Immunotherapy in mastitis: state of knowledge, research gaps and way forward.

Mastitis is an inflammatory condition that affects dairy cow's mammary glands. Traditional treatment approaches with antibiotics are increasingly leading to challenging scenarios such as antimicrobial resistance. In order to mitigate the unwanted side effects of antibiotics, alternative strategies such as those that harness the host immune system response, also known as immunotherapy, have been implemented. Immunotherapy approaches to treat bovine mastitis aims to enhance the cow's immune response against pathogens by promoting pathogen clearance, and facilitating tissue repair. Various studies have demonstrated the potential of immunotherapy for reducing the incidence, duration and severity of mastitis. Nevertheless, majority of reported therapies are lacking in specificity hampering their broad application to treat mastitis. Meanwhile, advancements in mastitis immunotherapy hold great promise for the dairy industry, with potential to provide effective and sustainable alternatives to traditional antibiotic-based approaches. This review synthesizes immunotherapy strategies, their current understanding and potential future perspectives. The future perspectives should focus on the development of precision immunotherapies tailored to address individual pathogens/group of pathogens, development of combination therapies to address antimicrobial resistance, and the integration of nano- and omics technologies. By addressing research gaps, the field of mastitis immunotherapy can make significant strides in the control, treatment and prevention of mastitis, ultimately benefiting both animal and human health/welfare, and environment health.

Evaluation of the efficacy of a new inactivated vaccine against Staphylococcus aureus, Echerchia coli and Mycoplasma bovis mastitis in cows.

Staphylococcus aureus, Escherichia coli and Mycoplasma bovis are the most commonly isolated mastitis pathogens. The aim of this study was to evaluate the efficacy of a new mixed vaccine against mastitis caused by  Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis. For this purpose, a mixed inactivated vaccine was administered subcutaneously to 24 heifers as one dose (2 mL) on the 45th day before birth and the second dose 21 days later. In 9 heifers, 2 mL of PBS was administered as placebo instead of vaccine. Then, heifers were divided into 3 groups as 7 vaccinated and 3 unvaccinated animals. Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis were administered to the groups through intramammary route. Three vaccinated heifers were considered the common control without bacteria in all groups. The parameters considered to assess the effect of vaccination were clinical findings, bacterial count in milk, somatic cell count, and antibody titers. Clinical signs were observed only in the unvaccinated placebo group. Bacteria count and somatic cell count in milk increased in vaccinated and unvaccinated heifers. However, this increase was less in vaccinated animals and gradually returned to the normal level. In the unvaccinated heifers, it was ever high. Serum antibody titers were measured before and after vaccination. Antibody titers were high in vaccinated heifers after vaccination and were negative in unvaccinated heifers. In conclusion, the mixed vaccine had beneficial effect against Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis mastitis and stimulated the immune response of vaccinated heifers.

Exploring the impact of biosecurity measures on somatic cell score in mountain dairy farms considering the CLASSYFARM system.

Biosecurity plays a crucial role in preventing the introduction of infectious diseases to a herd as well as the spread of diseases within or between animals and herds. In particular, biosecurity measures are crucial for maintaining animal health and reducing the need for the application of antibiotic substances for fighting the rising antibiotic resistance. The object of this cross-sectional study was to investigate the presence of biosecurity measures and their association with milk quality parameters, with a special focus on somatic cell count (SCC) - an indicator for udder health -, in small-scale mountain dairy farms. Therefore, for the very first time, the CLASSYFARM system was considered, which is a computer platform integrated into the Italian national veterinary portal, that processes a significant amount of data from various sources collected in the field or from other information systems (e.g. animal welfare, health status, biosecurity, antimicrobial use, slaughterhouse information). A total of 169 dairy farms were included in the study. Biosecurity measures, based on 15 questions required in the CLASSYFARM welfare assessment protocol, as well as information about husbandry systems, milking systems and pasture practices were gathered and combined with milk yield data, provided by the South Tyrolean dairy association. Farms only scored 44.00 points on average in a scoring system from 0.00 to 100.00 points that was be able to summarize 15 different biosecurity measures in one index. Our results show a clear negative correlation (-0.713) between the biosecurity index and somatic cell score (SCS) indicating that a higher level of biosecurity, which reflects the presence of biosecurity measures within a farm, is associated with lower SCC levels. Furthermore, we found significant correlations between SCS and milk production (-0.629), confirming that udder health is linked to higher milk production. Fat, protein, and the fat-to-protein ratio showed a positive correlation with SCS (0.281, 0.146, 0.106), likely to be caused by a concentration shift effect (dilution effect). Husbandry system, breed, milking system, and pasture practices seem to have an impact as well, but the main factor was the biosecurity score. This study highlights the importance of implementing biosecurity measures for ensuring animal health and thus productivity and quality in milk production, even in small-scale farms, which are characterized by limited structure availability and smaller herds compared to big dairy enterprises in the lowlands.

