(1976), 72, 225-243With 6 plates and 8 figures in the text
Schellackia landauae sp.nov.(Eimeriorina: Lankesterellidae) in the Brazilian lizard Polychrus marmoratus (Iguanidae): experimental transmission by Culex pipiens fatigans
LAINSON, J. J. SHA W and R. D. W ARD
The WellcomeBelém, Parasitology Pará State, Unit, Brazil Caixa Postal ' 3, I
Received 19 September1975)
SUMMARY A new haemogregarine, Schellackia landauae sp.nov., is described in the Brazilian lizard Polychrus marmoratus (Linn) from Pará State, north Brazil. Sporozoites are found principally in the red blood cells (840;0) in the peripheral blood but algo occur in lymphocytes and monocytes. Experimental transmission to three uninfected P. marmoratus was achieved after feeding them with laboratory-bred Oulex pipiens fatigans which had engorged on an infected lizard 14 days previously. The cycle of development in the smáll intestine of P. marmoratus takes approximately 30 days: schizogony, gametogony and fertilization of the macro- gametocytes is in the epithelial cells of the gut, with zygotes penetrating the lamina propria, where the mature oocysts develop. Living oocysts average 14-3 x 13.3 /lm, and are approximately 10-0/lm as seen in histological sections. During the period of intestinal development, the parasite algo undergoes asexual multiplication within cells of the spleen and liver by a process which appears to be endodyogeny. The exact time required before the first invasion of the peripheral blood by the sporozoites remains to be ascertained, but is some time within 30-45 days after the lizard ingests the infected mosquitoes. Morphology of the sporozoite in the vertebrate host is variable and depends on the host cell occupied; there may be 1 or 2 refractile bodies. Reduction or fusion of these to a single refractile body in those sporozoites within the gut cells of the infected mosquito suggests that the bodies may represent some form of energy source that is used up during this latent phase in the insect vector. Accumulation of sporozoites takes place in the reticulo-endothelial cells of the viscera, in particular the pigment- laden cells of the liver and lung. Attempts to infect other species of lizards, Tropidurus torquatus (Iguanidae) and Ameiva a.meiva (Teiidae) failed. Other specics of Schellackia are comparcd with S. landauae. There is a gradation of predilection for the gut epithelium among the known species which supports the hypothesis of evolution of the 'higher coccidia' (Eimeria, Isospora, etc.) from a line originating from a faecally transmitted, octozoic parasite such as a Tyzzeria.
225R.Parasitolf!gY 226 LAINSON, J. J. SHAW AND R. D. WARD
INTRODUCTION Polychrus marmoratus (Linn) is a beautifully marked iguanid, commonly found in low trees and shrubs. Over the last 10 years we have examined blood films from 148 specimens, most from Capanéma, Pará, north Brazil, and 17 (11.5%) of these \Vere infected with the new species of Schellackia described below. It is named Schellackia landauae sp.nov., in honour of our good friend Irene Landau, who has contributed much to our better understanding of the haemogregarines.
MATERIALS AND METHODS Methods ofmaintenance and bleeding oflizards from the orbital sinus have been given elsewhere (Lainson, Landau & Shaw, 1971; Lainson, Shaw & Landau, 1975). We might add, here, that the insect diet of P. marmoratus was conveniently sup- plemented by hand-feeding with small pieces of freshly killed baby mice. Blo()d films were air-dried as rapidly as possible, fixed in absolute methanol and stained for 90 min in double-strength Giemsa stain (30 drops of stain to 15 ml buffered distilIed water, pH 7-4). Tissue smears of liver, spleen, lung, kid- ney, bone-marrow and brain were treated in the garoe manner or were fixed in aqueous .Bouin's fixative and stained by a modified Giemsa method (Lainson, 1959). Pieces of tissue were fixed in Oarnoy's fluid and histological sections were cut at 4 pm: these were stained by the Giemsa-colophonium method (Bray & Garnham, 1962) Drawings and photographs of the living parasites were made using phase- contrast ilIumination, and photomicrography employed the use of a Zeiss W.L. research microscope, using Adox KB 14 and 17 and Agfa Isopan IF, 35 mm films. Ali measurements are given in micrometres. Mosquitoes used in transmission experiments were from a local, laboratory-bred colony of Oulex pipiens fatigans. FolIowing the infective blood-meal they were maintained on a 50 % sucrose solution, at a temperature of from 24-26 °0 and a humidity of about 85-95 %.
Transmission experiments In March 1974 we obtained a P. marmoratus which was showing unusually abundant haemogregarines in the peripheral blood. Luckily we had, at the same time, three clean P. marmoratus, the blood of which had been consistently nega- tive during a period of about 3 months. Approximately 200 mosquitoes were en- gorged on the infected lizard and, 14 days later, batchesof 20 of these insects were hand-fed to each ofthe clean lizards. Blood films were taken from the experimental animaIs after 8, 15, 20, 25, 28, 29, 30 and 45 days. One of the P. marmoratus (L.2917) was killed and autopsied on the 23rd day after its meal of infected mosquitoes, another (L.2871) on day 30, and the last (L.2918) on day 45. In a separare experiment, similarly infected mosquitoes were fed to two other species of lizards -a Tropidurus torquatu~ (Iguanidae) and an Ameiva ameiva lTeiidae) . R. Figs. 1-5. Schellackia landauae sp.nov., from the lizard Polychrus marmoratus. Developing oocysts as seen in fresh scrapings from the small intestine of an experi- mentally infected lizard. Fif!;. 6. Four freed sporozoites from rttptured oocysts: note variable number of refractile bodies. Bouin-fixed, Giemsa-stained. 228 R.LAINSON,J.J.SHAW AND R.D.WARD
RESULTS P. marmoratus (L. 2917): 23-day-old infection No parasites could be found in smears of the liver, spleen, lung, kidney, bone- marrow or brain of the animal killed on the 23rd day. Smears and sections of the small intestine, however, showed enormous numbers of mature and developing schizonts and smaller numbers of immature microgametocytes and macro- gametocytes, alI within the epithelial cells of the gut: no mature oocysts wereseen.
