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Abstract Background The periparturient period in dairy cows is marked by immunosuppression which increases the likelihood of infectious disorders, particularly also mastitis. An in-depth understanding of peripartum leukocyte biology is vital for the implementation of highly successful post-partum disease prevention measures. Immune checkpoint molecules, such as programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), are critical inhibitory receptors expressed on immune cells, particularly T cells, that drive immunosuppressive signaling pathways. However, the potential role of immune checkpoint molecules expression in T-cells on udder health has never been explored. Thus, the association between the occurrence of new postpartum intramammary infections (IMIs) and the expression of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on blood T-cells during the peripartum period was investigated. Results In this study, the incidence of IMIs by any pathogen in early lactation was not associated with a higher expression of PD-1 and CTLA-4 in the periparturient period. However, the incidence of IMIs by major pathogens throughout the first month of lactation was significantly associated with higher expression of PD-1 at 14 days before calving (P = 0.03) and CTLA-4 at parturition (P = 0.03) by blood T-cells. Also, the expression of CTLA-4 at D0 (P = 0.012) by T-cells was associated with the occurrence of persistent IMIs during the first month of lactation. Conclusions To our knowledge, this is the first report to investigate the expression of PD-1 and CTLA-4 by blood T-lymphocytes during the periparturient period in dairy cows and to explore their relationship with the incidence of new IMIs in the postpartum period. Thus, a comprehensive understanding of leukocyte biology during peripartum would appear to be a prerequisite for the identification of resilient dairy cows or targets innovative (immunological) non-antibiotic approaches in the transition period.

ABSTRACT: Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with ferritin iron supplementation. Only Staphylococcus hominis strains originating from quarter milk were able to significantly utilize ferritin as an iron source to reverse the growth inhibition caused by chelating agent 2,2ʹ-bipyridyl in varying degrees. Both S. chromogenes strains (IM and TA) and all S. hominis strains were unable to significantly use lactoferrin as an iron source for growth recovery.

Abstract Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

ABSTRACT: Non-aureus staphylococci and the closely related mammaliicoccal species (NASM) are the most common causes of bovine subclinical mastitis on modern dairy farms and are highly prevalent in bulk-tank milk. The purpose of this study was to determine the distribution of NASM in both composite cow milk (CCM) and bulk-tank milk (BTM) samples collected in tandem in commercial Flemish dairy herds and to estimate the origin of the different (subgroups of) NASM species present in BTM by applying strain typing (random amplification of polymorphic DNA or random amplified DNA [RAPD]). A single cross-sectional sampling was performed over 5 herds that volunteered to participate in the study. Composite cow milk samples (n = 356) were collected from all lactating cows (except those with clinical mastitis) during a milking in tandem with 6 BTM samples per herd sequentially collected immediately post that milking (n = 30). In total, 421 and 80 NASM isolates were recovered and identified by MALDI-TOF mass spectrometry from the CCM and BTM samples, respectively and a total of 21 and 12 different NASM species were identified from CCM and BTM samples, respectively. Staphylococcus cohnii was the most prevalent NASM species found in BTM followed by Staphylococcus haemolyticus, Staphylococcus epidermidis, Mammaliicoccus lentus, and Staphylococcus equorum, whereas from CCM samples the most common species were S. hemolyticus, S. cohnii, S. equorum, S. epidermidis, and Staphylococcus chromogenes. The prevalent NASM species in both CCM and BTM samples was distinct for each herd, corroborating other studies observing a herd-specific NASM microbiota. Random amplified DNA analysis was performed on 9 NASM species (S. chromogenes, S. epidermidis, S. haemolyticus, S. equorum, Mammaliicoccus sciuri, Staphylococcus xylosus, S. cohnii, Staphylococcus debuckii, and M. lentus) because these species were isolated from both sample types in a herd. The same RAPD types were found in both sample types for all NASM species selected for strain typing in varying degrees. When assessing the distribution of NASM species, differences within NASM species should be examined meaning a closer look should be taken at the strain level rather than at the species level only.

Abstract This study investigated whether cell-free supernatants (SN) from four bovine non-aureus staphylococcal (NAS) isolates prevent Staphylococcus aureus adhesion to and internalization into bovine mammary epithelial cells (MAC-T cells) and if so, to determine whether such effects were potentially associated with the S. aureus accessory gene regulator (agr) system. Overall, we demonstrated that all SN obtained from the NAS isolates promoted adhesion of a S. aureus agr + strain to, yet reduced the internalization into MAC-T cells, while similar effects were not observed for its agr − mutant strain. Our findings provide novel anti-virulence strategies for treating and controlling bovine S. aureus mastitis.

