Sabrina Schmidt

Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe

For a long time hantaviruses were believed to be exclusively rodent-borne pathogens. Recent findings of numerous shrew- and mole-borne hantaviruses raise important questions on their phylogenetic origin. The objective of our study was to prove the presence and distribution of shrew-associated Seewis virus (SWSV) in different Sorex species in Central Europe. Therefore, a total of 353 Sorex araneus, 59 S.minutus, 27 S. coronatus, and one S. alpinus were collected in Germany, the Czech Republic, and Slovakia. Screening by hantavirus-specific L-segment RT-PCR revealed specific amplification products in tissues of 49 out of 353 S. araneus and four out of 59 S. minutus. S-segment sequences were obtained for 45 of the L-segment positive S. araneus and all four L-segment positive S. minutus. Phylogenetic investigation of these sequences from Germany, the Czech Republic, and Slovakia demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria, and other sites in Germany. The low intra-cluster sequence variability and the high inter-cluster divergence suggest a long-term SWSV evolution in isolated Sorex populations. In 28 of the 49 SWSV S-segment sequences, an additional putative open reading frame (ORF) on the opposite strand to the nucleocapsid protein-encoding ORF was identified. This is the first comprehensive sequence analysis of SWSV strains from Germany, the Czech Republic, and Slovakia, indicating its broad geographical distribution and high genetic divergence. Future studies have to prove whether both S. araneus and S. minutus represent SWSV reservoir hosts or spillover infections are responsible for the parallel molecular detection of SWSV in both species.

Autochthonous dengue virus infection in Japan imported into Germany, September 2013.

In September 2013, dengue virus (DENV) infection was diagnosed in a German traveller returning from Japan. DENV-specific IgM and IgG and DENV NS1 antigen were detected in the patient’s blood, as were DENV serotype 2-specific antibodies. Public health authorities should be aware that autochthonous transmission of this emerging virus may occur in Japan. Our findings also highlight the importance of taking a full travel history, even from travellers not returning from tropical countries, to assess potential infection risks of patients.

Leptospira spp. in Rodents and Shrews in Germany

Leptospirosis is an acute, febrile disease occurring in humans and animals worldwide. Leptospira spp. are usually transmitted through direct or indirect contact with the urine of infected reservoir animals. Among wildlife species, rodents act as the most important reservoir for both human and animal infection. To gain a better understanding of the occurrence and distribution of pathogenic leptospires in rodent and shrew populations in Germany, kidney specimens of 2973 animals from 11 of the 16 federal states were examined by PCR. Rodent species captured included five murine species (family Muridae), six vole species (family Cricetidae) and six shrew species (family Soricidae). The most abundantly trapped animals were representatives of the rodent species Apodemus flavicollis, Clethrionomys glareolus and Microtus agrestis. Leptospiral DNA was amplified in 10% of all animals originating from eight of the 11 federal states. The highest carrier rate was found in Microtus spp. (13%), followed by Apodemus spp. (11%) and Clethrionomys spp. (6%). The most common Leptospira genomospecies determined by duplex PCR was L. kirschneri, followed by L. interrogans and L. borgpetersenii; all identified by single locus sequence typing (SLST). Representatives of the shrew species were also carriers of Leptospira spp. In 20% of Crocidura spp. and 6% of the Sorex spp. leptospiral DNA was detected. Here, only the pathogenic genomospecies L. kirschneri was identified.

Multiple infections of rodents with zoonotic pathogens in Austria

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the hosts immune system, the impact of the host-pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.

