ERYTHRODERMATITIS OF CARP, CYPRINUS CARPIO (t.): AN ELECTROPHORETIC STUDY OF BLOOD SERUM PROTEIN FRACTION LEVELS

~ehulka J.: Erythrodermatitis of Carp, Cyprinus carpio (L.): An Electrophoretic Study of Blood Serum Protein Fraction Levels. Acta vet. Bmo, 00, 1993: 187-197. Paper electrophoresis was used during the growing season from May to October to analyze the dynamics of total proteinaemia of blood serum and its fractions in clinically healthy carp, Cyprinuscarpio L., and in carp diseased with erythroderma titis (CE). Four basic fractions were detected including ,,-globulins (2 subfractions), fi-globulins (3 subfractions) and albumins. The physiological state of carp after overwintering was found to be optimum at a minimum total protein level of 25 gIl, with a relative albumin proportion of 0.25 and at an A/G quotient of 0.35. When fish were subsequently given favourable conditions and full-value food, the physiological state was stabilised at total protein levels above 33 gIl, albumins 8 to 12 gIl (relatively 0.25 to 0.35), and at an A/G quotient of 0.35 to 0.50. The severe form of CE, in fish reared under conditions without supplementary feeding, was accompanied by a decline in total protein (10 to 16 vs 32 to 34 gIl), by a relative and absolute decline in albumins (0.121 to 0.197 vs 0.276 to 0.293 and 1.21 to 3.16 vs 8.84 to 9.97 gIl), and by a low A/G ratio (0.15 to 0.25 vs 0.38 to 0.41). Erythrodermatitis, carp, paper !lectrophoresis, total serum protein, serum protein fraction levels, experimental conditions, growing season .. In order to rear carp, Cyprinus carpio (L.), intensively with minimal losses, it is necessary to be aware of the state of the health of the fish. Better diagnostics are required to enhance our understanding of pathogenesis and to control the course of diseases, evaluate the therapy and state the prognoses. To improve the diagnosis of caJ:P diseases, it was decided to study the total protein of the blood serum of clinically healthy and diseased fish by electrophoretic analysis in order to distinguish changes from normal conditions in the different fractions and to derive criteria for evaluation of the state of health. These changes depend on a number of e;Kternal and internal factors, especially nutrition, environmental conditions, method of rearing, stock weight and seasonal effects (Sorvachev 1957; Lysak and W6jcik 1960; Sadykhov and Petrenko 1969; VIa.; sov 1974; R6nyai et a1. 1982). It has, however, also been demonstrated that a significant contri,. bution to changes in the fractions that constitute the protein spectrum can be made by pathological state (Ranke and Ranke 1955; Offhaus et a1. 1955; Flemming 1958; Liebmann et a1. 1960; Riedmuller 1965, 1966; Gulyaev et al. 1966; Reichenbach-Klinke 1966, 1973; Kulow 1969; Ivasik and Karpenko 1971; Golovnev et al. 1983) including parasitoses (Jaraand Szerow 1981; Jara et a1. 1981). The majority of authors, quoted above, used paper electrophoresis, so this technique was adopted in the first stage of our research. In later experiments it is proposed to follow up the results obtained by those authors who has used more advanced tech~ niqued of separation in electrophoretic analysis, including the use of agar (Ivasik and Karpenko 1971), cellulose acetate paper (Hattingh 1972; Boon et a1. 1986), polyacrylamide gels (R6nyai et al. 1982) and cellogel microelectrophoresis (Fahic and Patackova 1990). The present paper reports on experiments carried out under field conditions. The results represent the initial stage of long-term studies on the biochemistry of the common carp. Materials and Methods Fish The trials were conducted· in the Fish Culture and Hydrobiology Research Institute's experimental pond at Klimkovice for 154 days from May to October. The experimental fish were scaly

