Whole Blood Transcriptome Analysis of Mycoplasma mycoides Subsp. mycoides-Infected Cattle Confirms Immunosuppression but Does Not Reflect Local Inflammation

Abstract

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm), is a severe respiratory disease of cattle responsible for major economic losses in sub-Saharan Africa. Disease control relies mainly on the use of empirically attenuated vaccines that provide limited protection. Thus, understanding the virulence mechanisms used by Mmm as well as the role of the host immune system in disease development, persistence, and control is a prerequisite for the development of new, rationally designed control strategies. The aim of this study was to assess the use of whole blood transcriptome analysis to study cattle-Mmm interactions, starting by the characterization of the bovine response to Mmm infection during the acute form of the disease. For that purpose, we compared the transcriptome profile of whole blood from six cattle, before challenge by contact with Mmm-infected animals and at the appearance of first clinical signs, using a bovine microarray. Functional analysis revealed that 680 annotated genes were differentially expressed, with an overwhelming majority of down-regulated genes characterizing an immunosuppression. The main bio-functions affected were "organismal survival", "cellular development, morphology and functions" and "cell-to cell signaling and interactions". These affected functions were consistent with the results of previous in vitro immunological studies. However, microarray and qPCR validation results did not highlight pro-inflammatory molecules (such as TNFα, TLR2, IL-12B and IL-6), whereas inflammation is one of the most characteristic traits of acute CBPP. This global gene expression pattern may be considered as the result, in blood, of the local pulmonary response and the systemic events occurring during acute CBPP. Nevertheless, to understand the immune events occurring during disease, detailed analyses on the different immune cell subpopulations, either in vivo, at the local site, or in vitro, will be required. Whole blood transcriptome analysis remains an interesting approach for the identification of bio-signatures correlating to recovery and protection, which should facilitate the evaluation and validation of novel vaccine formulations.

Fig 1. Experimental protocol and cELISA CBPP kinetics.

Healthy zebus were put in contact with Mmm-infected cattle (time = 0). Mmm-specific antibody responses were assessed twice before contact, monthly after contact, and on the day of slaughter, using the CBPP cELISA test (IDEXX, Montpellier, France). The seropositivity threshold, defined by inhibition percentage values > 50%, is represented by the red horizontal line. For transcriptome analyses, blood samples were collected before the introduction of infected cattle (①, time = -10 weeks) and subsequently, blood was sampled from six newly infected zebus when CBPP clinical signs appeared (②, time = 10 weeks for 724, 727 and 732 and 11 weeks for animals 747, 757 and 764). ° Animal euthanized because of severe clinical signs. * Animal slaughtered at the end of the experiment.

Fig 2. Hierarchical combined tree.

Hierarchical cluster dendrogram, grouped by GeneSpring according to the similarity in the expression of 1115entities. Red lines represent increased mRNA level and blue lines represent decreased mRNA level, with Benjamini Hochberg False Discovery Rate corrected p-value < 5 10⁻² and absolute fold change ≥ 2.

Fig 3. Top functions related to immune response differentially expressed in whole blood of Mmm-infected cattle.

The ten most modulated top functions related to immune response were categorized by Ingenuity Pathway Analysis software according to p-values and z-scores. The number of differentially expressed genes in each top function is given.