High prevalence of Rickettsia helvetica in wild small mammal populations in Germany

Since the beginning of the 21st century, spotted fever rickettsioses are known as emerging diseases worldwide. Rickettsiae are obligately intracellular bacteria transmitted by arthropod vectors. The ecology of Rickettsia species has not been investigated in detail, but small mammals are considered to play a role as reservoirs. Aim of this study was to monitor rickettsiae in wild small mammals over a period of five years in four federal states of Germany. Initial screening of ear pinna tissues of 3939 animals by Pan-Rick real-time PCR targeting the citrate synthase (gltA) gene revealed 296 rodents of seven species and 19 shrews of two species positive for rickettsial DNA. Outer membrane protein gene (ompB, ompAIV) PCRs based typing resulted in the identification of three species: Rickettsia helvetica (90.9%) was found as the dominantly occurring species in the four investigated federal states, but Rickettsia felis (7.8%) and Rickettsia raoultii (1.3%) were also detected. The prevalence of Rickettsia spp. in rodents of the genus Apodemus was found to be higher (approximately 14%) than in all other rodent and shrew species at all investigated sites. General linear mixed model analyses indicated that heavier (older) individuals of yellow-necked mice and male common voles seem to contain more often rickettsial DNA than younger ones. Furthermore, rodents generally collected in forests in summer and autumn more often carried rickettsial DNA. In conclusion, this study indicated a high prevalence of R. helvetica in small mammal populations and suggests an age-dependent increase of the DNA prevalence in some of the species and in animals originating from forest habitats. The finding of R. helvetica and R. felis DNA in multiple small mammal species may indicate frequent trans-species transmission by feeding of vectors on different species. Further investigations should target the reason for the discrepancy between the high rickettsial DNA prevalence in rodents and the so far almost absence of clinical apparent human infections.

Occurrence and distribution of Giardia species in wild rodents in Germany

BackgroundGiardiasis is an important gastrointestinal parasitic disease in humans and other mammals caused by the protozoan Giardia duodenalis. This species complex is represented by genetically distinct groups (assemblages A-H) with varying zoonotic potential and host preferences. Wild rodents can harbor potentially zoonotic assemblages A and B, and the rodent-specific assemblage G. Other Giardia spp. found in these animals are Giardia muris and Giardia microti. For the latter, only limited information on genetic typing is available. It has been speculated that wild rodents might represent an important reservoir for parasites causing human giardiasis. The aim of this study was to investigate the occurrence and distribution of Giardia spp. and assemblage types in wild rodents from different study sites in Germany.ResultsScreening of 577 wild rodents of the genera Apodemus, Microtus and Myodes, sampled at eleven study sites in Germany, revealed a high overall Giardia prevalence. Giardia species determination at the SSU rDNA gene locus revealed that Apodemus mice, depending on species, were predominantly infected with one of two distinct G. muris sequence types. Giardia microti was the predominant parasite species found in voles of the genera Microtus and Myodes. Only a few animals were positive for potentially zoonotic G. duodenalis. Subtyping at the beta-giardin (bg) and glutamine dehydrogenase (gdh) genes strongly supported the existence of different phylogenetic subgroups of G. microti that are preferentially harbored by distinct host species.ConclusionsThe present study highlights the preference of G. muris for Apodemus, and G. microti for Microtus and Myodes hosts and argues for a very low prevalence of zoonotic G. duodenalis assemblages in wild rodents in Germany. It also provides evidence that G. muris and G. microti subdivide into several phylogenetically distinguishable subgroups, each of which appears to be preferentially harbored by species of a particular rodent host genus. Finally, the study expands the database of sequences relevant for sequence typing of G. muris and G. microti isolates which will greatly help future analyses of these parasites’ population structure.

Puumala hantavirus infections in bank vole populations: host and virus dynamics in Central Europe

BackgroundIn Europe, bank voles (Myodes glareolus) are widely distributed and can transmit Puumala virus (PUUV) to humans, which causes a mild to moderate form of haemorrhagic fever with renal syndrome, called nephropathia epidemica. Uncovering the link between host and virus dynamics can help to prevent human PUUV infections in the future. Bank voles were live trapped three times a year in 2010–2013 in three woodland plots in each of four regions in Germany. Bank vole population density was estimated and blood samples collected to detect PUUV specific antibodies.ResultsWe demonstrated that fluctuation of PUUV seroprevalence is dependent not only on multi-annual but also on seasonal dynamics of rodent host abundance. Moreover, PUUV infection might affect host fitness, because seropositive individuals survived better from spring to summer than uninfected bank voles. Individual space use was independent of PUUV infections.ConclusionsOur study provides robust estimations of relevant patterns and processes of the dynamics of PUUV and its rodent host in Central Europe, which are highly important for the future development of predictive models for human hantavirus infection risk.

