Substrate elasticity does not impact DNA methylation changes during differentiation of pluripotent stem cells.

BACKGROUND AIMS: Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness affects the differentiation of induced pluripotent stem cells (iPSCs) and whether this is also reflected by epigenetic modifications. METHODS: We cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Young's modulus (0.2 kPa, 16 kPa or 64 kPa) to investigate the sequel on growth and differentiation toward endoderm, mesoderm and ectoderm. RESULTS: Immunofluorescence and gene expression of canonical differentiation markers were hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of undifferentiated iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP we did observe epigenetic differences, particularly on mesodermal differentiation. CONCLUSIONS: Stiffness of PDMS substrates did not affect directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters.

CTCF deletion alters the pluripotency and DNA methylation profile of human iPSCs.

Pluripotent stem cells are characterized by their differentiation potential toward endoderm, mesoderm, and ectoderm. However, it is still largely unclear how these cell-fate decisions are mediated by epigenetic mechanisms. In this study, we explored the relevance of CCCTC-binding factor (CTCF), a zinc finger-containing DNA-binding protein, which mediates long-range chromatin organization, for directed cell-fate determination. We generated human induced pluripotent stem cell (iPSC) lines with deletions in the protein-coding region in exon 3 of CTCF, resulting in shorter transcripts and overall reduced protein expression. Chromatin immunoprecipitation showed a considerable loss of CTCF binding to target sites. The CTCF deletions resulted in slower growth and modest global changes in gene expression, with downregulation of a subset of pluripotency-associated genes and neuroectodermal genes. CTCF deletion also evoked DNA methylation changes, which were moderately associated with differential gene expression. Notably, CTCF-deletions lead to upregulation of endo-mesodermal associated marker genes and epigenetic signatures, whereas ectodermal differentiation was defective. These results indicate that CTCF plays an important role in the maintenance of pluripotency and differentiation, especially towards ectodermal lineages.

YAP1 is essential for self-organized differentiation of pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) form aggregates that recapitulate aspects of the self-organization in early embryogenesis. Within few days, cells undergo a transition from epithelial-like structures to organized three-dimensional embryoid bodies (EBs) with upregulation of germ layer-specific genes. However, it is largely unclear, which signaling cascades regulate self-organized differentiation. The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. YAP1 has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human iPSCs remains to be elucidated. To gain insights into the function of YAP1 in early cell-fate decisions, we generated YAP1 knockout (YAP-/-) iPSC lines with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. YAP-/- iPSCs showed increased expression of several YAP1 targets and of NODAL, an important regulator of cell differentiation. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies towards endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in undirected self-organized YAP-/- EBs germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the undirected self-organization of EBs, and this is at least partly attributed to activation of the NODAL signaling.

Signalling and responding to zoonotic threats using a One Health approach: a decade of the Zoonoses Structure in the Netherlands, 2011 to 2021.

In the Netherlands, the avian influenza outbreak in poultry in 2003 and the Q fever outbreak in dairy goats between 2007 and 2010 had severe consequences for public health. These outbreaks led to the establishment of an integrated human-veterinary risk analysis system for zoonoses, the Zoonoses Structure. The aim of the Zoonoses Structure is to signal, assess and control emerging zoonoses that may pose a risk to animal and/or human health in an integrated One Health approach. The Signalling Forum Zoonoses (SO-Z), the first step of the Zoonoses Structure, is a multidisciplinary committee composed of experts from the medical, veterinary, entomology and wildlife domains. The SO-Z shares relevant signals with professionals and has monthly meetings. Over the past 10 years (June 2011 to December 2021), 390 different signals of various zoonotic pathogens in animal reservoirs and humans have been assessed. Here, we describe the Zoonoses Structure with examples from signals and responses for four zoonotic events in the Netherlands (tularaemia, Brucella canis, West Nile virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). This may serve as an example for other countries on how to collaborate in a One Health approach to signal and control emerging zoonoses.

A one health glossary to support communication and information exchange between the human health, animal health and food safety sectors.

