Rapp. P.-v. Réun. Cons. int. Explor. Mer, 182: 106—110. 1983.

Spring ulcer disease in eel (Anguilla anguilla)

N. Juul Jensen1, J. L. Larsen, and N. O. Christensen1 Laboratory of Aquatic Pathobiology Ambulatory Clinic and Clinical Central Laboratory Royal Veterinary and Agricultural University Biilowsvej 13, DK-1870 Copenhagen V, Denmark

In eels two types of diseases characterized by lesions of the skin have been described. Previously “red disease” proper is one type. This disease occurs in marine environ­ ments during the summer and is caused by Vibrio anguillarum. The other type is characterized by the occurrence of distinct ulcers in the skin and is most frequent at cold temperatures in both fresh water, brackish water, and to a certain degree, sea water. This latter has been studied closely by us in Danish fjords. The symptom complex of the disease, for which we have proposed the name “spring ulcer disease” of eel, seems to have a typical sequence of events, starting with small white spots in the region of the lateral line, which develop into larger haemorrhagic and necrotic ulcers. The etiology of the disease is complex. Three species of bacteria - Aeromonas, Alcaligenes, and Vibrio - have been isolated. A possible primary viral etiology has been postulated but virological studies have, till now, been without success. Healthy eels, transferred to an abnormal environment may develop the disease which seems also to be transmitted by contact.

local fishery districts, and replies disclosed that the dis­ Introduction ease was rather widespread in Denmark and, further­ Infectious disease in eels was first described by Canes- more, another disease was observed locally, especially trini (1892-1893) and the symptoms mentioned were after long and cold winter periods. similar to those characteristic of “red disease”, caused Hansen and Bonde (1973) studied repeated out­ by Vibrio anguillarum. A corresponding disease was breaks of disease of the latter type, which they called observed in Denmark by Feddersen (1896 a, b, 1897 a, furunculosis. The disease was characterized by an initial b), but no reports on isolation of bacteria from these occurrence of small haemorrhages in the skin, pro­ eels were given. Bruun and Heiberg (1932) studied an gressing to large ulcers with whitish-yellow necrotic epizootic among eels with “red disease” . The symptoms tissue centrally and with a diameter of 3 to 4 cm. were as follows: The first external symptoms are usually Aeromonas hydrophila and Alkaligenes bookerii were a red coloration at the end of the tail or on the body isolated from the lesions, and the prevalence of A. along the dorsal and anal fins; along with this the eels hydrophila was high in the water, where diseased eels become less lively and swim, if stimulated, with stiff were caught. wriggling movements — not so powerful, however, that During the last few years Japanese research workers the fish cannot easily be taken in the hands again. If have published papers on two bacterial diseases in eels: undisturbed it rests quietly on the bottom for the most 1) “red spot disease” caused by Pseudomonas anguilli- part. The red coloration gradually increases along the septica (Muroga et al., 1973, 1975, 1977). The predo­ fins until it appears as dark red streaks over longer or minant symptoms of this disease were petechial hae­ shorter distances, and a diffuse reddening spreads over morrhages in the skin occurring when the water tem­ most of the body. The lymph heart in the tail works perature was approximately 14°C; 2) vibriosis, caused more and more slowly until at last, it ceases to beat. The by Vibrio anguillarum, with the same symptoms as lis­ eels often die before the red coloration is well de­ ted previously (Muroga et al., 1976 a, 1976 b; Nishibu- veloped. Bruun and Heiberg (1932) made inquiries at chi and Muroga, 1977). The present work is a continuation of the work star­ ted by Hansen and Bonde (1973) and was performed on ’Present address: Institute of Toxicology, National Food In­ the request of the Danish Ministry of Fisheries, as a high stitute, Mørkhøj Bygade 19, DK 2860 Søborg, Denmark. frequency of diseased eels was reported from Randers ^ Deceased 26 May 1983 Fjord in the early spring of 1978.

106 Transmission experiments in aquaria

Hadsund Healthy eels from Mariager Fjord and diseased eels from Randers Fjord were placed in the same aquaria at various water temperatures.

Hobr« Results Mariager Pathological investigations The primary lesions in the eels appear as pin-point light spots along the lateral line, which can hardly be pal­ pated. Hyperaemia occurs later on and a haemorrhagic ulcer develops, which extends circularly from the pro­ cesses in the lateral line. A chequered pattern may be observed in the haemorrhagic zone giving the ulcers an irregular appearance. As the haemorrhages diminish, this pattern becomes more pronounced. Randej Later the ulcers have a greyish centre surrounded by a haemorrhagic marginal zone, which gradually grows narrower and finally disappears. In this later stage the ulcers are either completely greyish or the colour may change to greyish white or Figure 40. Map of the sampling sites in Randers and Mariager porcelain white with a weak yellow tint. A further de­ Fjords. velopment is the occurrence of small perforations in the centre of the greyish area often spreading in a star- shape from the centre, uncovering the underlying red tissue. Independently or simultaneously haemorrhagic or greyish lesions may be observed in the tail of the eel. Materials and methods The occurrence of real healing stages is subject to doubt. However, some fishermen have reported prob­ Eels lems with the flaying of eels which might be due to an A comparative study of eels trapped in Randers and increase in connective tissue in and beneath the skin. Mariager Fjords (Fig. 40) was performed during 1979. Fishermen from another region of Denmark have re­ Eels were investigated on board fishing vessels, and ported the occurrence of flattened non-pigmented ir­ prevalence of the disease was calculated. Bacteriologi­ regular changes in the skin of eels, which also might cal examination was done immediately after landing ac­ represent healing stages. cording to methods of Larsen and Jensen (1979) and pathological and histopathological examinations were also carried out. Attempts were made to isolate a virus Bacteriological investigations (Table 23) after the methods of Jensen et al. (1979). Seventy-nine % of the eels had infected skin lesions and 52 % infected kidneys. An infection was diagnosed,

Water and sediment Table 23. Bacterial infection in ulcers and kidneys of eel with Water and sediment samples from the locations listed in spring ulcer disease (Randers Fjord). Figure 40 were examined bacteriologically (Larsen and O/ Jensen, 1977) and the medium of Shotts and Rimler /o (1973) was used for specific isolation of A. hydrophila. No. of diseased e e l...... 124 100 Infection in ulcers...... 98 79 Aeromonas hydrophila ...... 20 20 Vibrio sp...... 23 23 Transmission experiments 100 Alcaligenes sp...... 52 53 Healthy eels were transferred in a tank from Mariager Miscellaneous ...... 3 3 Fjord to Randers Fjord and were combined in a spe­ Infection in kidneys...... 65 52 cially constructed 100-1 well box. This box was an­ Aeromonas hydrophila ...... 4 6 Vibrio sp...... 32 49 chored so near the bottom that it contained both sedi­ 100 Alcaligenes sp...... 28 43 ment and water, simulating the conditions in the fjord Miscellaneous ...... 1 2 and allowing the eels to come in contact with both.

107 when massive growth of a bacterium was found, and showed that only 10 had skin lesions. In eight of these when this bacterium occurred in pure culture or pre­ eels the lesions were found on the tail and only two had dominantly so. Three bacterial species seem involved in more disseminated ulcers. It was not possible to decide this disease: 1) A. hydrophila, 2) Alcaligenes spp., and if it was the same disease as that in Randers Fjord. 3) Vibrio spp.

Transmission experiments Virological investigations of spring ulcer disease Nineteen apparently healthy eels were transferred from in eels Mariager Fjord to the tank in Randers Fjord. After one Due to lack of uniformity in the bacteriological findings month only 14 eels were left alive. Three had typical it was found appropriate to perform virological investi­ spring ulcer disease, while four had non-specific skin gations. Organ pools from diseased eels were filtered lesions. (145 m|x) and passed three times of five cell-lines (RTG2, FHM, EPC, GM, and PS). No cytopathogenic effect was observed. Therefore it is still undecided Aquaria experiments (Table 24) whether a virus is involved in the disease. Transferring eels from Randers to aquarium conditions resulted in a high mortality rate among these animals. In a mixed population of diseased eels from Randers Fjord Prevalence of spring ulcer disease among eels in and healthy ones from Mariager Fjord kept at 8°C, one Randers and Mariager Fjords eel from Mariager Fjord showed skin changes but it was In Randers Fjord an increase in percentages of diseased not possible to decide for sure that it was a case of eels from 69 % to 86 % was observed from March to spring ulcer disease. the beginning of May, simultaneously with a rise in wa­ ter temperature. At the end of May when the water temperature reached 10°C, the prevalence of spring Microbiological investigations of water and ulcer disease was 26%. In July the disease was not sediment from Randers Fjord observed. While the haemorrhagic ulcers dominated in March, the white stages were most frequent at the end The monthly prevalences listed in the figures (Figs. 42 of May (Fig. 41). and 43) show some characteristic features of the fre­ An investigation of 2387 eels in Mariager Fjord quency and distribution of Vibrio and Aeromonas in the

