Association between days post conception and lactation persistency in dairy cattle.

Determining the optimal insemination moment for individual cows is complex, particularly when considering the impact of pregnancy on milk production. The effect of pregnancy on the absolute milk yield has already been reported in several studies. Currently, there is limited quantitative knowledge about the association between days post conception (DPC) and lactation persistency, based on a lactation curve model, and, specifically, how persistency changes during pregnancy and relates to the days in milk at conception (DIMc). Understanding this association might provide valuable insights to determine the optimal insemination moment. This study, therefore, aimed to investigate the association between DPC and lactation persistency, with an additional focus on the influence of DIMc. Available milk production data from 2005 to 2022 were available for 23,908 cows from 87 herds located throughout the Netherlands and Belgium. Persistency was measured by a lactation curve characteristic decay, representing the time taken to halve milk production after peak yield. Decay was calculated for 8 DPC (0, 30, 60, 90, 120, 150, 180 and 210 d after DIMc) and served as the dependent variable. Independent variables included DPC, DIMc (< = 60, 61-90, 91-120, 121-150, 151-180, 181-210, > 210), parity group, DPC × parity group, DPC × DIMc and variables from 30 d before DIMc as covariates. The results showed an increase in decay, i.e., a decrease in persistency, during pregnancy for both parity groups, albeit in different ways. Specifically, from DPC 150 to DPC 210, multiparous cows showed a higher decline in persistency compared with primiparous cows. Furthermore, a later DIMc (cows conceiving later) was associated with higher persistency. Except for the early DIMc groups (DIMc < 90), DIMc does not impact the change in persistency by gestation. The findings from this study contribute to a better understanding of how DPC and DIMc during lactation influence lactation persistency, enabling more informed decision-making by farmers who wish to take persistency into account in their reproduction management.

Structure-function effects of different pectin chemistries and its impact on the gastrointestinal immune barrier system.

The gastrointestinal immune system is crucial for overall health, safeguarding the human body against harmful substances and pathogens. One key player in this defense is dietary fiber pectin, which supports the gut's immune barrier and fosters beneficial gut bacteria. Pectin's composition, including degree of methylation (DM), RG-I, and neutral sugar content, influences its health benefits. This review assesses how pectin composition impacts the gastrointestinal immune barrier and what advantages specific chemistries of pectin has for metabolic, cardiovascular, and immune health. We delve into recent findings regarding pectin's interactions with the immune system, including receptors like TLRs and galectin 3. Pectin is shown to fortify mucosal and epithelial layers, but the specific effects are structure dependent. Additionally, we explore potential strategies for enhancing the gut immune barrier function. Understanding how distinct pectin chemistries affect the gastrointestinal immune system is vital for developing preventive and therapeutic solutions for conditions related to microbiota imbalances and immune issues. Ultimately, this review offers insights into strategies to boost the gut immune barrier's effectiveness, fostering better overall health by using specific pectins in the diet.

Type of intrinsic resistant starch type 3 determines in vitro fermentation by pooled adult faecal inoculum.

Resistant starch (RS) results in relatively high health-beneficial butyrate levels upon fermentation by gut microbiota. We studied how physico-chemical characteristics of RS-3 influenced butyrate production during fermentation. Six highly resistant RS-3 substrates (intrinsic RS-3, 80-95 % RS) differing in chain length (DPn 16-76), Mw distribution (PI) and crystal type (A/B) were fermented in vitro by pooled adult faecal inoculum. All intrinsic RS-3 substrates were fermented to relatively high butyrate levels (acetate/butyrate ≤ 2.5), and especially fermentation of A-type RS-3 prepared from polydisperse α-1,4 glucans resulted in the highest relative butyrate amount produced (acetate/butyrate: 1). Analysis of the microbiota composition after fermentation revealed that intrinsic RS-3 stimulated primarily Lachnospiraceae, Bifidobacterium and Ruminococcus, but the relative abundances of these taxa differed slightly depending on the RS-3 physico-chemical characteristics. Especially intrinsic RS-3 of narrow disperse Mw distribution stimulated relatively more Ruminococcus. Selected RS fractions (polydisperse Mw distribution) obtained after pre-digestion were fermented to acetate and butyrate (ratio ≤ 1.8) and stimulated Lachnospiraceae and Bifidobacterium. This study indicates that especially the α-1,4 glucan Mw distribution dependent microstructure of RS-3 influences butyrate production and microbiota composition during RS-3 fermentation.

