First Detections of Culiseta longiareolata (Diptera: Culicidae) in Belgium and the Netherlands.

Culiseta (Allotheobaldia) longiareolata (Macquart) (Diptera: Culicidae) is an ornithophilic mosquito species that occurs in the southern Palaearctic Region from the Azores to Central Asia, the Ethiopian Region, India, and Pakistan. Although it has a widespread distribution range, the species was only recently reported in Western and Central Europe. Between 2017 and 2020, larvae, pupae, and adults of Cs. longiareolata (n = 161) were found at 13 distinct locations in Belgium (n = 4) and The Netherlands (n = 9). Collected mosquitoes were morphologically identified and the identification was then validated by COI DNA barcoding. These are the first records of the species in the above-mentioned countries. The present results suggest that Cs. longiareolata could be increasing its distribution range in temperate regions, indicating a warming climate. As the species might be a potential vector of bird pathogens (e.g., West Nile virus), its spread in Western Europe is noteworthy.

Imported Hyalomma ticks in the Netherlands 2018-2020.

BACKGROUND: Ticks of the genus Hyalomma, which are vectors for several tick-borne diseases, are occasionally found in areas outside their endemic range including northern parts of Europe. The objective of this study was to analyse adult Hyalomma ticks that were recently found in the Netherlands. METHODS: Hyalomma ticks were morphologically identified. Cluster analysis, based upon sequence data (cox1 barcoding) for molecular identification, and pathogen detection were performed. Additionally, a cross-sectional survey of horses was conducted to actively search for Hyalomma ticks in summer 2019. Analysis of temperature was done to assess the possibility of (i) introduced engorged nymphs moulting to adults and (ii) establishment of populations in the Netherlands. RESULTS: Seventeen adult Hyalomma ticks (one in 2018, eleven in 2019, five in 2020) were found by citizens and reported. Fifteen ticks were detected on horses and two on humans. Twelve were identified as H. marginatum, one as H. rufipes and four, of which only photographic images were available, as Hyalomma sp. No Crimean-Congo haemorrhagic fever virus or Babesia/Theileria parasites were detected. One adult tick tested positive for Rickettsia aeschlimannii. In the cross-sectional horse survey, no Hyalomma ticks were found. Analysis of temperatures showed that engorged nymphs arriving on migratory birds in spring were able to moult to adults in 2019 and 2020, and that cumulative daily temperatures in the Netherlands were lower than in areas with established H. marginatum populations. CONCLUSIONS: Our results show that Hyalomma ticks are regularly introduced in the Netherlands as nymphs. Under the Dutch weather conditions, these nymphs are able to develop to the adult stage, which can be sighted by vigilant citizens. Only one human pathogen, Rickettsia aeschlimannii, was found in one of the ticks. The risk of introduction of tick-borne diseases via Hyalomma ticks on migratory birds is considered to be low. Establishment of permanent Hyalomma populations is considered unlikely under the current Dutch climatic conditions.

Seasonal patterns and spatial variation of Borrelia burgdorferi (sensu lato) infections in Ixodes ricinus in the Netherlands.

BACKGROUND: The incidence of Lyme borreliosis varies over time and space through as yet incompletely understood mechanisms. In Europe, Lyme borreliosis is caused by infection with a Borrelia burgdorferi (s.l.) genospecies, which is primarily transmitted by a bite of Ixodes ricinus nymphs. The aim of this study was to investigate the spatial and temporal variation in nymphal infection prevalence of B. burgdorferi (s.l.) (NIP), density of questing nymphs (DON) and the resulting density of infected nymphs (DIN). METHODS: We investigated the infection rates in I. ricinus nymphs that were collected monthly between 2009 and 2016 in 12 locations in the Netherlands. Using generalized linear mixed models, we explored how the NIP, DON and DIN varied during the seasons, between years and between locations. We also determined the genospecies of the Borrelia infections and investigated whether the genospecies composition differed between locations. RESULTS: The overall NIP was 14.7%. A seasonal pattern in infection prevalence was observed, with higher estimated prevalences in the summer than in the spring and autumn. This, combined with higher nymphal densities in summer, resulted in a pronounced summer peak in the estimated DIN. Over the 7.5-year study period, a significant decrease in infection prevalence was found, as well as a significant increase in nymphal density. These two effects appear to cancel each other out; the density of infected nymphs, which is the product of NIP × DON, showed no significant trend over years. Mean infection prevalence (NIP, averaged over all years and all months) varied considerably between locations, ranging from 5 to 26%. Borrelia genospecies composition differed between locations: in some locations almost all infections consisted of B. afzelii, whereas other locations had more diverse genospecies compositions. CONCLUSION: In the Netherlands, the summer peak in DIN is a result of peaks in both NIP and DON. No significant trend in DIN was observed over the years of the study, and variations in DIN between locations were mostly a result of the variation in DON. There were considerable differences in acarological risk between areas in terms of infection prevalence and densities of ticks as well as in Borrelia genospecies composition.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

