Rodentibacter haemolyticus sp. nov. isolated from laboratory rodents.

Nine strains of a Rodentibacter-related bacterium were isolated over a period of 38 years from a laboratory mouse (Mus musculus), seven laboratory rats (Rattus norvegicus) and a Syrian hamster (Mesocricetus auratus) in Düsseldorf and Heidelberg, Germany. The isolates are genotypically and phenotypically distinct from all previously described Rodentibacter species. Sequence analysis of 16S rRNA and rpoB gene sequences placed the isolates as a novel lineage within the genus Rodentibacter. In addition to the single-gene analysis, the whole genome sequence of the strain 1625/19T revealed distinct genome-to-genome distance values to the other Rodentibacter species. The genomic DNA G+C content of strain 1625/19T was 40.8 mol% within the range of Rodentibacter. At least six phenotypic characteristics separate the new isolates from the other Rodentibacter species, with Rodentibacter heylii being the most closely related. In contrast to the latter, the new strains display ß-haemolysis and are ß-glucuronidase, d-mannitol and sorbitol positive, but fail to produce lysine decarboxylase and trehalose. The genotypic and phenotypic differences between the novel strains and the other closely related strains of the genus Rodentibacter indicate that they represent a novel species within the genus Rodentibacter, family Pasteurellaceae, for which the name Rodentibacter haemolyticus sp. nov. is proposed. The type strain 1625/19T, (=DSM 111151T=CCM 9081T), was isolated in 2019 from the nose of a laboratory mouse (Mus musculus) in Düsseldorf, Germany.

Differentiation among the most important Rodentibacter species by multiplex PCR assays targeting the ITSile+ala sequences of the rRNA operons.

Screening for the Rodentibacter species is part of the microbiologic quality assurance programs of laboratory rodents all over the world. Nevertheless, currently there are no PCR amplification techniques available for the diagnostic of R. ratti, R. heidelbergensis and of a Rodentibacter related ß-haemolytic taxon. The aim of this study was to utilize the differences in the sequence of the Internal Transcribed Spacer (ITS) regions of R. pneumotropicus, R. heylii, R. ratti, R. heidelbergensis and of the ß-haemolytic Rodentibacter taxon for the design of specific PCR assays for these species. The ITSile+ala sequence variations allowed the design of specific forward and reverse primers for each species included, that could be combined in different multiplex assays. The performance characteristics specificity and sensitivity registered for each primer pair against a diverse collection of Pasteurellaceae isolated from rats and mice and of further non-Pasteurellaceae strains was 100% for all five Rodentibacter species included. In addition, the PCR assays displayed high limits of detection and could be successfully used for detection of Rodentibacter spp. DNA in clinical swabs of laboratory mice and rats. Overall, the assays described here represent the first PCRs able to diagnose R. ratti, R. heidelbergensis and the ß-haemolytic Rodentibacter taxon, whose diagnostic to species level could further facilitate better understanding of their geographic distribution, prevalence, and biology in the future.

Differentiation Among Rodentibacter Species Based on 16S-23S rRNA Internal Transcribed Spacer Analysis.

The internal transcribed spacer (ITS) regions of Rodentibacter pneumotropicus, R. heylii, R. rarus, R. ratti, and R. heidelbergensis and of a Rodentibacter- related ß-hemolytic Pasteurellaceae taxon isolated from laboratory rodents were studied for their feasibility to discriminate among these species. The 6 species analyzed showed species-specific ITS patterns that were shared by the type strains and clinical isolates and that allowed their identification. Nevertheless, differentiating between the ITS band patterns of R. pneumotropicus and R. ratti is visually challenging. In all species tested, sequence analysis of the ITS fragments revealed a larger ITSile+ala, which contained the genes for tRNAIle(GAU) and tRNA Ala(UGC), and a smaller ITSglu with the tRNAGlu(UUC) gene. The ITS sequences varied among the 6 species evaluated, displaying identity levels ranging from 62% to 86% for ITSile+ala and 68% to 90% for ITSglu. Overall, ITS amplification proved to be a reliable method to differentiate among these important Pasteurellaceae species of laboratory rodents. Moreover, the ITS sequence variations recorded here might facilitate the design of probes for specific identification of these species. The ability to diagnose these organisms to the species level could increase our understanding of their clinical significance.

