Bull Vet Inst Pulawy 51, 547-551, 2007 STUDY ON RAINBOW TROUT NODULAR GILL DISEASE DETECTED IN POLAND JERZY ANTYCHOWICZ Department of Fish Diseases, National Veterinary Research Institute, 24-100 Pulawy, Poland antych@piwet.pulawy.pl Received for publication September 10, 2007 Abstract In recent years in Poland a very distinctive form of proliferative gill disease of rainbow trout, accompanied by the presence of amoeba-like cells, was observed. The disease appeared to be the nodular gill disease for the first time diagnosed in Poland. Amoeba-like cells seemed to be related to pathological gill lesions, whereas other potential agents such as bacteria and parasites were excluded. It has been suggested that those cells are inland free living amoebae. Key words: rainbow trout, nodular gill disease, pathology, Poland. In recent years in Poland a very distinctive form of proliferative gill disease of rainbow trout was observed. The cases of this disease were accompanied with a high mortality rate. The disease was characterised by nodular hyperplasia of branchial epithelium associated with the presence of amoeba-like cells of an unknown origin. A similar disease was described in inland rainbow trout fingerlings by Daoust and Ferguson (1, 2) and by many other authors in marine cultured salmonids infected with Paramoeba pemaquidensis (6). In the gills of freshwater fish as well as in sea fish the lesions were multifocal and uniquely severe in distal regions of affected filaments. Characteristic proliferative cell nodules contrasted with other more common forms of proliferative gill disease found in trout. The aetiology of inland rainbow trout nodular disease was not clarified. Up to now, nodular gill disease was not reported in Poland. The purpose of this work is to compare the detected changes in the gills of the rainbow trout in Poland with those described in others countries and diagnosed as nodular gill disease. The character of amoeba-like cells which usually accompany this kind of proliferative changes in the gills will be discussed. Material and Methods Three groups of rainbow trout fingerlings originating from three salmonid farms were delivered to our laboratory for the purpose of diagnosing the cause of the severe hypoxia symptoms, which have appeared in the fish. The clinical symptoms and gross pathological lesions were described and discussed. Five fish from each farm with distinctive gill lesions were collected for microscopic examination. The fish were killed with anaesthetic overdose and within 12-15 s, the samples of the gills were collected and fixed in buffered formalin solution for histological examination. The fresh squash preparations of the gills were also made for microscopic examinations. Preparations made from the gills of clinically healthy rainbow trout fingerlings originating from disease free farm were used as controls. The routine histological methods were applied. The preparations were stained with haematoxylin and eosin. Results The majority of fish delivered from the farms showed the symptoms of severe hypoxia. The gills of these fish were swollen, non-uniformly coloured, and their distal regions were protruding from under the gill covers. Distal regions of the primary gill filaments showed clubbing. Diffuse whitish discolouration of the gills was suggesting the presence of diffuse epithelial hyperplasia, besides the excess of mucous production was the symptom of gill irritation and hypoxia. Microscopic examination revealed that focal massive branchial epithelium hyperplasia had resulted in fusion of two or more secondary lamellae most often in apical region of primary filaments, which caused their clubbing (Figs 1 and 2). The interlamellar spaces were filled with proliferating cells, and the structure of the gills was pathologically changed.
548 Fig. 1. Clubbing of the distal ends of primary gill filaments of the rainbow trout fingerling, fresh preparation. 100x. Fig. 2. Normal primary gill filaments of the rainbow trout fingerling, fresh preparation. 100x. Fig. 3. Amoeba-like cells (in the centre) between two proliferative masses of degenerated gill cells (upper right and lower right). 100x, HE.
549 Fig. 4. Amoeba-like cells in the gills of diseased rainbow trout, fresh preparation. 500x. Fig. 5. High magnification of isolated amoeba-like cells originating from the case of nodular gill disease, fresh preparation. 1000x. Fig. 6. Pathological changes in distal ends of primary gill filaments with numerous amoeba-like cells, fresh preparation. 100x.
