Short communicationNanotransformation of the haemotrophic Mycoplasma suis during in vitro cultivation attempts using modified cell free Mycoplasma media
Introduction
Mycoplasma suis is a member of the haemotrophic mycoplasmas (HMs) which attach to or invade host erythrocytes of many mammals (Hoelzle, 2008, Groebel et al., 2009). In all these animals HMs are considered host-specific and to cause acute or chronic anaemia. Currently, the zoonotic potential of HM infections is intensely discussed (Dos Santos et al., 2008, Hu et al., 2009, Bosnic et al., 2010, Sykes et al., 2010, Steer et al., 2011).
HMs are highly specialised bacteria with a high degree of host adaptation reflected by its specific cell tropism, persistent infection, immune modulation, and, finally, uncultivability in vitro. All attempts to cultivate HMs in vitro have failed so far. Only a short-term maintenance of M. suis has been described using a petri dish erythrocyte culture system (Nonaka et al., 1996). Therefore, to date, HM research relies on the propagation in splenectomised animals, a method connected with serious ethical concerns. Moreover, cultivation of bacteria is an important feature of microbiology since Robert Koch. Substantial amounts of pure bacteria are the precondition for the analyses of bacterial characteristics and would enable the development of strategies for therapy and prophylaxis of HM infections.
In this study we investigated the application of modified standard culture techniques for mycoplasmas for the cultivation of M. suis. Furthermore, we investigated the ultra-structure of M. suis by scanning electron microscopy (SEM) after sub-cultivation on agar plates, and studied the in vitro interaction of culture-derived M. suis cells with porcine erythrocyte.
Section snippets
Animal experiments and blood collection
In this study a splenectomised M. suis pig model was used (Hoelzle et al., 2003, Hoelzle et al., 2006). Na-citrate anti-coagulated blood was drawn at maximum bacteraemia (>90% of the erythrocytes parasitised). For in vitro interaction studies erythrocytes from M. suis-negative pigs were taken. The animal experiments were performed with the approval of the Veterinary Office of Zurich, Switzerland, under the registration no 55/2007.
Mycoplasma suis culture
Standard SP-4 Mycoplasma medium (ATCC medium 988) was prepared
Mycoplasma suis-cultivation in liquid cultures
To date, iron-acquisition systems used by HMs are unknown. Iron bound in the host's blood to haemin, haemoglobin or transferrin could be one major limiting factor for the growth of HMs in pure culture. Therefore, SP-4 medium supplemented with haemin, haemoglobin or transferrin, respectively, was inoculated with M. suis containing porcine blood (f.c. 5 × 104 M. suis/ml medium). The results of the qLC-PCR quantification of the culture aliquots taken weekly are shown in Fig. 1A and Table 1. After
Discussion
Despite a long known significance of haemotrophic mycoplasmas no in vitro cultivation system has been established so far (Hoelzle, 2008). To the best of our knowledge, to date no systematic experimental studies concerning HM propagation in cell-free media have been published. In the present study we attempted to propagate M. suis in vitro based initially on two working hypotheses: First, we claimed that iron-containing proteins i.e. haemin, haemoglobin or transferrin are essential for in vitro
Acknowledgements
This study was supported by the 3R Research Foundation Switzerland (Reduction, Refinement and Replacement of animal experimentation).
References (23)
- et al.
Rare zoonosis (hemotrophic mycoplasma infection) in a newly diagnosed systemic lupus erythematosus patient followed by a Nocardia asteroides pneumonia
Braz. J. Infect. Dis.
(2010) Haemotrophic mycoplasmas: recent advances in Mycoplasma suis
Vet. Microbiol.
(2008)- et al.
Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood
Vet. Microbiol.
(2003) - et al.
First Light-Cycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples
J. Microbiol. Methods
(2007) - et al.
Detection of Candidatus Mycoplasma haemobos in cattle with anaemia
Vet. J.
(2011) - et al.
In vitro maintenance of Eperythrozoon suis
Vet. Parasitol.
(1996) - et al.
Hemin-dependent growth and hemin binding of Bartonella henselae
FEMS Microbiol. Lett.
(2000) - et al.
Hemin-binding surface protein from Bartonella quintana
Infect. Immun.
(2000) - et al.
Mycoplasma adaptation to adverse growth conditions: nanotransformation and phytopathogenicity of Acholeplasma laidlawii PG8
Dokl. Biochem. Biophys.
(2007) - et al.
Proteomic characterization of Mycoplasma gallisepticum nanoforming
Biochemistry (Mosc.)
(2010)
Hemoplasma infection in HIV-positive patient
Brazil. Emerg. Infect. Dis.
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