Molecular Epidemiology of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in Wild, Captive and Laboratory Rats: Effect of Habitat on the Nasal S. aureus Population.

Rats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Free-living wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S.aureus lineages-many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S.aureus, respectively.

Development of multi locus sequence typing (MLST) of Rodentibacter pneumotropicus.

The aim of the investigation was to develop a definitive typing system for Rodentibacter pneumotropicus. A total of 79 strains including the type strain of R. pneumotropicus, all associated with rodents were used to develop a multi-locus sequence typing scheme (MLST). Primers were designed for conserved regions of seven house-keeping genes (atpG, frdB, gdh, pgi, pmi, recA, zwf) and internal fragments of 399-839 bp were sequenced for all strains. The genes were also extracted in full length from whole genomic sequences of 14 strains of which 10 were sequenced in the current study. The number of alleles at the different loci ranged from 5 to 7 and a total of 20 allelic profiles or sequence types were recognized amongst the 79 strains. Analysis of the MLST data showed that some STs have been stable over many years probably circulating in the same colonies and probably transferred between colonies. We assume that this MLST scheme may provide a high level of resolution and might be an excellent tool for studying the population structure and epidemiology of R. pneumotropicus. Further development of the scheme is expected by including more genes and more strains and involve whole genomic sequencing.

Reclassification of Bisgaard taxon 5 as Caviibacterium pharyngocola gen. nov., sp. nov. and Bisgaard taxon 7 as Conservatibacter flavescens gen. nov., sp. nov.

A total of 29 strains mainly from guinea pigs were investigated by a polyphasic approach that included previously published data. The strains were classified as Bisgaard taxa 5 and 7 by comparison of phenotypic characteristics and the strains showed typical cultural characteristics for members of family Pasteurellaceae and the strains formed two monophyletic groups based on 16S rRNA gene sequence comparison. Partial rpoB sequence analysis as well as published data on DNA-DNA hybridization showed high genotypic relationships within both groups. A new genus with one species, Caviibacterium pharyngocola gen. nov., sp. nov., is proposed to accommodate members of taxon 5 of Bisgaard, whereas members of taxon 7 are proposed as Conservatibacter flavescens gen. nov., sp. nov. The two genera are clearly separated by phenotype from each other and from existing genera of the family Pasteurellaceae. The type strain of Caviibacterium pharyngocola is 7.3T (=CCUG 16493T=DSM 105478T) and the type strain of Conservatibacter flavescens is 7.4T (=CCUG 24852T=DSM 105479T=HIM 794-7T), both were isolated from the pharynx of guinea pigs.

Global spread of mouse-adapted Staphylococcus aureus lineages CC1, CC15, and CC88 among mouse breeding facilities.

We previously reported that laboratory mice from all global vendors are frequently colonized with Staphylococcus aureus (S. aureus). Genotyping of a snap sample of murine S. aureus isolates from Charles River, US, showed that mice were predominantly colonized with methicillin-sensitive CC88 strains. Here, we expanded our view and investigated whether laboratory mice from other global animal facilities are colonized with similar strains or novel S. aureus lineages, and whether the murine S. aureus isolates show features of host adaptation. In total, we genotyped 230 S. aureus isolates from various vendor facilities of laboratory mice around the globe (Charles River facilities in the USA, Canada, France, and Germany; another US facility) and university- or company-associated breeding facilities in Germany, China and New Zealand. Spa typing was performed to analyse the clonal relationship of the isolates. Moreover, multiplex PCRs were performed for human-specific virulence factors, the immune-evasion cluster (IEC) and superantigen genes (SAg). We found a total of 58 different spa types that clustered into 15 clonal complexes (CCs). Three of these S. aureus lineages had spread globally among laboratory mice and accounted for three quarters of the isolates: CC1 (13.5%), CC15 (14.3%), and CC88 (47.0%). Compared to human colonizing isolates of the same lineages, the murine isolates frequently lacked IEC genes and SAg genes on mobile genetic elements, implying long-term adaptation to the murine host. In conclusion, laboratory mice from various vendors are colonized with host-adapted S. aureus-strains of a few lineages, predominantly the CC88 lineage. S. aureus researchers must be cautioned that S. aureus colonization might be a relevant confounder in infection and vaccination studies and are therefore advised to screen their mice before experimentation.