Collaboration across sectors, disciplines and countries is a key concept to achieve the overarching One Health (OH) objective for better human, animal and environmental health. Differences in terminology and interpretation of terms are still a significant hurdle for cross-sectoral information exchange and collaboration within the area of OH including One Health Surveillance (OHS). The development of the here described glossary is a collaborative effort of three projects funded within the One Health European Joint Programme (OHEJP). We describe the infrastructure of the OHEJP Glossary, as well as the methodology to create such a cross-sectoral web resource in a collaborative manner. The new OHEJP Glossary allows OH actors to identify terms with different or shared interpretation across sectors. Being aware of such differences in terminology will help overcome communication hurdles in the future and consequently support collaboration and a more inclusive development of OHS. The OHEJP Glossary was implemented as a web-based, user-friendly and searchable infrastructure that complies with the Findable, Accessible, Interoperable, Reusable (FAIR) data principles. Maintenance, enrichment and quality control of the OHEJP Glossary is supported through a flexible and updatable curation infrastructure. This increases the uptake potential and exploitation of the OHEJP Glossary by other OH initiatives or tools and services.

Prolonged carriage of (livestock-associated) MRSA in individuals without professional livestock contact.

OBJECTIVES: To investigate prolonged carriage of MRSA in adults from the general population living in a livestock-dense area, using WGS. METHODS: A cross-sectional study during 2014-15 among 2492 adults without professional livestock contact identified 14 (0.6%) nasal MRSA carriers, 10 of which carried livestock-associated (LA)-MRSA of multiple-locus variable-number tandem repeat analysis (MLVA) complex (MC) 398. Two years later, 12 MRSA-positive and 88 MRSA-negative participants provided a second nasal swab and filled in a short questionnaire. Isolates from persons who were MRSA positive at both timepoints were compared using MLVA and isolates with the same MLVA type were sequenced. The WGS data were used for core-genome MLST (cgMLST) and resistome analysis, including sequenced isolates from the national MRSA surveillance. RESULTS: All MRSA-negative persons tested negative again, while 6 of the 12 initially MRSA-positive persons tested positive again. MLVA revealed that isolate pairs from five individuals had the same MLVA type, of which three were LA-MRSA. cgMLST showed that the distance between these isolate pairs ranged between 3 and 13 genes, while the minimum distance to unrelated isolates from the national MRSA surveillance was 38 genes. Moreover, the resistome present in the five isolate pairs was identical within each pair. None of the prolonged carriers was hospitalized during the 3 months before the sampling moment and none of them with LA-MRSA had contact with livestock in this period. CONCLUSIONS: Prolonged carriage of MRSA, including LA-MRSA, can be demonstrated after more than 30 months in persons without professional livestock contact.

Insights into Livestock-Related Microbial Concentrations in Air at Residential Level in a Livestock Dense Area.

Microbial air pollution from livestock farms has raised concerns regarding public health. Little is known about airborne livestock-related microbial levels in residential areas. We aimed to increase insights into this issue. Air measurements were performed in 2014 and 2015 at 61 residential sites in The Netherlands. Quantitative-PCR was used to assess DNA concentrations of selected bacteria (commensals: Escherichia coli and Staphylococcus spp.; a zoonotic pathogen: Campylobacter jejuni) and antimicrobial resistance (AMR) genes ( tetW, mecA) in airborne dust. Mixed models were used to explore spatial associations (temporal adjusted) with livestock-related characteristics of the surroundings. DNA from commensals and AMR genes was detectable even at sites furthest away from farms (1200 m), albeit at lower levels. Concentrations, distinctly different between sites, were strongly associated with the density of farms in the surroundings especially with poultry and pigs. C. jejuni DNA was less prevalent (42% of samples positive). Presence of C. jejuni was solely associated with poultry (OR: 4.7 (95% CI: 1.7-14), high versus low poultry density). Residential exposure to livestock-related bacteria and AMR genes was demonstrated. Identified associations suggest contribution of livestock farms to microbial air pollution in general and attribution differences between farm types. This supports the plausibility of recent studies showing health effects in relation to residential proximity to farms.

A bead-based suspension array for the detection of Salmonella antibodies in pig sera.

