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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 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 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: 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: 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: 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.

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.

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.

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.

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.

Despite considerable efforts to control bovine mastitis and explain its causes, it remains the most costly and common disease of dairy cattle worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are not entirely understood. These Gram-positive bacteria have become the most frequently isolated group of bacteria in milk samples of dairy cows and are associated with (mild) clinical and subclinical mastitis. Different species and strains of NAS differ in their epidemiology, pathogenicity, virulence, ecology and host adaptation, and antimicrobial resistance profiles. They have distinct relationships with the microbiome composition of the udder and may also have protective effects against other mastitis pathogens. Some appear to persist on the skin and in the teat canal and udder, while others seem to be transient residents of the udder from the environment. Analyzing genotypic and phenotypic differences in individual species may also hold clues to why some appear more successful than others in colonizing the udder. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.

Staphylococcus chromogenes is a common skin commensal in cattle and has been identified as a frequent cause of bovine mastitis and intramammary infections. We have developed a seven locus Multilocus Sequence Typing (MLST) scheme for typing S. chromogenes. Sequence-based typing systems, such as MLST, have application in studies of genetic diversity, population structure, and epidemiology, including studies of strain variation as a factor in pathogenicity or host adaptation. The S. chromogenes scheme was tested on 120 isolates collected from three geographic locations, Vermont and Washington State in the United States and Belgium. A total of 46 sequence types (STs) were identified with most of the STs being location specific. The utility of the typing scheme is indicated by a discrimination power of 95.6% for all isolates and greater than 90% for isolates from each of the three locations. Phylogenetic analysis placed 39 of the 46 STs into single core group consistent with a common genetic lineage; the STs in this group differ by less than 0.5% at the nucleotide sequence level. Most of the diversification in this lineage group can be attributed to mutation; recombination plays a limited role. This lineage group includes two clusters of single nucleotide variants in starburst configurations indicative of recent clonal expansion; nearly 50% of the isolates sampled in this study are in these two clusters. The remaining seven STs were set apart from the core group by having alleles with highly variable sequences at one or more loci. Recombination had a higher impact than mutation in the diversification of these outlier STs. Alleles with hypervariable sequences were detected at five of the seven loci used in the MLST scheme; the average sequence distances between the hypervariable alleles and the common core alleles ranged from 12 to 34 nucleotides. The extent of these sequence differences suggests the hypervariable alleles may be remnants of an ancestral genotype.

In this paper, we describe a new quantitative method to evaluate and quantify in vitro growth inhibition of mastitis-related bacteria. Colony-forming units of Staphylococcus (S.) aureus (n = 10), Escherichia (E.) coli (n = 10), and Streptococcus (S.) uberis (n = 10) were quantified after their growth on top of layers of trypticase soy agar (TSA) containing six different concentrations (varying from 102 to 107 CFU/mL) of bovine non-aureus staphylococci (NAS), i.e., S. chromogenes (n = 3) and S. simulans (n = 3) isolates. Growth inhibition of the mastitis-related major bacterial pathogens, including E. coli, was confirmed by all NAS, an effect that varied highly among NAS isolates and was not evident from the semiquantitative method with which the new method was compared. By subsequent application of the new method on a larger set of 14 bovine NAS isolates, we observed that S. simulans and NAS originating from teat apices (especially S. epidermidis) required lower concentrations to inhibit both methicillin-sensitive (MSSA) (n = 5) and methicillin-resistant S. aureus (MRSA) isolates (n = 5) originating from milk. Therefore, the new assay is a promising tool to precisely quantify the intra- and inter-species differences in growth inhibition between NAS.

Mastitis is a complex, multifactorial disease. Pathogens, cows and farmers (via management) all play a role. It is costly and annoying for the farmer and threatens the image of the entire dairy industry. Prevention and control of mastitis is based on multiple principles that have been known for a long time. To implement them successfully, they should be put forward by a motivated and motivating advisor that transfers the existing knowledge to the farmer. When the changes are data-driven, applied by an encouraged farmer through a farm-specific implementation, prevention and control of mastitis will be successful and result in happy cows, happy farmers, happy advisors, happy consumers, and a happy industry. Nationwide projects focussing on communication and transfer of existing knowledge in prevention and control are very helpful in reaching high numbers of farmers and advisors and harmonizing the message brought by different parties. This paper gives an overview of multifactorial approach of mastitis management and prevention with a focus on milking, bedding and data-analysis.

Mastitis affects a high proportion of dairy cows throughout the world and is one of the greater problems faced by the dairy industry today. The disease is still a major cause of economic loss on a dairy farm. Mastitis poses not only negative consequences for the dairy farmer but also for the dairy industry as a number of issues threaten the reputation of milk as a healthy product from healthy animals. The use of antimicrobials is one of those concerns and threats. Antimicrobial usage on dairy farms is most often related to udder health as most medicines are used in prevention and control of mastitis. Antimicrobials remain vital for treatment of bacterial infections in dairy cattle, but in light of the upcoming debate instigated by the potential link between the use of antimicrobial products in animal husbandry and the development of antimicrobial resistance in both animal and human pathogens, there is an urgent need for innovation and alternatives to antibiotic therapy for mastitis treatment and control. Alternative approaches include vaccination, probiotics or beneficial microorganisms and inhibitory substances, immunomodulation, bacteriophages, homeopathy, and plant-derived inhibitory substances, yet only when scientifically-proven evidence is available indicating these alternatives are effective.

Since 2007, mortality associated with a previously unreported haemorrhagic disease has been observed in young calves in several European countries. The syndrome, which has been named 'bovine neonatal pancytopenia' (BNP), is characterised by thrombocytopenia, leukocytopenia and a panmyelophthisis. A herd-level case-control study was conducted in four BNP affected countries (Belgium, France, Germany and the Netherlands) to identify herd management risk factors for BNP occurrence. Data were collected using structured face-to-face and telephone interviews of farm managers and their local veterinarians. In total, 363 case farms and 887 control farms were included in a matched multivariable conditional logistic regression analysis. Case-control status was strongly associated with the odds of herd level use of the vaccine PregSure® BVD (PregSure, Pfizer Animal Health) (matched adjusted odds ratio (OR) 107.2; 95% CI: 41.0-280.1). This was also the case for the practices of feeding calves colostrum from the calf's own dam (OR 2.0; 95% CI: 1.1-3.4) or feeding pooled colostrum (OR 4.1; 95% CI: 1.9-8.8). Given that the study had relatively high statistical power and represented a variety of cattle production and husbandry systems, it can be concluded with some confidence that no other herd level management factors are competent causes for a sufficient cause of BNP occurrence on herd level. It is suggested that genetic characteristics of the dams and BNP calves should be the focus of further investigations aimed at identifying the currently missing component causes that together with PregSure vaccination and colostrum feeding represent a sufficient cause for occurrence of BNP in calves.

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Bovine neonatal pancytopenia (BNP), a high fatality condition causing haemorrhages in calves aged less than 4 weeks, was first reported in 2007 in Germany and subsequently observed at low incidence in other European countries and New Zealand. A multi-country matched case-control study was conducted in 2011 to identify calf-level risk factors for BNP. 405 BNP cases were recruited from 330 farms in Belgium, France, Germany and the Netherlands by laboratory confirmation of farmer-reported cases. Up to four calves of similar age from the same farm were selected as controls (1154 calves). Risk factor data were collected by questionnaire. Multivariable modelling using conditional logistic regression indicated that PregSure®BVD (PregSure, Pfizer Animal Health) vaccination of the dam was strongly associated with BNP cases (adjusted matched Odds Ratio - amOR 17.8 first lactation dams; 95% confidence interval - ci 2.4, 134.4; p = 0.005), and second or more lactation PregSure-vaccinated dams were more likely to have a case than first lactation vaccinated dams (amOR 2.2 second lactation; ci 1.1, 4.3; p = 0.024; amOR 5.3 third or more lactation; ci 2.9, 9.8; p =

The study aimed to compare reciprocal behavior during interaction with a virtual-player in a computer game between children with typical development (TD) and children with specific-learning-disabilities (SLD) and/or with attention-deficit/hyperactivity disorder (ADHD), and to examine its correlation with social skills. A total of 120 children (43 SLD/ADHD, 77 TD) aged 9-11 years participated. Participants completed self-reported questionnaires focusing on social skills and reciprocity and played a computer game in which such social situations arose. Results indicated no difference between the groups in self-reported social skills or reciprocity. However, the children's actual reciprocal behavior during gameplay revealed different results: the SLD/ADHD group exhibited higher levels of selfish (helping others for personal gain) and lower levels of altruistic reciprocity (helping others for their benefit) compared to the TD group. Furthermore, a correlation was found between self-reported social skills and reciprocity, as well as with the reciprocal-patterns observed in the gameplay., (© 2024. The Author(s).)