Autochthone Dobrava-Belgrad-Virus-Infektion in einem Nichtendemiegebiet in Mitteldeutschland

Ein 21-jahriger mannlicher Patient aus Borna, Sachsen, wurde der Nephrologie des Universitatsklinikums Leipzig mit einem akuten Nierenversagen und den Symptomen Ubelkeit, Erbrechen, Abdominalschmerzen sowie Diarrho aus dem Kreiskrankenhaus Borna zur weiteren Abklarung zugewiesen. Serologische Untersuchungen durch indirekte in-house IgM- und IgG-ELISAs, kommerziell erhaltliche Immunfluoreszenz- und Streifen-Immunoassays sowie Chemilumineszenz- Fokusreduktionsneutralisationstests bestatigten eine akute Infektion mit dem Dobrava-Belgrad-Virus (DOBV). Serologische und RT-PCR-Analysen von Brandmausen (Apodemus agrarius), die in der Nahe der Wohnregion des Patienten daraufhin gefangen wurden, zeigten eine Infektion mit DOBV, Genotyp Kurkino. Dies ist der erste dokumentierte Fall einer autochthonen Infektion mit dem DOBV bei einem Patienten, der nicht im bekannten Endemiegebiet Norddeutschland lebt. Dies zeigt, dass auch Arzte in Regionen, in denen der entsprechende Wirt lebt, die aber nicht als typische Hantavirus- Endemiegebiete gelten, eine solche Infektion nicht von vornherein ausschliesen sollten. * Erstpublikation der englischen Fassung in Clinical Nephrology Band 83, Nr. 2/2015, S. 111-116

Autochthonous Dobrava-Belgrade virus infection in Eastern Germany.

A 21-year-old male patient from Borna, Saxony, in Eastern Germany, suffered from acute kidney injury (AKI) and symptoms typical for a hantavirus infection. These symptoms included nausea, vomiting, abdominal pain, diarrhea, and acute renal failure. Serological investigations by indirect IgM and IgG in-house ELISAs, commercial immunofluorescence and line assays, as well as chemiluminescence focus reduction neutralization assay confirmed an acute Dobrava-Belgrade virus (DOBV) infection of the patient. Serological and RT-PCR analyses of striped field mouse (Apodemus agrarius) trapped in a neighboring region of the residence of the patient identified an infection by DOBV, genotype Kurkino. This is the first report of an autochthonous DOBV infection in a German patient living far from the known endemic region in the north of the country. This finding has implications for the awareness of physicians in areas which are not recognized as hantavirus endemic regions but where the reservoir host of the virus is present.