The level and distribution of methyl-esters influence the impact of pectin on intestinal T cells, microbiota, and Ahr activation.

Pectins are dietary fibres that modulate T cell immunity, microbiota composition, and fermentation profiles, but how this is influenced by the degree of methyl-esterification (DM) and degree-of-blockiness (DB) of pectin is unknown. Here, we demonstrate that supplementation of DM19(high-DB), DM49(low-DB) and DM43(high-DB) pectins at a low dose increased the frequencies of intestinal T-helper (Th)1 and Th2 cells after 1 week of pectin supplementation in mice, whereas DM18(low-DB) did not. After 4 weeks of supplementation with those pectins, Th1 and Th2 frequencies returned to control levels, whereas Rorγt+ regulatory T-cell frequencies increased. These structure-dependent effects could derive from induced shifts in microbiota composition that differed between DM18(low-DB) pectin and the other pectins. T-cell-modulating effects were not short-chain-fatty acid-dependent, but rather through an increase in Aryl-hydrocarbon-receptor-activating components. Thus, pectins with a specific combination of DM and DB have an impact on intestinal T cell-immunity in mice, when supplemented at a low dose.

Sex-specific differences and how to handle them in early psoriatic arthritis.

OBJECTIVES: The prevalence of psoriatic arthritis (PsA) is the same in men and women; however, the latter experience a higher burden of disease and are affected more frequently by polyarthritis. Here, we performed an early PsA cohort analysis to assess sex-related differences in demographics, disease characteristics, and evolution over 1 year including applied treatment strategies. METHODS: Our study is embedded in the Dutch south-west Early Psoriatic Arthritis cohoRt. We described patient characteristics and treatment decisions. For the comparison across sexes and baseline and 1 year follow-up, appropriate tests depending on the distribution were used. RESULTS: Two hundred seventy-three men and 294 women with no significant differences in age and ethnicity were included. Women reported significantly longer duration of symptoms before diagnosis and significantly higher tender joint count, a higher disease activity, higher levels of pain, and lower functional capacity. Although minimal disease activity (MDA) rates increased over time for both sexes, MDA remained significantly more prevalent among men at 1 year (58.1% vs 35.7%, p < 0.00). Initially, treatment strategies were similar in both sexes with methotrexate being the most frequently used drug during the first year. Women received methotrexate for a shorter period [196 (93-364) vs 306 (157-365), p < 0.00] and therefore received a lower cumulative dose compared to men. Retention time was shorter for all DMARDs, and women had a delayed start on b-DMARDs. CONCLUSION: After 1 year of standard-of-care treatment, women did not surpass their baseline disadvantages. Despite the overall improvement, they still presented higher disease activity, higher levels of pain, and lower functional capacity score than men. The nature of these findings may advocate a need for sex specific adjustment of treatment strategies and evaluation in early PsA patients.

ß(2→6)-Type fructans attenuate proinflammatory responses in a structure dependent fashion via Toll-like receptors.

Graminan-type fructans (GTFs) have demonstrated immune benefits. However, mechanisms underlying these benefits are unknown. We studied GTFs interaction with Toll-like receptors (TLRs), performed molecular docking and determined their impact on dendritic cells (DCs). Effects of GTFs were compared with those of inulin-type fructans (ITFs). Whereas ITFs only contained ß(2→1)-linked fructans, GTFs showed higher complexity as it contains additional ß(2→6)-linkages. GTFs activated NF-κB/AP-1 through MyD88 and TRIF pathways. GTFs stimulated TLR3, 7 and 9 while ITFs activated TLR2 and TLR4. GTFs strongly inhibited TLR2 and TLR4, while ITFs did not inhibit any TLR. Molecular docking demonstrated interactions of fructans with TLR2, 3, and 4 in a structure dependent fashion. Moreover, ITFs and GTFs attenuated pro-inflammatory cytokine production of stimulated DCs. These findings demonstrate immunomodulatory effects of GTFs via TLRs and attenuation of cytokine production in dendritic cells by GTFs and long-chain ITF.

Reproductive hormone use and its association with herd-level factors on Dutch dairy farms.

