Histopathology of experimental columnaris disease in young salmon

J. COMP.

PATH.

1967.

VOL.

77.

419

HISTOPATHOLOGY

OF DISEASE

EXPERIMENTAL IN

YOUNG

COLUMNARIS SALMON

BY

Department

R. E. PACHA* and E. J. ORDAL of Microbiology, Univer& of Washington, School of Medicine,

Seatik,

U.S.A.

INTRODUCTION

Columnaris disease, due to the aquatic myxobacterium Chondrococcus columnaris, was first described among warm water fishes of the Mississippi River Valley by Davis in 1922. This disease was easily recognised by the appearance of greyish-white or yellow areas of erosion usually surrounded by a reddish hyperaemic wne on the body surfaces of the fish or in the gills. Epidemics of columnaris disease affecting a number of different species of fish, have been reported by a number of investigators (Nigrelli, 1943; Ordal and Rucker, 1944; Nigrelli and Hutner, 1945; Davis, 1949; Johnson and Brice, 1952; Borg, 1960) in many parts of the United States. Rucker, Earp and Ordal (1953) reported wide variations in virulence among cultures of C. columnaris isolated from fishes taken from waters of the Pacific Northwest. When tested at comparable water temperatures, strains of C. columnaris fell into two categories with respect to virulence. The strains of low virulence reduced slow progressive infections at water temperatures above 21OC. and no ImiJ y caused massive tissue damage before death occurred. Strains of high virulence caused fulminating infections and killed young salmon in 12 to 24 hours at 20°C. when introduced into the water in which the fish were held in the experimental hatchery. Ordinarily, young salmon infected with strains of C. columnaris of high virulence showed no gross tissue damage at the time of death. Apparently, death occurred before gross external manifestations of the disease appeared. The histopathology of young fish experimentally infected with strains of C. column&s of high virulence is described in this report. MATERIALS

AND

METHODS

Bacterial strains. The cultures of C. columnaris used in this study were obtained from fish taken at various locations in the Columbia River Basin. The cultures were isolated by streaking material from surface and gill lesions on Cytophaga agar, a medium composed of 0.05 per cent. tryptone, 0.05 per cent. yeast extract, 0.02 per cent. beef extract, 0.02 per cent. sodium acetate, and 1.1 per cent. agar (Difco), adjusted to pH 7.2 to 7.4. The plates were incubated for 2 to 5 days at room temperature and the colonies of C. columnaris were isolated and checked for purity and identity. Colonies of C. columnaris were ordinarily easy to recognise by their spreading, rhizoid, greenish-yellow growth. Isolates were preserved by lyophihsation. Analysis of virulence. The medium employed consisted of 0.4 per cent. tryptone and 3-O per cent. yeast infusion broth. Screw cap tubes containing 5 ml. of this medium were inoculated with a loopful of growth from a deep culture of the strain to be tested and incubated on a reciprocal shaker at room temperature for 24 hours. The 5 ml. culture was then used to inoculate 50 ml. of the broth in a 250 ml. * Present

address:

Department

of Microbiology,

Oregon

State University,

Corvallis,

Oregon,

U.S.A.

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DISEASE

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SALMON

Erlenmeyer flask. After standing at room temperature for 16 to 18 hours, the flask culture was placed on a reciprocal shaker for 1 to 2 hours before use to ensure that actively growing cells were present in the inoculum. Tests for virulence were carried out using sockeye salmon or chinook salmon, depending on which fish were available. These fish were 6 to 14 months of age and ranged from 6 to 9 cm. in length. The contact method’ of infection was used, in which the experimental fish were allowed to swim for 2 minutes in a small receptacle containing a 1 in 20 dilution of a broth culture having an optical density of 0.1 as determined with a Coleman Nepho-Colorimeter using a 525 rnp filter. Following exposure, the fish were poured into a container through which water at 19.5 to 20*5OC. flowed at the rate of 35 to 40 litres per hour. The fish were inspected at intervals following infection and those found dead or near death were removed and the number recorded. Preparation of tissuefor histological study. Normal fish and moribund fish infected by the contact method with strains of C. columnar& of high virulence were examined ,histoIogically. The fish were fixed in Bouin’s solution for 24 hours and then stored in 70 per cent. alcohol. Before fixation, the abdomen of the fish was carefully opened to allow the fixative to reach the internal organs immediately. Paraffin sections were prepared from the organs and stained with haematoxylin and eosin or Giemsa stains. RESULTS

