21 pathogenesis of carp erythrodermatitis (ce)

8/13/2019 21 Pathogenesis of Carp Erythrodermatitis (CE)

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Pathogenesis of Carp Erythrodermatitis CE):Role of Bacterial Endo- and Exotoxin1.M.A. POL, R. BOOTSMA, and 1.M. v d BERG-BLOMMAERT 1

ntroductionCarp 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 hemorrhagicinflammatory process then develops between the epidennis and the dermis. As the infection spreads the red inflammatory zone gradually extends; tissue breakdown leadsto 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 entityfrom 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 eromonas species. During laterexperiments [3 9] it was demonstrated that the CE bacterium should be assigned tothe eromonas 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it was repeatedly noted that onlylow numbers of the CE bacterium can be found in the skin lesion (Bootsma andBlommaert, unpublished). This rmding was confirmed by examining skin sectionsusing the FAT technique (Vos-Maas, unpublished). In several cases, bacteremia couldonly be demonstrated during premortal stages of the disease. These findings led to thehypothesis that some toxic factor released by the CE bacterium could account for themarked inflammation, the tissue necrosis and, possibly, for the generalized edemas.

In the present paper the pathologic effects of endotoxin extracted from the CEbacterium, and cell-free culture supernatant were studied. Carp and mice were used astest animals. In addition, a few preliminary tests were carried out in order to characterize the toxic factor in the culture supernatant.

Materials and MethodsTest AnimalsBalb/c mice (TNO, Zeist, The Netherlands), 4 weeks old, were housed in macrolonplastic 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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Pathogenesis of Carp Erythrodermatitis CE): Role of Bacterial Endo- and Exotoxin 121were 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 aeratedand 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 forseveral months in aquaria without showing any clinical sign of disease.The BacteriumCE isolate V 76/134 was used throughout this study. Cultures were maintained in asemisolid medium at 12C, as described previously [2].EndotoxinBacterial cells were grown in 200 petri dishes 10 cm C/ on a solid medium composedof 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 phenolextraction method [8].Total LPS yield was 140 mg dry weight. Next the LPS was dissolved in pyrogen.free phosphatebuffered saline (PBS). The LPS solution (6 mg/ml) was inoculated intravenously IV) and intraperitoneally IP) into mice, and IP intocarp.

Cellfree Culture SupernatantBacterial cells were grown in liquid media containing 1 tryptose, supplementedwith either 0.5 synthetic seasalt or 10 (v/v) horse blood serum. After 3x24 hat 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 ofbacterial cells were made by incubating samples at 27C. Next the supernatant wassupplemented with kanamycin 50 p.g/ml (MIC=2 p.g/ml) and stored at 4 c Toxicitytests 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 SupernatantPreliminary information on the molecular size of the toxic factor was obtainedby transferring the tryptoseseasalt supernatant to a dialysis bag (cellulose tube,pore size 40 A, Visking, Zuid Holland Ltd, The Hague, The Netherlands) andconcentrating the volume ten times in a vacuum container. To remove excess ofsalt, the content of the bag was then dialyzed against running tap water. Subsequently, 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 byexposing different batches of tryptose-serum supernatant to heat 30 min,60C), a low pH, formalin treatment, and ammonium sulfate precipitation,


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122 J. M. A. Pol et al.followed by IP inoculation into carp. Acidification to pH 2.0 was performed byadding 1 N HCl. After 3 min the pH was adjusted to 7.0 with 1 N NaOH,followed by dialysis against running tap water to remove excess of salt. Formalintreatment was carried out by supplementing supernatant with 10 formaldehydeto obtain a final concentration of 2 . After 24 h exposure at 4 the formalinwas removed by dialysis against running tap water. Ammonium sulfate, saturatedsolution, was slowly added to supernatant, to obtain a final concentration of50 . After stirring the precipitate was sedimented by centrifugation for 3 minat 1,700 G. The remaining supernatant and a solution of the precipitate in waterwere separately dialyzed against running tap water, to remove excess of ammoniumsulfate. The supernatant and the dissolved precipitate were inoculated IP intocarp.

Clinical and Pathological ExaminationsHematocrit values were determined using heparized glass capillaries. Immediatelyafter filling the capillaries were centrifuged at 15,000 G for 5 min. Protein concentrations of blood plasma and oedematous fluids were determined using anAtago SPRt2 refractometer.