Dietary anti-inflammatory and anti-bacterial medicinal plants and its compounds in bovine mastitis associated impact on human life.

Bovine mastitis (BM) is the most common bacterial mediated inflammatory disease in the dairy cattle that causes huge economic loss to the dairy industry due to decreased milk quality and quantity. Milk is the essential food in the human diet, and rich in crucial nutrients that helps in lowering the risk of diseases like hypertension, cardiovascular diseases and type 2 diabetes. The main causative agents of the disease include various gram negative, and positive bacteria, along with other risk factors such as udder shape, age, genetic, and environmental factors also contributes much for the disease. Currently, antibiotics, immunotherapy, probiotics, dry cow, and lactation therapy are commonly recommended for BM. However, these treatments can only decrease the rise of new cases but can't eliminate the causative agents, and they also exhibit several limitations. Hence, there is an urgent need of a potential source that can generate a typical and ideal treatment to overcome the limitations and eliminate the pathogens. Among the various sources, medicinal plants and its derived products always play a significant role in drug discovery against several diseases. In addition, they are also known for its low toxicity and minimum resistance features. Therefore, plants and its compounds that possess anti-inflammatory and anti-bacterial properties can serve better in bovine mastitis. In addition, the plants that are serving as a food source and possessing pharmacological properties can act even better in bovine mastitis. Hence, in this evidence-based study, we particularly review the dietary medicinal plants and derived products that are proven for anti-inflammatory and anti-bacterial effects. Moreover, the role of each dietary plant and its compounds along with possible role in the management of bovine mastitis are delineated. In this way, this article serves as a standalone source for the researchers working in this area to help in the management of BM.

Post-milking application of a Lacticaseibacillus paracasei strain impacts bovine teat microbiota while preserving the mammary gland physiology and immunity.

Bovine mastitis (BM) is a major disease in dairy industry. The current approaches - mainly antibiotic treatments - are not entirely effective and may contribute to antimicrobial resistance dissemination, rising the need for alternative treatment. The present study aims to evaluate the impact of post-milking application of Lacticaseibacillus paracasei CIRM BIA 1542 (Lp1542) on the teat skin (TS) of 20 Holstein cows in mid lactation, in order to reinforce the barrier effect of the microbiota naturally present on the teat. Treatment (Lp1542, iodine or no treatment) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples, and TS swabs were collected at day (D)1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15E) to evaluate Lp1542 impact at the microbial, immune and physiological levels. Lp1542 treatment resulted in a higher lactic acid bacteria and total microbial populations on TS and in foremilk (FM) at D15(E) compared with iodine treatment. Metabarcoding analysis revealed changes in the composition of TS and FM microbiota, beyond a higher Lacticaseibacillus abundance. This included a higher abundance of Actinobacteriota, including Bifidobacterium, and a lower abundance of Pseudomonadota on TS of Lp1542 compared with iodine-treated quarters. In addition, Lp1542 treatment did not trigger any major inflammatory response in the mammary gland, except interleukin 8 production and expression which tended to be slightly higher in Lp1542-treated cows compared with the others. Finally, Lp1542 treatment had no impact on the mammary epithelium functionality (milk yield and composition) and integrity (epithelial cell exfoliation into milk and milk Na+/K+ ratio). Altogether, these results indicate that a topical treatment with Lp1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.

Phage cocktail superimposed disinfection: A ecological strategy for preventing pathogenic bacterial infections in dairy farms.