Analysis of Virulence Three strains of C. columnaris of high virulence were used. The results of analyses of virulence on these organisms using the contact method of infection are shown in Fig. 1. For comparative purpases the results of previous virulence analyses on strains of C. cuEumnmis of lower virulence are also included. Strains 2-057-4fk, 2-M57-23(M) and 2-B58-5b killed 100 per cent. of the experimental Fig.

‘2-B

1.

5a-5b

5 B 3 200

3-o 57-37 I 1 I I I1 90 120 150 30 60 Hours after initial exposure

Cumulative mortalities of fish infected varying degrees of virulence.

by the contact

method

with

strains

of C. columnaris

of

R.

E.

PACHA

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ORDAL

421

fish in less than 24 hours, thus satisfying the definition of high virulence put forward by Rucker et al. (1953). Strains 4-M57-4, 3-057-37 and 6-B58-17 were of lower virulence (Fig. 1) and failed to kill 100 per cent. of the experimental fish within 120 hours. Gross Pathology The gross pathology observed in the fish experimentally infected with strains of C. columnaris of high virulence was usually very limited. Ordinarily, the fish showed no evidence of gross tissue damage at the time of death. However, some of the last f%h to die showed areas of grey discoloration at some site on the body surface. These lesions occurred most frequently around the dorsal fin in the form of an area much like a “saddle”. Other sites at which areas of grey discoloration were observed included the skin around the adipose fin or on the head. Grmly the internal organs of these fish showed no evidence of damage or destruction. Microscopic Pathology The tissues examined included the gill, muscle underlying an area of grey discoloration and the internal organs. The most extensive damage was noted in the gill. The blood vessels supplying the gill were congested, the surface epithelium of the lamellae was dissociated from the capillary bed probably due to the accumulation of oedematous fluid (Fig. 2), and scattered areas of haemorrhage were noted (Fig. 3). Occasionally globose masses of blood cells were observed in the gill lamellae (Fig. 4) but these lesions did not seem to be true haemorrhages since the blood appeared to remain in circulation. Undoubtedly, this is one of the alterations occurring just prior to true haemorrhage. The infected gill tissues shown in the above figures differ from the section of normal gill tissue (Fig. 5) in which the epithelium of the lamellae is closely adherent to the capillary bed. Sections of gill tissue from infected f%h were stained with Giemsa and examined for bacteria. In many instances bacterial cells resembling C. columnaris were found in scattered masses throughout the tissue. However, these were embedded not in the tissue itself, but in a structureless, lightly staining material located between the individual gill filaments. Skin and muscle tissue taken from an area of grey discoloration was also examined histologically. Large numbers of bacteria were observed under the scales; in the more severe lesions the muscle fibres underlying the abnormal skin were observed to be necrotic. Giemsa stained sections containing necrotic muscle fibres showed bacteria in the muscle tissue, but in very small numbers. Degeneration of muscle fibres, similar to that observed in muscle tissue underIying areas of grey discoloration, also occurred following the intramuscular injection of C. columnaris, but the extent of muscle necrosis and invasion into the tissue was far greater than that following contact infection. Lesions in the kidney were confined to the glomerulus. As shown in Fig. 6, they consisted of an enlargement of Bowman’s capsule and eosinophilic material surrounding the glomerular capillaries. In the normal kidney the space between Bowman’s capsule and the glomerular capillaries was small and free of eosinophilic material (Fig. 7).

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DISEASE

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SALMON

Giemsa stained sections of kidney prepared from infected fish failed to reveal bacteria. However, on culture, C. columnuris could be recovered from the kidney in small numbers. The other internal organs showed no apparent pathological alterations and Giemsa stained sections failed to reveal bacteria. DISCUSSION

Major pathological alterations in fish experimentally infected with strains of C. column&s of high virulence were noted on the external surfaces. Extensive tissue damage was apparent in the gills and undoubtedly contributed significantly to death of the fish. It was noted that prior to death the respiratory rate of the infected fish was much greater than that of the control fish. This observation, together with the histopathological findings, indicates that the fish were in respiratory distress. It was noted by Pacha and Ordal (1962) that the route of infection is an important factor in determining the disease-producing capacity of strains of C. column& of high virulence. Strains of high virulence were found to be more lethal by the contact method of infection than by intramuscular or intraperitoneal injection. Since the gill is extensively damaged by strains of C. cdumnaris of high virulence, it is not surprising that routes of infection which afford easy access to this organ are highly effective. Wood and Yasutake (1956), showed that the myxobacterium responsible for “cold-water” disease of fish had a marked affinity for the renal glomeruli and frequently solid masses of bacteria were found at this site. Although C. columnar6 could be cultured from the kidneys of our experimental fish following infection with a highly virulent strain, Giemsa stained sections of kidney failed to demonstrate large numbers of bacteria. The necrosis noted in the muscle tissue underlying the grey skin lesions suggests that a diffusible toxin or proteolytic enzyme may be involved in the production of these lesions. Extensive microbial gmwtb had occurred in the surface tissues since large numbers of bacterial cells were noted on the skin overlying the necrotic muscle fibres. However, the small number of microbial cells in the underlying muscle tissue itself did not appear sufficient to account for the ohserved necrosis. This suggests that a diffusable, necrotising substance was produced during growth of the bacteria on the skin. Further studies are now needed to corroborate these observations. SUMMARY

The histopathology of columnaris disease in young salmon is described. The gill was the major site of damage in fish experimentally infected with strains of C. columrwris of high virulence. Extensive alterations were noted in this organ and just prior to death the fish appeared to be suffering from acute respiratory distress. Lesions observed in the musculature and internal organs of the infected fish are also described. ACKNOWLEDGMENT

This investigation Energy Commission.

was supported

by contract

AT(45-l)-1727

from

the Atomic

R.

Fig.

2.

Fig.

3.

E.

Gill filament from an infected bed(c). H.E. x 160. Gill section showing an area filaments. H.E. x 184.

PACHA

fish.

The

AND

surface

of haemorrhage.

E.

J.

ORDAL

epithelium Blood

cells

(e) is separated (b)

are

located

from

the czapillary

between

the

gill

To face page 422

EXPERIMENTAL

Fig. 4.

Gill

filament

Fig. 5.

Normal

from

gill filament.

COLUMNARIS

an infected The

fish.

surface

DISEASE

A mass of blood epithelium

IN

SALMON

(b) is located

(e) is closely

adherent

within

a lamella. H.E. x 360. to the cap&ryxb;zdc). . .

R.

Fig.

6.

Fig.

7.

Kidney material Normal material.

E.

PACHA

AND

E.

J.

ORDAL

from an infected fish. Bowman’s capsule (c) is enlarged and an eosinophilic (e) is present in the renal sinus. H.E. x 272. kidney. Note the normal size of Bowman’s capsule and the lack of eosinophilic H.E. x 320.

staining staining

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ORDAL

REFERENCES

Borg, A. (1960). Wildlife Diease, 8, 1. Davis, H. S. (1922). U.S. Bur. Fisheries Bull., 38, 261; (1949). Trans. Amer. Fisheries sm., 77, 102. Johnson, H. E., and Brice, R. F. (1952). Progressive Fish-Culturist, 14, 104. Nigrellj, R. F. (1943). Zoologica, 28, 203. Nigrelh, R. F., and Hutner, S. H. (1945). Ibid., 30, lQ1. Ordal, E. J. and Rucker, R. R. (1944). Proc. Sot. exp. Biol. Med., 56, 15. Pacha, R. E., and Ordal, E. J. (1962). Bacterial. Proc., 62, 20. Rucker, R. R., Earp, B. J., and Ordal, E. J. (1953). Trans. Amer. Fisheries Sot.,

83, 297.

Wood,

E. M., and Yasutake, [Received

W. T. (1956). for

publication,

Progressive january

Fish-C&u&t, 23rd,

19671

18, 58.