Glial Fibrillary Acidic Protein and Ionized Calcium-Binding Adapter Molecule 1 Immunostaining Score for the Central Nervous System of Horses With Non-suppurative Encephalitis and Encephalopathies.

Like humans, horses are susceptible to neurotropic and neuroinvasive pathogens that are not always readily identified in histological sections. Instead, alterations in astrocytes and microglia cells can be used as pathological hallmarks of injured nervous tissue in a variety of infectious and degenerative diseases. On the other hand, equine glial cell alterations are poorly characterized in diseases. Therefore, in this study, we provide a statistically proved score system to classify astrogliosis and microgliosis in the central nervous system (CNS) of horses, based on morphological and quantitative analyses of 35 equine cases of encephalitis and/or encephalopathies and four non-altered CNS as controls. For this system, we used glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1) immunohistochemistry, allied to statistical analysis to confirm that the scores were correctly designated. The scores of alterations ranged from 0 (non-altered) to 3 (severely altered) and provided a helpful method for describing astrocytic and microglial alterations in horses suffering from inflammatory and degenerative lesions. This system could be a template for comparative studies in other animal species and could aid algorithms designed for artificial intelligence methods lacking a defined morphological pattern.

Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses.

A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin-embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10% unbuffered formalin. Two deparaffinization methods and three proteinase K-based lysis step were tested and translated into three protocols. After detailed statistical analysis it was determined that a longer period and increase in volume of proteinase K incubation provide higher yields and purity of RNA (P < 0.01) of archived samples. Alongside, amplification of equid-housekeeping gene up to 298 bp was successful with the protocol adaptations. For different formalin-fixation timepoints, it was demonstrated that the right choice for treatment and formalin-fixation period is organ-related (P ≤ 0.05). Essentially, little alterations to pre-existing extraction protocols unwound the RNA of up to 11-year-old samples, enabling the use of FFPE tissue archives or e.g. harshly fixed material needed in infection research under high biosafety levels for a variety of molecular analysis.

Pathology and causes of death in stranded humpback whales (Megaptera novaeangliae) from Brazil.

This study describes the pathologic findings of 24 humpback whales (Megaptera novaeangliae) found stranded along the Brazilian coast from 2004 to 2016. Eighteen (75%) animals evaluated were found stranded alive. From these, 13 died naturally on shore and five were euthanized. Six died at sea and were washed ashore. Of the 24, 19 (79.2%) were calves, four (16.7%) were juveniles, and one (4.2%) was an adult. The most probable cause of stranding and/or death (CSD) was determined in 23/24 (95.8%) individuals. In calves, CSD included neonatal respiratory distress (13/19; 68.4%), infectious disease (septicemia, omphaloarteritis and urachocystitis; 3/19; 15.8%), trauma of unknown origin (2/19; 10.5%), and vehicular trauma (vessel strike; 1/19; 5.3%). In juveniles and adult individuals, CSD was: emaciation (2/5; 40%), sunlight-thermal burn shock (1/5; 20%); and discospondylitis (1/5; 20%). In one juvenile, the CSD was undetermined (1/5; 20%). This study integrates novel findings and published case reports to delineate the pathology of a South-western Atlantic population of humpback whales. This foundation will aid in the assessment of the population health and establish a baseline for development of conservation policies.