Identification, molecular characterization, and experimental transmission of a new hemoplasma isolate from a cat with hemolytic anemia in Switzerland

Abstract

Recently, there has been a growing interest in hemotropic mycoplasmal species (also known as the hemoplasmas), the causative agents of infectious anemia in several mammalian species. In felids, two different hemoplasma species have been recognized: Mycoplasma haemofelis (formerly Haemobartonella felis) and "Candidatus Mycoplasma haemominutum." Recently developed molecular methods have allowed sensitive and specific identification and quantification of these agents in feline blood samples. In applying these methods to an epidemiological study surveying the Swiss pet cat population for hemoplasma infection, we discovered a third novel and unique feline hemoplasma isolate in a blood sample collected from a cat that had exhibited clinical signs of severe hemolytic anemia. This agent was readily transmitted via intravenous inoculation to two specific-pathogen-free cats. One of these cats was immunocompromised by the administration of methylprednisolone acetate prior to inoculation, and this cat developed severe anemia. The other immunocompetent cat showed a moderate decrease in packed cell volume. Additionally, an increase in red blood cell osmotic fragility was observed. Sequencing of the entire 16S rRNA gene of the new hemoplasma isolate and phylogenetic analysis showed that the isolate was most closely related to two rodent hemotropic mycoplasmal species, M. coccoides and M. haemomuris. A quantitative real-time PCR assay specific for this newly discovered agent was developed, which will be a prerequisite for the diagnosis of infections with the new hemoplasma isolate.

FIG. 1.

Transmission experiment. The charts depict the loads of the newly described isolate (log copy number of DNA template per ml of blood, left y axes, black triangles) and the courses of PCV (%, right y axes, open squares) in cat 1 (A) and cat 2 (B) over 100 days p.i. (x axes). The reference ranges of PCV are shaded grey. The lower detection limits of the real-time PCR assay (100 copies/ml blood) are indicated by dotted lines.

FIG. 2.

Course of RBC OF in cat 2 over 31 days after experimental infection with the newly described isolate. The absorbance measured after incubation in 0.3% NaCl solution was defined as 100% hemolysis; the absorbance measured in 0.9% NaCl solution was defined as 0% hemolysis. The curves were fitted to the data using sigmoid regression (SigmaPlot Regression Wizard, SSPS, Chicago, Illinois). The shift in RBC OF from days 0 to 31 p.i. is indicated by an arrow. The complete time courses of the OF cannot be given for cats 1 and 946 because this assay had not been established during their early infection periods.

FIG. 3.

Phylogenetic tree demonstrating the relationship of the new isolate to other hemoplasma species using a neighbor joining method. M. coccoides (AY171918), M. haemomuris (U82963), M. haemocanis (AY150973), M. haemofelis (AY150985), M. fastidiosum (AF125878), “Candidatus Mycoplasma haemominutum” (AY297712), “Candidatus Mycoplasma kahanei” (AF338269), “Candidatus Mycoplasma haemoparvum” (AY532390), M. wenyonii (AF016546), and M. suis (AY492086) are shown. The numbers at the nodes were generated from 1,000 bootstrap resamplings; values of <500 are not shown. The bar represents the mean number of differences per 100 sites.