Molecular Studies on Haemotropic Mycoplasmas of Mammals

There has been limited genomic and proteomic study of the uncultivable haemoplasmas, causative agents of infectious anaemia. Accurate diagnosis and characterisation of infection is reliant on DNA amplification and sequencing. Diagnostic assays comprising species-specific canine haemoplasma quantitative PCRs (qPCRs) duplexed with an internal host DNA control qPCR were developed. Prevalence varied widely from 0-20% for Mycoplasma haemocanis and 0-3.8% for “Candidatus Mycoplasma haematoparvum” between geographically distinct canine populations. The first UK clinical case of M. haemocanis infection was confirmed and monitored over time. A novel human haemoplasma detected in the blood of a haemolytic anaemia patient was characterised and a generic haemoplasma assay used to monitor response to antibiotic therapy. To identify potential mechanisms of pathogenicity and predict growth requirements, feline haemoplasma Mycoplasma haemofelis strain Langford 1 was shotgun genome sequenced and annotated. The genome comprises a 1.15 Mbp circular chromosome with a G+C content of 38.9%. Of the 1,545 protein-coding sequences predicted, 328 (21.2%) matched characterised proteins, from which it was inferred that M. haemofelis metabolises glucose as a sole energy source and is reliant on the host for nutrients including amino acids, co-factors and ribonucleotide precursors. Clustered uncharacterized hypothetical protein paralogs (n=1,122) encoding putatively surface expressed proteins are suspected of playing a role in immune evasion. Comparison of in silico predicted proteins to mass spectrometry data confirmed in vivo expression of characterized and uncharacterized proteins (including clustered paralog proteins). Recombinant M. haemofelis chaperone protein DnaK, applied to an ELISA format, was used to demonstrate seropositivity following experimental haemoplasma infection, with the greatest antibody response in those cats inoculated with M. haemofelis. Diagnostic qPCR assays have detected and monitored haemoplasma infection in novel species and novel populations. Whole genome sequencing has provided clues to aid attempts of in vitro cultivation and targets for serological study.