Screening of wild deer populations for exposure to SARS-CoV-2 in the United Kingdom, 2020-2021.

Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small.

Red and fallow deer determine the density of Ixodes ricinus nymphs containing Anaplasma phagocytophilum.

BACKGROUND: The density of Ixodes ricinus nymphs infected with Anaplasma phagocytophilum is one of the parameters that determines the risk for humans and domesticated animals to contract anaplasmosis. For this, I. ricinus larvae need to take a bloodmeal from free-ranging ungulates, which are competent hosts for A. phagocytophilum. METHODS: Here, we compared the contribution of four free-ranging ungulate species, red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), and wild boar (Sus scrofa), to A. phagocytophilum infections in nymphs. We used a combination of camera and live trapping to quantify the relative availability of vertebrate hosts to questing ticks in 19 Dutch forest sites. Additionally, we collected questing I. ricinus nymphs and tested these for the presence of A. phagocytophilum. Furthermore, we explored two potential mechanisms that could explain differences between species: (i) differences in larval burden, which we based on data from published studies, and (ii) differences in associations with other, non-competent hosts. RESULTS: Principal component analysis indicated that the density of A. phagocytophilum-infected nymphs (DIN) was higher in forest sites with high availability of red and fallow deer, and to a lesser degree roe deer. Initial results suggest that these differences are not a result of differences in larval burden, but rather differences in associations with other species or other ecological factors. CONCLUSIONS: These results indicate that the risk for contracting anaplasmosis in The Netherlands is likely highest in the few areas where red and fallow deer are present. Future studies are needed to explore the mechanisms behind this association.

A combined transcriptomic approach to identify candidates for an anti-tick vaccine blocking B. afzelii transmission.

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

Tripartite Interactions among Ixodiphagus hookeri, Ixodes ricinus and Deer: Differential Interference with Transmission Cycles of Tick-Borne Pathogens.

For the development of sustainable control of tick-borne diseases, insight is needed in biological factors that affect tick populations. Here, the ecological interactions among Ixodiphagus hookeri, Ixodes ricinus, and two vertebrate species groups were investigated in relation to their effects on tick-borne disease risk. In 1129 questing ticks, I. hookeri DNA was detected more often in I. ricinus nymphs (4.4%) than in larvae (0.5%) and not in adults. Therefore, we determined the infestation rate of I. hookeri in nymphs from 19 forest sites, where vertebrate, tick, and tick-borne pathogen communities had been previously quantified. We found higher than expected co-occurrence rates of I. hookeri with deer-associated Anaplasma phagocytophilum, and lower than expected rates with rodent-associated Borrelia afzelii and Neoehrlichia mikurensis. The prevalence of I. hookeri in nymphs varied between 0% and 16% and was positively correlated with the encounter probability of ungulates and the densities of all life stages of I. ricinus. Lastly, we investigated the emergence of I. hookeri from artificially fed, field-collected nymphs. Adult wasps emerged from seven of the 172 fed nymphs. From these observations, we inferred that I. hookeri is parasitizing I. ricinus larvae that are feeding on deer, rather than on rodents or in the vegetation. Since I. hookeri populations depend on deer abundance, the main propagation host of I. ricinus, these wasps have no apparent effect on tick populations. The presence of I. hookeri may directly interfere with the transmission cycle of A. phagocytophilum, but not with that of B. afzelii or N. mikurensis.

Presence of Roe Deer Affects the Occurrence of Anaplasma phagocytophilum Ecotypes in Questing Ixodes ricinus in Different Habitat Types of Central Europe.