BACKGROUND: Slaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Nearly all Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). ELISAs for Salmonella serology are usually based on serogroup B and C1 LPS, often combined with serogroup D or E LPS. Although C2 LPS may improve serology, use of C2 LPS in a broad ELISA was never achieved. RESULTS: To enable detection of serum antibodies against Salmonella in pigs, a bead-based suspension array was developed with five LPS variants (B, 2× C1, C2, D1), each conjugated to a different bead set using triazine chemistry. Reactivity of the beads was confirmed with rabbit agglutination sera and with experimental pig sera. With a mixture of bead sets, 175 sera from slaughter pigs were investigated for the presence of antibodies against Salmonella. With a combination of ROC analysis (B and D LPS) and a prevalence estimation based on historic data (C LPS), individual cut-offs were defined for each LPS-conjugated bead set, and assay performance was evaluated. Results of the suspension array (BC1C1C2D) suggest that more pigs are seroconverted than indicated by a commercial BC1D1-ELISA, and that most of these extra seropositive samples give a signal on one of the beads with C LPS. These results show that expansion of a standard panel with more C LPS variants improves antibody detection. CONCLUSIONS: A suspension array for Salmonella serology in pigs was developed, that detects more seropositive sera than ELISA, which is achieved by expanding the panel of Salmonella LPS variants, including C2 LPS. The results demonstrate that bead-based suspension arrays allow for testing of pig sera, with the advantage of being able to set cut-offs per antigen. Ultimately, this type of assay can be applied in routine veterinary serology to test for antibodies against multiple Salmonella serovars (or other pathogens) in one single serum sample, using up-to-date antigen panels.

Residential proximity to livestock farms is associated with a lower prevalence of atopy.

OBJECTIVES: Exposure to farm environments during childhood and adult life seems to reduce the risk of atopic sensitisation. Most studies have been conducted among farmers, but people living in rural areas may have similar protective effects for atopy. This study aims to investigate the association between residential proximity to livestock farms and atopy among non-farming adults living in a rural area in the Netherlands. METHODS: We conducted a cross-sectional study among 2443 adults (20-72 years). Atopy was defined as specific IgE to common allergens and/or total IgE ≥100 IU/mL. Residential proximity to livestock farms was assessed as 1) distance to the nearest pig, poultry, cattle or any farm, 2) number of farms within 500 m and 1000 m, and 3) modelled annual average fine dust emissions from farms within 500 m and 1000 m. Data were analysed with multiple logistic regression and generalised additive models. RESULTS: The prevalence of atopy was 29.8%. Subjects living at short distances from farms (<327 m, first tertile) had a lower odds for atopy compared with subjects living further away (>527 m, third tertile) (OR 0.79, 95% CI 0.63 to 0.98). Significant associations in the same direction were found with distance to the nearest pig or cattle farm. The associations between atopy and livestock farm exposure were somewhat stronger in subjects who grew up on a farm. CONCLUSIONS: Living in close proximity to livestock farms seems to protect against atopy. This study provides evidence that protective effects of early-life and adult farm exposures may extend beyond farming populations.

Spatial Variation of Endotoxin Concentrations Measured in Ambient PM10 in a Livestock-Dense Area: Implementation of a Land-Use Regression Approach.

BACKGROUND: Results from studies on residential health effects of livestock farming are inconsistent, potentially due to simple exposure proxies used (e.g., livestock density). Accuracy of these proxies compared with measured exposure concentrations is unknown. OBJECTIVES: We aimed to assess spatial variation of endotoxin in PM10 (particulate matter ≤10µm) at residential level in a livestock-dense area, compare simple livestock exposure proxies to measured endotoxin concentrations, and evaluate whether land-use regression (LUR) can be used to explain spatial variation of endotoxin. METHODS: The study area (3,000 km2) was located in Netherlands. Ambient PM10 was collected at 61 residential sites representing a variety of surrounding livestock-related characteristics. Three to four 2-wk averaged samples were collected at each site. A local reference site was used for temporal variation adjustment. Samples were analyzed for PM10 mass by weighing and for endotoxin by using the limulus amebocyte lysate assay. Three LUR models were developed, first a model based on general livestock-related GIS predictors only, followed by models that also considered species-specific predictors and farm type-specific predictors. RESULTS: Variation in concentrations measured between sites was substantial for endotoxin and more limited for PM10 (coefficient of variation: 43%, 8%, respectively); spatial patterns differed considerably. Simple exposure proxies were associated with endotoxin concentrations although spatial variation explained was modest (R2<26%). LUR models using a combination of animal-specific livestock-related characteristics performed markedly better, with up to 64% explained spatial variation. CONCLUSION: The considerable spatial variation of ambient endotoxin concentrations measured in a livestock-dense area can largely be explained by LUR modeling based on livestock-related characteristics. Application of endotoxin LUR models seems promising for residential exposure estimation within health studies. https://doi.org/10.1289/EHP2252.

