Worldwide reports on clinical disease and serological surveys from horses with encephalitis and encephalopathies reveal the increase in emergent, re-emergent, and neglected diseases, mostly due to an infectious agent. Diseases caused by arboviruses or by henipaviruses are known zoonoses, while equine herpesvirus 1 (EHV-1) or protozoal encephalitis are detrimental to animal s performance. However, while most of diagnostics tools are adjusted to fresh tissues, formalin-fixed paraffin-embedded (FFPE) tissues are vastly used in pathology. Therefore, this study used FFPE tissue from the central nervous system (CNS) of 35 horses from Brazil suffering from non-suppurative encephalitis or encephalopathies as study model. The main aim was to provide adequate diagnostic tools adopted to FFPE tissues for morphologic, antigenic, and molecular detection as well as metagenomic screening of relevant pathogens. Samples available for the study consisted of a mixed population of male and female animals, mostly > 4 years-old, predominantly of mixed breeds. Most of the clinical signs (21/35) pointed to a multifocal distribution of lesions in the CNS, and most cases (20/35) occurred in fall. Histologically, 28/35 cases had inflammation in at least one CNS region or ganglion, and 7/35 had degenerative lesions. Score systems established to assess the degree of inflammatory cell infiltration and glial reaction patterns, revealed the predominance of mild lymphocytic inflammation, and 3 statistically distinguishable grades of astrocytic and microglial activation throughout the cohort. In 20/35 cases, an infectious agent could be related to disease - rabies virus/coccidia coinfection (1/35), alphavirus (6/35), flavivirus (4/35), alphavirus/EHV-1 coinfection (3/35), coccidia (5/35) detection, and helminth larvae migration (1/35). For other 15/35 cases, an unknown infectious etiology was suggested, or degenerative lesions could have mimicked an infection. Concurrently, 4/35 cases were also considered pathogen carriers of Babesia sp. or Elizabethkingia anopheles. The long-term stored FFPE materials used in this study provided valuable epidemiological information and identification of pathogens important for animal and human health. Missing CNS regions, environmental contamination, and protein cross-links bound during fixation might have hindered pathogen identification. In this regard, optimization of techniques such as multiplex sequencing assays using target enrichment could further improve the sensitivity of pathogen detection and decrease host/environment background. Moreover, the methods optimized in this study enable zoonotic pathogen assessment in humans and can also be transferred to important livestock species, like ruminants and pigs, which share susceptibility to several infectious and zoonotic agents. It is expected that during an outbreak e.g., basic laboratories located near affected areas will be able to provide an etiology using the methods quickly and precisely, with the routine procedures of tissue fixation.
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