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Memórias do Instituto Oswaldo Cruz, Vol. 104, No. 2, March, 2009, pp. On a new protozoan in gundis (Toxoplasma N. Gen)+ Messrs C Nicolle and L Manceaux Code Number : oc09023 This study is based on the examination of three naturally infected gundis and five animals of the same species in which infection was experimentally induced. The most important points were published in two reports submitted by ourselves to the Academy of Sciences (Cf. Comptes rendus, 26 October 1908 and 8 February 1909). I. Natural infection To date, the infection has only be encountered in one species in Tunisia: the gundi (Ctenodactylus gondi), a rodent belonging to the Octodontidae family, which is very interesting due to its similarities with the guinea pig and is commonly found in the southern part of Africa Minor. In this same species, one of us has already reported two blood infections: piroplasmosis due to P. quadrigeminum and spirillosis due to Sp. gondi. Piroplasmosis has been observed almost constantly in animals captured in various parts of Southern Tunisia: Matmata, Djerid, the Gafsa region. It does not cause any symptoms and does not appear to lead to any clearly noticeable lesions. Its pathogenic agent, P. quadrigeminum, is characterized by its multiplication mode (quadripartition) and by the frequent presence, outside the nucleus, of a second chromatic body (centrosome?). It appears to constitute an intermediate between piroplasma and Leishmania. For this reason, Nuttal thought he had to create a new genus for it: g. Nicollia. This seems premature to us. To designate this parasite, we will retain the name of P. quadrigeminum, which was the name one of us used to make it known. The three gundis in which we observed the new infection that is the subject of this study came from Matmata (Southern Tunisia). One had been captured in May 1907 and another in July 1908 and the third was part of a batch of 45 gundis captured in November of the same year. It is the last animal that was used for the experimental research; the other two animals had been dead for a few hours at the time of our examination. Gross lesions - We do not know anything about the symptoms of the natural infection; our observations of the experimentally-induced disease appear to indicate that it is likely to lead to death in a number of cases. The lesions consist primarily of hypertrophy, often very marked, of the spleen and noticeable hypertrophy of the liver. In healthy gundis, the spleen weighs 0.8 g on average and the liver weighs 10 to 11 g for a 250 to 300 g animal. The weight of the spleen in gundi 3 reached 5 g with dimensions of 5.5 x 3.3 x 0.45 and the organ was very brittle; the liver weighed 16 g, but had normal characteristics. No other lesions were observed in this animal. In gundi 1, considerable hypertrophy of the spleen was also observed (the weight was not recorded) and also congestion of the lungs and slight pleural effusion. Gundi 2 demonstrated a slight increase in spleen volume (weight not recorded) as the only lesion. Gundi 1 presented chronic piroplasma infection observed several times throughout its lifetime (5, 11, 12 and 15 June), but which was not detectable at its death (28 June) despite repeated tests on blood taken from the heart, liver and spleen. Gundis 2 and 3 did not present piroplasma during their lifetimes or at autopsy. Parasite morphology. Distribution in various organs We found the parasite in large numbers in the three naturally infected animals in the spleen, liver and mesenteric lymph nodes; in lower numbers, although still relatively frequent, in the lungs and kidneys; in rare or exceptional cases in heart blood and bone marrow. We will use spleen smears as the standard for our morphological description. In all cases, this is the most significantly affected organ. Spleen smears - The parasites are found both free and incorporated into cells or cell debris; the latter (matrices) are of the same type as the elements described under this name by Messrs. Laveran and Mesnil in the organs of patients suffering from Kala-Azar disease (visceral Leishmaniasis) and for which we have demonstrated a leukocytic origin. Sometimes the free forms predominate and sometimes the incorporated forms. They are both extremely abundant generally. The parasitized cells are either mononuclear leukocytes of all sizes, the biggest of which can host up to forty protozoa and the smallest of which have the characteristics of lymphocytes, or - in much rarer cases - genuine polynuclear leukocytes. The appearance of the large mononuclear leukocytes filled with parasites is exactly reminiscent of that of elements of the same type carrying Leishmania in Kala-Azar disease and Oriental sore. We have never encountered any parasites in red blood cells. The matrices contain variable numbers of parasites. At first sight, when the matrix has taken on a spherical shape by coiling up in the blood and when it contains relatively evenly arranged parasites (these parasites are generally grouped together in pairs), the element could be mistaken for a merozoite cyst, and we believe that a similar arrangement misled Splendore, then Mesnil with respect to the method of reproduction of the parasite, which is very similar to ours, discovered by the former of these authors