A cross sectional study on Dutch layer farms to investigate the prevalence and potential risk factors for different Chlamydia species.

In poultry several Chlamydia species have been detected, but Chlamydia psittaci and Chlamydia gallinacea appear to be most prevalent and important. Chlamydia psittaci is a well-known zoonosis and is considered to be a pathogen of poultry. Chlamydia gallinacea has been described more recently. Its avian pathogenicity and zoonotic potential have to be further elucidated. Within the Netherlands no data were available on the presence of Chlamydia on poultry farms. As part of a surveillance programme for zoonotic pathogens in farm animals, we investigated pooled faecal samples from 151 randomly selected layer farms. On a voluntary base, 69 farmers, family members or farm workers from these 151 farms submitted a throat swab. All samples were tested with a generic 23S Chlamydiaceae PCR followed by a species specific PCR for C. avium, C. gallinacea and C. psittaci. C. avium and psittaci DNA was not detected at any of the farms. At 71 farms the positive result could be confirmed as C. gallinacea. Variables significantly associated with the presence of C. gallinacea in a final multivariable model were 'age of hens,' 'use of bedding material' and 'the presence of horses.' The presence of C. gallinacea was associated with neither clinical signs, varying from respiratory symptoms, nasal and ocular discharges to diarrhoea, nor with a higher mortality rate the day before the visit. All throat swabs from farmers, family members or farm workers tested negative for Chlamydia DNA, giving no further indication for possible bird-to-human (or human-to-bird) transmission.

Long-term Carriage of Extended-Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in the General Population in The Netherlands.

Background: This longitudinal study aimed to investigate (risk factors for) persistence of carriage and molecular characteristics of extended-spectrum and plasmid-encoded AmpC ß-lactamase-producing (ESBL/pAmpC) Escherichia coli and Klebsiella pneumoniae (ESBL-E/K) in adults in the Dutch community. Methods: Following a cross-sectional study (ESBL-E/K prevalence, 4.5%), a subset of ESBL-E/K-positive (n = 76) and -negative (n = 249) individuals volunteered to provide 5 monthly fecal samples and questionnaires. ESBL-E/K was cultured using selective enrichment/culture, and multilocus sequence types (MLSTs) were determined. ESBL/pAmpC-genes were analyzed using polymerase chain reaction (PCR) and sequencing. Plasmids were characterized and subtyped by plasmid MLST. Risk factors for persistent carriage were analyzed using logistic regression. Results: Of the initially ESBL-E/K-positive participants, 25 of 76 (32.9%) remained positive in all subsequent samples; 51 of 76 persons (67.1%) tested ESBL-E/K negative at some time point during follow-up, of which 31 (40.8%) stayed negative throughout the longitudinal study. Carriers often carried the same ESBL gene and plasmid, but sometimes in different ESBL-E/K strains, indicative for horizontal transfer of plasmids. Of the 249 initially ESBL-E/K-negative participants, the majority (n = 218 [87.6%]) tested negative during 8 months of follow-up, whereas 31 of 249 (12.4%) participants acquired an ESBL-E/K. Escherichia coli phylogenetic group B2 and D and travel to ESBL high-prevalence countries were associated with prolonged carriage. Conclusions: ESBL-E/K carriage persisted for >8 months in 32.9% of the initially ESBL-positive individuals, while 12.4% of initially negative individuals acquired ESBL-E/K during the study. A single positive test result provides no accurate prediction for prolonged carriage. Acquisition/loss of ESBL-E/K does not seem to be a random process, but differs between bacterial genotypes.