As the lizard had never shown coccidial oocysts in its faeces during its captivity, we felt confident that these developmental stages were those of the haemogregarine and that we were probably dealing with a Schellackia species.
P. marmoratus (L. 2871): 30-day-old infection Smears and sections of the intestine showed an increased number of mature male and female gametocytes in the epithelial cells and, in addition, occasional mature oocysts in the sub-epithelial tissue: intestinal schizogony remained at a high leveI.. Impression smears of the spleen and liver showed a process of asexual division within the lymphocytes and monocytes, which we interpret as endodyogeny.
P. marmoratus (L. 2918): 45-day-old infection Unfortunately we were unable to take blood films from this lizard between day 30 (smears negative) and day 45, when it was killed and examined. On the 45th day, however, sporozoites were present in scanty numbers in the red and white cells of the peripheral blood and in smears of the viscera. Smears and sections of the intestine revealed a massive infection, mainly composed of developing and mature oocysts and innumerable free and intracellular sporozoites in the sub- epithelial tissues. Fresh preparations of scrapings from the gut wall provided large numbers of developing oocysts, typical of Schellackia, as illustrated in Figs. 1-5: from this experiment we could only deduce that the time required for the sporozoites to reach the peripheral blood was between 30 and 45 days.
Fig. 7. Diagrammatic representation of the life.cycle of Schellackia landauae sp.nov. (A) Sporozoite in epithelial cell of mosquito stomach, 14 days after infective blood- meal. (B) Sporozoites penetrare epithelial cells of small intestine of lizard, Polychrus marmoratus, after ingestion ofinfected mosquitoes. (C, D) Development ofmicroschi- zonts and micromerozoites. (E, F) Development of macroschizonts and macromero. zoites. (G) Asexual division in monocytes and lymphocytes of spleen and liver, apparently by endodyogeny, producing groups of tachyzoites, and probably iJlitiated by micromerozoites. (H, I) Development ofmicrogametes andfertilization of macrogametes in epithelial. cells of small intestine. (J, K) Entry of fertilized macrogamete into lamina propria and development of oocysts containing eight sporozoites. (L) Liberation of sporozoites from rupturing oocysts: entry of reticulo..endothelial cells and penetration of capillaries. (M, N) Entry of white and red cells of peripheral blood: note rolled-up form in erythrocyte. (O) Infective, diapausing sporozoites in the reticulo.endothelial cells of liver, lung and other viscera. Schellackia landauac sp.nov. 229
~
Fig. 7. For legend see opposite. 230 R.LAINSON,J.J.SHAW AND R.D.WARD
Tropidurus torquatus and Ameiva ameiva N"either of these lizards showed any evidence of infection when they were killed on the 45th day follo,ving their ingestion of the infected mosquitoes. The following description is based on material from both experimental and natural infections in P. marmoratu8. The life-cycle is shown diagrammatical1y in Fig.7. Schellackia landauae sp.nov. The sporozoites The morphology oí the sporozoitevaries considerably, dependingon whether or not it is within a host cell, and on the type oí host cell it eventually occupies. ln the intact oocyst, or freshly emerged(Fig. 4; PIso 5D, E; 6B) The living sporozoite is a slender, curved body measuring approximately 13.0 x 3.0, with one end broader and more acutely pointed than the other: when released from the oocyst it may undergo brief gliding movements. The nucleus of stained speci~ens is often band-form, made up of a small number of densely staining granules and usually placed centrally in the organism: it may sometimes, however, be located at either extrernity. The sporozoite of S. landauae may have either one or two refractile bodies. Of 100 stained examples selected at random in the present study, 70% had two, almost always of unequal size. The position of the refractile bodies in relation to the nucleus is variable -sometimes with one posterior and one anterior, or on other occasions with both together at one extremity. They stain a flat, bluish-grey colour, appear completely structureless, and may measure as much as 4.5 x 3.0 or as little as 1.0 x 1.0. The cytoplasm of the sporozoite stains poorly: it is finely vacuolated and sometimes shows a small number of randomly scattered azurophilic granules.
I n lhe u'hite cells o/ lhe peripheral blood (PI. 1 C) The oocysts of S. landauae are short-lived and soon rupture at their site of development in the sub-epithelial tissue of the lizard's intestine. Mter penetra- tion of the capillaries, a small number of the released sporozoites invade mono- cytes or lymphocytes (about 16%), in which cells they gain entry to the peripheral blood circulation. Within the white cells they quickly lose their elongated forro and assume a stumpy bean or sausage-shape: they now average only about 9.5 x 5.5, but in alI other respects their morphology remains the garoe.
