Abstract

This report describes a rickettsia-like organism (RLO) in the tissues of the blue-eyed plecostomus, Panaque suttoni. Although there are limited reports of rickettsia-like organisms in fishes (sea raised salmon), this represents the first known reported case of an RLO infection in a tropical freshwater fish. Both histopathology and cytopathology revealed numerous, small, discrete, basophilic organisms within the cytoplasm of the macrophages in the spleen, heart, kidney, and sometimes liver as well as within the cytoplasm of circulating monocytes.

Discussion

Blue-eyed plecostomus are popular fish among dedicated fresh water aquarists. These fish are imported from South America and often arrive in the United States in poor condition. High mortality occurs within the three week period following transcontinental shipping. Surviving fish are often anorectic, cachectic and frequently succumb to secondary bacterial infections.

Two moribund blue-eyed plecostomus from Columbia were examined in an attempt to determine the cause of morbidity. Blood samples were drawn from both fish by intracardiac puncture. The samples were centrifuged and cytological preparations of the buffy coats were made and stained with Giemsa (Wohlbach's modification) (Sheenan and Hrapchak, 1980). The fish were euthanized with an overdose of tricane methanesulfonate (300mg/L) and tissue samples were fixed in 10% neutral buffered formalin and in Trump's fixative. Large numbers of intracytoplasmic organisms resembling rickettsia were found in the cells of the mononuclear phagocytic system.

Examination of the buffy coat smears revealed moderate numbers of monocytes filled with numerous small (0.5um or less) basophilic intracytoplasmic organisms. These organisms appeared to replace the foamy cytoplasm of the monocytes and peripherally displaced the nucleus. Viewing the organisms at higher magnification revealed that many of the organisms had a dumb-bell shape.

Histopathological examination of tissues showed that the most significant changes were in the spleen, heart and kidney. There was a diffuse, severe granulomatous splenitis, pericarditis, and a mild to moderate granulomatous interstitial nephritis. The normal splenic architecture was replaced by islands and sheets of large, round to polyhedral, vacuolated cells that are separated by mats of pale eosinophilic fibrillar material. These vacuolated macrophages were markedly distended by small basophilic organisms (with H&E stain; Sheenan, 1980) within the vacuoles. These organisms were negative with Gram stain (Raphael, et al., 1976) and Fite's acid fast stain (tuna, 1968). Results of the Macchiavello's stain (Brancroft and Stevens, 1982) and Pinkerton's stain (tuna, 1968) for rickettsia were equivocal, with some organisms staining bright red (positive) and others blue. Large numbers of similarly affected macrophages were found peripheral to the epicardium and interspersed within the pericardial stroma. Individual macrophages or small aggregates containing the organisms were scattered within the renal interstitium, along with occasional small tufts of fibrin only a few Kupffer cells in the liver were found to contain the small basophilic organisms.

Transmission electron microscopy revealed macrophages within the renal interstitium that contained organisms and had characteristics suggesting metabolic activity. These cells were characterized by moderate numbers of mitochondria and had prominent interdigitating membranes. Degenerative changes such as swollen mitochondrial membranes, small vacuoles and sometimes karyorrhectic nuclei were occasionally associated with these cells.

The organisms within the macrophages occurred singly or in groups of four in the center of membrane bound vacuoles which had a thin outer membrane followed by a thicker, electron-lucent layer with a fine lacy network. The organisms had an adherent thick trilaminar cell wall. Within the cell wall there was a 0.07 to 0.09 um thick electron dense layer with an irregular poorly delineated inner border subjacent to the trilaminar membrane. Electron dense areas were found in the center of the organisms and occasionally electron-lucent vacuoles connected by fibrillar strands were seen. Several organisms appeared to be degenerate and consisted of roughly concentric rings of electron dense membranes.

Few reports of RLOs in fishes can be found in the literature, and most of these reports are of infections in marine salmonids (Fryer, Lannan, Garces, Larenas and Smith, 1990; Garces, Larenas, Smith, Sandino, Lannan and Fryer, 1991; Branson and Nieto Diaz-Munoz, 1991; Rodger and Drinan, 1993; Speare and Armstrong, 1992). There is one report of RLO in dragonets, Callionymus 1yra L. (Davis, 1986) and another in Tetrodon fahaka (Mohamed, 1939). All of these reports indicate that significant pathological lesions are caused by the presence of the rickettsial organisms.

The histopathology seen in these fish differed somewhat from the histopathology reported with salmonid rickettsia. No vasculitis or thrombosis was seen in the blue-eyed plecostomus but these lesions have been reported in salmonid rickettsial disease (Turnbull, 1993; Speare and Armstrong, 1992; Branson and Nieto Diaz-Munoz, 1991).

Lesions similar to those seen with salmonid rickettsial disease included the granulomatous inflammation in the spleen, kidney and pericardium. Necrosis and effacement of the hematopoietic elements seen in these fish have also been reported in salmonid rickettsial septicemia. Staining characteristics of the organisms in the plecostomus were similar to those that have been reported for rickettsia in salmonids, that is, Gram stain, acid fast and Macchiavello's negative and staining blue with Giemsa and hematoxylin and eosin.

Conclusions

The morphological and staining characteristics of the organisms found in the mononuclear phagocytic cells of these fish are consistent with descriptions of rickettsia identified in other fishes. It is possible that infection with this rickettsia-like organism is responsible for the morbidity seen in these fish. Further research will include attempts to isolate the organism and determine its clinical significance.

Acknowledgements

The authors would like to express their thanks to Dr. John Fryer, Dr. Michael Levy, and Dr. Ed Noga for their advice and assistance.

References

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