ABSTRACT: Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.

Abstract Background Older people with multifaceted care needs often require treatment and complex care across different settings. However, transitional care is often inadequately managed, and older people and their informal caregivers are not always sufficiently heard and/or supported in transitional care decision‐making. Objective To explore older people's and informal caregivers' experiences with, views on, and needs concerning empowerment in transitional care decision‐making. Methods A qualitative descriptive study was conducted in the TRANS‐SENIOR consortium's collaborative research using semistructured in‐depth interviews between October 2020 and June 2021 in Flanders, Belgium. A total of 29 people were interviewed, including 14 older people and 15 informal caregivers who faced a transition from home to another care setting or vice versa. Data were analysed according to the Qualitative Analysis Guide of Leuven. Findings Five themes were identified in relation to the participant's experiences, views and needs: involvement in the decision‐making process; informal caregivers' burden of responsibility; the importance of information and support; reflections on the decision and influencing factors. Conclusions Overall, older people and informal caregivers wished to be more seen, recognised, informed and proactively supported in transitional care decision‐making. However, their preferences for greater involvement in decision‐making vary and are affected by several factors that are both intrinsic and extrinsic. Therefore, healthcare systems might seek out age‐tuned and person‐centred empowerment approaches focusing on older people's and informal caregivers' empowerment. For future studies, we recommend developing specific strategies for such empowerment. Patient or Public Contribution Older persons' representatives were involved in designing the TRANS‐SENIOR programme of research, including the current study. Healthcare professionals and nursing care directors were involved in the study design and the selection and recruitment of participants.

ABSTRACT: We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the “commensal” Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4+ and CD8+ subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T- or B-cell proliferation. Furthermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-γ production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multiparous cows also produced significantly more IL-17A and IFN-γ. In contrast to concanavalin A, phytohemagglutinin M-form selectively stimulated T-cell proliferation.

Abstract Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Abstract Background Care transitions across different settings necessitate careful decision-making for all parties involved, yet research indicates that older people and informal caregivers do not have a strong voice in such decisions. Objective To provide a systematic overview of the literature about interventions designed to empower older people and informal caregivers in transitional care decision-making. Design A systematic review (Prospero Protocol CRD42020167961; funded by the EU’s Horizon 2020 program). Data sources Five databases were searched: PubMed, EMBASE, Web of Science, PsycINFO, and CINAHL. Review methods The review included evaluations of empowerment in decision-making interventions for older people and informal caregivers facing care transitions, that were published from the inception of the databases up until April 2022. Data extractions were performed by two independent researchers and the quality of studies was assessed with the relevant JBI-critical appraisal tools. A narrative descriptive analysis of the results was performed. Findings Ten studies, reporting on nine interventions, and including a total of 4642 participants, were included. Interventions included transition preparation tools, support from transition coaches, shared decision-making interventions, and advance care planning. Designs and outcomes assessed were highly diverse and showed a mix of positive and lacking effects. Conclusions There is a lack of research on how to empower older people and their informal caregivers in transitional care decision-making. Empowerment in decision-making is usually not central in transitional care interventions, and effects on actual empowerment are mostly not assessed. Conclusions on how to empower older people and informal caregivers in transitional care decision-making cannot be drawn.

ABSTRACT: Treatment of clinical mastitis (CM) contributes to antimicrobial use on dairy farms. Selective treatment of CM based on bacterial diagnosis can reduce antimicrobial use, as not all cases of CM will benefit from antimicrobial treatment, e.g., mild and moderate gram-negative infections. However, impacts of selective CM treatment on udder health and culling are not fully understood. A systematic search identified 13 studies that compared selective versus blanket CM treatment protocols. Reported outcomes were synthesized with random-effects models and presented as risk ratios or mean differences. Selective CM treatment protocol was not inferior to blanket CM treatment protocol for the outcome bacteriological cure. Noninferiority margins could not be established for the outcomes clinical cure, new intramammary infection, somatic cell count, milk yield, recurrence, or culling. However, no differences were detected between selective and blanket CM treatment protocols using traditional analyses, apart from a not clinically relevant increase in interval from treatment to clinical cure (0.4 d) in the selective group and higher proportion of clinical cure at 14 d in the selective group. The latter occurred in studies co-administering nonsteroidal anti-inflammatories only in the selective group. Bias could not be ruled out in most studies due to suboptimal randomization, although this would likely only affect subjective outcomes such as clinical cure. Hence, findings were supported by a high or moderate certainty of evidence for all outcome measures except clinical cure. In conclusion, this review supported the assertion that a selective CM treatment protocol can be adopted without adversely influencing bacteriological and clinical cure, somatic cell count, milk yield, and incidence of recurrence or culling.