I n lhe erythrocytesof lhe peripheral blood (PI. 1 A, B) Up to 840;0 of the sporozoitcs invading the blood enter the red blood cells and it is here that the most striking change in form takcs place. The sporozoite becomes curled up into an oval or spherical body which averages 6.5 x 5.5: it might indeed now be easily mistaken for the gametocyte of some non-pigmented hacmospori- dian, were it not for the conspieuous refractile bodies. Schellackialandauae sp.nov. The sporozoite almost always occupiesa polar or near-polar position in the erythrocyte. ln most casesthere is no visible space betweenit and the host-cell cytoplasmbut, when the parasiteis less tightly rolled up, a small colourlesscleft is sometimesvisible betweenits two ends (Pl. iR). The peripheral cytoplasm of the sporozoiteappears more dense,and usually showsnumerous reddish-staining granules which give the organisma cyst-like outline: no evidencewas ever seen, however, of any enclosingsheath about the sporozoite. AIthough the morphology of these sphericalforros in the erythrocytes is diffi- cult to make out, the parasites can be made to emergefrom the red blood cells ir blood is kept for some time at room temperature; and the blood forros may be almost entirely extracellular in blooa films prepared from lizards which have been dead for some hours. Outside the red blood cells the sporozoites regain their slender, elongate forro and are essentially similar to those from newly ruptured oocysts (Pl. i D).
In lhe viscera (PI. 6C, D). Rupture of the oocysts in the lamina propria results in heavy concentrations of freed sporozoites (PIs. 5F; 6A, B). As discussed above, some gain entrance to the peripheral blood after invading the red and white cells of neighbouring capil- laries: others are phagocytosed by macrophages, or actively penetrare them, and are thus transported to various organs of the body. Here they may be found within the reticulo-endothelial cells of the liver, spleen and lung: the pigment-Iaden cells of the liver and lung are those most frequently parasitized. Morphologically. these sporozoites. 'stock-piled' in the viscera, resemble those seen circulating in the white cells of the blood -short, stumpy forms about 9-5 x 5-5.
In the mosquito(PI. 1E) The sporozoitesremam quiescentwithin the epithelial celIs of the insect's mid- gut. The exact processof their entry is uncertain but, from their ready exit from the red bIood celIs describedabove, it seemsIikeIy that they quickly Ieave the ingestederythrocytes and actively penetratethe gut celIs of the invertebrate host. In form they are elongateand measureapproximateIy 13.0x 3.0. Interestingly, stained sporozoites,from mosquitoesinfected 14days previously, alI showedonIy a single refractile body, suggestingthat the refractile bodies representsome forro of energysource that is used up during this Iatent phasein the insect vector.
A sexualdivision Intestinal schizogony Events leading up to the intensive intestinal schizogony seen on the 23rd day of infection remain to be studied. At this time, however, two distinctly different types of schizonts are present. One (the microschizont) produces tiny merozoites, and the other (the macroschizont) gives rise to large merozoites which are twice thissize. Developing microschizonts can be differentiated at an early stage from young 231 232 R.LAINSON,J.J.SHAW AND R.D.WARD macroschizonts by their fine, more delicately stained cytoplasm which is devoid of conspicuous vacuoles: their nuclei are smaller and more compact, measuring about 2.0 x 1.5. Quite early on the nuclei become localized at the periphery of the parasite, where they eventually migrate into cytoplasmic protrusions which are pushed out from the surface (PIs. 1,G, H; 2A). The resulting merozoites even- tually are attached by only a fine stalk of cytoplasm, breaking away to leave a bulky spherical or irregularly shaped mass of residual cytoplasm. In some of the larger microschizonts the cytoplasmic mass may be thrown into a network of finger- like protrusions, from which the merozoites are budded off. This is very reminiscent of the type of schizont we have recently descriped for the saurian haemosporidian, Garnia multiformis (Lainson et ai. 1975). There is a great variation in the number of micromerozoites produced -from 10 to 50, and the size of the microschizont varies accordingly: in the Bouin-fixed smears they ranged from 10.0 x 10.0 to 25.0 x 15,0, but appeared somewhat smaller in sections. The individual micromerozoites average only 4.5 x 1.5: they are slightly crescentic, with one end more acutely pointed and less intensely stained than the other. The nucleus is very compact, stains densely, and is usually located centrally or slightly towards the more rounded end. Young, uninucleate macroschizonts are difficult to distinguish from growing macrogametocytes: they are more or less spherical, and may measure up to 11.0 x 9.0 before the first nuclear division is completed. The coarsely granular cytoplasm stains a deep, bright blue and shows a small number of vacuoles which have a sharply punched-out appearance. During subsequent division the large, diffuse nuclei may measure up to 3.5 in diameter and appear scattered at random in the macroschizont. Once again, the macroschizonts produce a very variable number of macromero- zoites but, unlike the microschizonts, there is little or no residual cytoplasm left at the end of the division processoSmaller parasites, measuring about 12.0 x 10,0, may give rise to only 6-10 macromerozoites (PI. 2E-G), hut others reach up to 30.0 x 25 in diameter and produce as many as 100 (PI. 2H, I). In the smaller macroschizonts the nuclei become arranged in a median line within the cytoplasm, so that subsequent longitudinal division leaves the macromerozoites lying free like the staves of a disruptedbarrel (PI. 2E-G). In Bouin-fixed smears the macromerozoites are gently curved bodies, averaging 11.0 x 2.5: one end is broader than the other and tapers to a point. There is a dis- tinctly different staining reaction in the cytoplasm oí the pointed extremity, which takes on a pinkish hue compared with the grey-blue cytoplasm of the rest of the macromerozoite. Small numbers of densely stained granules are scattered in the cytoplasm, particularly in the anterior two-thirds oí the organism, in front of the nucleus. The latter is compact, composed oí a small number of separate granules, and located centrally or slightly towards the more rounded end of the parasite. Schellackialandauae sp.nov. 233