We studied the use of the 3 commonly used reproductive hormones, namely prostaglandins, GnRH, and progesterone, and associated herd-level factors on 760 Dutch dairy farms from 5 veterinary clinics. From 2017 to 2019 we collected data on the sales of reproductive hormones, converted this data into the number of reproductive hormone doses conducted, and expressed this as the annual number of reproductive hormone doses per 100 adult dairy cows. Additional herd-level information was available for 2019. Due to the excess of zeros in the data set (i.e., a substantial number of farms did not use any hormones), we used a zero-inflated negative binomial model to identify related herd-level factors for the use of reproductive hormones. In the entire study period of 2017 to 2019, 5.8% of the dairy farms did not use any reproductive hormones, with the proportion of nonusers varying between 0.0 and 10.3% per veterinary clinic. This proportion was around 13.5% on an annual basis. Prostaglandins were the most frequently used reproductive hormone in Dutch dairy cows (62.9%), followed by GnRH (33.1%) and progesterone (4.0%). Furthermore, participating in a veterinary herd health management program had a significant effect on reproductive hormone use. These farms used more reproductive hormones than farms that did not participate in a herd health management program and were less represented in the group of nonuser farms. Technologies, such as pedometers and automatic milking systems, also had an effect on reproductive hormone use. The presence of pedometers or activity monitors did not reduce the use of the reproductive hormones but was associated with a greater frequency of users. Farms with an automatic milking system used more reproductive hormones than farms with a conventional milking system. With this study, we have made a first step in achieving transparency in the Dutch dairy industry by providing an objective overview of reproductive hormone use on Dutch dairy farms and identifying associations with some herd-level factors.

Digestibility of resistant starch type 3 is affected by crystal type, molecular weight and molecular weight distribution.

Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14-76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.

Traumatic Cerebral Microbleeds in the Subacute Phase Are Practical and Early Predictors of Abnormality of the Normal-Appearing White Matter in the Chronic Phase.

BACKGROUND AND PURPOSE: In the chronic phase after traumatic brain injury, DTI findings reflect WM integrity. DTI interpretation in the subacute phase is less straightforward. Microbleed evaluation with SWI is straightforward in both phases. We evaluated whether the microbleed concentration in the subacute phase is associated with the integrity of normal-appearing WM in the chronic phase. MATERIALS AND METHODS: Sixty of 211 consecutive patients 18 years of age or older admitted to our emergency department ≤24 hours after moderate to severe traumatic brain injury matched the selection criteria. Standardized 3T SWI, DTI, and T1WI were obtained 3 and 26 weeks after traumatic brain injury in 31 patients and 24 healthy volunteers. At baseline, microbleed concentrations were calculated. At follow-up, mean diffusivity (MD) was calculated in the normal-appearing WM in reference to the healthy volunteers (MDz). Through linear regression, we evaluated the relation between microbleed concentration and MDz in predefined structures. RESULTS: In the cerebral hemispheres, MDz at follow-up was independently associated with the microbleed concentration at baseline (left: B = 38.4 [95% CI 7.5-69.3], P = .017; right: B = 26.3 [95% CI 5.7-47.0], P = .014). No such relation was demonstrated in the central brain. MDz in the corpus callosum was independently associated with the microbleed concentration in the structures connected by WM tracts running through the corpus callosum (B = 20.0 [95% CI 24.8-75.2], P < .000). MDz in the central brain was independently associated with the microbleed concentration in the cerebral hemispheres (B = 25.7 [95% CI 3.9-47.5], P = .023). CONCLUSIONS: SWI-assessed microbleeds in the subacute phase are associated with DTI-based WM integrity in the chronic phase. These associations are found both within regions and between functionally connected regions.

Pectin limits epithelial barrier disruption by Citrobacter rodentium through anti-microbial effects.

