Impact of different immunization protocols against Mycoplasma hyopneumoniae and Porcine Circovirus (PCV-2) on the swine respiratory microbiota

• Grant number: 25/02007-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: May 01, 2025
• End date: April 30, 2030
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal Investigator: Luís Guilherme de Oliveira
• Grantee: Isabela de Andrade
• Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil

Abstract

Mycoplasma (M.) hyopneumoniae and porcine circovirus type 2 (PCV-2) are important respiratory pathogens present on farms and that cause great economic losses to the swine industry worldwide. This project aims to evaluate different immunization protocols for these pathogens, assessing the impact of vaccination, including commercial vaccines and a new oral vaccine, with booster doses in the finishing phase, on the respiratory microbiota of pigs and comparing it with unvaccinated animals. To carry out this study, 80 piglets, 21 days old, from a farm certified as free of M. hyopneumoniae will be selected. These piglets will be divided into eight experimental groups, each subjected to different vaccination protocols, as follows: G1 (control): Piglets that will not receive any type of vaccine; G2: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, without booster dose; G3: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, receiving a booster dose for M. hyopneumoniae of the same vaccine at 65 days of age; G4: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, revaccinated at 65 days of age against M. hyopneumoniae with a vaccine different 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 of age only against PCV-2; G7: Piglets vaccinated at 25 days of age against M. hyopneumoniae and PCV-2, receiving a booster dose with oral vaccine at 65 days of age; G8: Piglets vaccinated at 25 days of age against M. hyopneumoniae with oral vaccine and PCV-2, receiving a booster dose with oral vaccine at 65 days of age. Fifteen days after administration of the second dose of vaccines, two animals from each group will be euthanized for initial data collection, while the other eight will be challenged with 10^6 CCU/mL of M. hyopneumoniae, via the endotracheal route. These remaining eight animals will be euthanized 45 days after receiving the last dose of the vaccine protocol. Regular physical examinations, weighing and collection of biological samples will be performed, starting on the day of the first vaccination (D0), when nasal swabs will be collected. The clinical evaluation will include cough monitoring and weighing of the animals to calculate daily weight gain, at D15, D40, D55, D65, D75 and D85. The remaining animals in each group will be euthanized and necropsied on D85, with bronchoalveolar lavage (BAL) collection and evaluation by the Pneumonia Index (PPI). Nasal swab and BAL samples will be used to analyze the respiratory microbiota, which will undergo metagenomic DNA isolation, followed by amplification and sequencing of the V3-V4 region of the 16S-rDNA gene on a high-performance platform (Nanopore), for later metataxonomic analyses, with taxonomic affiliation and diversity analyses. With the results obtained, statistical analyses will be performed to compare the groups, internally within each group and the variations along the respiratory tract (upper vs. lower) of the pigs analyzed. The normality of the variables will be verified by the Shapiro-Wilk and Bartlett tests. If the assumptions are met, analysis of variance (ANOVA) and Tukey's test will be applied for multiple comparison. If there is no normality, non-parametric tests (Kruskal-Wallis) will be used. To analyze the possible correlation between the quantitative variables, Pearson's or Spearman's correlation will be applied, depending on whether the variable meets the assumptions or not.