Development of a multiplex serological assay reveals a worldwide distribution of murine astrovirus infections in laboratory mice.

Laboratory mice play a tremendous role in biomedical research in studies on immunology, infection, cancer and therapy. In the course of standardization of mice used in animal experiments, health monitoring constitutes an important instrument towards microbiological standardization. Infections with murine astroviruses (MuAstV) were only recently discovered and are, therefore, still relatively unknown in laboratory animal science. In rodent health monitoring viral infections within a population are commonly assessed in terms of specific antibodies by serological testing, as active infection and excretion of virus is often temporary and can easily be missed. So far only ongoing infections with astroviruses can be detected by PCR. The objective of this work was the development of a sensitive and specific MuAstV multiplex serological assay with a high-throughput capability to be used in routine testing of laboratory mice. Four different MuAstV proteins were recombinantly expressed and used as antigens. The best reacting antigen, the capsid spike protein VP27, was selected and tested with a panel of 400 sera of mice from units with a known MuAstV status. Assay sensitivity and specificity resulted in 98.5% and 100%, respectively, compared to RT-PCR results. Eventually this assay was used to test 5529 serum samples in total, during routine diagnostics at the German Cancer Research Center (DKFZ) in Heidelberg between 2015 and 2017. High sero-prevalence rates of up to 98% were detected in units with open cages indicating that the virus is highly infectious and circulates within these populations virtually infecting all animals regardless of the mouse strain. In addition, data collected from 312 mice purchased from commercial breeders and from 661 mice from 58 research institutes in 15 countries worldwide allowed the conclusion that MuAstV is widespread in contemporary laboratory mouse populations.

Rodentibacter gen. nov. including Rodentibacter pneumotropicus comb. nov., Rodentibacter heylii sp. nov., Rodentibacter myodis sp. nov., Rodentibacter ratti sp. nov., Rodentibacter heidelbergensis sp. nov., Rodentibacter trehalosifermentans sp. nov., Rodentibacter rarus sp. nov., Rodentibacter mrazii and two genomospecies.

