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The effects of chloramphenicol and oxytetracycline on haematopoiesis in the European eel (Anguilla anguilla)
Aquaculture, 10 (1977) 323-334 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
323
THE EFFECTS OF CHLORAMPHENICOL AND OXYTETRACYCLINE ON HAEMATOPOIESIS IN THE EUROPEAN EEL (ANGUILLA ANGUILLA)
HANNE-LORE
KREUTZMANN
Department of Biology, fchthyological (German Democratic Republic)
Research Group, Wilhelm-Pieck-Uniuersitiit,
Restock
(Received 7 December 1976; revised 21 January 1977)
ABSTRACT Kreutzmann, H.L., 1977. The effects of chloramphenicol and oxytetracycline poiesis in the European eel (Anguilla anguilla). Aquaculture, 10: 323-334.
on haemato-
Disturbances were observed in the erythropoiesis of the eel (Anguilla anguilla) after treatment with chloramphenicol and - to a lesser extent - with oxytetracycline. They are characterized by a vacuolation in the plasma, nuclear changes, a large decrease in the number of erythroblasts, and disturbances in the fat metabolism of erythrocytes.
INTRODUCTION
Investigations on the occurrence of side effects produced by medicaments have been performed to an increasing extent in both the human and veterinary medical fields in recent years. Special attention in this respect has been paid to the antibiotics chloramphenicol (CAP) and oxytetracycline (OTC). Among the side effects produced by these antibiotics, disturbances in the cellular blood components and blood forming organs are particularly characteristic (Kautz, 1960; KZhler, 1962; Libansky, 1970; Remmele, 1972). When discussing medicament-induced blood damage, it is essential to include the blood forming organs in the assessment, since they, together with the blood, form an inseparable functional entity of which each component affects the other under both normal and pathological conditions. Changes in the cell content of the peripheral blood leads to reactive modifications in the blood forming organs and, conversely, disturbances in cell proliferation in the blood forming organs will affect the peripheral blood. Medicament-induced damage to the blood and blood forming organs may become apparent in the metabolism of the cell and the cellular functions, in the morphology of the cell and in the numbers of cells. Medicament-induced aberrations in the cell morphology can assume various forms in the peripheral blood and also in the blood forming organs. They may occur as toxic granulation of the leucocytes, the occurrence of internal bodies in erythrocytes and vacuolation of the red and white blood
324
corpuscles. The proportions in which the blood cell components are present may change substantially. If, when medicaments are administered, such phenomena are observed and correctly interpreted, they provide important pointers for further treatment and for prevention of medicament-induced damage. CAP is one of the pharmaceuticals which must be regarded as potentially toxic with regard to its side effects on the blood, whereas OTC belongs to the category of medicaments with only slight toxic side effects (Bock, 1972). The side effects of these two antibiotics, which are frequently used in fish culture, on the blood and blood forming organs of fish is investigated in European eel (Anguilh anguilh) in the following paper. The results of these investigations are intended to form a basis for guideline? to the use, dosage and rate of application of these antibiotics in order to avoid therapeutic damage when combatting fish diseases. MATERIAL
AND METHODS
The animals used in the experiments (Anguilla anguih) were taken from the Strelasund and adapted to aquarium conditions. The eels were about 30-40 cm long. All experiments were performed in 100-I basins and all fish were exposed to identical light and temperature conditions. The water temperature was held at 18 + 1°C. The animals were fed daily with liver ad libitum. Animals with a mean body weight of 90 g in group I served as controls. The eels in experimental group II (mean body weight: 95 g) received an intraperitoneal injection of 2 mg/lOO g body weight (BW) of CAP at the start of the experiment. The eels in experimental group III (mean body weight: 95 g) were treated with 2 mg/lOO g BW of OTC by intraperitoneal injection at the start of the experiment. A few animals in groups II and III were injected again with the same dose after 7 days. Blood was extracted from the eels by cardiocentesis (Kreutzmann, 1976) 24 h after the injection. Apart from blood smears, contact kidney and spleen preparations were also made in a few cases, these being air dried and stained immediately. Further cardiocenteses were undertaken during the 7 days following the first injection and the 14 days following the second one. The histological staining techniques used were taken from Romeis’ (1968) manual and modified for the investigation of fish blood (Kreutzmann, 1974): (1) panoptic staining (Romeis, 1968, no. 1406); (2) staining after Unna-Ziehl (Kreutzmann, 1974); (3) staining after Fey (1966); (4) Staining of &granulation (Romeis, 1968, no. 1412). The following cytochemical techniques were used: (1) test for non-specific esterase (Kreutzmann, 1974); (2) test for peroxidase after Graham-Knoll (Romeis, 1968); (3) test for alkaline phosphatase (Plenert, 1958); (4) test for acidic phosphatase (Kreutzmann and Lehmitz, 1976); (5) test for lipids using Sudan black B (Kreutzmann and Lehmitz, 1976); (6) test for iron after Griine berg (Stobbe, 1974).
325
The statistical method used for evaluating the data obtained by measurement and counting was that described by Cavalli-Sforza (1965). Student’s t-test was used to determine the statistical significance. RESULTS
Modifications in the ery throcy te series The first modifications in the erythrocyte series were observed 48 h after the initial CAP or OTC injection. The number of erythroblasts in peripheral blood and in the contact preparation of kidneys had dropped to such an extent that it could be described in terms of erythroblastopenia. At this time, however, occasional severely vacuolated erythrocytes with enlarged nuclei were observed only in experimental group II (Table I). Cytochemical examination revealed small lipid droplets in the vacuoles (Fig. 1). None of the other cytochemical tests used revealed differences from the controls.