ABSTRACT: Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates (“IM” and “TA”) were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen–derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

ABSTRACT: Administering intramammary antimicrobials to all mammary quarters of dairy cows at drying-off [i.e., blanket dry cow therapy (BDCT)] has been a mainstay of mastitis prevention and control. However, as udder health has considerably improved over recent decades with reductions in intramammary infection prevalence at drying-off and the introduction of teat sealants, BDCT may no longer be necessary on all dairy farms, thereby supporting antimicrobial stewardship efforts. This narrative review summarizes available literature regarding current dry cow therapy practices and associated impacts of selective dry cow therapy (SDCT) on udder health, milk production, economics, antimicrobial use, and antimicrobial resistance. Various methods to identify infections at drying-off that could benefit from antimicrobial treatment are described for selecting cows or mammary quarters for treatment, including utilizing somatic cell count thresholds, pathogen identification, previous clinical mastitis history, or a combination of criteria. Selection methods may be enacted at the herd, cow, or quarter levels. Producers' and veterinarians' motivations for antimicrobial use are discussed. Based on review findings, SDCT can be adopted without negative consequences for udder health and milk production, and concurrent teat sealant use is recommended, especially in udder quarters receiving no intramammary antimicrobials. Furthermore, herd selection should be considered for SDCT implementation in addition to cow or quarter selection, as BDCT may still be temporarily necessary in some herds for optimal mastitis control. Costs and benefits of SDCT vary among herds, whereas impacts on antimicrobial resistance remain unclear. In summary, SDCT is a viable management option for maintaining udder health and milk production while improving antimicrobial stewardship in the dairy industry.

Automated milking systems (AMSs) already incorporate a variety of milk monitoring and sensing equipment, but the sensitivity, specificity, and positive predictive value of clinical mastitis (CM) detection remain low. A typical symptom of CM is the presence of clots in the milk during fore-stripping. The objective of this study was the development and evaluation of a deep learning model with image recognition capabilities, specifically a convolutional neural network (NN), capable of detecting such clots on pictures of the milk filter socks of the milking system, after the phase in which the first streams of milk have been discarded. In total, 696 pictures were taken with clots and 586 pictures without. These were randomly divided into 60/20/20 training, validation, and testing datasets, respectively, for the training and validation of the NN. A convolutional NN with residual connections was trained, and the hyperparameters were optimized based on the validation dataset using a genetic algorithm. The integrated gradients were calculated to explain the interpretation of the NN. The accuracy of the NN on the testing dataset was 100%. The integrated gradients showed that the NN identified the clots. Further field validation through integration into AMS is necessary, but the proposed deep learning method is very promising for the inline detection of CM on AMS farms.

BackgroundDespite numerous attempts to improve interprofessional collaboration and integration (IPCI) in primary care, patients, care providers, researchers, and governments are still looking for tools and guidance to do this more efficiently. To address these issues, we decided to develop a generic toolkit, based on sociocracy and psychological safety principles, to guide care providers in their collaboration within and outside their practice. Finally, we reasoned that, in order to obtain integrated primary care, different strategies should be combined.MethodsDevelopment of the toolkit consisted of a multiyear co-development process. Data originating from 65 care providers, through 13 in-depth interviews and five focus groups were analysed and subsequently evaluated in eight co-design workshop sessions, organised with a total of 40 academics, lecturers, care providers and members of the Flemish patient association. Findings from the qualitative interviews and co-design workshops were gradually, and inductively adapted and transformed into the content for the IPCI toolkit.ResultsTen themes were identified: (i) awareness of the importance of interprofessional collaboration, (ii) the need for a self-assessment tool to measure team performance, (iii) preparing a team to use the toolkit, (iv) enhancing psychological safety, (v) developing and determining consultation techniques, (vi) shared decision making, (vii) developing workgroups to tackle specific (neighbourhood) problems, (viii) how to work patient-centred, (ix) how to integrate a new team member, and (x) getting ready to implement the IPCI toolkit. From these themes, we developed a generic toolkit, consisting of eight modules.ConclusionIn this paper, we describe the multiyear co-development process of a generic toolkit for the improvement of interprofessional collaboration. Inspired by a mix of interventions from in and outside healthcare, a modular open toolkit was produced that includes aspects of Sociocracy, concepts as psychological safety, a self-assessment tool and other modules concerned with meetings, decision-making, integrating new team members and population health. Upon implementation, evaluation and further development and improvement, this compounded intervention should have a beneficial effect on the complex problem of interprofessional collaboration in primary care.

