REPRODUCTION OF THE CYCLE OF COCCIDIA EIMERIA ACERVULINA (TYZZER, 1929) IN CELL CULTURES OF CHICKEN KIDNEYS

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1 REPRODUCTION OF THE CYCLE OF COCCIDIA EIMERIA ACERVULINA (TYZZER, 1929) IN CELL CULTURES OF CHICKEN KIDNEYS Muriel NaciriBontemps To cite this version: Muriel NaciriBontemps. REPRODUCTION OF THE CYCLE OF COCCIDIA EIMERIA AC ERVULINA (TYZZER, 1929) IN CELL CULTURES OF CHICKEN KIDNEYS. Annales de Recherches Vétérinaires, INRA Editions, 1976, 7 (3), pp <hal > HAL Id: hal Submitted on 1 Jan 1976 HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 REPRODUCTION OF THE CYCLE OF COCCIDIA EIMERIA ACERVULINA (TYZZER, 1929) IN CELL CULTURES OF CHICKEN KIDNEYS Muriel NACIRIBONTEMPS Laboratoire de Parasitologie, 1. N. R. A.. Centre de Recherches de Tours, Nouzilly, Monnaie SUMMARY The cycle of Eimeria acervuldna was grown in primary culture of cells of threeweekoldchicken kidneys. EHT medium (Eagle, Hydrolysate of Lactalbumine, BactoTryptose phosphate) allowed the development of this coccidium. 44 hours after infestation of the cells with sporozoites, the first schizonts appeared. The merozoites of first generation were released after 54 hours. They invaded the neighbouring cells and developed in them into schizonts of second generation mature after 68 hours. After 72 and g3 hours, a third and a fourth generation of schizonts were noticed. in vitro, we inoculated the Merozoites IV create the Gamogony. To obtain the gamogony ultures with merozoites IV recovered from axenic animals. The oocysts were released into the hours after inoculation. nedium INTRODUCTION These last ten years, several authors have been able to reproduce the cycle of!rtain species of coccidia, either in embryos or in cell cultures. It was rather easy for the chicken s Eime yia, except for Eime yia acervulina and Eimeria!naxima. In embryos, it was not possible to obtain development after inoculations of sporozoites of Eimeria acervulina (LONG, i966 ; SHIBALOVA, 1969, 1970, 1972). Recently, LONG (1973 ) has considered that Eime yia mivati is only a variety of E. acervulina and that it is the only one capable of multiplying in embryos. In cell cultures, STROUT et al. (19 65) only obtained a beginning of development of E. acervulina to the stage of immature first generation schizonts. ITAGA! et al. (1974 ) also observed the penetration of the sporozoites without further development.

3 the the Obtaining Obtaining fnoculatiox Irtoeudation MATERIALS Cell The The present importance of intestinal species and especially of E. acervulina incited us to return to this problem. These cultures are indeed of great importance for pharmacological or immunological studies. To avoid the problems of cell division linked to the duration of the parasitic cycle, we tried to obtain the asexual and sexual stages separately, starting either from the sporozoite or the merozoite obtained in axenic animals. II. AND METHODS 1. cultures Primary cell cultures of chicken kidneys are used. They are obtained from the trypsinized kidneys of three week old animals. The cells are suspended in PBS and their number is estimated with Thoma cell. The cultures are established on coverslips in Petri dishes from o cells/ml or 7 X Io6 per dix. The growth medium contains 5 p. 100 hydrolysate of lactalbumine, 75,6 p. Too deionized water, 8,4 p. 100 Earle (X 10 ), 5 p. Too actotryptose phosphate, 5 p. Too calf serum, 10 UI per ml Penicillin and 100 Lg per ml Streptomycin. The ph is adjusted to 7,2 with sodium bicarbonate. The Petri dishes are placed at 37 C in an atmophere of 5 p. 100 C0 2. The variations of the ph are estimated with phenol red. 2. parasite 2.r. rjbtaining the parasitic stvai?!. The strain was purified by inoculation of fourweek oldchickens with a single oocyst isolated by micromanipulation. Four days later, the oocysts are recovered in the duodenum. We used two infective germs : 2.2. sporozoite merozoite the sporozoites. The oocyst sterilization is achieved by passage in sodium hypochlorite (NaOCl 45, 8 p. ioo) for half an hour. The sporocysts are released by grinding. Excystation is obtained by placing the sporocysts in an agitated medium of trypsin pig bile, at ph 7,6, at 41 C. The released sporozoites are suspended in PBS and purified by passage in a leukopak column containing synthetic fibres (Muriel BONTEMPS, P. YvoR>:, 1974) the merazoites. They are obtained in fourweekold axenic chickens. The animals are given oocysts, sterilized by successive passage in sodium hypochlorite 45 at 10 p. 100 and in formol at 10 p The duodenal contents are recovered 89 hours later and placed in a trypsin solution at 0,25 p. Too. After digestion, the cell debris are eliminated by passage in the leukopak column of synthetic fibres. We thus obtain numerous clean and sterile merozoites. 3. of the cell cultures and maintenance 3.1. of the sporozoites. After 48 hours, the cells form a 25 p. 100 confluent cellular monolayer. It is then inoculated with the sporozoites. The inoculation medium is similar to the growth medium and contains 150 ooo sporozoites of E, acervulina per ml. The Petri dishes are placed at 4oC and 4 hours later the inoculum is removed and replaced by a maintenance medium containing only 2 p. 100 bactotryptose phosphate and z p. 100 calf serum. Every 24 hours, the medium is changed to maintain the ph at 7,2.