Fig.1. Vacuolated erythrocytes with enlarged nuclei (left), and erythrocytes lipid droplets in the vacuoles after CAP injection (right).
with small
Vacuolation of the erythrocytes from the animals in group II was observed in 40% of all erythrocytes 72 h after the injection, whereas similar vacuolation
7.21 f 0.62
7.81 f 0.61
7.14 + 0.71
Group III 3 days after OTC injection 5 days after OTC injection
7.15 f 0.34
8.21 * 0.28
7.34 f 0.31
9.97 f 0.85
7.04 + 0.28
Large nucleus diameter (pm)
nucleus diameter (rm)
Group II 3 days after CAP injection 7 days after CAP injection
Proerythrocyte
Erythroblast
Group I (control animals)
Experimental group
5.61 + 0.32
6.18 * 0.31
5.42 f 0.28
7.34 f 0.42
5.02 + 0.37
Small nucleus diameter (flm)
7.14 f 0.31
8.41 f 0.28
7.84 f 0.85
10.03 f 0.51
7.02 + 0.28
Large nucleus diameter (ccm)
3.74 + 0.34
6.21 * 0.41
3.92 f 0.72
7.84 ?r 0.37
3.84 f 0.31
Small nucleus diameter (rm)
Mature erythrocytes
Nucleus diameters (mean I standard error) of AnguilZa anguilla erythrocytes in control animals and animals which had been treated with CAP or OTC (n = 30)
TABLE I
g Q,
327
was observed in 4% of the erythrocytes from group 111. All severely vacuolated erythrocytes contained an enlarged nucleus with a membrane which was partly or fully detached. The initial coarsening in the structure of the nucleus which progressed to a stage similar in appearance to the prophase was conspicuous. These properties were observed most frequently in eels after CAP injection and were found only occasionally in fish after the administration of OTC. The lipid test was positive in both experimental groups, but the indication was considerably stronger in the vacuolated erythrocytes of the eels from group II (Fig. 1). The lipid droplets observed after 48 h had united to form compact drops. Control preparations produced no positive Sudan black B reaction after extraction of lipids. Further characteristic modifications were revealed 72 h after the injection, when erythrocyte fragments occurred and the erythrocyte diameter fluctuated considerably. Whereas nucleus size, cytoplasm structure and the cell distribution 5 days after OTC injection and 7 days after CAP injection corresponded to those of the control animals, the anisocytosis was still in evidence at that time. After a second injection of CAP, the blood of the eels in group II did not become approximately normal until 14 days after injection. One animal died 21 days after the second CAP injection. The skin of this animal was severely infested by fungi and was very necrotic; its liver was pale and spongy. Blood investigations revealed toxic modification of the blood cells. Several characteristics corresponded to those we had observed after a single CAP injection, but they were of greater severity. In experimental group III, the second injection of OTC produced no major change to the results observed after the first injection. Side effects of the antibiotics CAP and OTC on the leucocytes and thrombocy tes Initial vacuolation of the monocytes and, occasionally, of the heterophiles were observed in the fish of experimental group II 48 h after application of the antibiotics. A larger number of vacuolated cells was observed in the contact spleen and liver preparations in particular. At the same time, the nuclei of these cells were slightly enlarged. After 72 h, these phenomena were present to a greater degree in experimental group II. Stimulated phagocytosis of the monocytes was observed in the peripheral blood smears and, especially, in the contact preparations of the spleen from these animals (Fig. 2). Apart from slightly vacuolated monocytes, the fish from group III exhibited no changes in comparison to the control animals. The investigations on the percentage distribution of white blood cells in eels which had been injected with CAP revealed that, after 3 days, the numbers of heterophiles and thrombocytes dropped and the numbers of monocytes, basophiles and lymphocytes rose, particularly in the kidney, but also in the peripheral blood. Isolated plasma cells were observed in kidney
Fig.2. Stimulated phagocytosis of the monocytes
(macrophage)
after CAP injection.
contact preparations. In the experimental animals from group III, the number of basophiles was also found to have risen after 72 h, whereas the numbers of monocytes and lymphocytes rose only slightly. As in the fish from group II, the proportion of thrombocytes was smaller, whereas no significant changes were noticed with respect to the heterophiles. The white blood components of the eels returned to their normal state 5 days after treatment with OTC and 7 days after CAP application. None of the cytochemical investigations revealed differences when compared with blood from the controls. After a second injection, the observed modifications occurred in a more severe and enduring form only in eels from group II. DISCUSSION
Several medical investigations on the side effects of medicaments in humans have shown that chloramphenicol takes first place among the pharmaceuticals with a definite toxic effect (Remmele, 1972). This preparation, which was the first synthetically produced antibiotic, is highly effective and is frequently used for a variety of infections. Oxytetracyline also belongs to the medicaments which can, under certain circumstances, lead to a bone marrow insufficiency (Lawkowicz, 1965). Since, however, both antibiotics are also applied for several fish diseases, such as bacterial fin rot and infectious ascites of the stomach or swollen kidney
329
330
(Amlacher, 1961), any side effects which might occur must be known and taken into consideration. As far as we know, no investigations have been conducted on the side effects of these medicaments in fish in the past.