Detection of Exotic Mosquito Species (Diptera: Culicidae) at International Airports in Europe.

In Europe, the air-borne accidental introduction of exotic mosquito species (EMS) has been demonstrated using mosquito surveillance schemes at Schiphol International Airport (Amsterdam, The Netherlands). Based upon these findings and given the increasing volume of air transport movements per year, the establishment of EMS after introduction via aircraft is being considered a potential risk. Here we present the airport surveillance results performed by the Centre for Monitoring of Vectors of the Netherlands, by the Monitoring of Exotic Mosquitoes (MEMO) project in Belgium, and by the Public Health England project on invasive mosquito surveillance. The findings of our study demonstrate the aircraft mediated transport of EMS into Europe from a wide range of possible areas in the world. Results show accidental introductions of Aedes aegypti and Ae. albopictus, as well as exotic Anopheles and Mansonia specimens. The findings of Ae. albopictus at Schiphol airport are the first evidence of accidental introduction of the species using this pathway in Europe. Furthermore, our results stress the importance of the use of molecular tools to validate the morphology-based species identifications. We recommend monitoring of EMS at airports with special attention to locations with a high movement of cargo and passengers.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Effect of rodent density on tick and tick-borne pathogen populations: consequences for infectious disease risk.

BACKGROUND: Rodents are considered to contribute strongly to the risk of tick-borne diseases by feeding Ixodes ricinus larvae and by acting as amplifying hosts for pathogens. Here, we tested to what extent these two processes depend on rodent density, and for which pathogen species rodents synergistically contribute to the local disease risk, i.e. the density of infected nymphs (DIN). METHODS: In a natural woodland, we manipulated rodent densities in plots of 2500 m2 by either supplementing a critical food source (acorns) or by removing rodents during two years. Untreated plots were used as controls. Collected nymphs and rodent ear biopsies were tested for the presence of seven tick-borne microorganisms. Linear models were used to capture associations between rodents, nymphs, and pathogens. RESULTS: Investigation of data from all plots, irrespective of the treatment, revealed a strong positive association between rodent density and nymphal density, nymphal infection prevalence (NIP) with Borrelia afzelii and Neoehrlichia mikurensis, and hence DIN's of these pathogens in the following year. The NIP, but not the DIN, of the bird-associated Borrelia garinii, decreased with increasing rodent density. The NIPs of Borrelia miyamotoi and Rickettsia helvetica were independent of rodent density, and increasing rodent density moderately increased the DINs. In addition, NIPs of Babesia microti and Spiroplasma ixodetis decreased with increasing rodent density, which had a non-linear association with DINs of these microorganisms. CONCLUSIONS: A positive density dependence for all rodent- and tick-associated tick-borne pathogens was found, despite the observation that some of them decreased in prevalence. The effects on the DINs were variable among microorganisms, more than likely due to contrasts in their biology (including transmission modes, host specificity and transmission efficiency). The strongest associations were found in rodent-associated pathogens that most heavily rely on horizontal transmission. Our results draw attention to the importance of considering transmission mode of a pathogen while developing preventative measures to successfully reduce the burden of disease.

Detection of Aedes flavopictus (Yamada, 1921), Netherlands, June 2019.

In June 2019, a single specimen collected at a used tyre company was identified as Aedes flavopictus (Yamada, 1921), a sibling species of Ae. albopictus. Ae. flavopictus has not been recorded outside Japan and South Korea. Although it has only shown dengue virus vector competence under laboratory conditions, its detection demonstrates the value of active surveillance at risk locations and molecular tools for timely intervention against exotic mosquitoes with potential future public health impact.

