Martin Pfeffer

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Chikungunya Fever in Travelers: Clinical Presentation and Course

BACKGROUND An outbreak of chikungunya virus infection emerged in the southwest Indian Ocean islands in 2005, spread out to India, and resulted in an ongoing outbreak that has involved >1.5 million patients, including travelers who have visited these areas. METHODS Our study investigated 69 travelers who developed signs and symptoms compatible with chikungunya fever after returning home from countries involved in the epidemic. Twenty cases of infection that were confirmed by serological analysis, polymerase chain reaction, and/or cell culture were investigated. RESULTS All patients experienced flulike symptoms with fever and joint pain. No serious complications were observed, but 69% of the patients had persistent arthralgia for >2 months, and 13% had it for >6 months. Viral RNA could be detected in blood samples using reverse-transcriptase polymerase chain reaction in 4 of 4 patients who presented to a health care facility during their first week of illness, and the virus was successfully isolated from blood samples obtained from 2 of these patients. Chikungunya virus-specific immunoglobulin M and/or immunoglobulin G antibodies were detected in all patients. However, initial testing of serum samples yielded negative results for 3 of 5 patients during the first week. CONCLUSIONS Chikungunya fever must be considered in travelers who develop fever and arthritis after traveling to areas affected by an ongoing epidemic. Related arthritis mainly affects smaller joints and often persists for extended periods. Serological testing may have negative results during the first week of the disease; diagnosis using polymerase chain reaction appears to be more reliable during this time. Travelers to areas of epidemicity should be informed of the risk of infection and of adequate preventive measures, such as protection against mosquitos.

Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of Orthopoxvirus species by polymerase chain reaction.

A PCR protocol was established that not only allows the detection of, but also the differentiation of species of the genus Orthopoxvirus. This assay was accomplished by the selection of oligonucleotides located within the gene that encodes the A-type inclusion protein of cowpox virus. The primer pair flanked a region exhibiting distinct and specific DNA deletions in the corresponding sequences of vaccinia, mousepox, monkeypox and camelpox virus. For this reason, PCR resulted in DNA fragments of different sizes. The presented PCR protocol, combined with BglII restriction digests, allowed the unequivocal assignment of 42 orthopoxvirus (OPV) strains and isolates to the correct OPV species. The resulting classification corresponded exactly with known biological data for the OPV strains investigated. Furthermore, 13 out of 22 cowpox virus isolates could be subtyped by the presence or absence of a small BglII fragment. DNA sequencing showed that the lack of this BglII fragment was caused by a deletion of 72 nucleotides.

Usutu virus activity in Austria, 2001–2002

Usutu virus (USUV), a member of the mosquito-borne clade within the Flaviviridae family, was responsible for avian mortality in Austria in 2001. In 2002, the virus continued to kill birds, predominantly blackbirds. High numbers of avian deaths were recorded within the city of Vienna and in surrounding districts of the federal state of Lower Austria, while single die-offs were noticed in the federal states of Styria and Burgenland. A total of 72 birds were submitted for laboratory examination, 30 of which tested positive for USUV by immunohistochemistry and/or polymerase chain reaction. Laboratory-confirmed cases of USUV infection originated from the federal states of Vienna and Lower Austria only. The data show that (i) USUV has managed to overwinter and has been able to establish a transmission cycle in Austria, (ii) the virus seems to have become a resident pathogen of Austria with a tendency to spread to other geographic areas, and (iii) the surveillance of dead blackbirds is a useful sentinel system for monitoring USUV activity.

Isolation of Brucella microti from Mandibular Lymph Nodes of Red Foxes, Vulpes vulpes, in Lower Austria

From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.

Re-emergence of tularemia in Germany: Presence of Francisella tularensis in different rodent species in endemic areas

BackgroundTularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent Francisella (F.) tularensis. No systematic study on the reservoir situation of F. tularensis has been published for Germany so far.MethodsWe investigated three areas six to ten months after the initial tularemia outbreaks for the presence of F. tularensis among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation.ResultsA total of 386 small mammals were trapped. F. tularensis was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for F. tularensis. We were able to demonstrate F. tularensis-specific DNA in one of 28 water samples taken in one of the outbreak areas.ConclusionThe findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany.

Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum–Brucella group by recA and 16S rRNA gene-based comparative sequence analysis

The genetic diversity and phylogenetic interrelationships among 106 Ochrobactrum strains (O. anthropi: 72, O. intermedium: 22, O. tritici: 5, O. oryzae: 2, O. grignonense: 2, O. gallinifaecis: 1, O. lupini: 2), the type strains of the eight Brucella species and other closely related taxa were studied by recA and rrs gene (16S rRNA) comparative sequence analysis. Both markers correctly delineated the various Ochrobactrum species; however, resolution at the subspecies level was considerably higher in the recA gene-based approach. Phylogenetic analyses using neighbor-joining, parsimony, and maximum likelihood algorithms generated trees with similar topologies but the overall branching order, and also the order of the subclades, were not stable in either assay, which could be explained by generally high recA and rrs sequence similarities. Ochrobactrum and Pseudochrobactrum formed separate clades distinct from other Alphaproteobacteria with Bartonella, Agrobacterium, and Rhizobium as the closest relatives. O. gallinifaecis was the most distinct member, when compared to the type species O. anthropi, with rrs and recA similarities of 96.2% and 81.4%. Brucella species were indistinguishable, exhibiting high rrs and recA gene similarities of 98.6% and 85.5% compared with Ochrobactrum intermedium. At the protein level, all RecA sequences among the various Ochrobactrum species and between Ochrobactrum and Brucella were highly similar with only a few amino acid substitutions. O. anthropi and O. tritici were indistinguishable by means of their RecA proteins. A set of initially biochemically classified strains did not cluster within their assigned species and they either grouped within other known species or grouped as potential novel Ochrobactrum species. In further investigations, these strains were reclassified and described as novel species. In summary, Ochrobactrum is a highly diverse genus comprising several novel species. We recommend recA- in addition to rrs gene-analysis for correct species allocation and subtyping of novel Ochrobactrum isolates.

Spotted Fever Group Rickettsiae in Ticks, Germany

To explore increased risk for human Rickettsia spp. infection in Germany, we investigated recreational areas and renatured brown coal surface-mining sites (also used for recreation) for the presence of spotted fever group rickettsiae in ticks. R. raoultii (56.7%), R. slovaca (13.3%), and R. helvetica (>13.4%) were detected in the respective tick species.

Phylogeographic Structure and Evolutionary History of Sindbis Virus

Sindbis (SIN) virus, Alphavirus, is a mosquito-borne and bird-associated virus with large geographic distribution in the Old World. We investigated the genetic diversity of 59 SIN strains after limited sequencing of their E2 glycoprotein genes. The SIN strains showed maximal diversity of 22.2% at the amino acid (aa) level, and formed five tentative genotypes. The SIN-I genotype included strains from Europe and Africa. Strains from Australia and East Asia formed SIN-II and SIN-III with about 12% and 15% aa divergence from SIN-I. The only isolate from New Zealand was distinct, and constitutes the SIN-V genotype. Isolates from Azerbaijan and China formed genotype SIN-IV with 15.6%-19.1% aa divergence from SIN-I to III and SIN-V. Phylogenetic analyses indicated that Aura virus was present before the recombinant alphavirus lineage arose. This is consistent with a South American origin of the SIN complex, and argue for a spread in North America before reaching Asia and Australia, followed by westward radiation into Africa and Europe. High levels of sequence identities were observed for geographic regions belonging to the same north-south axis, whereas the east-west genetic exchange appears to be limited. The observed phylogeographic structure was confirmed by distinct aa patterns within two-thirds of the structural protein-coding region of SIN virus strains from Saudi Arabia, Asia, and Australia. The present geography of the five SIN genotypes and subclusters within SIN-I correlate with major bird migration patterns.

Genetic Evidence for Tula Virus in Microtus arvalis and Microtus agrestis Populations in Croatia

To determine the threat of hantavirus infection to U.S. Forces, small mammals were sampled from training areas within Croatia. Of the 152 samples, 20 were positive for Tula virus (TUL), 12 common voles (Microtus arvalis) and eight field voles (Microtus agrestis). Sequences from M. agrestis were found in five and sequences from M. arvalis were found in six of seven sequence groups. The high percentage of the same TUL sequences in M. agrestis and M. arvalis suggests the co-occurrence of this virus in both Microtus species is not an accident. If M. agrestis field voles were accidentally infected with TUL, the percentage of polymerase chain reaction-positive animals should be lower than that of M. arvalis. Because the infection rate in M. arvalis (11.8%) was less than half of that found in M. agrestis (27.6%), it is unlikely that the predominance of positive M. agrestis could be due to accidental exposure. It is much more likely that the Croatian virus is circulating between both rodent species.