Prevalence and predictors of vector-borne pathogens in Dutch roe deer.

BACKGROUND: The main objective of this study was to determine the prevalence of nine vector-borne pathogens or pathogen genera in roe deer (Capreolus capreolus) in the Netherlands, and to identify which host variables predict vector-borne pathogen presence in roe deer. The host variables examined were the four host factors 'age category', 'sex', 'nutritional condition' and 'health status', as well as 'roe deer density'. METHODS: From December 2009 to September 2010, blood samples of 461 roe deer were collected and analysed by polymerase chain reaction (PCR) for the presence of genetic material from Anaplasma phagocytophilum, Bartonella spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.), Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia spp., and epizootic haemorrhagic disease virus (EHDV), and by commercial enzyme-linked immunosorbent assay (ELISA) for antibodies against bluetongue virus (BTV). The possible associations of host factors and density with pathogen prevalence and co-infection, and in the case of A. phagocytophilum with bacterial load, were assessed using generalized linear modelling. RESULTS AND CONCLUSION: Analysis revealed the following prevalence in roe deer: A. phagocytophilum 77.9%, Bartonella spp. 77.7%, Babesia spp. 17.4%, Rickettsia spp. 3.3%, B. burgdorferi sensu lato 0.2%. Various co-infections were found, of which A. phagocytophilum and Bartonella spp. (49.7% of infected roe deer) and A. phagocytophilum, Bartonella spp. and Babesia spp. (12.2% of infected roe deer) were the most common. Anaplasma phagocytophilum, Babesia spp., and co-infection prevalence were significantly higher in calves than in adult roe deer, whereas the prevalence of Bartonella spp. was lower in roe deer in good nutritional condition than in deer in poor nutritional condition. Local roe deer density was not associated with pathogen presence. The high prevalence of A. phagocytophilum, Bartonella spp., and Babesia spp. is evidence for the role of roe deer as reservoirs for these pathogens. Additionally, the results suggest a supportive role of roe deer in the life-cycle of Rickettsia spp. in the Netherlands.

Dual-Use Quickscan: A Web-Based Tool to Assess the Dual-Use Potential of Life Science Research.

Research on pathogenic organisms is crucial for medical, biological and agricultural developments. However, biological agents as well as associated knowledge and techniques, can also be misused, for example for the development of biological weapons. Potential malicious use of well-intended research, referred to as "dual-use research", poses a threat to public health and the environment. There are various international resources providing frameworks to assess dual-use potential of the research concerned. However, concrete instructions for researchers on how to perform a dual-use risk assessment is largely lacking. The international need for practical dual-use monitoring and risk assessment instructions, in addition to the need to raise awareness among scientists about potential dual-use aspects of their research has been identified over the last years by the Netherlands Biosecurity Office, through consulting national and international biorisk stakeholders. We identified that Biorisk Management Advisors and researchers need a practical tool to facilitate a dual-use assessment on their specific research. Therefore, the Netherlands Biosecurity Office developed a web-based Dual-Use Quickscan (www.dualusequickscan.com), that can be used periodically by researchers working with microorganisms to assess potential dual-use risks of their research by answering a set of fifteen yes/no questions. The questions for the tool were extracted from existing international open resources, and categorized into three themes: characteristics of the biological agent, knowledge and technology about the biological agent, and consequences of misuse. The results of the Quickscan provide the researcher with an indication of the dual-use potential of the research and can be used as a basis for further discussions with a Biorisk Management Advisor. The Dual-Use Quickscan can be embedded in a broader system of biosafety and biosecurity that includes dual-use monitoring and awareness within organizations. Increased international attention to examine pathogens with pandemic potential has been enhanced by the current COVID-19 pandemic, hence monitoring of dual-use potential urgently needs to be encouraged.

The Vulnerability Scan, a Web Tool to Increase Institutional Biosecurity Resilience.