Temporal-Spatial Variation in Questing Tick Activity in the Netherlands: The Effect of Climatic and Habitat Factors.

Longitudinal studies are fundamental in the assessment of the effect of environmental factors on tick population dynamics. In this study, we use data from a 10-year study in 11 different locations in the Netherlands to gauge the effects of climatic and habitat factors on the temporal and spatial variation in questing tick activity. Marked differences in the total number of ticks were found between locations and between years. We investigated which climatic and habitat factors might explain this variation. No effects of climatic factors on the total number of ticks per year were observed, but we found a clear effect of temperature on the onset of tick activity. In addition, we found positive associations between (1) humus layer thickness and densities of all three stages, (2) moss and blackberry abundance and larval densities, and (3) blueberry abundance and densities of larva and nymphs. We conclude that climatic variables do not have a straightforward association with tick density in the Netherlands, but that winter and spring temperatures influence the onset of tick activity. Habitats with apparently similar vegetation types can still differ in tick population densities, indicating that local composition of vegetation and especially of wildlife is likely to contribute considerably to the spatial variation in tick densities.

Isolation and Characterization of Different Mesenchymal Stem Cell Populations from Rat Femur.

Purified mesenchymal stem cells (MSCs) may be used for a multitude of applications, from the study of biological processes such as cell division and coordinated gene expression to tissue engineering and regenerative medicine. However, although highly similar, MSCs isolated and purified from different tissues may be biologically different in the ability of the cells to respond to environmental cues that instigate and propagate changes in cell fate such as differentiation, proliferation, apoptosis, and senescence. Selecting which MSC subtype to study may therefore profoundly influence the outcome of the investigation. Here we outline the isolation, purification, and differentiation of three different MSC subtypes derived from various depots within rat bone. These include MSCs from bone marrow, compact bone, and the proximal femur. Osteoblastic and adipogenic differentiation exemplify differences between these cells.

The buzz about bees and poverty alleviation: Identifying drivers and barriers of beekeeping in sub-Saharan Africa.

The potential of beekeeping to mitigate the exposure of rural sub-Sahara African farmers to economic stochasticity has been widely promoted by an array of development agencies. Robust outcome indicators of the success of beekeeping to improve household well-being are unfortunately lacking. This study aimed to identify the key drivers and barriers of beekeeping adoption at the household level, and quantified the associated income contribution in three agro-ecological zones in Uganda. Beekeepers were generally the most economically disadvantaged people in the study areas and tended to adopt beekeeping following contact with non-government organisations and access to training. Whilst incomes were not statistically lower than their non-beekeeping counterparts; their mean household well-being scores were significantly lower than non-beekeeping households. The inability of beekeeping to significantly improve well-being status can in part be attributed to a lack of both training in bee husbandry and protective equipment provision such as suits, gloves and smokers. These are critical tools for beekeepers as they provide the necessary confidence to manage honey bees. Rather than focussing solely on the socio-economic conditions of farmers to effectively adopt beekeeping, future research should also attempt to evaluate the effectiveness of development agencies' provision to the beekeeping sector.

Acarological Risk of Borrelia burgdorferi Sensu Lato Infections Across Space and Time in The Netherlands.

A longitudinal investigation on tick populations and their Borrelia infections in the Netherlands was undertaken between 2006 and 2011 with the aim to assess spatial and temporal patterns of the acarological risk in forested sites across the country and to assess variations in Borrelia genospecies diversity. Ticks were collected monthly in 11 sites and nymphs were examined for Borrelia infections. Tick populations expressed strong seasonal variations, with consistent and significant differences in mean tick densities between sites. Borrelia infections were present in all study sites, with a site-specific mean prevalence per month ranging from 7% to 26%. Prevalence was location-dependent and was not associated with tick densities. Mean Borrelia prevalence was lowest in January (4%), gradually increasing to reach a maximum (24%) in August. Borrelia afzelii represented 70% of all infections, with Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia valaisiana represented with 4%, 8%, and 10%, respectively. The density of infected nymphs and the proportional distribution of the four Borrelia genospecies, were significantly different between sites. The results show a consistent and significant spatial and temporal difference in acarological risk across the Netherlands.

