM during different seasons through milk somatic cell count (SCC: reference test using 200 × 103 cells/ml as cut off value), California mastitis test (CMT) and differential electrical conductivity (DEC) test at an organized research farm. SCM positive milk samples (n = 34) were inoculated in selective media for Coliform sp., Streptococcus sp. and Staphylococcus sp. and DNA was isolated (n = 10) for species confirmation by 16s rRNA method. Both bivariate and multivariate models were used for risk assessment. We found the cumulative prevalence of 31 and 65% SCM in Deoni and crossbred cows, respectively. Screening of 328 crossbred cows under field conditions revealed point prevalence of 55% SCM. Multivariate analysis revealed stage of lactation (SOL), milk yield in previous lactation and test day milk yield in Deoni cows, as well as parity and mastitis treatment history in current lactation in HF crossbred cows as risk factors. SOL was a significant factor under field conditions. Receiver operated characteristic curve analysis revealed better accuracy of CMT than DEC. We found more mixed infections due to Staphylococcus sp. and Streptococcus sp. in culture, while 16s rRNA based molecular method revealed lesser-known pathogens associated with SCM. It is concluded that SCM prevalence rate is higher in crossbred than indigenous cows and these breeds have different risk factors for SCM. HF crossbred cows had similar SCM prevalence rate under different farming conditions, where CMT can be used for SCM diagnosis with excellent accuracy. The 16s rRNA method is useful for specific identification of lesser known and emerging mastitis pathogens.

Antimicrobial activity of essential oils against Staphylococcus aureus and Staphylococcus chromogenes isolated from bovine mastitis.

Staphylococcus aureus and Staphylococcus chromogenes are pathogens frequently detected in bovine mastitis. Treatment and prevention of this disease have been usually carried on with antimicrobials. However, the emergence of bacterial isolates with antimicrobial resistance has aroused interest in new therapeutic alternatives. Plant essential oils (EOs) have been largely studied as antibacterial treatments. In the present study, EOs from five plants were evaluated for their antibacterial activities against S. aureus and S. chromogenes. Bacterial isolates were obtained in a previous study of clinical cases of bovine mastitis. EOs from lemongrass, eucalyptus, lavender, peppermint, and thyme were obtained by hydrodistillation and their chemical compositions were evaluated by gas chromatography (GC). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated for all EOs. The results demonstrated that citral (40.9%), myrcene (24.7%), and geraniol (1.9%) were detected in lemongrass EO; 1,8-cineole (76.9%), α-pinene (8.2%), and ledene (5.1%) in eucalyptus EO; 1,8-cineole (45.2%), camphor (18.2%), and fenchone (14.6%) in lavender EO; L-menthol (38.5%), menthofuran (16.3%), and citronellal (10.6%) in peppermint EO; and thymol (44.2%), p-cymene (24.6%) and 1,8-cineole (9.9%) in thyme EO. More effective antibacterial activities were observed only with the use of lemongrass (MIC and MBC ranging from 0.39 to 3.12 mg/mL and 0.39 to 6.35 mg/mL, respectively) and thyme (MIC and MBC ranging from 0.39 to 1.56 mg/mL and 0.39 to 3.12 mg/mL, respectively). Peppermint, lavender and eucalyptus EOs did not show bactericidal activities. In conclusion, lemongrass and thyme EOs are promising antibacterial alternatives against Staphylococcus species associated with bovine mastitis.

Reservoirs of Corynebacterium spp. in the Environment of Dairy Cows.

