Crystalline Inclusions Associated with Vomeronasal Organ Pathology in Red-Eyed Tree Frogs (Agalychnis callidryas)
The vomeronasal organ (VNO), located in the ventral portion of the nasal septum, plays an important role in chemoreception. Odorant particles from inhaled air and the oral cavity reach the VNO via the incisive duct. Once there, the particles come into contact with two kinds of epithelium. The lateral aspect of the VNO is lined by pseudostratified columnar respiratory epithelium. The medial portion is lined by specialized sensory epithelium composed of basal, supporting, and bipolar nerve cells.2 The neurons deliver chemical messages via the vomeronasal nerve to the olfactory bulb. Pheromones detected in this manner are believed to serve as cues for maternal and male and female sexual behavior.1 Any pathology of the VNO could potentially interfere with normal reproductive behavior and therefore warrants investigation.
Nine red-eyed tree frogs (Agalychnis callidryas) from the collection at the Wildlife Conservation Society died over a 5-yr period with similar lesions in the VNO. The frogs were from a single breeding colony. All but one were captive bred. One frog was obtained from a now defunct dealer and in all likelihood was imported from the wild.
All nine frogs shared a dramatic histologic finding on routine hematoxylin and eosin (H&E) sections. Triangular to polyhedral inclusion bodies were seen in the nuclei of cells in the VNO chemosensory epithelium. The inclusions were brightly eosinophilic, refractile, and had a crystalline appearance. In most cases the inclusions had a base width of approximately 2 microns and were readily visible. In others, the inclusions were smaller, less distinct and were only discerned by focusing in and out of the plane of section.
The inclusions were colorless on unstained sections, intensely eosinophilic on H&E sections and were intensely basophilic with Toluidine blue stains. The inclusions did not stain with periodic acid-Schiff (PAS) or Gomori’s methenamine silver (GMS) stains.
Five out of nine cases had mild to marked, acute to chronic inflammation associated with the VNO. Transmission electron microscopy was performed on two cases. Ultrastructural evaluation revealed greater variety in the size, shape and location of the inclusions than was appreciable at the light microscopic level. The majority of inclusions were intranuclear, but some were found in the cytoplasmic compartment as well. Triangular shapes predominated but rectangular and rhomboid forms were also present. Multiple inclusions were found in some nuclei. These were attached and arranged in a haphazard manner reminiscent of toy building blocks. Individually or when multiple, the inclusions were surrounded by a fine limiting membrane. Internally the inclusions were composed of a highly ordered pattern of closely spaced parallel dense lines approximately 60–70 angstroms diameter. In addition to these, smaller discrete globular amorphous bodies were seen projecting and/or separate from the larger inclusions. When separate, these were surrounded by a multilayered membrane ranging from 66–160 nm in thickness.
A third identifiable structure within the nuclei of cells with inclusions consisted of honeycomb arrays of roughly spherical particles measuring 22 nm in diameter. These particles studded the surface of the inclusions and were also dispersed throughout the nucleoplasm. Additional ultrastructural changes included mitochondrial swelling, dilatation of smooth endoplasmic reticulum, the formation of myelin figures and/or cellular apoptosis. These changes were seen only in cells containing inclusions. Adjacent non-affected cells were normal.
The nature and significance of these inclusions remains unclear. Crystalline inclusions of both viral3,7 and non-viral4-6,8 origin have been described in nearly all compartments of the cell3 in both vertebrates and invertebrates. The presence of honeycombed structures, in association with the inclusions, degenerative cellular changes in only those cells containing inclusions, and the associated inflammation suggest these findings are not innocuous.
Additional studies are being pursued. These include: infrared spectral analysis of the crystals that may provide information as to their chemical composition; immunostaining specific for the neurons of the VNO that may better define the cell type involved; and polymerase chain reaction (PCR) and immunostaining against nuclear polyhedrosis viruses, insect pathogens known to cause similar inclusions.
We sincerely thank Mr. Alfred Ngbokoli for the extensive slide preparation required in this study.
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