Efficacy and immune response in vaccination protocols against Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV-2) with booster doses during the finishing phase in experimentally challenged pigs

• Grant number: 24/17201-3
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: December 01, 2024
• End date: November 30, 2029
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal Investigator: Luís Guilherme de Oliveira
• Grantee: Geovana Coelho Ferreira
• Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil

Abstract

The Swine Respiratory Disease Complex is one of the main health challenges in intensive pig farming, with emphasis on infections caused by Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV-2). These co-infections lead to significant production losses, and current vaccination protocols, while reducing clinical signs and lesions, do not fully prevent infection and pathogen spread. This project aims to evaluate the efficacy and immune response of different vaccination protocols, with booster doses during the finishing phase. Sixty piglets, 21 days old, from a farm certified free of M. hyopneumoniae, will be selected and divided into 6 experimental groups, each subjected to different vaccination protocols: G1 (control): Piglets will not receive any vaccines; G2: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, without a booster dose; G3: Piglets vaccinated at 25 days of age against M. hyopneumoniae, receiving a booster dose of the same vaccine at 65 days of age; G4: Piglets vaccinated at 25 days of age against M. hyopneumoniae and revaccinated at 65 days with a different vaccine from the initial one; G5: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, receiving booster doses of both vaccines at 65 days of age; G6: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, being revaccinated at 65 days only against PCV-2. This design will allow comparison of the efficacy of different vaccination approaches, evaluating immune response, reduction in lung lesions, and decreased pathogen shedding. Fifteen days after the second vaccine dose, two animals from each group will be euthanized for initial data collection, while the remaining eight will be challenged with 106 CCU/mL of M. hyopneumoniae by the endotracheal route on D55. These eight animals will be euthanized 45 days after receiving the last vaccination dose. Regular physical exams, weighing, and collection of biological samples will be conducted starting on the day of the first vaccination (D0), including blood samples, nasal and laryngeal swabs. Clinical evaluation will monitor cough and weigh the animals to calculate daily weight gain at time points D15, D40, D55, D65, D75, D85. Additionally, blood samples will be collected for cytokine analysis (IFN-¿, IL-6, IL-10) by ELISA at different time points: D0, D3, D40, D43, D55, D58. These same cytokines will also be analyzed in the bronchoalveolar lavage fluid (BALF). Furthermore, blood samples and nasal swabs will be collected for IgG and IgA detection on days D0, D15, D40, D55, D65, D75, D85. The collected serum will be used for porcine circovirus type 2 (PCV-2) viremia analysis via qPCR. After the challenge with M. hyopneumoniae, laryngeal swabs will be collected on days D58, D65, D75, D85 to assess pathogen shedding. The Pneumonia Index evaluation and collection of lung, spleen, and lymph node fragments, as well as BALF, will occur during animal necropsies. Spleen samples will be used for cytokine expression analysis (IFN-¿, IL-6, IL-10, IL-17, TNF-¿). Lymph node fragments will be used for PCV-2 detection. Lung fragments will undergo histopathological analysis and qPCR for both agents. These detailed analyses will allow a precise evaluation of vaccine efficacy and their influence on the animals' immune responses. The normality of variables will be checked using the Shapiro-Wilk and Bartlett tests. If assumptions are met, analysis of variance (ANOVA) and Tukey's test will be applied for multiple comparisons. If normality is not observed, non-parametric tests (Kruskal-Wallis) will be used. To analyze possible correlations between quantitative variables, Pearson or Spearman correlation will be applied, depending on whether the variable meets the assumptions or not.