Pathogenesis of Carp Erythrodermatitis (CE): Role of Bacterial Endo- and Exotoxin

1.M.A. POL, R. BOOTSMA, and 1.M. v. d. BERG-BLOMMAERT 1

Introduction
Carp erythrodennatitis (CE) is a subacute to chronic contagious disease of the skin. The infection frequently starts at the site of an injury to the epidennis. A hemorrhagic inflammatory process then develops between the epidennis and the dermis. As the infection spreads the red inflammatory zone gradually extends; tissue breakdown leads to the fonnation of a central ulcer. In tenninal stages a generalized edema may occur. Fijan (1972) suggested separation of the disease as a distinct pathological entity from the infectious dropsy of carp (IDC) complex, and gave it the present name. The etiology ofCE was established by Bootsma et al. (1977). The causative bacterium was preliminarily characterized as a nonmotile Aeromonas species. During later experiments [3,9] it was demonstrated that the CE bacterium should be assigned to the Aeromonas salmonicida complex but, using the present taxonomic subdivision [6], it cannot be identified at a subspecies level. At bacteriological examination of diseased carp it was repeatedly noted that only low numbers of the CE bacterium can be found in the skin lesion (Bootsma and Blommaert, unpublished). This rmding was confirmed by examining skin sections using the FAT technique (Vos-Maas, unpublished). In several cases, bacteremia could only be demonstrated during premortal stages of the disease. These findings led to the hypothesis that some toxic factor released by the CE bacterium could account for the marked inflammation, the tissue necrosis and, possibly, for the generalized edemas. In the present paper the pathologic effects of endotoxin extracted from the CE bacterium, and cell-free culture supernatant were studied. Carp and mice were used as test animals. In addition, a few preliminary tests were carried out in order to characterize the toxic factor in the culture supernatant.

Materials and Methods

Test Animals Balb/c mice (TNO, Zeist, The Netherlands), 4 weeks old, were housed in macrolon plastic cages. Room temperature was 23C; air humidity was maintained at 50% saturation. Pelleted food (Muracon, Trouw Ltd, Putten, The Netherlands) and water
Department of Special Animal Pathology, Utrecht, Netherlands

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

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were administered ad libitum. One-summer-old mirror carp (Cyprinus carpio L.), weighing 100-200 g each, were kept in 20-2001 aquaria. The aquaria were aerated and continuously supplied with running tap water of 25C. The carp were fed a pelleted trout food (Trouvit, Trouw Ltd) 4-5 times a day. All test fish had been kept for several months in aquaria without showing any clinical sign of disease. The Bacterium CE isolate V 76/134 was used throughout this study. Cultures were maintained in a semisolid medium at 12C, as described previously [2]. Endotoxin Bacterial cells were grown in 200 petri dishes (10 cm C/) on a solid medium composed of tryptose (Difco) 1% and agar 1.5%, supplemented with horse blood serum 10% (v/v). Approximately 30 g of cells (wet weight) was harvested after 3x24 h at 27C. Endotoxin (lipopolysaccharide, LPS) was extracted from the cells using the phenol extraction method [8]. Total LPS yield was 140 mg dry weight. Next the LPS was dissolved in pyro gen.free phosphatebuffered saline (PBS). The LPS solution (6 mg/ml) was in oculated intravenously (IV) and intraperitoneally (IP) into mice, and IP into carp. Cellfree Culture Supernatant Bacterial cells were grown in liquid media containing 1% tryptose, supplemented with either 0.5% synthetic seasalt or 10% (v/v) horse blood serum. After 3x24 h at 27C cells were removed by centrifugation for 30 min at 3,000 G. The supernatant was filtered through a 11Onm Millipore filter. Tests for the presence of bacterial cells were made by incubating samples at 27C. Next the supernatant was supplemented with kanamycin 50 p.g/ml (MIC=2 p.g/ml) and stored at 4 c. Toxicity tests were made by IP, IV, and subcutaneous (SC) inoculation into mice, and IP, intracardial, and subepidermal inoculation into carp. Preliminary Characterization of Toxic Factor(s) in the Culture Supernatant Preliminary information on the molecular size of the toxic factor was obtained by transferring the tryptoseseasalt supernatant to a dialysis bag (cellulose tube, pore size 40 A, Visking, Zuid Holland Ltd, The Hague, The Netherlands) and concentrating the volume ten times in a vacuum container. To remove excess of salt, the content of the bag was then dialyzed against running tap water. Subse quently, the contents of the dialYSis bag and of the vacuum container were separately inoculated IP into carp. Indications for a protein nature of the toxic factor were obtained by exposing different batches of tryptose-serum supernatant to heat (30 min, 60C), a low pH, formalin treatment, and ammonium sulfate precipitation,

