Strengthening public health by understanding the epidemiology of rodent-borne diseases: Pathogen behavior and epidemiology of Leptospira

The joint project RoBoPub aims at an interdisciplinary and synergistic OneHealth approach focussing on two reportable zoonoses both endemic in Germany, the hantavirus disease and leptospirosis. For both diseases there are a number of similarities that can be expected to result in strong synergy effects during the project. The aim of the RoBoPub joint project is to generate knowledge about the pathogens, their distribution, host association, environmental stability and transmission. In addition, host-specific factors such as population fluctuations and the underlying environmental and climatic parameters as well as the seroprevalence in human population cohorts are to be investigated. Finally, the risk perception and behavior of the population as well as the sensitization of resident doctors will be considered. In summary, the RoBoPub consortium intends to use new knowledge about these two endemic pathogens to develop public health measures for the prevention of rodent-borne infections. Therefore, the consortium aims to improve the prevention options for both pathogens by creating a risk management plan for the public health service (ÖGD) and developing detailed risk maps, early warning modules, management strategies and prevention recommendations. At the same time, the risk awareness of the general population and of the risk groups, as well as the awareness of doctors in private practice in endemic regions will be increased. The knowledge gained will not only be translated into measures of the ÖGD, but also enable conclusions to be drawn about other rodent-borne zoonotic pathogens, such as orthopox viruses.

AS1: Pathogen Geography Milestone 1.1. Molecular biological analysis of the rodent samples sent in by the FLIshort forFriedrich Loeffler Institute (live and dead fish)- In this sub-project, molecular biological investigations were carried out on the rodent samples sent in by the FLIshort forFriedrich Loeffler Institute and LFB Brandenburg. All samples received (n = 55 +19) were examined using PCR, SLST and MLST and the results forwarded to the project partners.
Milestone 1.2. Cultivation of Leptospira from organs of rodent samples (live catches)  Leptospira from organs of the rodent samples (live catches) sent in by JKIshort forJulius Kühn Institute / LAVES were cultivated: Two isolates were obtained from a total of 12 samples. The isolated strains are used in work package AS2-AP4.
Milestone 1.3. Protocol for metagenomic analysis for direct detection and typing of Leptospira from primary samples. After participating in the workshop "Introduction to Whole Genome Sequencing and Analysis for Microbial Diagnostics" in October 2017 in Denmark, it became apparent that the sensitivity and costs for the routine detection and typing of primary samples using metagenome analysis are currently too low or too high. Instead, a real-time PCR for the quantitative detection of Leptospira was established and validated according to ISO 17025. This involves the validation of a method for the detection of pathogenic Leptospira in body fluids (EDTA blood, serum, urine, liquor) and organ materials (e.g. kidney tissue). The analysis takes place via the detection of the lipL32 gene. To determine the limit of detection, limit of quantification and PCR efficiency, serial dilutions of the DNA of the strain L. interrogans serogroup Icterohaemorrhagiae Serovar Icterohaemorrhagiae were evaluated. The limit of detection (LOD95%) is 5,551 genome units (95% confidence interval from 3,652 to 8,451 genome units). In addition, the inclusivity using 29 other strains of pathogenic Leptospira was investigated. To test the exclusivity, 2 non-pathogenic Leptospira strains of the species L. yanagawae and L. biflexa, as well as the strain Leptonema illini were used. In addition, DNA from other spirochetes wasused, which, due to their relationship, can produce false-positive results. The sensitivity of the method in urine and blood was also determined by means of spiking tests. The detection limit was 102 Leptospira / mlshort formillilitre.
