Journal of Fish Diseases 1990, 13, 345-353

Progressive panophthalmitis in chinook salmon,Oncorhynchus tshawytscha (Walbaum): a case report

S. BACKMAN,^ H. W. FERGUSON,' J. F. PRESCOTT^ & B. P. WILCOCK'Departments of'Pathology, and 'Veterinary Microbiology and Immunology, Ontario Veterinary College,

University of Guelph, Ontario, Canada

Abstract. This report describes the gross and histopathological appearance as well as thebacteriological findings in an outbreak of panophthalmitis in farmed chinook salmon. In allcases, the bacteria were either Myeobacterium neoaurum or an unidentified Rhodococcusspp.; in some fish, both types were present in roughly equal numbers, while in others, onetype dominated. Despite occasionally recovering the bacteria from kidney or spleen, nolesions were found in these or in any other organ, suggesting that the localization to the eyewas not incidental, but a specific tropism. Nevertheless, the possibility of predisposingcausc(s) must be considered and these arc discussed.

Introduction

Ocular diseases of fish are common and represent a significant problem within the aquacultureindustry. The decreased visual acuity associated with ophthalmic diseases in species such assalmonids, some of which rely heavily on sight for feeding, may produce poor growth rates andfeed wastage (Hughes 1985). In addition, the gross lesions are readily apparent, detractingfrom the aesthetic appearance of the fish and thereby lowering their value. Various aetiologiesare described, including nutritional imbalance (Richardson, Higgs & Beames 1986; Hughes,Riis, Nickum & Rumsey 1981; Blazer & Wolke 1983; Ketola 1979; Poston, Riis, Rumsey &Ketola 1977), parasitic infestation (Shariff 1981; Shariff, Richards & Sommerville 1980; Riis,Georgi, Leibovitz & Smith 1981), bacterial infection (Bruno 1986; Llewellyn 1980; Shah &Tyagi 1986; Lee, Roberts & Shepherd 1976), viral infection (Dukes & Lawler 1975), gas super-saturation (Langdon 1988; Dehadrai 1966), traumatic injury (Ubels & Edelhauser 1987),excessive exposure to ultra violet light (Steucke, Allison, Piper & Robertson 1968) andhormonal aberrations (Matty, Menzel & Bardach 1958).

This paper describes, in intensively reared chinook salmon, an outbreak of ocular diseaseassociated with Gram-positive bacteria distinct from Renibacterium salmoninarum^ which inCanada would represent the major differential diagnosis for such organisms.

Materials and methods

History

Six chinook salmon, Oncorhynchus tshawytscha (Walbaum), were submitted for examinationto the Fish Pathology Laboratory, Ontario Veterinary College, Canada. Five fish exhibitedunilateral exophthalmia and one was bilaterally affected. Smears taken from within the eyesrevealed the presence of large numbers of Gram-positive and of weakly acid-fast bacteria. A

Correspondence: Dr H. W. Ferguson, Department of Pathology, Ontario Veterinary College, Utiiversity ofGuelph, Ontario, Canada NIG 2W1.

346 S. Backman ct al.

subsequent visit to the farm showed that approximately 2000 chinook salmon from the affectedgroup remained. These fish were kept in vinyl-lined raceways supplied with a mixture of wellwater and surface water. The raceways were located inside a metal-sheathed barn, withtranslucent panels and attenuated incandescent lights as the only light sources. Water tempera-Uirc at the tail of the raceway was 7-3'̂ C; dissolved oxygen was 6-0ppm (measured with theHI8543 D.O. meter*); ammonia was 0-15ppm (Nessler's method^) and the pH was 8-1(measured with the HI 8414 stick/ATC pH meter 1). The present owner had just takenpossession of the farm and could supply little information about previous Josses, or aboutmanagement practices. The observed morbidity was approximately 20% and the mortalityreported to be 15-20 fish per day.

Investigation

Another 14 fish exhibiting various clinical stages of disease were collected for study andtransported to the laboratory in plastic bags. On arrival, they were killed using tricainemethane sulphonate (MS-222, Crescent Research Chemicals Inc.) and the lesions were photo-graphed. The eyes and adjacent tissues were excised; some were sampled for bacteriology andthe rest were prepared for histopathological examination by placing them in Bouin's fixativefor 12 h. Kidney and spleen were also taken for bacteriology. Sections of skin, skeletal muscle,liver, kidney, spleen, brain, intestine, pyloric caeca, pancreas and heart were removed andplaced in Bouin's fixative. Following fixation, the eyes were bisected in the vertical plane andembedded in paraffin wax for sectioning. Five micron sections were cut and stained withhaematoxylin and eosin (H&E), Zichl-Nielsen, Nocardia oil decolourized acid fast, periodicacid-Schiff, and Brown and Brenn Gram stains. The remaining tissues were embedded inparaffin wax and standard histologica! sections prepared using H&E stain.

Bacteriology

All samples for bacterial isolation were streaked on blood and MacConkey's agar; plates wereincubated in air at room temperature for 2 weeks and checked daily. Blood agar plates werealso incubated under 10% CO2 in air and under anaerobic conditions.

Cell wall chemotaxonomic analysis of bacterial isolates was carried out using standardmethods by Dr T. Kudo, Japan Collection of Microorganisms, RIKEN, Wako-shi, Saitama,Japan (Kudo, Hatai & Seino 1988). Aryl sulphatase production by isolate 2 was carried outusing standard methods by the Tuberculosis Section, Ontario Ministry of Health, Toronto,Canada. Biochemical tests on isolated bacteria were done using standard methods (MacFaddin1980; Carter 1984). Isolate 2, a Mycobacterium was identified to species by Dr M. Tsukumura,The National Chubu Hospital, Japan, using methods described (Tsukumura, Mizrino &Tsukumura 1981; Wayne & Kubica 1986).

* Hanna Instruments, Laval Lab Inc., Luva), Qc. Canada.

^ Hach Chemical Co., Ames, Iowa, USA.

Panophthalmitis in chinook salmon 347

Results

Clinical examination

Affected fish were dark and in poor body condition. Although occasionally bilateral, the ocularlesions were most often unilateral. Marked chemosis, proptosis and peripheral corneal oedemaappeared as the earliest signs (stage 1). The condition progressed with the development ofcataraet, hyphema and hypopyon (stage 2). Corneal rupture, evacuation of ocular contents andextensive scarring of the remnants (phthisis) constituted the final stage (stage 3).

Gross pathology

Stage 1. On external examination, gross lesions were restricted to the eye(s). The most strikinglesions were those of the marked exophthalmos and swelling of periocular soft tissues. Oncloser examination, slight corneal opacity was seen around the periphery and haemorrhage wasevident at the corneal-scleral junction. No additional gross lesions were apparent when theeyes were seetioned. Examination of heart, liver, spleen, kidney, brain, epaxial muscle, skin orgills did not reveal any abnormalities. The absence of ingesta within the lumen of the stomaeh.

Figure 1. Normal eye fTom hea\thy chinook saimon.

Figure 2. The anterior chamber contains relatively mild cloudy white exudatc. Iris is still visible through atransparent peripheral cornea.

Figure 3. Abundant exudatc mixed with blood fills the anterior chamber in this chinook salmon with stage 2disease.

Figure 4. Extensive opaque white exudatc with some haemorrhage fills the anterior chamber (stage 2).

348 \ . liackn]an ct M,

intestine and pyloric caccii was Ihc only abnormality detected.

Stage 2. External lesions were confined to the eye. The exophthalmos and swelling remainedsevere, and the haemorrhage around the corneal-seleral junction became more pronounced.Within the anterior chamber, there was blood and inflammatory exudate (Figs 1-4). Therewas an apparent increase in iris vasculature (rubeosis iridis). On section, the lens was opaqueand in some instances it appeared as an irregular yellow caseous mass. The vitreal chambercontained large amounts of inflammatory exudate and the retina was detached. On internalexamination, the spleen was large and dark red. The heart, kidney, brain, liver, gill andskeletal muscle appeared normal. No abnormalities were detected in the digestive tract otherthan the absence of ingesta.

Stage 3. Gross lesions were confined to the eyes which were collapsed, with an opaque andwrinkled cornea. On cut section, the sclera was thickened and mottled grey/black. The vitrealcavity was reduced and no lens was found (aphakic). No gross lesions were apparent oninternal examination.

Histopathology

Stage I. There was generalized periocular oedema. The ocular musculature exhibited multifocalmyofibrillar degeneration with loss of striations, migration of leucocytes within the perimysiumand satellite cell proliferation. Within the corneal epithelium, there was mild hydropic de-generation of the polyhedral and basal cells. Epithelial sloughing, stromal oedema andseparation of Descemet's membrane from the stromal lamellae were observed. Neutrophilswere occasionally apparent within the stroma near the corneoscleral junction, limbus and inthe ciliary body. In five out of seven eyes examined, there was histological evidence of cataractformation marked by posterior migration of lenticular epithelium. No bacterial, ftmgal,protozoan or metazoan organisms were seen. No lesions were apparent in any other organ.