Background : Ultrasound (US)-guided fine needle aspiration (FNA) cytology is the gold standard for the evaluation of thyroid nodules. However, up to 30% of FNA results are indeterminate, requiring further testing. In this study, we present a machine-learning analysis of indeterminate thyroid nodules on ultrasound with the aim to improve cancer diagnosis. Methods : Ultrasound images were collected from two institutions and labeled according to their FNA (F) and surgical pathology (S) diagnoses [malignant (M), benign (B), and indeterminate (I)]. Subgroup breakdown (FS) included: 90 BB, 83 IB, 70 MM, and 59 IM thyroid nodules. Margins of thyroid nodules were manually annotated, and computerized radiomic texture analysis was conducted within tumor contours. Initial investigation was conducted using five-fold cross-validation paradigm with a two-class Bayesian artificial neural networks classifier, including stepwise feature selection. Testing was conducted on an independent set and compared with a commercial molecular testing platform. Performance was evaluated using receiver operating characteristic analysis in the task of distinguishing between malignant and benign nodules. Results: About 1052 ultrasound images from 302 thyroid nodules were used for radiomic feature extraction and analysis. On the training/validation set comprising 263 nodules, five-fold cross-validation yielded area under curves (AUCs) of 0.75 [Standard Error (SE) = 0.04; P < 0.001 ] and 0.67 (SE = 0.05; P = 0.0012 ) for the classification tasks of MM versus BB, and IM versus IB, respectively. On an independent test set of 19 IM/IB cases, the algorithm for distinguishing indeterminate nodules yielded an AUC value of 0.88 (SE = 0.09; P < 0.001 ), which was higher than the AUC of a commercially available molecular testing platform (AUC = 0.81, SE = 0.11; P < 0.005 ). Conclusion: Machine learning of computer-extracted texture features on gray-scale ultrasound images showed promising results classifying indeterminate thyroid nodules according to their surgical pathology., (© 2022 Society of Photo-Optical Instrumentation Engineers (SPIE).)

Background: The 2015 American Thyroid Association (ATA) clinical practice guidelines (CPGs) on management of thyroid nodules (TNs) and differentiated thyroid cancer (DTC) in adults were developed to inform clinicians, patients, researchers, and health policy makers about the best available evidence, and its limitations, relating to management of these conditions. Methods: We conducted a cross-sectional electronic survey of ATA members' perspectives of these CPGs, using a standardized survey (Clinician Guidelines Determinant Questionnaire) developed by the Guidelines International Network. A survey link was electronically mailed to members in February of 2019, with reminders sent to nonrespondents 2 and 5 weeks later. Data were descriptively summarized, after excluding missing responses. Results: The overall response rate was 19.8% (348/1761). The effective response rate was 20.2% (348/1720), after excluding a recently deceased member and individuals who had either invalid e-mail addresses or whose e-mails were returned. Of the respondents, 37.9% (132/348) were female, 60.4% (209/346) were endocrinologists, 27.5% (95/346) were surgeons, and 3.5% (12/346) were nuclear medicine specialists. The majority of respondents (71.9%; 250/348) were at a mid- or advanced-career level, and more than half were in academia (57.5%; 195/339). The majority (69.8%; 243/348) practiced in North America. The vast majority of respondents indicated that the CPGs explained the underlying evidence (92.3%; 298/323) and 92.9% (300/323) agreed or strongly agreed with the content. Most respondents stated that they regularly used the CPGs in their practice (83.0%; 268/323). Most respondents (83.0%; 268/323) also agreed or strongly agreed that the recommendations were easy to incorporate in their practice. The most popular CPG format was an electronic desktop file (78.8%; 252/320). Shorter more frequent CPGs were favored by 55.0% (176/320) of respondents, and longer traditional CPGs were favored by 39.7% (127/320). Conclusions: The clinical content and evidence explanations in the adult TN and DTC CPGs are widely accepted and applied among ATA survey respondents. Future ATA CPG updates need to be optimized to best meet users' preferences regarding format, frequency, and length.

