Early detection of intramammary infection (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 utilize 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 somatic cell count (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 5756 quarter milk samples (QMS) 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 d in milk (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 Staph. 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 intramammary infection (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 Strep. dysgalactiae, followed by Strep. simulans, Strep. 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.

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.

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.

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.

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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.
CONCLUSIONS: 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.

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.

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).

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.

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.