The way in which European genetic variants of Anaplasma phagocytophilum circulate in their natural foci and which variants cause disease in humans or livestock remains thus far unclear. Red deer and roe deer are suggested to be reservoirs for some European A. phagocytophilum strains, and Ixodes ricinus is their principal vector. Based on groEL gene sequences, five A. phagocytophilum ecotypes have been identified. Ecotype I is associated with the broadest host range, including strains that cause disease in domestic animals and humans. Ecotype II is associated with roe deer and does not include zoonotic strains. In the present study, questing I. ricinus were collected in urban, pasture, and natural habitats in the Czech Republic, Germany, and Slovakia. A fragment of the msp2 gene of A. phagocytophilum was amplified by real-time PCR in DNA isolated from ticks. Positive samples were further analyzed by nested PCRs targeting fragments of the 16S rRNA and groEL genes, followed by sequencing. Samples were stratified according to the presence/absence of roe deer at the sampling sites. Geographic origin, habitat, and tick stage were also considered. The probability that A. phagocytophilum is a particular ecotype was estimated by a generalized linear model. Anaplasma phagocytophilum was identified by genetic typing in 274 I. ricinus ticks. The majority belonged to ecotype I (63.9%), 28.5% were ecotype II, and both ecotypes were identified in 7.7% of ticks. Ecotype II was more frequently identified in ticks originating from a site with presence of roe deer, whereas ecotype I was more frequent in adult ticks than in nymphs. Models taking into account the country-specific, site-specific, and habitat-specific aspects did not improve the goodness of the fit. Thus, roe deer presence in a certain site and the tick developmental stage are suggested to be the two factors consistently influencing the occurrence of a particular A. phagocytophilum ecotype in a positive I. ricinus tick.

Impact of vertebrate communities on Ixodes ricinus-borne disease risk in forest areas.

BACKGROUND: The density of questing ticks infected with tick-borne pathogens is an important parameter that determines tick-borne disease risk. An important factor determining this density is the availability of different wildlife species as hosts for ticks and their pathogens. Here, we investigated how wildlife communities contribute to tick-borne disease risk. The density of Ixodes ricinus nymphs infected with Borrelia burgdorferi (sensu lato), Borrelia miyamotoi, Neoehrlichia mikurensis and Anaplasma phagocytophilum among 19 forest sites were correlated to the encounter probability of different vertebrate hosts, determined by encounter rates as measured by (camera) trapping and mathematical modeling. RESULT: We found that the density of any tick life stage was proportional to the encounter probability of ungulates. Moreover, the density of nymphs decreased with the encounter probability of hare, rabbit and red fox. The density of nymphs infected with the transovarially-transmitted B. miyamotoi increased with the density of questing nymphs and the encounter probability of bank vole. The density of nymphs infected with all other pathogens increased with the encounter probability of competent hosts: bank vole for Borrelia afzelii and N. mikurensis, ungulates for A. phagocytophilum and blackbird for Borrelia garinii and Borrelia valaisiana. The negative relationship we found was a decrease in the density of nymphs infected with B. garinii and B. valaisiana with the encounter probability of wood mouse. CONCLUSIONS: Only a few animal species drive the densities of infected nymphs in forested areas. There, foxes and leporids have negative effects on tick abundance, and consequently on the density of infected nymphs. The abundance of competent hosts generally drives the abundances of their tick-borne pathogen. A dilution effect was only observed for bird-associated Lyme spirochetes.

Anaplasma phagocytophilum evolves in geographical and biotic niches of vertebrates and ticks.

BACKGROUND: Anaplasma phagocytophilum is currently regarded as a single species. However, molecular studies indicate that it can be subdivided into ecotypes, each with distinct but overlapping transmission cycle. Here, we evaluate the interactions between and within clusters of haplotypes of the bacterium isolated from vertebrates and ticks, using phylogenetic and network-based methods. METHODS: The presence of A. phagocytophilum DNA was determined in ticks and vertebrate tissue samples. A fragment of the groEl gene was amplified and sequenced from qPCR-positive lysates. Additional groEl sequences from ticks and vertebrate reservoirs were obtained from GenBank and through literature searches, resulting in a dataset consisting of 1623 A. phagocytophilum field isolates. Phylogenetic analyses were used to infer clusters of haplotypes and to assess phylogenetic clustering of A. phagocytophilum in vertebrates or ticks. Network-based methods were used to resolve host-vector interactions and their relative importance in the segregating communities of haplotypes. RESULTS: Phylogenetic analyses resulted in 199 haplotypes within eight network-derived clusters, which were allocated to four ecotypes. The interactions of haplotypes between ticks, vertebrates and geographical origin, were visualized and quantified from networks. A high number of haplotypes were recorded in the tick Ixodes ricinus. Communities of A. phagocytophilum recorded from Korea, Japan, Far Eastern Russia, as well as those associated with rodents had no links with the larger set of isolates associated with I. ricinus, suggesting different evolutionary pressures. Rodents appeared to have a range of haplotypes associated with either Ixodes trianguliceps or Ixodes persulcatus and Ixodes pavlovskyi. Haplotypes found in rodents in Russia had low similarities with those recorded in rodents in other regions and shaped separate communities. CONCLUSIONS: The groEl gene fragment of A. phagocytophilum provides information about spatial segregation and associations of haplotypes to particular vector-host interactions. Further research is needed to understand the circulation of this bacterium in the gap between Europe and Asia before the overview of the speciation features of this bacterium is complete. Environmental traits may also play a role in the evolution of A. phagocytophilum in ecotypes through yet unknown relationships.

