Detection of humoral response using a recombinant heat shock protein 70 (dnaK) of Mycoplasma haemofelis in experimentally and naturally hemoplasma infected-cats

'Hemoplasmas' is the trivial name given to a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. Of the feline hemoplasmas, Mycoplasma haemofelis (Mhf) is the most pathogenic whilst 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicensis' (CMt) are less pathogenic. Shotgun libraries of fragmented Mhf genomic DNA were constructed and random colonies selected for DNA sequencing. In silico translated amino-acid sequences of putative open reading frames were compared to mass-spectrometry data from Mhf protein spots identified as being immunogenic by two-dimensional gel electrophoresis and western blotting. Three of the spots matched the predicted sequences of a heat shock protein 70 (dnaK)-homolog, elongation factor-Ts and a fragment of phosphoglycerate kinase found during library screening. A full length copy of the Mhf dnaK gene was cloned into Escherichia coli and recombinantly-expressed. Recombinant Mhf dnaK was purified, and then used in western blots and an ELISA to investigate the humoral immune response during acute infection in cats experimentally infected with Mhf, CMhm or CMt. The recombinant Mhf dnaK ELISA was also used to screen clinical samples submitted for hemoplasma PCR testing to a commercial laboratory (n = 254). Experimentally-infected cats became seropositive following infection, with a greater and earlier antibody response seen in cats inoculated with Mhf, compared to CMhm or CMt, on both western blot and ELISA. Of the clinical samples 31.1 % had antibodies detected by the ELISA but only 9.8 % were positive by PCR for one or more hemoplasmas.