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Production and characterization of a polyclonal antiserum against Spiroplasma mirum (ATCC 29335)
B Tenckhoff1, H W Kölmel, V Wolf
1Immanuel-Krankenhaus GmbH, Rheumaklinik, Berlin (Wannsee), Germany.
Abstract:
Spiroplasma mirum (ATCC 29335), an American tick isolate, was cultivated and its purity was controlled using electron microscopy and Tricine-SDS-PAGE. In the SDS-PAGE analysis, the protein pattern of the antigen used revealed a polypeptide profile (35 bands) with an approximate molecular weight of 161 kDa to 10.5 kDa. This strain was used to immunize two rabbits. The produced antisera did not cross-react with Mycoplasma pneumoniae, Salmonella minnesota, Borrelia burgdorferi, Treponema pallidum but did still react in Western blot analysis with Spiroplasma mirum at a dilution of 1:128.000. This specific and sensitive antiserum was used to examine 72 midgut smears of Berlin ticks (lxodes ricinus) individually by direct immunofluorescence. In 26 samples, we could demonstrate fluorescent structures, but we were not able to culture spiroplasmas from these samples.
Insights
Researchers developed a specific antiserum for Spiroplasma mirum, an American tick isolate. This antiserum detected Spiroplasma mirum in 26% of Ixodes ricinus tick midguts, aiding in pathogen detection.
Area of Science:
- Microbiology
- Immunology
- Parasitology
Background:
- Spiroplasma mirum is a tick-borne bacterium.
- Accurate detection methods are crucial for understanding its prevalence.
Purpose of the Study:
- To develop a specific antiserum against Spiroplasma mirum.
- To evaluate the antiserum's utility in detecting Spiroplasma in tick midguts.
Main Methods:
- Cultivation and characterization of Spiroplasma mirum using electron microscopy and Tricine-SDS-PAGE.
- Production of polyclonal antibodies in rabbits.
- Western blot analysis for antibody specificity.
- Direct immunofluorescence assay on Ixodes ricinus midgut smears.
Main Results:
- SDS-PAGE revealed a distinct protein profile for Spiroplasma mirum.
- The generated antiserum showed high specificity, with no cross-reactivity against other tested bacteria.
- The antiserum detected Spiroplasma mirum in 26 out of 72 (36%) Ixodes ricinus midgut samples.
- Spiroplasma culture was not successful from the positive samples.
Conclusions:
- A specific and sensitive antiserum for Spiroplasma mirum was successfully produced.
- Direct immunofluorescence is a viable method for detecting Spiroplasma mirum in tick vectors.
- The study highlights the presence of Spiroplasma mirum in Ixodes ricinus ticks.