Swollen Ampulla as an Indicator of Successful Pregnancy in B6C3F1 Recipient Mice used for Assisted Reproduction.

In vitro fertilization (IVF), embryo cryopreservation, and embryo transfer (ET) are assisted reproductive technologies (ARTs) that are used extensively for the maintenance of mouse models in animal research. Inbred mouse strains with different genetic backgrounds vary in their reproductive performance. Cryopreservation can affect embryo quality and viability, and the genetic background of ET recipients can influence the ET result. In this retrospective study, we analyzed the out- comes of ETs performed in our facility during the last 6 y. We found that B6C3F1 mice with swollen ampullae show almost 3-fold higher pregnancy rates than mice with nonswollen ampullae when either freshly isolated or frozen-thawed embryos are implanted. Implantation of freshly collected embryos in recipients with swollen ampullae led to significantly higher pregnancy rates in comparison to implantation of frozen-thawed embryos, regardless of whether the latter were fertilized in vivo or in vitro. Moreover, we found a significant effect of genetic background on the birth rate; C57BL/6J mice and mice with a mixed genetic background had 34% higher birth rates than did C57BL/6N mice. Within the C57BL/6J group, the birth rates were significantly higher when using fresh in vivo-fertilized embryos, and cryopreservation negatively affected both in vivo- and in vitro-fertilized embryos. The success rate of obtaining one living pup was not significantly different between frozen-thawed and fresh embryos. Overall, a swollen ampulla is a strong indicator for a successful pregnancy, together with the embryo manipulation and genetic background. A better understanding of the factors that affect the reproductive outcome might lead to optimization of the ART protocols and contribute to a reduction in the number of mice used for these procedures.

Genomic and pathogenic characterization of RTX toxin producing Rodentibacter sp. that is closely related to Rodentibacter haemolyticus.

Rodentibacter spp. are opportunistic pathogens that are often isolated from the upper respiratory tracts of laboratory rodents. In particular, R. pneumotropicus and R. heylii require considerable caution in rodent colonies, as they cause lethal pneumonia in rodents. A new species, R. haemolyticus, has recently been classified in the genus, and a very closely related strain, Rodentibacter sp. strain JRC, has been isolated in Japan. This study focused on strain JRC by performing genomic and pathogenic analyses. Draft genome sequencing of strain JRC identified several genes coding for putative virulent proteins, including hemolysin and adhesin. Furthermore, we found a new RTX (repeats-in-structural toxin) toxin gene in the genome, which was predicted to produce a critical virulence factor (RTXIA) similar to Enterobacteriaceae. The concentrated culture supernatant containing RTX toxin (RTXIA) showed cytotoxicity toward RAW264.7 cells. Pre-incubation with anti-CD11a attenuated the cytolysis, suggesting that the concentrated culture supernatant containing RTXIA is cell surface LFA-1 mediated cytolysin. Experimental infection of strain JRC intranasally with 5 female BALB/c-Rag2-/- mice showed 60% lethality and was not significantly different from those of R. pneumotropicus ATCC 35149T using the log-rank test. Combined with our finding that RTXIA has an almost identical amino acid sequence (98% identity) to that of R. haemolyticus 1625/19T, these results strongly suggest that RTXIA-producing strain JRC (and related R. haemolyticus) is pathogenic to immunodeficient rodents, and both agents should be excluded in laboratory rodent colonies.

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.

Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain.

Rodentibacter (R.) heylii is frequently detected in laboratory rodents. Repeats in toxin (RTX) toxins are considered important virulence factors of this major murine pathogen. We evaluated the virulence of a R.heylii strain negative for all known RTX toxin genes and Muribacter (M.) muris, a commensal in mice, in experimental infections of C57BL/6 and BALB/c mice. Experimental intranasal infection with 108 CFU of the pnxI-, pnxII- and pnxIII- R. heylii strain resulted in 75% and 100% mortality in C57BL/6 and BALB/c mice, respectively. In early losses, multiple internal organs were infected and purulent bronchopneumonia was the main pathology. Intranasal application of M. muris did not result in mortality or severe weight loss. Immunoproteomics led to the identification of a surface-associated and specific immunogen, which was designated as R. heylii immunogen A (RhiA) and which was exclusively recognised by sera obtained from mice infected with this R. heylii pathotype. RhiA is a 262.6 kDa large protein containing long imperfect tandem repeats and C-terminal RTX consensus sequences. Immunohistochemical analysis confirmed that this R.heylii pathotype expresses RhiA in the lower respiratory tract. In summary, this study describes a specific immunogen in a virulent R. heylii, strain which is an excellent antigen for pathotype-specific serological screenings and which might carry out RTX-related functions.