Although Corynebacterium spp. can be regularly associated with subclinical and clinical mastitis cases in dairy cows, knowledge on their reservoirs in dairy farms is sparse. Therefore, samples were collected at 10 visits with 14 day intervals from bedding material (n = 50), drinking troughs (n = 20), different walking areas (n = 60), cow brushes (n = 8), fly traps (n = 4), the passage to pasture (n = 9) as well as milking liners (n = 80) and milker gloves (n = 20) in one dairy cow farm. Additionally, quarter foremilk samples from all lactating cows (approximately 200) were collected at each visit. All samples underwent microbiological examination and cultured isolates were identified using MALDI-TOF MS. Most Corynebacterium spp. that were cultivated from milk were also isolated from the housing environment and milking-related niches (C. amycolatum, C. confusum, C. stationis, C. variabile, C. xerosis) or from milking-related niches only (C. frankenforstense, C. pilosum, C. suicordis). C. bovis was not cultivated from any environmental niche, while being the dominant species in milk samples. This study demonstrates that many Corynebacterium spp. present in milk samples can also be isolated from the cows' environment. For C. bovis, the most relevant Corynebacterium species with regard to intramammary infections, it indicates that environmental reservoirs are of little relevance.

The microbiome of common bedding materials before and after use on commercial dairy farms.

BACKGROUND: Bovine mastitis is one of the most economically important diseases affecting dairy cows. The choice of bedding material has been identified as an important risk factor contributing to the development of mastitis. However, few reports examine both the culturable and nonculturable microbial composition of commonly used bedding materials, i.e., the microbiome. Given the prevalence of nonculturable microbes in most environments, this information could be an important step to understanding whether and how the bedding microbiome acts as a risk factor for mastitis. Therefore, our objective was to characterize the microbiome composition and diversity of bedding material microbiomes, before and after use. METHODS: We collected 88 bedding samples from 44 dairy farms in the U.S. Unused (from storage pile) and used (out of stalls) bedding materials were collected from four bedding types: new sand (NSA), recycled manure solids (RMS), organic non-manure (ON) and recycled sand (RSA). Samples were analyzed using 16S rRNA sequencing of the V3-V4 region. RESULTS: The overall composition as well as the counts of several microbial taxa differed between bedding types, with Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes dominating across all types. Used bedding contained a significantly different microbial composition than unused bedding, but the magnitude of this difference varied by bedding type, with RMS bedding exhibiting the smallest difference. In addition, positive correlations were observed between 16S rRNA sequence counts of potential mastitis pathogens (bacterial genera) and corresponding bedding bacterial culture data. CONCLUSION: Our results strengthen the role of bedding as a potential source of mastitis pathogens. The consistent shift in the microbiome of all bedding types that occurred during use by dairy cows deserves further investigation to understand whether this shift promotes pathogen colonization and/or persistence, or whether it can differentially impact udder health outcomes. Future studies of bedding and udder health may be strengthened by including a microbiome component to the study design.

Antibacterial activity and mechanism of essential oils in combination with medium-chain fatty acids against predominant bovine mastitis pathogens.

Bovine mastitis has become a significant economic importance for the dairy industry. Concerns regarding poor milk quality and emergence of bacterial resistance have necessitated to develop an alternative therapeutic approach to antibiotics for the treatment of mastitis. Saturated medium-chain fatty acids (MCFAs) and essential oils (EOs) are known natural antimicrobials, but their combined effect has not been investigated extensively. The objective of the present investigation was to examine the bactericidal effect of various combined treatments of eight EOs and three saturated MCFAs to inactivate predominant mastitis pathogens, including Staphylococcus aureus ATCC 29213; Escherichia coli ATCC 25922; Klebsiella pneumoniae ATCC 27736 and Streptococcus agalactiae ATCC 27956. The minimum inhibitory concentration (MIC) values confirmed that all the tested pathogens were variably susceptible to both EOs and saturated MCFAs. Among essential oils, carvacrol (CAR), trans-cinnamaldehyde (TC) and thymol (TM) showed the highest inhibitory activity at concentration 0·38-1·32 mg/mL. Carvacrol exhibited effective additive antibacterial activity in combined treatment with octanoic acid (OA) in terms of its fractional inhibitory index (0·63-0·88) and time-kill effect in reducing about 6 log CFU/mL bacterial cells in less than 5 min. The effort was also made to elucidate the mechanism of antibacterial action of CAR and OA against selected mastitis pathogens by observing changes in cell microstructure, permeability and integrity of cell membrane and their membrane potential. After adding CAR and OA at MIC level, there were obvious changes in cell morphology, leakage of small electrolytes and macromolecules at the initial few hours of treatment i.e. within 1-2 h were observed. Our results indicated that CAR and OA could be evaluated as alternatives or adjuncts to antibiotics as intramammary infusion or topical application to treat bovine mastitis, significantly improving the microbiological safety of milk.

Minimum inhibitory concentrations of chlorhexidine- and lactic acid-based teat disinfectants: An intervention trial assessing bacterial selection and susceptibility.

Teat disinfection is a recommended preventive tool to improve udder health and to prevent new intramammary infections. However, side effects are discussed, such as bacterial selection of less-susceptible bacteria with the application of certain teat disinfectants. The objective of this study was to assess the species composition and bacterial in vitro susceptibility by means of an interventive trial. For this purpose, 3 different postmilking teat treatments (disinfection with 0.215% chlorhexidine or 3.5% lactic acid, or control group with no dipping) were applied to 28 cows in a 6-d intervention approach using a split-udder design. Milk samples were taken before and after intervention. Bacteria were cultured and differentiated to species or genus level by MALDI-TOF mass spectrometry. Minimum inhibitory concentrations (MIC) were determined, and MIC changes over time were recorded. Susceptibilities to chlorhexidine and lactic acid were compared between species of the genera Staphylococcus, Streptococcus, Corynebacterium, and others. Species composition changed during the intervention. Under the treatment of chlorhexidine and lactic acid, the proportion of coagulase-negative staphylococci (CNS) decreased. An increased proportion of species belonging to the genus Corynebacterium was observed especially under the application of lactic acid. Although both teat disinfectants were basically effective, isolates differed in their susceptibility to both teat disinfectants. Populations of CNS, Staphylococcus aureus, and Corynebacterium spp. showed significantly lower absolute MIC values for chlorhexidine. Compared with other species, Corynebacterium spp. showed the lowest susceptibility for chlorhexidine as well as for lactic acid. A significant increase in MIC values after 6 d of intervention was observed with the lactic acid treatment in all isolates, as well as in CNS. This increase can be interpreted as either adaptation of isolates or displacement of more-susceptible species by less-susceptible species. Further studies using long-term intervention might reveal more pronounced effects on MIC values and species composition.

Spatio-Temporal Variation in the Prevalence of Major Mastitis Pathogens Isolated From Bovine Milk Samples Between 2008 and 2017 in Ontario, Canada.

An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of mastitis. However, in the absence of an active surveillance program such information is not readily available. In this retrospective study we analyzed passive surveillance data from a diagnostic laboratory with an aim to describe the spatio-temporal trend of major mastitis pathogens between 2008 and 2017 in Ontario dairy cattle. Data for all milk culture samples submitted to the Animal Health Laboratory (AHL) at the University of Guelph between 2008 and 2017 was accessed. Descriptive analyses were conducted to identify the major pathogens and Chi-square goodness-of-fit tests were used to compare between multiple proportions. Likewise, univariable logistic regression analysis was performed to determine if there was a change in the probability of isolating the major mastitis pathogens depending on geography or time. Seasonality was assessed by calculating the seasonal relative risk (RR). Of a total of 85,979 milk samples examined, more than half of the samples (61.07%) showed no growth and the proportion of samples that showed no growth almost halved during the study period. Of the samples (36.21%, n = 31,133) that showed any growth, the major bacterial pathogens were Staphylococcus aureus (15.60%), Non-aureus Staphylococci (NAS) (5.04%), Corynebacterium spp. (2.96%), and Escherichia coli (2.00%). Of the NAS, the major species reported were Staphylococcus chromogenes (69.02%), Staphylococcus simulans (14.45%), Staphylococcus epidermidis (12.99%), and Staphylococcus hyicus (2.13%). A temporal change in the prevalence of contagious pathogens like S. aureus and Corynebacterium spp. was observed with an increasing odds of 1.06 and 1.62, respectively. Likewise, except for Trueperella pyogenes, the prevalence of all the major environmental mastitis pathogens increased during the study period. The isolation of most of the pathogens peaked in summer, except for S. aureus, T. pyogenes, and Streptococcus dysgalactiae which peaked in spring months. Interestingly, a regional pattern of isolation of some bacterial pathogens within Ontario was also observed. This study showed a marked spatio-temporal change in the prevalence of major mastitis pathogens and suggests that a regional and seasonal approach to mastitis control could be of value.