Rodentibacter gen. nov. is proposed based on isolation and phenotypic characterization of strains, predominantly from rodents. The strains showed 86 % or higher rpoB gene sequence similarity and indicated a genus-level relationship within Pasteurellaceae. The strains compared at 16S rRNA gene sequence level showed 93.8 % or higher similarity, and their genus-level relationship within Pasteurellaceae was confirmed by phenotypic analysis. The type species Rodentibacter pneumotropicus comb. nov. is reclassified from [Pasteurella] pneumotropica with type strain NCTC 8141T (=CCUG 12398T). Whole genomic comparison allowed the estimation of DNA-DNA renaturation. Rodentibacter heylii sp. nov. was proposed for a group that included the biovar Heyl of [Pasteurella] pneumotropica with the type strain ATCC 12555T (=CCUG 998T). A group was proposed as Rodentibacter ratti sp. nov., which included the taxon 22 of Bisgaard; the type strain is F75T (=CCUG 69665T=DSM 103977T). Taxon 41 of Bisgaard was proposed as Rodentibacter myodis sp. nov. with type strain Ac151T (=CCUG 69666T=DSM 103994T). Rodentibacter heidelbergensis sp. nov. included the type strain 1996025094T (=Ac69T) (=CCUG 69667T=DSM 103978T). A group strains of was proposed as Rodentibacter trehalosifermentans sp. nov. with type strain H1987082031T (=CCUG 69668T=DSM 104075T). Two strains including the reference strain of taxon 17 of Bisgaard that showed 16S rRNA gene similarity of 97.3 % were proposed as Rodentibacter rarus sp. nov. 2325/79T (=CCUG 17206T=DSM 103980T). Rodentibacter mrazii sp. nov. was proposed with type strain Ppn418T (Bisgaard taxon 21) (=CCUG 69669T=DSM 103979T). The eight species could be separated based on phenotypic characteristics such as NAD requirement, ornithine decarboxylase and indole formation, α-glucosidase, ß-galactosidase and in acid formation from (+)-l-arabinose, (-)-d-ribose, (+)-d-xylose, myo-inositol, (-)-d-mannitol, lactose, melibiose and trehalose. Forty-six strains including taxon 48 of Bisgaard formed a monophyletic group by rpoB and 16S rRNA gene sequence analysis, but could not be separated phenotypically from R. pneumotropicus and R. heylii, and it was left as an unnamed genomospecies 1 of Rodentibacter with reference strain Ppn416. Another taxon that included 13 strains, mainly isolated from Apodemus sylvaticus, could not be separated phenotypically from R. pneumotropicus or R. heylii and was designated as genomospecies 2. Strain Ppn85 with 95 % or less rpoB gene sequence similarity and with 16S rRNA gene sequence similarity of 97 % or less to the other members of Rodentibacter was left as an unnamed singleton.

Acute Tetraplegia Caused by Rat Bite Fever in Snake Keeper and Transmission of Streptobacillus moniliformis.

We report acute tetraplegia caused by rat bite fever in a 59-year old man (snake keeper) and transmission of Streptobacillus moniliformis. We found an identical characteristic bacterial pattern in rat and human samples, which validated genotyping-based evidence for infection with the same strain, and identified diagnostic difficulties concerning infection with this microorganism.

Phylogenetic and comparative genomics of the family Leptotrichiaceae and introduction of a novel fingerprinting MLVA for Streptobacillus moniliformis.

BACKGROUND: The Leptotrichiaceae are a family of fairly unnoticed bacteria containing both microbiota on mucous membranes as well as significant pathogens such as Streptobacillus moniliformis, the causative organism of streptobacillary rat bite fever. Comprehensive genomic studies in members of this family have so far not been carried out. We aimed to analyze 47 genomes from 20 different member species to illuminate phylogenetic aspects, as well as genomic and discriminatory properties. RESULTS: Our data provide a novel and reliable basis of support for previously established phylogeny from this group and give a deeper insight into characteristics of genome structure and gene functions. Full genome analyses revealed that most S. moniliformis strains under study form a heterogeneous population without any significant clustering. Analysis of infra-species variability for this highly pathogenic rat bite fever organism led to the detection of three specific variable number tandem analysis loci with high discriminatory power. CONCLUSIONS: This highly useful and economical tool can be directly employed in clinical samples without laborious prior cultivation. Our and prospective case-specific data can now easily be compared by using a newly established MLVA database in order to gain a better insight into the epidemiology of this presumably under-reported zoonosis.

Approved and novel strategies in diagnostics of rat bite fever and other Streptobacillus infections in humans and animals.