The genetic diversity of Borrelia afzelii is not maintained by the diversity of the rodent hosts.

BACKGROUND: Small mammals are essential in the enzootic cycle of many tick-borne pathogens (TBP). To understand their contribution to the genetic diversity of Borrelia afzelii, the most prevalent TBP in questing Ixodes ricinus, we compared the genetic variants of B. afzelii at three distinct genetic loci. We chose two plasmid loci, dbpA and ospC, and a chromosomal one, IGS. RESULTS: While the larvae that fed on shrews (Sorex sp.) tested negative for B. afzelii, those fed on bank voles (Myodes glareolus) and wood mice (Apodemus sylvaticus) showed high infection prevalences of 0.13 and 0.27, respectively. Despite the high genetic diversity within B. afzelii, there was no difference between wood mice and bank voles in the number and types of B. afzelii haplotypes they transmit. CONCLUSIONS: The genetic diversity in B. afzelii cannot be explained by separate enzootic cycles in wood mice and bank voles.

Assessing the risk of human trichinellosis from pigs kept under controlled and non-controlled housing in Europe.

To support risk-based approach to prevent human trichinellosis, we estimated the human incidence for pigs originating from controlled and non-controlled housing, using a quantitative microbial risk assessment model for Trichinella (QMRA-T). Moreover, the effect of test sensitivity on human trichinellosis incidence from pigs from non-controlled housing was quantified. The estimated annual risk from pigs from non-controlled housing was 59,443 human trichinellosis cases without testing at meat inspection and 832 (95%CI 346-1410) cases with Trichinella testing, thus preventing 98.6% of trichinellosis cases per year by testing at meat inspection. Using the QMRA-T, a slight decrease in test sensitivity had a significant effect on the number of human trichinellosis cases from this housing type. The estimated annual risk for pigs from controlled housing was <0.002 (range 0.000-0.007) human cases with- and <0.010 (0.001-0.023) cases without Trichinella testing at meat inspection, which does not differ significantly (p = 0.2075). In practice, this means no cases per year irrespective of Trichinella testing. Thus controlled housing effectively prevents infection and Trichinella testing does not contribute to food safety for this housing type. Not testing for Trichinella requires evidence based full compliance with regulations for controlled housing.

High Prevalence of Tula Hantavirus in Common Voles in The Netherlands.

Tula virus (TULV) is a zoonotic hantavirus. Knowledge about TULV in the Netherlands is very scarce. Therefore in 2014, 49 common voles (Microtus arvalis) from a region in the south of the Netherlands, and in 2015, 241 common voles from regions in the north of the Netherlands were tested with the TULV quantitative RT-PCR. In the southern region, prevalence of TULV was 41% (20/49). In the northern regions, prevalence ranged from 12% (4/34) to 45% (17/38). Phylogenetic analysis of the obtained sequences showed that the regions fall within different clusters. Voles from the south were also tested on-site for the presence of hantavirus antibodies, but serology results were poorly associated with qRT-PCR results. These findings suggest that TULV may be more widespread than previously thought. No human TULV cases have been reported thus far in the Netherlands, but differentiation between infection by TULV or the closely related Puumala virus is not made in humans in the Netherlands, thus cases may be misdiagnosed.

