Amoebic gill infections of rainbow trout in freshwater ponds

Gill disease of fry and fingerlings associated with extensive mortality in rainbow trout freshwater farms in Denmark have been observed on an increasing number of occasions in the latest years. Clinical symptoms of hyperventilation indicate respiratory distress. Gross-pathological observations of gills show pale nodules and patches on gill filaments. Histological and scanning electron microscopic investigations were conducted on gills from trout with these symptoms. It was found that extensive hyperplasia of gill filaments, clubbing and fusion of filaments and lamellae occurred at high frequency. Heavy colonization of affected gills with amoeba-like protozoans was observed. The protozoans were approximately 10-20 micrometers in diameter and the density on gill tissue varied but could reach 50 cells per mm surface section. The gill infection resembled nodular gill disease previously described from Canada, a disease connected to concomitant infections with bacteria and amoebae. However, in the present study no colonization with filamentous bacteria could be detected. The amoebae could not be ascribed to the known freshwater amoeba genera recorded in rainbow trout. The amoeboid organisms co-occurred with a few specimens of Trichodina. Formaldehyde treatments of the affected tank were shown to alleviate the acute situation. Introduction Parasite infections in freshwater trout farms in Denmark have been relatively well studied (Buchmann & Bresciani 1997). Both metazoan and protozoan organisms are known to occur regularly. Thus, various flagellates and ciliates are common findings on rainbow trout gills during fish examinations. However, in the latest years gill disease problems associated with mortalities have been registered. All cases have shown hyperplasia of gill filaments, clubbing and lamellar fusion. In order to elucidate the disease problem the present investigation was undertaken on affected gills from two trout farms in Denmark where outbreaks occurred in May 2003. Materials and methods Rainbow trout fingerlings (body length 10-15 cm) from two freshwater trout farms in Jutland (western part of Denmark) were sampled in May 2003. Fish were clinically affected and showed respiratory distress associated with high mortality. Gills were dissected and fixed in neutral formaldehyde and brought to the laboratory for further investigation. Whole mount preparations Formalin fixed gill filaments were rinsed in distilled water and stained with haematoxylin for 5 min and subsequently embedded in glycerine-gelatine. Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 88 Scanning electron microscopy Formalin fixed gills were post-fixed in 2.5 % neutral glutaraldehyde (24 h), washed in distilled water, immersed in 2 % tannic acid, dehydrated, critically point-dried, sputtered with gold and studied in a JEOL JSM 840 scanning electron microscope. Histology Formalin fixed gill filaments were dehydrated, embedded in paraffin wax , sectioned (5 micrometer sections), stained with Giemsa, haematoxylin/eosin or haematoxylin/alcian blue and subsequently mounted on slides with Depex as embedding medium. Results Farm observations Fish in the two freshwater farms were showing signs of respiratory distress, lethargy and surface swimming associated with mortality. Gross pathology The gill tissue appeared swollen and irregularly structured with whitish or pale patches spread on the gill filaments. Whole mounts When studied at magnification 100 x , 200 x and 400 x the extensive hyperplasia of gill filaments was evident. The surface of the gills was in certain areas covered with uni-cellular nucleated amoeboid organisms. A few specimens of Trichodina sp. were detected at low densities (approximately one parasite per gill filament). The size of the amoeba-like organisms varied from 10-20 micrometer. Bacterial aggregations were not detected. Scanning electron microscopy The extensive hyperplasia of gill filaments caused clubbing and fusion of gill lamellae (Fig. 1). Amoeba-like organisms were firmly attached to the gill surface. In certain areas these protozoans produced a carpet-like covering of the epithelial substrate (Fig. 2). The shape of individual cells was roundish to ovoid (10-15 micrometers) with the ectoplasma forming blebs (Fig. 3), folds or ridges (Fig. 4). A few Trichodina parasites were also found on the gills (Fig. 4). No extensive aggregations of bacteria were detected on the surface of affected areas. Histology Extensive hyperplasia of gill epithelia and mucous cells was found. The gill structure was highly disorganized. The epithelial surface