4 Penetration First Fixation 3.2. Inoculation of the mevozoites. The inoculum contains I million merozoites per ml medium. It is left on the cell layer during 20 hours, then the medium is changed daily. 4. and staining Samples are taken every 12 hours. The cells are fixed with AceticMethanol (IV/ 3V), then they are stained with MayGrunwaldGiemsa. I.I. III RESULTAT9 i. Sehizogony of thesporozoites into the cells. The microscopic examination of a dish after inoculation of the sporozoites of E. acervulina, without any staining, enables one to note mobile parasites above the cells. These parasites show a rather indistinct central nucleus, a very clear refringent posterior globule and sometimes a small refringent anterior globule. They penetrate the cells the first minutes after inoculation and they invariably settle near the nucleus of the hostcell. After fixation and staining with MayGrunwald Giemsa, the intracellular sporozoites can be distinguished from those which have not yet penetrated the cells by the presence of a peripheral vacuole in the cytoplasm of the hostcell (Pl. I, ph. z). They measure about 7 to 10!./2 to 3!1 and are easily recognizable thanks to their eosinophile refringent globule that will persist during the total duration of the first schizogony. =.2, T!O!/KM <M <gs. From the 20 th to the 3oth hour, the intracellular sporozoites develop into trophozoites ;They are very similar to sporozoites but they are much larger, particularly about their median parts. Their nuclei have indeed become bigger and their posterior refringent globules more spherical. They measure about 7,8 tk/3,6!1. This shape is predominant at 30 hours. At 44 hours, 2 types of trophozoites can be observed : some are almost totally spherical (Pl. I, ph. 2) and measure about 7,9!1/6, S!1 ; others show a cytoplasmic outgrowth that often contains the refringent globule and they look like the trophozoites (B type) described by DOR AN and V>;TT>~RI, ING (19 68) or type III described by IT AGAK et al. (sg 74). i.3. generation schizonts. 2 days after inoculation, numerous first generation schizonts are present in the cultures. The nucleus of the trophozoite divides and produces a young binucleate first generation schizont that measures about 9!t/6!1. Then the size of the schizont and the number of. nuclei increase. The refringent globule, smaller than a nucleus, is still visible but becomes less clear. This globule, characteristic of the ist generation, disappears as soon as the ist generation merozoites start appearing. The immature

