Dataset Information

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.

SUBMITTER: Fingas F

PROVIDER: S-EPMC6380038 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

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

Fingas Felix F Volke Daniela D Hassert Rayk R Fornefett Juliane J Funk Sophie S Baums Christoph Georg CG Hoffmann Ralf R

BMC microbiology 20190218 1

<h4>Background</h4>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.<h4>Results</h4>Here, we report a ...[more]

PMID: 30777007

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Project description:BACKGROUND: Mycoplasma pneumoniae is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of M. pneumoniae to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes. METHODS: A 609 bp fragment P116(N-27), corresponding to the N-terminal region of M. pneumoniae P116 gene was cloned and expressed. A C-terminal fragment P1(C-40), of P1 protein of M. pneumoniae was also expressed. Three IgM ELISA assays based on P116(N-27), P1(C-40) and (P116 (N-27) + P1(C-40)) proteins were optimized and a detailed analysis comparing the reactivity of these proteins with a commercial kit was carried out. Comparative statistical analysis of these assays was performed with the SPSS version 15.0. RESULTS: The expressed P116(N-27) protein was well recognized by the patient sera and was immunogenic in rabbit. P1(C-40) of M. pneumoniae was also immunogenic in rabbit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116(N-27), P1(C-40) and (P116(N-27) + P1(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, and there was a good correlation and association between them. CONCLUSION: This study shows that an N-terminal fragment of P116 protein holds a promise for serodiagnosis of M. pneumoniae infection. The IgM ELISA assays based on the recombinant proteins seem to be suitable for the use in serodiagnosis of acute M. pneumoniae infections. The use of short recombinant fragments of P116 and P1 proteins as specific antigens may eliminate the risk of cross-reactions and help to develop a specific and sensitive immunodiagnostic assay for M. pneumoniae detection.

Project description:Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5α. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 µM, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 µM. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs.IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.

Dataset Information

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

Background

Results

Conclusion

Publications

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

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