H,C
OH
OH
H
OH
N
NH-COCHCI, 6H
Chloramphenicol
(CAP)
0
Oxy,tetracycline
Fig.3. Chemical formulae of chloramphenicol
6H
0 (OTC)
and oxytetracycline.
In a medical paper on medicament-induced damage to the haematopoietic apparatus, Remmele (1972) discussed the pathogenetic mechanisms, the affects of which can, to a certain extent, be categorized into groups. He distinguishes between the direct and indirect toxic effects of a medicament which may affect a blood forming organ. The term “toxic” is defined here as meaning that the deleterious effect will, depending on the dose, be present in all individuals treated with the medicament. With regard to the toxic group of CAP, Libinskjl (1970) is of the opinion that this group is the nitrobenzol core, whereas others maintain that it is the dichloracetyl group which is toxic. A further mechanism may be triggered by disturbances in the detoxification of the medicament and may also lead to damage to the blood and the blood forming organs. Destruction of the peripheral blood cells and damage to the blood forming organs can also be caused by an allergic immunoreaction The medicament, as a hapten, unites with the protein of the body to form a total antigen against which the organism forms antibodies. The antigen-antibody complex leads to cell agglutination and, when the complement is added, to lysis of the cell. Apart from these three mechanisms, medicament-induced cell damage or even some genetically caused enzyme defect may occur. Remmele (1972) characterized this mechanism by the term “idiosyncrasy”. Such cell damage occurs primarily in erythrocytes, but has also been observed in all other blood cells. Many medicaments act on the blood and blood forming organs by several of the above mechanisms. CAP can, on the one hand, cause toxic damage to the blood and blood forming organs (Flenker and Remmele, 1972) and may, on the other hand, produce irreversible side effects which are probably based on immunological mechanisms (Libanskjr, 1970). The antibiotics were applied intraperitoneally to the eels we investigated and the dosage was selected as 2 mg/lOO g BW corresponding to that used for practical therapeutic purposes (see Amlacher, 1961). All animals in experimental groups II and III were first treated with the antibiotics CAP and OTC,
331
respectively. In a follow-up investigation, a few animals were injected again 7 days after the first injection, after which the blood was checked. With the method of administration we used, the antibiotics enter the blood plasma indirectly by resorption and exist in the plasma partly in free form and partly combined with plasma protein. The degree of protein combination is 50% for CAP and 20% for OTC (Remmele, 1972). In our investigations we have taken not only morphological but also cytochemical modifications of the blood cells into consideration. We have also assessed deviations from the normal cell distribution and from the normal cell and nucleus size. The modifications in the erythrocyte series after a single application of CAP are particularly conspicuous. Initial changes occurred 48 h after the injection. Erythroblastopenia, vacuolation of the plasma and modifications in the structure and size of the nucleus could be discerned as characteristic differences from the blood pattern of the control animals. These forms of damage reached their climax 72 h after the injections. Rosenbach et al. (1960) investigated the effects of CAP on anaemic subjects and found, just as we did, that maturation of the erythroblasts in the bone marrow was inhibited and that the plasma of the erythrocytes was vacuolated. The pronounced coarsening of the erythrocyte nucleus structure might be explained by the blockage of protein synthesis and cell division discussed by Giese (1963). The lipid droplets found in the vacuoles indicate a disturbance in the lipid metabolism of the erythrocytes. Small fat droplets unite to form larger ones, thus greatly inflating the cell body. This may be the explanation for the anisocytosis with numerous macrocytes which we also observed. The precise mechanism through which CAP affects erythropoeisis is still unknown. Apart from modifications to the erythrocytes, characteristic symptoms also occurred in the white blood cells. In this case too, increased vacuolation of the heterophiles and monocytes was observed 72 h after injection of the CAP. Comparison of the percentage distribution of the white blood cells of fish which had been treated with CAP with those of the control animals revealed a considerable reduction in the numbers of heterophiles and thrombocytes and, conversely, a rise in the numbers of monocytes, basophiles and lymphocytes in the peripheral blood and kidney. The heterophilic granulocytopenia we found corresponds to the neutropenia occurring after treatment with CAP as described by Lawkowicz (1965). In our experimental animals, the number of heterophiles returned to normal 7 days after injection. However, in the peripheral blood at this time, mainly myelocytes and metamyelocytes were observed. This shift to the left can be ascribed to the more intense proliferation of cells in the blood forming organs. At this point, the CAP no longer has a deleterious effect on granulopoiesis. In the literature it is frequently stated that leucopenia is often compensated by monocytosis. Our investigations also indicated a conspicuous rise in the number of monocytes after treatment with CAP. Substantial changes were observed in the structure of these cells, which were produced in greater numbers than normal. The cells were excessively vacuolated; their nuclei were coarsely structured