5 Second Third Fourth ist generation schizonts are very polymorphous. The sizes are therefore variable. The schizont (Pl. II, ph. 7) is slighly oblong and measures about 15,6 fl/ioa!,. When the nuclear division is over, the cytoplasm surrounds each nucleus and the merozoites appear. At 54 hours, numerous mature ist generation schizonts can be observed. They measure 10 to 19 fl/15 to i8!, and contain, on an average, 16 ist generation merozoites measuring 4 to 5 {!1,5 to 2!, turning around a residual body generation schizonts. From 54 hours, a few ist generation merozoites released from the schizonts can be observed. They are mobile but they do not move much over the surface of the culture, invading the neighbouring cells to give 2nd generation trophozoites that develop into schizonts (Pl. II, ph. io). These schizonts are smaller than ist generation schizonts. They are usually spherical and measure about 12 [L/ io!,. When they are mature, after 68 hours, they contain 10 2nd generation merozoites on an average. There is no residual body (Pl. II, ph. II ) generation schixonts. The 2nd generation merozoites have the same size that the ist generation merozoites. They penetrate the cells and develop in them into 3rd generation schizonts about 19 [A/15 V. which, when mature, contain about 6 3rd generation merozoites larger than the ist or 2nd generation schizonts, 6 to 8 long. After 73 hours, these merozoites are released into the medium (Pl. II, ph. 12 ) generation schizonts. From 73 hours, we observe nests of 4th generation schizonts (Pl. III, ph. 13) that can reach very big sizes, 5i!,/20!. (Pl. III, ph. 14 ).Some schizonts have compartmented cytoplasm and can contain 4th generation merozoites 9fL to 10 fl long that will give gamogony. But it is not rare, even at II9 hours, to meet mature 4th generation schizonts. 2. Gamogorcy The inoculation of our cell cultures with fourth generation merozoites, recovered 89 hours after infestation of axenic chickens, enabled us to obtain the gamogony of E. aceruulina. These merozoites penetrate the cells rapidly and develop in them into trophozoites that develop into either male microgamonts or female macrogamonts Mic yogametogenesis. Until 21 hours, it is not possible to distinguish the trophozoite (Pl. II, ph. 10) that will give a microgametocyte, from the trophozoite that will develop into macrogametocyte. From 21 to 28 hours, the young microgametocytes are identified by the presence of a great number of small masses of nuclear material scattered in the cytoplasm. These masses, more or less spherical or ovoid, come from the intensive division of the nucleus of the trophozoite. As the microgametocyte matures, they become oblong and finally commashaped. Then they progressively separate from the central cytoplasmic mass and appear at the periphery (Pl. IV, ph. 17 ). From 28 to 45 hours, numerous mature microgamonts, of variable sizes, with sometimes indistinct walls, measuring about 18 V/12!t (Pl. IV, ph. 18), contain

6 DISCUSSION microgametes which are too numerous to be counted. These microgametes are released into the peripheral vacuole and then released into the cell. They measure 2 to 2,5 li, are mobile and move thanks to their flagella. The flagella are hardly visible under the optical microscope. The microgametes fecundate the macrogamonts that develop almost simultaneously in the neighbouring cells. 2. Macrogametogenesis. Unlike in microgametogenesis, the nucleus of the sporozite does not divide but on the contrary increases in breadth and, after staining with MayGrunwald Giemsa, appears like a large light patch with a very dark slightly eccentric nucleole (Pl. IV, ph. 19). The young macrogametocyte is often eggshaped. It measures 13!,!7,8!, at 21 hours and contains a fine granulous cytoplasm. Then, as it grows old, the nucleus becomes spherical and its volume decreases whereas the nucleole becomes bigger and sometimes spreads over the whole nuclear space. After 28 hours, we can observe old macrogametes whose cytoplasm is full of big plastinoid granules that appear like vacuoles at the periphery (Pl. IV, ph. 20 ). At this stage, they can be fecundated. After the fecundation, a zygote appears. The nucleus of the macrogamete becomes less clear and disappears. The cytoplasmic inclusions gather at the periphery and help to the forming of the shell of the oocyst. The cytoplasm retracts and becomes spherical, forming a cytoplasmic mass which is still visible through the thin and transparent shell of the young oocyst. Then this shell becomes thicker and opaque and it can no longer be stained with MayGrunwaldGiemsa (PI. IV, ph. 2z), At about 45 hours, the cell release the oocyst thus formed into the medium. It measures about 16 to 18!./r3 to 15 [1.. IV. AND CONCLUSION If we compare the cycle obtained in cell cultures to the normal development of E. acervulina in the chicken (V! r r!xr.mtg and DORAN, 19 66), we note they are very similar. Indeed, the four schizogonies occur in the same lengths of time as in vivo. The schizonts are usually larger in vitro but, on an average, they contain the same number of merozoites, except for the 4th generation schizonts which are much bigger and produce a hundred merozoites instead of 32. Gamogony also takes place in 40 hours. The gamonts and the oocysts obtained have the same size that those met in vivo. After 45 hours, no parasite can be seen in our cells. It is therefore possible to reproduce the cycle of E. ace yvulina in cell cultures. It is not easy to recognize the different stages ; an important number of schizogonies (4 generations) appear indeed in a little more than 48 hours (from 40 hours after inoculation to 95 hours). Moreover, it is not rare to meet at the same time young 1st generation schizonts with their refringent globules and 3rd generation schizonts. In vitro, we also find these different generations present at the same time, just as they can be found in the animal. This contrasts with the development of E. te!aella. Each generation of E. tenella occurs indeed slower and is more distinct.