Remarkable spatial variation in the seroprevalence of Coxiella burnetii after a large Q fever epidemic.

BACKGROUND: Prior to the 2007-2010 Q fever epidemic in the Netherlands, the seroprevalence of antibodies against Coxiella burnetii in the general population was 1.5%, which is low compared to other countries. We aimed to determine the seroprevalence after the Q fever epidemic among people living in the affected area, compare the seroprevalence with the incidence of Q fever notifications during the 2007-2010 Q fever epidemic, and to identify farm exposures associated with having antibodies against C. burnetii. METHODS: During the period March 2014-February 2015, residents aged 18-70 years from two provinces were invited by general practitioners to complete a questionnaire on their symptoms and personal characteristics and to submit a blood sample. We used the mandatory provincial database of livestock licences to calculate distance to farms/farm animals for each participant. To compare ELISA-positive participants for C. burnetii antibodies with those who were negative, we calculated prevalence ratios (PR) using binominal regression. We compared the C. burnetii seroprevalence in the period March 2014-February 2015 with the incidence of Q fever notifications during the 2007-2010 Q fever epidemic at municipal level by calculating the Spearman correlation coefficient. RESULTS: Of the 2296 participants (response rate: 34%), 6.1% (n = 139, 95% CI 5.1-7.1%) had C. burnetii antibodies (range in municipalities: 1.7-14.1%). C. burnetii seroprevalence was higher in individuals living within 1000 m of goat farms (PR 3.0; 95% CI 1.4-6.4) or within 1000 m of > 50 goats (PR 1.9; 95% CI 1.2-3.0). Seroprevalence increased with decreasing distance to the closest goat farm that was infected during the epidemic years (< 500 m, PR 9.5, 95% CI 2.8-32; 500-1000 m, PR 4.5, 95% CI 2.6-7.7; 1000-1500 m, PR 2.2, 95% CI 1.1-4.3, 1500-2000 m, PR 1.2, 95% CI 0.6-2.5; > 2000 reference group). There was no significant correlation between C. burnetii seroprevalence and Q fever incidence during the 2007-2010 epidemic (r s = 0.42, p = 0.156). CONCLUSIONS: Results showed a remarkable spatial variation in C. burnetii seroprevalence in a relatively small livestock dense area. It confirms previous evidence that the Q fever epidemic was primarily the result of airborne C. burnetii transmission from Q fever affected goat farms.

Air Pollution from Livestock Farms Is Associated with Airway Obstruction in Neighboring Residents.

RATIONALE: Livestock farm emissions may not only affect respiratory health of farmers but also of neighboring residents. OBJECTIVES: To explore associations between spatial and temporal variation in pollutant emissions from livestock farms and lung function in a general, nonfarming, rural population in the Netherlands. METHODS: We conducted a cross-sectional study in 2,308 adults (age, 20-72 yr). A pulmonary function test was performed measuring prebronchodilator and post-bronchodilator FEV1, FVC, FEV1/FVC, and maximum mid-expiratory flow (MMEF). Spatial exposure was assessed as (1) number of farms within 500 m and 1,000 m of the home, (2) distance to the nearest farm, and (3) modeled annual average fine dust emissions from farms within 500 m and 1,000 m of the home address. Temporal exposure was assessed as week-average ambient particulate matter <10 µm in diameter and ammonia (NH3) concentrations before lung function measurements. Data were analyzed with generalized additive models (smoothing). MEASUREMENTS AND MAIN RESULTS: A negative association was found between the number of livestock farms within a 1,000-m buffer from the home address and MMEF, which was more pronounced in participants without atopy. No associations were found with other spatial exposure variables. Week-average particulate matter <10 µm in diameter and NH3 levels were negatively associated with FEV1, FEV1/FVC, and MMEF. In a two-pollutant model, only NH3 remained associated. A 25-µg/m3 increase in NH3 was associated with a 2.22% lower FEV1 (95% confidence interval, -3.69 to -0.74), FEV1/FVC of -1.12% (-1.96 to -0.28), and MMEF of -5.67% (-8.80 to -2.55). CONCLUSIONS: Spatial and temporal variation in livestock air pollution emissions are associated with lung function deficits in nonfarming residents.