332
and enlarged. The increased tendency of the monocytes towards phagocytosis was remarkable. We frequently found very large cells which had phagocytosed the remains of two or three erythrocytes. The occurrence of a large number of erythrocyte fragments can stimulate phagocytosis, so that phagocytosis is intensified considerably by secondary CAP. Jung (1947) came to similar conclusions when assessing the effects of dinitrobenzol on the blood in humans. The relative increase in the number of lymphocytes frequently runs parallel to neutropenia. The fact that we found transformed forms of these cells, so-called lymphoid plasma cells, even in the peripheral blood 3 days after the injection of CAP is noteworthy. These cells are characterized by numerous vacuole-like lighter areas in the plasma. The nucleus structure is similar to that of the lymphocytes and contains strong chromatin beams. The drop in the numbers of thrombocytes might be an indication of incipient thrombocytopenia. Remmele (1972) also describes such a drop in the number of thrombocytes in humans after CAP administration, and states that this thrombocytopenia may be accompanied by purpura. Except in the case of one eel, no signs of capillary damage or hemorrhagic diathesis were found in any of our experimental animals. However, in the one eel which died 21 days after the second CAP injection we found hemorrhagic modifcations of the fins and intestine as well as necroses of the skin. These might be a consequence of a persistent thrombocytopenia or heterophile granulatocytopenia (Gross et al., 1967). The liver modifications found in this eel may have increased the toxic effect of the medicament and might thus have led to death(Libtiskjr, 1970). Our observations on animals belonging to group III show clearly that the effect of the medicament OTC on erythropoiesis is considerably less pronounced, Whereas vacuolated erythrocytes account for 40% of the total 72 h after the injection of CAP, they account for only 4% 72 h after OTC injection. All other symptoms are the same as those exhibited by fish after CAP treatment, but the symptoms in eels are substantially less pronounced after OTC treatment. The characteristics of the red blood components in these animals were normal 5 days after the injection of OTC. The modifications to the white blood components are also considerably less severe after OTC treatment; for example, no significant drop in the number of heterophile granulocytes was observed. At the dosage levels we used, OTC appears to have no effect on granulopoiesis. The drop in the number of thrombocytes after OTC application, however, is noteworthy. This effect on thrombocytopoiesis must be permanently monitored, since, depending on the severity, it may lead to petechial or lethal massive hemorrhages. After final comparison of the two antibiotics with regard to their side effects on the blood and the blood forming organs of fish, we may conclude that the administration of OTC at a dose of 2 mg/lOO g BW has a relatively slight effect on haematopoiesis. It should therefore be used in preference to CAP for therapeutic purposes, Further application of CAP makes closer monitoring of the blood of the treated animals necessary in order to detect
333
and interpret slight modifications at an early stage. If only a single injection is to be performed, the CAP concentration should not exceed 2 mg/lOO g BW when treating fish. Simultaneously, the changes in the blood observed by ourselves in the course of our investigations after treatment with antibiotics must be taken into consideration and correctly interpreted when investigating the blood of diseased fish. SUMMARY
Erythropoiesis in eels (Anguillu anguilla) was disturbed after treatment with chloramphenicol and, to a lesser degree, after treatment with oxytetracycline. The disturbances include characteristic vacuolation of the plasma, modifications of the nucleus, a considerable drop in the number of erythroblasts and disturbances in the fat metabolism of the erythrocytes. Deleterious effects of chloramphenicol and, to a lesser degree, of oxytetracycline were observed in leucocytes and thrombocytes. The most conspicuous characteristics of these effects are heterophile granulocytopenia, thrombocyto penia accompanied by monocytosis and a relative lymphocytosis. After a single administration of the antibiotics at a dose of 2 mg/lOO g body weight, all changes in the blood are reversible. The blood returns to normal 5 days after oxytetracycline treatment, but not until 7 days after administration of chloramphenicol. A second application of chloramphenicol intensifies the haematopoietic disturbances. The blood does not approach its normal state again until after 14 days. Comparison of these two antibiotics indicates that oxytetracycline has a relatively slight effect on haematopoiesisand should therefore be used in preference to chloramphenicol for therapeutic purposes on fish. ACKNOWLEDGEMENTS
This study was conducted under the direction of Professor Dr Spannhof. I express my gratitude to Professor Spannhof for his interest in my work and for his critical review of the manuscript. I wish to thank Mrs I. Wegener for assisting in conducting the experiments.
REFERENCES Amlacher, E., 1961. Taschenbuch der Fischkrankheiten. Gustav Fischer Verlag, Jena, 236 pp. Bock, H.E., 1972. Nebenwirkung der Arzneitherapie. Verh. Dtsch. Ges. Pathol., 56: 34-57. Cavalli-Sforza, L., 1965. Grundbegriffe der Biometrie. Gustav Fischer Verlag, Jena, 209 pp.