For histological examination tissue samples were fixed in Bouin Hollandeand processed using standard techniques. The samples were embedded in Paraplast (Sherwood Med Ind, St. Louis, Missouri, USA). Sections were stained withMayer s hematoxylin and eosin.

ResultsEndotoxinLPS was toxic to mice. At 20 mg/kg IV the mice showed clinical signs of fever anddistress, i.e., a rough hair-coat, loss of appetite, and apathy. After 3 days the symptomsgradually disappeared and the mice recovered. At 40 mg/kg IV the same effects werenoted, but they occurred more acutely, followed by death within 24 h. The same doseIP was also lethal.

LPS had no perceptible effect on carp at dosages of 4 and 8 mg/kg IP.

Cell-free Culture Supernatant: Toxicity to MiceIV 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 thoracicand in the abdominal cavity. At 0.1 ml IP the same symptoms were noted, but themice survived and had fully recovered after 14 days. Autopsy, performed 4 days afterinoculation, revealed subperitoneal hemorrhages. SC inoculation of 0.05 ml resultedin a local inflammatory response with vasodilatation and hemorrhages.


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Pathogenesis of Carp Erythrodermatitis (CE) : Role of Bacterial Endo- and Exotoxin 123Cell-free Culture Supernatant: Toxicity to CarpSubepidennal injection of 0.01 ml tryptose-serum supernatant produced a local CE-like lesion: subepidermal vasodilatation and hemorrhages, followed by necrosis andulceration.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, wererecorded. Some of the fish only showed blackening of the skin. All IP inoculated carpdied after a maximum of 29 days.

In case of severe hemorrhages hematocrit values had dropped to 20 , whereasvalues of 45 -55 were found in control fish. In edematous fish serum protein valueshad decreased to 1.5-2.5 g , as compared with 4.5-6.5 g in control fish. Asciticfluid contained 1.5-2.5 g protein; edematous fluids from the eye orbits and scalepockets contained 1.0-1.5 g protein. Histologically, the findings closely resembledthose occurring with natural CE (Frederix-Wolters, unpublished). t is noteworthythat after IP inoculation CE-like lesions were also found subepiderma1ly (Fig. I).

Pathologic effects produced by tryptose-seasalt supernatant were essentially thesame 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 dermisright)


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124 I.M.A. Pol et aI.higher, but after IP inoculation generalized vasodilatation and edemas occurred within24 h, followed by death within 48 h. After supplementing tryptose-seasalt supernatantwith 10 v/v) horse blood serum and incubating the mixture for 24 h at 27C theoccurrence 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 SupernatantThe toxic factor did not pass the wall of the dialysis bag, indicating aMW


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Pathogenesis of Carp Erythrodermatitis (CE): Role of Bacterial Endo- and Exotoxin 125scarification [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 mediuminto carp will result in higher mortality and less specific disease symptoms.

ImmunizationBecause of the important role of exotoxin in the pathogenesis of CE, an antitoxicimmunity may be equally or even more protective than immunity against the bacteriumitself. This point should be investigated further when conSidering the preparation of avaccine.Acknowledgment The authors wish to express their sincere thanks to Mr. DJ. Kool, Mr C. Dekker,and Mr. B. de Graaf for technical assistance.

References1 Berczi I, Bertok L Bereznai T (1966) Comparative studies of the toxicology of Escherichia coli

LPS endotoxin in various animal species. Can MicrobioI12:1070-10712. Bootsma R, Fijan NN, Blommaert (1977) Isolation and preliminary identification of thecausative agent of Carp Erythrodermatitis. Vet Arhiv 47 6):291-3023. Bootsma R, Blommaert (1978) Zur Aetiologie der Erythrodermatitis beim Karpfen Cyprinuscarpio 1. In: Neuere Erkenntnisse liber Fischinfektionen. Gustav Fischer, Stuttgart New York,

S 20-274. Fijan NN (1972) Infectious dropsy in carp - a disease complex. In: Mawdesley-Thomas LE (ed)Diseases of fish. Academic Press, London New York, pp 39-515. 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: Generalmicrobiology. MacMillan Press Ltd, London, pp 786-7898. Westphal 0, Llideritz 0, Bister F (1952) Ober die Ekstraktion von Bakterien mit Phenol/Wasser.Forschung 7b: 148-1559. Wiedemann H (1979) Erythrodermatitis der Karpfen - zur Isolierung und Klassifizierung desErregers. Dtsch Tierarzti Wochenschr 86: 176 -181

21 pathogenesis of carp erythrodermatitis (ce)

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