In order to rear carp, Cyprinus carpio (L.), intensively with minimal losses, it is necessary to be aware of the state of the health of the fish.Better diagnostics are required to enhance our understanding of pathogenesis and to control the course of diseases, evaluate the therapy and state the prognoses.To improve the diagnosis of caJ:P diseases, it was decided to study the total protein of the blood serum of clinically healthy and diseased fish by electrophoretic analysis in order to distinguish changes from normal conditions in the different fractions and to derive criteria for evaluation of the state of health.These changes depend on a number of e;Kternal and internal factors, especially nutrition, environmental conditions, method of rearing, stock weight and seasonal effects (Sorvachev 1957;Lysak and W6jcik 1960;Sadykhov and Petrenko 1969;VIa.;sov 1974;R6nyai et a1. 1982).It has, however, also been demonstrated that a significant contri,.bution to changes in the fractions that constitute the protein spectrum can be made by pathological state (Ranke and Ranke 1955;Offhaus et a1. 1955;Flemming 1958;Liebmann et a1. 1960;Riedmuller 1965Riedmuller , 1966;;Gulyaev et al. 1966;Reichenbach-Klinke 1966, 1973;Kulow 1969;Ivasik and Karpenko 1971;Golovnev et al. 1983) including parasitoses (Jaraand Szerow 1981;Jara et a1. 1981).The majority of authors, quoted above, used paper electrophoresis, so this technique was adopted in the first stage of our research.In later experiments it is proposed to follow up the results obtained by those authors who has used more advanced tech~ niqued of separation in electrophoretic analysis, including the use of agar (Ivasik and Karpenko 1971), cellulose acetate paper (Hattingh 1972;Boon et a1. 1986Boon et a1. ), polyacrylamide gels (R6nyai et al. 1982) and cellogel microelectrophoresis (Fahic and Patackova 1990).
The present paper reports on experiments carried out under field conditions.The results represent the initial stage of long-term studies on the biochemistry of the common carp.

Fish
The trials were conducted• in the Fish Culture and Hydrobiology Research Institute's experimental pond at Klimkovice for 154 days from May to October.The experimental fish were scaly (2 +) common carp, Cyprinus calpio (L.), from one of the State Fishery's wintering ponds.These • fish were subjected to a veterinary check-up at the end of hibernation and were found to be pres disposed to erythrodermatitis (CE).Early in May, ISO fish were picked at random and put in groupof 30 into 6 test cages 2.5 x 5 m in size, installed onto the bottom of a pond 1 m deep.During the experimental period the carp were given feed pellets.In the first stage of the experiment (61 days from May 7 to July 8) the amount of the feed corresponded.to a maintenance ration.Thereafter (93 days from July 8 to October 9) the ration was a production diet.The growth dynamics of the fish during the experiment are shown in Fig. 1

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The state of health of the fish was evaluated post-mortem by dissection and parasitological, histological and bacteriological examination.Tissue was embedded in 10 % neutral buffered formalin for histological examination and the paraffin slices were stained with haematoxylin and eosin (H & E).The objective of the bacteriological and mycological examination of the fish was to detect the causative agents of skin lesions.Tissue smears were prepared and stained and nutrient media were inoculated.The cultivation media included the blood, Ordal, Mueller-Hinton, • Sabouraud and sweet-wort agars.The incubation temperature for examination of the growth and biochemistry of the causative microorganism was 20 to 25°C.

Serum protein
Four blood samples were taken during the experiment (on May 7, July 8, August 23 and October 9) from twenty fish on each date (7 to 10 fish with signs of CE and 7 to 10 fish without signs of CE).Blood was collected from the severed caudal vein in the morning immediately after removing the fish from the experimental cages.The fish were stunned before the blood was collected.The blood serum was separated after centrifugation at 5 800 rpm for 15 min at 4°C and stored for future analysis at 4° C. Total protein was determined by the Biuret method, photometrically, at a wavelength of 545 nm, using the BM: StlUldard.This was done within 4 hours of blood collection.The physicochemical properties of the water at dates of blood sampling are presented in Table 1.