334 Fey, F., 1966. Vergleichende H$mozytologie niederer Vertebraten. III. Granulozyten. Folia Haematol. (Leipzig), 86: l-20. Flenker, H. and Remmele, W., 1972. Beitrag zur pathologischen Anatomie der Panmyelophthise nach Chloramphenikol-Medikation. Beitr. Pathol., 145: 8-24. Giese, A.C., 1963. Cell Physiology. W.B. Saunders, Philadelphia, Pa., London, 592 pp. Gross, R.H., Horstmann, J., Vogel, J. and Zach, J., 1967. Zur Epidemiologie und Klinik der medikamentos allergischen Agranulozytose. Med. Welt, 1767-1779. Jung, F., 1947. Uber toxische Schldigungen an Erythrozyten. Klin. Wochenschr., 29: 459-468. K%hler, H.J., 1962. Kritische Beurteilung der Blutkrankheiten nach Anwendung von Chloramphenikol. Wissenschaftliche Verlagsgesellschaft, Stuttgart, 362 pp. Kautz, H.D., 1960. Blood dyscrasias associated with chloramphenicol (chloromycetin) therapy. J. Am. Med. Assoc., 172: 2044-2056. Kreutzmann, H.L., 1974. Anleitung zur Durchfilhrung routinemiissiger Blutuntersuchungen an Fischen in der Praxis. Z. Binnenfisch. DDR, 21: 376-385. Kr&tzmann, H.L., 1976. Untersuchungen zur Morphologie des Blutes vom europiischen Aal (Anguilka anguilla) I. Die Erythrozyten und ihre Entwicklungsstadien. Folia Haematol. (Leipzig), 103: 226-235. Kreutzmann, H.L. and Lehmitz, R., 1976. Der Einfluss von Vitamin B,-Mange1 auf physiologische und morphologische Blutparameter bei Aalen (Anguilla onguik). Arch. Tiererniihr., 26: 293-303. Lawkowicz, W., 1965. Differentialdiagnose hlmathologischer Erkrankungen. Georg Thieme Verlag, Stuttgart, 564 pp. Libanskjr, J., 1970. Stiirungen der Erythropoese postmedikamentosen Ursprungs. Fortschr. Hlimatol., 1: 125-155. Plenert, W., 1958. Cytochemische Studie iiber das Verhalten der alkalischen Phosphatase der Leukozyten beim Neugeborenen. Folia Haematol. (Leipzig), 75: 378-385. Remmele, W., 1972. ArzneischPden am Knochenmark. Verh. Dtsch. Ges. Pathol., 56: 291-310. Romeis, B., 1968. Mikroskopische Technik. Oldenburg, Miinchen, Wien, 2nd edn, 757 pp. Rosenbach, L., Gariles, A. and Mitur, W., 1960. Chloramphenicol toxicity. Reversible vacuolization of erythroid cells. N. Engl. J. Med., 263: 724-731. Stobbe, H., 1974. Untersuchungen von Blut und Knochenmark. VEB Verlag Volk und Gesundheit, Berlin, 319 pp.
323
THE EFFECTS OF CHLORAMPHENICOL AND OXYTETRACYCLINE ON HAEMATOPOIESIS IN THE EUROPEAN EEL (ANGUILLA ANGUILLA)
HANNE-LORE
KREUTZMANN
Department of Biology, fchthyological (German Democratic Republic)
Research Group, Wilhelm-Pieck-Uniuersitiit,
Restock
(Received 7 December 1976; revised 21 January 1977)
ABSTRACT Kreutzmann, H.L., 1977. The effects of chloramphenicol and oxytetracycline poiesis in the European eel (Anguilla anguilla). Aquaculture, 10: 323-334.
on haemato-
Disturbances were observed in the erythropoiesis of the eel (Anguilla anguilla) after treatment with chloramphenicol and - to a lesser extent - with oxytetracycline. They are characterized by a vacuolation in the plasma, nuclear changes, a large decrease in the number of erythroblasts, and disturbances in the fat metabolism of erythrocytes.
INTRODUCTION
Investigations on the occurrence of side effects produced by medicaments have been performed to an increasing extent in both the human and veterinary medical fields in recent years. Special attention in this respect has been paid to the antibiotics chloramphenicol (CAP) and oxytetracycline (OTC). Among the side effects produced by these antibiotics, disturbances in the cellular blood components and blood forming organs are particularly characteristic (Kautz, 1960; KZhler, 1962; Libansky, 1970; Remmele, 1972). When discussing medicament-induced blood damage, it is essential to include the blood forming organs in the assessment, since they, together with the blood, form an inseparable functional entity of which each component affects the other under both normal and pathological conditions. Changes in the cell content of the peripheral blood leads to reactive modifications in the blood forming organs and, conversely, disturbances in cell proliferation in the blood forming organs will affect the peripheral blood. Medicament-induced damage to the blood and blood forming organs may become apparent in the metabolism of the cell and the cellular functions, in the morphology of the cell and in the numbers of cells. Medicament-induced aberrations in the cell morphology can assume various forms in the peripheral blood and also in the blood forming organs. They may occur as toxic granulation of the leucocytes, the occurrence of internal bodies in erythrocytes and vacuolation of the red and white blood
324
corpuscles. The proportions in which the blood cell components are present may change substantially. If, when medicaments are administered, such phenomena are observed and correctly interpreted, they provide important pointers for further treatment and for prevention of medicament-induced damage. CAP is one of the pharmaceuticals which must be regarded as potentially toxic with regard to its side effects on the blood, whereas OTC belongs to the category of medicaments with only slight toxic side effects (Bock, 1972). The side effects of these two antibiotics, which are frequently used in fish culture, on the blood and blood forming organs of fish is investigated in European eel (Anguilh anguilh) in the following paper. The results of these investigations are intended to form a basis for guideline? to the use, dosage and rate of application of these antibiotics in order to avoid therapeutic damage when combatting fish diseases. MATERIAL
AND METHODS
The animals used in the experiments (Anguilla anguih) were taken from the Strelasund and adapted to aquarium conditions. The eels were about 30-40 cm long. All experiments were performed in 100-I basins and all fish were exposed to identical light and temperature conditions. The water temperature was held at 18 + 1°C. The animals were fed daily with liver ad libitum. Animals with a mean body weight of 90 g in group I served as controls. The eels in experimental group II (mean body weight: 95 g) received an intraperitoneal injection of 2 mg/lOO g body weight (BW) of CAP at the start of the experiment. The eels in experimental group III (mean body weight: 95 g) were treated with 2 mg/lOO g BW of OTC by intraperitoneal injection at the start of the experiment. A few animals in groups II and III were injected again with the same dose after 7 days. Blood was extracted from the eels by cardiocentesis (Kreutzmann, 1976) 24 h after the injection. Apart from blood smears, contact kidney and spleen preparations were also made in a few cases, these being air dried and stained immediately. Further cardiocenteses were undertaken during the 7 days following the first injection and the 14 days following the second one. The histological staining techniques used were taken from Romeis’ (1968) manual and modified for the investigation of fish blood (Kreutzmann, 1974): (1) panoptic staining (Romeis, 1968, no. 1406); (2) staining after Unna-Ziehl (Kreutzmann, 1974); (3) staining after Fey (1966); (4) Staining of &granulation (Romeis, 1968, no. 1412). The following cytochemical techniques were used: (1) test for non-specific esterase (Kreutzmann, 1974); (2) test for peroxidase after Graham-Knoll (Romeis, 1968); (3) test for alkaline phosphatase (Plenert, 1958); (4) test for acidic phosphatase (Kreutzmann and Lehmitz, 1976); (5) test for lipids using Sudan black B (Kreutzmann and Lehmitz, 1976); (6) test for iron after Griine berg (Stobbe, 1974).