Extra-intestinal division Spleenand liver smearsfrom the P. marmoratuswith the 30-day-old infection (L. 2871)showed a processofasexual division within lymphocytesand monocytes (PI. 3A-J). The great frequencyof paired organismsin thesecells, and particulàrly the division stagessuch as thoseillustrated in PI. 3C, led us to concludethat these stagesrepresent endodyogeny of S. landauae,although no such phase of division has hitherto been described in the life-cycle of Schellackiaor other members of the LankesterelIidae.AlI attempts to demonstrareother, concomitant protozoal infection to account for these extra-intestinal division forms failed. The stagesare clearly not sporozoites,for they measureonly 5.0 x 2.5, possessno refractile bodies and stain completelydifferently. A prolonged searchof smearsof the liver revealed a smalI intracelIular group of parasites, alI of which appearedto be undergoingdivision (PI. 3F). Three ex- amples of celIs containing 8, 10 and 16 organismswere found in the spleen. As no evidence could be found of undivided schizonts, it is concluded that these aggregationsderived from repeated endodyogeny(PI. 3G-I). Whether singly, in pairs, or in groups, th~ parasites always lie in a conspicuousvacuole within the cytoplasm of the host celI: there is no evidenceof any limiting cyst or membrane about them. The question arises as to the origin of the extra-intestinal phase of division. Its appearanceat the height of intestinal schizogony,before sporozoiteshave yet reachedthe blood or viscera,and the sizeand form of the undivided extra-intestinal parasites alI indicare that they are micromerozoitesderived from the intestinal schizogon)Tdescribed above. Their role in the life-cycle of S. landauaeremains undetermined,but they might possiblyserve as a cryptic sourceof parasiteswhich can periodicalIy invade the gut and initiate new phasesof the intestinal develop- ment. Gametogony Like the schizonts, both male and female gametocytes develop in the apical portion of the epithelial cells of the small intestine, above the nucleus. They are surroundedby a conspicuousvacuole in the host-cell cytoplasm.
The microgametocytesand microgametes(PIs. 3K, L; 4A-E; 5A). Early stages are distinguished from the young asexual stages by their delicately stained, fine cytoplasm and their small nuclei, which stain so densely that they have an almost refractile appearance. The nuclei are at first oval or fusiform in shape and soon assume a position at the periphery of the microgametocyte, where they later elongate into the typical 'comma' forro of the eimêriid microgamete. The mature or near-mature microgametocyte is most frequently oval in shape, its size depending on the number of microgametes produced. Thus, those giving rise to as few as ,.., 20 may measure only 11.0 in diameter, as seen in sections, whereas other producing ,.., 150 microgametes may reach up to 27.0 x 10.0. Shedding of the microgametes from the surface of the microgametocyte leaves a bulky cytopIasmic residuum (PI. 4E). As seen in impression smears, the freed 234 LAINSON, J. J. SHAW AND R. D. \VARD are about 10.0-11.0 long, appearing to be composed entirely of chromatin: they may maintain the comma-shape, but are often straightened out or twisted into other forms. Two flagella are visible in stained films, these being of equallength and about 10.0 long (PI. 40).
The macrogametocytes(PI. 4D; 5A-C) The young macrogametocyte is round to oval, with a highly vacuolated and delicately staining cytoplasm. Its nucleus is from 3.8 to 5.0 in diameter: it has a dense, centrally placed nucleolus surrounded by a faintly pink-staining nucleo- plasm, bounded by a distinct nuclear membrane. Apparently-mature female gametocytes reach up to 15.0 x 12.5, as seen in sections, and contain the usual, peripherally disposed 'wall- forming bodies' which are seen in other eimiorine coccidia: with growth the female gametocytes migrate towards the basal region of the epithelial cell, displacing and deforming the host-cell nucleus.
Fertilization The development and fertilization of the mature macrogametocyte is in the epithelial cell of th.e small intestine, and the zygote subsequently migrates into the lamina prupria where sporogony takes place (PIs. 4D; 5A-E). The infections studied were extremely heavy ones, but the only stages seen in the sub-epithelial tissues were developing or mature oocysts and ne:ver deveJoping macrogameto- cytes, microgametocytes or free microgametes. The fertilized female parasite pushes its way through the basal part of the epi- thelial cell, which is by now reduced to a mere wisp of cytoplasm. Sometimes the degenerating host-cell nucleus is left within the epithelial cell-remnant but, on other occasions, it may be pushed out with the zygote and can be seen adhering to its surface. By now, marked changes have taken place in the fertilized parasite. The nuclear membrane and nucleolus are no longer distinct, the nucleus appearing as a small cluster of reddish granules. The cytoplasm becomes full of vacuoles and there develop a number of pinkish-staining, structureless bodies which possibly represent the developing refractile bodies. A distinct wall now becomes visible about the parasite and the contents of the resulting oocyst are poorly stained and seen with difficulty. The ensuing process of sporogony can now be followed accurately only by the examination of fresh material in scrapings made from thé intestinal wall; although histological sections, do occasionally show the sporozoites and residual body of the mature oocyst (PI. 5D, E).