Silver and Copper Nanoparticles Inhibit Biofilm Formation by Mastitis Pathogens.

Bovine mastitis is a common bovine disease, frequently affecting whole herds of cattle. It is often caused by resistant microbes that can create a biofilm structure. The rapidly developing scientific discipline known as nanobiotechnology may help treat this illness, thanks to the extraordinary properties of nanoparticles. The aim of the study was to investigate the inhibition of biofilms created by mastitis pathogens after treatment with silver and copper nanoparticles, both individually and in combination. We defined the physicochemical properties and minimal inhibitory concentration of the nanoparticles and observed their interaction with the cell membrane, as well as the extent of biofilm reduction. The results show that the silver-copper complex was the most active of all nanomaterials tested (biofilm was reduced by nearly 100% at a concentration of 200 ppm for each microorganism species tested). However, silver nanoparticles were also effective individually (biofilm was also reduced by nearly 100% at a concentration of 200 ppm, but at concentrations of 50 and 100 ppm, the extent of reduction was lower than for the complex). Nanoparticles can be used in new alternative therapies to treat bovine mastitis.

Milk Lactose as a Biomarker of Subclinical Mastitis in Dairy Cows.

Bovine subclinical mastitis can cause great harm to dairy herds because of its negative impact on milk production and quality and cow health. Improved diagnostic tools are needed to maximise the control of subclinical mastitis distribution and ensure the high quality of milk as an industrial product. Between 2015 and 2020, seventy-two dairy herds were screened for bovine subclinical mastitis causative agents to identify the relationship between seasons, lactose levels and subclinical mastitis infection. The predominant species found in the milk samples were mixed microbiota, coagulase-negative Staphylococcus and Staphylococcus aureus. Yeasts were found exclusively in autumn, while Enterococcus spp. and Escherichia coli were only found in summer and autumn. A negative correlation was detected between milk lactose and number of somatic cells in milk (-0.471; p < 0.001). The lactose levels in milk were closely associated with the prevalence (%) of subclinical mastitis pathogens, such as Streptococcus agalactiae (y = -1.8011x + 10.867, R2 = 0.9298), Staph. aureus (y = -3.5216x + 25.957, R2 = 0.8604) and other Streptococci (y = -0.5956x + 7.6179, R2 = 0.6656). These findings suggest that milk lactose may be used as a biomarker of suspected udder inflammation in modern health prevention programmes for cows to reduce the prevalence of subclinical mastitis pathogens in dairy cattle herds.

Antimicrobial Activity of Sorghum Phenolic Extract on Bovine Foodborne and Mastitis-Causing Pathogens.

Antimicrobial resistance in bacterial pathogens associated with bovine mastitis and human foodborne illnesses from contaminated food and water have an impact on animal and human health. Phenolic compounds have antimicrobial properties and some specialty sorghum grains are high in phenolic compounds, and the grain extract may have the potential as a natural antimicrobial alternative. The study's objective was to determine antimicrobial effects of sorghum phenolic extract on bacterial pathogens that cause bovine mastitis and human foodborne illnesses. Bacterial pathogens tested included Escherichia coli, Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Staphylococcus aureus, and Enterococcus faecalis. Antibacterial activities of sorghum phenolic extracts were determined by agar-well diffusion assay. Sorghum phenolic extract was added to the wells in concentrations of 0, 100, 200, 500, 1000, or 4000 µg/mL. The control wells did not receive phenolic extract. Plates were incubated for 18-24 h, and the diameter of each zone of inhibition was measured. The results indicated that sorghum phenolic extract had inhibitory effects on Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, and Campylobacter coli.