Vanadate Impedes Adipogenesis in Mesenchymal Stem Cells Derived from Different Depots within Bone.

Glucocorticoid-induced osteoporosis (GIO) is associated with an increase in bone marrow adiposity, which skews the differentiation of mesenchymal stem cell (MSC) progenitors away from osteoblastogenesis and toward adipogenesis. We have previously found that vanadate, a non-specific protein tyrosine phosphatase inhibitor, prevents GIO in rats, but it was unclear whether vanadate directly influenced adipogenesis in bone-derived MSCs. For the present study, we investigated the effect of vanadate on adipogenesis in primary rat MSCs derived from bone marrow (bmMSCs) and from the proximal end of the femur (pfMSCs). By passage 3 after isolation, both cell populations expressed the MSC cell surface markers CD90 and CD106, but not the hematopoietic marker CD45. However, although variable, expression of the fibroblast marker CD26 was higher in pfMSCs than in bmMSCs. Differentiation studies using osteogenic and adipogenic induction media (OM and AM, respectively) demonstrated that pfMSCs rapidly accumulated lipid droplets within 1 week of exposure to AM, while bmMSCs isolated from the same femur only formed lipid droplets after 3 weeks of AM treatment. Conversely, pfMSCs exposed to OM produced mineralized extracellular matrix (ECM) after 3 weeks, compared to 1 week for OM-treated bmMSCs. Vanadate (10 µM) added to AM resulted in a significant reduction in AM-induced intracellular lipid accumulation and expression of adipogenic gene markers (PPARγ2, aP2, adipsin) in both pfMSCs and bmMSCs. Pharmacological concentrations of glucocorticoids (1 µM) alone did not induce lipid accumulation in either bmMSCs or pfMSCs, but resulted in significant cell death in pfMSCs. Our findings demonstrate the existence of at least two fundamentally different MSC depots within the femur and highlights the presence of MSCs capable of rapid adipogenesis within the proximal femur, an area prone to osteoporotic fractures. In addition, our results suggest that the increased bone marrow adiposity observed in GIO may not be solely due to direct effect of glucocorticoids on bone-derived MSCs, and that an increase in femur lipid content may also arise from increased adipogenesis in MSCs residing outside of the bone marrow niche.

Mosquito Attraction: Crucial Role of Carbon Dioxide in Formulation of a Five-Component Blend of Human-Derived Volatiles.

Behavioral responses of the malaria mosquito Anopheles coluzzii (An. gambiae sensu stricto molecular 'M form') to an expanded blend of human-derived volatiles were assessed in a dual-port olfactometer. A previously documented attractive three-component blend consisting of NH3, (S)-lactic acid, and tetradecanoic acid served as the basis for expansion. Adding 4.5% CO2 to the basic blend significantly enhanced its attractiveness. Expansion of the blend with four human-derived C4-volatiles was then assessed, both with and without CO2. Only when CO2 was offered simultaneously, did addition of a specific concentration of 3-methyl-1-butanol or 3-methyl-butanoic acid significantly enhance attraction. The functional group at the terminal C of the 3-methyl-substituted C4 compounds influenced behavioral effectiveness. In the absence of CO2, addition of three concentrations of butan-1-amine caused inhibition when added to the basic blend. In contrast, when CO2 was added, butan-1-amine added to the basic blend strongly enhanced attraction at all five concentrations tested, the lowest being 100,000 times diluted. The reversal of inhibition to attraction by adding CO2 is unique in the class Insecta. We subsequently augmented the three-component basic blend by adding both butan-1-amine and 3-methyl-1-butanol and optimizing their concentrations in the presence of CO2 in order to significantly enhance the attractiveness to An. coluzzii compared to the three- and four-component blends. This novel blend holds potential to enhance malaria vector control based on behavioral disruption.

Field evaluation of a novel synthetic odour blend and of the synergistic role of carbon dioxide for sampling host-seeking Aedes albopictus adults in Rome, Italy.

BACKGROUND: Despite the expanding worldwide distribution of Aedes albopictus and its increasing relevance as arboviral vector, current methods to collect adult specimens are not optimal. Improved approaches are thus needed to monitor their density and pathogen infections, and to establish baseline data for control interventions. A widely used device is the BG-Sentinel (BG-trap) which mostly targets host-seeking females attracted by release of CO2 and/or a synthetic odour blend (the BG lure). We compared the attractiveness of this blend to that of the Mbita (MB5) lure, a new synthetic blend of proven efficiency in attracting Afrotropical malaria vectors, and evaluated the additional effect of CO2 to the two odour baits. FINDINGS: We carried out 6x6 Latin square experiments in two Ae. albopictus-infested areas in Rome, baiting the BG-traps as follows: CO2, BG lure, MB5 lure, BG lure + CO2, MB5 lure + CO2, no bait. CO2 was derived from yeast-fermented sugar. Overall, 949 females and 816 males were collected. Baited traps collected significantly more females than unbaited ones. Traps baited with either lures in combination with CO2 were more effective than those baited with CO2 alone. No significant differences were observed in female captures between traps baited with any of the two lures, nor between the two lures, independently from the addition of CO2. The use of BG lure + CO2 significantly increased males catches compared to unbaited traps. CONCLUSIONS: The results suggest a broad significance of the MB5 lure for sampling medically important mosquito species and highlight the high efficacy of the combination of lures + CO2 for female Ae. albopictus and of BG lure + CO2 for males, leading to consider CO2 as an essential additional cue for the sampling of this species.

Circumstantial evidence for an increase in the total number and activity of Borrelia-infected Ixodes ricinus in the Netherlands.

BACKGROUND: Between 1994 and 2009, a threefold increase has been observed in consultations of general practitioners for tick bites and Lyme disease in The Netherlands. The objective of this study was to determine whether an increase in the number of questing ticks infected with B. burgdorferi sensu lato is a potential cause of the rise in Lyme disease incidence. METHODS: Historic data on land usage, temperature and wildlife populations were collected and analyzed together with data from two longitudinal field studies on density of questing ticks. Effective population sizes of Borrelia burgdorferi s.l. were calculated. RESULTS: Long-term trend analyses indicated that the length of the annual tick questing season increased as well as the surface area of tick-suitable habitats in The Netherlands. The overall abundances of feeding and reproductive hosts also increased. Mathematical analysis of the data from the field studies demonstrated an increase in mean densities/activities of questing ticks, particularly of larvae between 2006 and 2009. No increase in infection rate of ticks with Borrelia burgdorferi sensu lato was found. Population genetic analysis of the collected Borrelia species points to an increase in B. afzelii and B. garinii populations. CONCLUSIONS: Together, these findings indicate an increase in the total number of Borrelia-infected ticks, providing circumstantial evidence for an increase in the risk of acquiring a bite of a tick infected with B. burgdorferi s.l. Due to the high spatiotemporal variation of tick densities/activities, long-term longitudinal studies on population dynamics of I. ricinus are necessary to observe significant trends.

Culicoides obsoletus extract relevant for diagnostics of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an allergic dermatitis in horses caused by the bites of Culicoides species. The aim of the present study was to evaluate the applicability of whole body extracts of C. obsoletus (the main species found feeding on horses in the Netherlands), C. nubeculosus (rarely found in The Netherlands) and C. sonorensis (typical for North America) for diagnosis of IBH in horses in The Netherlands. Blood and serum samples of 10 clinically confirmed IBH affected and 10 healthy control horses were used to evaluate the IgE titers (ELISA) against the Culicoides whole body extracts of the three Culicoides species. Basophil degranulation was assessed by histamine release test (HRT) after stimulation with these extracts at 5, 0.5 and 0.05 µg/ml. IBH affected horses had significantly higher IgE titers against C. obsoletus than against C. nubeculosus and C. sonorensis. Furthermore, C. obsoletus induced significantly higher histamine release in whole blood of IBH affected horses compared to the other extracts at 0.5 µg/ml. Western blot data revealed IgE binding to many proteins in C. obsoletus extract. This interaction was absent or weak in C. nubeculosus and C. sonorensis extracts for IBH affected horses. Results on individual level indicate that the HRT is more sensitive than ELISA in diagnosing IBH. However, ELISA is more practical as a routine test, therefore the ELISA was further evaluated using C. obsoletus extract on 103 IBH affected and 100 healthy horses, which resulted in a test sensitivity and specificity of 93.2% and 90.0%, respectively. The IgE ELISA readings enabled the analysis of the predicted probability of being IBH affected. From an optical density 450nm value of 0.33 onwards, the probability of IBH affected was more than 0.9. The results presented in this paper show that the use of native Culicoides spp. that feed on horse, is important for improved diagnosis and that the described ELISA based on C. obsoletus can be used routinely to diagnose IBH in countries where this species is the main Culicoides feeding on horses.