Background: Requiring indications for inpatient medication orders may improve the quality of prescribing and allow for easier placement of diagnoses on the problem list. Indications for inpatient medication orders are also required by some regulators., Objective: This study assessed a clinical decision support (CDS) system designed to obtain indications and document problems during inpatient computerized physician order entry (CPOE) of medications frequently used off-label., Methods: A convenience sample of three medications frequently used off-label were selected: the PPI lansoprazole; intravenous immune globulin, and recombinant Factor VIIa. Alerts triggered when a medication was ordered without an FDA approved indication in the problem list. The alerts prompted clinicians to enter either a labeled or off-label indication for the order. Chart review was used as the gold standard to assess the accuracy of clinician entered information., Results: The PPI intervention generated 873 alerts during 60 days of operation; IVIG 55 alerts during alerts during 93 days; Factor VIIa 25 alerts during 175 days. Agreement between indications entered and chart review was 63% for PPI, 49% for IVIG, and 29% for Factor VIIa. The alerts for PPI, IVIG and Factor VIIa alerts produced accurate diagnoses for the problem list 9%, 16% and 24% respectively. Rates of off-label use measured by chart review were 87% for PPI, and 100% for IVIG and factor VIIa, which were higher than if measured using the ordering clinicians' indications., Conclusion: This trial of indication-based prescribing using CDS and CPOE produced less than optimal accuracy of the indication data as well as a low yield of accurate problems placed on the problem list. These results demonstrate the challenge inherent in obtaining accurate indication information during prescribing and should raise concerns over potential mandates for indication based prescribing and motivate further study of appropriate mechanisms to obtain indications during CPOE.

Objective: The problem list is a key and required element of the electronic medical record (EMR). Problem lists may contribute substantially to patient safety and quality of care. Physician documentation of the problem list is often lower than desired. Methods are needed to improve accuracy and completeness of the problem list., Design: An automated clinical decision support (CDS) intervention was designed utilizing a commercially available EMR with computerized physician order entry (CPOE) and CDS. The system was based on alerts delivered during inpatient medication CPOE that prompted clinicians to add a diagnosis to the problem list. Each alert was studied for a 2-month period after implementation., Measurements: Measures included alert validity, alert yield, and accuracy of problem list additions., Results: At a 450 bed teaching hospital, the number of medication orders which triggered alerts during all 2-month study periods was 1011. For all the alerts, the likelihood of a valid alert (an alert that occurred in patients with one of the predefined diagnoses) was 96+/-1%. The alert yield, defined as occuring when an alert led to addition of a problem to the problem list, was 76+/-2%. Accurate problem list additions, defined as additions of problems when the problem was determined to be present by expert review, was 95+/-1%., Conclusion: The CDS problem list mechanism was integrated into the process of medication order placement and promoted relatively accurate addition of problems to the EMR problem list., (Copyright 2008 Elsevier Ireland Ltd. All rights reserved.)

The development of hyperparathyroidism after radioactive iodine (RAI) therapy has been reported in 38 cases in the literature. However, the development of a parathyroid adenoma after RAI therapy for a hyperfunctioning multinodular goiter has not been reported. This report describes the pathologic and operative finding on a patient with both hyperthyroidism and hyperparathyroidism, which was diagnosed after previous RAI therapy for a toxic, multinodular goiter.

Oral iodinated radiographic contrast agents such as ipodate and iopanoic acid form an important part of the armamentarium used to treat hyperthyroidism. They rapidly and dramatically reduce serum triiodothyronine (T3) levels by inhibiting conversion of thyroxine (T4) to T3 in the periphery and by blocking secretion from the thyroid. Potential risks from the large iodine load resulting from their use limit their widespread applicability. In addition, they are ineffective when used alone on a long-term basis. However, these agents may be especially useful in treating thyrotoxic patients preoperatively, in neonatal Graves' disease, in massive levothyroxine ingestion, and when other conventional antithyroid drugs are unsuccessful or contraindicated.

Radiation remains the only factor that has been shown unequivocally to cause (nonmedullary) thyroid cancer. Recent advances include the analysis of the dose-response relationship using data pooled from multiple studies. This analysis confirms that radiation-induced thyroid cancers continue to occur, with a maximum risk at approximately 30 years after exposure. Physicians asked to evaluate patients with a history of radiation exposure should attempt to estimate the dose from the history and should be familiar with the other risk factors. For some individuals, screening should include thyroid imaging, but the results of such imaging, especially with thyroid ultrasound, should be interpreted with caution. The treatment of radiation-induced thyroid cancers is based on the observation that they appear to be no more aggressive than thyroid cancers not associated with radiation. In the future, more information should emerge about the role of cancer genes and susceptibility factors in radiation-induced thyroid cancer.