Abstract Background Loneliness is associated with severe consequences for both the physical and mental health of older people. Research has shown that mobility limitations are an important risk factor for the emergence and maintenance of feelings of loneliness. The aim of this study was to explore the experiences of loneliness among community-dwelling older people with reduced mobility and its underlying dynamics. Methods This study employed a qualitative, descriptive design, inspired by a grounded theory approach. Participants were purposively recruited in collaboration with home health care providers. The main inclusion criteria were as follows: aged 75 years or older, being mobile impaired, experiencing feelings of loneliness, and living at home and being cognitively able to be interviewed. Data analysis of 15 semi-structured, in-depth interviews was conducted based on the Qualitative Analysis Guide of Leuven (QUAGOL). Results Loneliness was expressed through all the stories, but it appeared as an elusive, intangible phenomenon. Both indicating the presence of loneliness and describing what this phenomenon means were difficult to express for most participants. Loneliness was experienced as an inherent part of the ageing process characterised by losses, limitations and meaninglessness. Participants described how they have experienced losing grip on the world in which they live and feel isolated in a literally and figuratively shrinking world. Loneliness is described as the feeling that one is unable to address the situation that results in deep sadness and the feeling of no longer being of value to their environment. Conclusions This study shows that loneliness among community-dwelling older persons with reduced mobility is embedded in experiences of loss related to ageing, among which reduced mobility plays a significant role. The results suggest the existence of a more profound experience of loneliness than might appear at first glance. How to recognise experiences of loneliness and how to support a meaningful existence for community-dwelling older persons should be given priority in health care. The findings of this study can increase professional caregivers’ sensitivity to implicit signals of loneliness. Further research is necessary to refine the outcomes and to further explore the role of reduced mobility in the experience of loneliness.

ABSTRACT Aim Design Methods Results Conclusions Implications to Patient Care Reporting Method Patient or Public Contribution To capture older people's, informal caregivers' and health professionals' ideas on potential interventions for empowering older people and informal caregivers in transitional care decision‐making.A descriptive qualitative design was adopted.The study was conducted between February and May 2022 in the region of Flanders, Belgium, as part of the TRANS‐SENIOR consortium's collaborative research. Data were collected using focus groups, including older people, informal caregivers and healthcare professionals involved in any physical relocation of the older person across home, hospital or nursing home settings. Thematic data analysis was performed based on Braun and Clarke's six‐step method.A total of 40 people participated in the focus groups. Four main themes were identified, which describe ideas on how to empower older people and informal caregivers in transitional care: Providing clear and timely information, preparing people for what is to come, person‐centredness and providing professional and peer support for informal caregivers.Healthcare (professionals) should facilitate older people's and informal caregivers' empowerment in transitional care decision‐making by setting them at the core and inception of the decision‐making process. While informal caregivers support their loved ones in decision‐making processes, they should also be supported and monitored for burdensome issues.Multicomponent, well‐planned and personalised interventions are needed to empower older people and informal caregivers in transitional care decision‐making. The ideas raised by all stakeholders who participated in this study can inform these interventions.Adhered to consolidated criteria for reporting qualitative research checklist.Organisations advocating for the interests of older people and informal caregivers played a pivotal role in shaping the TRANS SENIOR project. Furthermore, the study benefitted from the collaborative input of AGE Platform Europe, which amplified the voices and representation of older people during the project design phase. [ABSTRACT FROM AUTHOR]