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.

Differentiation of Rodentibacter pneumotropicus, Rodentibacter heylii and Muribacter muris by MALDI-TOF MS.

The pathogens Rodentibacter (R.) pneumotropicus and R. heylii as well as the commensal Muribacter (M.) muris are frequently isolated in mice. In this study, a MALDI-TOF MS database was extended with spectra of well characterized strains of these species. Compared to a multiplex PCR, all examined out-of-sample isolates were correctly identified.

Current Distribution of Rodentibacter Species Among the Mice and Rats of an Experimental Facility.

The uncertain taxonomy of [Pasteurella] pneumotropica and other rodent Pasteurellaceae has hindered the acquisition of knowledge on the biology and disease for this group of bacteria. Recently, these organisms have been reclassified within the new genus Rodentibacter. In this study, we documented which of the new described Rodentibacter spp. are present in the mouse and rat microbiologic units of an experimental facility. Screening all of the microbiologic units populated with mice and rats yielded 51 Rodentibacter isolates. Molecular and phenotypic diagnosis indicated the colonization of mice by R. pneumotropicus and R. heylii, whereas R. ratti and R. heylii were found in rats. Overall, we document the association of laboratory rodents with 3 of the newly described Rodentibacter. Diagnostics of the Rodentibacter spp. at the species level can decisively contribute to the progress of knowledge on these bacteria.

Leucobacter muris sp. nov., isolated from the nose of a laboratory mouse.

A Gram-stain-positive, rod-shaped, aerobic, non-motile, white, opaque bacterial isolate, designated 924/12T, was isolated from the nose of a laboratory mouse in Düsseldorf, Germany. The 16S rRNA gene sequence analyses indicated the phylogenetic position of the strain within the genus Leucobacter. Similarity levels over 97 % were recorded between the 16S rRNA gene sequence of strain 924/12T and the type strains of the species Leucobacter chironomi DSM 19883T (99.5 %), followed by Leucobacter celersubsp. astrifaciens CBX151T (97.6 %), Leucobacter celersubsp. celer NAL101T (97.5 %), 'Leucobacter kyeonggiensis' F3-P9 (97.5 %), Leucobacter zeae CC-MF41T (97.3 %), Leucobacter chromiiresistens JG31T (97.1 %), Leucobacter triazinivorans JW-1T (97.1 %), Leucobacter corticis 2 C-7T (97.0 %) and Leucobacter aridicolis CIP108388T (97.0 %). DNA-DNA hybridization and whole genomic comparison, mandatory to taxonomically separate strain 924/12T from the type strain of L. chironomi, revealed similarity values of 40.4 and 30.8 %, respectively, thus below the threshold of 70 % recommended differentiating between species. The cell-wall amino acids of the novel isolate were diaminobutyric acid, alanine, glycine, threonine and glutamic acid. The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unknown lipid, whereas the predominant menaquinones were MK-11 and MK-10. The genomic DNA G+C content of strain 924/12T was 70.6 mol%. Phylogenetic analyses based on the 16S rRNA gene sequences and the phenotypical differences between strain 924/12T and the other closely related type strains of the genus Leucobacter indicated that strain 924/12T represents a novel species within the genus Leucobacter, family Microbacteriaceae, for which the name Leucobacter muris sp. nov. is proposed. The type strain is 924/12T (=DSM 101948T=CCM 8761T).

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.

Acinetobacter species in laboratory mice: species survey and antimicrobial resistance.