SCOPE: C. rodentium is the murine equivalent of Enteropathogenic Escherichia. coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) which induce damage to the intestinal epithelial barrier that results in diarrhea and intestinal inflammation. Dietary fibre intake can be an effective approach to limit epithelial damage by these enteric pathogens. Therefore, the protective effect of dietary fibre pectin against dysfunction of epithelial barrier integrity upon C. rodentium infection was investigated. METHODS AND RESULTS: Pectins that structurally differed in the degree and distribution of methylesters were tested on barrier protective effects on epithelial cells against C. rodentium by measuring transepithelial electrical resistance and lucifer yellow fluxes. All three pectins protected the epithelial barrier from C. rodentium induced damage in a structure-independent manner. These barrier protective effects were also independent of pectin-induced TLR2 activation. Furthermore, the pectins induced anti-adhesive effects on C. rodentium by interacting with C. rodentium and not with epithelial cells. This may be explained by antimicrobial effects of pectins on C. rodentium and not on other enteric bacteria including Lactobacillus plantarum and E. coli. A competition ELISA for binding of C. rodentium to pectin supported this finding as it showed that pectin interacts strongly with C. rodentium, whereas it interacts weakly or not with L. plantarum or E. coli. CONCLUSION: These findings demonstrate that pectin protects the epithelial barrier from C. rodentium induced damage by inducing anti-microbial effects.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Microencapsulated islet allografts in diabetic NOD mice and nonhuman primates.

OBJECTIVE: Our goal was to assess the efficacy of encapsulated allogeneic islets transplanted in diabetic NOD mice and streptozotocin (STZ)-diabetic nonhuman primates (NHPs). MATERIALS AND METHODS: Murine or NHP islets were microencapsulated and transplanted in non-immunosuppressed mice or NHPs given clinically-acceptable immunosuppressive regimens, respectively. Two NHPs were treated with autologous mesenchymal stem cells (MSCs) and peri-transplant oxygen therapy. Different transplant sites (intraperitoneal [i.p.], omental pouch, omental surface, and bursa omentalis) were tested in separate NHPs. Graft function was monitored by exogenous insulin requirements, fasting blood glucose levels, glucose tolerance tests, percent hemoglobin A1c (% HbA1c), and C-peptide levels. In vitro assessment of grafts included histology, immunohistochemistry, and viability staining; host immune responses were characterized by flow cytometry and cytokine/chemokine multiplex ELISAS. RESULTS: Microencapsulated islet allografts functioned long-term i.p. in diabetic NOD mice without immunosuppression, but for a relatively short time in immunosuppressed NHPs. In the NHPs, encapsulated allo-islets initially reduced hyperglycemia, decreased exogenous insulin requirements, elevated C-peptide levels, and lowered % HbA1c in plasma, but graft function diminished with time, regardless of transplant site. At necropsy, microcapsules were intact and non-fibrotic, but many islets exhibited volume loss, central necrosis and endogenous markers of hypoxia. Animals receiving supplemental oxygen and autologous MSCs showed improved graft function for a longer post-transplant period. In diabetic NHPs and mice, cell-free microcapsules did not elicit a fibrotic response. CONCLUSIONS: The evidence suggested that hypoxia was a major factor for damage to encapsulated islets in vivo. To achieve long-term function, new approaches must be developed to increase the oxygen supply to microencapsulated islets and/or identify donor insulin-secreting cells which can tolerate hypoxia.

Mitochondrial function in immune cells in health and disease.

One of the main functions of mitochondria is production of ATP for cellular energy needs, however, it becomes more recognized that mitochondria are involved in differentiation and activation processes of immune cells. Upon activation, immune cells have a high need for energy. Immune cells have different strategies to generate this energy. In pro-inflammatory cells, such as activated monocytes and activated T and B cells, the energy is generated by increasing glycolysis, while in regulatory cells, such as regulatory T cells or M2 macrophages, energy is generated by increasing mitochondrial function and beta-oxidation. Except for being important for energy supply during activation, mitochondria also induce immune responses. During an infection, they release mitochondrial danger associated molecules (DAMPs) that resemble structures of bacterial derived pathogen associated molecular patterns (PAMPs). Such mitochondrial DAMPS are for instance mitochondrial DNA with hypomethylated CpG motifs or a specific lipid that is only present in prokaryotic bacteria and mitochondria, i.e. cardiolipin. Via release of such DAMPs, mitochondria guide the immune response towards an inflammatory response against pathogens. This is an important mechanism in early detection of an infection and in stimulating and sustaining immune responses to fight infections. However, mitochondrial DAMPs may also have a negative impact. If mitochondrial DAMPs are released by damaged cells, without the presence of an infection, such as after a trauma, mitochondrial DAMPs may induce an undesired inflammatory response, resulting in tissue damage and organ dysfunction. Thus, immune cells have developed mechanisms to prevent such undesired immune activation by mitochondrial components. In the present narrative review, we will describe the current view of mitochondria in regulation of immune responses. We will also discuss the current knowledge on disturbed mitochondrial function in immune cells in various immunological diseases.