325
The statistical method used for evaluating the data obtained by measurement and counting was that described by Cavalli-Sforza (1965). Student’s t-test was used to determine the statistical significance. RESULTS
Modifications in the ery throcy te series The first modifications in the erythrocyte series were observed 48 h after the initial CAP or OTC injection. The number of erythroblasts in peripheral blood and in the contact preparation of kidneys had dropped to such an extent that it could be described in terms of erythroblastopenia. At this time, however, occasional severely vacuolated erythrocytes with enlarged nuclei were observed only in experimental group II (Table I). Cytochemical examination revealed small lipid droplets in the vacuoles (Fig. 1). None of the other cytochemical tests used revealed differences from the controls.
Fig.1. Vacuolated erythrocytes with enlarged nuclei (left), and erythrocytes lipid droplets in the vacuoles after CAP injection (right).
with small
Vacuolation of the erythrocytes from the animals in group II was observed in 40% of all erythrocytes 72 h after the injection, whereas similar vacuolation
7.21 f 0.62
7.81 f 0.61
7.14 + 0.71
Group III 3 days after OTC injection 5 days after OTC injection
7.15 f 0.34
8.21 * 0.28
7.34 f 0.31
9.97 f 0.85
7.04 + 0.28
Large nucleus diameter (pm)
nucleus diameter (rm)
Group II 3 days after CAP injection 7 days after CAP injection
Proerythrocyte
Erythroblast
Group I (control animals)
Experimental group
5.61 + 0.32
6.18 * 0.31
5.42 f 0.28
7.34 f 0.42
5.02 + 0.37
Small nucleus diameter (flm)
7.14 f 0.31
8.41 f 0.28
7.84 f 0.85
10.03 f 0.51
7.02 + 0.28
Large nucleus diameter (ccm)
3.74 + 0.34
6.21 * 0.41
3.92 f 0.72
7.84 ?r 0.37
3.84 f 0.31
Small nucleus diameter (rm)
Mature erythrocytes
Nucleus diameters (mean I standard error) of AnguilZa anguilla erythrocytes in control animals and animals which had been treated with CAP or OTC (n = 30)
TABLE I
g Q,
327
was observed in 4% of the erythrocytes from group 111. All severely vacuolated erythrocytes contained an enlarged nucleus with a membrane which was partly or fully detached. The initial coarsening in the structure of the nucleus which progressed to a stage similar in appearance to the prophase was conspicuous. These properties were observed most frequently in eels after CAP injection and were found only occasionally in fish after the administration of OTC. The lipid test was positive in both experimental groups, but the indication was considerably stronger in the vacuolated erythrocytes of the eels from group II (Fig. 1). The lipid droplets observed after 48 h had united to form compact drops. Control preparations produced no positive Sudan black B reaction after extraction of lipids. Further characteristic modifications were revealed 72 h after the injection, when erythrocyte fragments occurred and the erythrocyte diameter fluctuated considerably. Whereas nucleus size, cytoplasm structure and the cell distribution 5 days after OTC injection and 7 days after CAP injection corresponded to those of the control animals, the anisocytosis was still in evidence at that time. After a second injection of CAP, the blood of the eels in group II did not become approximately normal until 14 days after injection. One animal died 21 days after the second CAP injection. The skin of this animal was severely infested by fungi and was very necrotic; its liver was pale and spongy. Blood investigations revealed toxic modification of the blood cells. Several characteristics corresponded to those we had observed after a single CAP injection, but they were of greater severity. In experimental group III, the second injection of OTC produced no major change to the results observed after the first injection. Side effects of the antibiotics CAP and OTC on the leucocytes and thrombocy tes Initial vacuolation of the monocytes and, occasionally, of the heterophiles were observed in the fish of experimental group II 48 h after application of the antibiotics. A larger number of vacuolated cells was observed in the contact spleen and liver preparations in particular. At the same time, the nuclei of these cells were slightly enlarged. After 72 h, these phenomena were present to a greater degree in experimental group II. Stimulated phagocytosis of the monocytes was observed in the peripheral blood smears and, especially, in the contact preparations of the spleen from these animals (Fig. 2). Apart from slightly vacuolated monocytes, the fish from group III exhibited no changes in comparison to the control animals. The investigations on the percentage distribution of white blood cells in eels which had been injected with CAP revealed that, after 3 days, the numbers of heterophiles and thrombocytes dropped and the numbers of monocytes, basophiles and lymphocytes rose, particularly in the kidney, but also in the peripheral blood. Isolated plasma cells were observed in kidney
Fig.2. Stimulated phagocytosis of the monocytes
(macrophage)
after CAP injection.
