Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment

Rickettsiae Infection in Farmed Variously Colored Abalone Haliotis diversicolor Reeve (Mollusca, Gastropoda) from Daya Bay, Guangdong in China

Xinzhong Wu


The artificial rearing of the variously colored abalone Haliotis diversicolor (Reeve) along the coasts of Guangdong Province, China, has developed in recent years. With the development of abalone culture, mass mortality of the abalone has also occurred (natural mortality rate has been about 30–50%). The deaths generally have occurred during the summer months. Extensive foot ulcerations and atrophy of abalone foot muscle and mantle, visible to the naked eye, are the main disease symptoms.

To investigate disease occurrence in a cultured abalone population, suspected pathogenic organisms were surveyed. The present study describes an extensive rickettsial infection and histopathological findings associated with the diseased abalone population.

Approximately 100 diseased variously colored abalone (about 3–5 cm in shell length) were collected from a culture facility in Daya Bay, Shen Zhen City, Guangdong Province, in August 1997. Twelve of these animals were killed and dissected. Portions of mantle, digestive gland, digestive tract (including stomach and intestine), foot, and gill tissue were fixed in 4% neutral Formalin fixative and Bouin’s fixative and sliced into 4-µm sections for examination by light microscopy.

Small fragments (about 1 mm long) of the dissected tissues were fixed in cold 2% glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide for 1.5 h at 4°C, dehydrated in a series of ethanol solutions, and embedded in Epon 812 resin. First, semithin sections (2 µm) were sliced and stained with 1% toluidine blue for orientation. Next, ultrathin (60-nm) sections were cut, stained with uranyl acetate and lead citrate, and examined with a Philip CM 10 Hitachi transmission electron microscope at 60 kV.

Light microscopy revealed infections with a rick-ettsia- like bacteria in 12 animals. Large numbers of eosinophilic, intracytoplasmic inclusions in the epithelial cells of epipodium and mantle as well as the connective tissue cells of mantle and gonad were observed on the sections stained with Harris’ hematoxylin and eosin (H&E) stain (Figures 1–4). Only a few inclusions were observed in the epithelial cells of digestive tract and gill. In the digestive gland and foot muscle, no inclusions were observed. Many fine round eosinophilic granules were involved within inclusions. These inclusions appeared round or ellipsoidal in all tissues and averaged 9.05 × 6.02 µm (n = 50), as measured in sectioned materials, in the foot and 8.41 × 5.57 µm (n = 45) in the mantle.

Ultrastructural observations of the mantle and foot revealed that rickettsiae are sparsely free-living in the cytoplasm of epithelial cells of the variously colored abalone (Figure 5). They usually appear round and ellipsoid-shaped, sometimes long spindled-shaped (Figure 6), and measured 1244.2 × 723.7 nm (n = 20) as in transverse sections of electron microscope. Of the rickettsia organisms observed, the largest one was 3,300 nm long. The rickettsiae showed the rippled cell wall, which consists of two trilaminar membranes (Figure 7) and dot- or filament-like nucleoid structures (Figure 8) of the family Rickettsiaceae. Each prokaryotic organism was filled with electron-dense ribosomal bodies in the cytoplasm (Figures 6–8).