Investigation and response to a large outbreak of leptospirosis in field workers in Lower Saxony, Germany.

Between June and August 2014, 45 cases of leptospirosis were notified among workers on two strawberry farms in North-West Germany. We describe the characteristics of the outbreak and the actions taken to prevent further cases. The activities of the local, federal and national public health and veterinary authorities included collection of case data, laboratory testing of human specimens and of small mammals trapped on the fields, investigation of weather data, as well as information provided to farmers, field workers, physicians and to the authorities in Poland and Romania. Of the 45 identified cases (median age 22, 60% male), 47% were hospitalized. Characteristic symptoms were fever ≥38.5°C, generalized muscle pain and an increase in renal or liver enzymes. Thirteen cases were laboratory confirmed by serological and/or molecular methods. ELISA tests for Leptospira IgG and IgM-antibodies were positive in those samples taken >5 days after hospitalization. The probable causative agent was identified as Leptospira kirschneri serovar Grippotyphosa. Leptospira-specific DNA was found in kidneys of 67% of 64 trapped small mammals and was further identified as Leptospira kirschneri multi locus sequence type 110. During the estimated time period of human infections, the affected region faced warm weather with heavy rainfalls. The results of this investigation are in accordance with the theory of a chain of infection from mice to field workers during warm and humid weather. In 2015, a campaign was initiated to inform physicians, farmers and workers to enhance prevention measures, such as the use of personal protective equipment and early consultation of physicians in case of illness. Since then, no further outbreak occurred.

Cluster of human Puumala orthohantavirus infections due to indoor exposure?-An interdisciplinary outbreak investigation.

Puumala orthohantavirus (PUUV) is the most important hantavirus species in Europe, causing the majority of human hantavirus disease cases. In central and western Europe, the occurrence of human infections is mainly driven by bank vole population dynamics influenced by beech mast. In Germany, hantavirus epidemic years are observed in 2- to 5-year intervals. Many of the human infections are recorded in summer and early autumn, coinciding with peaks in bank vole populations. Here, we describe a molecular epidemiological investigation in a small company with eight employees of whom five contracted hantavirus infections in late 2017. Standardized interviews with employees were conducted to assess the circumstances under which the disease cluster occurred, how the employees were exposed and which counteractive measures were taken. Initially, two employees were admitted to hospital and serologically diagnosed with hantavirus infection. Subsequently, further investigations were conducted. By means of a self-administered questionnaire, three additional symptomatic cases could be identified. The hospital patients' sera were investigated and revealed in one patient a partial PUUV L segment sequence, which was identical to PUUV sequences from several bank voles collected in close proximity to company buildings. This investigation highlights the importance of a One Health approach that combines efforts from human and veterinary medicine, ecology and public health to reveal the origin of hantavirus disease clusters.

Influence of Season, Population and Individual Characteristics on the Prevalence of Leptospira spp. in Bank Voles in North-West Germany.

Leptospirosis is a worldwide zoonotic disease with more than 1 million human cases annually. Infections are associated with direct contact to infected animals or indirect contact to contaminated water or soil. As not much is known about the prevalence and host specificity of Leptospira spp. in bank voles (Clethrionomys glareolus), our study aimed to evaluate Leptospira spp. prevalence and genomospecies distribution as well as the influence of season, host abundance and individual characteristics on the Leptospira prevalence. Bank voles, which are abundant and widely distributed in forest habitats, were collected in the years 2018 to 2020 in North-West Germany, covering parts of North Rhine-Westphalia and Lower Saxony. The DNA of 1817 kidney samples was analyzed by real-time PCR targeting the lipl32 gene. Positive samples were further analyzed by targeting the secY gene to determine Leptospira genomospecies and multilocus sequence typing (MLST) to determine the sequence type (ST). The overall prevalence was 7.5% (95% confidence interval: 6.4-8.9). Leptospira interrogans (83.3%), L. kirschneri (11.5%) and L. borgpetersenii (5.2%) were detected in bank voles. Increasing body weight as a proxy for age increased the individual infection probability. Only in years with high bank vole abundance was this probability significantly higher in males than in females. Even if case numbers of human leptospirosis in Germany are low, our study shows that pathogenic Leptospira spp. are present and thus a persisting potential source for human infection.

