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New approaches aimed at rapid and differential diagnosis of equine pythiosis.


Caused by the oomycete fungus-like organism Pythium insidiosum, pythiosis is an emerging disease that occurs in tropical and subtropical countries, affecting mainly horses, dogs and humans. The disease has a difficult diagnosis and is often confused with other diseases that induce granulomatous lesions in horses. Treatment of pythiosis is also difficult, as the pathogen does not respond satisfactorily to the available antifungal drugs due to the absence of ergosterol in its citoplasmatic membrane, leading to surgical procedures. In this scenario, we propose the standardization and validation of the qPCR and qPCR-HRM techniques for an early and differential diagnosis between granulomatous lesions caused by P. insidiosum and / or zygomycetes fungi. In the samples that are negative for pythiosis and / or zygomycosis, a differential diagnosis will be made for habronemosis and equine sarcoid. For the development of this stage of the study, a qPCR for the specific diagnosis of P. insidiosum will be standardized using primers Pi1 and Pi2 derived from the ITS1 region of rDNA. To perform a rapid and differential diagnosis by qPCR-HRM for molecular differentiation of P. insidiosum and zygomycetes fungi, primers derived from the 18S rRNA region will be designed and employed. Negative samples in qPCR-HRM will be submitted to molecular analysis for Habronema spp. and bovine papillomavirus 1, 2 and 13 (etiological agents of equine sarcoid), the main differential diagnosis of pythiosis in horses. In addition, the characterization of total proteins of this oomycete will be carried out, as well as the evaluation of the immunodominant protein (s) of P. insidosum in equine pythosis, using analytical techniques such as two-dimensional electrophoresis, Western blotting, by mass spectrometry and bioinformatics. From the characterization of the immunodominant protein (s) it is expected the identification of an specific antigen (s) that may allow in the diagnosis of equine pythosis, as well as in the development of a new immunotherapeutic for treatment. (AU)

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