AS2: Stability and host association An intensive literature research on the survival of LeptospirA SPP: in environmental matrices was carried out, the data were presented as poster  "Survival of Leptopira spp. In soil and water: A literature review" at the ELS Scientific Meeting 2018 in Italy from May 24, 2018 to May 26, 2018 . The conclusion of the review was that environmental parameters such as pH, humidity and UV radiation have an influence on the survival of the leptospira outside the host. However, not enough data are available to compare and determine the survival time of the Leptospira depending on these parameters. The review shows tthat new data is needed. Based on this, the first studies on the survival in soil were carried out in cooperation with the University of Leipzig. The results of the literature research were used in the publication on the survival of Leptospira in urine in collaboration with the University of Leipzig. Investigations on the survival of Leptospira on strawberries were carried out by means of spiking experiments with reference and field strains of L. kirschneri and temporal kinetics were determined by microscopy, PCR and cultivation of Leptospira. The counting procedures for the tenacity studies were established after a training stay at the reference laboratory in Amsterdam. The tenacity studies were initially carried out with a Leptospira kischneri serovar Grippotyphosa laboratory strain and were repeated with a field strain (Leptospira kischneri serovar Grippotyphosa Sequence Type 110) that was isolated from a field vole during a 2007 strawberry field outbreak. No significant difference between the strains was detected (z = 0, p = 1). Like the first strain, the field strain showed that temperature affects survival. Higher temperatures (> 21 ° C) support the survival time significantly (z = 4.10, p <0.001). Tenacity decreased significantly with increasing incubation time (z = -7.01, p <0.001). Leptospira kirschneri can survive on strawberries for up to 6 hours. The risk for the consumer increases if the strawberry consumption takes place in the field and if supportive conditions such as high temperatures and frequent precipitation coincide. The data was published in PlosOne. Milestone 2.2. Hospitals recruited and protocol available for transport and isolation of Leptospira from blood cultures.
In order to provide a protocol for the hospitals, DNA extraction experiments, qPCR examinations and cultivation experiments from blood cultures were carried out. In addition, hospitals were recruited and the transport medium as well as the established protocol were distributed. Milestone 2.3. Cultivation of Leptospira from blood cultures completed; isolated strains to be used in work package AS2-AP4. Lleptospira isolates were not obtained during the project period. The probability of obtaining an isolate is low due to the low incidence of leptospirosis and the difficult cultivation of leptospirosis. In order to increase the probability, a cooperation was begun  with the Bernard Nocht Institute, which, compared to other hospitals, treats more cases of leptospirosis. Milestone 2.4. Comparative genome analysis  The strains described in AS1-AP2 and further 25 Leptospira strains from the strain collection of the National Consultant Laboratory were cultivated and sequenced with the MiSeq system available at the BfRshort forGerman Federal Institute for Risk Assessment. In addition, a protocol for creating a genome library using the Nextera XT Library Prep Kit was established and applied.  During the second funding phase, the application of methods for assembling the genome sequences using SPADES or Velvet, searching and comparing virulence and resistance genes using e.g. a virulence finder database or CGE tools is planned. Further training in other evaluation methods should should have taken place at the end of the first funding phase in cooperation with the Pasteur Institute in Paris. This stay had to be canceled due to the corona crisis.
AS3: epidemiology Milestone 3.1. Serological testing of the serum samples provided by the NLGA. A collaboration with the NLGA regarding the serological analysis of human sera from risk groups (forest workers) took place. Furthermore, a serological comparison of methods between the BfRshort forGerman Federal Institute for Risk Assessment in-house ELISA and the commercially available ELISA from Virion / Serion as well as the gold standard test MAT was carried out. A total of 146 sera were examined. These new data were subjected to a statistical analysis together with previous data (a total of 525 samples). With the help of the Cohen’s Kappa test, significant differences between the tests were detected. A Bayesian modeling of the data is to be published, the manuscript is in progress. AS6: Public Health Translation Milestone 6.1. Transfer of the results into measures of the public health service. All results were made available to the network partners for further processing, presentation and use in the implementation of measures for the ÖGD. The results were also presented in the project meetings and presented in an ÖGD workshop. • Participation in the RoBoPub project meeting on May 14th, 2018 in Berlin • Participation in the RoBoPub project meeting on October 17th, 2018 in Berlin • Participation in the RoBoPub project meeting on May 13th, 2019 in Berlin • Participation in the RoBoPub project meeting on October 16, 2019 in Berlin • Participation in the RoBoPub project meeting, on May 28th, 2020 virtually • Participation in the workshop rodent-borne zoonoses of the National Research Platform for Zoonoses, on November 27th - November 30th, 2018 in Berlin • Participation in the workshop of the network "Rodent-borne pathogens", on 28.-30. November, Berlin To educate the medical profession and the ÖGD, the manuscript: "Leptospirosis in Germany: Current findings on pathogens, reservoir hosts and diseases in humans and animals" was published.