Sporogony In fresh preparations, the living oocyst of S. landauae is spherical or sub- spherical and averages 14.3 x 13.3; range 13.7 x 12-3 to 15.0 x 15-0, for 50 oocysts measured (Figs. 1-5). It has a delicate, smooth, colourless wall which is about 0'5thick and apparently composed of a single layer. There is no visible microp~'"le, and the undivided contents form a highl)'" vacuolated, spherical mass approximately 12.0 in diameter. At an early stage in sporogony, a large vacuole is formed in the cytoplasm and microgametesR. Schellackia landauae sp.nov. 235 cight sporozoites then arrear as finger-like processespushed out from the surface oí' the parasite. The Cate of the vacuole remains uncertain: possibly it is this structure which, by division, gives rise to the refractile body, or bodies, of each sporozoite. When fully formed the sporozoites remain for some time attached to a bulky, round, oval or irregularly shaped residuum: they then break away, to lie free within the oocyst. Few details can be made out in the living sporozoite, as seen by normallight or phase-contrast illumination. The oval, less-denseareas (Fig. 4) probably represent the refractile bodies rather than the nuclei. Living sporozoites measure approximately 13.0 x 3.0.
DEFINITION Schellackia landauae sp. novo Sporozoites in the red blood cells (84%) or lymphocytes and monocytes (16%) of the peripheral blood, with moderare accumulation within the reticulo-endo- thelial cells in the viscera, in particular the pigment-cells of the liver and lung. Sporozoites in erythrocytes rolled up on themselves into oval or spherical bodies measuring '" 6.5 x 5.5: extended, they measure '" 13.0 x 2.7. Forms within the white cells are shorter and broader, averaging 9.5 x 5.5. Oneor two refractile bo~ies present in the blood forms, these being variable in position in the sporozoite. Sporozoites in the infected mosquito host remaining quiescent within the epithelial cells of the midgut, when they latterly show only a single refractile body. Transmission to vertebrate host after the ingestion of the infected vector. Schizogony takes place in the apical portion of the epithelial cells of the small intestine of the lizard host. Microschizonts produce from 10 to 50 small micro- merozoites, which average only 4:5 x 1.5, and leave a prominent residual body: macroschizonts produce from 6 to 100 large macromerozoites, measuring about 11.0 x 2.5, and leave little or no residual cytoplasm. Extra-intestinal asexual division by endodyogeny in the lymphocytes and monocytes of the spleen and liver, producing small intracellular groups of up to 16 individuaIs measuring about 5.0 x 2.5. No production of a cyst wall about these aggregations. Microgametocytes and macrogametocytes developing in the apical portion of the gut epithelial cells. Microgametocyte producing from 20 to 150 microgametes which are about 10.0-11.0 ~ong and possesstwo ftagella approximately 10.0 longo Mature macrogametocytes subspherical, up to 15.0 x 12.5, and tending to migrate to the basal part of the host ccll. Fertilization is within the epithelial cells of the small intestine, with the zygotes rupturing into the sub-epithelial tissues to form the oocysts. Living ooeysts averaging 14.3 x 13.3, range 13.7 x 12.3 to 15.0 x 15-0, as seen in fresh gut-scrapings. Wall smooth, colourless, single-layered and 0-5 thick. No visible micropyle. Parasite budding off eight naked sporozoites, measuring 13-0 x 3.0, and leavin~ a bulky cytoplasmic residuum. Oocysts rapidly breaking dO\Vll to rcleasc sporozoitcs, which appear in the peripheral blood of the lizard bet\veen 30 and 45 da~'s following ingestion of the infected vector. 236 LAINSON, J. J. SHAW AND R. D. WARD
HOST. The lizard (Iguanidae) Polychru8 marmoratu8 (Linn). NATURALVECTOR. Unknown. EXPERIMENTALVECTOR. Culex pipiens fatigans. TYPE LOCALITYOF HOST.Low trees and shrubs, Capanéma, Pará State, north Brazil. TYPE MATERIAL. The Wellcome Parasitology Unit, The Instituto Evandro Chagas, Belém, Pará, Brazil.
DISCUSSION Schellackia bolivari was the name given by Reichenow (1919) to a haemo- gregarine of the Spanish lizards Acanthodactylus vulgaris and Psammodromas hispanicus. Transmission was sho,,'ll to be by the ingestion of infected mites, Liponyssus saurarum, in which the ,sporozoites remain passively in the cells of the stomach epithelium. Reichenow described schizogony and microgametogony in the apical part ofthe epithelial cells ofthe lizard's small intestine and development of the macrogametocytes in reticuloendothelial cells of the underlying lamina propria. They were fertilized by microgametes which actively migrated there, so that mature oocysts were only to be found in the sub-epithelial tissues. They con- tained eight naked sporozoites, with a prominent residual body, and soon rup- tured: the released sporozoites penetrated the capilIaries and entered the erythrocytes. It is remarkable that Reichenow's observations remained the only adequare description of Schellackia until very recently, when Landau (1973) traced the development of S. brygooi of Madagascar lizards and experimentalIy transmitted this haemogregarine to clean lizards by feeding them with infected Culex pipiens fatigans and C. p. pipiens. Her findings were very similar to those of Reichenow, but in addition she stressed the importance of the maintenance of infection in the lizard by naked, non-dividing sporozoites which lie dormant in the reticulo- endothelial celIs of the viscera. A parasite of the lizard Lacerta ocellatawas described by França (1909) as H aemo- gregarina minuta, and later re-described and transferred to the genus Schellackia by Reichenow (1921): it is of very doubtful validity, in the absence of definite information on its life-cycle. S. occidentalis Bonorris & BalI (1955) of Californian lizards, algo needs further investigation: its life-history is poorly known and, although immature 'cysts' were described in the lamina propria, the mature oocysts have not been described. In 1974-5 the French workers described two further species of Schellackia. S. balli, the first species recorded in an amphibian, was described in the toad Bufo marinus fromFrench Guyana (Le Bail & Landau, 1974). It was exceptional in that ali the development in the vertebrate host took place in the epithelial celIs of the smalI intestine, including maturation of the oocysts. The liberated sporo- zoites then invaded the lamina propria. S. golvani was recorded in the anolid lizard Anolis marmoratus from Guadeloupe (Rogier & Landau, 1975): its develop- ment folIowed closely that of S. bolivari and S. brygooi, with development of both macrogametoc~"t('s and oocysts in the lamina propria.