in rabbits. We will return to this point at the end of this article. The parasites are variable in shape; sometimes they are round, sometimes - and more usually - they are oblong. The typical shape in circulating form is that of a crescent, in which one of the ends may be more tapered than the other. Examined in fresh state, this protozoan appears to be immobile. In circulating forms, it measures 5 to 5.5μm by 3 to 4 μm, on average. The intracellular forms are always a little smaller. However, there are some large individuals, that can reach 5 by 7 μm ; but these are extreme dimensions On preparations stained with Giemsa, the structure of the protozoan is shown. It is an extremely simple structure since all that can be seen is a nucleus and a protoplasm; there are no flagella or centrosomes. The nucleus is a genuine nucleus that is oval or round, located in the protoplasm, at a variable point, but which almost always borders the centre of the element; it is composed of a relatively loose chromatic network, the arrangement of which varies; it measure 2 to 3 μm. The protoplasm presents a honeycomb structure. There is no centrosome. The exceptional figures attributed to this body in our first description (Comptes rendus, 26 August 1908) relate to the early stages of division of the nucleus. The multiplication forms are extremely common. Any individual that loses its crescent appearance to become oval or round already demonstrates the beginnings of nucleus segmentation. Division is by bipartition. In the protoplasm of infected cells, the parasites are generally arranged in two or in pairs; the same is true, as we have already said, in matrices produced by rupture of the cell protoplasm. Liver smears - Always numerous parasites, but fewer than in the spleen; free, intracellular or in matrices. Up to twenty per cell can be counted. The parasitised elements are generally mononuclear leukocytes; however polynuclear leukocytes containing a few microorganisms are also encountered, and not only in exceptional cases. However, there is a constant absence of parasites in the hepatic cells and red blood cells. Frequent division figures. Mesenteric lymph nodes - Very numerous parasites. Nothing of note. Kidneys and lungs - Relatively numerous parasites. Nothing of note. Bone marrow. - Few parasites. Cardiac blood - Rare parasites, almost always intracellular or contained in matrices. II. Experimentally-induced infection of gundi Using the spleen of the third naturally infected gundi, a few hours after death, we inoculated via the peritoneal cavity five rodents of the same species and various other animals that we will discuss later. The five gundis contracted the infection and died as a result of it after seven to thirteen days. On autopsy, we observed similar lesions: abundant peritoneal effusion with false membranes on the liver, spleen and intestines. Hypertrophic (4 to 5 g) and brittle spleen; enlarged liver. Same locations and same forms of the parasite as in natural infection. The peritoneal fluid demonstrates numerousmacrophages and polynuclear leukocytes filled with protozoa; in two animals, there is coexisting streptococcal infection. One gundi (46) also presented an acute episode of piroplasmosis, probably caused by inoculation. Below are the details of observation of these gundis. They were inoculated on 10 December 1908. Gundi 46 - Found dying on 17 December; sacrificed on the same day, at 4 p.m. Autopsy - Abundant peritoneal effusion with perihepatitis and a few intestinal adhesions; hypertrophic, brittle spleen, weighing 4 g; the mesenteric lymph nodes are enlarged. The liver, spleen, mesenteric lymph nodes and peritoneal fluid contain very numerous parasites, with their usual characteristics; in the lymph nodes, many polynuclear leukocytes are infected. Examination of the blood also reveals very intense piroplasmosis. Gundi 47 - Found dying on 18 December; sacrificed on the same day. Autopsy - Very abundant peritoneal effusion with false membranes, perihepatitis, persplenitis, a few intestinal adhesions. Hypertrophic, slightly soft spleen. Parasite locations same as for gundi 46. Gundi 49 - Found in a state of torpor on 12 December, with convulsive agitation when turned over and unable to find its balance. Sacrificed on the same day. Autopsy - Very enlarged inguinal lymph nodes. Intense peritonitis; perihepatitis, congested liver. Very hypertrophic (4 g), brittle spleen; slight congestion of the meninges without ventricular hydrops. The peritoneal fluid contains numerous polynuclear and mononuclear leukocytes, filled with parasites, along with free individuals. Numerous free or incorporated streptococci. Blood - No piroplasma; a few leukocytes (mononuclear and polynuclear) containing parasites; red blood cells unaffected. Liver - Numerous parasites and streptococci. Spleen - Same observations: some cells contain up to thirty parasites. Brain substance - Absence of parasites. Gundi 50 - Died on 21 December. Autopsy - Congested zone at the inoculation site. Inguinal and mesenteric lymph nodes very obvious; little ascitis. Enlarged (5 g), brittle and congested spleen; congested liver (12 g); slight pleural effusion. Numerous parasites in the liver, spleen, lymph nodes and peritoneal fluid; rare in the blood. No piroplasma. Gundi 51 - Died on 22 December. Autopsy - Peritoneal effusion containing white and red blood cells, streptococci. Congested, brittle spleen weighing 5 g, congested liver. No pleural effusion. Numerous parasites in the spleen, liver, lymph nodes and peritoneal fluid; rare and intracellular in the blood. III. Experimental inoculation of laboratory animals These inoculations were conducted with the virus1 (spleen and peritoneal fluid) from gundi 3, which was naturally infected, and from gundis 46, 47 and 51, in which infection was experimentally induced. 