Friedrich Loeffler Institute FLI

Greifswald, Germany

Friedrich Loeffler Institute FLI

Greifswald, Germany
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Kuczius T.,University of Munster | Wohlers J.,University of Munster | Karch H.,University of Munster | Groschup M.H.,Friedrich Loeffler Institute FLI
Experimental Neurology | Year: 2011

A human form of a prion disorder is the Creutzfeldt-Jakob disease. A hallmark of the disease is the accumulation of misfolded prion proteins (PrPSc), which exist as heterogeneous subtypes. PrPSc is formed by protein conversion from the host-encoded cellular prion (PrPC), which is expressed and modified to various isoforms. Little is known about variation in PrPC; however, it is assumed that PrPC types play important roles in the formation of PrPSc. In this study, we separated distinct human PrPC subtypes on the basis of differential protein solubilities in detergent solutions. Single and sequential application of the detergents Triton X-100, octyl-glucopyranoside and CHAPS facilitated high solubility of glycosylated PrPC isoforms, whereas high proportions of nonglycosylated PrPC remained non-soluble. Most proteins became highly soluble with laurylsarcosine and sodium dodecyl sulphate. Our findings demonstrate that the solubility characteristics of heterogeneous PrPC overlap in human brains and convey distinct solubility subtypes. Differentiation by solubility experiments can therefore provide valuable information on prion protein composition, facilitate the separation of subtypes, and offer new prospects for conversion specificity of distinct isoforms. © 2010 Elsevier Inc.


Mayer-Scholl A.,Federal Institute for Risk Assessment BfR | Draeger A.,Federal Institute for Risk Assessment BfR | Luge E.,Federal Institute for Risk Assessment BfR | Ulrich R.,Friedrich Loeffler Institute FLI | Nockler K.,Federal Institute for Risk Assessment BfR
Current Microbiology | Year: 2011

In this study we compared two routine PCR systems for the detection of Leptospira spp. and assessed their performance when directly applied to kidney samples from small mammals. Although the kappa value of 0.9 indicated a high level of agreement between the tests, the outer membrane lipoprotein gene lipl32 based PCR was more robust and showed a higher number of positive kidney samples. © 2010 Springer Science+Business Media, LLC.


Banyard A.C.,Animal Health and Veterinary Laboratories Agency AHVLA Weybridge | Horton D.L.,Animal Health and Veterinary Laboratories Agency AHVLA Weybridge | Freuling C.,Friedrich Loeffler Institute FLI | Muller T.,Friedrich Loeffler Institute FLI | And 2 more authors.
Antiviral Research | Year: 2013

Dogs are the source of more than 99% of human rabies virus infections in endemic regions. Without postexposure prophylaxis, almost all cases are fatal, making rabies the most lethal infectious disease. Tens of thousands of deaths are reported annually, but the official figures are believed to be gross underestimates. Controlling canine rabies, especially in free-ranging dogs, is the first priority to reduce the burden of human disease. Because of their limited medical infrastructure, most endemic countries lack the laboratory facilities needed to diagnose human cases of viral encephalitis. Moreover, the veterinary sectors are often unable to undertake systematic surveillance and reporting of rabies in animals. Without an adequate and functioning risk assessment system that is primed for use, rabies will remain a 'neglected' and omnipresent disease, especially in poverty-stricken regions of the world. Fortunately, experience with the elimination of canine rabies from many industrialized countries has shown that these barriers are not insurmountable. Successful rabies prevention and control strategies that prove the absence of the disease depend on laboratory-based surveillance, rapid data reporting and an adequate system of risk assessment. Future control and prevention programmes should therefore coordinate the development of these key factors, creating synergies to eliminate rabies at its animal source. This article forms part of a symposium in Antiviral Research on the global elimination of canine rabies. © 2013.


Freuling C.M.,Friedrich Loeffler Institute FLI | Muller T.F.,Friedrich Loeffler Institute FLI | Mettenleiter T.C.,Friedrich Loeffler Institute FLI
Veterinary Microbiology | Year: 2017

Aujeszkýs disease (AD, pseudorabies) is a notifiable herpesvirus infection of pigs causing substantial economic losses to swine producers. AD in pigs is controlled by the use of vaccination with inactivated and attenuated live vaccines. Starting with classically attenuated live vaccines derived from low virulent field isolates, AD vaccination has pioneered novel strategies in animal disease control by the first use of genetically engineered live virus vaccines lacking virulence-determining genes, and the concept of DIVA, i.e. the serological differentiation of vaccinated from field-virus infected animals by the use of marker vaccines and respective companion diagnostic tests. The basis for this concept has been the molecular characterization of PrV and the identification of so-called nonessential envelope glycoproteins, e.g. glycoprotein E, which could be eliminated from the virus without harming viral replication or immunogenicity. Eradication of AD using the strategy of vaccination-DIVA testing has successfully been performed in several countries including Germany and the United States. Furthermore, by targeted genetic modification PrV has been developed into a powerful vector system for expression of foreign genes to vaccinate against several infectious diseases of swine, while heterologous vector systems have been used for expression of major immunogens of PrV. This small concise review summarizes the state-of-the-art information on PrV vaccines and provides an outlook for the future. © 2016


Roedig J.V.,Max Planck Institute for Dynamics of Complex Technical Systems | Rapp E.,Max Planck Institute for Dynamics of Complex Technical Systems | Hoper D.,Friedrich Loeffler Institute FLI | Genzel Y.,Max Planck Institute for Dynamics of Complex Technical Systems | And 2 more authors.
PLoS ONE | Year: 2011

The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit. © 2011 Roedig et al.


