Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Abstract Background Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. Methods The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. Results The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. Conclusions The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens

Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice

Abstract Background Rodentibacter (R.) pneumotropicus colonizes the respiratory and urogenital tracts of laboratory mice with a reported moderate serological prevalence from 4 to 13%. Thus, regular tests to identify this pathogen in mice are recommended for animal facilities. However, a recent study indicated that current serological assays are partly insensitive, as C57BL/6 and BALB/c mice infected with R. pneumotropicus were incorrectly screened as seronegative. Results Here, we report a systematic analysis of protein and lipopolysaccharides antigens by immunoblot and ELISA that allowed establishing a sensitive test system able to differentiate between R. pneumotropicus and the closely related species R. heylii. Furthermore, the main immunogen, designated as ‘characteristic antigen for Rodentibacter of laboratory origin 1’ (CARLO-1), was identified by two-dimensional gel electrophoresis followed by immunoblot and tandem mass spectrometry in a preparation of outer membrane proteins. An indirect ELISA relying on the recombinantly expressed protein provided high sensitivity, specificity, and selectivity. The corresponding carlo1 gene was highly conserved (> 97%) among 21 isolates of R. pneumotropicus and R. heylii. Conclusion The newly identified protein CARLO-1 is well suited for the sensitive and specific serological detection of Rodentibacter infections in mice. Indirect differentiation of R. pneumotropicus and R. heylii infections may be possible using an ELISA based on a whole-cell antigen preparation. All four established ELISA systems using a whole-cell preparation, lipopolysaccharides, outer-membrane proteins and protein CARLO-1 as antigen, respectively, outperformed a commercial ELISA in terms of sensitivity

Additional file 5: of Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Excel Sheet Raw data of ELISA validation. Sheet 1: Raw data of repeatability, intermediate precision and calculation of the limit of detection. Sheet 2: Raw data of stability experiments. Sheet 3: Raw data of ROC analysis. (XLSX 21 kb

Additional file 4: of Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Temperature-dependent stability of Strep-rσ-1-His-ELISA. Freshly coated ELISA plates were stored at 4 °C (black) and 37 °C (red), respectively, including all material needed for testing (controls, buffers, conjugate, substrate, sulfuric acid). Positive control (solid line, anti-His antibody) and negative control (dashed line, pool of reovirus type-3 [−] sera) were assayed in replicates of three at a total of nine time points for each storage temperature. Mean values and standard deviations are indicated. (TIF 178 kb

Additional file 1: of Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Alignment of σ-1 amino acid sequences shows conservation within and diversity across reovirus serotypes. The following GenBank accession numbers were used for the BLASTp alignment: HM159619.1 (T3D), GU589583.1 (T3A), M35964.1 (T2J) and M35963.1 (T1L). Absolute and percentage values of identity, positives and gaps as well as percentage of query cover are provided. Information modified after [36]. (XLSX 11 kb

Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described

Biocompatible Silicon Surfaces through Orthogonal Click Chemistries and a High Affinity Silicon Oxide Binding Peptide

Multifunctionality is gaining more and more importance in the field of improved biomaterials. Especially peptides feature a broad chemical variability and are versatile mediators between inorganic surfaces and living cells. Here, we synthesized a unique peptide that binds to SiO₂ with nM affinity. We equipped the peptide with the bioactive integrin binding c­[RGDfK]-ligand and a fluorescent probe by stepwise Diels–Alder reaction with inverse electron demand and copper­(I) catalyzed azide–alkyne cycloaddition. For the first time, we report the generation of a multifunctional peptide by combining these innovative coupling reactions. The resulting peptide displayed an outstanding binding to silicon oxide and induced a significant increase in cell spreading and cell viability of osteoblasts on the oxidized silicon surface

Additional file 2: of 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

Table S2. Scoring of semiquantitive bacteriological findings in R. pneumotropicus infected mice of the indicated strains surviving until the end of the observation period (4 weeks). (PDF 33 kb

Additional file 5: of 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

Table S5 Scoring of catarrhal - purulent inflammations in mice infected with R. pneumotropicus. (PDF 4 kb

Additional file 4: of 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

Table S4 R. pneumotropicus and R. heylii strains used in this study. (PDF 10 kb