Livestock-associated risk factors for pneumonia in an area of intensive animal farming in the Netherlands.

Previous research conducted in 2009 found a significant positive association between pneumonia in humans and living close to goat and poultry farms. However, as this result might have been affected by a large goat-related Q fever epidemic, the aim of the current study was to re-evaluate this association, now that the Q-fever epidemic had ended. In 2014/15, 2,494 adults (aged 20-72 years) living in a livestock-dense area in the Netherlands participated in a medical examination and completed a questionnaire on respiratory health, lifestyle and other items. We retrieved additional information for 2,426/2,494 (97%) participants from electronic medical records (EMR) from general practitioners. The outcome was self-reported, physician-diagnosed pneumonia or pneumonia recorded in the EMR in the previous three years. Livestock license data was used to determine exposure to livestock. We quantified associations between livestock exposures and pneumonia using odds ratios adjusted for participant characteristics and comorbidities (aOR). The three-year cumulative frequency of pneumonia was 186/2,426 (7.7%). Residents within 2,000m of a farm with at least 50 goats had an increased risk of pneumonia, which increased the closer they lived to the farm (2,000m aOR 1.9, 95% CI 1.4-2.6; 500m aOR 4.4, 95% CI 2.0-9.8). We found no significant associations between exposure to other farm animals and pneumonia. However, when conducting sensitivity analyses using pneumonia outcome based on EMR only, we found a weak but statistically significant association with presence of a poultry farm within 1,000m (aOR: 1.7, 95% CI 1.1-2.7). Living close to goat and poultry farms still constitute risk factors for pneumonia. Individuals with pneumonia were not more often seropositive for Coxiella burnetii, indicating that results are not explained by Q fever. We strongly recommend identification of pneumonia causes by the use of molecular diagnostics and investigating the role of non-infectious agents such as particulate matter or endotoxins.

Spirometry, questionnaire and electronic medical record based COPD in a population survey: Comparing prevalence, level of agreement and associations with potential risk factors.

BACKGROUND: COPD-diagnosis is confirmed by post-bronchodilator (BD) spirometry. However, epidemiological studies often rely on pre-BD spirometry, self-reports, or medical records. This population-based study aims to determine COPD-prevalence based on four different operational definitions and their level of agreement, and to compare associations between COPD-definitions and risk factors. METHODS: COPD-prevalence in 1,793 adults from the general Dutch population (aged 18-70 years) was assessed based on self-reported data, Electronic Medical Records (EMR), and post-BD spirometry: using the FEV1/FVC below the lower limit of normal (LLN) and GOLD fixed cut-off (FEV1/FVC <0.70). Using spirometry as a reference, sensitivity was calculated for self-reported and EMR-based COPD. Associations between COPD and known risk factors were assessed with logistic regression. Data were collected as part of the cross-sectional VGO study (Livestock Farming and Neighboring Residents' Health Study). RESULTS: The highest prevalence was found based on spirometry (GOLD: 10.9%, LLN: 5.9%), followed by self-report (4.6%) and EMR (2.9%). Self-reported or EMR-based COPD identified less than 30% of all COPD-cases based on spirometry. The direction of association between known risk factors and COPD was similar across the four definitions, however, magnitude and significance varied. Especially indicators of allergy were more strongly associated with self-reported COPD compared to the other definitions. CONCLUSIONS: COPD-prevalence varied depending on the used definition. A substantial number of subjects with spirometry-based COPD cannot be identified with questionnaires or medical records which can cause underestimation of COPD-prevalence. The influence of the different COPD-definitions on associations with known risk factors was limited.

Hepatitis E virus seroprevalence among the general population in a livestock-dense area in the Netherlands: a cross-sectional population-based serological survey.

