Hidden in plain sight: novel molecular data reveal unexpected genetic diversity among paramphistome parasites (Digenea: Paramphistomoidea) of European water frogs.

Parasites might represent a helpful tool in understanding the historical dispersion and phylogeography of their hosts. In order to reveal whether the migration routes and diversification of hosts can be traceable in the genetic structures of their parasites, we investigated the diversity of paramphistomoid trematodes of Pelophylax frogs in 2 geographically distant European regions. Water frogs belonging to the genus Pelophylax represent a striking example of a species with a high variety of ecological adaptations and a rich evolutionary history. The parasites were collected from 2 Balkan endemic species, P. epeiroticus and P. kurtmuelleri, and 2 species in Slovakia, P. esculentus and P. ridibundus. While in Slovakia, Pelophylax frogs harboured 2 species, the diplodiscid Diplodiscus subclavatus and the cladorchiid Opisthodiscus diplodiscoides, only the former was recorded in the south-western Balkans. Remarkably high genetic diversity (16 unique mitochondrial cox1 haplotypes, recognized among 60 novel sequences) was observed in D. subclavatus, and subsequent phylogenetic analyses revealed a strong population-genetic structure associated with geographical distribution. We also evidenced the existence of 2 divergent D. subclavatus cox1 haplogroups in the south-western Balkans, which might be associated with the historical diversification of endemic water frogs in the regional glacial microrefugia.

Hepatozoon in Eurasian red squirrels Sciurus vulgaris, its taxonomic identity, and phylogenetic placement.

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species.

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by 'Candidatus B. rudakovii' and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

The Role of Peridomestic Animals in the Eco-Epidemiology of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an important tick-borne zoonotic agent of human granulocytic anaplasmosis (HGA). In Europe, the Ixodes ticks are the main vector responsible for A. phagocytophilum transmission. A wide range of wild animals is involved in the circulation of this pathogen in the environment. Changes in populations of vertebrates living in different ecosystems impact the ecology of ticks and the epidemiology of tick-borne diseases. In this study, we investigated four species, Western European hedgehog (Erinaceus europaeus), northern white-breasted hedgehog (Erinaceus roumanicus), Eurasian red squirrel (Sciurus vulgaris), and the common blackbird (Turdus merula), to describe their role in the circulation of A. phagocytophilum in urban and periurban ecosystems. Ten different tissues were collected from cadavers of the four species, and blood and ear/skin samples from live blackbirds and hedgehogs. Using qPCR, we detected a high rate of A. phagocytophilum: Western European hedgehogs (96.4%), northern white-breasted hedgehogs (92.9%), Eurasian red squirrels (60%), and common blackbirds (33.8%). In the groEL gene, we found nine genotypes belonging to three ecotypes; seven of the genotypes are associated with HGA symptoms. Our findings underline the role of peridomestic animals in the ecology of A. phagocytophilum and indicate that cadavers are an important source of material for monitoring zoonotic pathogens. Concerning the high prevalence rate, all investigated species play an important role in the circulation of A. phagocytophilum in municipal areas; however, hedgehogs present the greatest anaplasmosis risk for humans. Common blackbirds and squirrels carry different A. phagocytophilum variants some of which are responsible for HGA.

Hedgehogs, Squirrels, and Blackbirds as Sentinel Hosts for Active Surveillance of Borrelia miyamotoi and Borrelia burgdorferi Complex in Urban and Rural Environments.

Lyme borreliosis (LB), caused by spirochetes of the Borrelia burgdorferi sensu lato (s.l.) complex, is one of the most common vector-borne zoonotic diseases in Europe. Knowledge about the enzootic circulation of Borrelia pathogens between ticks and their vertebrate hosts is epidemiologically important and enables assessment of the health risk for the human population. In our project, we focused on the following vertebrate species: European hedgehog (Erinaceus europaeus), Northern white-breasted hedgehog (E. roumanicus), Eurasian red squirrel (Sciurus vulgaris), and Common blackbird (Turdus merula). The cadavers of accidentally killed animals used in this study constitute an available source of biological material, and we have confirmed its potential for wide monitoring of B. burgdorferi s.l. presence and genospecies diversity in the urban environment. High infection rates (90% for E. erinaceus, 73% for E. roumanicus, 91% for S. vulgaris, and 68% for T. merula) were observed in all four target host species; mixed infections by several genospecies were detected on the level of individuals, as well as in particular tissue samples. These findings show the usefulness of multiple tissue sampling as tool for revealing the occurrence of several genospecies within one animal and the risk of missing particular B. burgdorferi s.l. genospecies when looking in one organ alone.

Multiple Lineages of Usutu Virus (Flaviviridae, Flavivirus) in Blackbirds (Turdus merula) and Mosquitoes (Culex pipiens, Cx. modestus) in the Czech Republic (2016-2019).

Usutu virus (USUV) is a flavivirus (Flaviviridae: Flavivirus) of an African origin transmitted among its natural hosts (diverse species of birds) by mosquitoes. The virus was introduced multiple times to Europe where it caused mortality of blackbirds (Turdus merula) and certain other susceptible species of birds. In this study, we report detection of USUV RNA in blackbirds, Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 new Czech USUV strains from carcasses of blackbirds in cell culture. Multiple lineages (Europe 1, 2 and Africa 3) of USUV were found in blackbirds and mosquitoes in the southeastern part of the country. A single USUV lineage (Europe 3) was found in Prague and was likely associated with increased mortalities in the local blackbird population seen in this area in 2018. USUV genomic RNA (lineage Europe 2) was detected in a pool of Cx. pipiens mosquitoes from South Bohemia (southern part of the country), where no major mortality of birds has been reported so far, and no flavivirus RNA has been found in randomly sampled cadavers of blackbirds. The obtained data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.