Mycoplasma hyopneumoniae infection: pathogenic factors, mechanisms of response and alternative strategies of immunological stimulation

Abstract

Mycoplasma hyopneumoniae, the main etiological agent of Swine Enzootic Pneumonia, is a widespread microorganism in the world swine production, which prevention is of high interest to the productive system since its colonization in the lung tissue leads to intense productive losses. The dynamics of the agent and the mechanisms of stimulation of the immune system, as well as the formation of lung injury, are still poorly understood. Similarly, commercially available vaccines are known to attenuate the clinical manifestation of the disease and lung lesions, but lead to a weak mucosal immune response and do not prevent bacterial colonization. Therefore, we seek to develop two parallel and complementary studies, aiming to (i) better understand the dynamics of the host and the stimulation of the immune system, and (ii) to develop a specific immunostimulatory carrier for disease, administered orally, by encapsulating proteins of the agent. It is intended to increase the immune response of the respiratory mucosa by stimulating the intestinal mucosa, and consequently, to decrease the number/extent of lung lesions at slaughter. For both studies, 74 piglets will be used. The first will use 24 animals, 16 will be challenged with 5 mL of culture medium containing 106 CCU/mL of M. hyopneumoniae, administered via the tracheal route, while eight will compose the control group, inoculated with only 5mL of pure culture medium. Blood and nasal swab samples will be taken weekly to assess serological response and monitor excretion of the agent. Fortnightly, four infected animals and two controls will be euthanized for macroscopic evaluation of lung lesions and lung, trachea and tracheobronchial lavage samples for bacterial load quantification and gene expression of cytokines. The experiment will take 56 days after infection. The second study will use 50 piglets, divided into five groups of 10 animals. Each group will receive a weaning vaccine protocol, with Group 1 (G1) having piglets vaccinated with a single-dose commercial vaccine; Group 2 (G2) will have piglets exposed to one dose of immunostimulant; in Group 3 (G3) the piglets will be vaccinated with commercial single dose vaccine and immunostimulant booster after 4 weeks; in Group 4 (G4) the piglets will receive two doses of the immunostimulant, one at weaning and the other 4 weeks later; and Group 5 (G5) will constitute the control group, without vaccination. All animals in this study will be challenged at 70 days of age, with the same dose and via as previously described. From day one, oral fluid samples will be collected from each pen for IgA response monitoring every three days. Nasal and oral swabs will be weekly collected to monitor mucosal antibody levels by ELISA, and laryngeal swabs after challenge to assess qPCR excretion of the agent. Fortnightly, blood samples will be taken to monitor serum antibody levels. Thirty days after the challenge, 50% of the animals in each group will be euthanized, the lungs of all animals will be evaluated for the extent of the lesions, and tracheobronchial lavage will be collected for ELISA antibody measurement and bacterial DNA detection by qPCR, as well as lung fragments for bacterial load quantification by qPCR and histopathology (HE). The remaining animals will be euthanized 60 days after the challenge, about 130 days of age, and will undergo the same evaluations as the previous ones. The data obtained in both studies will be evaluated for the normality of the variables by the Shapiro-Wilkins test. If there is normality, analysis of variance (ANOVA), simple T-test and paired T-test will be applied to compare groups at different times. If there is no normality, non-parametric tests (Wilcoxon) will be used. In order to evaluate the presence of correlation between lung lesions and the investigated variables (antibody titer and bacterial load), Pearson or Spearman correlation tests and linear regression analysis will be performed. (AU)