Resistance to thyroid hormone (RTH) is a condition of impaired tissue responsiveness to thyroid hormone characterized by elevated free thyroid hormone levels in serum accompanied by nonsuppressed TSH. RTH has been associated with mutations in the thyroid hormone receptor (TR) beta gene. We report studies carried out in 9 members of a family (F94) of Jewish ethnic origin and a single subject of Mexican origin. All subjects fulfilling the criteria of RTH (6 of family F94 and one of family F27) had the same point mutation in the T3-binding domain on one of the two alleles of the TR beta gene. This mutation resulted in the replacement of the normal proline-453 with serine (P453S). Nevertheless, the clinical characteristics of affected members of each of the two families differed as did the severity of hormonal resistance in terms of responses to the administration of L-T3. Genetic studies indicate that the same mutation occurred independently in each of the two families.

Thyroxine-binding globulin (TBG) is a liver glycoprotein that transports thyroid hormone in serum. In 1989, a variant TBG was reported with reduced binding affinity for thyroxine (T4) and triiodothyronine (T3) which results in low serum T4 and T3 levels. This variant, TBG-San Diego (TBG-SD), also displays reduced heat stability but has a normal isoelectric focusing pattern. We now report the sequence of the entire coding region of TBG-San Diego. It reveals two nucleotide substitutions: one located in exon 1 which results in the replacement of the normal Ser-23 (TCA) with threonine (ACA) and the other, located in exon 3, changes the normal codon 283 of TTG (leucine) with that of TTT, (phenylalanine). Allele specific amplification was used to search for both nucleotide substitutions in four affected members of the family. Results confirmed the co-segregation of these nucleotide substitutions with the TBG-SD phenotype. The substitution in codon 283 has been previously described and exists as a polymorphism in some ethnic groups or in combination with other TBG variants with different physical characteristics. Thus, it appears that the replacement of Ser-23 with threonine is responsible for the observed alterations in physical properties of TBG-San Diego.

Serum TSH and PRL levels and their response to TRH were measured in 11 patients with generalized resistance to thyroid hormone (GRTH), 6 euthyroid subjects, and 6 patients with primary hypothyroidism. TSH and PRL levels and their response to TRH were also measured after the consecutive administration of 50, 100, and 200 micrograms T3 daily, each for a period of 3 days. Using a sensitive TSH assay, all GRTH patients had TSH values that were elevated or within the normal range. On the basis of a normal or elevated TSH level, GRTH patients were classified as GRTH-N1 TSH (5 patients) or GRTH-Hi TSH (6 patients), respectively. Only GRTH patients with previous thyroid ablative therapy had basal TSH values greater than 20 mU/L. TSH responses, in terms of percent increment above baseline, were appropriate for the basal TSH level in all subjects. No GRTH patient had an elevated basal PRL level. PRL responses to TRH were significantly increased only in the hypothyroid controls compared to values in all other groups. On 50 micrograms T3, 7 of 12 (58%) nonresistant (euthyroid and hypothyroid) and 1 of 11 (9%) resistant subjects had a greater than 75% suppression of the TSH response to TRH. On the same T3 dose, 2 of 12 (17%) nonresistant and 4 of 11 (36%) resistant subjects had a greater than 50% suppression of the PRL response to TRH. On 200 micrograms T3, all subjects, except for 1 with GRTH, had a greater than 75% suppression of the TSH response to TRH. On the same T3 dose, while 11 of 12 (92%) nonresistant subjects had a greater than 50% reduction of the PRL response to TRH, only 3 of 10 (30%) resistant patients showed this degree of suppression (P less than 0.005). Without previous ablative therapy, serum TSH in patients with GRTH is usually normal or mildly elevated. The TSH response to TRH is proportional to the basal TSH level and is suppressed by exogenous T3. However, on 200 micrograms T3 basal TSH was not detectable (less than 0.1 mU/L) in all euthyroid subjects, but it was measurable in three of four GRTH patients with normal TSH levels before T3 treatment. PRL levels in GRTH are normal even when TSH is elevated. The PRL response to TRH is not increased in GRTH. In all subjects, exogenous T3 suppresses the PRL response to TRH to a lesser degree than the TSH response, but this difference is much greater in patients with GRTH.