abstract

Conjunctiva-associated lymphoid tissue (CALT) is a portion of the immune system most closely associated with the eye, forming its first line of defense against potential pathogenic invasion. In most species, CALT is located within the upper and lower palpebrae as well as the nictitating membrane and has a close association with tears and their formation. As manatees have been found to possess the thickest tear film of any sea mammal, and possibly of any animal¹, we have begun to investigate the relationship of tears and CALT in the Florida manatee. While tear analysis with regard to CALT has been used in human ophthalmology for the past decade or so, it is in its early stages in veterinary medicine. Our preliminary investigations have revealed that the manatees' thick, mucous tear film contains proteins, including antibodies (IgG) that would prevent bacteria and other pathogens from causing disease.

For this study, samples from 12 animals were collected from the Marine Mammal Pathobiology Laboratory in St. Petersburg, Florida and placed in 10% buffered formalin, embedded in paraffin, and sectioned at 5 μm. Sections were stained using either hematoxylin and eosin, Masson's trichrome or periodic acid Schiffs (PAS). Cell populations had begun to be evaluated by immunohistochemistry (IHC). Localization for macrophages was performed by using the AM-3K antibody (mouse monoclonal anti-human), which has excellent cross-reactivity of macrophages of a wide range of species¹.

All specimens revealed well-developed CALT within the upper eyelid that extended from the fornix to the margin of the gray line of the eyelid. The CALT consisted of both diffuse and nodular lymphatic tissue, which laid immediately beneath the epithelium, that for the most part was stratified squamous but at times became reduced to simple squamous with intermittent areas of pseudostratified columnar epithelium. These latter areas were confluent with the ducts of the adjacent accessory mucous glands. The ducts were most developed in the regions between adjacent nodules and were often serpentine. IHC localization of macrophages revealed their presence within these ducts and indicated that duct-associated lymphoid tissue (DALT) occurs in the upper eyelids of this species. In the nictitating membrane, the CALT consisted of both diffuse and nodular forms as well. The diffuse form was closely associated with the glandular tissue, whereas the nodular form appeared to be less associated with the ducts than seen in the eyelids.

The CALT of the Florida manatee is highly developed, consisting of a prominent lymphoid layer along the superficial conjunctiva of the upper eyelid and bulbar conjunctiva of the nictitating membrane. The occurrence of numerous Peyer's patch-like large nodules within the upper eyelid, a feature only previously described in the guinea pig, is associated in a way comparable to the DALT of parotid glands. Variations in the development of the CALT were seen and may be indicative of the health of the animal. By sampling manatees' tear film in addition to performing other standard tests, we hope to more efficiently evaluate manatees' immune system function and better determine strategies for rescue, treatment and rehabilitation.

acknowledgements

This work was supported by a grant from the Florida Fish and Wildlife Commission.

References

1.  Komohara Y, et al. 2006. AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with anti-inflammatory macrophage phenotype. J. Histochem. Cytochem. 54:763-771.