A new duplex qPCR-based method to quantify Mycoplasma mycoides in complex cell culture systems and host tissues.

Bacterial pathogen-host interactions are a complex process starting with adherence and colonization followed by a variety of interactions such as invasion or cytotoxicity on one hand and pathogen recognition, secretion of proinflammatory/antibacterial substances and enhancing the barrier function of epithelial layers on the other hand. Therefore, a variety of in vitro, ex vivo and in vivo models have been established to investigate these interactions. Some in vitro models are composed of different cell types and extracellular matrices such as tissue explants or precision cut lung slices. These complex in vitro models mimic the in vivo situation more realistically, however, they often require new and more sophisticated methods for quantification of experimental results. Here we describe a multiplex qPCR-based method to quantify the number of bacteria of Mycoplasma (M.) mycoides interacting with their hosts in an absolute manner as well as normalized to the number of host cells. We choose the adenylate kinase (adk) gene from the pathogen and the Carcinoembryonic antigen-related cell adhesion molecule 18 (CEACAM18) gene from the host to determine cell numbers by a TaqMan-based assay system. Absolute copy numbers of the genes are calculated according to a standard containing a defined number of plasmids containing the sequence which is amplified by the qPCR. The new multiplex qPCR therefore allows the quantification of M. mycoides interacting with host cells in suspension, monolayer, 3D cell culture systems as well as in host tissues.

Transcriptional Host Responses to Infection with Streptococcus suis in a Porcine Precision-Cut Lung Slice Model: Between-Strain Differences Suggest Association with Virulence Potential.

Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.

Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria-host interactions.

Pasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air-liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

Host-Pathogen Interactions of Mycoplasma mycoides in Caprine and Bovine Precision-Cut Lung Slices (PCLS) Models.

Respiratory infections caused by mycoplasma species in ruminants lead to considerable economic losses. Two important ruminant pathogens are Mycoplasma mycoides subsp. Mycoides (Mmm), the aetiological agent of contagious bovine pleuropneumonia and Mycoplasma mycoides subsp. capri (Mmc), which causes pneumonia, mastitis, arthritis, keratitis, and septicemia in goats. We established precision cut lung slices (PCLS) infection model for Mmm and Mmc to study host-pathogen interactions. We monitored infection over time using immunohistological analysis and electron microscopy. Moreover, infection burden was monitored by plating and quantitative real-time PCR. Results were compared with lungs from experimentally infected goats and cattle. Lungs from healthy goats and cattle were also included as controls. PCLS remained viable for up to two weeks. Both subspecies adhered to ciliated cells. However, the titer of Mmm in caprine PCLS decreased over time, indicating species specificity of Mmm. Mmc showed higher tropism to sub-bronchiolar tissue in caprine PCLS, which increased in a time-dependent manner. Moreover, Mmc was abundantly observed on pulmonary endothelial cells, indicating partially, how it causes systemic disease. Tissue destruction upon prolonged infection of slices was comparable to the in vivo samples. Therefore, PCLS represents a novel ex vivo model to study host-pathogen interaction in livestock mycoplasma.

Identification of targets of monoclonal antibodies that inhibit adhesion and growth in Mycoplasma mycoides subspecies mycoides.

Mycoplasma mycoides subspecies mycoides (Mmm) adhesion is tissue and host specific. Inhibition of adhesion will prevent Mmm from binding to lung cells and hence prevent colonization and disease. The aim of this study was to develop a panel of Mmm monoclonal antibodies against Mmm and use these antibodies to investigate their inhibitory effect on the adherence of Mmm to bovine lung epithelial cells (BoLEC), and to further identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoides subsp. mycoides (AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that the antibodies bound to proteins of variable sizes. AMMY 10, a previously characterized Mmm- capsular polysaccharide (CPS) specific antibody, inhibited growth of Mmm in vitro and also caused agglutination of Mmm total cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion of Mmm to BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune response in vivo.