BackgroundTularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.ResultsCultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.ConclusionsF. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RHDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.

Red foxes served as a biological indicator for the temporal development of environmental contamination with polychlorinated biphenyls (PCB). The concentration of PCB congeners nos. 28, 49, 52, 101, 138, 153, and 180 were analyzed in the body fat of 80 foxes (Canis vulpes) from Germany. The samples were from animals that had been submitted for examination in 1983, 1987, and 1991. Throughout this time period, a reduction was seen in the concentration of the highly chlorinated biphenyls 138, 153, and 180, whereas the concentration of the low-chlorinated congeners PCB nos. 28, 49, and 52 increased. No change in contamination with congener 101 was observed. These results show a trend toward reduction of environmental contamination with highly-chlorinated biphenyls since 1983, while contamination with low-chlorinated congeners is apparently increasing. An interesting observation is the disproportionately higher amount of 2,2',3,4,4',5,5'-hepatachlorobiphenyl (PCB 180) over that of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) in body-fat samples from all foxes analyzed. This phenomenon was also observed in 10 dogs examined as controls. Based on evidence from other authors of experimental toxicological studies on beagles, it appears that the fox possesses a similar cytochrome P-450 isoenzyme that can degrade 2,4,5-trichloro-substituted aromatic compounds. As a consequence, in canines, PCB 180 which is additionally meta-chloro substituted is accumulated to a greater degree than is PCB 153.

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Ninety-one reptiles were examined for the presence of yeasts by standard protocols and pathohistological methods. Yeasts were isolated from 42 of the animals. Representatives of herbivorous families (Testudinidae and Iguanidae) carried yeasts more often than animals belonging to carnivorous taxa (Boidae and Emydidae). Yeasts were most often isolated from the gastrointestinal tract, and in 24-6 per cent of cases they could be cultured from the oral cavity and/or cloaca of living animals. Postmortem examination revealed that the intestines of 80-6 per cent of the animals carried yeasts. In all, 56 isolates, belonging to the genera Candida (32), Trichosporon (11), Torulopsis (9) and Rhodotorula (3), and one perfect yeast were obtained. The species included taxa potentially pathogenic to man. However, no sufficiently reliable criteria could be established to prove that positive culture results were associated with disease. In the reptiles examined postmortem, three cases of dermatomycosis were detected histologically. No case of organ mycosis was identified.