The extra-hospital epidemiology of Acinetobacter infections is a subject of debate. In recent years, the prevalence of animal multidrug-resistant Acinetobacter infections has increased considerably. The goal of the present study was to specify Acinetobacter species isolated from laboratory mice and to test them for their antimicrobial susceptibility. During routine microbiological monitoring of laboratory mice, 12 Acinetobacter spp. were isolated. By means of 16S rRNA and rpoB gene sequencing, seven of the isolates were identified as Acinetobacter radioresistens, three isolates belonged to Acinetobacter genomospecies 14BJ, one isolate was classified as Acinetobacter pitii and one as Acinetobacter sp. ANC 4051. The distribution of the minimal inhibitory concentration (MIC) values was uniform for 21 of the 23 antimicrobial agents tested, whereas a broad MIC distribution was recorded for tulathromycin and streptomycin. The MIC values recorded were low for the majority of the antibiotics tested. Nevertheless, very high MIC values, which will probably render a therapeutic approach using these substances unsuccessful, were recorded for florfenicol, tiamulin, tilmicosin and cephalothin in most of the isolates. In conclusion, we document colonization of laboratory mice with different Acinetobacter species, displaying similar antibiotic susceptibility profiles, with possible implications in the Acinetobacter epidemiology as well as in the husbandry and experimentation of the colonized animals.

Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain.

BACKGROUND: Mice are a natural host for Rodentibacter (R.) pneumotropicus. Despite specific monitoring, it is still one of the most important infectious agents in laboratory animals. The objective of this study was to determine the virulence of a prevalent pathotype of R. pneumotropicus and characterize the host response in a new animal model. RESULTS: Intranasal infection of C57BL/6 and BALB/c mice with a R. pneumotropicus strain (JF4Ni) bearing the genes of the three known repeats in toxin (RTX) toxins resulted in an unprecedented high mortality and morbidity above 50 and 80%, respectively. Morbidity was associated with severe weight loss as well as conjunctivitis and dyspnea. A main pathology was a catarrhal purulent to necrotic bronchopneumonia. Specific immune globuline (Ig) A was detected in tracheonasal lavages of most surviving mice which were still colonized by R. pneumotropicus. Furthermore, all surviving animals showed a distinct production of IgG antibodies. To differentiate T-helper cell (Th) 1 and Th2 immune responses we used subclasses of IgGs as indicators. Mean ratios of IgG2b to IgG1 were below 0.8 in sera drawn from both mice strains prior infection and from BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 1.6 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice associated with a tenfold higher bacterial load in the lung. In accordance with a Th1 response high antigen-specific IgG2c titers were detected in the majority of surviving C57BL/6 mice. CONCLUSIONS: R. pneumotropicus JF4Ni is a highly virulent strain causing severe pneumonia and septicemia after intranasal infection of C57BL/6 and BALB/c mice. Persisting infections in the two mice strains are associated with Th1 and Th2 immune responses, respectively, and differences in the bacterial burden of the lung. The described model is ideally suited for future vaccination studies using the natural host.

From the [Pasteurella] pneumotropica complex to Rodentibacter spp.: an update on [Pasteurella] pneumotropica.

The species [Pasteurella] pneumotropica has been reclassified into the new genus Rodentibacter, within the family Pasteurellaceae. Along with the type species (Rodentibacter pneumotropicus) of the new genus, seven new species have been named. These organisms were formerly mainly known as the [P.] pneumotropica complex and [P.] pneumotropica was considered as the most important Pasteurellaceae species colonizing laboratory rodents. The aim of this review is to update the veterinary relevant aspects of clinical manifestations, pathogenesis, virulence and diagnostics of members of Rodentibacter with a focus on the most important species from a veterinary perspective. The organisms are obligate commensals of the mucous membranes and members of Rodentibacter are not able to persist for long in the environment. Members of Rodentibacter spp. are responsible for the most prevalent bacterial infections in laboratory mice and rats, but are also common in rodents outside laboratory settings. Some Rodentibacter spp. produce mainly localised disease in connection with favouring factors and seldomly act as primary pathogens in healthy immunocompetent animals. The subclinical infection with Rodentibacter spp. can affect the results of certain types of research using contaminated animals thus placing them on a list of microbes which are often not tolerated in experimental rodent facilities. The presences of RTX toxins, YadA-like proteins and a capsule with possible role in the pathogenesis have been described. Some species of Rodentibacter are able to form robust biofilms which might be involved in colonisation and persistence within the host. Current possibilities for diagnostics and differentiation among Rodentibacter spp. are outlined and options for treatment and control are provided.