was colonized by a carpet-like covering of densely packed amoeba-like organisms of sizes between 10 and 24 micrometers (length) (Figs. 5 and 6). In certain areas at least two layers of these parasites were found over gill cells. Mucus associated with aggregations of amoebae was observed between gill filaments. The colonizing cells contained a nucleus and showed highly varying shapes in cross sections. Discussion The present investigation was based on formalin-fixed material and it should be stressed that further studies on fresh preparations and cultures should be conducted. Likewise, specific diagnosis needs additional transmission electron microscopy studies. However, the work suggested strongly that the causative agent of the gill disease in the Danish trout farms is an amoeba-like parasite. A disease Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 89 Fig. 1. Scanning electron microscopy of rainbow trout gills showing extensive hyperplasia and clubbing of gill filaments and fusion of gill filaments (arrow) associated with colonization of numerous amoeba-like protozoans. Scale bar 1mm. Fig. 2. Scanning electron microscopy of rainbow trout gill filament heavily colonized with amoebae forming a carpet-like covering (arrow). Scale bar 100 micrometers. Fig. 3. Scanning electron microscopy of amoebae on gill epithelium showing pseudopodia and membrane blebs. Scale bar 10 micrometers. (Arrows?) Fig. 4. Scanning electron microscopy of amoebae associated with Trichodina sp. on the gills of rainbow trout. Scale bar 10 micrometers. Fig. 5. Histological section of amoeba-infected gill filament. Giemsa stain. Scale bar 100 micrometers. Fig. 6. Histological section of amoeba-infected tips of gill filament. Haematoxylin stain. Scale bar 100 micrometers. Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 90 with similar clinical findings was previouslydetected in rainbow trout and described asnodular gill disease (NGD) by Daoust &Ferguson (1985). NGD has been recorded in anumber of other hosts including charrSalvelinus alpinus (Speare 1999). However,NGD is normally caused by amoebae in asso-ciation with heavy colonization byfilamentous bacteria. Such bacteria were notfound on the gills of Danish trout in thesecases, although previously, bacterial coloni-zation in connection with these particular dis-ease symptoms has been recorded in Danishtrout farms (own observations). It should benoted that the fixation method used in thestudy reported herein, could have obscuredthe presence of bacteria. The presence of palenodules in the gill material due to extensivehyperplasia and the presence of clubbed gillfilaments and fused gill lamellae presents apicture much like NGD. Certain areas of in-fected gills were colonized by dense layers ofamoebae. Such observations have been donein other infected fish. As early as 1909 and1910 Chatton described colonization of labridfish gills (Symphodus tinca) with amoebae.Likewise, Sawyer et al. (1974) found thatThecamoeba covered the gill surface of rainbowtrout. Later studies have shown that infectionsby amoebae occur even in recirculated fishproduction systems (Bullock et al. 1994, No-ble et al. 1997). The amoeba infecting gills ofsalmonid fish have been assigned to the gen-era Thecamoeba (Sawyer et al. 1974) in fresh-water, and Neoparamoeba (formerly Paramoeba)in saltwater (Kent et al. 1988, Roubal et al.1989, Clark & Nowak 1999). Other marinefishes such as turbot and sea-bass also sufferfrom gill diseases associated with coloniza-tion by Paramoeba and Neoparamoeba (Dykovaet al. 1998, 2000). Gross pathology changes re-ported for NGD have many similarities to thefindings in the present investigation. It is notclear to what extent other associated parasitesplay a role in the amoebic proliferative gilldisease. Trichodinids in association withamoebae as found in the present study werealso mentioned by Chatton (1909, 1910) andSawyer et al. (1974) but the importance, if any,needs further investigation. At least the den-sity of trichodinids on the gill tissue was verylow compared to the density of amoebae. Al-though the clinical and histological workpresents similarities to NGD and Thecamoeba,specific diagnosis of the organism found inthe Danish rainbow trout farms await furthercharacterization by transmission electronmicroscopy and in vitro cultivation studiesfollowed by molecular diagnostic work. ReferencesBuchmann, K., Bresciani, J. (1997). Parasiticinfections in pond reared rainbow troutOncorhynchus mykiss in Denmark. Dis. Aquat.Org. 28, 125-138. Bullock, G., Herman, R., Heinen, J., Noble, A.,Weber, A., Hankins, J. (1994). 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