Rat bite fever (RBF), a worldwide occurring and most likely under-diagnosed zoonosis caused by Streptobacillus moniliformis, represents the most prominent disease of Streptobacillus infections. Recently, novel members have been described, from which a reservoir in rats and other animal species and a zoonotic potential can be assumed. Despite regularly published case reports, diagnostics of RBF continues to represent a 'diagnostic dilemma', because the mostly applied 16S rRNA sequence analysis may be uncertain for proper pathogen identification. Virtually nothing is known regarding prevalence in humans and animal reservoirs. For a realistic assessment of the pathogen's spread, epidemiology and virulence traits, future studies should focus on the genomic background of Streptobacillus. Full genome sequence analyses of a representative collection of strains might facilitate to unequivocally identify and type isolates. Prevalence studies using selective enrichment mechanisms may also enable the isolation of novel strains and candidate species of this neglected group of microorganisms.

Streptobacillus ratti sp. nov., isolated from a black rat (Rattus rattus).

An indole-, oxidase- and catalase-negative, non-motile bacterium, strain OGS16T, was isolated from an oral swab of a feral black rat (Rattus rattus) in 2007 in Japan. It stained Gram-negative and had pleomorphic, rod-shaped, non-spore-forming cells. Based on 16S rRNA gene sequence analyses, strain OGS16T was assigned to the genus Streptobacillus, with 16S rRNA gene sequence similarities of 99.3, 99.0, 98.6 and 95.5% to the type strains of Streptobacillus moniliformis, Streptobacillus notomytis, Streptobacillus felis and Streptobacillus hongkongensis, respectively. Strain OGS16T could also be differentiated clearly from other species of the genus Streptobacillus by rpoB, groEL and recA nucleotide and deduced amino acid sequence analysis. DNA-DNA relatedness as obtained by average nucleotide identity was 89.10% between strain OGS16T and Streptobacillus moniliformis DSM 12112T. Chemotaxonomic and physiological data for strain OGS16T were congruent with results for other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in discriminating strain OGS16T unequivocally from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus ratti sp. nov., with the type strain OGS16T (=JCM 31098T=DSM 101843T). The G+C content of the DNA of the type strain is 25.9 mol% and the genome size is 1.50 Mbp.

Streptobacillus notomytis sp. nov., isolated from a spinifex hopping mouse (Notomys alexis Thomas, 1922), and emended description of Streptobacillus Levaditi et al. 1925, Eisenberg et al. 2015 emend.

A pleomorphic, Gram-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium was isolated in 1979 from the heart of a spinifex hopping mouse (Notomys alexis Thomas, 1922) with septicaemia and stored as Streptobacillus moniliformis in the strain collection of the Animal Health Laboratory, South Perth, Western Australia (AHL 370-1), as well as under CCUG 12425. On the basis of 16SrRNA gene sequence analyses, the strain was assigned to the genus Streptobacillus, with 99.4 % sequence similarity to the type strain of Streptobacillus moniliformis, 95.6 %sequence similarity to the type strain of Streptobacillus hongkongensis and 99.0 %sequence similarity to the type strain of Streptobacillus felis. The clear differentiation of strain AHL 370-1T from Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis was also supported by rpoB, groEL and recA nucleotide and amino acid sequence analysis. Average nucleotide identity was 87.16 % between strain AHL 370-1T and Streptobacillus moniliformis DSM 12112T. Physiological data confirmed the allocation of strain AHL 370-1T to the family Leptotrichiaceae, considering the very similar profiles of enzyme activities and fatty acids compared to closely related species. Within the genus Streptobacillus,isolate AHL 370-1T could also be separated unambiguously from the type strains of Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis by MALDI-TOF mass spectrometry. Two further strains (KWG2 and KWG24) isolated from asymptomatic black rats in Japan were highly similar to AHL 370-1T. On the basis of these data, we propose the novel species Streptobacillus notomytis sp. nov., with the type strain AHL370-1T (=CCUG 12425T=DSM 100026T=CCM 8593T=EF 12425T).

Maintaining and Monitoring the Defined Microbiota Status of Gnotobiotic Rodents.

