Advances in the identification and development of biomarkers for health and welfare assessmenta One- Health perspective

Abstract

The main objective of the present PhD Thesis was to increase the knowledge about the identification, assessment, and validation of novel biomarkers of health and wellness that can be used under the One-Health (OH) context. Besides, the use of non-invasive samples such as saliva was employed whenever it was possible. Under this premise, different species were employed: humans; dogs since they are often used as animal models to humans’ diseases; cows that are prone to suffer from mastitis, and condition with high importance in the OH concept; and fish and mussels that, although they are phylogenetically very different, they are commonly used in environmental biomonitoring programs and thus are of high interest for OH. The specific objectives of this doctoral thesis were to: (1) Revise the state-of-the-art about the search and validation of novel biomarkers in different species and sample types, under the applications of the OH concept. (2) Perform proteomic and biochemical studies in various clinical or experimental situations for the identification of novel biomarkers. Studies in humans and mussels were focused on muscular damage, while studies in dogs and cows were related to diseases of interest from the OH point of view due to their prevalence or clinical relevance. An environmental evaluation model was also performed using fish and mussels since these species are commonly employed as sentinels of environmental quality. (3) Identify a common biomarker through the OH application and to develop a rapid-test for its measurement. For the consecution of objective 1, two literature reviews were performed describing the potential benefits from the adoption of the OH concept from a clinical analysis’ laboratory point of view, as well as the main analytical techniques used in saliva in both humans and animals. Besides, the OH level of knowledge of the University of Murcia’s second-year veterinary students before and after a flipped-classroom OH module was evaluated, and the changes in salivary biomarkers after filtration and alpha-amylase depletion were assessed. For the consecution of objective 2, biochemical and proteomic studies were performed in serum and saliva to identify biomarkers of different situations. The muscular damage model was based on a damage-induced mussels model to determine if this species could be used as an animal model and the possible muscular damage that occurred by exercise in humans. Changes in the expression of different proteins were detected by proteomics and biochemical analyses in both cases. The disease model included canine leishmaniosis, parvovirosis, pyometra, mammary tumors and diabetes mellitus in dogs, and mastitis in cows. These studies allowed the identification of more than 6700 proteins, revealing differences of statistical relevance in more than 400 proteins compared to their healthy control group. In the third model, focused on environmental biomonitoring, the studies were centered on validating nine automated methods for the detection of biomarkers and evaluate if they could be useful in detecting the effects caused by pollutants or hypoxia. In fish, cortisol, esterase activity (EA), total oxidant status (TOS), and total antioxidant capacity (TAC) were validated, and they were employed to detect the effects of pollutants derived from the pharmaceutical industry. For the consecution of objective 3 of the present PhD Thesis, CRP protein was identified and purified in mussels for the first time, a commercial assay for the measurement of human CRP was analytically validated, and its usefulness was proven for environmental biomonitoring. A battery of monoclonal antibodies against CRP was produced with the aim of developing a rapid system for detecting inflammation. From the discussion of results obtained in this PhD doctoral Thesis, six conclusions can be derived. 1. The One-Health concept can be applied to laboratories’ conditions by using the same biomarker in different species, sample types, and diseases. 2. The non-invasive samples such as saliva and skin mucus are biofluids of high analytical value since they can show changes in their composition after muscular damage, diseases, or hypoxia. 3. The studies related to muscular damage allowed identifying a potential panel of biomarkers for its diagnosis in human saliva and mussel hemolymph, including lactate, actin, tropomyosin, and cystatin-B. 4. The proteomic studies performed in dogs and cows showed that serum and saliva could provide valuable clinical information in different diseases such as parvovirosis, leishmaniosis, pyometra, diabetes mellitus, mammary tumors, and mastitis. More than 6700 proteins were identified in these studies, and more than 400 were differentially modulated in these diseases. 5. Certain biomarkers employed in human medicine to evaluate oxidative stress can be applied in other species, including fish and mussels, for the biomonitoring of the physiological effects associated with pollution. The characteristics of these biomarkers (fast, economical, sensitive, accessible, automated, and required low sample volume) make them suitable for their use in biomonitoring programs related to pollution or hypoxia. 6. The C-reactive protein has been identified and described in mussels for the first time in this PhD Thesis. Besides, a series of monoclonal antibodies with affinity to CRP were produced, which could be an initial step for creating a rapid and portable system for CRP measurement in different species for the detection of inflammation.