550 The proliferation of the gill cells and focal fusions of secondary lamellae formed nodules. The cells in the nodules showed anisokaryosis and variable amount of pale cytoplasm (Fig. 3). Among the cells, small dark nuclei were also present. In fresh squash preparations as well as in histological preparations unidentified amoeba-like cells were seen (Figs 3, 4, and 5). They appeared as hyperplastic masses focally attached to the surface of the gill in single, double or triple layers (Fig. 6). In fresh preparations, the amoeba-like cells were round with one or two nuclei-like bodies. Their vacuolated plasma contained numerous small refractive granules. In histological preparations these cells were angular and distinctly darker than the proliferative gill cells (Fig. 3). The size of these cells was between 15µm and 25µm. The amoeba-like cells had slightly basophilic cytoplasm, whereas the gill cells were eosinophilic. In some places, the congregation of the amoeba-like cells detached from the gill tissue, probably due to the process of tissue conservation (Fig. 3). Discussion The three cases of proliferative gill disease presented in rainbow trout fingerlings in Poland are identical to the cases nodular gill diseases described by other authors. The lesions were considered severe enough to have contributed to clinical symptoms, which were observed in fish and could cause mortality. The proliferating process destroyed gill structure, obliterated spaces between the secondary filaments, and impaired gas and ion exchange between environment and blood. The gill lesions presented in this paper in some aspect differ from the epithelial hypertrophy and hyperplasia, which usually appear in fish in acute or chronic intoxication by pollutants or are associated with various microorganisms. According to Daoust and Ferguson (1, 2), in all those cases lesions appeared to be diffused along primary filaments, whereas in the cases of nodular gill disease they appear only in some regions of the gill being especially severe in distal parts of the affected primary filaments. Toxic lesions and the pathological changes caused by hypoxia or stress are characterised by the oedema of individual secondary gill filaments as well as detachment of epidermal layer and, in some cases, also by haemorrhages. In ammonia intoxication, the proliferation of eosinophilic granular cells is the usual symptom. In the studied case of the gill disease, none of the above mentioned characteristics was detected and neither filamentous bacteria nor parasites were present. The identity of the so-called amoeba-like cells was not yet determined and is still a matter of speculation. It has been suggested that these cells are inland free living amoebae, similar to saltwater amoebae causing the condition known as amoebic gill disease (AGD) in marine cultured salmonids in Tasmania, and increasingly in other fish species of different geographic areas (3, 7, 8). Karlsson Norrgren (4, 5) suggested they could be proliferating chloride cells, which migrate and accumulate on the surface of other proliferating gill cells. The migration of chloride cells could be induced by aluminium poisoning. According to Daoust and Ferguson (1, 2), a marked epithelial hyperplasia accompanied usually by the fusion of adjacent lamellae and obliteration of the interlamellar space is connected with free living amoeba infection. This hypothesis is not confirmed and the significance of the large cells detected in nodular gill disease (called also A cells) is not clear. The varied size and the lack of degenerative changes in these cells as well as the absence of bacterial decomposition could suggest that they are live cells and the possibility that they are amoebae cannot be discounted. These cells are distributed focally on the mass of proliferating gill cells but the congregations of these amoeba-like cells are distinctly marked. Sometimes, in histological preparations, they are seen detached from proliferating gill cells. These phenomena suggest that they are possibly not transformed gill cells (Fig. 3). Moreover, inland fish chloride cells are only occasionally seen among lamellar epithelial cells, so they probably could not achieve such a great number even due to the proliferation. The cytoplasm of amoeba-like cells appeared to be slightly basophilic, whereas the cytoplasm of chloride cells is acidophilic. According to Taylor (9), free living amoebae are capable of parasitizing freshwater fish. Some genera appear to be casual facultative dwellers that become entrapped in mucus of the gills where they feed on bacteria. Under some conditions, fish can become heavily infected by amoeba and then it results in fish mortality. In the cases detected in Poland, the amoeba-like cells seemed to be related to pathological lesions in the gill, whereas other potential agents such as bacteria or parasites were excluded. The investigations of the aetiology of nodular gill disease are important and should be continued with special emphasis on the study of eventual infectivity and pathogenicity of the amoebalike cells. Account should be taken of the fact that nodular gill disease could be dangerous for trout and is a new emerging disease for the first time reported in Poland. References 1. Daoust P.Y., Ferguson H. W.: Gill diseases of cultured salmonids in Ontario. Can J comp Med 1983, 47, 358-362. 2. Daoust P.Y., Ferguson H. W.: Nodular gill disease: a unique form of proliferative gill disease in rainbow trout, Salmo gairdneri Richardson. J Fish Dis 1985, 8, 511-522. 3. Dykova I., Figueras A., Novoa B.: Epizootic amoebae from the gills of turbot Scopthhalmus maximus. Dis Aquat Org 1999, 38, 33-38. 4. Karlsson Norrgren L., Dickson W., Ljungberg O., Runn P.: Acid water and aluminium exposure: gill lesions and
551 aluminium accumulation in farmed brown trout Salmo trutta L. J Fish Dis 1986, 9, 1-10. 5. Karlsson Norrgren L., Bjőrklund I., Ljungberg O., Runn P.: Acid water and aluminium exposure: experimentally induced gill lesions in brown trout, Salmo trutta L. J Fish Dis 1986, 9, 11-26. 6. Kent M. L.: Diseases of seawater netpen-reared salmonid fishes in the Pacific Northwest, Can Spec Publ Aquatic Sci 1992, 116, 17-19. 7. Munday B. L., Foster C. K., Roubal F. R., Lester R. G. J.: Paramoebic gill infection and associated pathology of Atlantic salmon, Salmo salar and rainbow trout Salmo gairdnari in Tasmania. Pathology in Marine Science. F. O. Perkins, T. C. Cheng Academic Press, San Diego, 1990. 8. Rodger H.D., Mc Ardle J. F.: An outbreak of amoebic gill disease in Ireland. Vet Rec 1996, 139, 348-349. 9. Taylor P. W.: Izolation and experimental infection of free-living amebae in freshwater fishes. J Parasit 1977, 2, 232-237.