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followed by IP inoculation into carp. Acidification to pH 2.0 was performed by adding 1 N HCl. After 30 min the pH was adjusted to 7.0 with 1 N NaOH, followed by dialysis against running tap water to remove excess of salt. Formalin treatment was carried out by supplementing supernatant with 10% formaldehyde to obtain a final concentration of 2%. After 24 h exposure at 4 c the formalin was removed by dialysis against running tap water. Ammonium sulfate, saturated solution, was slowly added to supernatant, to obtain a final concentration of 50%. After stirring the precipitate was sedimented by centrifugation for 30 min at 1,700 G. The remaining supernatant and a solution of the precipitate in water were separately dialyzed against running tap water, to remove excess of ammonium sulfate. The supernatant and the dissolved precipitate were inoculated IP into carp. Clinical and Pathological Examinations Hematocrit values were determined using heparized glass capillaries. Immediately after filling the capillaries were centrifuged at 15,000 G for 5 min. Protein concentrations of blood plasma and oedematous fluids were determined using an Atago SPRt2 refractometer. For histological examination tissue samples were fixed in Bouin Hollande and processed using standard techniques. The samples were embedded in Paraplast (Sherwood Med Ind, St. Louis, Missouri, USA). Sections were stained with Mayer's hematoxylin and eosin.

Results
Endotoxin LPS was toxic to mice. At 20 mg/kg IV the mice showed clinical signs of fever and distress, i.e., a rough hair-coat, loss of appetite, and apathy. After 3 days the symptoms gradually disappeared and the mice recovered. At 40 mg/kg IV the same effects were noted, but they occurred more acutely, followed by death within 24 h. The same dose IP was also lethal. LPS had no perceptible effect on carp at dosages of 40 and 80 mg/kg IP. Cell-free Culture Supernatant: Toxicity to Mice IV inoculation of 0.1 ml tryptose-serum supernatant produced clinical signs of fever, followed by death after 4 days. At autopsy hemorrhages were found in the thoracic and in the abdominal cavity. At 0.1 ml IP the same symptoms were noted, but the mice survived and had fully recovered after 14 days. Autopsy, performed 4 days after inoculation, revealed subperitoneal hemorrhages. SC inoculation of 0.05 ml resulted in a local inflammatory response with vasodilatation and hemorrhages.

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Cell-free Culture Supernatant: Toxicity to Carp Subepidennal injection of 0.01 ml tryptose-serum supernatant produced a local CElike lesion: subepidermal vasodilatation and hemorrhages, followed by necrosis and ulceration. Tryptose-serum supernatant was lethal to carp at a dose of 0.5 ml/lOO g IP or intracardially. After IP inoculation, some of the fish showed extensive vasodilatation, hemorrhages and edemas. These effects were first observed around the site of injection, suggesting an initial spread of the toxic factor through the abdominal wall. After 2-3 days generalized vasodilatation and edemas, suggestive of a toxemia, were recorded. Some of the fish only showed blackening of the skin. All IP inoculated carp died after a maximum of 29 days. In case of severe hemorrhages hematocrit values had dropped to 20%, whereas values of 45%-55% were found in control fish. In edematous fish serum protein values had decreased to 1.5-2.5 g%, as compared with 4.5-6.5 g% in control fish. Ascitic fluid contained 1.5-2.5 g% protein; edematous fluids from the eye orbits and scale pockets contained 1.0-1.5 g% protein. Histologically, the findings closely resembled those occurring with natural CE (Frederix-Wolters, unpublished). It is noteworthy that after IP inoculation CE-like lesions were also found subepiderma1ly (Fig. I). Pathologic effects produced by tryptose-seasalt supernatant were essentially the same as those produced by tryptose-serum supernatant. The lethal dose was a little

Fig. 1. Histologic section through the skin of a mirror carp injected IP with 0.5 ml culture supernatant, HE stain X750. Accumulation of blood cells and fluid between epidermis (left) and dermis
(right)