Correction to: Safety and efficacy of Cerebrolysin in acute brain injury and neurorecovery: CAPTAIN I-a randomized, placebo-controlled, double-blind, Asian-Pacific trial.

The above article was published online with incorrect abbreviations in Figures 2 and 3 last sentence of the legend. HDA should be corrected to HADS.

Safety and efficacy of Cerebrolysin in acute brain injury and neurorecovery: CAPTAIN I-a randomized, placebo-controlled, double-blind, Asian-Pacific trial.

OBJECTIVE: To evaluate the safety and efficacy of Cerebrolysin as an add-on therapy to local standard treatment protocol in patients after moderate-to-severe traumatic brain injury. METHODS: The patients received the study medication in addition to standard care (50 mL of Cerebrolysin or physiological saline solution daily for 10 days, followed by two additional treatment cycles with 10 mL daily for 10 days) in a prospective, randomized, double-blind, placebo-controlled, parallel-group, multi-centre phase IIIb/IV trial. The primary endpoint was a multidimensional ensemble of 14 outcome scales pooled to be analyzed by means of the multivariate, correlation-sensitive Wei-Lachin procedure. RESULTS: In 46 enrolled TBI patients (Cerebrolysin 22, placebo 24), three single outcomes showed stand-alone statistically significant superiority of Cerebrolysin [Stroop Word/Dots Interference (p = 0.0415, Mann-Whitney(MW) = 0.6816, 95% CI 0.51-0.86); Color Trails Tests 1 and 2 (p = 0.0223/0.0170, MW = 0.72/0.73, 95% CI 0.53-0.90/0.54-0.91), both effect sizes lying above the benchmark for "large" superiority (MW > 0.71)]. While for the primary multivariate ensemble, statistical significance was just missed in the intention-to-treat population (pWei-Lachin < 0.1, MWcombined = 0.63, 95% CI 0.48-0.77, derived standardized mean difference (SMD) 0.45, 95% CI -0.07 to 1.04, derived OR 2.1, 95% CI 0.89-5.95), the per-protocol analysis showed a statistical significant superiority of Cerebrolysin (pWei-Lachin = 0.0240, MWcombined = 0.69, 95% CI 0.53 to 0.85, derived SMD 0.69, 95% CI 0.09 to 1.47, derived OR 3.2, 95% CI 1.16 to 12.8), with effect sizes of six single outcomes lying above the benchmark for "large" superiority. Safety aspects were comparable to placebo. CONCLUSION: Our trial suggests beneficial effects of Cerebrolysin on outcome after TBI. Results should be confirmed by a larger RCT with a comparable multidimensional approach.

The role of autoimmunity in women with type 1 diabetes and adverse pregnancy outcome: A missing link.

The incidence of pregnancy complications in women with type 1 Diabetes Mellitus (T1D) is greater than in healthy pregnant women. This has mostly been attributed to hyperglycemia. However, despite the implementation of stricter guidelines regarding glycemic control, pregnancy complications remain more common in women with T1D. This may suggest that other etiological factors are involved. We suggest that the immune response may play a role, since the immune response has to adapt during pregnancy in order to facilitate implantation, placental and fetal development, and aberrant immunological adaptations to pregnancy are involved in various pregnancy complications. Since T1D is an autoimmune disorder, the question rises whether the immune response of women with T1D is able to adapt properly during pregnancy. Here we review the current proof and views on the role of aberrant immunological adaptations in pregnancy complications and whether such aberrant adaptations could be involved in the pregnancy complications of T1D patients.

In vitro maturation of class I oocytes of bovine during different tropical seasons.

The aims of this study were to determine the effects of seasonality on yields and quality of bovine abattoir-derived oocytes and their in vitro maturation (IVM) competence in Thailand, a tropical country. Ovaries were collected monthly from a slaughterhouse, from January to June 2017, separated into cool (January to February), summer (March to April), and rainy (May to June) seasons. Cumulus oocyte complexes (COCs) were obtained by follicular aspiration, and subsequently, oocyte yield and quality were examined. Selected class I and II COCs were cultured under IVM condition, and their maturation capacity was also evaluated. Results showed that average numbers of COCs and class I COCs per ovary were highest in summer. However, no significant seasonal difference in oocyte maturation rates was found. The higher numbers of COCs and class I COCs in summer might be caused by their follicular development during the cool season, and might be a reason why the pregnancy rate in summer is better than in the rainy season.