Enzootic origins for clinical manifestations of Lyme borreliosis.

Both early localized and late disseminated forms of Lyme borreliosis are caused by Borrelia burgdorferi senso lato. Differentiating between the spirochetes that only cause localized skin infection from those that cause disseminated infection, and tracing the group of medically-important spirochetes to a specific vertebrate host species, are two critical issues in disease risk assessment and management. Borrelia burgdorferi senso lato isolates from Lyme borreliosis cases with distinct clinical manifestations (erythema migrans, neuroborreliosis, acrodermatitis chronica atrophicans, and Lyme arthritis) and isolates from Ixodes ricinus ticks feeding on rodents, birds and hedgehogs were typed to the genospecies level by sequencing part of the intergenic spacer region. In-depth molecular typing was performed by sequencing eight additional loci with different characteristics (plasmid-bound, regulatory, and housekeeping genes). The most abundant genospecies and genotypes in the clinical isolates were identified by using odds ratio as a measure of dominance. Borrelia afzelii was the most common genospecies in acrodermatitis patients and engorged ticks from rodents. Borrelia burgdorferi senso stricto was widespread in erythema migrans patients. Borrelia bavariensis was widespread in neuroborreliosis patients and in ticks from hedgehogs, but rare in erythema migrans patients. Borrelia garinii was the dominant genospecies in ticks feeding on birds. Spirochetes in ticks feeding on hedgehogs were overrepresented in genotypes of the plasmid gene ospC from spirochetes in erythema migrans patients. Spirochetes in ticks feeding on hedgehogs were overrepresented in genotypes of ospA from spirochetes in acrodermatitis patients. Spirochetes from ticks feeding on birds were overrepresented in genotypes of the plasmid and regulatory genes dbpA, rpoN and rpoS from spirochetes in neuroborreliosis patients. Overall, the analyses of our datasets support the existence of at least three transmission pathways from an enzootic cycle to a clinical manifestation of Lyme borreliosis. Based on the observations with these nine loci, it seems to be justified to consider the population structure of B. burgdorferi senso lato as being predominantly clonal.

Parasite to patient: A quantitative risk model for Trichinella spp. in pork and wild boar meat.

Consumption of raw or inadequately cooked pork meat may result in trichinellosis, a human disease due to nematodes of the genus Trichinella. In many countries worldwide, individual control of pig carcasses at meat inspection is mandatory but incurs high costs in relation to absence of positive carcasses from pigs reared under controlled housing. EU regulation 2015/1375 implements an alternative risk-based approach, in view of absence of positive findings in pigs under controlled housing conditions. Moreover, Codex Alimentarius guidelines for the control of Trichinella spp. in meat of suidae have been published (CAC, 2015) and used in conjunction with the OIE terrestrial Animal health code, to provide guidance to governments and industry on risk based control measures to prevent human exposure to Trichinella spp. and to facilitate international pork trade. To further support such a risk-based approach, we model the risk of human trichinellosis due to consumption of meat from infected pigs, raised under non-controlled housing and wild boar, using Quantitative Microbial Risk Assessment (QMRA) methods. Our model quantifies the distribution of Trichinella muscle larve (ML) in swine, test sensitivity at carcass control, partitioning of edible pork parts, Trichinella ML distribution in edible muscle types, heat inactivation by cooking and portion sizes. The resulting exposure estimate is combined with a dose response model for Trichinella species to estimate the incidence of human illness after consumption of infected meat. Paramater estimation is based on experimental and observational datasets. In Poland, which served as example, we estimated an average incidence of 0.90 (95%CI: 0.00-3.68) trichinellosis cases per million persons per year (Mpy) due to consumption of pork from pigs that were reared under non-controlled housing, and 1.97 (95%CI: 0.82-4.00) cases per Mpy due to consumption of wild boar. The total estimated incidence of human trichinellosis attributed to pigs from non-controlled housing and wild boar in Poland, is similar to the incidence of human trichinellosis in that country reported by EFSA. Overall, in Europe, we estimated an upper incidence limit of 5.3×10-4 cases per Mpy, or less than one predicted case of trichinellosis in the European Union every 4years, due to consumption of pork from controlled housing. Therefore, Trichinella testing of pigs under controlled housing is not adding any value to protect human health. We suggest applying our farm-to-fork QMRA model to further support decision making on the global scale.