Gnotobiotic (germfree, defined colonized) rodents have become powerful tools to advance our understanding of the host-microbiome relationship. However, the maintenance and ultimately the monitoring of gnotobiotic rodents is a critical, labor-intensive, and costly process (e.g., sterility, not absence of specific pathogens, must be demonstrated in germfree animals). Here, we provide information on the housing and maintenance of gnotobiotic animals, elucidate prophylactic measurements to avoid contamination, and make specific recommendations for sampling procedures, sampling frequencies, and test methods.

Reclassification of Actinobacillus muris as Muribacter muris gen. nov., comb. nov.

To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains, mainly from mice and rats, were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole-genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intragroup similarities of 96.7 and 97.2 % for the 16S rRNA and rpoB genes, respectively. The highest 16S rRNA gene sequence similarity to a taxon with a validly published name outside the group was 95.9 %, to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium', with 88.4 % similarity. A new genus and a new combination, Muribacter muris gen. nov., comb. nov., are proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons, with major divergence from the existing genera of the family Pasteurellaceae. The new genus has the characteristics of [A.] muris with the emendation that acid formation from ( - )-d-mannitol and hydrolysis of aesculin are variable, while the α-glucosidase test is positive. There is no requirement for exogenously supplied NAD (V factor) for the majority of strains investigated; however, one strain was found to require NAD. The major fatty acids of the type strain of Muribacter muris were C14 : 0, C14 : 0 3-OH/iso-C16 : 1 I, C16 : 1ω7c and C16 : 0, which is in line with most genera of the Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T).

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus.

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Streptobacillus felis sp. nov., isolated from a cat with pneumonia, and emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis.

A pleomorphic, Gram-stain-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium (strain 131000547(T)) was isolated from the lungs of a cat with pneumonia. On the basis of 16S rRNA gene sequence analyses the strain was assigned to the genus Streptobacillus with 97.6% sequence similarity to the type strain of Streptobacillus moniliformis and 94.6% to that of Streptobacillus hongkongensis. The clear differentiation of strain 131000547(T) from Streptobacillus moniliformis and Streptobacillus hongkongensis was also supported by gyrB, groEL, and recA nucleotide and amino acid sequence analysis. DNA-DNA hybridization demonstrated ≤ 19.9% (reciprocal 28.7%) DNA-DNA relatedness between strain 131000547(T) and Streptobacillus moniliformis DSM 12112(T). Physiological data confirmed the allocation of strain 131000547(T) to the family Leptotrichiaceae. Strain 131000547(T) has a unique profile of enzyme activities allowing differentiation from the most closely related species. Within the genus Streptobacillus, isolate 131000547(T) could also unambiguously be separated from Streptobacillus moniliformis and Streptobacillus hongkongensis by both matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Fourier transform-infrared spectroscopy. On the basis of these data, a novel species of the genus Streptobacillus, Streptobacillus felis sp. nov., is proposed with the type strain 131000547(T) ( = DSM 29248(T) = CCUG 66203(T) = CCM 8542(T)). Emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis are also given.

Stability of cryopreserved samples of mutant mice.

Genetically modified animals are unique models with enormous scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if a sufficient number of samples have been cryopreserved. To maintain the opportunity to recover a line, it is mandatory to assess the quality of the cryopreserved samples and to assure safe long-term storage conditions. Here, we investigated the revitalization rate of cryopreserved pre-implantation embryos stored in-house up to 158 months, of imported (and shipped) embryos, and of embryos received after in vitro fertilization. The storage period did not affect the revitalization rate, whereas the recovery of imported embryos was significantly reduced, possibly due to shipment conditions. The genotypes of genetically modified pups received following embryo-transfer were slightly smaller than expected by Mendelian laws. Intensive investigations of the hygienic state of the cryopreserved samples and the equipment used never showed microbiological contamination of a sample within a cryo-tube. However, environmental organisms were found frequently in the permanent freezers and dry shippers used. Since such contamination cannot be completely excluded and an embryo-transfer might not lead in all cases to a secure rederivation, foster mothers and revitalized pups should be housed in an intermediate facility and their health assessed before introducing them into the target facility.

A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.