contact preparations. In the experimental animals from group III, the number of basophiles was also found to have risen after 72 h, whereas the numbers of monocytes and lymphocytes rose only slightly. As in the fish from group II, the proportion of thrombocytes was smaller, whereas no significant changes were noticed with respect to the heterophiles. The white blood components of the eels returned to their normal state 5 days after treatment with OTC and 7 days after CAP application. None of the cytochemical investigations revealed differences when compared with blood from the controls. After a second injection, the observed modifications occurred in a more severe and enduring form only in eels from group II. DISCUSSION
Several medical investigations on the side effects of medicaments in humans have shown that chloramphenicol takes first place among the pharmaceuticals with a definite toxic effect (Remmele, 1972). This preparation, which was the first synthetically produced antibiotic, is highly effective and is frequently used for a variety of infections. Oxytetracyline also belongs to the medicaments which can, under certain circumstances, lead to a bone marrow insufficiency (Lawkowicz, 1965). Since, however, both antibiotics are also applied for several fish diseases, such as bacterial fin rot and infectious ascites of the stomach or swollen kidney
329
330
(Amlacher, 1961), any side effects which might occur must be known and taken into consideration. As far as we know, no investigations have been conducted on the side effects of these medicaments in fish in the past.
H,C
OH
OH
H
OH
N
NH-COCHCI, 6H
Chloramphenicol
(CAP)
0
Oxy,tetracycline
Fig.3. Chemical formulae of chloramphenicol
6H
0 (OTC)
and oxytetracycline.
In a medical paper on medicament-induced damage to the haematopoietic apparatus, Remmele (1972) discussed the pathogenetic mechanisms, the affects of which can, to a certain extent, be categorized into groups. He distinguishes between the direct and indirect toxic effects of a medicament which may affect a blood forming organ. The term “toxic” is defined here as meaning that the deleterious effect will, depending on the dose, be present in all individuals treated with the medicament. With regard to the toxic group of CAP, Libinskjl (1970) is of the opinion that this group is the nitrobenzol core, whereas others maintain that it is the dichloracetyl group which is toxic. A further mechanism may be triggered by disturbances in the detoxification of the medicament and may also lead to damage to the blood and the blood forming organs. Destruction of the peripheral blood cells and damage to the blood forming organs can also be caused by an allergic immunoreaction The medicament, as a hapten, unites with the protein of the body to form a total antigen against which the organism forms antibodies. The antigen-antibody complex leads to cell agglutination and, when the complement is added, to lysis of the cell. Apart from these three mechanisms, medicament-induced cell damage or even some genetically caused enzyme defect may occur. Remmele (1972) characterized this mechanism by the term “idiosyncrasy”. Such cell damage occurs primarily in erythrocytes, but has also been observed in all other blood cells. Many medicaments act on the blood and blood forming organs by several of the above mechanisms. CAP can, on the one hand, cause toxic damage to the blood and blood forming organs (Flenker and Remmele, 1972) and may, on the other hand, produce irreversible side effects which are probably based on immunological mechanisms (Libanskjr, 1970). The antibiotics were applied intraperitoneally to the eels we investigated and the dosage was selected as 2 mg/lOO g BW corresponding to that used for practical therapeutic purposes (see Amlacher, 1961). All animals in experimental groups II and III were first treated with the antibiotics CAP and OTC,
331
respectively. In a follow-up investigation, a few animals were injected again 7 days after the first injection, after which the blood was checked. With the method of administration we used, the antibiotics enter the blood plasma indirectly by resorption and exist in the plasma partly in free form and partly combined with plasma protein. The degree of protein combination is 50% for CAP and 20% for OTC (Remmele, 1972). In our investigations we have taken not only morphological but also cytochemical modifications of the blood cells into consideration. We have also assessed deviations from the normal cell distribution and from the normal cell and nucleus size. The modifications in the erythrocyte series after a single application of CAP are particularly conspicuous. Initial changes occurred 48 h after the injection. Erythroblastopenia, vacuolation of the plasma and modifications in the structure and size of the nucleus could be discerned as characteristic differences from the blood pattern of the control animals. These forms of damage reached their climax 72 h after the injections. Rosenbach et al. (1960) investigated the effects of CAP on anaemic subjects and found, just as we did, that maturation of the erythroblasts in the bone marrow was inhibited and that the plasma of the erythrocytes was vacuolated. The pronounced coarsening of the erythrocyte nucleus structure might be explained by the blockage of protein synthesis and cell division discussed by Giese (1963). The lipid droplets found in the vacuoles indicate a disturbance in the lipid metabolism of the erythrocytes. Small fat droplets unite to form larger ones, thus greatly inflating the cell body. This may be the explanation for the anisocytosis with numerous macrocytes which we also observed. The precise mechanism through which CAP affects erythropoeisis is still unknown. Apart from modifications to the erythrocytes, characteristic symptoms also occurred in the white blood cells. In this case too, increased vacuolation of the heterophiles and monocytes was observed 72 h after injection of the CAP. Comparison of the percentage distribution of the white blood cells of fish which had been treated with CAP with those of the control animals revealed a considerable reduction in the numbers of heterophiles and thrombocytes and, conversely, a rise in the numbers of monocytes, basophiles