Bovine mastitis (BM) is mainly caused by bacterial infection that has a highly impact on dairy production, affecting both economic viability and animal well-being. A cross-sectional study was conducted in dairy farms to investigate the prevalence and antimicrobial resistance patterns of bacterial pathogens associated with BM. The analysis revealed that Staphylococcus (49%), Escherichia (16%), Pseudomonas (11%), and Klebsiella (6%) were the primary bacterial pathogens associated with mastitis. A significant proportion of Staphylococcus strains displayed multiple drug resistance. The use of disinfectants is an important conventional measure to control the pathogenic bacteria in the environment. Bacteriophages (Phages), possessing antibacterial properties, are natural green and effective disinfectants. Moreover, they mitigate the risk of generating harmful disinfection byproducts, which are commonly associated with traditional disinfection methods. Based on the primary bacterial pathogens associated with mastitis in the investigation area, a phage cocktail, named SPBC-SJ, containing seven phages capable of lysing S. aureus, E. coli, and P. aeruginosa was formulated. SPBC-SJ exhibited superior bactericidal activity and catharsis effect on pollutants (glass surface) compared to chemical disinfectants. Clinical trials confirmed that the SPBC-SJ-based superimposed disinfection group (phage combined with chemical disinfectants) not only cut down the dosage of disinfectants used, but significantly reduced total bacterial counts on the ground and in the feeding trough of dairy farms. Furthermore, SPBC-SJ significantly reduced the abundance of Staphylococcus and Pseudomonas in the environment of the dairy farm. These findings suggest that phage-based superimposed disinfection is a promising alternative method to combat mastitis pathogens in dairy farms due to its highly efficient and environmentally-friendly properties.

[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.

Understanding barriers to reducing antimicrobials on Australian dairy farms: A qualitative analysis.

INTRODUCTION: Reducing antibiotic use in production animal systems is one strategy which may help to limit the development of antimicrobial resistance. To reduce antimicrobial use in food-producing animals, it is important to first understand how antibiotics are used on farm and what barriers exist to decreasing their use. In dairy production systems, mastitis is one of the most common reasons for administering antimicrobials. Therefore, it is important to understand the motivations and behaviours of dairy farmers in relation to the diagnosis, treatment and prevention of mastitis. MATERIALS AND METHODS: In this study, we interviewed a sample of dairy farmers and dairy industry professionals from the major dairying regions of eastern Australia regarding their current practices used to diagnose, treat, and control subclinical and clinical mastitis. Inductive thematic analysis was used to code interview transcripts and identify the recurrent themes. RESULTS: Four overarching themes were identified: (1) the challenges associated with the detection and diagnosis of clinical mastitis, including with laboratory culture, (2) the motivations behind treatment decisions for different cases, (3) decisions around dry cow therapy and the role of herd recording, and (4) concerns regarding the development of antimicrobial resistance. DISCUSSION: This study identifies several challenges which may limit the ability of Australian dairy farmers to reduce antimicrobial use on farm, such as the need for rapid and reliable diagnostic tests capable of identifying the pathogenic causes of mastitis and the difficulties associated with conducting herd recording for the implementation of selective dry cow therapy. Industry professionals were concerned that farmers were not using individual cow records to aid in treatment decisions, which could result in unnecessary antimicrobial use. The results of this study can act as the basis for future research aimed at assessing these issues across the broader Australian dairy industry.

Concentrations of antimicrobial components in milk at dry off and postpartum and their relationships to new high somatic cell counts at quarter level in dairy cows.

We investigated the antimicrobial components in cow milk at dry off and postpartum and their contribution in preventing new high SCC at quarter level. Milk samples from 72 quarters of 19 lactating cows were collected at last milking before dry off and at 7 d after parturition. Milk yield of each cow was recorded and SCC, IgG, IgA, lactoferrin, lingual antimicrobial peptide (LAP), and S100A7 concentrations in each quarter milk sample were measured. The postpartum milk yield was significantly higher than that at dry off. The IgG, IgA and lactoferrin concentrations in milk at dry off were significantly higher than those at postpartum, whereas the LAP concentration was lower. Quarters with SCC < 300 000 cells/ml at both dry off and postpartum were classified as persistent low SCC (PL) whereas those that rose above that same threshold postpartum were classified as new high SCC (NH). At dry off, IgG and LAP concentrations in milk were significantly higher in PL than in NH. These results suggest that high LAP concentrations during the dry period may contribute toward the prevention of new high SCC.

Understanding mastitis: Microbiome, control strategies, and prevalence - A comprehensive review.

Mastitis significantly affects the udder tissue in dairy cattle, leading to inflammation, discomfort, and a decline in both milk yield and quality. The condition can be attributed to an array of microbial agents that access the mammary gland through multiple pathways. The ramifications of this ailment are not merely confined to animal welfare but extend to the financial viability of the livestock industry. This review offers a historical lens on mastitis, tracing its documentation back to 1851, and examines its global distribution with a focus on regional differences in prevalence and antimicrobial resistance (AMR) patterns. Specific microbial genes and communities implicated in both mastitis and AMR are explored, including Staphylococcus aureus, Streptococcus agalactiae,Streptococcus dysagalactiae, Streptococcus uberis Escherichia coli, Klebsiella pneumoniae, Mycoplasma bovis, Corynebacterium bovis, among others. These microorganisms have evolved diverse strategies to elude host immune responses and neutralize commonly administered antibiotics, complicating management efforts. The review aims a comprehensive overview of the current knowledge and research gaps on mastitis and AMR, and to highlight the need for a One Health approach to address this global health issue. Such an approach entails multi-disciplinary cooperation to foster judicious antibiotic use, enhance preventive measures against mastitis, and bolster surveillance and monitoring of AMR in pathogens responsible for mastitis.

Efecto ecotóxico y preventivo del uso de aceites esenciales en la mastitis bovina / Ecotoxic and preventive effect of the use of essential oils in bovine mastitis

The essential oils of Lippia citriodora (Ort.) and Lippia origanoides (Kunth) have shown antimicrobial activity associated with mastitis. The objective of this study was to evaluate its ecotoxic effect with the Artemia salina bioassay and the prevention of mastitis through an in vivo test in cattle (n=20) with a product based on these oils using a conventional product as a control. Contact hypersensitivity, the effect on somatic cells, and residuality in mil k samples were evaluated. The results of the Artemia salina bioassay were 10.05 and 19.36 (µg/mL) respectively. No negative effects or contact hypersensitivity were observed, and no residual metabolites were found in post - test milk. The somatic cell count showed 75% effectiveness in the prevention of mastitis with essential oils compared to 62.5% with the conventional product. The evaluated formulation could be used in the prevention of bovine mastitis safely, further investigation is required.

Los aceites esenciales de Lippia citriodora (Ort.) y Lippia origanoides (Kunth), han mostrado acti vidad antimicrobiana asociada a la mastitis. El objetivo de este estudio fue evaluar su efecto ecotóxico con el bioensayo Artemia salina y la prevención de mastitis mediante un ensayo in vivo en bovinos (n=20) con un producto a base de estos aceites utiliz ando como control un producto convencional. Se evaluó la hipersensibilidad de contacto, efecto en células somáticas y residualidad en muestra de leche. Los resultados del bioensayo de Artemia salina fueron 10,05 y 19,36 (µg/mL) respectivamente. No se obser varon efectos negativos, ni hipersensibilidad de contacto, y no se encontraron metabolitos residuales en leche posterior al ensayo. El conteo de células somáticas mostró efectividad en la prevención de mastitis del 75% con aceites esenciales frente al 62.5 % del producto convencional. La formulación evaluada podría ser utilizada en la prevención de la mastitis bovina de forma segura, se requiere profundizar en la investigación.

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.

Milk as diagnostic fluid for udder health management.

BACKGROUND: Mastitis is the major disease affecting milk production of dairy cattle, and milk is an obvious substrate for the detection of both the inflammation and its causative infectious agents at quarter, cow, or herd levels. In this review, we examine the use of milk to detect inflammation based on somatic cell count (SCC) and other biomarkers, and for the detection of mastitis pathogens through culture-based and culture-free methods. FINDINGS: The use of SCC at a cow or bulk milk level to guide udder health management in lactation is well-established, and SCC is increasingly used to guide selective dry cow treatment. Other markers of inflammation include electrical conductivity, which is used commercially, and markers of disease severity such as acute phase proteins but are not pathogen-specific. Some pathogen-specific markers based on humoral immune responses are available, but their value in udder health management is largely untested. Commercial pathogen detection is based on culture or polymerase chain reaction, with other tests, for example, loop-mediated isothermal amplification or 16S microbiome analysis still at the research or development stage. Matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) is increasingly used for the identification of cultured organisms whilst application directly to milk needs further development. Details of test sensitivity, specificity, and use of the various technologies may differ between quarter, cow, and bulk milk applications. CONCLUSIONS: There is a growing array of diagnostic assays that can be used to detect markers of inflammation or infection in milk. The value of some of these methods in on-farm udder health improvement programs is yet to be demonstrated whilst methods with proven value may be underutilised.

Improvement of mammary gland health in 81 'sentinel herds' in England and Scotland between 2012 and 2021.

