(Spelerpes fuscus) B. BABUDIERI. Received for publication 29 February after treatment of the preparation with deoxyribonuclease

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INFECTiON AND IMMUNITY, JUly 1972, p. 77-82 Copyright 1972 American Society for Microbiology Vol. 6, No. 1 Printed in U.S.A. Mycoplasma-Like Organism, Parasite of Red Blood Cells of an Amphibian, Hydromantes italicus (Spelerpes fuscus) B.

1 INFECTiON AND IMMUNITY, JUly 1972, p Copyright 1972 American Society for Microbiology Vol. 6, No. 1 Printed in U.S.A. Mycoplasma-Like Organism, Parasite of Red Blood Cells of an Amphibian, Hydromantes italicus (Spelerpes fuscus) B. BABUDIERI Laboratori di Microbiologia, Istituto Superiore di Saniti, Roma, Italia Received for publication 29 February 1972 In the present investigation we studied a parasite of red blood cells in the amphibian Hydromantes italicus. The parasite, a globular microorganism having a diameter of 0.25 to 0.3 Am, was found in large numbers in vacuoles of red blood cells. As seen by electron microscopy, it possesses no cell wall and has the morphological appearance of a mycoplasma. The parasite has great motility by mechanisms not yet elucidated. It is possible to transmit the infection experimentally from infected to healthy amphibians. Culture of the microorganism, named Chondrostagon haematicum, in artificial media has not yet been successful. Over 40 years ago, in 1930 and 1931, we described some globular inclusions containing a large quantity of minute granules in the protoplasm of blood cells of some specimens of Hydromantes (1, 2). We named this possible parasite Chondrostagon haematicum and we thought it to be akin to the Chlamidozoa. At that time we did not take into consideration any possible relationship with the mycoplasmas, because the morphological characteristics of this group of microorganisms had been described only shortly before. After such a long time, we thought it interesting to resume the study of the parasite, employing modern methods and techniques and, particularly, electron microscopy. MATERIALS AND METHODS The Hydromantes employed in the study of the parasite came from small, damp grottos of the Ligurian Appennines, near Genoa, and of the Tuscan Appennines, not far from Florence. The percentage of infected animals differed in the various batches, from 10 to 60%. The Hydromantes were maintained in small enclosures, containing damp moss, and kept in a cool and moderately lighted place. The blood samples were drawn either by cutting the tip of the tail, or directly from the heart; in the latter case, the animals were killed. The blood was examined either at once, directly, or after treatment with vital dyes, in dark or bright field, or after fixation of smears and staining with various histological stains. For the detection of nucleic acids we used both Feulgen's method and staining with acridine orange, after treatment of the preparation with deoxyribonuclease or ribonuclease. For observation by electron microscopy we used either the negative staining technique with phosphotungstic acid or ultrathin sections of samples embedded in araldite; these sections were examined directly or after treatment with uranyl acetate and after staining by the method of Karnovsky (3). In some cases, the samples were not fixed; in other cases they were fixed with osmic acid or with potassium permanganate. We tried to culture the parasite at 37 C and at room temperature in the most common liquid or solid culture media, and also in those more specifically suggested for the mycoplasmas (PPLO medium, Difco), substituting also the special serum fraction with amphibian serum (frog, toad). RESULTS Morphology by light microscopy. The parasite inclusions can have two different morphological appearances. In some animals only a small number of red blood cells contained the inclusions (chronic infection?) (Fig. 1). In these cases the parasite was large (5-16,m), and only one or a few (2-5) of them could be found in one blood cell (Fig. 2). When in contact with one another they did not coalesce. In other animals, however, nearly all of the blood cells were infected, and the inclusions, which had a relatively small diameter (1-6 um), were very numerous (acute infection?) (Fig. 3). In these cases also, if the inclusions came in contact with one another, they did not coalesce. Beside these inclusions, some of the infected 77

78 BABUDIERI INFECT. IMMUNITY FIG. 1. Blood smear ofan infected Hydromantes italicus (methanol, Giemsa staining). FIG. 2. Infected red blood cells (methanol, Giemsa staining). J,OOOX.

We tried to establish whether the parasites could be found in smears and in sections of various organs of the amphibian.

2 78 BABUDIERI INFECT. IMMUNITY FIG. 1. Blood smear ofan infected Hydromantes italicus (methanol, Giemsa staining). FIG. 2. Infected red blood cells (methanol, Giemsa staining). J,OOOX. red blood cells showed another type of smaller inclusions, of 1.5 to 0.5,um in diameter, generally clustered in small groups of four or five. We tried to establish whether the parasites could be found in smears and in sections of various organs of the amphibian. However, we found them only in the splenic macrophages of Hydromantes with acute infection. In this case, a great number of parasites was found in the protoplasm of these cells; as usual, the parasites were enclosed in round, small vacuoles, similar to those present in red blood cells. However, when stained with Giemsa they took up color to a lesser extent. When the red blood cells were examined immediately after sampling, under bright or, even better, under dark field, it was possible to observe an enormous number of small, refractile granules ( ,um) within the inclusions; these granules were in constant movement,

VOL. 6, 1972 PARASITE OF AMPHIBIAN RED BLOOD CELLS 79 FIG. 3. Infected red blood cells (methanol, Giemsa staining). J,OOOX. giving the inclusion the appearance of boiling water.

3 VOL. 6, 1972 PARASITE OF AMPHIBIAN RED BLOOD CELLS 79 FIG. 3. Infected red blood cells (methanol, Giemsa staining). J,OOOX. giving the inclusion the appearance of boiling water. Among these minute granules, it was possible to detect few granules of larger size (up to 1,um). The lively motility of the granules could be stopped by adding to the live preparation even trace amounts of mercury bichloride, ethyl alcohol, ether, copper sulfate, sodium taurocholate, or saponin, or by contact with osmic acid vapors. Granules were not visible in the small inclusions which clustered in groups; these inclusions had a homogeneous appearance. In live preparations, the granules took up methylene blue, however, they were not stained by neutral red when they were in motion, but they were stained by this dye as soon as their movement stopped. When smears were fixed in methanol and stained with Giemsa, the inclusions maintained their granular appearance and became light violet. At times, as a consequence of fixation, the inclusions of smaller size assumed a circular appearance, with an empty core. The smaller inclusions clustered in groups were stained homogeneously and with greater intensity than the others. The inclusions were feulgen-positive, and if they were observed by fluorescent microscopy after treatment with ribonuclease or deoxyribonuclease and staining with acridine orange, they showed the presence of ribonucleic and deoxyribonucleic acid (RNA, DNA). Morphology by electron microscopy. The electron microscopic examination of ultrathin sections of infected red blood cells revealed two different morphological appearances of the parasite. The inclusions of large and medium size and a part of those having small diameter contained a great number of round or elliptical organisms of practically equal size (Fig. 4). Their diameter was 250 to 300 nm. The organisms were enclosed by a very evident cytoplasmic membrane, which in some cases appeared three-layered. The outer and inner layers were opaque, and the middle one was transparent to the electron beam. The thickness of the sheet was about 10 nm (Fig. 5). On the outer surface the sheet had a crown of minute "pins." The central part of each microorganism had a fine and irregular reticular structure, very similar to that of the "nuclear" zone of the schizomycetes. The central zone was surrounded by the opaque protoplasm, sometimes irregularly granular or reticular, with a tendency to cluster at the poles of the parasite. In the protoplasm we did not observe any formation resembling ribosomes. Furthermore, we could not observe any structure reminiscent of any phase of a reproductive cycle. The vacuoles containing the cluster of microorganisms were limited by a well defined "sheet," or membrane with no particular structure, which was probably of host origin. This "sheet" was very resistant, and even when the infected red blood cells were lysed with anti-hydromantes immune serum, the "sheet" was not affected,

: wse~~~~~~~~~~~~~~..i., Wi. N.i 9 4O,r.*'k <KS*7sj > FIG. 4. Vacuoles containing parasitic bodies in a red blood cell (stained withl uranyl acetate).

4 hj :.,... ^F...* * *,^i. v' <s. * t Z. *4i..8fiit Ur~ %W~~~~st4#.Vi*4 '-.. s s.@*.....,;."x.<y, 4, 4, *. S. ;Vsf,&, A ;w. *4?.ie- e 4 > /* > s $ 9*t S~~~~iA te@>^e eegt ^ * ^ X * t * i0 ; 'ds4}i <d*x * < 1 t X t ' * i.: wse~~~~~~~~~~~~~~..i., Wi. N.i 9 4O,r.*'k <KS*7sj > FIG. 4. Vacuoles containing parasitic bodies in a red blood cell (stained withl uranyl acetate). 9,300X. -'s;~'-.1- AN- :,Fy : FIG. 5. Vacuole containing parasitic bodies in a red blood cell (Karnovsky staining). 31,OOOX. 80

5 VOL. 6, 1972 PARASITE OF AMPHIBIAN RED BLOOD CELLS 8I and it was possible to obtain free saccules, filled with parasites. The content of some small vacuoles, especially those existing in experimentally infected Hydromantes, and others probably corresponding to the small inclusions clustered in groups, was somewhat different. In this case, the vacuoles did not contain the globular organisms previously mentioned, but one or a few large parasites (up to 1 or 2,um) having an extremely polymorphous appearance (Fig. 6). They could have globular or rod-like shape, and were sometimes constricted as if they were going to divide. Aside from the size and the irregularity of shape, their inner structure corresponded to that of the organisms mentioned above. In these cases, the sheet bordering the vacuole was only partially visible, and sometimes the parasites were included in the protoplasm of red blood cells and not surrounded by a vacuole. Some of the parasites extracted from the infected red blood cells by means of a short ultrasonic treatment had the appearance of long, repeatedly elbow-bent filaments. Similar morphology is relatively common in mycoplasmas. Aftempts of cultivation. We repeatedly tried to culture the parasite. Infected blood cells, both intact or lysed with anti-hydromantes serum or disrupted by ultrasonic treatment were seeded in the most common solid and liquid microbiology media, and also those most specifically used for mycoplasmas cultivation were tried (PPLO agar, Difco, with the addition of either the Difco serum fraction or amphibian serum: frog, toad). They were incubated at 37 C and at room temperature. The results of these tests, however, were constantly negative. The inclusions and the granular corpuscles remained in good condition even for a few weeks. but it was impossible to observe a proliferation of the parasite or the formation of colonies. Experimental infection. Finally, we tried to transmit the infection experimentally from infected to healthy Hydromantes. For this purpose, we inoculated a small quantity of heavily parasitized red blood cells into the coelomic cavity of five healthy Hydromantes. The number of Hydromantes employed for these tests was small, because these animals are rare and their capture is difficult. FIG. 6. Large anid polymorphic bodies contained in a red blood cell (uranyl acetate staining). 46,000X.

6 82 BABUDIERI After 4 to 5 months, we observed a certain number of small typical inclusions in the red blood cells of the inoculated amphibians. In the following days, the inclusions increased in number and size. Four Hydromantes were not inoculated with the parasite, but were kept in a medium in contact with other infected animals, and two out of the four animals became infected. On the other hand, we were not able to infect frogs, tree-frogs, toads, newts, or salamanders by inoculation of infected blood or oral administration of fragments of tissues, or feces from infected Hydromantes. DISCUSSION The inclusions found in red blood cells of some Hydromantes are of parasitic and not of degenerative nature, as demonstrated by the presence of DNA and RNA in the inclusions, by the experimental transmission of infection to other animals, and by the structure of the contents of the vacuoles, studied by electron microscopy. With this instrument it has been possible to identify the "granules" as a parasitic agent. In fact, these granules are tiny microorganisms, having no cell wall and a structure similar to that of mycoplasma. A characteristic feature of mycoplasmas is also the giant or polymorphous form we previously described in some small INFECT. IMMUNITY inclusions, and the filament-like shape of the parasite. However, we prefer to characterize this parasite as a "mycoplasma-like organism" rather than as a mycoplasma, because we were not able to culture the microorganism on solid media to obtain the "fried egg" colonies peculiar to the genus Mycoplasma. Also, other species of Mycoplasma have proved very difficult to culture in artificial media. At any rate, the capacity of the parasite to invade red blood cells and to multiply in them and its property of forming large clusters in vacuoles defined by a sheet may justify, in our opinion, the assignment of the parasite to an autonomous genus, i.e. Chondrostagon, which we proposed 40 years ago. ACKNOWLEDGMENTS We thank very much F. Tangucci, of the Laboratories of Physics of the Istituto Superiore di Sanits, for the preparation of the samples for electron microscopy, and Prof. Lanza, Director of the Institute of Zoology of the University of Florence, who kindly supplied us with some of the animals used in this study. LITERATURE CITED 1. Babudieri, B Su di un parassita degli eritrociti dello Spelerpes. Monit. Zool. Ital. 41: Babudieri, B Chondrostagoni haema:icum n. g., n. sp. nuovo enigmatico emoparassita di Spelerpes fuscus. Arch. Zool. Ital. 97: Karnovsky, M. J Simple methods for "staining with lead" at high ph in electron microscopy. J. Biophys. Biochem. Cytol. 11:729.

GROWTH OF CELLULAR FORMS IN CULTURES OF CHROMIATIN BODIES

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