Kronefeld M.,Friedrich Loeffler Institute FLI | Werner D.,Leibniz Center for Agricultural Landscape Research | Kampen H.,Friedrich Loeffler Institute FLI
Parasitology Research | Year: 2014

Based primarily on nucleotide polymorphisms in the internal transcribed spacer 2 (ITS2) of the ribosomal DNA, Anopheles daciae was recently described as an additional member of the Maculipennis Group of species, separate from Anopheles messeae with which it had previously been confused due to morphological and genetic similarity. Species differentiation between A. messeae and A. daciae was possible only by ITS2 polymerase chain reaction (PCR) amplification followed by DNA sequencing or RFLP analysis. In addition to its siblings, Anopheles maculipennis, Anopheles atroparvus and A. messeae, A. daciae has been shown to occur in Germany, although with limited distribution. We here describe additional collection sites for this species in Germany, showing concentrations in East Germany and the northern Upper Rhine Valley in Southwest Germany. A species-specific multiplex PCR assay is presented that is able to differentiate the four Maculipennis Group sibling species occurring in Germany plus Anopheles sacharovi, Anopheles melanoon and Anopheles labranchiae. The correct identification and detailed knowledge of the biology of A. daciae are of relevance since it might be a vector of disease agents, as suggested by the vector potential of its siblings and the recent finding of an A. daciae female infected with Dirofilaria repens in southern Germany. © 2014 Springer-Verlag.


Lehmann K.,Humboldt University of Berlin | Werner D.,Leibniz Center for Agricultural Landscape Research | Hoffmann B.,Friedrich Loeffler Institute FLI | Kampen H.,Friedrich Loeffler Institute FLI
Parasites and Vectors | Year: 2012

Background: Biting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV) in northern and central Europe during the 2006 outbreak of bluetongue disease (BT). Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV) in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI) gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus) plus the isomorphic species C. dewulfi. Methods. Biting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer. Results: The newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic band for each species, could be realized with high specificity and sensitivity. Conclusion: To elucidate the biological characteristics of potential vectors of disease agents, such as ecology, behaviour and vector competence, and the role of these haematophagous arthropods in the epidemiology of the diseases, simple, cost-effective and, most importantly, reliable identification techniques are necessary. The PCR assay presented will help to identify culicoid vector species and therefore add to bluetongue and Schmallenberg disease research including vector control and monitoring. © 2012 Lehmann et al.; licensee BioMed Central Ltd.


Hubner K.,Friedrich Loeffler Institute FLI | Hubner K.,University of Melbourne | Phi-van L.,Friedrich Loeffler Institute FLI
Biochimica et Biophysica Acta - Gene Regulatory Mechanisms | Year: 2010

The 5′-flanking region of the chicken glioma-amplified sequence (GAS) 41 gene is close to the 3′ end of the lysozyme gene and contains no typical TATA box, but several GC boxes. In this study, we have localized the GAS 41 promoter to this narrow region. Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses revealed that Sp1 and Sp3 bind to this promoter. Mapping by a technique of indirect end labeling demonstrated that the Sp1-binding sites contained in this region exactly co-map with two previously identified DNase I hypersensitive (HS) sites, which suggests the important role of Sp1 binding in maintaining an open chromatin structure of the GAS41 promoter. We further found that Sp1 and Sp3 strongly activate CAT expression controlled by the putative GAS41 promoter in Drosophila Schneider S2 cells and that deletion of the Sp1 sites resulted in a loss of promoter activity in chicken HD11 cells. The results indicate that transcription factors of the Sp family play an important role in the transcriptional regulation of the chicken GAS41 gene. © 2010 Elsevier B.V.


Wernike K.,Institute of Diagnostic Virology | Breithaupt A.,Friedrich Loeffler Institute FLI | Keller M.,Institute of Novel and Emerging Infectious Diseases | Hoffmann B.,Institute of Diagnostic Virology | And 2 more authors.
PLoS ONE | Year: 2012

Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Europe in late 2011. It causes mild and transient disease in adult ruminants, but fetal infection can lead to abortion or severe malformations. There is considerable demand for SBV research, but in vivo studies in large animals are complicated by their long gestation periods and the cost of high containment housing. The goal of this study was to investigate whether type I interferon receptor knock-out (IFNAR-/-) mice are a suitable small animal model for SBV. Twenty IFNAR-/- mice were inoculated with SBV, four were kept as controls. After inoculation, all were observed and weighed daily; two mice per day were sacrificed and blood, brain, lungs, liver, spleen, and intestine were harvested. All but one inoculated mouse lost weight, and two mice died spontaneously at the end of the first week, while another two had to be euthanized. Real-time RT-PCR detected large amounts of SBV RNA in all dead or sick mice; the controls were healthy and PCR-negative. IFNAR-/- mice are susceptible to SBV infection and can develop fatal disease, making them a handy and versatile tool for SBV vaccine research. © 2012 Wernike et al.


Pietsch C.,University of Basel | Kersten S.,Friedrich Loeffler Institute FLI | Burkhardt-Holm P.,University of Basel | Valenta H.,Friedrich Loeffler Institute FLI | Danicke S.,Friedrich Loeffler Institute FLI
Toxins | Year: 2013

The control of mycotoxins is a global challenge not only in human consumption but also in nutrition of farm animals including aquatic species. Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZEN), are common contaminants of animal feed but no study reported the occurrence of both mycotoxins in fish feed so far. Here, we report for the first time the occurrence of DON and ZEN in samples of commercial fish feed designed for nutrition of cyprinids collected from central Europe. A maximal DON concentration of 825 μg kg-1 feed was found in one feed whereas average values of 289 μg kg-1 feed were noted. ZEN was the more prevalent mycotoxin but the concentrations were lower showing an average level of 67.9 μg kg-1 feed. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

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