BACKGROUND: Achieving a reduction in mastitis in dairy cows is a common industry goal, but there is no recent peer-reviewed record of progress in the UK. METHODS: A convenience sample of 125 herds in England and Scotland was recruited based on the quality of records in 2016, willingness to participate and representative geographical distribution. Individual cow somatic cell counts and clinical mastitis data from 2012 to 2021 were summarised annually, and temporal changes were analysed. Eighty-one herds had sufficient data for comparison between 2012 and 2021, for one or more parameters. RESULTS: Over this period, the median incidence rate of clinical mastitis decreased from 40.0 to 21.0 cases per 100 cows per year (p < 0.001), with improvement in both lactation and dry period indicators. Lactation new infection rate calculated from individual cow somatic cell counts fell from 8.75% to 5.95% (p < 0.001), dry period new infection rate fell from 16.8% to 14.1% (p < 0.05) and proportion of cows over 200,000 cells/mL fell from 20.0% to 14.3% (p < 0.001). LIMITATIONS: Data were necessarily from herds with good records and do not provide absolute values for the industry. CONCLUSION: The findings reflect good progress over a 10-year period in a cohort of well-recorded herds and align with other national datasets.

Evaluation of the Efficacy of a Lactobacilli-Based Teat Detergents for the Microbiota of Cows Teats Using an Untargeted Metabolomics Approach.

Teat cleaning pre- and post-milking is important for the overall health and hygiene of dairy cows. The purpose of this research was to evaluate the effectiveness of a teat detergents based on lactic acid bacteria according to changes in somatic cell count and cow-milk metabolites. Sixty-nine raw milk samples were collected from 11 Holstein-Friesian cows in China during 12 days of teat cleaning. An ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry-based untargeted metabolomic approach was applied to detect metabolomic differences after treatment with lactic acid bacteria and chemical teat detergents in cows with subclinical mastitis. The results suggest that the lactobacilli-based teat detergents could reduce somatic cell count and improve microhabitat of cow teat apex by adjusting the composition of metabolites. Furthermore, the somatic cell count could be decreased significantly within 10 days following the cleaning protocol. Lactic acid bacteria have the potential to be applied as a substitution to teat chemical detergents before and after milking for maintenance of healthy teats and breasts. Further, larger scale validation work is required to support the findings of the current study.

Preparation and Antimicrobial Activity of a Film-Forming Polyhexamethylene Biguanide Teat Disinfectant.

Bovine mastitis caused by infectious pathogens can lead to a decline in production performance and an increase in elimination rate, resulting in huge losses to the dairy industry. This study aims to prepare a novel dairy cow teat disinfectant with polyhexamethylene biguanide (PHMB) as the main bactericidal component and to evaluate its bactericidal activity in vitro and its disinfection effect in dairy cow teats. PHMB disinfectant with a concentration of 3 g/L was prepared with PVA-1788, propylene glycol and glycerol as excipients. When the dilution ratio is 1:4800 and the action time is 5 min, the PHMB teat disinfectant can reduce the four types of bacteria (S. agalactiae ATCC 12386, S. dysgalactiae ATCC 35666, S. aureus ATCC 6538, and E. coli ATCC 8099) by 99.99%. PHMB teat disinfectant applied on the skin of rabbits with four bacteria types achieved an average log10 reduction greater than 4. After 30 s of PHMB teat disinfectant dipping, the bacteria of cow teats were counted prior to disinfection. The mean log10 reduction in bacteria on the skin surface of 12 cows ranged from 0.99 to 3.52 after applying the PHMB teat disinfectant for 10 min. After 12 h, the PHMB teat disinfectant achieved an average log10 reduction in bacteria from 0.27 to 0.68 (compared with that prior to disinfection). These results suggested that PHMB teat disinfection has the potential to prevent and treat mastitis-causing bacteria in dairy herds.

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.

Teat-apex colonizer Bacillus from healthy cows antagonizes mastitis-causing Staphylococcus aureus biofilms.

Staphylococcus aureus is the most frequent causal agent of bovine mastitis, which is largely responsible for milk production losses worldwide. The pathogen's ability to form stable biofilms facilitates intramammary colonization and may explain disease persistence. This virulence factor is also highly influential in the development of chronic intramammary infections refractory to antimicrobial therapy, which is why novel therapies that can tackle multiple targets are necessary. Since udder microbiota have important implications in mastitis pathogenesis, they offer opportunities to develop alternative prophylactic and therapeutic strategies. Here, we observed that a Bacillus strain from the teat apex of lactating cows was associated to reduce colonization by S. aureus. The strain, identified as Bacillus sp. H21, was able to antagonize in-formation or mature S. aureus biofilms associated to intramammary infections without affecting cell viability. When exploring the metabolite responsible for this activity, we found that a widespread class of Bacillus exopolysaccharide, levan, eliminated the pathogenic biofilm under evaluated conditions. Moreover, levan had no cytotoxic effects on bovine cellular lines at the biologically active concentration range, which demonstrates its potential for pathogen control. Our results indicate that commensal Bacillus may counteract S. aureus-induced mastitis, and could therefore be used in novel biotechnological strategies to prevent and/or treat this disease.

Usefulness of the total and differential somatic cell count based udder health group concept for evaluating herd management practices and udder health in dairy herds.

Subclinical mastitis and associated economic losses are a steady challenge in the dairy industry. The combination of the well-established somatic cell count (SCC) parameter and the new differential SCC (DSCC) opens up the possibility to categorise cows into four different udder health groups (UHG) based on results from a single milk recording/dairy herd improvement (DHI) test: UHG A: healthy/normal, ≤ 200,000 cells/mL and DSCC ≤ 65 %; B: suspicious, ≤ 200,000 cells/mL and DSCC > 65 %; C: (subclinical) mastitis, > 200,000 cells/mL and DSCC > 65 %; D: chronic/persistent mastitis, > 200,000 cells/mL and DSCC ≤ 65 %. The objectives of this study were to investigate 1) herd management practises among herds in different UHG categories and 2) herd performance parameters depending on the proportion of cows in UHG A. A total number of 41 herds in Styria, Austria, and Thuringia, Germany, were visited and interviewed for the first part of the study. The herds were categorised into 3 UHG categories depending on the proportion of cows in UHG A: I = >65 %; II = 55-65 %; and III = <55 %. Those with good udder health and best herd performance (+9 % milk yields, +11 % longevity, -35 % antibiotic treatments) applied distinct preventive measures, in particular excellent cubicle management and early antibiotic treatment (P < 0.05 each). However, preventive measures were applied to a lower extent in other herds. Herds were categorised differently using the UHG concept compared to SCC alone as the UHG-based categorisation allowed to clearer distinguish herds with medium-good from those with good udder health. A total number of 129,812 regular milk recording/DHI test day results of 890 Austrian and 183 German herds was used for the second part of the study. Results revealed a trend of increasing daily production as proportions of cows in UHG A increase. In conclusion, the UHG concept allowed clearer distinction of herds with good, medium-good, and poor udder health and could be used to promote practises leading to better animal health, less antibiotic treatments, and higher milk quality.

Molecular regulatory mechanism of key LncRNAs in subclinical mastitic cows with folic acid supplementation.

BACKGROUND: Folic acid is a water-soluble B vitamin (B9), which is closely related to the body's immune and other metabolic pathways. The folic acid synthesized by rumen microbes has been unable to meet the needs of high-yielding dairy cows. The incidence rate of subclinical mastitis in dairy herds worldwide ranged between 25%~65% with no obvious symptoms, but it significantly causes a decrease in lactation and milk quality. Therefore, this study aims at exploring the effects of folic acid supplementation on the expression profile of lncRNAs, exploring the molecular mechanism by which lncRNAs regulate immunity in subclinical mastitic dairy cows. RESULTS: The analysis identified a total of 4384 lncRNA transcripts. Subsequently, differentially expressed lncRNAs in the comparison of two groups (SF vs. SC, HF vs. HC) were identified to be 84 and 55 respectively. Furthermore, the weighted gene co-expression network analysis (WGCNA) and the KEGG enrichment analysis result showed that folic acid supplementation affects inflammation and immune response-related pathways. The two groups have few pathways in common. One important lncRNA MSTRG.11108.1 and its target genes (ICAM1, CCL3, CCL4, etc.) were involved in immune-related pathways. Finally, through integrated analysis of lncRNAs with GWAS data and animal QTL database, we found that differential lncRNA and its target genes could be significantly enriched in SNPs and QTLs related to somatic cell count (SCC) and mastitis, such as MSTRG.11108.1 and its target gene ICAM1, CXCL3, GRO1. CONCLUSIONS: For subclinical mastitic cows, folic acid supplementation can significantly affect the expression of immune-related pathway genes such as ICAM1 by regulating lncRNAs MSTRG.11108.1, thereby affecting related immune phenotypes. Our findings laid a ground foundation for theoretical and practical application for feeding folic acid supplementation in subclinical mastitic cows.