Advances in the study of inflammation biomarkers in the saliva of pig

Resumo

Sepsis is a severe and life-threatening condition characterized by a systemic inflammatory response triggered by an infectious agent that can finally lead to organ dysfunction and even death. Biomarkers can allow an earlier intervention and better treatment of sepsis, improving the health outcomes and welfare of patients, and reducing development of antibiotic resistances and economic losses. In human medicine, several biomarkers are routinely used to early detect bacterial infections and guide in antibiotic stewardship, like procalcitonin. In veterinary medicine, even though inflammatory and infectious diseases lead to many health problems and economic losses in farms, this field is poorly explored. This PhD thesis seeks to contribute to the field of sepsis diagnosis and characterization in pigs by deepen and widen our understanding of inflammatory biomarkers, their behavior, and their relevance in sepsis in pigs. The specific objectives are: • Objective 1. Bibliographic research about sepsis and the current most common biomarkers used to diagnose and monitor it in veterinary medicine. • Objective 2. Identification of new potential biomarkers using proteomics techniques. • Objective 3. Validation and measurement of various biomarkers of inflammation, oxidative stress, welfare, or muscle damage, with potential application in septic inflammation and study of their possible changes in sepsis and other conditions. • Objective 4. Development and validation of new assays to diagnose sepsis: procalcitonin and presepsin. To this end, the methods that were used were: • Samples from pigs were collected mainly in saliva, using polypropylene sponges. Saliva is considered a non-invasive sample that ensures welfare in pigs and allows for serial sample collections, even in the same day, and by non-trained personnel. Blood was collected with comparative purposes, through jugular vein puncture. • Proteomic gel-based and gel-free analyses of pig samples were conducted to identify new biomarkers, including SDS-page and mass spectrometry. • Various methods, including automated commercial kits, ELISA, and chemiluminescent immunoassays, were employed to analyze the studied biomarkers, covering stress, inflammation, redox status, and muscle damage. • Polyclonal and monoclonal antibodies for the detection of two sepsis biomarkers, procalcitonin and presepsin, were developed: polyclonal antibodies through immunization of a rabbit and a goat; and monoclonal antibodies using mice as the chosen species for immunization, with the subsequent production of hybridomas. • The technology used for assays development with the previous antibodies was AlphaLISA, that has several advantages such as minimal sample usage and no need for washing steps.Overall, this research contributed to the development of valuable tools for diagnosing infections and inflammatory conditions, with a comprehensive approach that involved antibody production, sample collection, assay development, and biomarker analysis. The specific conclusions of this PhD these are: 1. The biomarkers with a potential in the diagnosis and monitoring of sepsis can be classified into three main categories: (1) acute phase proteins and cytokines, which have been traditionally used in veterinary medicine for evaluation of inflammation; (2) PCT, PSE, and other more recent proteins that are more specific of bacterial infections; and (3) other markers that can provide complementary information. 2. In the proteomic studies, saliva and serum showed different patterns in response to septic inflammation in a model induced by LPS and meningitis due to Streptococcus suis. In addition, changes in proteins in saliva were found in other septic diseases, such as diarrhoea caused by Escherichia coli. . Sepsis produces changes in salivary analytes related to stress, redox status, inflammation, and muscle damage, which opens the possibility of using them as potential biomarkers for this process in the pig. In this line, further studies are necessary to define the ability and applications of these biomarkers to diagnose and monitor sepsis, as well as establish accurate cut-off values. 4. Procalcitonin was measured for the first time in porcine saliva and showed higher increases in pigs in different septic conditions, such as in an LPS-induced model, Streptococcus suis meningitis, and a tail-biting outbreak, than in non-septic inflammatory processes like a turpentine-induced model.