Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis

Abstract

ABSTRACTMycoplasma haemofelisinfection frequently causes anemia in cats. Despite an intense immune response and/or antibiotic treatment, cats often remain asymptomatic carriers following infection. Our hypothesis is that detection of antibodies toM. haemofelisis a sensitive approach for identifying infected cats, particularly carriers. To date, no immunoassay has been developed. This is due largely to the inability to cultureM. haemofelis in vitro; hence, a source of antigen is not readily available. The objective of this study was to identify, express, and purify immunogenic proteins ofM. haemofelis. To accomplish this, two whole-genomic expression libraries were created in the Lambda ZapII vector and immunoscreened with preimmune plasma, plasma from specific-pathogen-free cats, and pooled acute- and convalescent-phase plasma from experimentally infected cats. The inserts from 21 immunoreactive clones were sequenced, resulting in the identification of 60 genes coding for putative proteins necessary for diverse cellular functions, along with several novel genes ofM. haemofelis. Fragments of selected genes based on bioinformatic analyses were PCR amplified, cloned into a high-level protein expression system, and subsequently expressed inEscherichia colias a His6-fusion protein. The recombinant fusion proteins ofM. haemofeliswere purified and evaluated as an antigen in a Western blot to verify the findings of previous immunoscreening. Together with bioinformatics analyses of individual genes, this approach provided several putative candidate antigens. Five antigens ofM. haemofeliswere reactive by Western blotting against the immune plasma and negative against nonimmune plasma; these antigens might be useful serologic or even vaccine targets.