Culicoides species associated with sheep in the Netherlands and the effect of a permethrin insecticide.

Culicoides spp. act as vectors for a number of viral diseases of animals including bluetongue in sheep. The aims of this study were to determine: (1) which Culicoides spp. are associated with sheep in The Netherlands; (2) the time of the day when they are most active; and (3) the effect of treatment of animals with a permethrin insecticide. Two pairs of sheep were each housed within mosquito tents of either one or two layers of netting and all trapped Culicoides spp. were identified microscopically. For the permethrin insecticide study, one of two pairs of sheep was treated with 3.6% permethrin and all animals were housed in tents of similar design. Of the 6210 midges captured, 54.1% were identified as C. chiopterus and 42.7% as C. obsoletus. C. imicola was not identified. The average insect feeding rate was 35-40% and midge activity was greatest around sunset. Permethrin treatment reduced the number of midges captured by 50% and also resulted in a decrease in the percentage of midges that had fed. The findings provide useful information on the behaviour and distribution of Culicoides spp. that will facilitate the development of appropriate control strategies to minimise the risk of insect-vector borne virus diseases such as bluetongue.

Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in The Netherlands.

In a countrywide investigation of the ecological factors that contribute to Lyme borreliosis risk, a longitudinal study on population dynamics of the sheep tick Ixodes ricinus and their infections with Borrelia burgdorferi sensu lato (s.l.) was undertaken at 24 sites in The Netherlands from July 2006 to December 2007. Study sites were mature forests, dune vegetations, or new forests on land reclaimed from the sea. Ticks were sampled monthly and nymphal ticks were investigated for the presence of Borrelia spp. I. ricinus was the only tick species found. Ticks were found in all sites, but with significant spatial and temporal variations in density between sites. Peak densities were found in July and August, with lowest tick numbers collected in December and January. In some sites, questing activities of I. ricinus nymphs and adults were observed in the winter months. Mean monthly Borrelia infections in nymphs varied from 0% to 29.0% (range: 0%-60%), and several sites had significantly higher mean nymphal Borrelia infections than others. Four genospecies of Borrelia burgdorferi s.l. were found, with B. afzelii being dominant at most sites. Borrelia infection rates in nymphal ticks collected in July, September, and November 2006 were significantly higher (23.7%, p<0.01) than those in the corresponding months of 2007 (9.9%). The diversity in Borrelia genospecies between sites was significantly different (p<0.001). Habitat structure (tree cover) was an effective discriminant parameter in the determination of Borrelia infection risk, as measured by the proportion of nymphal ticks infected with B. burgdorferi s.l. Thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities. The study shows that Borrelia-infected ticks are present in many forest and dune areas in The Netherlands and suggests that in such biotopes, which are used for a wide variety of recreational activities, the infection risk is high.

Transfer of sulfamethazine from contaminated beeswax to honey.

A liquid chromatographic tandem mass spectrometric method for the determination of sulfa drugs in beeswax was developed. When performing residue control on beeswax intended for the fabrication of wax foundations, residues of sulfonamides were found. A migration test was set up to study whether sulfonamide-containing beeswax could lead to the contamination of honey. The higher the concentration of sulfamethazine doped in the wax, the higher was the concentration of sulfamethazine found in the honey. The maximum transfer was 15.6, 56.9, and 29.5% of the initial amount spiked in the wax foundation. In a second experiment, the percentage of sulfamethazine migrating from medicated winter feed to beeswax in relation to the concentration in the syrup and the contact time was studied. The maximum transfer of sulfamethazine from medicated sucrose syrup to beeswax was 3.1%.