Elsevier

Veterinary Microbiology

Volume 160, Issues 1–2, 9 November 2012, Pages 227-232
Veterinary Microbiology

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

https://doi.org/10.1016/j.vetmic.2012.05.022Get rights and content

Abstract

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 (0.2–0.6 μm) 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.

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).

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