Stage 2. This stage was characterized by extensive intra- and periocular inflammation withhaemorrhage and the presence of bacteria. There was severe keratitis marked by ukeration,stromal oedema and vascularization. In some eyes, there was attempted corneal healingcharacterized by corneal epithelial hyperplasia with formation of rete ridges, thickening of theepithelial basement membrane and stromal scarring. Within the uvea there was markedchoroidai congestion and telangiectasis. Throughout the uveal tract, there was an accumulationof a pleomorphic population of leucocytes, including many neutrophils, proteinaceous exudateand haemorrhage (Fig. 5). Exudation into the adjacent tissues produced hypopyon, hyphema,irido-corneal adhesions (anterior synechia) and retinal detachment (Fig. 6). The detachedretina was extensively folded, and had marked gliosis within the inner nuclear and plexiformlayers. The inflammatory response sometimes involved the optic nerve, producing an opticneuritis and papillitis. The epithelium of the lens was sometimes hyperplastic and attemptedlens repair resulted in posterior migration of the epithelium. In some fish, there was dis-integration of lenticular fibres and the formation of bladder cells, while in others there waslenticular rupture and collapse (Fig. 7). Two of the eyes were aphakic and several had corneaiperforation, iris prolapse and even vitreal prolapse through the corneal defect.

In some fish, large numbers of short Gram-positive bacteria and fewer beaded Gram-positive, PAS-positive, weakly acid-fast bacterial rods were present throughout the uvea and

Panophthalmitis in chinook salmon 349

Figure 5. Histology of stage 2 eye showing antenor uveitisnumerous leucocytes in an oedematous stroma (H&E, x 220).

characterized by dilated iris vessels sunounded by

urc 6 In this more severely affected stage 2 eye, the anterior chamber is obliterated by inflammatory' exudatewrch effectively adheres the iris (arrow) to the inner aspect of the cornea; this itself is markedly mflamed and .slargely denuded of epithelium; X - lens capsule (H&H, x 50).

350 S. liackman ct al.

Figure 7. Stage 2 eye with ruptured and collapsed lens leaving little more than the capsule {X); anterior chambercontains large quantities of inflammatory exudate. Numerous eolonies of baeteria are scattered throughout theexudate (arrows) (H&E, x 50).

within the ocular chambers. They were found within maerophages and free in colonies. Inother fish, the acid-fast bacteria predominated.

The livers of these fish exhibited a generalized pattern of macrovesicular lipid depositionand moderate hepatocellular dissociation. Anisocytosis was moderate and occasional apoptoticcells were observed. The kidneys were congested and there was a mild interstitial proliferation.The spleen was congested and there was hypertrophy of ellipsoidal reticular cells. No otherlesions were apparent other than decreased granulation of pancreatic zymogen cells andincreased numbers of apoptotic enterocytes within the intestinal mucosa.

Stage 3. End stage eyes were observed in two fish. Changes were characterized by extremedisorganization of ocular tissues, fibrosis and global shrinkage. There was thickening of thescleral cartilage with mineralization and ossification. The retina was detached and completelydisorganized, and the pigment epithelium was distributed randomly throughout the uveal andvitreal space. The cornea was oedematous and scarred. Within the liver there was mildgeneralized hepatocellular dissociation and atrophy. There was also multifocal nephrocalcinosisand frequent apoptotic cells within the intestinal mucosa. No other lesions were apparent.

Bacteriology

Two types of bacteria were recovered from the affected eyes, in some cases as mixtures inapproximately equal quantities, and in other cases with one type dominating. Both organismswere isolated from kidney and spleen, but only in low numbers and only from severely affected

fish.Isolate 1 was a Gram-positive, non-acid fast, slightly club-shaped rod (2-3 x 0-6/im),

I

Panophthalmitis in chinook salmon 35

which grew aerobically on blood agar at room temperature, but not at 37°C, in 3-4 days.Colonies at 8 days were 0-5mm, slightly domed, round, smooth, dry and deep creamy yellowin colour. It was identified as an unidentified Rhodococctis species, since cell wall chemotypewas IV A (containing w^.w-diaminopimelic acid, arabinose and galactose), cell wall muramicacid residues were N-glycolated, the predominant isoprenoid menaquinone was MK-8 (H2)and the cell wall contained mycolic acids (Kudo, Hatai & Seino 1988; Goodfellow 1986). Theorganism was facultatively anaerobic, catalase and cytochrome oxidase negative, and ureasepositive; it fermented xylose but not other commonly tested carbohydrate substrates includingdextrose, lactose, maltose and sucrose, and it produced acid from m^Ao-inositol but notmannitol, dulcitol or sorbitol. It grew on 5% salt agar and was sensitive to penicillin.