Survival of bacteria of laboratory animal origin on cage bedding and inactivation by hydrogen peroxide vapour.

This study aims to determine the ability of laboratory animal bacteria to resist desiccation and inactivation by hydrogen peroxide vapour (HPV) on paper bedding pieces. Bedding pieces were saturated with bacterial suspensions in water or 2% (w/v) bovine serum albumin (BSA) in water, and held in a mouse facility. Viable counts showed variable survival rates over time for the bacterial species used ([ Pasteurella] pneumotropica, Muribacter muris, Pseudomonas aeruginosa, Acinetobacter redioresistens, Escherichia coli, Klebsiella oxytoca, Bordetella bronchiseptica, Bordetella hinzii, Enterococcus faecalis, ß-haemolytic Streptococcus spp., Staphylococcus aureus and Staphylococcus xylosus). Overall, BSA increased bacterial survival in the bedding pieces. The survival rates of Bacillus safensis were not influenced by BSA but depended on sporulation. When bedding pieces and Petri dishes inoculated with E. coli, P. aeruginosa and S. aureus were subjected to HPV disinfection, all bacterial species on the bedding pieces inoculated with bacterial suspensions in water were readily inactivated. By contrast, S. aureus and P. aeruginosa, but not E. coli cells survived HPV treatment in high numbers when inoculated on bedding pieces as a BSA suspension. Notably, all three bacterial species were readily inactivated by HPV even in the presence of BSA when smeared on smooth surfaces. In conclusion, the suspension medium and the carrier can influence the environmental survival and susceptibility of bacterial species to HPV. Our results may help to develop standard protocols that can be used to ensure the microbiological quality of experimental rodent housing.

Synergistic effects of HBO and PRP improve bone regeneration with autologous bone grafting.

Bone defects remain a challenge for patients and orthopaedic surgeons. Autologous transfer of cancellous bone grafts remains the standard of care. However, in recent years various osteoinductive substitute materials, such as platelet rich plasma (PRP) and hyperbaric oxygen therapy (HBO) have been shown to improve bone healing. This study evaluates the effects of a combined application of PRP and HBO with autologous bone grafting in an animal model. In 48 New Zealand White rabbits bone defects at the radius were filled with autologous bone harvested at the iliac crest. This was combined with application of autologous PRP and/or HBO treatment for the duration of this study. After 3 and 6 weeks histomorphometric, immunohistochemical and radiologic evaluations were performed. All animals tolerated the treatment well. Improved bone regeneration was shown in all groups at 6 weeks compared to 3 weeks. Additional application of PRP and HBO resulted in an increase in new bone formation and increased neovascularization at 3 and 6 weeks. There was no statistical significant difference between PRP and HBO application in these regards. A combinatory use of PRP and HBO resulted in an increased bone regeneration and neovascularization compared to all other groups. This study provides evidence for an improvement of bone regeneration with the combinatory application of PRP and HBO to autologous cancellous bone grafts in a model of weight bearing bone defects in rabbits. Also synergistic effects of these two measures on angiogenesis were evident.

Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin.

[Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of ß-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of ß-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms were less sensitive to treatment with amoxicillin and enrofloxacin than planktonic bacteria. Taken together, these findings provide a first step in understanding of the biofilm mechanisms in [P.] pneumotropica, which might contribute to elucidation of colonization and pathogenesis mechanisms for these obligate inhabitants of the mouse mucosa.

Spontaneous bacterial and fungal infections in genetically engineered mice: Is Escherichia coli an emerging pathogen in laboratory mouse?

