Erythrodermatitis of Carp: Studies of the Mode of Infection

D. SCHULZl

Erythrodermatitis is characterized by inflammatory lesions of the skin and by necrosis leading to ulceration. The disease is common in the majority of carp fish farms in Europe (Fijan, 1972). It is a contagious disease of the skin of carp with varying severity and lethality. The etiology of this disease has not yet been established. In spite of the fact that a number of scientists has studied this disease, there are still problems (Schaperclaus, 1930, 1967; Schaperclaus and Mann, 1939; Schubert, 1960, 1962, 1963, 1964 a,b, 1967; Brunner, 1961; Heuschmann-Brunner, 1965, 1970, 1971, 1978; Bootsma et aI., 1977). An outbreak of clinical erythrodermatitis in a carp pond in West Germany was reported to our laboratory within the Division of Zoonoses and Epizootics in 1976. A group of 67 two-year-old mirror carps from this farm pond were examined bacteriologically. The pond carps were killed electrically, their skin branded with a red-hot iron, and incisions made with sterile scissors in order to obtain material from the following organs: heart, liver, kidney, swim bladder, gill. Material from the ulcers was not taken because it was thought to be contaminated by other water bacteria. A loopful of the tissue fluid of sulfide) medium incubated at 30C for 24 h, nutrient agar and trypticase soy agar incubated at 22C for 48 h]. Colonies in pure culture' were isolated and identified by the bacteriological methods described in Bergey's Manual (1974). Data on the isolates are given in Table 1. In total, 92 strains were isolated. From these, 45 Aeromonas strains belonged to the hydrophila punctata group, 27 strains to the genus Pseudomonas, and 20 strains were from other bacterial species. For pathogenicity tests, mirror carps weighing approximately 60 g each were used. These fish had been kept in aquaria or in laboratory-owned ponds for several months without showing any clinical signs of disease. The aquaria were continuously supplied with tap water of 20 c. The carps were automatically fed with commercial pelleted trout food 5 times during 24 h. Before experimental infection, the carps were narcotized electrically and scarified with a flamed vaccinostyle that had been dipped into the culture medium. The unsealed skin was scarified by perforating the epidermis but not the dermis. In the first pathogenicity test involving different strains of bacteria that had been isolated, 24-h culture medium from blood agar was used. After 3 months had passed and no clinical signs of erythrodermatitis were observed, experimental conditions were changed: the water temperature was lowered to 12C and the fish put under stress
1 Division of Zoonoses and Epizootics, Institute for Veterinary Medicine, Federal Health Office, Postfach, 0-1000 Berlin 33

W. Ahne (ed.), Fish Diseases Springer-Verlag Berlin Heidelberg 1980

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Table 1. Isolation of bacteria from a clinical outbreak of erythrodermatitis

Isolated bacteria from the organs of diseased fish Aeromonas: (hydrophila punctata group) A. hydrophila subsp. hydrophila biotype I A. hydrophila subsp. hydrophila biotype II A. hydrophila subsp. anaerogenes biotype I A. hydrophila subsp. anaerogenes biotype II A. punctata subsp. punctata A. punctata subsp. caviae A. VPR-neg. anaerogenes Pseudomonas: P. P. P. P. P. P. P. maltophila stutzeri alcaligenes diminuta vesicularis cepacia putrefaciens 92 strains 45 grains 10 strains 19 strains 3 strains 3 strains 4 strains 4 strains 3 strains 27 strains
1 strain 1 strain 2 strains 4 strains 3 strains 1 strain 15 strains

Vibrio: V. a/ginolyticus Plesiomonas: P. shigelloides Brucella: Pasteurella Moraxella Flavobacterium Achromobacter Mucosus Group: Enterobacteriacea: Citrobacter: Klebsiella: Enterobacter: S. liquefaciens: Proteus providencia group:

3 strains
1 strain

3 strains 2 strains

2 strains 2 strains

1 strain 1 strain 1 strain 3 strains 1 strain

by administration of prednisolone acetate in a dosis of 20 mg/kg body weight. The fishes were again infected with bacteria by scarification. Two months after infection, the fish did not show any effect except a lower body weight than the controls. Therefore, the method of infection was changed again: a flamed dissection needle was thrust into the skin (Fig. 1) between epidermis and dermis to prepare a channel in the tissue. By means of a straight platinum wire pure culture material was introduced into this channel. In addition, the opening of the channel was closed with Histoacryl tissue plaster, which is used for kidney and liver operations because it is resistant to fluids.

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Fig. 1. Mirror carp experimentally infected with Klebsiella (strain 884 I), showing erythrodermatitis lesions (a hemorrhagic inflammatory process develops between the epidermis and the dermis); picture taken 4 days after infection. Infection was performed by a flamed dissection needle which was thrust into the skin to prepare a channel between epidermis and dermis. With a straight platinum wire, pure culture material was introduced into the channel. After this the channel was closed with Histoacryl tissue plaster

The culture medium was also changed and a mixture of tryptose (1 %), serum (10 vol. %) and agar (1.2%) was used. Mortality was recorded every 24 h. Reisolation of all strains was performed approximately 10 days after infection using the method and isolation technique described before. In spite of the fact that now the fish showed symptoms of erythrodermatitis in several cases, often other bacterial species were found. It was even not quite established that when A eromonas hydrophila was isolated, these bacteria were identical with those that had been inoculated into the fish. Therefore, the bacteria were rendered resistant with nalidixic acid before infecting the fish. N ot all of the bacteria used could be made resistant: 19 strains did not become resistant and thus could not be used in the experiments. The results of pathogenicity testing are presented in Table 2. Mortality was highest in carps infected with Aeromonas hydrophila subspecies hydrophila biotope II. The ulcers which occurred were not always found on the site of infection. Sometimes they were situated on the opposite side of the body and sometimes on the abdomen. Very severe symptoms were caused by Aeromonas subspeciesanaerogenes biotope I and by anaerogenic aeromonads in the Voges-Proskauer test. In the cases first an extended inflammation was observed, which was followed by an extended necrosis. From the genus Pseudomonas, P. putre!aciens was identified as the pathogen in 24% of the cases, causing symptoms of erythrodermatitis and mortality. Vibrio alginolyticus was also pathogenic for mirror carps. Klebsiella was highly pathogenic for carps and Serratia lique!aciens gave only a weak reaction. No pathological changes were caused by Pseudomonas maltophila,

P. alcaligenes, P. diminuta, Plesiomonas shigelloides, Moraxella, Achromobacter mucosus group, En terobacter , and Proteus providencia group (see Figs. 2-6).

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Table 2. Results of infection experiments

Strain No. of infected fishes Mortalities Symptom of erythrodermatitis 13 37 5 6 6 1 5

A. A. A. A. A. A. A.

hydrophila subsp. hydrophila biotype I hydrophila subsp. hydrophila biotype II hydrophila subsp. anaerogenes biotype I hydrophila subsp. anaerogenes biotype II punctata subsp. punctata punctata subsp. caviae VPR-neg. anaerogenes

20 59 8 9 8 7 7 6 6 6 6 6 6 37 6 7 11 6 7 6 6 6 6 6 6 6 6 6

22
7 5 2

Pseudomonas maltophila P. stutzeri P. alcaligenes P. diminuta P. vesicu laris P. cepacia P. putrefaciens Vibrio alginolyticus Plesiomonas shigelloides Pasteurella Moraxella Flavobacterium Achromobacter mucosus group Citrobacter Klebsiella Enterobacter Serratia liquefaciens Proteus providencia group

2
3 3 8 3

3 4 5 3

Ref. strain: Bootsma V 76/134 Ref. strain: Soc. Microbiol. Gottingen Ref. strain: Animal Health Servo Grub

6 6 6

Comparative studies with reference strains were made using an Aeromonas hydrophila strain from the Society of Microbiology in Gbttingen, an "erythrodermatitis" strain from Fijan/Bootsma (V 76/134) and two "erythrodermatitis" strains from the Fish Health Service, Grub (445/78 and 314/78). Under the same experimental conditions the reference strains showed a low pathogenicity. In conformity with the findings of Heuschmann-Brunner (1978), most of the isolated strains belonged to the Aeromonas hydrophila group. This group is found not only in diseased fish but also in fish without symptoms of a disease, in surface water,

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Fig. 2. Mirror carp experimentally infected with Klebsiella (strain 884 I), showing ery throdermatitis lesions; picture taken 4 days after infection. Experimental infection was performed by methods of skin scarification. The skin was scarified by perforating the epidermis but not the dermis

Fig. 3. Mirror carp experimentally infected with Aeromonas VPR-negative anaerogenic (strain 878 I) showing erythrodermatitis lesions (the inflammatory zone gradually extends; tissue breakdown leads to the formation of an ulcer with central necrosis); picture taken 3 days after infection. Infection was performed by the method mentioned in Fig. 1

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Fig. 4. Mirror carp experimentally infected with Aeromonas VPR-negative anaerogenic (strain 878 I) showing erythrodermatitis lesions as in Fig. 3. Infection was performed by the method mentioned in Fig. 2. Picture taken 3 days after infection

Fig. 5. Mirror carp experimentally infected with Proteus providencia group (strain 875 I) showing erythrodermatitis lesions (a hemorrhagic inflammatory process develops between the epidermis and the dermis); picture taken 4 days after infection. Infection was performed by the method mentioned in Fig. 1

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Fig. 6. Mirror carp experimentally infected with Proteus providencia group (strain 875 1) showing erythrodermatitis lesions; picture taken 4 days after infection. Infection was performed by the method mentioned in Fig. 2

and in sewage. In polluted water, the number of Aeromonas may amount to between several thousand and 10,000 bacteria per milliliter of water. Therefore, it is troublesome to estimate the infectivity which is transmitted from the water to the fish. The fish-pathogenic strains of A. hydrophila subspecies hydrophila in Schubert's collection had been isolated from surface water. Our experiments have shown that different species of bacteria may cause the symptoms of erythrodermatitis and that for their pathogenicity the culture media and the route of experimental infection are important. The method of scarification is inexact because a number of bacteria may be washed out into the surrounding water or, vice versa, may settle on the injured skin and thus mask the true character of the infection. On the other hand, there are different possibilities for the fish to get injuries of the epidermis, by ectoparasites, or by detergents, etc. These injuries may lead to openings in the skin, providing portals of entry for the bacteria. A number of diseases of freshwater fish is due to complexes of responsible injurious factors. A use of SPF fish in experiments to elucidate the etiology of this disease would be most helpful.
Acknowledgements: I would like to thank Prof. Dr. Bulling, Chief of the Division of Zoonoses
and Epizootics, Federal Health Office Berlin for supporting these studies and Mrs. Gabriele Schulz and Mrs. Eleonore Rademacher for their very skilful technical assistance. I am also grateful to Dr. Bootsma, Utrecht and Dr. Wiedemann, Grub for providing reference strains.

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D. Schulz: Erythrodermatitis of Carp: Studies of the Mode of Infection

References
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