title: Identification, characterization, and application of a recombinant antigen for the serological investigation of feline hemotropic Mycoplasma infections.
abstract: In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, size-exclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, "Ca. Mycoplasma haemominutum," and "Ca. Mycoplasma turicensis," respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.
authors.affiliation: Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.
authors.forename: Godelind A
authors.initials: GA
authors.lastname: Wolf-Jäckel
authors.forename: Christian
authors.initials: C
authors.lastname: Jäckel
authors.forename: Kristina
authors.initials: K
authors.lastname: Museux
authors.forename: Katharina
authors.initials: K
authors.lastname: Hoelzle
authors.forename: Séverine
authors.initials: S
authors.lastname: Tasker
authors.forename: Hans
authors.initials: H
authors.lastname: Lutz
authors.forename: Regina
authors.initials: R
authors.lastname: Hofmann-Lehmann
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D002371:Cat Diseases / Q000175:diagnosis* / Q000382:microbiology; D002415:Cats; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007118:Immunoassay / Q000379:methods; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics / Q000276:immunology*; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000662:veterinary*; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins