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Selim A.M.,Benha University | Gaede W.,State Institute for Consumer Protection of Saxony Anhalt
Asian Journal of Animal and Veterinary Advances | Year: 2015

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne´s disease) in ruminants. The infected and carrier animals shed the microorganism intermittently in faeces. In order to ensure the sensitive identification of MAP-shedders by the examination of faecal samples. Real-time PCR assays were compared which amplify the insertion sequences IS900 and ISMAV2 and furthermore the genomic element F57. The assays were designed as duplex-PCR including the amplification of PUC19-plasmid as internal control. The analytical sensitivity of the assays was determined using DNA of 6 different isolates of MAP in broad linear range (50 ng-5 fg μLG-1). The specificity was validated using 23 known species and subspecies of the Mycobacteriacea and 18 other non-Mycobacteriacea pathogens. The sensitivity for detection of MAP-DNA was 5 fg/ reaction targeting IS900. Reproducible detection limit for real-time PCR targeting ISMAV2 and F57 was 50 fg reaction. All Mycobacteriacea different from MAP and non-Mycobacteriacea gave negative results for ISMAV2 and F57 sequence. For IS900 weak positive signals were observed with highly concentrated DNA (5 ng μLG-1) of 3 Mycobacterium avium subsp. avium strains from cattle and poultry but not with low concentrated DNA (5 fg μLG-1). Thus false-positive results should not be found if analyzing ruminant faeces directly with IS900-PCR. ISMAV2-PCR and F57-PCR has to be preferred to IS900-PCR if real-time PCR is intended for the specification of cultured Mycobacteria. © 2015 Academic Journals Inc. Source


Granzow H.,Institute of Infectology | Fichtner D.,Institute of Infectology | Schutze H.,Institute of Infectology | Lenk M.,Friedrich Loeffler Institute | And 3 more authors.
Journal of Fish Diseases | Year: 2014

Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100-350 nm after negative staining and 100-300 nm in ultrathin sections, covered by short projections of 8-10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family. © 2013 John Wiley & Sons Ltd. Source


Ziegler U.,Friedrich Loeffler Institute | Angenvoort J.,Friedrich Loeffler Institute | Klaus C.,Friedrich Loeffler Institute | Nagel-Kohl U.,Food and Veterinary Institute Braunschweig Hanover | And 8 more authors.
International Journal of Environmental Research and Public Health | Year: 2013

West Nile virus (WNV) is a mosquito-borne viral pathogen of global importance and is considered to be the most widespread flavivirus in the World. Horses, as dead-end hosts, can be infected by bridge mosquito vectors and undergo either subclinical infections or develop severe neurological diseases. The aim of this study was to detect WNV specific antibodies in horses in Germany as an indicator for an endemic circulation of WNV. Sera from more than 5,000 horses (primarily fallen stock animals) were collected in eight different federal states of Germany from 2010 to 2012. Sera were screened by a competitive ELISA and positive reactions were verified by an indirect IgM ELISA and/or by virus neutralization tests (VNT) for WNV and Tick-borne encephalitis virus (TBEV) in order to exclude cross-reacting antibody reactions. In essence WNV specific antibodies could not be detected in any of the horse sera. Not surprisingly, a small number of sera contained antibodies against TBEV. It is noteworthy that equine sera were often collected from horse carcasses and therefore were of poor quality. Nonetheless, these sera were still suitable for WNV ELISA testing, i.e., they did not produce a high background reaction which is a frequently observed phenomenon. According to these data there is no evidence for indigenous WNV infections in horses in Germany at present. © 2013 by the authors; licensee MDPI, Basel, Switzerland. Source


Hoffmann B.,Institute of Diagnostic Virology | Tappe D.,Friedrich Loeffler Institute | Tappe D.,German Center for Infection Research | Hoper D.,Institute of Diagnostic Virology | And 15 more authors.
New England Journal of Medicine | Year: 2015

Between 2011 and 2013, three breeders of variegated squirrels (Sciurus variegatoides) had encephalitis with similar clinical signs and died 2 to 4 months after onset of the clinical symptoms. With the use of a metagenomic approach that incorporated next-generation sequencing and real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), the presence of a previously unknown bornavirus was detected in a contact squirrel and in brain samples from the three patients. Phylogenetic analyses showed that this virus, tentatively named variegated squirrel 1 bornavirus (VSBV-1), forms a lineage separate from that of the known bornavirus species. Copyright © 2015 Massachusetts Medical Society. Source


Selim A.,Benha University | El-Haig M.,Suez Canal University | Galila E.S.,Benha University | Gaede W.,State Institute for Consumer Protection of Saxony Anhalt
Animal Science Papers and Reports | Year: 2013

The study aimed at direct detection of Mycobacterium avium subsp. Paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng - 5 fg/μl). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriaceae were tested. Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/μl and 50 fg/μl respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/μl of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/μl). To maximize the assay's detection sensitivity, an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large-scale analysis of milk samples and other clinical specimens from man and animals. Source

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