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Abortion among dairy cattle is one of the major causes of economic losses in the livestock industry. This study describes a 1-step multiplex real-time polymerase chain reaction (PCR) to detect Brucella spp., Leptospira spp. and Campylobacter foetus, these are significant bacteria commonly implicated in bovine abortion. ß-actin was added to the same PCR reaction as an internal control to detect any extraction failure or PCR inhibition. The detection limit of multiplex real-time PCR using purified DNA from cultured organisms was set to 5 fg for Leptospira spp. and C. foetus and to 50 fg for Brucella spp. The multiplex real-time PCR did not produce any non-specific amplification when tested with different strains of the 3 pathogens. This multiplex real-time PCR provides a valuable tool for diagnosis, simultaneous and rapid detection for the 3 pathogens causing abortion in bovine. © 2014, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G.Caporale". All right reserved. Source

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

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

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

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

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

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