Paper electrophoresh
Four hours after blood collection, serum proteins were separated by two-dimensional paper electrophoresis.Barbital acetate buffer was used at a pH of 9 and ionic strength of 0.06.•The protein separation time lasted 16 hours at a temperature of 20 to 22° C at a current intensity of 10 rnA (2 rnA per one paper strip) and at a voltage of 120V.Serum amounts ofO.02ml were applied and albumins (a).Statistical significance of the differences between the means for the selected sets was verified by the t -test at the significance levels of P = 0.05 and P = 0.01.

Results
Early in May, 20 days after being taken from the storage pond, the clinically healthy experimental fish, free of patho-anatomical findings, had a total protein level of 28 gil of blood serum; however, there were also fish show4tg cachexia, local anemic foci at the bases of gill filaments, and a slightly icteric hep'atopancreas.Parasitological examination revealed sporadic Trichodina sp. and Apiosoma sp. on the skin; in 30% of hearts there was sporadic Sanguinicola inermis, and in 50% of intestines there was a moderate to severe infestation of the wall by Goussia subepithelialis.Histology revealed necrosis of the gill lamellae, hyperaemia of the spleen, and hyperplastic reticular cells.Within 25 days, in 30% of carp emaciation gradually increased, visceral oedema was registered, and inflammatory lesions began appearing on the skin signalling the onset of CEo Aeromonas hydrophila subsp.hydrophila, Flavobacterium sp., Schewanella putrefaciens and Pseudomonas maltophila were isolated from• erythrodermatitis lesions.All these species were isolated in the cases ofCE as described by Schulz (1980) and some of them were experimentally confirmed to be causative agents of CEo This latter study supports our interpretation of the bacterial aetiology of the skin lesions, as described, which occurred in our experimental fish.
In the first ten-day period of July, classical alterations due to CE, in the form of epidermal necrosis and ulcerations in muscle tissue, were registered in 80% of fish (Figs. 2, 3).The lesions ranged from 1 to 10 cm 2 in size.In 80% of affected cases there was mild peritonitis and an increase in water retention by organs, while in 20%. of cases, severe peritonitis and moderate water retention were recorded.Histologically, there were inflammatory alterations in the interstitial tissue of muscles, sometimes even degenerative changes in muscle fibres.In cases where the epidermis tended to re-epithelize, the oedematous skin was intensively invaded by a cellular inflammatory reaction which penetrated the subcutis and epidermis.Histology of the hepatopancreas indicated increased activity of RES, presence of inflammatory elements in portal fields, and hyperaemia in some regions.In the third ten-day period of August, the lesions had resolved in 60 % of fish.The remaining 40% possessed both progressing and regressing lesions.In the former case, there were ulcerations up to 4.5 ems in dimension; in the latter case, there were fiat, shallow, dark pigmented scars up to 23 cm 2 , almost devoid of scales.Severe peritonitis was registered in both groups.
The final examination in October showed advanced regeneration manifested by a dark pigmentation of the epidermis and, occasionally, even by minute bases of new scales (Figs. 4,5).At this stage, histology illustrated regenerated epidermis with residual signs of inflammation.In most carp, considerable amounts of glycogen were evident in hepatocytes.
According to the level of total proteins and the absolute and relative changes in protein fractions which occurred in the course of the experiment, the state of health of the common carp was classified as follows: fish in good condition, manifesting no clinical symptoms 14 days after taking them out of the storage pond, had total serum protein ranging from 25 to 28 gil (Fig. 6); in carp which were emaciated and displayed a clear exudate in the body cavity and infiamma-• tion foci on the skin, the values were substantially lower, 14 to 20 gil (P < 0.01) (Fig. 7).This reduction in total protein in diseased fish was associated with an increased d-fraction (/31 = 0.13 to 0.21) and with a decrease in relative and absolute levels o( albumin (0.11 to 0.205 and 1.9 to 3.9 gil, respectively) (P < 0.05 and P<O.OI).In healthy carp, thed-fraction amounted to 0.053 toO.112 (p < 0.05) and the mean relative and absolute levels of albumin were 0.293 (0.24 to 0.34) (P < 0.05) and 8 gil (7.6 to 8.5 gil) (p < 0.01), respectively.

