Highly Pathogenic Avian Influenza Virus (H5N1) in Frozen Duck Carcasses, Germany, 2007

Article summary line: Phylogenetic and epidemiologic evidence shows incursion of HPAIV into the food chain

A Conserved Insertion in Protein-primed DNA Polymerases is Involved in Primer Terminus Stabilisation

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or α-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of ø29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3′–5′ exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in ø29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.This investigation has been aided by research grant 5R01 GM27242-23 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigación Cientı́fica y Técnica, by grant ERBFMRX CT97 0125 from the European Union, and by an institutional grant from Fundación Ramón Areces to the Centro de Biologı́a Molecular “Severo Ochoa”. E.D. was a post-doctoral fellow of the European Union.Peer reviewe

A potential novel Brucella species isolated from mandibular lymph nodes of red foxes in Austria

International audienceThe wild red fox (Vulpes vulpes) is a known indicator species for natural foci of brucellosis. Here, we describe phenotypic and molecular characteristics of two atypical Brucella strains isolated from two foxes hunted 2008 in Eastern Austria. Both strains agglutinated with monospecific anti-Brucella A serum and were positive in ELISA with monoclonal antibodies directed against various Brucella lipopolysaccharide epitopes. However, negative nitrate reductase- and negative oxidase-reaction were atypical traits. Affiliation to the genus Brucella was confirmed by 16S rRNA gene sequencing and by detection of the Brucella specific insertion element IS711 and gene bcsp31 using real-time PCR. Both fox strains showed identical IS711 Southern blot profiles but were distinct from known brucellae. The number of IS711 copies detected was as high as found in B. ovis or marine mammal Brucella strains. Molecular analyses of the recA and omp2a/b genes suggest that both strains possibly represent a novel Brucella species

Оксана Козловская работает по специальности

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a and omp2b gene sequences, and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by R/C B. microti exhibited high metabolic activities similar to those closely related soil organisms such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be matter of gene regulation rather than a matter of gene configuration

Brucella inopinata sp. nov., isolated from a breast implant infection

International audienceA Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1(T)) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1(T) to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1(T) harboured four to five copies of the Brucella-specific insertion element IS 711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1(T) reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1(T) showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1(T) differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1(T) displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1( T) was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1(T) (=BCCN 09-01(T)=CPAM 6436(T)) is proposed