7 . REPRODUCTION The conditions of culture and the medium have a great effect on the development of the parasite in the cells. In an EHT medium, in a CO, enriched atmosphere, B type trophozoites can be observed (Doxnrr and VE TTERLING, 19 68) (Pl. III and IV). They develop in a normal way into schizonts which, once mature, keep their cytoplasmic outgrowth (Pl. I, ph. 6). The first generation schizonts produced by these B type schizonts are identical to those produced by the normal schizonts (A type). Yet, no B type schizont can be found in cultures made in EHT + Hepes (1). Besides, in trials carried out with EagleTris f io p. 100 calf serum we could not obtain a development. Yet, when we used foetal calf serum we could observe a few ist generation schizonts. Their development was delayed and the schizonts did not reach maturity. This shows the importance of the culture medium and explains perhaps why other authors failed to obtain development. Reçu pour pubdication en mai RÉSUMÉ DU CYCLE DE LA COCCIDIE ElME Rf A ACERVULINA (TYZZER, 1929) SUR CULTURES DE CELLULES DE REIN DE POULET Le cycle d Eimeria acervulina est réalisé sur culture primaire de cellules de rein de poulet de trois semaines. Le milieu qui nous a permis d obtenir le développement de cette coccidie est le milieu EHT (Eagle, Hydrolysat de lactalbumine, BactoTryptose phosphate). Après infestation des cellules (temps o) avec des sporozoïtes, les premiers schizontes apparaissent vers 44 heures. Les mérozoïtes de re génération sont libérés vers 54 heures. Ils envahissent les cellules voisines et s y développent, donnant des schizontes de 2e génération mûrs vers 68 heures. A partir de 72 heures et 93 heures se déroulent respectivement une 3e et une 4e génération de schizontes. Les mérozoïtes IV donnent naissance à la Gamogonie. Pour obtenir celleci in vitro, nous avons inoculé les cultures avec des mérozoïtes IV récupérés sur animaux axéniques. Les oocystes sont libérés dans le milieu 45 heures après l inoculation. RÉFÉRENCES BIBLIOGRAPHIQUES BorrTEMrs M., YVORE P., Technique de purification de suspensions de sporozoites d Eimeria sur colonne de fibres synthétiques. Ann. Rech, vét., 5, rogrr 3. DORAN D. J., VETTERLING S. M., Survival and development of Eimeria meleagrimitis Tyzzer 1929 in bovine kidney and turkey intestine ceh cultures. J. Protozool., 15, 796c8 0z. IT AGAK K., UIRAY AMA N., TSUBOK URA M., OTSUKI K., Development of Eimeria tenella E. brunetti and E. acervulina in cell cultures. Jap. J. Vet. Sci., 36, LONG P L., The growth of some species of 1neria Ei in avian embryos. J, I. Parasitol., 56, LONG P L., Studies on the relationship between E. acervulina and E. mivati. Parasitology, 67, SxIaAI,ovA T. A., Koxol,nv A. M., SOBCHAK 1. A., Cultivation of chicken coccidia in chick embryos (in Russian). l eterinariya, Moskva, 11, 68 7,. SxI SA r.ova T. A., rg7o. Cultivation of the endogenous stages of chicken coccidia in embryos and tissue cultures. J. Parasitol.,56 (4, sect. II, part. I), 3153i6! ) Acide [(hydroxy2 4piperaziiiyl i éthyl) j 2 éthane sulfonique C8 H&dquo; N20!S P.M. : 23 8, 30. Thus, the use of C02 is not necessary

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