Abstract
Bovine laminitis disorder results in animal welfare and economic concerns in dairy and beef farms worldwide. However, the affected metabolic pathways, pathophysiologic characteristics, and inflammatory mechanisms remain unclear, hampering the development of new diagnostics. Using cerumen (earwax) as a source of volatile metabolites (cerumenomic) that carry valuable biological information has interesting implications for veterinary medicine. Nonetheless, up to now, no applications of veterinary cerumenomic assays have been made to identify bovine laminitis. This work aims to develop a veterinary cerumenomic assay for bovine laminitis identification that is non-invasive, robust, accurate, and sensitive to detecting the metabolic disturbances in bovine volatile metabolome. Twenty earwax samples (10 from healthy/control calves and 10 from laminitis calves) were collected from Nellore cattle, followed by Headspace/Gas Chromatography-Mass Spectrometry (HS/GC–MS) analysis and biomarker selection in two multivariate approaches: semiquantitative (intensity data) and semiqualitative (binary data). Following the analysis, cerumen volatile metabolites were indicated as candidate biomarkers for identifying bovine laminitis by monitoring their intensity or occurrence. In the semiquantitative strategy, the p-cresol presented the highest diagnostic figures of merit (area under the curve: 0.845, sensitivity: 0.700, and specificity: 0.900). Regarding the binary approach, a panel combining eight variables/volatiles, with formamide being the most prominent one, showed an area under the curve, sensitivity, and specificity of 0.97, 0.81, and 0.90, respectively. In summary, this work describes the first veterinary cerumenomic assay for bovine laminitis that indicates new metabolites altered during the inflammatory condition, paving the way for developing laminitis early diagnosis by monitoring the cerumen metabolites.
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Data availability
The GC–MS raw data files (.mzXML extension) and MZmine project are freely available on the Mendeley Data repository: https://www.doi.org/10.17632/ngkkjk9t74.1
Code availability
The datasets and R script used in this work are freely available on GitHub: https://github.com/Barbosa-JMG/Data-repository-for-Bovine-laminitis-protocol.git
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Funding
This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior— Brazil (CAPES)— Finance Code 001—for fellowship to J.M.G.B. (Process Number: 88882.386430/2019–01); A.L.R.R.C. (Process Number: 88887.819733/2023–00); L.C.D. (Process Number: 88887.805333/2023–00); I.N.C. (Process Number: 88887.819729/2023–00), and the management of financial resources by the Agência Nacional do Petróleo, Gás Natural e Biocombustíveis (ANP), Fundação de Apoio à Pesquisa (FUNAPE), Fundação Rádio e TV (RTVE) and Universidade Federal de Goiás.
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N.R.A.F. is responsible for the original idea and the analysis conditions for using cerumen for clinical diagnoses in humans and animals. J.M.G.B., P.H.J.C., J.J.R.F., and N.R.A.F. the work conceptualization, visualization, and study design. F.S.G., D.R.M., M.T.V., R.D.S.C., J.J.R.F., A.R.C.F., I.M.V., and P.H.J.C. performed the biochemical exams and clinical evaluation of the animals to confirm laminitis or control condition. J.M.G.B. and A.L.R.R.C. realized the earwax sample collection. J.M.G.B., A.L.R.R.C., and N.M.M., performed the HS/GC–MS analysis. J.M.G.B. and A.L.R.R.C. conducted the software analysis and interpretation. J.M.G.B. wrote the original draft. J.M.G.B., A.L.R.R.C., L.C.D., I.N.C., J.J.R.F., P.H.J.C. and N.R.A.F. performed the formal investigation, data curation, and writing review and editing. N.R.A.F. provided the funding acquisition and project administration. All authors approved the final version of the manuscript.
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This study was approved by the ethical standards of the local ethical committee on animal research at the Universidade Federal de Goiás, Brazil (protocol number: 027/16). All applicable international, national, and institutional guidelines for the care and use of animals were strictly followed.
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Barbosa, J.M.G., Cunha, A.L.R.R., David, L.C. et al. A veterinary cerumenomic assay for bovine laminitis identification. Vet Res Commun 48, 1003–1013 (2024). https://doi.org/10.1007/s11259-023-10271-2
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DOI: https://doi.org/10.1007/s11259-023-10271-2