Katsuhisa Takumi

Circulation of four Anaplasma phagocytophilum ecotypes in Europe

BackgroundAnaplasma 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.MethodsThe 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.ResultsDNA 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.ConclusionFour 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.

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.

Circumstantial evidence for an increase in the total number and activity of borrelia-infected ixodes ricinus in the Netherlands

BackgroundBetween 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.MethodsHistoric 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.ResultsLong-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.ConclusionsTogether, 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.

Geodemographic analysis of Borrelia burgdorferi sensu lato using the 5S-23S rDNA spacer region.

BACKGROUND Lyme borreliosis is the predominant tick-borne disease in the Northern hemisphere, with considerable heterogeneity in clinical manifestations. Here, we evaluated one genetic marker for its use in population genetic based analysis. For that we collected molecular and epidemiological records of Borrelia burgdorferi sensu lato isolates from ticks, animals and humans at various sites in The Netherlands and worldwide. METHODS The 5S-23S rDNA (rrfA-rrlB) intergenic spacer region (IGS) from 291 Dutch Borrelia positive ticks was sequenced and compared to Borrelia sequences from GenBank. We estimated several population genetic measures to test the neutrality of the marker. We also assessed the ability of this marker to discriminate between Eurasian Borrelieae at a finer geographical resolution, and to detect population expansion per genospecies. RESULTS The most prevalent genospecies in The Netherlands was Borrelia afzelii, whereas Borrelia garinii, B. burgdorferi sensu stricto, Borrelia spielmanii and Borrelia valaisiana were found less frequently. The result of the Ewens-Watterson-Slatkin test was consistent with neutral selection of IGS region. Estimated pairwise fixation indices (Fst) were significantly different from zero between The Netherlands, the rest of Europe, Russia and Asia for B. afzelii and Borrelia garinii. Estimated Fus Fs were significantly negative for B. afzelii and B. garinii. CONCLUSIONS At least seven B. burgdorferi s.l. genospecies circulate in Ixodes ricinus population in The Netherlands. The population genetic analyses of IGS region can resolve subpopulations within a genospecies and detect a large excess of rare genetic variants at the genospecies level. A genetic trace of population expansion for B. afzelii and B. garinii is consistent with the reported increase in Lyme borreliosis incidence in European countries.

Ability to cause erythema migrans differs between Borrelia burgdorferi sensu lato isolates

BackgroundLyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato. The variety of characteristic and non-specific clinical manifestations is partially explained by its genetic diversity. We investigated the ability of B. burgdorferi sl isolates to cause erythema migrans.MethodsThe genetic constellation of isolates from ticks was compared to isolates found in erythema migrans. PCR and sequence analysis was performed on the plasmid-encoded ospC and the chromosomal 5S-23S rDNA spacer region (IGS).ResultsSeven different B. burgdorferi sl genospecies were identified in 152 borrelia isolates from ticks and erythema migrans biopsies. B afzelii (51%) and B. garinii (27%) were the most common in ticks. From the 44 sequences obtained from erythema migrans samples 42 were B. afzelii, one B. garinii and one B. bavariensis. Significant associations with erythema migrans formation were found for four IGS and two ospC types. Five from 45 ospC types were associated with more than one genospecies.ConclusionsB. burgdorferi sl isolates differ in their propensity to cause erythema migrans. These differences were also found within genospecies. In other words, although B. afzelii was mostly associated with erythema migrans, some B. afzelii isolates had a low ability to cause erythema migrans. Our data further support the occurrence of plasmid exchange between borrelia genospecies under natural conditions.

Transmission risk of human trichinellosis

Trichinella is a food-borne parasitic zoonoses and human cases are still reported in Europe mainly due to the consumption of pig meat originating from small backyard farms. Infections originating from industrialized pig farming have not been reported for decades in Europe, due to control measures to prevent the transmission of Trichinella from wildlife by indoor housing and good management practices. Therefore, risk-based monitoring programs might replace individual carcass control in industrialized pig farming as described in EU legislation SANCO 2075/2005. Transmission of Trichinella species between wildlife and the risk that may pose to humans via consumption of contaminated pork meat has not been studied quantitatively. One pathway by which human trichinellosis can occur is the rat-pig-human route. To evaluate the transmission risk though this pathway the dose responses of rat, pig, and human were studied. Experimental T. spiralis infection was performed in rats with doses of as few as 10 parasites and the data set was analysed using a newly developed dose response model that describes larvae per gram (LPG). Experimental T. spiralis infection in pig was analysed in a similar way. Furthermore nine published outbreaks of human trichinellosis were analysed to determine the dose response in humans. The risk of human trichinellosis via the rat-pig-human transmission was simulated by the Monte Carlo method. A pair of female and male parasites representing the lowest infection pressure from the environment, led to the probability of human trichinellosis by consumption of 100g of raw pork meat equal to 5% via the studied rat-pig-human pathway. In the absence of rodent control near the farm, a low infection pressure from wildlife presents a relatively high risk of human trichinellosis via consumption of uncooked pork meat.

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.

Population-based analyses of Giardia duodenalis is consistent with the clonal assemblage structure

BackgroundGiardia duodenalis is a common protozoan parasite of humans and animals. Genetic characterization of single loci indicates the existence of eight groups called assemblages, which differ in their host distribution. Molecular analyses challenged the idea that G. duodenalis is a strictly clonal diplomonad by providing evidence of recombination within and between assemblages. Particularly, inter-assemblage recombination events would complicate the interpretation of multi-locus genotyping data from field isolates: where is a host infected with multiple Giardia genotypes or with a single, recombined Giardia genotype.MethodsPopulation genetic analyses on the single and multiple-locus level on an extensive dataset of G. duodenalis isolates from humans and animals were performed.ResultsOur analyses indicate that recombination between isolates from different assemblages are apparently very rare or absent in the natural population of Giardia duodenalis. At the multi-locus level, our statistical analyses are more congruent with clonal reproduction and can equally well be explained with the presence of multiple G. duodenalis genotypes within one field isolate.ConclusionsWe conclude that recombination between G. duodenalis assemblages is either very rare or absent. Recombination between genotypes from the same assemblage and genetic exchange between the nuclei of a single cyst needs further investigation.

Transmission dynamics of Echinococcus multilocularis; its reproduction number, persistence in an area of low rodent prevalence, and effectiveness of control

On the basis of high prevalences of Echinococcus multilocularis in the growing fox populations in Central Europe, its total biomass may have increased significantly in the past 20 years. E. multilocularis is now also found in areas outside the known endemic area in Central Europe. Therefore, E. multilocularis, the causative agent of a serious parasitic zoonosis, might be of major concern for public health and a challenge to control. Some experimental field trials to control E. multilocularis using an anti-worm drug reduced parasite burden in a contaminated region during the control campaign, but failed to eradicate the parasite completely. It was our aim to develop a mathematical model describing the biomass of egg, larval, and adult worm stages of the E. multilocularis life-cycle, and simulate a hypothetical control campaign. Additionally, we derived the reproduction number of this parasite and explored conditions for the persistence of the parasites life-cycle. Our model shows that while control campaigns rapidly reduce the worm burden in the definitive host, and consequently eggs in the environment, the pool of larvae in the intermediate host remains large. The parasites life-cycle persists in a region where prevalence in the intermediate host is low (approximately 1%). Therefore, we conclude that the parasite is likely to re-emerge if control is discontinued on the basis of reduced worm population. Continued treatment of the definitive host is required to eradicate the larval stage of the parasite from the intermediate host population.

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.