The importance of vigilance within organizations working with high-risk biological material receives increasing attention. However, an in-depth and comprehensive tool, dedicated to increase awareness of potential risks and to assess an organization's current biosecurity vulnerabilities, has not been available yet. We developed the "Biosecurity Vulnerability Scan," a web tool that identifies biosecurity gaps in an organization based on eight biosecurity pillars of good practice. Although the tool aims primarily to assist biosafety and biosecurity officers, it can also be useful to researchers working with dangerous pathogens, their principal investigators, management, or those responsible for security issues in the life sciences. Results are only stored locally and are provided in an "overview report," which includes information on relevant risks and control measures. This can support well-substantiated decision-making on strengthening biosecurity measures within a specific organization. With this article, we aim to support institutes to increase their overall security resilience and to improve institutional biosecurity in particular by providing practical recommendations. The Biosecurity Vulnerability Scan is available at www.biosecurityvulnerabilityscan.nl.

Detection of Anaplasma phagocytophilum, Candidatus Neoehrlichia sp., Coxiella burnetii and Rickettsia spp. in questing ticks from a recreational park, Portugal.

Tick-borne agents with medical relevance have been recorded in Portugal but little is known about their occurrence in urban outdoor leisure areas. This study aimed to investigate ticks and tick-borne agents in three public parks of Lisbon's metropolitan area. A total of 234 questing ticks belonging to eight species were found in Parque Florestal de Monsanto (PFM). Ixodes ventalloi represented 40% of collections. Mitochondrial genes confirmed Ixodes morphological identification, evidencing the intraspecific variability of I. ricinus and particularly I. frontalis populations. Regarding tick-borne agents, Rickettsia massiliae DNA were found in 21 (9.0%) ticks, Coxiella burnetii in 15 (6.4%), Anaplasma phagocytophilum in five (2.1%), an agent closely related to Candidatus Neoehrlichia mikurensis in two (0.9%), Rickettsia sibirica mongolitimonae and Rickettsia monacensis each in one (0.4%). Active enzootic cycles were suggested for these agents by the detection of positives in different time periods. Five tick species were founded with C. burnetii, including I. ventalloi which seems to be a new association record. This tick was also the only species found positive for A. phagocytophilum and the Candidatus Neoehrlichia mikurensis-like agent. Two A. phagocytophilum variants were detected in PFM, one of them representing a potentially new ecotype already found in I. ventalloi from another Portuguese area. To the authors´ knowledge, this is also the first report of such a Candidatus Neoehrlichia mikurensis-like microorganism. These data show an interesting diversity of ticks and tick-borne agents with potential public health relevance in PFM, an urban recreational area commonly frequented by humans and their pets.

A lifelong study of a pack Rhodesian ridgeback dogs reveals subclinical and clinical tick-borne Anaplasma phagocytophilum infections with possible reinfection or persistence.

BACKGROUND: Various tick-borne infections often occur without specific clinical signs and are therefore notoriously hard to diagnose separately in veterinary practice. Longitudinal studies over multiple tick seasons performing clinical, serological and molecular investigations in parallel, may elucidate the relationship between infection and disease. In this regard, six related Rhodesian Ridgeback dogs living as a pack became subject of lifetime studies due to ongoing tick infestations and recurring clinical problems. Blood samples for diagnostic tests were obtained throughout the years 2000 to 2009. METHODS: Data collected from clinical observations, hemograms, serology and detection of Anaplasma phagocytophilum, either by microscopy or by DNA amplification and typing, were placed in a time line. This dataset essentially presents as a prospective study enabling the association of the Anaplasma infections with occurring disease. RESULTS: All six dogs were infected, and two of them developed particular clinical symptoms that could be associated with Anaplasma infections over time. More specifically, episodes of general malaise with fever and purpura with thrombocytopenia and bacterial inclusions in granulocytes, were found concurrently with Anaplasma DNA and specific antibodies in peripheral blood samples. DNA from A. phagocytophilum variant 4 (of 16S rRNA) was found in multiple and sequential samples. DNA-sequences from variant 1 and the human granulocytic ehrlichiosis (HGE) agent were also detected. CONCLUSIONS: In this study two lifelong cases of canine anaplasmosis (CGA) are presented. The data show that dogs can be naturally infected concurrently with A. phagocytophilum variant 1, variant 4 and the HGE agent. The ongoing presence of specific antibodies and Anaplasma DNA in one dog indicates one year of persisting infection. Treatment with doxycycline during recurring clinical episodes in the other dog resulted in transient clinical improvement and subsequent disappearance of specific antibodies and DNA suggesting that re-infection occurred.

Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated Neorickettsia sp. in Europe.

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.

Pathogen communities of songbird-derived ticks in Europe's low countries.

BACKGROUND: Birds play a major role in the maintenance of enzootic cycles of pathogens transmitted by ticks. Due to their mobility, they affect the spatial distribution and abundance of both ticks and pathogens. In the present study, we aim to identify members of a pathogen community [Borrelia burgdorferi (s.l.), B. miyamotoi, 'Ca. Neoehrlichia mikurensis', Anaplasma phagocytophilum and Rickettsia helvetica] in songbird-derived ticks from 11 locations in the Netherlands and Belgium (2012-2014). RESULTS: Overall, 375 infested songbird individuals were captured, belonging to 35 species. Thrushes (Turdus iliacus, T. merula and T. philomelos) were trapped most often and had the highest mean infestation intensity for both Ixodes ricinus and I. frontalis. Of the 671 bird-derived ticks, 51% contained DNA of at least one pathogenic agent and 13% showed co-infections with two or more pathogens. Borrelia burgdorferi (s.l.) DNA was found in 34% of the ticks of which majority belong to so-called avian Borrelia species (distribution in Borrelia-infected ticks: 47% B. garinii, 34% B. valaisiana, 3% B. turdi), but also the mammal-associated B. afzelii (16%) was detected. The occurrence of B. miyamotoi was low (1%). Prevalence of R. helvetica in ticks was high (22%), while A. phagocytophilum and 'Ca. N. mikurensis' prevalences were 5% and 4%, respectively. The occurrence of B. burgdorferi (s.l.) was positively correlated with the occurrence of 'Ca. N. mikurensis', reflecting variation in susceptibility among birds and/or suggesting transmission facilitation due to interactions between pathogens. CONCLUSIONS: Our findings highlight the contribution of European songbirds to co-infections in tick individuals and consequently to the exposure of humans to multiple pathogens during a tick bite. Although poorly studied, exposure to and possibly also infection with multiple tick-borne pathogens in humans seems to be the rule rather than the exception.

Detection of Coxiella burnetii in Ambient Air after a Large Q Fever Outbreak.

One of the largest Q fever outbreaks ever occurred in the Netherlands from 2007-2010, with 25 fatalities among 4,026 notified cases. Airborne dispersion of Coxiella burnetii was suspected but not studied extensively at the time. We investigated temporal and spatial variation of Coxiella burnetii in ambient air at residential locations in the most affected area in the Netherlands (the South-East), in the year immediately following the outbreak. One-week average ambient particulate matter < 10 µm samples were collected at eight locations from March till September 2011. Presence of Coxiella burnetii DNA was determined by quantitative polymerase chain reaction. Associations with various spatial and temporal characteristics were analyzed by mixed logistic regression. Coxiella burnetii DNA was detected in 56 out of 202 samples (28%). Airborne Coxiella burnetii presence showed a clear seasonal pattern coinciding with goat kidding. The spatial variation was significantly associated with number of goats on the nearest goat farm weighted by the distance to the farm (OR per IQR: 1.89, CI: 1.31-2.76). We conclude that in the year after a large Q fever outbreak, temporal variation of airborne Coxiella burnetii is suggestive to be associated with goat kidding, and spatial variation with distance to and size of goat farms. Aerosol measurements show to have potential for source identification and attribution of an airborne pathogen, which may also be applicable in early stages of an outbreak.

Presence of zoonotic agents in engorged ticks and hedgehog faeces from Erinaceus europaeus in (sub) urban areas.