Imbalanced presence of Borrelia burgdorferi s.l. multilocus sequence types in clinical manifestations of Lyme borreliosis.

In this study we used typing based on the eight multilocus sequence typing scheme housekeeping genes (MLST) and 5S-23S rDNA intergenic spacer (IGS) to explore the population structure of Borrelia burgdorferi sensu lato isolates from patients with Lyme borreliosis (LB) and to test the association between the B. burgdorferi s.l. sequence types (ST) and the clinical manifestations they cause in humans. Isolates of B. burgdorferi from 183 LB cases across Europe, with distinct clinical manifestations, and 257 Ixodes ricinus lysates from The Netherlands, were analyzed for this study alone. For completeness, we incorporated in our analysis also 335 European B. burgdorferi s.l. MLST profiles retrieved from literature. Borrelia afzelii and Borrelia bavariensis were associated with human cases of LB while Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana were associated with questing I. ricinus ticks. B. afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. The samples in our study belonged to 251 different STs, of which 94 are newly described, adding to the overall picture of the genetic diversity of Borrelia genospecies. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and Borrelia spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). We found six multilocus sequence types that were significantly associated to clinical manifestations in humans and five IGS haplotypes that were associated with the human LB cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs.

Molecular characterization of human Cryptosporidium spp. isolates after an unusual increase in late summer 2012.

BACKGROUND: During the late summer 2012, a number of medical microbiological laboratories (MMLs) reported an unusual increase in cases of cryptosporidiosis, a gastrointestinal infection caused by the protozoan parasites Cryptosporidium spp. Prompted by this signal, the National Institute of Public Health and the Environment (RIVM) started an epidemiological investigation into possible causes. Simultaneously, samples diagnosed at MMLs were sent to RIVM for genotyping, aiming to further identify the possible source of the increase. METHODS: Genotyping was performed by sequencing a fragment of the GP60 gene. Additional genotyping was performed on a subset of samples using six microsatellite markers. Population genetic analysis was performed using BEAST. RESULTS: The majority of the samples were typed as C. hominis, and a single GP60 genotype (IbA10G2) largely predominated. Genotyping microsatellite markers further supported the circulation of a single genetic type. Population genetic analysis with genotypes found in previous years is inconsistent with a decrease in effective population size. CONCLUSIONS: The conclusion of this finding is that the rise reflects more an overall increase and not a common source outbreak.

Hypothesis: Cryptosporidium genetic diversity mirrors national disease notification rate.

BACKGROUND: Cryptosporidiosis is a gastrointestinal disease affecting many people worldwide. Disease incidence is often unknown and surveillance of human cryptosporidiosis is installed in only a handful of developed countries. A genetic marker that mirrors disease incidence is potentially a powerful tool for monitoring the two primary human infected species of Cryptosporidium. METHODS: We used the molecular epidemiological database with Cryptosporidium isolates from ZoopNet, which currently contains more than 1400 records with their sampling nations, and the names of the host species from which the isolates were obtained. Based on 296 C. hominis and 195 C. parvum GP60 sequences from human origin, the genetic diversities of Cryptosporidium was estimated for several nations. Notified cases of human cryptosporidiosis were collected from statistics databases for only four nations. RESULTS: Genetic diversities of C. hominis were estimated in 10 nations in 5 continents, and that of C. parvum of human origin were estimated in 15 nations. Correlation with reported incidence of human cryptosporidiosis in four nations (the Netherlands, United States, United Kingdom and Australia) was positive and significant. A linear model for testing the relationship between the genetic diversity and incidence produced a significantly positive estimate for the slope (P-value < 0.05). CONCLUSIONS: The hypothesis that genetic diversity at GP60 locus mirrors notification rates of human cryptosporidiosis was not rejected based on the data presented. Genetic diversity of C. hominis and C. parvum may therefore be an independent and complementary measure for quantifying disease incidence, for which only a moderate number of stool samples from each nation are sufficient data input.