1. Monkeys - Two macaque monkeys (M. cynomolgus) were inoculated, one under the skin and the other in the peritoneal cavity, with virus from gundi 3. Nil results. 2. Guinea pigs - Twelve guinea pigs were inoculated in the peritoneal cavity or directly into the hepatic tissue: four with the virus from gundi 3, three with the virus from gundi 46, two with the virus from gundi 47 and three with the virus from gundi 51. Only one contracted a mild infection which could not then be transmitted serially to other guinea pigs. (Three guinea pigs inoculated with no result.) Below are the observations made for this animal: Guinea pig 12 - inoculated in the liver with virus from gundi 51; died on the fourth day after inoculation: hypertrophic spleen, liver and mesenteric lymph nodes, peritonitis with effusion. Numerous parasites, generally circulating freely in the spleen, liver and peritoneal fluid; in exceptional cases, up to ten, twenty or thirty protozoa can be counted in the mononuclear leukocytes. There are numerous division forms. 3. Rats - Three white rats were unsuccessfully inoculated in the peritoneal cavity with virus from gundi 3. IV. Culture tests Cultures were attempted several times, without results, using virus from various infected gundis on ordinary or simplified Novy-MacNeal medium (NNN medium). V. Nature of the parasite The parasites that we have just described cannot be considered to be forms of P. quadrigeminum. The coexistence of piroplasmosis was only observed in one of our two gundis. Furthermore, 70 other gundis infected with P. quadrigeminum did not demonstrate similar forms. Finally, the following characteristics can be used to differentiate between the two parasites: Dimensions, 2 μm on average for P.; 5 μm to 5.5 μm by 2.5μm to 4 μm for the other protozoan. Young forms of P. have a maximum diameter of 1 μm; for our microorganism, we did not find any forms measuring less than by 4 μm by 2.5μm. Nucleus: The nucleus of P. is vesicular with a constant, compact karyosome that is part of the parasite’s contour, and a small inconstant karyosome. The nucleus of our protozoan is a genuine, round nucleus, located in the protoplasm, which is not part of the cell contour and is composed of a non-compact chromatic substance, but which is arranged in a network. - Centrosome. Always absent in our parasite; seems to exist sometimes in P. - Division mode: quadripartition is the usual mode for P., bipartition for the other microorganism. - Habitat: P. is a parasite of red blood cells: the new protozoan is a parasite of mononuclear white blood cells. There are free forms in both cases but these are always very rare for P.; in contrast, they can be very common for our protozoan. P. is a parasite of the blood, while the other is a parasite of certain organs (spleen, liver, lymph nodes). Similar reasons distinguish our new parasite from other piroplasma. It also differs from Leishmania, which it still resembles morphologically and with which it also has similarities in terms of location and abundance in white blood cells, the absence of centrosomes and its inability to grow on Novy and Neal medium, and it also differs from haemogregarina through its division mode and inoculability. We believe that it belongs to a genus not yet described, more distant from trypanosomes than Leishmania and we propose giving this new genus the name of Toxoplasma. The name of the gundi parasite will therefore be T. gondii. Close to this parasite and belonging to the same genus is another protozoan discovered in rabbits in Brazil by Mr. Splendore. This parasite, which we were able to study on preparations kindly sent to us by our colleague, is morphologically identical to the protozoan that we have just described. This similarity leads us to regret not having attempted to inoculate rabbits with virus from our gundis; the close relationship between gundis and guinea pigs led us to preferentially select guinea pigs over other animals as the animal for our experiments. We will definitely attempt this inoculation in rabbits whenever we are lucky enough to find a gundi suffering from natural toxoplasmosis again. We mentioned above that, according to Splendore and Mesnil (loc. cit.), Toxoplasma from rabbits may have, in addition to bipartition, a multiplication mode in the form of cyst with merozoites. We studied this theory, with examination of our preparations and those of Splendore, and only revealed leukocyte debris or matrices in these claimed cysts, with a regular arrangement of these incorporated parasites.
1: Translation note: The term “virus” was commonly used in the beginning of the XXth century to designate any transmissible agent. Here it should be understood as “parasite”. Copyright 2009 - Instituto Oswaldo Cruz - Fiocruz |
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