Obesity in COPD: Comorbidities with Practical Consequences?

COPD and obesity often coexist and there is a complex interaction between them. Our aim was to evaluate the prevalence of obesity in a secondary care COPD population. Furthermore, the presence of comorbidities in obese (COPDOB) and non-obese COPD (COPDNO) individuals was studied. In 1654 COPD patients (aged ≥18 years) who visited a pulmonologist between January 2015 and December 2015, patient characteristics, pulmonary function tests and comorbidities were obtained from the medical records. Subjects were categorized according their BMI as underweight (<18.5 kg/m2), normal weight (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2) or obese (BMI ≥30.0 kg/m2). The Charlson comorbidity index and COTE index were used to quantify comorbidities. The prevalence of obesity was 21.8% in our COPD population. Obesity was significantly less common in GOLD stage IV (10.1%) compared to GOLD I (20.5%), II (27.8%) and III (18.9%). COPDOB had different comorbidities compared with COPDNO. Hypertension, diabetes mellitus, atrial fibrillation and congestive heart failure were significantly more prevalent in COPDOB compared with COPDNO. Osteoporosis and lung cancer were significantly more common in COPDNO compared with COPDOB. Obesity is common in patients with COPD and is most prevalent in COPD GOLD I-II and least prevalent in COPD GOLD IV. Obese COPD patients have different comorbidities than non-obese COPD patients. Cardiovascular and metabolic comorbidities, especially hypertension and diabetes mellitus, are highly prevalent in obese COPD patients. Active screening for these conditions should be a priority for physicians treating obese COPD patients.

Epidemiological aspects of drowning and non-fatal drowning in the waters of Amsterdam.

We examined who, when, and where people non-fatally drowned of drowned in Amsterdam between January 2011 and December 2015 so that in the future more targeted and effective measures can be taken to prevent drowning. Data on non fatal drownings (N = 515, fire department database) were combined with the forensic medicine data on drowning fatalities (N=88). Who drowns in Amsterdam? The majority of those who drowned were men (82%). A high percentage of the people who drowned were under the influence of alcohol or drugs (up to 55%). No children drowned in the Canal District during the study period. The majority of those who drowned in the Canal District (67%) were not official inhabitants of Amsterdam (e.g., tourists and homeless people.) When do people non-fatally drown in Amsterdam? Forty-seven percent of the non-fatal drownings in the Canal District occurred on Saturday and Sunday nights between midnight and six o'clock in the morning. No specific hot times could be defined for other parts of Amsterdam. Where do people non-fatally drown or drown in Amsterdam? Compared to the non-fatal drownings, there are fewer drowning casus in the Canal District than in other parts of Amsterdam. Given the high number of non-fatal drownings and the remaining drownings in the Canal District, further prevention is necessary and should focus on hot times and hotspots. Additional measures must be taken to enable those who fall into the canals to climb out.

Unbiased random mutagenesis contributes to a better understanding of the virulent behaviour of Paenibacillus larvae.

AIMS: American foulbrood, caused by the Gram-positive bacteria Paenibacillus larvae, is one of the most severe bacterial diseases of the European honey bee. The bacterium has been known for long, but only the last decade the mechanisms used by the pathogen to cause disease in its host are starting to unravel. In this study, the knowledge of this virulent behaviour is expanded and several possible virulence factors are suggested. METHODS AND RESULTS: Identification of possible virulence factors has been done by random mutagenesis to ensure an unbiased approach. A library of mutants was tested for a significant difference in virulence using in vitro exposure assays. Affected loci were characterized and their potential to contribute in virulence of the pathogen was assessed. CONCLUSIONS: The identified mutated loci dacB, dnaK, metN, ywqD, lysC, serC and gbpA are known to encode for virulence factors in other bacteria and are suggested to play a similar role in P. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The study identified new possible virulence factors for P. larvae genotype ERIC I in an unbiased way. This contributes to the knowledge and understanding of the possible mechanisms used by this pathogen to colonize and kill its host.