Heterogeneous Puumala orthohantavirus situation in endemic regions in Germany in summer 2019.

Puumala orthohantavirus (PUUV) causes most human hantavirus disease cases in Europe. PUUV disease outbreaks are usually synchronized Germany-wide driven by beech mast-induced irruptions of its host (bank vole, Myodes glareolus). Recent data indicate high vole abundance, high PUUV prevalence and high human incidence in summer 2019 for some regions, but elsewhere values were low to moderate. This significant lack of synchrony among regions in Germany is in contrast to previous studies. Health institutions need to be informed about the heterogeneous distribution of human PUUV infection risk to initiate appropriate actions.

Occurrence of Gastrointestinal Parasites in Small Mammals from Germany.

An increase in zoonotic infections in humans in recent years has led to a high level of public interest. However, the extent of infestation of free-living small mammals with pathogens and especially parasites is not well understood. This pilot study was carried out within the framework of the "Rodent-borne pathogens" network to identify zoonotic parasites in small mammals in Germany. From 2008 to 2009, 111 small mammals of 8 rodent and 5 insectivore species were collected. Feces and intestine samples from every mammal were examined microscopically for the presence of intestinal parasites by using Telemann concentration for worm eggs, Kinyoun staining for coccidia, and Heidenhain staining for other protozoa. Adult helminths were additionally stained with carmine acid for species determination. Eleven different helminth species, five coccidians, and three other protozoa species were detected. Simultaneous infection of one host by different helminths was common. Hymenolepis spp. (20.7%) were the most common zoonotic helminths in the investigated hosts. Coccidia, including Eimeria spp. (30.6%), Cryptosporidium spp. (17.1%), and Sarcocystis spp. (17.1%), were present in 40.5% of the feces samples of small mammals. Protozoa, such as Giardia spp. and amoebae, were rarely detected, most likely because of the repeated freeze-thawing of the samples during preparation. The zoonotic pathogens detected in this pilot study may be potentially transmitted to humans by drinking water, smear infection, and airborne transmission.

Detection of rat hepatitis E virus in wild Norway rats (Rattus norvegicus) and Black rats (Rattus rattus) from 11 European countries.

Rat hepatitis E virus (HEV) is genetically only distantly related to hepeviruses found in other mammalian reservoirs and in humans. It was initially detected in Norway rats (Rattus norvegicus) from Germany, and subsequently in rats from Vietnam, the USA, Indonesia, China, Denmark and France. Here, we report on a molecular survey of Norway rats and Black rats (Rattus rattus) from 12 European countries for ratHEV and human pathogenic hepeviruses. RatHEV-specific real-time and conventional RT-PCR investigations revealed the presence of ratHEV in 63 of 508 (12.4%) rats at the majority of sites in 11 of 12 countries. In contrast, a real-time RT-PCR specific for human pathogenic HEV genotypes 1-4 and a nested broad-spectrum (NBS) RT-PCR with subsequent sequence determination did not detect any infections with these genotypes. Only in a single Norway rat from Belgium a rabbit HEV-like genotype 3 sequence was detected. Phylogenetic analysis indicated a clustering of all other novel Norway and Black rat-derived sequences with ratHEV sequences from Europe, the USA and a Black rat-derived sequence from Indonesia within the proposed ratHEV genotype 1. No difference in infection status was detected related to age, sex, rat species or density of human settlements and zoological gardens. In conclusion, our investigation shows a broad geographical distribution of ratHEV in Norway and Black rats from Europe and its presence in all settlement types investigated.

High genetic structuring of Tula hantavirus.

Tula virus (TULV) is a vole-associated hantavirus with low or no pathogenicity to humans. In the present study, 686 common voles (Microtus arvalis), 249 field voles (Microtus agrestis) and 30 water voles (Arvicola spec.) were collected at 79 sites in Germany, Luxembourg and France and screened by RT-PCR and TULV-IgG ELISA. TULV-specific RNA and/or antibodies were detected at 43 of the sites, demonstrating a geographically widespread distribution of the virus in the studied area. The TULV prevalence in common voles (16.7 %) was higher than that in field voles (9.2 %) and water voles (10.0 %). Time series data at ten trapping sites showed evidence of a lasting presence of TULV RNA within common vole populations for up to 34 months, although usually at low prevalence. Phylogenetic analysis demonstrated a strong genetic structuring of TULV sequences according to geography and independent of the rodent species, confirming the common vole as the preferential host, with spillover infections to co-occurring field and water voles. TULV phylogenetic clades showed a general association with evolutionary lineages in the common vole as assessed by mitochondrial DNA sequences on a large geographical scale, but with local-scale discrepancies in the contact areas.