The impact of particular microbes on genetically engineered mice depends on the genotype and the environment. Infections resulting in clinical disease have an obvious impact on animal welfare and experimentation. In this study, we investigated the bacterial and fungal aetiology of spontaneous clinical disease of infectious origin among the genetically engineered mice from our institution in relation to their genotype. A total of 63 mice belonging to 33 different mice strains, from severe immunodeficient to wild-type, were found to display infections as the primary cause leading to their euthanasia. The necropsies revealed abscesses localized subcutaneously as well as in the kidney, preputial glands, seminal vesicles, in the uterus, umbilicus or in the lung. In addition, pneumonia, endometritis and septicaemia cases were recorded. Escherichia coli was involved in 21 of 44 (47.72%) of the lesions of bacterial origin, whereas [Pasteurella] pneumotropica was isolated from 19 of 44 (43.18%) cases. The infections with the two agents mentioned above included three cases of mixed infection with both pathogens. Staphylococcus aureus was considered responsible for five of 44 (11.36%) cases whereas Enterobacter cloacae was found to cause lesions in two of 44 (4.54%) mice. Overall, 16 of the 44 (36.36%) cases of bacterial aetiology affected genetically engineered mice without any explicit immunodeficiency or wild-type strains. The remaining 19 cases of interstitial pneumonia were caused by Pneumocystis murina. In conclusion, the susceptibility of genetically modified mice to opportunistic infections has to be regarded with precaution, regardless of the type of genetic modification performed. Beside the classical opportunists, such as [Pasteurella] pneumotropica and Staphylococcus aureus, Escherichia coli should as well be closely monitored to evaluate whether it represents an emerging pathogen in the laboratory mouse.

16S ribosomal DNA sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics.

Correct identification of bacteria is crucial for the management of rodent colonies. Some bacteria are difficult to identify phenotypically outside reference laboratories. In this study, we evaluated the utility of 16S ribosomal DNA (rDNA) sequencing as a means of identifying a collection of 30 isolates of rodent origin which are conventionally difficult to identify. Sequence analysis of the first approximate 720 to 880 bp of the 5'- end of 16S rDNA identified 25 isolates (83.33%) with ≥ 99% similarity to a sequence of a type strain, whereas three isolates (10%) displayed a sequence similarity ≥ 97% but <99% to the type strain sequences. These similarity scores were used to define identification to species and genus levels, respectively. Two of the 30 isolates (6.67%) displayed a sequence similarity of ≥ 95 but <97% to the reference strains and were thus allocated to a family. This technique allowed us to document the association of mice with bacteria relevant for the colonies management such as Pasteurellaceae, Bordetella hinzii or Streptococcus danieliae. In addition, human potential pathogens such as Acinetobacter spp., Ochrobactrum anthropi and Paracoccus yeei or others not yet reported in mouse bacterial species such as Leucobacter chironomi, Neisseria perflava and Pantoea dispersa were observed. In conclusion, the sequence analysis of 16S rDNA proved to be a useful diagnostic tool, with higher performance characteristics than the classical phenotypic methods, for identification of laboratory animal bacteria. For the first time this method allowed us to document the association of certain bacterial species with the laboratory mouse.

Development of a multiplex PCR assay based on the 16S-23S rRNA internal transcribed spacer for the detection and identification of rodent Pasteurellaceae.

The rodents Pasteurellaceae have to be excluded from the specified pathogen free experimental animal facilities. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals just a few molecular based methods are available for their detection and identification. The aim of the present investigation was to develop a multiplex PCR assay allowing detection of all rodent Pasteurellaceae and identification of [Pasteurella] pneumotropica biotype Jawetz, [P.] pneumotropica biotype Heyl and [Actinobacillus] muris, as the most prevalent members of the group. For this, a Pasteurellaceae common forward primer located on the 16S rRNA gene was used in conjunction with four different reverse primers specific for [P.] pneumotropica biotype Jawetz, [P.] pneumotropica biotype Heyl, [A.] muris and a common reverse primer for all rodent Pasteurellaceae, all targeting the 16S-23S rRNA internal transcribed spacer sequences. The performance characteristics of the assay were tested against 125 Pasteurellaceae isolates belonging to eleven different species and including 34 strains of [P.] pneumotropica biotype Jawetz, 44 strains of [P.] pneumotropica biotype Heyl and 37 strains of [A.] muris. Additionally, eight other mouse associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA gene sequencing. This multiplex PCR represents the first molecular tool able to detect and differentiate in a single assay among the Pasteurellaceae found in laboratory mouse and may become a reliable alternative to the present diagnostic methods.

Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods.