24 Erythrodermatitis of Carp

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Erythrodermatitis o CarpStudies o the Mode o InfectionD SCHULZ

Erythrodermatitis is characterized by inflammatory lesions of the skin and by necrosisleading to ulceration. The disease is common in the majority of carp fish farms in Europe (Fijan, 1972). t is a contagious disease of the skin of carp with varying severityand lethality. The etiology of this disease has not yet been established. In spite of thefact 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 carppond in West Germany was reported to our laboratory within the Division ofZoonoses and Epizootics in 1976. A group of 67 two-year-old mirror carps fromthis farm pond were examined bacteriologically. The pond carps were killedelectrically, their skin branded with a red-hot iron, and incisions made with sterilescissors 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 thoughtto be contaminated by other water bacteria. A loopful of the tissue fluid ofsulfide) medium incubated at 30C for 24 h, nutrient agar and trypticase soy agar in-cubated at 22C for 48 h]. Colonies in pure culture were isolated and identified bythe bacteriological methods described in Bergey s Manual (1974). Data on the isolatesare given in Table 1 In total, 92 strains were isolated. From these, 45 Aeromonasstrains belonged to the hydrophila punctata group, 27 strains to the genus Pseudomo-nas 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 monthswithout showing any clinical signs of disease. The aquaria were continuously suppliedwith tap water of20 c The carps were automatically fed with commercial pelleted troutfood 5 times during 24 h. Before experimental infection, the carps were narcotizedelectrically 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 thedermis.

In the first pathogenicity test involving different strains of bacteria that had beenisolated, 24-h culture medium from blood agar was used. After 3 months had passedand no clinical signs of erythrodermatitis were observed, experimental conditions werechanged: the water temperature was lowered to 12C and the fish put under stress1 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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138Table 1 Isolation of bacteria from a clinical outbreak of erythrodermatitisIsolated bacteria from the organs of diseased fish

Aeromonas:hydrophila punctata group)A hydrophila subsp. hydrophila biotype IA hydrophila subsp. hydrophila biotypeA hydrophila subsp. anaerogenes biotype IA hydrophila subsp. anaerogenes biotypeA punctata subsp. punctataA punctata subsp. caviaeA VPR-neg. anaerogenesPseudomonas:P maltophilaP stutzeriP alcaligenesP diminutaP vesicularisP cepaciaP putrefaciens

Vibrio:V a/ginolyticusPlesiomonas:P shigelloidesBrucella:PasteurellaMoraxellaFlavobacteriumAchromobacter Mucosus Group:Enterobacteriacea:Citrobacter:Klebsiella:Enterobacter:S liquefaciens:Proteus providencia group:

D. Schulz

92 strains

45 grains10 strains19 strains

3 strains3 strains4 strains4 strains3 strains

27 strainsstrainstrain2 strains

4 strains3 strains

strain15 strains

3 strainsstrain

3 strains2 strains2 strains2 strains

1 strain1 strain1 strain3 strains1 strain

y administration of prednisolone acetate in a dosis of 20 mg/kg ody weight. Thefishes were again infected with bacteria y 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 thrustinto 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 thischannel. n addition, the opening of the channel was closed with Histoacryl tissueplaster, which is used for kidney and liver operations because it is resistant to fluids.


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Erythrodermatitis of Carp: Studies of the Mode of Infection 139

Fig. 1 Mirror carp experimentally infected with lebsiella (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 whichwas 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 wasclosed 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 strainswas performed approximately 10 days after infection using the method and isolationtechnique described before.

In spite of the fact that now the fish showed symptoms of erythrodermatitis inseveral cases, of ten other bacterial species were found. t was even not quite establishedthat when Aeromonas hydrophila was isolated, these bacteria were identical with thosethat had been inoculated into the fish. Therefore, the bacteria were rendered resistant withnalidixic acid before infecting the fish. Not all of the bacteria used could be made resistant:19 strains did not become resistant and thus could not be used in the experiments. Theresults of pathogenicity testing are presented in Table 2. Mortality was highest in carps infected with Aeromonas hydrophila subspecies hydrophila biotope II. The ulcers whichoccurred were not always found on the site of infection. Sometimes they were situated onthe opposite side of the body and sometimes on the abdomen. Very severe symptomswere caused byAeromonas 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 oferythrodermatitis and mortality. Vibrio alginolyticus was also pathogenic for mirrorcarps. Klebsiella was highly pathogenic for carps and Serratia lique aciens gave only aweak reaction. No pathological changes were caused by Pseudomonas maltophila,P alcaligenes, P. diminuta, Plesiomonas shigelloides, Moraxella, Achromobacter muco-sus group, En terobacter, and Proteus providencia group (see Figs. 2-6) .


