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Parasitologia Hungarica 9. (Budapest, 1976)

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Parasitologia Hungarica 9. (Budapest, 1976) An Unidentifiable Extracellular Sporozoan Parasite from the Blood of the Carp Dr. György CSABA Central Veterinary Institute, Budapest "An unidentifiable extracellular sporozoan parasite from the blood of the carp". - Csaba, Gy. - Parasit. Hung. _9. 21-24. 1976. ABSTRACT. Description is given of an extracellular sporozoan parasite found in the blood of carp (Cyprinus carpio). In the plasma of these protozoa of white cell size 8 spindle-shaped developmental units are formed. After smash- up of the cell these elements develop further to another new ceU containing likewise 8 developmental forms each. In summer, 1975 while studying the corpuscular elements of carp blood I found an un­ known protozoon resembling the Haemosporidia. In the blood of freshwater fish unicellular parasites of the genera Haemogregarlna and Hepatozoon are known to occur most commonly (SHULMAN, 1962). The incidence of the Dactylosoma genus has also been reported by BECKER (1970) and MANWELL (1964). It was NAWROTZKY (1914) and BECKER (1962) who thoroughly dealt with the freshwa­ ter Haemogregarina. Considerably more data are available about blood protozoa of the marine fish (KOHL-YAKIMOFF and YAKIMOFF, 1915; LAIRD ana BULLOCK, 1969). Protozoological textbooks, however, record only intracellularly living sporozoa in the fish blood (BECKER, 1970; KUDO, 1954; OLLENSCHLAGER, 1975). Even the reptiles, being far richer in genera, are known to have only intraceUular sporozoa in their blood (LAISSON, LANDAU and SHAW, 1974). From the blood of the carp SMTRNOVA (1971) has described a sporozoan parasite as Haemogregarina cyprini. The aim of the present study is to introduce those extracellularly parasitizing sporozoan forms which I found in carp blood. Material and Method Between July, 1975 and end of January, 1976 I carried out the examination of about 400 carps of different age, coming from 15 fish farms. Some carps were investigated right after their collection, while others were subjected to subsequent several examinations in the course of their maintenance in aquarium of several months duration. In order to prepare smears, blood was taken by three methods: 1) by cutting the tall through at the end of the body, 2) after killing the fish, directly from the heart, 3) from the tail artery by heparinized glass capillary. The dry blood smears were fixed with 96% ethyl alcohol for 10 minutes, then stained according to Giemsa (100 ml boiled distilled water, 1,5 ml Giemsa stock-solution) for 16 hours. In almost every case native blood prepa­ ration was also examined under cover slip. Results The incidence of the said parasite was recorded in 9 of the 15 fish farms examined. Only fry proved to be infected, about 200 fishes altogether. Mean extensity of infection was 70%. Blood taking methods did not have much influence on the recovery rate of the parasite. In smears they could be found more frequently, in heavier infections, how­ ever, the amoeba-like forms of the white cell size moving relatively actively could be detected also in native preparations. They shoved locally dancing movement resembling a punctured rubber ball frequently changing the site of its dent. In these amoeba-like bodies 1 to 8 spindle-shaped elements occurred. In stained blood smears the parasites appeared as spherical, granular formulae, 3-15 u in diameter with purplish-red nucleus and blue cytoplasma. They included 1 to 8 spindle-shaped or rounded independent elements. These formulae multiply by bipartition within the cell resulting in considerable variability in the shape of the parasite (Fig. 1). Free spindle-shaped formulae never occured in the blood plasma. In no case was the parasite found in red or white blood-cells, in some instances, however, one or two pa­ rasites could be seen in phagocytic white blood-cells (Fig. 19). Phagocytized forms were mainly found in agonizing fish. Fig. 1: Tentative cycle of development of the unidentifiable extracellular spor. Figs. 2-5: A spindle-shaped structure develops in the parasite. Fig. 6: The inner structure becomes spherical. Fig. 7: The inner structure in the process of division. Figs. 8-10: Protozoa containing 2-4 spindle-shaped structures. (Photo Csaba) As to the size, the smallest protozoon I observed was 3 n, its purplish-red nucleus was 2, 5 ja in diameter. In the strikingly blue cytoplasma, next to the nucleus', there is a deep purplish-red dotlike granule which turnes into a pinlike thin stick in the protozoa of about 4 ju in size. This stick stains blue at both ends and purplish-red in the middle (Figs, lb, and 2-3). In protozoa of 5-6 u (max. up to 11 ju) the stick becomes spindle- shaped, is already 1 n in width and reaches through the cell almost in the total lenght of the diameter (Figs, le, d, e and 5). The spindle-shaped developmental form looks dark blue at both ends, the nucleus