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In carp severely affected by CE -which' occurred in its most intensive form in July under conditions without supplementary feeding -the decrease in total .protein to 10 to 16 vs 32 to 34 gil (p < 0.01) (Figs.8,9) was particularly evident.This state was connected with a prolonged increase of relative /31 -globulin (0.150 to 0.189) and a gradual decrease of relative and absolute albumin (0.121 to 0.197 vs 0.276 to 0.293 and 1.21 to 3.16 vs 8.84 to 9.97 gil) (P < 0.01), respectively.Abundant feeding with pelleted food containing 15 to 19% of crude protein • resulted in a substantial increase in total protein within one month.Total protein values subsequently remained at the level of 39 gil till the final examination in October and were accompanied, besides an increased level of albumin and the albumin/globulin quotient (A/G) (Fig. 12), by a satisfactory recuperation from CEo Electropherograms from these carp (Figs. 10,11) illustrate that albumins outbalanced, both relatively and absolutely (reI.0.276 to 0.299 and 10.5 to 11.7 gil) the e-fraction (f:J2' reI.0.200 to 0.230 and 7.8 to 8.9 gil).In fish suffering with CE the relation was reverse, as demonstrated by the.albuminl f:J2-globulin ratio (AI f:J2): in fish in good condition the A/ f:J2 values ranged from 1.2 to 1.6 while in sick fish it was significantly (P < 0.01) lower, (0.57 to 0.87) (Figs 7,8,10,11).Fish of the October sample also typically showed a decline in beta globulins (fractions f, e, d) (0.36 to 0.41), compared with the fish with symptoms of CE in the summer and spring seasons (0.48 to 0.55); gama globulins, represented by two subfractions (h, g) had a comparatively increasing tendency during the year (in relative terms), with fluctuating values in fish having CE in July (0.16 to 0.22) and with a stabilised culmination in October (0.20 to 0.21), compared with fish examined in May (0.14 to 0.15).