Population dynamics of bank voles and human Puumala virus infections in Germany

The main role of MADS-box transcription factors in plant developmental processes has been well described in the model plant Arabidopsis thaliana. However, little is known about their function in crops of important agricultural and commercial value. Our study aims to investigate their role in two agronomical relevant Rosaceae crops: apple (Malus x domestica Borkh.) and strawberry (Fragaria vesca). Expression studies using qPCR and RNA seq have identified two apple Dormancy Associated MADS-box (DAM) genes. They group with the StMADS11 clade, and were named MdDAM1 and MdDAM2, the last one discovered ex novo. Real time expression studies in dormant buds collected during the chilling period and chromatin immunoprecipitation (ChIP) analyses confirmed that the genes are downregulated by exposure to cold and MdDAM1 is epigenetically repressed, as it has been demonstrated for Arabidopsis FLC and peach DAM genes. In parallel we worked on strawberry MADS-box genes of known function involved in flower development. We chose three MADS-box genes that are homologs of Arabidopsis PISTILLATA and AGAMOUS to perform gene expression and functional analysis using a RNA interference approach to obtain post-transcriptional gene silencing. The positive transgenic lines of each transformation were evaluated at the molecular and phenotypic level. Single gene mutants does not show altered flower phenotype, suggesting a different mechanism of flower development in strawberry, probably due to the peculiar flower structure.The quarantine pest pine wood nematode (PWN) Bursaphelenchus xylophilus, native in North America, can be found in wood and root parts of infected conifer trees with focus on Pinus species. At the beginning of the 20 century this species was introduced into Japan supposedly with timber imports where it induced pine wilt expression on susceptible native pine species. Further outbreaks outside Europe are reported from China, Korea, Taiwan and Mexico. In 1999 PWN was found in Portugal, which today is declared as complete infested, since followed by first single tree outbreaks in Spain. The European Union (EU) member states have to undertake measures to prevent further spread of this nematode according to the emergency measures of the EU Commission.The box tree pyralid Cydalima perspectalis (Walker 1859) is an invasive alien moth from East Asia which occurs in Central Europe since 2007. It is an insect pest on plants of the genus Buxus, causing serious damage. Because of the rapid spread in Germany and nearby countries like Switzerland, one part of this study was testing different ways of eco-friendly regulation with commercially available beneficials and biological control agents.1 Julius Kühn-Institute, Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany. 2 Geisenheim Research Center, Section Phytomedicine, Geisenheim, Germany. 3 Área de Genética, Facultad de Ciencias, Universidad de Málaga, Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC), Málaga (Spain). 4 Suez Canal University, Faculty of Agriculture, Ismailia, Egypt. Email of corresponding author: namis.eltlbany@jki.bund.deExcessive use of mineral nitrogen (N) fertilizer is a common practice in rice-wheat rotations in southeastern China. At the same time N use efficiencies (NUEs) in these rice-based cropping system are very low (Peng et al., 2006). The consequences are high N losses from arable land to water bodies (surfaceand groundwater) and to the atmosphere (Richter and Roelcke, 2000). To investigate the scope and scale of reductions in mineral N fertilizer inputs, demonstration field experiments on farmers’ field sites were conducted for three consecutive winter wheat-summer rice double crop rotations in two counties of Jiangsu Province from 2008 to 2011. Results from the first two years are presented here. NUEsPolycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants which often persist in soil. The biochemistry of microbe-driven degradation of PAHs is well-studied though less is known about abiotic soil environmental factors influencing the microbe-phenanthrene interaction. Since minerals and charcoal are major soil components we matured different microbial communities in artificial soils based only on variation in the mineral content (montmorillonite, illite, ferrihydrite) and presence of charcoal for 2 years after addition of Luvisol microorganisms and sterile manure. Established microbial communities in soils were exposed to PAHs’ model compound phenanthrene (2 mg/g) to study microbial functionality depending on soil composition. Furthermore, litter which was previously shown to enhance microbial activity was added to the soils (1 wt%). Both treatments with corresponding controls were further incubated for 63 days with sampling on day 0, 7, 21 and 63. A natural Luvisol soil was treated similarly for control. Fingerprints by denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA or ITS gene fragments, respectively, amplified from extracted total community DNA were generated. The presence of phenanthrene-degradative genes was screened by PCR-Southern Blot detection and soil treatments were subjected to a chemical phenanthrene analysis. DGGE fingerprints revealed that the addition of phenanthrene and litter caused a shift in microbial community composition. Differences in bacterial response to phenanthrene were seen depending on litter addition and soil composition. The incubation time was determined as additional influencing factor. Furthermore, a dominance of a specific genotype for phenanthrene degradation is assumed based on PCR-Southern Blot analysis.1 Julius Kühn-Institute Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Messeweg 11-12, D-38104 Braunschweig 2 Institute of Crop Science and Resource Conservation, Soil Science and Ecology, University of Bonn, Nussallee 13, D-53115 Bonn 3 Institute of Bioand Geosciences 3, Agrosphere, Forschungszentrum Jülich GmbH, D-52425 Jülich Email of corresponding author: sven.jechalke@jki.bund.de