R. Schellackialandauae sp.nov. 237
J-oíô' ~ ~ 'i: o 4>t- .s >"~ M '&>0) ~c .~ .."O ~ ~ o~ 'õ ',0 ~ ~ e s oN '"C ~O~t:ooai. G) g. o oS '3 ~ .~2.ot-~~= (!) o r.. ~~ 5 ~ ~- o ~ ~ O -:= ...; ~.~ '-" '.. ~ '" ~ q)'~ix o -= ~'i O .. ~.~ ...~ ~"'~~S~-='..= ~ =' .S 2.g~~'io l]~ ~o ~ I C'I~] obiJ...aJoO o.~ (!) '" 1m ~ ~~ M ,..,. ~ d 0~.8 Q a ~W..~~(!) 00.~ t.. 1<:>0 ~.~ ...~ ~- ~ ..~.~.~ ~ ~ t:lj0i8 ~~ P ~ -C'lZ ~ .~ o ~ .., :=...'"t: ~.s ~ .d $..S .., ~ S.., i:: bO~ M 1 ~ 'õ ~ ~ ~ tU ~ o ..>, S.~ ~ -.o ~ ~ ~ ~ -rD N>'á)~ "$... .bO'" "" ..,~.>..O .'t3 ~~... o: ~ m..2m ~ ~.. ..,"5 F:.~ e~..2>.C!) ~ !~.S"g ~ &,§ ~ ,~.S.8 .~~ --:~ ~ i ~ e--:-~'i>.o>,. § ~ ~ã~t~~ ~"5 I s ~ ci'~ tU~.s ~ s s ~.~ ~ ~ ~ ~ x § 00~ N rIJ'=' ~"'.. o """ I ~Ol:-bOg.8o~~~á!~~g .~ ~ t- ., ~ = ~ ~ 8 '8 a5"52 ~ ~ ~ ~ § -; §' ti' ~ ~ ~ ~ ..g'f t: ~.~ O) ~ 5 ~ ~ .,; "- -""'" -.~ ~ "" IQ~Xe-+"eO ~ ...~ ã. aj o tU o.~ à) ~ ~ ~ o '" ~ ~ Q) = o C' o~~"C~~ h o -~.~ .S Mt:- -!. 1"-'~ :o~ e o I ~ w'~.~ S.c ~.N~~ p.o'" ~ p ,P-4 ~ ~ -.to"' Ç,) '- ..."'~ ~ ~ IX) ~ .~ S '" .e '" O) 11) .~ § ~ c;;- ~ ~~~ .E o+"e-:5°~~~.~ "'". o t- .; ~ ~ o.. O ..o .."$ 'õ -~~="'"'.. d'~ 11)'" ~ '" S Q) o~ O) ~ ., o~ o~ ~ ., N ~ ~ '" .. ~~ 08 ~~ ~ ...~ g. ~ ~ ":' "5 ~ i. O>., o = >,0> ~o~-;:."I;o~o-=-= "'.~ -= 11) ~ .~ ~ ~ CJ ~ ~ ~ ~.~ ~.~ o 'tj .~ '" o~ c ~ o 'i'~ e ~.~ d ~~.@ ~~'E o ~~~ ~ 8 e 8 o '" .. ~ ~ ~ .o",'i~~-= ~Q),. = ~ ..-c ~ -~ s ~ ~ c ~~ ,.~.;;"',,0+"0)- o ~ o+" r1I ~OOo ~ 0",:= ~~~ ~ ~G.E. '" o ~ II).~ 00 ~~ cc~ p Ç,) ~ fs J"'ô -""Oll)~~= ~ ...~z < OO~OO ~ '+c;- ~ -"' ~ ~ ~ "'"'" ..Ij .~ -a ~ ~ ~ s '" '" ~ d 00 "'"tI) ""'>'>.,e ..S.~ ~ ~ o~ ~ ~ ~ o-o~~ ~ ~ o~ o~ ao IX) ao 'õ o o.~ ~..~ ~ -oi8 .~ ~ o N o 8 Q).8~ ~ ~ ~.c ~ ~ t- C!) ,Q ~ ri >, ~ ..
'" ~ .. .-d~ - .. ri} o ~ ~"'" "'" d ... ~ d '"' o .-~ '" ri} Q .: .- .c ""'..'" Q) ~ -; C!J til >'>. o o ] ;> c;; $ o S .- ~~ ~",$,S ~§ o .-'" o o Q) ~""'~~~ ~.~ o d '; o ~ C!) :& ",ON"tj ~ 0'- "'" o !: .. " ...o Ó ~~ W N d <11 aJ .. = ~ o '" o == Z o~ .::: °;: ..> ~ d ~ ~ 0- s 8- o ~... ~.I:1 ~..s .~.: ""~~~O-""'Q) d= ..., ~ Z ~ 00 OO~ 1 ~ c3 238 R. LAINSON, J. J. SHA W AND R. D. W ARD
,
mi ep
I - ~ ~ I. ~ ," ~~ A feno -D ~ bl
sc~L7:'" 8'.1 c,&~~ @ ~ ~~ ~~~~~ i);.',~~~ mi eeee f,L\~f, 'o lu ..-lUJf@~ \ '
=~~.., ~~~~--~, ---I B;:::::::~ ~~ -~E. '. -," no:::; .sc 41.' . m; I e--~""" ""---
:::=:=: ,~4: no --'" -I
Fig. 8. Schematic illustration of increasing involvement of the intestinal epithelium in four species of Schellackia. Possible evolution of the higher coccidia through a genus such as Tyzzeria when development of a resistant oocyst, passing out with the faeces, replaced non-resistant oocysts liberating sporozoites into the blood. (A) Schellackia bolivari,. (B) S. landauae; (C) S. brygooi; (D) S. balli; (E) Tyzzeria sp. bl., invasion of blood by sporozoites in the lamina propria; ep., intestinal epithe- lium; fe., fertilization of macrogametocytes; lp., lamina propria; lu., lumen of intestine; mi., microgametocytes; no., non-resistant oocysts; ro., resistant oocyst; 8C.,schizogony.