BACKGROUND: Recent serological studies indicate that hepatitis E virus (HEV) is endemic in industrialised countries. The increasing trend in the number of autochthonous cases of HEV genotype 3 in Western European countries, stresses the importance to get insight in the exact routes of exposure. Pigs are the main animal reservoir, and zoonotic food-borne transmission of HEV is proven. However, infected pigs can excrete large amounts of virus via their faeces enabling environmental transmission of HEV to humans. This might pose a risk for of neighbouring residents of livestock farming. METHODS: Within a large study on the health of people living in the vicinity of livestock farming we performed a cross-sectional population-based serological survey among 2,494 non-farming adults from the general population in a livestock-dense area in the south of the Netherlands. Participants completed risk factor questionnaires and blood samples of 2,422 subjects (median age 58 years, range 20-72) were tested for anti-HEV IgG using an enzyme immune assay (Wantai). The aim of this study was to determine the HEV seroprevalence and to assess whether seropositivity in adults was associated with living in the vicinity of pig farms. RESULTS: The average seroprevalence of HEV was 28.7% (95% CI: 26.9-30.5). Determinants associated with an increased risk for HEV seropositivity were male gender and low level of education. There was a clear trend of increasing prevalence with increasing age (Chi-square test for linear trend, X2 = 83.1; p < 0.001). A high number of pigs within 1,000 m of the residential address was not a risk factor for seropositivity. CONCLUSIONS: This study confirmed the high HEV seroprevalence (29%) in the general population of the Netherlands, but presence of antibodies was not associated with residential proximity to pig farms. The prevalence increased with age from 10% in adolescents to 33% among those aged 50 and above, supporting the assumption of a cumulative lifetime exposure to HEV in the Netherlands as well as a higher infection pressure in the past. Our findings cannot refute the assumption that transmission is primarily food-borne.

MRSA in persons not living or working on a farm in a livestock-dense area: prevalence and risk factors.

Objectives: MRSA emerged in livestock and persons in contact with livestock is referred to as livestock-associated MRSA (LA-MRSA). We assessed the prevalence and risk factors for MRSA carriage in persons not living or working on a farm. Methods: A cross-sectional study was performed among 2492 adults living in close proximity of livestock farms. Persons working and/or living on farms were excluded. Nasal swabs were cultured using selective media. Participants completed questionnaires and the distance from the residential address to the nearest farm was calculated. The Mann-Whitney U -test was used to compare median distances. Risk factors were explored with logistic regression. Results: Fourteen persons carried MRSA (0.56%; 95% CI 0.32%-0.92%), 10 of which carried LA-MRSA of multiple-locus variable-number tandem repeat analysis complex (MC) 398 (0.40%; 95% CI 0.20%-0.71%). MRSA MC 398 carriers lived significantly closer to the nearest farm than non-carriers (median: 184 versus 402 m; P < 0.01). In bivariate analyses correcting for contact with livestock, this difference remained significant. Conclusions: Although the prevalence was low, living near farms increased the risk of MRSA MC 398 carriage for persons not living or working on a farm. Further research is necessary to identify the transmission routes.

Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus.

Ixodes ricinus transmits Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease. Previous studies have also detected Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis, and several Babesia species in questing ticks in The Netherlands. In this study, we assessed the acarological risk of exposure to several tick-borne pathogens (TBPs), in The Netherlands. Questing ticks were collected monthly between 2006 and 2010 at 21 sites and between 2000 and 2009 at one other site. Nymphs and adults were analysed individually for the presence of TBPs using an array-approach. Collated data of this and previous studies were used to generate, for each pathogen, a presence/absence map and to further analyse their spatiotemporal variation. R. helvetica (31.1%) and B. burgdorferi sensu lato (11.8%) had the highest overall prevalence and were detected in all areas. N. mikurensis (5.6%), A. phagocytophilum (0.8%), and Babesia spp. (1.7%) were detected in most, but not all areas. The prevalences of pathogens varied among the study areas from 0 to 64%, while the density of questing ticks varied from 1 to 179/100 m². Overall, 37% of the ticks were infected with at least one pathogen and 6.3% with more than one pathogen. One-third of the Borrelia-positive ticks were infected with at least one other pathogen. Coinfection of B. afzelii with N. mikurensis and with Babesia spp. occurred significantly more often than single infections, indicating the existence of mutual reservoir hosts. Alternatively, coinfection of R. helvetica with either B. afzelii or N. mikurensis occurred significantly less frequent. The diversity of TBPs detected in I. ricinus in this study and the frequency of their coinfections with B. burgdorferi s.l., underline the need to consider them when evaluating the risks of infection and subsequently the risk of disease following a tick bite.