ABSTRACT: Mastitis affects a high proportion of dairy cows and is still one of the greatest challenges faced by the dairy industry. Staphylococcal bacteria remain the most important cause of mastitis worldwide. We investigated how distinct staphylococcal species evade some critical host defense mechanisms, which may dictate the establishment, severity, and persistence of infection and the outcome of possible therapeutic and prevention interventions. Thus, the present study investigated variations among distinct bovine-associated staphylococci in their capability to resist phagocytosis and to trigger respiratory burst activity of blood and milk polymorphonuclear neutrophil leukocytes (PMNL) in dairy cows. To do so, PMNL of 6 primiparous and 6 multiparous dairy cows were used. A collection of 38 non-aureus staphylococci (NAS) and 12 Staphylococcus aureus were included. The phagocytosis and intracellular reactive oxygen species (ROS) production by blood and milk PMNL were analyzed by flow cytometry. Phagocytosis, by both blood and milk PMNL, did not differ between S. aureus and NAS as a group, although within-NAS species differences were observed. Staphylococcus chromogenes (a so-called milk-adapted NAS species) better resisted phagocytosis by blood PMNL than the so-called environmental (i.e., Staphylococcus fleurettii) and opportunistic (i.e., Staphylococcus haemolyticus) NAS species. Otherwise, S. haemolyticus was better phagocytosed by blood PMNL than S. aureus, S. fleurettii, and S. chromogenes. No influence of the origin of the isolates within the staphylococci species in the resistance to phagocytosis by blood and milk PMNL was found. Overall, both S. aureus and NAS did not inhibit intracellular ROS production in blood and milk PMNL. Non-aureus staphylococci induced fewer ROS by milk PMNL than S. aureus, which was not true for blood PMNL, although species-specific differences in the intensity of ROS production were observed. Staphylococcus chromogenes induced more blood PMNL ROS than S. fleurettii and S. haemolyticus, and as much as S. aureus. Conversely, S. chromogenes induced fewer milk PMNL ROS than S. aureus. The origin of the isolates within the staphylococci species did not affect the ROS production by blood and milk PMNL. In conclusion, our study showed differences in staphylococci species in evading phagocytosis and triggering ROS production, which may explain the ability of some staphylococci species (i.e., S. aureus and S. chromogenes) to cause persistent infection and induce inflammation.

Background High-risk medications use at home entails an increased risk of significant harm to the patient. While interventions and strategies to improve medications care have been implemented in hospitals, it remains unclear how this type of medications care is provided in the home care setting. The objective was to describe home care nurses’ management of high-risk medications. Methods A cross-sectional, descriptive design was set up in home care nurses in Flanders, Belgium. Participants were recruited through convenience sampling and could be included in the study if they provided medications care and worked as a home care nurses. Participants completed an online structured questionnaire. Questions were asked about demographic information, work experience, nurses’ general attitude regarding high-risk medications, contact with high-risk medications and the assessment of risk and severity of harm, specific initiatives undertaken to improve high-risk medications care and the use of additional measures when dealing with high-risk medications. Descriptive statistics were used. Results A total of 2283 home care nurses participated in this study. In our study, 98% of the nurses reported dealing high-risk medications. Home care nurses dealt the most with anticoagulants (96%), insulin (94%) and hypnotics and sedatives (87%). Most nurses took additional measures with high-risk medications in less than 25% of the cases, with the individual double check being the most performed measure for all high-risk medications except lithium. Nurses employed by an organization received support mostly in the form of a procedure while self-employed nurses mostly look for support through external organizations and information sources. Conclusions The study shows several gaps regarding high-risk medications care, which can imply safety risks. Implementation and evaluation of more standardized high-risk medications care, developing and implementing procedures or guidelines and providing continuous training for home care nurses are advised.

Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.

Abstract Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.

Abstract Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.

