Quantification of Mycoplasma hyopneumoniae in cell culture systems and host tissues by a new duplex qPCR-based method.

Introduction

Mycoplasma hyopneumoniae is the causative agent of Enzootic pneumonia (EP) in pigs, a highly prevalent, chronic respiratory disease, which causes considerable economic losses in the swine industry. The infection leads to damage of the ciliated swine respiratory epithelium, either by direct cytotoxic processes or indirectly, by the induction of a strong, damaging inflammatory response. Despite the medical and economical importance of EP, most pathomechanisms are not yet fully understood.

We describe here a duplex qPCR-based method to quantify the number of Mycoplasma hyopneumoniae cells interacting with the host, normalized to the number of host cells. This method allows a better comparison of pathogen burden in 3D organotypic tissue models, like tissue explants or Precision-cut lung slices (PCLS), with samples obtained from in-vivo infections.

Objective

Establishing a duplex qPCR to quantify M.°hyopneumoniae cells, normalized to the number of porcine host cells.

Materials & methods

Lungs were obtained from healthy slaughtered pigs. Accessory and cranial lobes were used for the preparation of PCLS. Slices were infected with freshly grown M.°hyopneumoniae for 4 h or 24 h. Tissue samples from experimentally infected pigs were obtained during in-house animal experiments. DNA was isolated from PCLS and tissue samples by standard methods. PCR methods were developed on Biometra Tone and Stratagene™ Mx3005P cyclers.

Results

We compared different porcine target genes to determine host cell numbers. Finally, we have chosen the porcine Carcinoembryonic antigen-related cell adhesion molecule 18 (CEACAM18) gene and combined it with the already established p102 gene from M.°hyopneumoniae in a duplex TaqMan-based assay system. Absolute copy numbers of the genes were calculated according to plasmid standards containing the cloned target sequences.

Conclusion

The new duplex qPCR is well suited quantify the pathogen burden normalized to host cell numbers. It allows a better comparison of 3D infection models with in-vivo samples.