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higher, but after IP inoculation generalized vasodilatation and edemas occurred within 24 h, followed by death within 48 h. After supplementing tryptose-seasalt supernatant with 10% (v/v) horse blood serum and incubating the mixture for 24 h at 27C, the occurrence of generalized effects was retarded as with tryptose-serum supernatant. This finding indicates that the toxic factor was probably bound to serum proteins. Preliminary Characterization of the Toxic Factor in the Culture Supernatant The toxic factor did not pass the wall of the dialysis bag, indicating a MW <15,000 daltons. The tenfold-concentrated tryptose-seasalt supernatant appeared to be lethal at dosages much lower than 0.5 ml/lOO g. The toxic factor was inactivated by heat, a low pH, and formalin treatment. IP inoculation of 1 ml supernatant/lOa g, after various treatments, had no perceptible effect on carp. These findings are suggestive of a protein nature. The toxic factor was removed from tryptose-serum supernatant by ammonium sulfate precipitation. The precipitate was as toxic as the initial supernatant. Possibly the protein-bound toxic factor was precipitated together with serum proteins. Attempts to precipitate the toxic factor in tryptose-seasalt supernatant failed.

Discussion
Endotoxin The negative results with bacterial LPS are in accordance with those obtained by others [1], who found that Escherichia coli LPS is not toxic to carp at 80 mg/kg. Apparently endotoxin is less toxic to carp than it is to certain mammals. Exotoxin If a toxic substance from a pathogenic bacterium is to be implicated as a determinant of virulence , it must be demonstrated to produce one or more of the specific symptoms of the disease. Furthermore the site of action and the effective concentration must be such that they could plaUSibly be obtained in the course of a natural infection [7]. All bacterial exotoxins that have been characterized chemically have been found to be proteins. Exposure of exotoxins to heat, acid, and formaldehyde eliminates toxicity [5]. In view of these criteria, the results obtained with culture supernatant strongly suggest that the lesions occurring with natural CE are partly or exclusively produced by a genuine bacterial exotoxin. The generalized edemas, frequently encountered during premortal stages of natural CE, would then be indicative of toxemia. Natural and Artificial Infection I t is assumed that natural infection takes place after an injury to the epidermis [4]. In addition, artificial infection should preferably be carried out using the method of skin

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scarification [2,4]. In such tests only the bacterium itself is introduced into the skin, the exotoxin being washed away. Inoculation of bacterial cells in liquid culture medium into carp will result in higher mortality and less specific disease symptoms. Immunization Because of the important role of exotoxin in the pathogenesis of CE, an antitoxic immunity may be equally or even more protective than immunity against the bacterium itself. This point should be investigated further when conSidering the preparation of a vaccine.
Acknowledgment. The authors wish to express their sincere thanks to Mr. DJ. Kool, Mr. C. Dekker, and Mr. B. de Graaf for technical assistance.

References
1. Berczi I, Bertok L, Bereznai T (1966) Comparative studies of the toxicology of Escherichia coli LPS endotoxin in various animal species. Can J MicrobioI12:1070-1071 2. Bootsma R, Fijan NN, Blommaert J (1977) Isolation and preliminary identification of the

causative agent of Carp Erythrodermatitis. Vet Arhiv 47(6):291-302 3. Bootsma R, Blommaert J (1978) Zur Aetiologie der Erythrodermatitis beim Karpfen Cyprinus carpio 1. In: Neuere Erkenntnisse liber Fischinfektionen. Gustav Fischer, Stuttgart New York, S 20-27 4. Fijan NN (1972) Infectious dropsy in carp - a disease complex. In: Mawdesley-Thomas LE (ed) Diseases of fish. Academic Press, London New York, pp 39-51 5. McCarty M (1973) Host - parasite relations in bacterial diseases. In: Davis BD, Dulbecco R, Eisen HN, Ginsberg HS, Wood WB (eds) Microbiology. 2nd edn. Harper & Row, Hagerstown, USA, pp 635-638 6. Schubert RHW (1974) Chapter on the genus Aeromonas. In: Buchanan RE, Gibbons NE (eds) Bergey's manual of determinative bacteriology, 8th edn. Williams and Wilkins Company, Baltimore, pp 345-348 7. Stanier R Y, Doudoroff M, Adelberg EA (1971) Chapter on bacterial exotoxins. In: General microbiology. MacMillan Press Ltd, London, pp 786-789 8. Westphal 0, Llideritz 0, Bister F (1952) Ober die Ekstraktion von Bakterien mit Phenol/Wasser. Forschung 7b: 148-155 9. Wiedemann H (1979) Erythrodermatitis der Karpfen - zur Isolierung und Klassifizierung des Erregers. Dtsch Tierarzti Wochenschr 86: 176 -181