BACKGROUND: European hedgehogs (Erinaceus europaeus) are hosts for Ixodes hexagonus and I. ricinus ticks, which are vectors for zoonotic microorganisms. In addition, hedgehogs may carry several enteric zoonoses as well. It is unclear to what extent a presence of pathogens in hedgehogs poses a risk to public health, as information on the presence of zoonotic agents in hedgehogs in urban areas is relatively scarce. METHODS: Engorged ticks and hedgehog faeces were collected from rehabilitating hedgehogs. Ticks were screened individually for presence of Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis using PCR-based assays. Faecal samples were screened for presence of Campylobacter, Salmonella, Giardia, Cryptosporidium, and extended-spectrum cephalosporin-resistant-Escherichia coli (ESC)-resistant E. coli, using both culture-based and PCR-based methods. RESULTS: Anaplasma phagocytophilum and Borrelia genospecies B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi sensu stricto were detected in both I. hexagonus and I. ricinus ticks. Despite their widespread distribution in the Netherlands, B. miyamotoi and Candidatus N. mikurensis were not detected in collected ticks. Analysis of hedgehog faecal samples revealed the presence of Salmonella enterica subspecies enterica and Campylobacter jejuni. In addition, ESC-resistant E. coli were observed in high prevalence in faecal samples, but no Shiga-toxin producing-E.coli were detected. Finally, potentially zoonotic protozoan parasites were observed in hedgehog faecal samples as well, including Giardia duodenalis assemblage A, Cryptosporidium parvum subtypes IIaA17G1R1 and IIcA5G3, and C. hominis subtype IbA10G2. CONCLUSIONS: European hedgehogs in (sub)urban areas harbor a number of zoonotic agents, and therefore may contribute to the spread and transmission of zoonotic diseases. The relatively high prevalence of B. burgdorferi s.l. and A. phagocytophilum in engorged ticks, suggests that hedgehogs contribute to their enzootic cycles in (sub)urban areas. To what extent can hedgehogs maintain the enteric zoonotic agents in natural cycles, and the role of (spill-back from) humans remains to be investigated.

Vertical transmission of Bartonella schoenbuchensis in Lipoptena cervi.

BACKGROUND: Lipoptena cervi (Diptera: Hippoboscidae) is a hematophagous ectoparasite of cervids, which is considered to transmit pathogens between animals and occasionally to humans. The principal life stage that is able to parasitize new hosts is a winged ked that just emerged from a pupa. To facilitate efficient transmission of pathogens between hosts, vertical transmission from female deer keds to their offspring is necessary. We investigated vertical transmission of several vector-borne pathogens associated with cervids. METHODS: Deer keds from several locations in Hungary were collected between 2009 and 2012. All life stages were represented: winged free-ranging adults, wingless adults collected from Capreolus capreolus and Cervus elaphus, developing larvae dissected from gravid females, and fully developed pupae. The presence of zoonotic pathogens was determined using qPCR or conventional PCR assays performed on DNA lysates. From the PCR-positive lysates, a gene fragment was amplified and sequenced for confirmation of pathogen presence, and/or pathogen species identification. RESULTS: DNA of Bartonella schoenbuchensis was found in wingless males (2%) and females (2%) obtained from Cervus elaphus, dissected developing larvae (71%), and free-ranging winged males (2%) and females (11%). DNA of Anaplasma phagocytophilum and Rickettsia species was present in L. cervi adults, but not in immature stages. DNA of Candidatus Neoehrlichia mikurensis was absent in any of the life stages of L. cervi. CONCLUSIONS: B. schoenbuchensis is transmitted from wingless adult females to developing larvae, making it very likely that L. cervi is a vector for B. schoenbuchensis. Lipoptena cervi is probably not a vector for A. phagocytophilum, Rickettsia species, and Candidatus N. mikurensis.

A probably minor role for land-applied goat manure in the transmission of Coxiella burnetii to humans in the 2007-2010 Dutch Q fever outbreak.