The considerabledifferences between S. landauaeand the previously described species of Schellackiaare given in Table 1. They leave no doubt as to the necessity of a new specific name. In addition, the development of S. landauaeprovides an interesting inter- mediatestage in a definite gradation of predilection for the gut epithelium, shown Schellackia landauae sp.nov. 239 among the known speciesof Schellackia.This seemsto support the view that the 'higher coccidia', such as Eimeria, Isospora, etc., may have evolved from a Tyzzeria-likeorganism after the acquisition of a resistant cyst-wall and the means of faecal transmission(Landau, 1974).We tend to favour this opinion, although opponents of this hypothesis might view the sequencewe show in Fig. 8 in the reversemanner. That is to say, that Schellackiadeveloped from a Tyzzeria- like parasite by increasing invasion of the sub-epithelial tissues from the gut epithelium, followed by utilization of the blood cells for transmissionby haemato- phagous invertebrate vectors. The discovery of the eimiorine nature of Toxoplasma,Sarcocystis and allied organisms, with their primary and secondaryhosts, Isospora-like oocysts and resistant tissue-cysts,has suggestedthat the life-cycles describedfor some other coccidia might not be so simpIe as at first thought. In bis recent review of advances in this direction, Frenkel (1974)utilized the above features in a re-definition of the Eimeriorina, and stated that 'This leaves undefined the apparently related generaLankesterella and Schellackia,the cyclesof which needto be reinvestigated and defined.' This would seem to us somewhat of an under-evaluation of the considerable knowledge already existent on the membersof the Lankesterellidae.The present study has revealedsome variations and additional features in the life-cycle of one speciesof Schellackia,and it is possiblethat similar observationsmay be made for the related generaLankesterella and Lainsonia. They do not in our opinion, how- ever, reveal the need for any great revision of the existing definition and position of the family Lankesterellidaewithin the Eimeriorina. The processof the penetration of the zygotes of S. landauaeinto the lamina propria needsto be studied further: it is, of course,inconsistent with the actual definition of the family Eimeriorina to accept an active migration of the zygote, which is always defined as 'not motile' (Levine, 1973).We reeI that the process, in the caseof S. landauae,is probably more of a mechanicalbursting of the zygote through the weakenedbase of the epithelial cell than a true motility comparable with that of the malarial zygote (ookinete). There are somenotable points regardingthe asexualcycle of S. landauae.Thus, previous descriptions of Schellackiaspecies have failed to record the remarkable dimorphism of the intestinal schizontswe have describedhere. It is not an un- commonfeature in other coccidia,however, and has beennoted in generasuch as Cyclospora,Eimeria and Lankesterella(Lainson, 1959, 1965,1968). Exactly what this dimorphismsignifies is still obscure.It is known that the schizontsof different generationsmay vary considerablyin size, time of developmentand the number and form of the merozoitesproduced; and the view is frequently expressedthat the dimorphism may representsexually determinedschizonts. We have given one interpretation with regardsS. landauae,in Fig. 7, but it is clear that the asexual developmentof this parasite needsto be followed in much more detail than was possible in the present study. The presenceof an extra-intestinal phase of asexual division, apparently by endodyogeny,in S. landauaeis an interesting new feature which needsconfirma-
16-2 240 R.LAINSON,J.J.SHAW AND R.D.WARD tion and further stud)' in relation to other genera in the Lankesterellidae. It is important to stress our opinion, here, that the division stagesare not within re- sistant cysts; they exactly fit the definition of Frenkel (1974)for the equivalent stages of Toxoplasma,i.e. 'Tachyzoites... which develop in groups within a vacuole'. We have failed, so far, to demonstrate any resistant tissue-cystfor Schellackia either in the experimental infections or in tissues from animaIs with old, chronic infections. Frenkel's reference (1974) to Landau's (1973) finding of resistant , tissue-cysts' in the LankestereIlidaeis erroneous,according to her own personal communication. It is presumed that confusion,vas made with her reference to the long-term 'diapause' sporozoites,which persist in reticulo-endothelial ceIls of the viscera and which are a characteristic feature of the lankestereIlids ('Ia diapause simple du sporozoitedes LankestereIlidaedans Ia ceIlule qui l'herberge comme étant un phénomeneplus primatif que l'enkystement avec division par endogenesedes autres Coccidies'). Records of non-specificity of Schellackia,at least in lizards (Reichenow,1919; Bonorris & BaIl, 1955; Landau, 1973),raise the possibility of direct transmission to predatory lizards (or snakes1) which may eat an infected lizard; although the failure of Rogier & Landau (1975)to infect other speciesof lizards fed with liver from Anolis marmoratusharbouring S. golvanidoes not support this idea. We, too, failed to infect other speciesof lizards fed with mosquitoesrich in sporozoitesof S. landauae. The natural vector of S. landauaeremains unknown. It might indeed be Oulex p.fatigans, ,vhich we found to feed so readily on P. marmoratusin the laboratory. The incrimination of mites for S. bolivari and S. occidentalis,however, is good reasonto expect an acarine vector for the Brazilian parasite; and mite-infested P. marmoratushave been recorded in this laboratory on two occasions.Further studies are in progresso The definition of the family LankestereIlidaemight now be slightly modified and amplified as follows, following the observationsof Madame Landau and her colleagues,and our present description.