Microscopic differential cell count and specific mastitis pathogens in cow milk from compost-bedded pack barns and cubicle barns.

Compost bedded pack barns (compost) as a new free walk housing system favorably influence udder health due to improved animal welfare and lying comfort. On the other hand, unfavorable effects on udder health are possible, due to the open bedded pack and the associated larger bacterial content in moisture. For in-depth farming system comparisons, the present study aimed to evaluate the specific cell fractions and mastitis pathogens in milk from cows kept in compost and in conventional cubical barns (cubicle). For milk sample collection we used a repeated measurement data structure of 2,198 udder quarters from 537 Holstein cows kept in six herds (3 in compost and 3 in cubicle). Differential cell counting was conducted including lymphocytes, macrophages and polymorphonuclear leukocytes (PMN). Specific mastitis pathogens comprised major and minor pathogens. Mixed models were applied to infer environmental and cow associated effects on cell fractions and on prevalences for pathogen infections, with specific focus on system × lactation stage, system × milk yield and system × somatic cell count effects. The interaction between system and lactation stage showed significant differences (P < 0.01) between the systems. A significantly smaller number of bacteriologically positive quarters and lower prevalences for minor pathogens were detected in compost compared to cubicle. Least squares means for pathogen prevalences indicated a quite constant proportion of bacteriologically negative udder quarters across milk yield levels in compost, but a slight increase with increasing milk yield in cubicle. Cell fraction responses in both systems differed in relation to the overall bacteriological infection status and farming system particularities. In conclusion, different cell fractions and specific mastitis pathogens should be considered as an indicator for udder health in different production systems, taking into account cow associated factors (lactation stage, milk yield).

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

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

Molecular characterization of antimicrobial resistance and virulence genes of Escherichia coli isolates from bovine mastitis.

BACKGROUND AND AIM: Mastitis is a common and economically important disease in dairy cattle. It remains one of the most common reasons for the extensive use of antimicrobials in dairy farms leading to the emergence of antimicrobial-resistant pathogens. The aim of this study was to determine the patterns of antimicrobial resistance of Escherichia coli isolates from bovine mastitis and to identify prominent antimicrobial resistance and virulence genes among isolated strains. MATERIALS AND METHODS: Antimicrobial susceptibility testing against six antibiotic groups, including tetracyclines, aminoglycosides, beta-lactams, macrolides, sulfonamides, and fluoroquinolones was performed using the disk diffusion method. PCR was performed on resistant isolates to detect resistance and virulence genes using commercially available primers. RESULTS: Out of 216 milk samples cultured, 14 samples yielded E. coli isolates. All isolates (100%) were resistant to ampicillin, amoxicillin, procaine penicillin, streptomycin, oxytetracycline, and sulfamethoxazole-trimethoprim. Only one isolate (7%) was sensitive to gentamicin, and all isolates (100%) were sensitive to enrofloxacin and ciprofloxacin. All isolates carried at least one resistance gene against one or more of the major antibiotic groups. All isolates carried the ereA, tetG, tetE, and tetB genes, followed by tetA (93%), ampC (86%), strA (86%), sul1 (78%), tetD (71%), tetC (57%), aadA (57%), and strB (36%). The lowest percentage of isolates carried bla1 (17%) and bla2 (12%) genes, and none of the isolates carried the qnrA gene. Most of the isolates (93%) carried the Shiga toxin 1 virulence gene, followed by complement resistance protein (79%), intimin (64%), Shiga toxin 2 (36%), cytotoxic necrotizing factor (35%), aerotaxis receptor (21%), and type 1 fimbriae (15%). CONCLUSION: Results of this study indicate that the high percentages of E. coli isolate from bovine mastitis are resistant to two or more of the major antibiotic groups, irrespective of the presence or absence of relevant resistance or virulence genes.