The Robert Koch Institute (RKI): main building in Berlin. more
Germany’s Public Health institute RKI monitors the population’s health and wellbeing. more
The Robert Koch Institute is the German government's central institution for the identification, surveillance and prevention of infectious diseases in the Portfolio of the German Ministry of Health. more
Andreas Kurth, Ph.D. (Dr.rer.nat.)
Silke Riesle-Sbarbaro, Ph.D.
Marcel Bokelmann, Ph.D. (Former Lab Member)
Kathryn Edenborough, Ph.D. (Former Lab Member)
Our research focuses on experimental approaches for zoonotic viruses in their (potential) reservoir species. For this we develop infection models to elucidate mechanisms of pathogenicity and transmission (contact, fomite, aerosol, foodborne or vertical transmission) in the context of abiotic (temperature, humidity, airflow) and biotic factors (routes of transmission, immune status, receptor distribution, amount of shed virus).
Presently, we focus on Angolan free-tailed bats (Mops condylurus) as a suspected reservoir host for Ebola virus. More than forty years after the first Ebola virus disease outbreak, there is still no convincing evidence identifying a natural reservoir host, nor the mechanisms of virus circulation in nature or spillover to humans. Circumstantial evidence has associated the insectivorous Angolan free-tailed bat as a spillover species triggering the so far largest Ebola virus disease in 2013–2016 in West Africa. Also, Angolan free-tailed bats are a natural reservoir host for Bombali virus which is closely related to Ebola virus. Studies on bats as natural reservoir hosts are globally very limited due to the very complicated and complex working conditions. For reaching our goals, we had to build up a bat husbandry in order to capture and keep the animals on-site in Africa, develop diagnostic tools (cell lines, serology, immunological markers) and finally the infection model for Ebola virus at the BSL-4 laboratory at Robert Koch Institute in Berlin with special emphasis on animal biology, welfare and health monitoring during captivity.
Rather than persecuting bats, we need to unravel the secrets of the success of this group of mammals in coping with this virus. Understanding how bats control viral replication may not only help to predict transmission events from bats into human and animal populations but would also assist in developing future therapeutics.
Why are live bats indispensable?
A large variety of cell lines from different animal species are susceptible to Ebola virus although these animal species themselves are not susceptible. This means that experiments with different cell lines unfortunately do not allow any reliable conclusions to be drawn as to which species could be a potential natural reservoir host of Ebola viruses. Animals that are competent as virus reservoirs would allow virus to replicate abundantly, shed virus and, in most cases, induce no clinical disease; there is no other way to investigate these features but with few living animals which previously have been tested Ebola virus-negative. Nevertheless, and with regard to the 3R-Principle, we had also developed various cell lines from Angolan free-tailed bats, e.g. kidney, lymph node, brain, spleen, skin, muscle, lung and liver, to address specific scientific questions on a cellular level such as Ebola virus cell receptor or tolerance and persistence of Ebola virus.
Established cell lines from Angolan free-tailed bats. Source: RKI
Get to know Angolan free-tailed bats
Angolan free-tailed bats are insectivores from the family Molossidae with an average body weight of 25-45g. These bats can be found in large parts of sub-Saharan Africa and in various habitats such as wet and dry savannah. Their roosts can be particularly easy to detect due to the very distinct smell (pheromones) they produce. They are a non-threatened species according to the Red List of the International Union for Conservation of Nature and Natural Resources. As there is no breeding facility for insectivorous bats available worldwide that could provide the required number of animals for the planned project, wild-caught animals had to be used. For the experiments, exclusively adult Angolan free-tailed bats are captured in Côte d'Ivoire and housed locally under quarantine conditions before some of them are brought to RKI’S BSL-4 laboratory. When catching bats in urban areas, we make sure to have the acceptance of the local community, the necessary permits for bat capture and an appropriate personal protection equipment for each individual situation.
