abstract
In 2007, dolphins in a large public exhibit facility developed proliferative lesions in new or pre-existing skin wounds.1 Raised nodules were associated with progressive erosion of the tissue, and the wounds developed into coalescing ulcers. Skin scrapings from the lesions revealed a diversity of organisms, including bacteria, fungi and ciliates. To understand the role of the fungi and ciliates in the pathogenesis of the skin lesions, we pursued a diversity of approaches to identify them, and to determine their relationship with the host.
For the ciliates, we took skin scrapings, preserved them in Bouins fixative, and then stained the whole mounts with Protargol silver protein, in order to show the key taxonomic features of the somatic and oral ciliature.2,3 Additional skin scrapings were preserved in Trumps fixative4, and processed for transmission electron microscopy (TEM) to show ultrastructural features of the ciliate--with particular attention being given to the cytoplasm, and examined for evidence of the kind of material that had been ingested.
Protargol impregnation showed that the flattened pyriform (pear-shaped) ciliates had two bands of cilia on their ventral surface close to the body margins, a longer arched right band, and a shorter straight left band; the dorsal surface had only a short kinety (row of cilia) at the anterior end of the body. Around the oral opening (cytostome) were three kineties. The cytostome led down into the inner cytoplasm of the cell via a conical cytopharynx. At the narrow anterior end of the cell, the cytoplasm was rather homogenous, in contrast to the broader middle and posterior regions of the cell, where the cytoplasm was heterogenous, with numerous inclusions. The ciliates divided by transverse binary fission. The morphological features of the ciliate were consistent with the genus Chilodonella, which contains some species that infect cetaceans, while others infect fishes or are free-living. The numerous individuals from a dolphin lesion were morphologically similar, suggesting that a single species of Chilodonella was present in the lesions.
The TEM studies showed fine detail of the ciliate, including the cell surface with pinocytotic vesicles, the infra-ciliature (the network of microtubules and fibres just beneath the cell surface), cytopharynx, diverse cytoplasmic organelles, and degenerate structures, which probably represented partially digested food items. Many of the degenerate structures were identified as cells, since they had a distinct cell membrane, a more or less amorphous cytoplasm, and a degenerate nucleus.
We suggest that the ingested cells were host cells, though we have not yet determined if they are inflammatory, epidermal, or dermal. Concurrent light microscopic (LM) studies1 showed that there was an infiltration of neutrophils. Necrotic cell debris occurred in the lesions, providing a source of nutrition. We did not see any structures recognizable as microbial agents in the cytoplasm of the ciliates, suggesting that the ciliates fed selectively on host tissue.
Extensive colonies of a fungus and bacteria occurred in some lesions. We have examined the fungus in fixed material using LK but have not yet identified it. Neither fungi nor bacteria produced colonies when fresh material was cultured using standard media. Their role in the pathogenesis of the lesions was not established.
References
1. Doescher B, Renner R, Clarke A, Poynton SL, Ma H, Overstreet R. 2008. Cutaneous ciliate protozoan infection in healthy Atlantic Bottlenose dolphins (Tursiops truncatus). Abst. Proc. IAAAM.
2. Lynn D, Small EB. 2000. Phylum Ciliophora. In: Lee, JJ., Leedale, GF., Bradbury, P. An Illustrated Guide to the Protozoa. Society of Protozoologists, Lawrence, Kansas. Pp. 371-656.
3. Lom J, Dykova I. 1992. Protozoan Parasites of Fishes. Elsevier, Amsterdam.
4. McDowell E, Trump B. 1976. Histological fixatives for diagnostic light and electron microscopy. Archives of Pathology and Laboratory Medicine 100: 405-414.