Isolate 2 was an acid-fast rod (with the acid-alcohol technique); it measured 3—4 x 0-6^^rn,and virtually failed to take up the Gram stain. The organism grew on blood agar at roomtemperature, but not at 37°C, in 5—7 days. Colonies at 8 days of incubation were variablysized, up to 0 3—0 4mm, smooth, round and clear, but were starting to develop a light yellowcolour. The organism was penicillin resistant and did not grow on 5% salt agar. It was arylsulphatase positive after 14 days of incubation. It was identified as a Mvcobacterium species, sincecell wall chemotype was IV A, cell wall muramic acid residues were glycolated, the predomi-nant menaquinone was MK-9 (H2) and mycolic acid was present and the organism was acidfast (Wayne & Kubica 1986). It was identified as M. neoaurum by Dr M. Tsukumura, usingover 100 properties as characteristics in numerical taxonomic analysis.

Discussion

These salmon had progressive eye disease characterized by a pyogranulomatous and necrotizingpanophthalmitis, associated in some cases with the presence of large numbers of Gram-positivebacteria and in other cases with acid-fast bacteria. The associated inflammatory reactionterminated in corneal rupture and the loss of most ocular contents. The observation andrecovery of large numbers of Gram-positive organisms from the eye (with very few organismselsewhere) make this an unusual case and an important differential for bacterial kidney disease(Bruno 1986) and other infections such as Streptococcus (Boomker, Imes, Cameron, Naude &Schoonbee 1979) which can also target the eye, although this latter disease has not beenreported from fish in Canada. Mycobacterium can also affect the eye, but it normally causesgranulomas in the choroid gland and is rarely a particularly destructive lesion: moreover,granulomas arc not normally restricted to the eye, with kidney, spleen, and heart beingfavoured sites. Myeobaeteriosis in farmed salmonids has, in any case, become much lesscommon since the practice of feeding trash fish has been largely superseded in favour ofcommercially compounded diets. Mycobacterium neoaurum has been isolated previously fromsoil (Wayne & Kubica 1986).

The tremendous inflammation and vascular damage observed in these eyes would easilyhave allowed entry of bacteria into the circulation causing bacteraemia, and indeed this wasconfirmed by positive cultures from kidney or spleen of a few severely affected fish. Despiterecovery of the bacteria, significant pathological lesions were not, however, seen in theseorgans. This suggests that the condition was primarily an eye disease rather than a generalizedsystemic infection with subsequent localization to the eye.

The pathological changes and progression of lesions in this outbreak resemble those of'Zomba Disease' described in rainbow trout, Oncorhynchus mykiss (Richardson), by Lee et al.(1976). They postulated that this condition was the result of a nutritional imbalance associated

352 S. Backman ct ill.

with the feeding ol animal renderings as the principle protein source. The ready availability ofcommercial fish feeds makes such haphazard diets less likely in Canada, but the presentauthors were nevertheless unable to ascertain the age, quality or amount of feed provided tothese fish prior to the new owner taking possession of the farm. It is probable, as with 'ZombaDisease', that the baeterial infection was largely responsible for the severe inflammatoryresponse which in turn caused destruction of the eye.

We have not yet determined the pathogenesis for this ocular disease, and the possibility ofpredisposing eauses such as malnutrition, toxicity or trauma must be considered. Steucke et al.(1 )̂68) reported lenticular damage resulting from exposure to excessive ultraviolet light. This isconsidered an unlikely aetiology in the present case, as the fish were maintained indoors undersubdued lighting conditions. Gas supersaturation is a common cause of exophthalmia inOntario where much of the water is pumped; such eyes frequently become traumatized andseeondarily infected. Although supersaturation was not detected in the present case at the timeof sampling, the marginal water quality and questionable nutritional status may neverthelesshave been suffieient to produce the initial exophthalmia. However, if this disease is purely theresult of opportunistic invasion through the breached defences of a damaged cornea (and thiswas not obvious histologically), the presence of a mixed population of organisms seems muchmore likely. The precise identity of the bacteria recovered, the reasons for their oculartropism, and their ability to experimentally reproduce eye disease, all remain to be determined.

Acknowledgments

We thank Dr T. Kudo, Japan Collection of Microorganisms, for cell wall analysis of theseisolates, and the Tuberculosis Section, Ontario Ministry of Health, for aryl sulphatase testingon isolate 2. We thank Dr M. Tsukumura for identifying the Mycobacterium species. The FishPathology Laboratory receives much of its funding from the Ontario Ministry of Agricultureand Food.

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