Predicting tick presence by environmental risk mapping.

Public health statistics recorded an increasing trend in the incidence of tick bites and erythema migrans (EM) in the Netherlands. We investigated whether the disease incidence could be predicted by a spatially explicit categorization model, based on environmental factors and a training set of tick absence-presence data. Presence and absence of Ixodes ricinus were determined by the blanket-dragging method at numerous sites spread over the Netherlands. The probability of tick presence on a 1 km by 1 km square grid was estimated from the field data using a satellite-based methodology. Expert elicitation was conducted to provide a Bayesian prior per landscape type. We applied a linear model to test for a linear relationship between incidence of EM consultations by general practitioners in the Netherlands and the estimated probability of tick presence. Ticks were present at 252 distinct sampling coordinates and absent at 425. Tick presence was estimated for 54% of the total land cover. Our model has predictive power for tick presence in the Netherlands, tick-bite incidence per municipality correlated significantly with the average probability of tick presence per grid. The estimated intercept of the linear model was positive and significant. This indicates that a significant fraction of the tick-bite consultations could be attributed to the I. ricinus population outside the resident municipality.

An experimental Toxoplasma gondii dose response challenge model to study therapeutic or vaccine efficacy in cats.

High numbers of Toxoplasma gondii oocysts in the environment are a risk factor to humans. The environmental contamination might be reduced by vaccinating the definitive host, cats. An experimental challenge model is necessary to quantitatively assess the efficacy of a vaccine or drug treatment. Previous studies have indicated that bradyzoites are highly infectious for cats. To infect cats, tissue cysts were isolated from the brains of mice infected with oocysts of T. gondii M4 strain, and bradyzoites were released by pepsin digestion. Free bradyzoites were counted and graded doses (1000, 100, 50, 10), and 250 intact tissue cysts were inoculated orally into three cats each. Oocysts shed by these five groups of cats were collected from faeces by flotation techniques, counted microscopically and estimated by real time PCR. Additionally, the number of T. gondii in heart, tongue and brains were estimated, and serology for anti T. gondii antibodies was performed. A Beta-Poisson dose-response model was used to estimate the infectivity of single bradyzoites and linear regression was used to determine the relation between inoculated dose and numbers of oocyst shed. We found that real time PCR was more sensitive than microscopic detection of oocysts, and oocysts were detected by PCR in faeces of cats fed 10 bradyzoites but by microscopic examination. Real time PCR may only detect fragments of T. gondii DNA without the presence of oocysts in low doses. Prevalence of tissue cysts of T. gondii in tongue, heart and brains, and anti T. gondii antibody concentrations were all found to depend on the inoculated bradyzoite dose. The combination of the experimental challenge model and the dose response analysis provides a suitable reference for quantifying the potential reduction in human health risk due to a treatment of domestic cats by vaccination or by therapeutic drug application.

Circulation of four Anaplasma phagocytophilum ecotypes in Europe.

BACKGROUND: Anaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved. METHODS: The presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses. RESULTS: DNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion. CONCLUSION: Four ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.

Increase in number of helminth species from Dutch red foxes over a 35-year period.

BACKGROUND: The red fox (Vulpes vulpes) is host to a community of zoonotic and other helminth species. Tracking their community structure and dynamics over decades is one way to monitor the long term risk of parasitic infectious diseases relevant to public and veterinary health. METHODS: We identified 17 helminth species from 136 foxes by mucosal scraping, centrifugal sedimentation/flotation and the washing and sieving technique. We applied rarefaction analysis to our samples and compared the resulting curve to the helminth community reported in literature 35 years ago. RESULTS: Fox helminth species significantly increased in number in the last 35 years (p-value <0.025). Toxascaris leonina, Mesocestoides litteratus, Trichuris vulpis and Angiostrongylus vasorum are four new veterinary-relevant species. The zoonotic fox tapeworm (E. multilocularis) was found outside the previously described endemic regions in the Netherlands. CONCLUSIONS: Helminth fauna in Dutch red foxes increased in biodiversity over the last three decades.

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.