Histopathology of nosemosis in honey bees: correlation with manual counting and comparison of staining methods

Nosemosis, caused by <i>Vairimorpha</i> (<i>Nosema</i>) <i>ceranae</i> or <i>V.</i> (<i>Nosema</i>) <i>apis</i>, is the main fungal disease affecting the Western honey bee (<i>Apis mellifera</i>). We evaluated the use of histology in the diagnosis of disease, identified the histologic patterns, and compared the efficacy of different staining techniques. We sampled 10 hives, collecting ~80 bees per hive. Spore counts were performed on 60 bees per sample using a hemocytometer in accordance with the standard procedure. Slides of whole bees were produced from the remaining bees, stained with 15 different techniques, and observed under a light microscope at 400×. Infection in the ventriculus was graded using hematoxylin-phloxine-saffron stain; prevalence and severity of the infection were determined; and an intra-class coefficient (ICC) was calculated to correlate the histologic results with the standard counting method. Based on contrast, specificity, and sensitivity, we found hot Gram chromotrope and Ziehl-Neelsen stains offered the best approach for highlighting <i>Vairimorpha</i> spores. These stains were optimized to find the ideal staining times for <i>Vairimorpha</i> by testing different immersion durations in key steps to enhance spore contrast. There was a notable association between histologic observations and spore count, with an ICC of 0.74 (95% CI [0.36, 0.91]) and 0.82 (95% CI [0.54, 0.93]) for the percentage of infected bees and histologic grade, respectively. Lesions included distension of ventricular epithelial cells, intracellular microsporidia, reduced ciliation, and disintegration of the peritrophic membrane. No spores were detected in extra-ventricular organs.