Nanotransformation of the haemotrophic Mycoplasma suis during in vitro cultivation attempts using modified cell free Mycoplasma media.

Mycoplasma suis belongs to haemotrophic mycoplasmas (HMs) which cause infectious anaemia in a large variety of mammals. To date, no in vitro cultivation system for M. suis or other HMs has been established. We hypothesised that M. suis could grow in classical Mycoplasma media supplemented with nutrients (e.g. glucose, iron-binding proteins) which are naturally available from its host environment, the porcine blood. Blood from experimentally M. suis-infected pigs was used to inoculate either standard SP-4 Mycoplasma medium supplemented with iron-binding proteins (transferrin, haemin, and haemoglobin) or glucose-enriched Hayflick Mycoplasma medium. A quantitative M. suis-specific real-time PCR assay was applied to determine and quantify M. suis loads weekly during 12 week-incubation. The first 2 weeks after inoculation M. suis loads decreased remarkably and then persisted at a stationary level over the observation time of 12 weeks in iron-binding protein- or glucose supplemented media variants. Scanning electron microscopic analysis of liquid M. suis sub-cultures on Hayflick agar showed small, densely-packed microcolonies of irregular M. suis cells of reduced size (0.2-0.6µm) indicating nanotransformation. The partial 16S rDNA sequence of these cultured M. suis nanocells was 99.9% identical to M. suis. M. suis cells derived from liquid cultures interact in vitro with porcine erythrocytes by fibril-like structures. We conclude, that the modified Mycoplasma media used for M. suis cultivation are obviously unfavourable for growth but lead to culture persistence. M. suis adapt to inappropriate culture conditions by alteration into nanoforms.

Use of recombinant antigens to detect antibodies against Mycoplasma suis, with correlation of serological results to hematological findings.

Porcine eperythrozoonosis is a disease with worldwide distribution caused by the unculturable hemotrophic bacterium Mycoplasma suis. Current serological testing utilizes crude M. suis antigens purified from the blood of experimentally infected pigs. These antigens show high variability and are restricted to specialized laboratories. We evaluated a novel serological assay based on two recombinant M. suis antigens (rMSG1 and rHspA1). Antigen specificity was proven by means of sera raised against nonhemotrophic mycoplasma and other relevant bacteria. Using experimental and convalescent-phase sera, rMSG1 and rHspA1 enzyme-linked immunosorbent assays (ELISAs) demonstrated sensitivities, specificities, and predictive values (94.0 to 100.0%) equal to or higher than those of the M. suis whole-cell ELISA. Field samples from 120 weaning piglets grouped by quantitative PCR results were used to evaluate the diagnostic capability of the new ELISA systems in comparison to that of the whole-cell ELISA. Assuming a 100.0% specificity of the PCR, the whole-cell ELISA, rHspA1 ELISA, and rMSG1 ELISA showed specificities of 84.8%, 83.8%, and 90.6% and sensitivities of 61.5%, 74.0% and 58.1%, respectively. Cohen's kappa coefficients comparing the recombinant ELISAs to the whole-cell ELISA indicate moderate to substantial agreement. The detection of anti-MSG1 and/or anti-HspA1 antibodies in pigs was significantly correlated with decreased hematocrit, erythrocyte numbers, and hemoglobin concentrations, indicating that a single seropositive result is connected with clinical and etiological significance. In conclusion, rMSG1 and rHspA1 are sensitive and specific serological and infection markers which are for the first time used independently of animal experiments. They are especially fit to be used in routine diagnosis, pathogenesis studies, and large-scale epidemiological investigations.