Objective To identify strategies and interventions used to improve interprofessional collaboration and integration (IPCI) in primary care.Design Scoping reviewData sources Specific Medical Subject Headings terms were used, and a search strategy was developed for PubMed and afterwards adapted to Medline, Eric and Web of Science.Study selection In the first stage of the selection, two researchers screened the article abstracts to select eligible papers. When decisions conflicted, three other researchers joined the decision-making process. The same strategy was used with full-text screening. Articles were included if they: (1) were in English, (2) described an intervention to improve IPCI in primary care involving at least two different healthcare disciplines, (3) originated from a high-income country, (4) were peer-reviewed and (5) were published between 2001 and 2020.Data extraction and synthesis From each paper, eligible data were extracted, and the selected papers were analysed inductively. Studying the main focus of the papers, researchers searched for common patterns in answering the research question and exposing research gaps. The identified themes were discussed and adjusted until a consensus was reached among all authors.Results The literature search yielded a total of 1816 papers. After removing duplicates, screening titles and abstracts, and performing full-text readings, 34 papers were incorporated in this scoping review. The identified strategies and interventions were inductively categorised under five main themes: (1) Acceptance and team readiness towards collaboration, (2) acting as a team and not as an individual; (3) communication strategies and shared decision making, (4) coordination in primary care and (5) integration of caregivers and their skills and competences.Conclusions We identified a mix of strategies and interventions that can function as ‘building blocks’, for the development of a generic intervention to improve collaboration in different types of primary care settings and organisations.

Abstract Communications via quorum sensing (QS) between non-aureus staphylococci (NAS) and Staphylococcus (S.) aureus in the bovine mammary gland remains largely unexplored. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices were able to regulate the QS of S. aureus, and if so, how in vitro growth inhibition of S. aureus by NAS, or NAS metabolites, or NAS cells themselves play a role in this process. In co-culture with S. aureus we observed that these 3 NAS species in general downregulated the expression of rnaIII, the effector molecule of the QS system, but this effect was more pronounced in S. chromogenes and S. simulans isolates than in S. epidermidis isolates. In vitro growth inhibition of S. aureus by NAS resulted in a small underestimation of the downregulating effect of NAS on rnaIII expression of S. aureus. Additionally, the culture supernatant of these NAS isolates and supernatant treated with proteinase K expressed greater regulatory activity over S. aureus virulence genes rnaIII, hla, and spa than washed NAS cells suspended in sterile water. These microbial interactions may influence S. aureus virulence and pathogenesis within the host. Isolation and identification of NAS metabolites affecting the QS system of S. aureus might help to develop alternative strategies for treatment and control of S. aureus mastitis.

The main objective of the study was to evaluate whether or not implementing selective dry cow therapy (SDCT) on commercial dairy farms reduces antimicrobial consumption without negatively affecting future performances when compared to blanket dry cow therapy (BDCT). Twelve commercial herds in the Flemish region of Belgium with overall good udder health management were enrolled in a randomized control trial, including 466 cows that were assigned to a BDCT (n = 244) or SDCT (n = 222) group within herds. Cows in the SDCT group were dried off with internal teat sealants combined or not with long-acting antimicrobials according to a predefined algorithm based on test-day somatic cell count (SCC) data. Total antimicrobial use for udder health between drying off and 100 days in milk was significantly lower in the SDCT group (i.e., a mean of 1.06 defined the course dose) compared to the BDCT group (i.e., a mean of 1.25 defined the course dose), although with substantial variation between herds. Test-day SCC values, milk yield, and the clinical mastitis and culling hazard in the first 100 days in milk did not differ between the BDCT and SDCT groups. SCC-based and algorithm-guided SDCT is suggested to decrease the overall use of antimicrobials without jeopardizing cows’ udder health and milk yield.

Objective: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. Methods: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. Results: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48 h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. Conclusions: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Abstract The presence of non-aureus staphylococci (NAS) in bovine rectal feces has recently been described. Similar to other mastitis causing pathogens, shedding of NAS in the environment could result in intramammary infection. The objective of this study was to investigate whether NAS strains present in feces can cause intramammary infection, likely via teat apex colonization. During a cross-sectional study in 5 dairy herds, samples were collected from the habitats quarter milk, teat apices, and rectal feces from 25%, 10%, and 25% of the lactating cows, respectively, with a cow serving as the source of one type of sample only. Samples from clinical mastitis cases were continuously collected during the 1-year study period as well. The 6 most prevalent NAS species, Staphylococcus (S.) chromogenes, S. cohnii, S. devriesei, S. equorum, S. haemolyticus, and S. hominis, were further subtyped by random amplification of polymorphic deoxyribonucleic acid polymerase chain reaction (RAPD-PCR), when the same NAS species was present in the same herd in the three habitats. For S. chromogenes, S. cohnii, S. devriesei, and S. haemolyticus, the same RAPD type was found in rectal feces, teat apices, and quarter milk, indicating that fecal NAS can infect the mammary gland. For S. hominis and S. equorum, we were unable to confirm the presence of the same RAPD types in the three habitats.

Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.