In 2007, Q fever started to become a major public health problem in the Netherlands, with small ruminants as most probable source. In order to reduce environmental contamination, control measures for manure were implemented because of the assumption that manure was highly contaminated with Coxiella burnetii. The aims of this study were 1) to clarify the role of C. burnetii contaminated manure from dairy goat farms in the transmission of C. burnetii to humans, 2) to assess the impact of manure storage on temperature profiles in dunghills, and 3) to calculate the decimal reduction time of the Nine Mile RSA 493 reference strain of C. burnetii under experimental conditions in different matrices. For these purposes, records on distribution of manure from case and control herds were mapped and a potential relation to incidences of human Q fever was investigated. Additionally, temperatures in two dunghills were measured and related to heat resistance of C. burnetii. Results of negative binomial regression showed no significant association between the incidence of human Q fever cases and the source of manure. Temperature measurements in the core and shell of dunghills on two farms were above 40°C for at least ten consecutive days which would result in a strong reduction of C. burnetii over time. Our findings indicate that there is no relationship between incidence of human Q fever and land applied manure from dairy goat farms with an abortion wave caused by C. burnetii. Temperature measurements in dunghills on two farms with C. burnetii shedding dairy goat herds further support the very limited role of goat manure as a transmission route during the Dutch human Q fever outbreak. It is very likely that the composting process within a dunghill will result in a clear reduction in the number of viable C. burnetii.

Smooth incidence maps give valuable insight into Q fever outbreaks in The Netherlands.

From 2007 through 2009, The Netherlands faced large outbreaks of human Q fever. Control measures focused primarily on dairy goat farms because these were implicated as the main source of infection for the surrounding population. However, in other countries, outbreaks have mainly been associated with non-dairy sheep and The Netherlands has many more sheep than goats. Therefore, a public discussion arose about the possible role of non-dairy (meat) sheep in the outbreaks. To inform decision makers about the relative importance of different infection sources, we developed accurate and high-resolution incidence maps for detection of Q fever hot spots. In the high incidence area in the south of the country, full postal codes of notified Q fever patients with onset of illness in 2009, were georeferenced. Q fever cases (n = 1,740) were treated as a spatial point process. A 500 x 500 m grid was imposed over the area of interest. The number of cases and the population number were counted in each cell. The number of cases was modelled as an inhomogeneous Poisson process where the underlying incidence was estimated by 2-dimensional P-spline smoothing. Modelling of numbers of Q fever cases based on residential addresses and population size produced smooth incidence maps that clearly showed Q fever hotspots around infected dairy goat farms. No such increased incidence was noted around infected meat sheep farms. We conclude that smooth incidence maps of human notifications give valuable information about the Q fever epidemic and are a promising method to provide decision support for the control of other infectious diseases with an environmental source.

Molecular typing of Coxiella burnetii from animal and environmental matrices during Q fever epidemics in the Netherlands.

BACKGROUND: The bacterium Coxiella burnetii has caused unprecedented outbreaks of Q fever in the Netherlands between 2007 and 2010. Since 2007, over 4000 human cases have been reported, with 2354 cases in 2009 alone. Dairy goat farms were identified as most probable sources for emerging clusters of human Q fever cases in their vicinity. However, identifying individual farms as primary source for specific clusters of human cases remains a challenge, partly due to limited knowledge of the different C. burnetii strains circulating in livestock, the environment and humans. RESULTS: We used a multiplex multi-locus variable number of tandem repeats analysis (MLVA) assay to investigate the genotypic diversity of C. burnetii in different types of samples that were collected nationwide during the Dutch Q fever outbreaks between 2007 and 2010. Typing was performed on C. burnetii positive samples obtained from several independent studies investigating C. burnetii presence in animals and the environment. Six different genotypes were identified on 45 farm locations, based on sequence-confirmed estimates of repeat numbers of six MLVA markers. MLVA genotype A was observed on 38 of the 45 selected farm locations in animals and in environmental samples. CONCLUSIONS: Sequence confirmation of the numbers of tandem repeats within each locus and consensus about repeat identification is essential for accurate MLVA typing of C. burnetii. MLVA genotype A is the most common genotype in animal samples obtained from goat, sheep, and rats, as well as in environmental samples such as (aerosolized) dust, which is considered to be the major transmission route from animals via the environment to humans. The finding of a single dominant MLVA genotype in patients, the environment, and livestock complicates accurate source-finding. Pinpointing individual sources in the Netherlands requires discrimination of genotypes at a higher resolution than attained by using MLVA, as it is likely that the dominant C. burnetii MLVA type will be detected on several farms and in different patients in a particular area of interest.

Q fever: single-point source outbreak with high attack rates and massive numbers of undetected infections across an entire region.

BACKGROUND: In early 2009, a dairy-goat annex care farm in South Limburg, the Netherlands, reported 220 Coxiella burnetii-related abortions in 450 pregnant goats. These preceded human cases and occurred in a region that was Q-fever free before 2009, providing a unique quasi-experimental setting for investigating regional transmission patterns associated with a Q-fever point source. METHODS: Index-farm residents/employees, visitors, and their household contacts were traced and screened for C. burnetii. Distribution of community cases was analysed using a geographic information system. True incidence, including undetected infections, was estimated regionwide by seroprevalence in a pre- versus postoutbreak sample, and near-farm by immunoglobulin M seroprevalence in a municipal population sample. Environmental bacterial load was repeatedly measured in surface and aerosol samples. RESULTS: Serological attack rate was 92% (24/26) in index-farm residents/employees, 56% (28/50) in visitors, and 50% (7/14) in household contacts, and the clinical attack rate (ie, the proportion of persons seropositive for acute infection who also had clinical illness) was ≥ 80%. Notified symptomatic community cases (n = 253) were scattered downwind from the index farm, following a significant exposure-response gradient. Observed incidence ranged from 6.3% (0-1 km) to 0.1% (4-5 km), and remained high beyond. True incidence of infections was estimated at 2.9% regionwide, extrapolating to 8941 infections; estimated near-farm incidence was 12%. Coxiella burnetii load was high on-farm (2009), and lower off-farm (2009-2010). CONCLUSIONS: Linking a single dairy-goat farm to a human Q-fever cluster, we show widespread transmission, massive numbers of undetected infections, and high attack rates on- and off-farm, even beyond a 5-km high-risk zone. Our investigation may serve as an essential case study for risk assessment in public health and related fields such as bioterrorism response and preparedness.

Detection of Coxiella burnetii DNA in inhalable airborne dust samples from goat farms after mandatory culling.

Coxiella burnetii is thought to infect humans primarily via airborne transmission. However, air measurements of C. burnetii are sparse. We detected C. burnetii DNA in inhalable and PM10 (particulate matter with an aerodynamic size of 10 µm or less) dust samples collected at three affected goat farms, demonstrating that low levels of C. burnetii DNA are present in inhalable size fractions.

Genotypic diversity of Coxiella burnetii in the 2007-2010 Q fever outbreak episodes in The Netherlands.

The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.

Coxiella burnetii (Q fever) in Rattus norvegicus and Rattus rattus at livestock farms and urban locations in the Netherlands; could Rattus spp. represent reservoirs for (re)introduction?

The Q fever outbreak in the Netherlands in 2007-2010 prompted government interventions to reduce the human incidence by reduction of Q fever shedding at dairy goat farms. Mandatory hygiene measures were taken, including the control of animal reservoirs. It has been postulated that brown rats, through their commensal nature, form an important factor in the persistent dissemination of endemic circulating Coxiella burnetii in nature to domestic animals, livestock and humans. Here, the occurrence of C. burnetii in rats captured at different types of location during the Q fever outbreak in the Netherlands, viz. urban areas, nature areas and various types of farm has been determined. This is a first step towards the elucidation of the reservoir status of rats in veterinary and human Q fever epidemiology. C. burnetii DNA was detected in the spleen of 4.9% of the brown rats (Rattus norvegicus) and 3.0% of the black rats (Rattus rattus). Evidence for C. burnetii infection was also found in liver, kidney, lung and intestinal tissue but not in heart, brain and pancreas. C. burnetii IgGs were detected in 15.8% of the brown rats. Positive rats were collected at goat, pig, cattle and poultry farms, and urban locations; including locations outside the designated 5km "increased-risk" zones around bulk milk positive goat farms. The percentage of rat-positive locations was the highest for goat farms (50%) and cattle farms (14.3%). The presence of actively infected rats outside the lambing season and at multiple environmental settings including urban locations might suggest that rats are not merely a spill-over host due to infection by a contaminated environment but might represent true reservoirs, capable of independent maintenance of C. burnetii infection cycles and thereby contributing to spread and transmission of the pathogen. If frequent (re)introduction of C. burnetii to small ruminant farms can be caused by rats as maintenance reservoirs, mandatory wildlife control and lifelong vaccination of herds will be necessary.

Interlaboratory comparison of real-time polymerase chain reaction methods to detect Coxiella burnetii, the causative agent of Q fever.

The bacterium Coxiella burnetii, which has a wide host range, causes Q fever. Infection with C. burnetii can cause abortions, stillbirth, and the delivery of weak offspring in ruminants. Coxiella burnetii infection is zoonotic, and in human beings it can cause chronic, potentially fatal disease. Real-time polymerase chain reaction (PCR) is increasingly being used to detect the organism and to aid in diagnosis both in human and animal cases. Many different real-time PCR methods, which target different genes, have been described. To assess the comparability of the C. burnetii real-time PCR assays in use in different European laboratories, a panel of nucleic acid extracts was dispatched to 7 separate testing centers. The testing centers included laboratories from both human and animal health agencies. Each laboratory tested the samples using their in-house real-time PCR methods. The results of this comparison show that the most common target gene for real-time PCR assays is the IS1111 repeat element that is present in multiple copies in the C. burnetii genome. Many laboratories also use additional real-time PCR tests that target single-copy genes. The results of the current study demonstrate that the assays in use in the different laboratories are comparable, with general agreement of results for the panel of samples.

The use of a geographic information system to identify a dairy goat farm as the most likely source of an urban Q-fever outbreak.

BACKGROUND: A Q-fever outbreak occurred in an urban area in the south of the Netherlands in May 2008. The distribution and timing of cases suggested a common source. We studied the spatial relationship between the residence locations of human cases and nearby small ruminant farms, of which one dairy goat farm had experienced abortions due to Q-fever since mid April 2008. A generic geographic information system (GIS) was used to develop a method for source detection in the still evolving major epidemic of Q-fever in the Netherlands. METHODS: All notified Q-fever cases in the area were interviewed. Postal codes of cases and of small ruminant farms (size >40 animals) located within 5 kilometres of the cluster area were geo-referenced as point locations in a GIS-model. For each farm, attack rates and relative risks were calculated for 5 concentric zones adding 1 kilometre at a time, using the 5-10 kilometres zone as reference. These data were linked to the results of veterinary investigations. RESULTS: Persons living within 2 kilometres of an affected dairy goat farm (>400 animals) had a much higher risk for Q-fever than those living more than 5 kilometres away (Relative risk 31.1 [95% CI 16.4-59.1]). CONCLUSIONS: The study supported the hypothesis that a single dairy goat farm was the source of the human outbreak. GIS-based attack rate analysis is a promising tool for source detection in outbreaks of human Q-fever.

Adaptation of the fungal parasite Zygorhizidium planktonicum during 200 generations of growth on homogeneous and heterogeneous populations of its host, the diatom Asterionella formosa.

We followed adaptation of the chytrid parasite Zygorhizidium planktonicum during 200 generations of growth on its host, the freshwater diatom Asterionella formosa, in a serial passage experiment. Evolution of parasite fitness was assessed both on a homogenous and heterogeneous host population, consisting of respectively a single new and ten different new host strains. These 10 host strains were genetically different and also varied in their initial susceptibility to the parasite. Parasite fitness increased significantly and rapidly on the new, genetically homogenous host population, but remained unaltered during 200 generations of growth on the heterogeneous host population. Enhanced parasite fitness was the result of faster and more efficient transmission, resulting in higher values of R0 (number of secondary infections). Consequently, parasites that evolved within the uniclonal host population infected significantly more of these hosts than did their ancestors. We thus provide experimental evidence for the widely held view that host genetic diversity restricts evolution of parasites and moderates their harmful effects. Genetically uniform host populations are not only at increased risk from fungal epidemics because they all share the same susceptibility, but also because new parasite strains are able to adapt quickly to new host environments and to improve their fitness.