Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

Occurrence of Rickettsia spp., Hantaviridae, Bartonella spp. and Leptospira spp. in European Moles (Talpa europaea) from the Netherlands.

The European mole (Talpa europaea) has a widespread distribution throughout Europe. However, little is known about the presence of zoonotic pathogens in European moles. We therefore tested 180 moles from the middle and the south of the Netherlands by (q)PCR for the presence of multiple (tick-borne) zoonotic pathogens. Spotted fever Rickettsia was found in one (0.6%), Leptospira spp. in three (1.7%), Bartonella spp. in 69 (38.3%) and Hantaviridae in 89 (49.4%) of the 180 moles. Infections with Anaplasma phagocytophilum, Babesia spp., Neoehrlichia mikurensis, Borrelia spp., Spiroplasma spp. and Francisella tularensis were not found. In addition, in a subset of 35 moles no antibodies against Tick-borne encephalitis virus were found. The obtained sequences of Bartonella spp. were closely related to Bartonella spp. sequences from moles in Spain and Hungary. The Hantaviridae were identified as the mole-borne Nova virus, with high sequence similarity to sequences from other European countries, and Bruges virus. Though the zoonotic risk from moles appears limited, our results indicate that these animals do play a role in multiple host-pathogen cycles.

Zoonotic Pathogens in Eurasian Beavers (Castor fiber) in the Netherlands.

Successful repopulation programs of Eurasian beavers (Castor fiber) have resulted in an increase in beaver populations throughout Europe. This may be of public health relevance because beavers can host multiple zoonotic pathogens. From March 2018 to March 2020, opportunistic testing of dead beavers was performed for hepatitis E virus, orthohantavirus, Anaplasma phagocytophilum, Bartonella spp., extended-spectrum-betalactamase or AmpC (ESBL/AmpC-)-producing Enterobacteriaceae, Francisella tularensis, Leptospira spp., Neoehrlichia mikurensis, Babesia spp., Echinococcus multilocularis, Toxoplasma gondii, and Trichinella spp. From the 24 beavers collected, three zoonotic pathogens were detected. One beaver was positive for T. gondii, one was positive for ESBL/AmpC-producing Enterobacteriaceae, and one was positive for N. mikurensis. The latter finding indicates that beavers can be bitten by Ixodes ricinus and be exposed to tick-borne pathogens. The detected ESBL/AmpC-gene was blaCMY-2 in an Escherichia coli ST6599. The findings suggest that the role of beavers in the spread of zoonotic diseases in the Netherlands is currently limited.

Seoul Virus in Pet and Feeder Rats in The Netherlands.

Seoul virus (SEOV) is a zoonotic orthohantavirus carried by rats. In humans, SEOV can cause hemorrhagic fever with renal syndrome. Recent human SEOV cases described in the USA, United Kingdom, France and the Netherlands were associated with contact with pet or feeder rats. The prevalence of SEOV in these types of rats is unknown. We collected 175 pet and feeder rats (Rattus norvegicus) from private owners, ratteries and commercial breeders/traders in the Netherlands. Lung tissue of the rats was tested using a SEOV real-time RT-qPCR and heart fluid was tested for the presence of antibodies against SEOV. In all three investigated groups, RT-qPCR-positive rats were found: in 1/29 rats from private owners (3.6%), 2/56 rats from ratteries (3.4%) and 11/90 rats from commercial breeders (12.2%). The seroprevalence was largely similar to the prevalence calculated from RT-qPCR-positive rats. The SEOV sequences found were highly similar to sequences previously found in domesticated rats in Europe. In conclusion, SEOV is spread throughout different populations of domesticated rats.

Circulation of Babesia Species and Their Exposure to Humans through Ixodes Ricinus.

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borrelia burgdorferi sensu lato and Neoehrlichia mikurensis in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of B. microti and B. venatorum in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for B. divergens and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.

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.

Genospecies of Borrelia burgdorferi sensu lato detected in 16 mammal species and questing ticks from northern Europe.

Lyme borreliosis is the most common vector-borne zoonosis in the northern hemisphere, and the pathogens causing Lyme borreliosis have distinct, incompletely described transmission cycles involving multiple host groups. The mammal community in Fennoscandia differs from continental Europe, and we have limited data on potential competent and incompetent hosts of the different genospecies of Borrelia burgdorferi sensu lato (sl) at the northern distribution ranges where Lyme borreliosis is emerging. We used qPCR to determine presence of B. burgdorferi sl in tissue samples (ear) from 16 mammalian species and questing ticks from Norway, and we sequenced the 5S-23 S rDNA intergenic spacer region to determine genospecies from 1449 qPCR-positive isolates obtaining 423 sequences. All infections coming from small rodents and shrews were linked to the genospecies B. afzelii, while B. burgdorferi sensu stricto (ss) was only found in red squirrels (Sciurus vulgaris). Red squirrels were also infected with B. afzelii and B. garinii. There was no evidence of B. burgdorferi sl infection in moose (Alces alces), red deer (Cervus elaphus) or roe deer (Capreolus capreolus), confirming the role of cervids as incompetent hosts. In infected questing ticks in the two western counties, B. afzelii (67% and 75%) dominated over B. garinii (27% and 21%) and with only a few recorded B. burgdorferi ss and B. valaisiana. B. burgdorferi ss were more common in adult ticks than in nymphs, consistent with a reservoir in squirrels. Our study identifies potential competent hosts for the different genospecies, which is key to understand transmission cycles at high latitudes of Europe.

Infection prevalence and ecotypes of Anaplasma phagocytophilum in moose Alces alces, red deer Cervus elaphus, roe deer Capreolus capreolus and Ixodes ricinus ticks from Norway.

BACKGROUND: The geographical expansion of the tick Ixodes ricinus in northern Europe is a serious concern for animal and human health. The pathogen Anaplasma phagocytophilum is transmitted by ticks and causes emergences of tick-borne fever (anaplasmosis) in livestock. The transmission dynamics of the different ecotypes of A. phagocytophilum in the ecosystems is only partly determined. Red deer and roe deer contribute to circulation of different ecotypes of A. phagocytophilum in continental Europe, while the role of moose for circulation of different ecotypes is not fully established but an important issue in northern Europe. METHODS: We determined infection prevalence and ecotypes of A. phagocytophilum in moose (n = 111), red deer (n = 141), roe deer (n = 28) and questing ticks (n = 9241) in Norway. RESULTS: As previously described, red deer was exclusively linked to circulation of ecotype I, while roe deer was exclusively linked to circulation of ecotype II. Surprisingly, we found 58% ecotype I (n = 19) and 42% of ecotype II (n = 14) in moose. Both ecotypes were found in questing ticks in areas with multiple cervid species present, while only ecotype I was found in ticks in a region with only red deer present. Hence, the geographical distribution of ecotypes in ticks followed the distribution of cervid species present in a given region and their link to ecotype I and II. CONCLUSIONS: Moose probably function as reservoirs for both ecotype I and II, indicating that the ecotypes of A. phagocytophilum are not entirely host-specific and have overlapping niches. The disease hazard depends also on both host abundance and the number of immature ticks fed by each host. Our study provides novel insights in the northern distribution and expansion of tick-borne fever.

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.