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Entry/Exit for bats into the roof of a family house. Source: RKI
Husbandry of Angolan free-tailed bats during captivity
Angolan free-tailed bats generally choose to roost in confined spaces that are also warm and dark. We have built a large enclosure (flight cage) to provide these animals with enough space to move, fly and socialize. To protect the space from light, we cover the cage with tarpaulins and provide several bat houses and other enrichments. The flight cage is located in a larger building that protects the bats from contact with other wild animals. We keep the bats in the flight cage for four weeks to train them for further handling, but more importantly to quarantine them so that we can assess the health of the animals and carry out a sampling to identify possible infections. Only the bats that react negatively to filoviruses (e.g. Ebola virus, Marburg virus, etc.) and other pathogens such as rabies are brought to our BSL-4 laboratory at the Robert Koch Institute.
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Bat House (LW 9 x 6 m) for quarantine captivity of Angolan free-tailed bats in Ivory Coast. Source: RKI
Diet of Angolan free-tailed bats during captivity
A molecular analysis of the bat feces from a colony in Côte d'Ivoire revealed a diet of various flies, moths and beetles. Although the constructed bat enclosure provides both sufficient space for bat flight and easy handling, it cannot hold these kinds of flying insects. Therefore, we train the bats to eat the non-volant larval form of beetles: mealworms (Tenebrio molitor). Switching the bat diet to mealworms is possible, but time-consuming. Importantly, we do not observe any changes in weight, social behavior or reproduction during or after the diet switch. They are also tolerant to changes in ambient temperature and humidity.
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Mealworms: alternative food for insectivorous bats since common food such as beatles and moths (flying insects) cannot be provided. Source: RKI
Working with bats in Maximum Containment
The air transportation of the living animals to Germany, which is strictly regulated by export and import regulations as well as the International Air Transport Association, was a challenging part of the project. Fortunately, the animals survived the journey well. To address best possible animal welfare and refinement for the bats, we specifically adjusted our laboratory design and handling procedures. The artificial animal housing during their time inside the BSL-4 is designed to allow the bats to live out their natural lifestyle and behavior as much as possible. They live together in a group with the best possible freedom of movement and permanent availability of food at a temperature of 27°C and relative humidity of approx. 70%. We only handle them under red light using cotton bags to minimize light stress and only when they are active (night time). The safety of staff handling infected bats is our top priority. The use of additional bite-resistant gloves when we remove them from their sleeping box, which is separated from the flight cage by a trap door, and the anaesthetization of the bats before any manipulation, as well as strict training of all staff, are basic principles for working safely in BSL-4.
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Inner transport box for bats with mealworms and cotton cloths.
Bat infection model for Ebola virus
For the animal infection experiments, bats are inoculated with a pre-determined virus dose and monitored for any clinical signs until they are finally euthanized under deep anesthesia for subsequent sample analyses, following approved animal study protocols. As expected and hypothesized, no bat showed any clinical symptoms (overall minimal burden) after infection. The performed investigation of the permissiveness of Angolan free-tailed bats to various filoviruses such as Ebola virus, Marburg virus, Taï Forest virus and Reston virus led to the following conclusions: 1) only bats inoculated with Ebola virus show high and disseminated viral replication and infectious virus shedding, while the other filoviruses fail to establish productive infections, 2) Ebola virus show placental-specific tissue tropism and a unique ability to traverse the placenta, infect and persist in foetal tissues, and 3) Ebola virus demonstrate a high genetic stability and therefore slow evolution when shifting hosts, indicating a readily virus replication in humans after spillover without requiring genetic adaptation. Our findings not only demonstrate plausible routes of horizontal and vertical transmission of Ebola virus in these bats, which are expectant of reservoir hosts, but may also reveal an ancillary transmission mechanism, potentially required for the maintenance of Ebola virus in small reservoir populations.
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For safety reasons all bats are anaesthetized before any manipulation. Source: RKI
Date: 01.02.2024
