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.

Keywords: Antigen; CARLO-1; ELISA; FELASA; Health monitoring; Pasteurella pneumotropica; Rodentibacter heylii; Rodentibacter pneumotropicus; Two-dimensional gel electrophoresis.

Fig. 1

Commercial (a) and WCA-based ELISA (b) results of sera obtained from C57BL/6 (full circles) and BALB/c mice (open circles) infected with R. pneumotropicus or R. heylii and uninfected animals. Test specific cut-off value for the commercial ELISA (0.3) is indicated as a dashed line. Similar results of a comparable WCA-ELISA for the R. pneumotropicus infected mice and respective controls shown in (b) have been published previously [4]

Fig. 2

SDS-PAGE (a) and ELISA (b) of a WCA preparation incubated (56 °C, overnight) in the absence or presence of proteinase K. a) Oriole stain of a WCA preparation obtained from R. pneumotropicus strain JF4Ni separated by SDS-PAGE before (−) and after (+) proteinase K digestion. b) Indirect ELISA using WCA (black) and digested WCA (hatched) probed with sera of experimentally infected C57BL/6 and BALB/c mice and a control serum

Fig. 3

SDS-PAGE and corresponding blots of protein and LPS preparations. a) SDS-PAGE of cytosolic (Cyt), sodium N-laurylsarcosinate (SLS)-soluble membrane (inner membrane, IM), SLS-insoluble membrane (outer membrane, OM) preparations, and the OM preparation digested with Proteinase K (OM + Prot. K). Proteins were stained with Oriole. Gel areas cut out to identify the proteins by mass spectrometry (MS) are indicated by rectangles. b) Immunoblots probed with sera obtained from C57BL/6 and BALB/c mice experimentally infected with R. pneumotropicus ([+]) and an uninfected BALB/c mouse (control [−]) c) SDS-PAGE of the LPS preparation stained with Pro-Q Emerald (Glyco) or Coomassie (Protein) and the corresponding immunoblot using serum of an infected C57BL/6 mouse (Immuno). Molecular weights (kDa) of the marker proteins are indicated left

Fig. 4

Indirect ELISA based on OM (a) and LPS preparations (b) probed with sera from C57BL/6 and BALB/c mice experimentally infected with R. pneumotropicus or R. heylii and uninfected SPF mice (Control). The LODs of OM- (0.16) and LPS-ELISA (0.08) based on the absorbance of control sera are indicated as dashed lines. No differences were observed between the used mouse strains

Fig. 5

2-DE of SCP of R. pneumotropicus stained with Oriole (a) and corresponding immunoblots probed with sera obtained from infected C57BL/6 (b) and BALB/c mice (c) and a non-infected C57BL/6 mouse (control, d). Molecular weights of the marker proteins are indicated on the left site. The gel spot area for protein identification by tandem MS is indicated by a rectangle

Fig. 6

Protein-based ELISA. Strep-rHP-His was coated and probed with sera from mice experimentally infected with R. pneumotropicus (R. pneu.), R. heylii, M. pulmonis (M. pul.), and S. moniliformis (S. mon.), sera of uninfected mice (Control), and with field sera. The dashed line indicates the LOD (0.47)