Caracterización y resistencia a antimicrobianos de aislados españoles de "Glaesserella parasuis". Valoración de la eficacia de una vacuna frente a la enfermedad de Glässer basada en una TbpB (proteína de unión a la transferrina) modificada

Abstract

The swine industry faces significant economic challenges, with Glässer's disease being one of the most relevant. This disease is caused by Glaesserella (Haemophilus) parasuis, a pathogen that colonizes the upper respiratory tract of pigs and, under stressful conditions such as weaning or transportation, can trigger severe systemic infections. In addition, this bacterium is one of the secondary pathogens included in the Porcine Respiratory Disease Complex. Piglets in the early stages of lactation and fattening are particularly vulnerable, leading to high morbidity and mortality rates. This bacterium acquires iron from porcine transferrin through a surface receptor composed of the transferrin-binding proteins TbpA and TbpB, facilitating its survival in iron-limited environments, such as mucosal surfaces. However, the virulence mechanisms of this bacterium are not yet fully understood, and the challenge is further exacerbated by the concerning rise in antimicrobial resistance, driven by its intensive use in pig production. This dual issue highlights the urgent need to deepen research into the pathogenic factors of G. parasuis and to develop strategies that reduce the use of these drugs without compromising disease control. This phenomenon undermines the effectiveness of treatments and poses an additional risk of resistance gene transmission to other bacteria in the environment. Nonetheless, in the absence of a commercially available universal vaccine to prevent this disease, antimicrobial treatment remains the primary control strategy despite the associated challenges. This underscores the necessity to develop accurate diagnostic tools, promote responsible antimicrobial use, and encourage research to advance the design of effective, broad-spectrum vaccines. The main objective of this doctoral thesis is to provide a detailed characterization of the virulence and antimicrobial resistance profiles of G. parasuis isolates from Spanish pig farms. In parallel, the genetic diversity of the TbpB protein in these isolates will be analyzed, with the aim of evaluating the effectiveness of a recombinant TbpB-based vaccine against a variety of clinical serovars representative of Glässer's disease in Spain. First, 68 clinical G. parasuis isolates collected between 2018 and 2021 from various regions of Spain were analyzed. These isolates were obtained from respiratory and systemic samples from pigs. For their characterization, a species-specific PCR targeting the tbpA gene and a multiplex PCR assay were used to identify the present serovars. Serovars 5, 10, 2, 4, and 1 were found to be the most prevalent, accounting for approximately 84% of the isolates, highlighting their high prevalence across the country. An analysis of the amino acid sequences of the TbpB protein in 59 of these isolates was subsequently conducted, allowing for the classification of the strains into ten distinct clades. This finding reflects significant genetic diversity among the strains, which is associated with both capsular types and the various anatomical and geographical origins of the isolates. To characterize the virulence of the isolates, virulence was assessed using multiplex PCR with leader sequence targeting the vtaA genes, and all isolates were identified as clinical (presumed virulent). Pangenome-based pathotyping revealed that the virulent gene HPS_22970 was the most frequent (83.1%). Various pathotype profiles were observed, with 29 unique gene combinations and two isolates carrying only potentially non-virulent pangenome genes. Subsequently, the antimicrobial resistance profiles of the isolates were analyzed, revealing widespread resistance in the antimicrobial resistance phenotyping, with 63.3% classified as multi-resistant, and high resistance to clindamycin (98.3%) and tylosin (93.3%). A statistically significant association was found between certain pathotype genes and antimicrobial resistance phenotypes, particularly between the virulent gene HPS_22970 and tetracycline resistance (p < 0.001). Finally, this study assessed in two phases the immunogenicity and protection of a two-dose oral vaccine based on a modified TbpB (TbpBY167A) in colostrum-deprived piglets. In the first phase (days 0-45), the immune response was analyzed, and in the second phase (days 45-52), heterologous protection was evaluated after challenging the piglets with four clinical isolates of G. parasuis from different serovars. The vaccine induced a robust immune response, with a significant increase in IgA and IgM. Following the challenge, improved survival rates were observed, along with a reduction in clinical signs and lesions, regardless of the serovar. Additionally, the vaccine reduced colonization in the respiratory tract and in systemic tissues such as joints, liver, and brain. Immunohistochemical analysis revealed a lower number of macrophages in the lungs of immunized piglets. The findings of this study highlight the genetic variability of G. parasuis and its complex pathogenicity, underscoring the need for precise characterization of clinical isolates to ensure appropriate antimicrobial treatments and effective prophylactic measures against virulent strains. Despite the genetic diversity observed in the serovars, in silico findings suggest that a vaccine based on the TbpB protein could provide broad protection against outbreaks of Glässer’s disease in Spain, emphasizing its potential as a control tool in the swine industry. Regarding the in vivo study, the oral administration of the subunit vaccine TbpBY167A in piglets has proven effective, offering heterologous protection against different virulent isolates, significantly reducing bacterial colonization and dissemination in tissues. This approach overcomes the limitations of traditional vaccines by counteracting the effects of maternal immunity, making it a promising alternative for universal vaccination against Glässer’s disease.