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140 D Schulzable 2 Results o infection experiments

Strain No. o infected Mortalities Symptom ofishes erythroder-matitis

A hydrophila subsp. hydrophila biotype I 20 6 13A hydrophila subsp. hydrophila biotype II 59 22 37A hydrophila subsp. anaerogenes biotype I 8 7 5A hydrophila subsp. anaerogenes biotype II 9 5 6A punctata subsp. punctata 8 6A punctata subsp. caviae 7 1A VPR-neg. anaerogenes 7 2 5Pseudomonas maltophila 6P stutzeri 6 2P alcaligenes 6P diminuta 6P vesicu laris 6 3P cepacia 6 3P putrefaciens 37 7 8Vibrio alginolyticus 6 3Plesiomonas shigelloides 7Pasteurella 11Moraxella 6Flavobacterium 7 2Achromobacter mucosus group 6Citrobacter 6 3Klebsiella 6 4Enterobacter 6Serratia liquefaciens 6 5Proteus providencia group 6 3Ref. strain: Bootsma V 76/134 6 2 6

Ref. strain: Soc. Microbiol. Gottingen 6 6Ref. strain: Animal Health Servo Grub 6 6

Comparative studies with reference strains were made using an Aeromonas hydro-phila strain from the Society o Microbiology in Gbttingen, an erythrodermatitisstrain from Fijan/Bootsma (V 76/134) and two erythrodermatitis strains from theFish 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 o Heuschmann-Brunner (1978), most o the iso-lated strains belonged to the Aeromonas hydrophila group. This group is found notonly in diseased fish but also in fish without symptoms o a disease, in surface water,


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

Fig. 3. Mirror carp experimentally infected with eromonas VPR-negative anaerogenic strain878 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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142 D Schulz

Fig. 4. Mirror carp experimentally infected with eromonas VPR-negative anaerogenic strain878 I) showing erythrodermatitis lesions as in Fig. 3 Infection was performed by the methodmentioned in Fig. 2 Picture taken 3 days after infection

Fig. 5 Mirror carp experimentally infected with roteus providencia group strain 875 I) showingerythrodermatitis lesions a hemorrhagic inflammatory process develops between the epidermis andthe 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 roteus providencia group (strain 875 1 showingerythrodermatitis lesions; picture taken 4 days after infection. Infection was performed y themethod mentioned in Fig. 2

and in sewage. In polluted water, the number of eromonas may amount to betweenseveral 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. Thefish-pathogenic strains ofA hydrophila subspecies hydrophila in Schubert s collectionhad been isolated from surface water.

Our experiments have shown that different species of bacteria may cause thesymptoms of erythrodermatitis and that for their pathogenicity the culture media andthe 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 injuriesof the epidermis, y ectoparasites, or by detergents, etc. These injuries may lead toopenings in the skin, providing portals of entry for the bacteria. A number of diseasesof freshwater fish is due to complexes of responsible injurious factors. A use of SPFfish 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 Zoonosesand Epizootics, Federal Health Office Berlin for supporting these studies and Mrs Gabriele Schulzand 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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144 D. Schulz: Erythrodermatitis of Carp: Studies of the Mode of Infection

eferencesBergey's (1974) Manual of determinative bacteriology, 8edn. Williams & Wilkins Comp,

BaltimoreBootsma R, Fijan N, Blommaert J (1977) Isolation and preliminary identification of the causative

agent of carp erythrodermatitis. Vet Arch 47:291-302Brunner G (1961) Neuere Erkenntnisse tiber die infektiose Bauchwassersucht des Karpfens. Fischwirt 11:240-241

Fijan NN (1972) Infectious dropsy of carp - a disease complex. In: Mawdesley-Thomas LE (ed)Diseases of fish. Academic Press, London New York, pp 39-51

Heuschmann-Brunner G (1965) Ein Beitrag zur Erregerfrage der infektiosen Bauchwassersuchtdes Karpfens. In: Festschrift: Der Fisch in Wissenschaft und Praxis. Sonder AUg Fischz 90:41-49

Heuschmann-Brunner G (1970) Die Aeromonaden in der Hydrobiologie. Z Wasser Abwasser Forsch3:40-41

Heuschmann-Brunner G (1971) Einige Bemerkungen aus bakteriologischer Sicht zu dem ThemaInfektiose Bauchwassersucht des Karpfens . Miinch Beitr Abwasser Fisch Flussbiol 20:67 -69Heuschmann-Brunner G (1978) Die Aeromonaden der Hydrophila-Punctata-Gruppe bei StiBwas

serfischen. Arch HydrobioI83:99-125Schaperclaus W (1930) Pseudomonas punctata als Krankheitserreger bei Fischen. Untersuchungen

tiber SiiBwasseraalrotseuche, Leibeshohlenwassersucht der Cypriniden, insbesondere des Karpfensund Fleckenseuche der Weilifische. Z Fisch 28:289-370

Schaperclaus W (1967) Probleme der Karpfenimmunitat gegeniiber Aeromonas punctata und Fragen der antigenen Struktur des Bakteriums. Z Fisch NF 15: 129-138

Scbaperclaus W Mann H (1939) Untersuchungen tiber die ansteckende Bauchwassersucht desKarpfens und ihre Bekampfung. Z Fisch 37:1-182

Schubert RHW (1960) Untersuchungen tiber die Merkmale der Gattung Aeromonas. ZentralblBakteriol I Orig 180: 310-327

Schubert RHW (1962) Uber die biochemischen Eigenschaften der anaerogenen Aeromonaden. Zentralbl Bakteriol I Orig 185:503-511Schubert RHW (1963) Uber die biochemischen Eigenschaften von Aeromonas hydrophila. Zentral

bl Bakteriol I Orig 188:62-69Schubert RHW 1964a) Zur Taxonomie der Voges-Proskauer-negativen hydrophila-ahnlichenAeromonaden. Zentralbl Bakteriol I Orig 193:482-490

Schubert RHW (1964 b) Zur Taxonomie der anaerogenen Aeromonaden. Zentralbl Bakteriol I Orig193:343-352Schubert RHW (1967) Das Vorkommen der Aeromonaden in oberirdischen Gewassern. Arch Hyg150:688-708

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