is purplish-red in its centre, and there is a tiny pale blue nucleolus in it (Fig le). In protozoa of about 10 u in size the spindle-shaped stru­ cture becomes spherical (Figs. If and 6), and begins to divide (Figs, lg, and 7). In the course of division the nucleus stretches, performs bipartition in the middle and soon the dividing nucleus is bridged merely by a purplish-red threadlike part. The division of the nucleus is followed by that of the cytoplasma as well. Repeated divisions within the protozoon result in parasites containing 2-4-6-8 spindle-shaped or rounded forms (Figs, lh-1 and 8-13) as well as in transitional forms (Fig. 18), When the parasites contain 6-8 divisional products and grow up to 10 to 15 m in size, the internal elements become rounded their inner structure undergoes transformation and the cell becomes similar to the smallest form described first. During the course of internal divisions, besides inner developmental forms also residual bodies appear in the plasma of the pro­ tozoon. They are deep purplish-red in colour, their number being 2, 3 and 4 in cells containing 6, 7 and 8 divisional products, respectively (Figs. Ik-1 and 11-13). In the next stage of development the parasites containing 8 elements fall into pieces (Figs, lm and 16) releasing the developmental forms of 3 ja, described first, and the whole cycle begins again. In heavily infected fish many immature red blood-cells and an in­ crease in the number of white blood-cells can be observed by microscopic examination. Discussion As judged by the type of its division the observed protozoon seems to be a sporozoan parasite, however, the question whether this is a vegetative or a sporogonic process remains to be cleared. In case of Haemosporidia generally gametogonic forms appear in the blood (KUDO, 1954). The division of the parasites in the present study rather resembles the vegetative phase at the start, however, during the last divisions (in cells containing 6-8 divisional products) residual bodies appear which are rather characteris­ tic of the sporogonic processes. This parasite differs from all known sporozoa of fish as far as its development takes place extracellularly. Similar parasites had been de­ scribed by SMIRNOVA (1971), these however, developed in white and red blood-cells and were considered to be schizogonic forms, the resulting schizonts falling into max. 16 pieces depending on their size. In the present study merely cells that fell into max. 8 units were found. Besides, in the blood of the same fish SMIRNOVA observed the fu­ sion of gametocytes as well, and discussed the division of the zygota in red blood-cells. In the course of my observations no similar sexual forms have occurred. The protozoon I found resembles at least to a certain extent (in case of more mature developmental forms) a white blood-cell attacked by parasites. However, the clear sequence of succes­ sive developmental stages and the increase in their size from 3 to 15 u appear to prove that these formulae, situated in the blood plasma, are by no means of host cell origin but independent extracellular protozoa. The developmental form attacking the nucleus of erythrocytes described by SMIRNOVA (1971) and erythrocyte necrosis recorded by LAIRD and BULLOCK (1969) were not present. The accurate taxonomic identification on the protozoon described here still requires further examinations. Figs. 11-13: Sphaerical inner structures and residual bodies. Figs. 14-16: Cells falling into 8 pieces. Figs. 17-18: Transitional developmental forms. Fig. 19: The parasite in phagocytic ceUs. (Photo Csaba) 2 3 Acknowledgement Thanks are due to dr. K. MOLNAR for his encouragement and valuable instructions rendered to me during this work. CSABA, Gy.: Ismeretlen extracelluláris spórás véglény pontyok vérében A szerző' a ponty (Cyprinus carpio) vérében talált extracellulárisan fejló'dó' spórás egy­ sejtűek leírását adja. A fehérvérsejt nagyságú protozoonok plazmájában 8 orsóalakú fej- ló'dési forma képződik, melyek a sejt szétesése után ugyancsak 8 fejló'dési alakot tar­ talmazó képletté növekednek. References BECKER, CD. (1962): A new haemogregarine from the blood of freshwater fish, Cato- stomus macrocheilius Giard. - J. Parasitol. 48. 596-600. BECKER, CD. (1970): A symposium on diseases of fishes and shellfishes. Special Publication No. 5, American Fisheries Society, Washington, D. C. KOHL-YAKIMOFF, N. - YAKIMOFF, W. L. (1915): Hámogregarinen der Seefische. - Zentr. Bakteriol. Parasitenk. Abt. I. Orig. 76. 135-146. KUDO, R. R. (1954): Protozoology, 4th ed., Charles C. Thomas. Springfield, Illinois, U. S.A. LAINSON, R. - LANDAU, I. - SHAW, J.J. (1974): Further parasites of the family Gar- niidae (Coccidia: Haemosporidiidea) in Brazilian lizards. Fallisia effusa gen. nov. sp. nov. and Fallisia modesta gen. nov., sp. nov. - Parasitology, 68. 117-125. LAIRD, M. - BULLOCK, W. L. (1969): Marine fish haematozoa from New Brunswick and New England. - J. Fish. Res.
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