Discussion
Our results are comparable to findings by some of the quoted authors, particularly Sadykhov and Petrenko (1969) and Sorvachev (1957), confirming the correlations between total protein and protein fractions on the one hand and quality of nutrition and density of population on the other.In addition to this, our results show that the albumin fraction, in absolute terms or relative to the level of globulins, presents a valuable indicator of nutritionl1 and physiological status in the carp.
In agreement with Sadykhov and Petrenko (1969) we found that if aphysiological minimum f.ssociated with a normal protein synthesis is to be maintained, it is necessary to provide sufficient food with adequate available crude protein.The fish in our experiments responded to the production ration, provided after July 8, and reached a total proteinaemia, an absolute level of albumin and A/G ratio, with a simultaneous slight increase in y-globulin.Like Vlasov (1974), we found that a-globulins are the labile fraction in relative terms and that y-globulins increase in autumn .Sorvachev (1957) found that maintaining the optimum level of total protein in the blood serum of carp yearlings in spring (March and April) is essential to ensure a good state of health in the subsequent period and to avoid losses.This is in good agreement with our results.A decrease in total protein to under 20 gil (10 to 16 gil) together with a decline in the absolute and relative levels of albumin (1.21 to 3.16 gil and 0.121 to 0.197, respectively) did not maintain a good physiological state in the carp.The optimum levels of total protein which we determined in spring and autumn (>25 and >33 gil, respectively) are in agreement with values published by Kulow (1967 as cited by Ivasik andKarpenko 1971).We also agree with the results published by Pravda (1985)  as the normal proteinaemia for carp fry.Pravda (1985) also draws attention to the results obtained by Cervinka (1971) and to unpublished results by Adamek (1977), who were the first in the Czech Republic to emphasize the importance of plasma proteins for maintaining an optimum development of carp fry.Spurny and MareS (1989) provided convincing evidence on the importance of spring feeding of carp fry for increasing the total protein level of blood plasma.Our results, intended primarily for evaluation of the state of ehalth of carp, can be compared with findings published by pioneers in the investigation of this problem, including the following German authors: Ranke and Ranke (1955), Offhaus et al. (1955), Flemming (1958), Liebmann et al. (1960), Riedmiiller (1965 and1966) and Kulow (1966 and1969).As to infectious ascites and CE, Ranke and Ranke (1955) and Flemming (1958) agree that both diseases are accompanied by marked decrease in total protein and level of albumin, which is in keeping with our findings.High importance is attached to the system worked out by Offhaus (quoted by Riedmiiller 1966) which takes into account the total protein level, albumin level and A/G ratio in order to classify the state of health of carp into four categories.R6nyai et al. (1982) conducted experiments with 3-year-old carp and interpreted the results of electrophoretic analysis on polyacrylamide gels.Unlike us, they recorded comparatively high levels of albumin (15.7 to 20.5 gIl) and reported that the A/G ratio (0.7 to 1.18) increased with stocking density.Ivasik and Karpenko (1971), who carried out electrophoresis on agar, put forward an interesting interpretation of values for relative albumin and A/G ratio.Comparing these conclusions with our results makes it advisable to try different carriers, for example, agarose and cellulose paper. .Among other factors affecting the onset of CE in carp -whose aetiology is understood in the Czech Republic in the same way as described by Fij an (1982) -a considerable role must be attributed to optimum supply, not only of proteins, but also of fatty acids (Csengeri et al. 1983).
From the practical point of view, our results, supported by the cited literature, indicate that if total protein drops below 25 gIl after overwintering, the physiological state of carp will be suboptimum and is likely to be severely impaired by the traditional handling and transferring/in the spring season.From many years of experience we know that CE, which affects carp of any age, is an extremely dangerous disease and that the particularly predisposed fish are those in poor conditions prior to storage or those weakened in its course.
The levels of protein and its fractions which provide the best prognosis after overwintering are at least 28 gIl for total protein and blood serum, a relative albumin over 0.25 (8 gIl) and an A/G ratio of 0.35.If fish are not offered appropriate rearing and feeding conditions during the period atfer transfer, the disease aggravates and combines mostly with spring viremia.If there is vigorous competition for food, as indicated by a decreased total protein (10 to 16 gIl) and if albumin is depressed below 3 gIl whilst p-globulins show a slight relative increase, CE develops in a severely progressive form.With adequate environmental conditions, the physiological state of carp may be stabilized at a total protein over 33 gIl, albumin 8 to 12 gIl (relative level 0.25 to 0.35), and an A/G ratio 0.35 to 0.5.These values represent si~cant criteria of the state of health.

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Figs 2 to 5. Development of erythrodermatitis lesions in (2+) carp during the growing season: severe form of erythrodermatitis in summer (2, 3) and residues of eryth:-odermatitis lesions in a stage of satisfactory regeneration (4) and with visible bases of • new scales (5) in October.•
IS4days Fig.1.Live weight growth dynamics of carp during the experimental period, as depending on time (expressed by linear regression).

Table 1
The physical aDd hydrochemical characteristic of the water at time• of blood..amplinas I May  I Jul8 I Aug 23 I Oct 9 ten minutes and stained with phenylene blue.They were evaluated by the Zeiss ERI densitometer and integrative curves were constructed to estimate the values for each fraction.In Figs 6 to 11, components of the serum protein spectrum are designated by lower-case letters; they were classified, for the purposes of comparison with the authors cited above,.as gamma globulins BereTa~HoHHblH nepHoA C MaSi no OKTSl6pb npoBoAHnH HccneAoBaHHSI AHHaMHKH 06LQero npoTeMHa H ee cppaK~HH Ha KnHHH4eCKH 3AOPOBOM H 3a60neBweM 3pHTpO-AepMaTHTOM nocaA04HOM MaTepHane Kapna.npH npHMeHeHHH 6ap6HTan--a~eTaTOBoro 6ycpepHoro pacTBopa pH 9, HOHHOH CHnbl 0,06, nepHoAa