Temperature 0°C 4°C lo 't 13 C 16 C 19 C

T O O . °/o

Month March April 9May 19 May June July

Number examined 48 187 734 681 608 600 Figure 41. Occurrence of spring Number diseased 33 43 632 173 16 0 ulcer disease in Randers Fjord Per cent diseased 69 76 86 25 2,6 0 in 1979.

108 Salinity No.germs/ml

104 20

1 0

10 10

10

10

Figure 42. Bacteriological find­ ings in the water in Randers 4 5 6 Fjord in May 1979. (v.c. = via­ Station 1 2 3 ble counts.) ’ total v.c. ; T'""* Aeromonas; Vibrio anguillarum;

Salinity No.germs/g °/oo • • 10* 20 / 1 0 s.

1 0 *.

1 0 s.

2 10 .

1 1 0 .

Figure 43. Bacteriological find­ ings in the sediment in Randers 4 5 6 Fjord in May 1979. (v.c. = via­ Station 1 2 3 ble counts.) •---•total v.c. ; A— A Aeromonas; “Vibrio anguillarum;

Table 24. Transmission experiments in aquaria with eels af­ fjord. As expected Vibrio occurred with the highest fected with spring ulcer disease, and apparently healthy eels 9 prevalence near the sea in contrast to Aeromonas with to 29 May 1979. the highest prevalence, where the salinity was lowest. t(°C) Mariage r Fjord Randers Fjord The water temperature greatly influenced the preval­ Healthy No. affected Diseased No. dead ence. Generally the total bacterial counts and the number of Aeromonas are highest in Randers Fjord. 7 5 0 30 25 8 5 l 1 36 35 13 5 0 40 39 23 5 0 5 5 Discussion 23 5 0 5 5 Spring ulcer disease in eels might be a specific syndrome 1This case could not with certainty be ascribed to spring ulcer with a primary localization along the lateral line. A disease. progressive development from small skin lesions to

109 large ulcers was observed under aquarium conditions References and the corresponding stages were found in eel catches. Bruun, A. F., and Heiberg, G. 1932. The "red disease” of the The bacterial involvement in the disease seems to be eel in Danish waters. Medd Kommn Danm. Fisk.- og Hav- complex, as three different species were isolated from unders., Serie: Fiskeri, Bind IX, no. 6. Canestrini, G. 1893. La malatia dominante delle anguilla. Atti, ulcers and kidneys. The most frequent bacterium found Del R. Instituto Veneto di Science, Lettere ed Arti (Tomo in ulcers was an Alcaligenes sp. (53 %), while Vibrio sp. LI) Serie Settima, Tomo Quarto Dispensa Sesta, Venezia and A. hydrophila were isolated in 23 % and 20 % re­ 1892-93. spectively. In kidneys Vibrio sp. were found in 49 %, Feddersen, A. 1896 a. En mærkelig aal. Dansk Fiskerifor­ enings Medlemsblad. København, 12 November. Alcaligenes sp. in 43 %, and A. hydrophila in 6 %. Feddersen, A. 1896 b. Den mærkelige aal. Dansk Fiskerifor­ A virus might possibly be the etiologic agent. A pre­ enings Medlemsblad. 19 November. liminary attempt to isolate virus in five different cell- Feddersen, A. 1897 a. Rødsygen. Dansk Fiskeriforenings lines, however, was unsuccessful. Aquarium experi­ Medlemsblad. 2 December. Feddersen, A. 1897 b. Rødsygen. Dansk Fiskeriforenings ments did not clarify the transmissibility of the disease, Medlemsblad. 2 December. but it was obvious from some of these experiments that Hansen, J. C., and Bonde, G. J. 1973. Aeromonas hydrophila the mortality of affected eels was exceptionally high. (s. liquefaciens) som mulig årsag til furunkulose hos ål. Field observations showed that spring ulcer disease was Nord. VetMed., 25: 121-130. Jensen, N. J., Block, B., and Larsen, J. L. 1979. The ulcus- an important problem in Randers Fjord, while among syndrome in cod (Gadus morhua). I. A pathological and 2387 eels in Mariager Fjord only eight were found to histopathological report. Nord. VetMed., 31: 436—442. have skin lesions, which could not with certainty be Larsen, J. L., and Jensen, N. J. 1977. Patologiske, mikrobio­ considered typical for spring ulcer disease. The preval­ logiske og fysisk-kemiske undersøgelser i relation til ulcus- syndromet hos torsk. Rapport for perioden 1/1 1976 - ence of the syndrome in Randers Fjord was high in 21/12 1976. KVL, Amb. Klinik m.v. og Inst. Vet. Mikro- March, April, and May with 69, 76, and 86 % of the biol. & Hyg., København 1977. eels affected. When the water temperature exceeded Larsen, J. L., and Jensen, N. J. 1979. The ulcus-syndrome in 10°C, the prevalence of diseased eels decreased. At the cod (Gadus morhua). II. A bacteriological investigation. Nord. VetMed., 31: 289-296. end of May the prevalence was 25 % at a water tempe­ Muroga, K., Jo, Y., and Yano, M. 1973. Studies on red spot rature of 13°C, the corresponding values for June were disease of pond-cultured eels — I. The occurrence of the 2 6 % and 16°C respectively, and in July the disease disease in eel culture ponds in Tokushima Prefecture in was not observed. 1972. Fish Pathol., 8(1): 9. Muroga, K., Jo, Y., and Sawada, T. 1975. Studies on red spot Bacteriological examinations of water and sediment disease of pond-cultured eels - II. Pathogenicity of the from the two fjords showed that the total aerobic bac­ causative bacterium, Pseudomonas anguilliseptica. Fish Pa­ terial counts and the A. hydrophila counts were higher thol., 9(2): 3. in Randers Fjord than in Mariager Fjord. Muroga, K., Nakai, T., and Sawada, T. 1977. Studies on red spot disease of pond-cultured eels - IV. Physiological cha­ It has recently been stated that the potential public racteristics of the causative bacterium, Pseudomonas an­ health significance of large numbers of Aeromonas bac­ guilliseptica. Fish Pathol., 12(1): 33-38. 6. teria in the aquatic environment should not be ignored, Muroga, K., Nishibuchi, M., and Jo, Y. 1976 a. Pathogenic and that this bacterium exists in high numbers in pollu­ Vibrio isolated from cultured eels. I. Characteristics and taxonomic status. Fish Pathol., 11(3): 12. ted, fresh, and brackish waters (Seidler et al., 1980). Muroga, K., Nishibuchi, M., and Jo, Y. 1976 b. Pathogenic Therefore we regard the high prevalence of A. hydro­ Vibrio isolated from cultured eels. II. Physiological charac­ phila as an indication of abundant nutrients, which might teristics and pathogenicity. Fish Pathol., 11(3): xx. be ascribed to the presence of sewage and waste water Nishibuchi, M., and Muroga, K. 1977. Pathogenic Vibrio iso­ lated from cultured eels - III. NaCl tolerance and flagella­ from a rendering plant at Randers. A concomitant drop tion. Fish Pathol., 12(2): 87-92. 9. in the oxygen content of the water should also be consi­ Shotts, E. B., and Rimler, R. 1973. Medium for the isolation of dered an important factor, as it is well known that such Aeromonas hydrophila. Appl. Microbiol., 26: 550-553. circumstances can trigger disease among fish. The fact Seidler, R. J., Allen, D. A., Lockman, H., Colwell, R. R., Joseph, S. W., and Daily, O. P. 1980. Isolation, enumera­ that eels transferred from Mariager Fjord to Randers tion, and characterization of Aeromonas from polluted wa­ Fjord develop spring ulcer disease indicates that en­ ter encountered in diving operations. Appl. Environ. Micro­ vironmental factors seem to be important. Therefore biol., 39: 1010-1018. further research should be aimed at clarifying all the determinants of this syndrome. If spring ulcer disease in the future should be used in monitoring programmes, this would be a necessary presupposition.

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