Spatiotemporal dynamics of Puumala hantavirus associated with its rodent host, Myodes glareolus.

Many viruses significantly impact human and animal health. Understanding the population dynamics of these viruses and their hosts can provide important insights for epidemiology and virus evolution. Puumala virus (PUUV) is a European hantavirus that may cause regional outbreaks of hemorrhagic fever with renal syndrome in humans. Here, we analyzed the spatiotemporal dynamics of PUUV circulating in local populations of its rodent reservoir host, the bank vole (Myodes glareolus) during eight years. Phylogenetic and population genetic analyses of all three genome segments of PUUV showed strong geographical structuring at a very local scale. There was a high temporal turnover of virus strains in the local bank vole populations, but several virus strains persisted through multiple years. Phylodynamic analyses showed no significant changes in the local effective population sizes of PUUV, although vole numbers and virus prevalence fluctuated widely. Microsatellite data demonstrated also a temporally persisting subdivision between local vole populations, but these groups did not correspond to the subdivision in the virus strains. We conclude that restricted transmission between vole populations and genetic drift play important roles in shaping the genetic structure and temporal dynamics of PUUV in its natural host which has several implications for zoonotic risks of the human population.

Complete genome of a Puumala virus strain from Central Europe.

Puumala virus (PUUV) is one of the predominant hantavirus species in Europe causing mild to moderate cases of haemorrhagic fever with renal syndrome. Parts of Lower Saxony in north-western Germany are endemic for PUUV infections. In this study, the complete PUUV genome sequence of a bank vole-derived tissue sample from the 2007 outbreak was determined by a combined primer-walking and RNA ligation strategy. The S, M and L genome segments were 1,828, 3,680 and 6,550 nucleotides in length, respectively. Sliding-window analyses of the nucleotide sequences of all available complete PUUV genomes indicated a non-homogenous distribution of variability with hypervariable regions located at the 3'-ends of the S and M segments. The overall similarity of the coding genome regions to the other PUUV strains ranged between 80.1 and 84.7 % at the level of the nucleotide sequence and between 89.5 and 98.1 % for the deduced amino acid sequences. In comparison to the phylogenetic trees of the complete coding sequences, trees based on partial segments revealed a general drop in phylogenetic support and a lower resolution. The Astrup strain S and M segment sequences showed the highest similarity to sequences of strains from geographically close sites in the Osnabrück Hills region. In conclusion, a primer-walking-mediated strategy resulted in the determination of the first complete nucleotide sequence of a PUUV strain from Central Europe. Different levels of variability along the genome provide the opportunity to choose regions for analyses according to the particular research question, e.g., large-scale phylogenetics or within-host evolution.

Distribution of rodenticide resistance and zoonotic pathogens in Norway rats in Lower Saxony and Hamburg, Germany.

BACKGROUND: Genetically based resistance to anticoagulants has led to increasing difficulties in the control of rodents over recent decades. The possible impact of rodenticide-resistant rats on the infection risk of humans and livestock by zoonotic pathogens is generally unknown. Hence, in a monitoring programme in the German federal states of Lower Saxony and Hamburg, more than 500 Norway rats were analysed for both Tyr139Cys polymorphisms within the VKORC1 gene and zoonotic agents. RESULTS: Evidence of resistance was almost completely restricted to the known resistance area in southern Lower Saxony. Homozygous mutations were only found in urban areas sampled owing to the occurrence of rat control problems and were missing in bycatches of rats by muskrat trappers in rural areas. In more than 25% of the rats, zoonotic bacteria (Leptospira, Salmonella, Yersinia and Coxiella) were detected. There was no obvious correlation between the occurrence of rats carrying zoonotic pathogens and anticoagulant resistance. CONCLUSION: Zoonotic agents and genetically based resistance conferred by the Tyr139Cys polymorphism are both unevenly distributed in Lower Saxony. The study provides the basis for further studies focusing on districts with high levels of pathogens and resistance to assess the potential health risk of their combined occurrence.