and lymphocytes in the peripheral blood and kidney. The heterophilic granulocytopenia we found corresponds to the neutropenia occurring after treatment with CAP as described by Lawkowicz (1965). In our experimental animals, the number of heterophiles returned to normal 7 days after injection. However, in the peripheral blood at this time, mainly myelocytes and metamyelocytes were observed. This shift to the left can be ascribed to the more intense proliferation of cells in the blood forming organs. At this point, the CAP no longer has a deleterious effect on granulopoiesis. In the literature it is frequently stated that leucopenia is often compensated by monocytosis. Our investigations also indicated a conspicuous rise in the number of monocytes after treatment with CAP. Substantial changes were observed in the structure of these cells, which were produced in greater numbers than normal. The cells were excessively vacuolated; their nuclei were coarsely structured
332
and enlarged. The increased tendency of the monocytes towards phagocytosis was remarkable. We frequently found very large cells which had phagocytosed the remains of two or three erythrocytes. The occurrence of a large number of erythrocyte fragments can stimulate phagocytosis, so that phagocytosis is intensified considerably by secondary CAP. Jung (1947) came to similar conclusions when assessing the effects of dinitrobenzol on the blood in humans. The relative increase in the number of lymphocytes frequently runs parallel to neutropenia. The fact that we found transformed forms of these cells, so-called lymphoid plasma cells, even in the peripheral blood 3 days after the injection of CAP is noteworthy. These cells are characterized by numerous vacuole-like lighter areas in the plasma. The nucleus structure is similar to that of the lymphocytes and contains strong chromatin beams. The drop in the numbers of thrombocytes might be an indication of incipient thrombocytopenia. Remmele (1972) also describes such a drop in the number of thrombocytes in humans after CAP administration, and states that this thrombocytopenia may be accompanied by purpura. Except in the case of one eel, no signs of capillary damage or hemorrhagic diathesis were found in any of our experimental animals. However, in the one eel which died 21 days after the second CAP injection we found hemorrhagic modifcations of the fins and intestine as well as necroses of the skin. These might be a consequence of a persistent thrombocytopenia or heterophile granulatocytopenia (Gross et al., 1967). The liver modifications found in this eel may have increased the toxic effect of the medicament and might thus have led to death(Libtiskjr, 1970). Our observations on animals belonging to group III show clearly that the effect of the medicament OTC on erythropoiesis is considerably less pronounced, Whereas vacuolated erythrocytes account for 40% of the total 72 h after the injection of CAP, they account for only 4% 72 h after OTC injection. All other symptoms are the same as those exhibited by fish after CAP treatment, but the symptoms in eels are substantially less pronounced after OTC treatment. The characteristics of the red blood components in these animals were normal 5 days after the injection of OTC. The modifications to the white blood components are also considerably less severe after OTC treatment; for example, no significant drop in the number of heterophile granulocytes was observed. At the dosage levels we used, OTC appears to have no effect on granulopoiesis. The drop in the number of thrombocytes after OTC application, however, is noteworthy. This effect on thrombocytopoiesis must be permanently monitored, since, depending on the severity, it may lead to petechial or lethal massive hemorrhages. After final comparison of the two antibiotics with regard to their side effects on the blood and the blood forming organs of fish, we may conclude that the administration of OTC at a dose of 2 mg/lOO g BW has a relatively slight effect on haematopoiesis. It should therefore be used in preference to CAP for therapeutic purposes, Further application of CAP makes closer monitoring of the blood of the treated animals necessary in order to detect
333
and interpret slight modifications at an early stage. If only a single injection is to be performed, the CAP concentration should not exceed 2 mg/lOO g BW when treating fish. Simultaneously, the changes in the blood observed by ourselves in the course of our investigations after treatment with antibiotics must be taken into consideration and correctly interpreted when investigating the blood of diseased fish. SUMMARY
Erythropoiesis in eels (Anguillu anguilla) was disturbed after treatment with chloramphenicol and, to a lesser degree, after treatment with oxytetracycline. The disturbances include characteristic vacuolation of the plasma, modifications of the nucleus, a considerable drop in the number of erythroblasts and disturbances in the fat metabolism of the erythrocytes. Deleterious effects of chloramphenicol and, to a lesser degree, of oxytetracycline were observed in leucocytes and thrombocytes. The most conspicuous characteristics of these effects are heterophile granulocytopenia, thrombocyto penia accompanied by monocytosis and a relative lymphocytosis. After a single administration of the antibiotics at a dose of 2 mg/lOO g body weight, all changes in the blood are reversible. The blood returns to normal 5 days after oxytetracycline treatment, but not until 7 days after administration of chloramphenicol. A second application of chloramphenicol intensifies the haematopoietic disturbances. The blood does not approach its normal state again until after 14 days. Comparison of these two antibiotics indicates that oxytetracycline has a relatively slight effect on haematopoiesisand should therefore be used in preference to chloramphenicol for therapeutic purposes on fish. ACKNOWLEDGEMENTS
This study was conducted under the direction of Professor Dr Spannhof. I express my gratitude to Professor Spannhof for his interest in my work and for his critical review of the manuscript. I wish to thank Mrs I. Wegener for assisting in conducting the experiments.
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