Family Lankesterellidae Nõller 1902 Schizogony, gametogony and sporogony alI within various cells of the same vertebrate host. Microgametocytes producing large numbers of bi-flagellate microgametes. Oocysts asporocystic, with a variable number ofnaked sporozoites: they are of a temporary nature, soon rupturing within the tissues of the host. Sporozoites enter white and/or red cells of circulating blood, where they are ingested with the blood-meal of the invertebrate vector. Other sporozoites enter into long-term diapause in cells of the reticulo-endothelial system of the viscera. No resistant tissue-cysts formed. Sporozoites remain passively in the vector, usually in the epithelial cells of the gut (mites, mosquitoes) and transmission is after ingestion of the vector by the vertebrate host: transmission may algo be by bite (leeches). Schellackia landauae sp.noY. 241
Genus Schellackia Reichenow 1919 Schizogony in cells of the intestinal epithelium, after ingestion of the infected vector (mites, mosquitoes): dimorphic microschizonts and macroschizonts presentin at least one species. Extra-intestinal, asexual division giving rise to groups oftachyzoites in lymphocytes and monocytes of the viscera in at least one species. Microgametocytes developing in intestinal epithelium. Macrogametocytes may algo develop in the garoe cells, in which case (a) the fertilized zygote later enters the lamina propria for the formation of the oocyst, or (b) oocysts mature in the epithelial cells and liberated sporozoites later migrare into the lamina propria. In other species macrogametocytes develop principally in the laminapropria and are fertilized there by microgametes which migrare from the epithelium. Oocysts with eight naked sporozoites. Liberated sporozoites enter red and white cells of the peripheral blood by the capillaries: others enter reticulo-endothelial cells of the viscera, in modest numbers, where they remain in infective diapause for long periods. Parasites of lizards (Old and New Worlds) and amphibia (New World).
GenusLankesterella Labbé 1899 Schizogonyin cells oí reticulo-endothelial cells oí the viscera, aíter ingestion oí iníected invertebrate vector (mites, mosquitoes)or by injection or bite (1) (leeches).Dimorphic microschizontsand macroschizontsin at least one species. Gametogonyand sporogonyalso in cellsoí the visceral reticulo-endothelialsystem. Oocystspolyzoic, with large numbers oí naked sporozoites.Liberated sporozoites enter red or white cells by way oí the capillaries, where they are infective to the haematophagousinvertebrate host (mites, mosquitoes(1), leeches).Other sporo- zoites enter reticulo-endothelialcells oí the viscera, in large numbers, where they remain in infective diapause.Parasites oí Old and New World amphibia and birds.
Genus Lainsonia Landau 1973 Schizogony, gametogony and sporogony in reticulo-endothelial cells of the viscera, in particular the pigmented cells. Oocysts with eight naked sporozoites. Sporozoites invade red and white cells of the peripheral blood by way of the capillaries, where they are available for the blood-sucking vector (as yet unknown). Large numbers of sporozoites enter recticulo-endothelial cells of the viscera to remain in infective diapause. Special mechanism for dissemination of parasites throughout the body of the vertebrate host, following detachment of the intact oocyst from the walls of the capillaries at gires of development, when they are carried to other tissues in the circulating blood. Parasites so far recorded only in New World lizards.
These studies were made under the auspices of the WellcorIle Trust, London; the Instituto Evandro Chagas of the Fundação Serviços de Saúde Pública, Belém, Pará, Brazil; and the World Health Organization, Geneva. Particular thanks are due to Roberto Daibes Naiff, Henrique da Silva Buna, and Aluizio de Figueiredo Silva for patient technical assistance. 242 LAINSON, J. J. SHA W AND R. D. W ARD
REFERENCES BONORRIS,J. S. & BALL, G. H. (1955). Schellackia occidentalis n.sp., a bIood-inhabiting coccidian found in Iizards in Southem CaIifomia. Journal of Protozoology2,31-4. BRAY,R. S. &GARNHAM,P. C. C. (1962).The Giemsa-Colophoniummethod forstainingproto- zoa in tissue sections. lndian Journal of Malariology 16, 153-5. FRANÇA,C. (1909). Sur Ies Hématozoaires des Sauriens. I. Hémogrégarines de Lacerta ocellata. Archivos do Real Instituto bacteriológicoCamara Pestana 2, 337-60. FRENXEL,J. K. (1974). Advances in the biology ofsporozoa. Zeitschriftfür Parasitenkunde 45, 125-62. LAINSON,R. (1959). Atoxoplasma Garnham 1950, as a synonym for LankesterellaLabbé 1889. Its Iife cycIe in the EngIish sparrow (Passer domesticusdomesticus Linn). Journal of Proto- zoology6, 360-71. LAINSON,R. (1965). Parasitological studies in British Honduras: lI. Cyclosporaniniae sp. novo (Eimeriidae, CycIosporinae)from the snake Ninia sebaesebae (Colubridae). Annals of Tropi- cal Medicine and Parasitology 59, 159-63. LAINSON,R. (1968). Parasitological studies in British Honduras: m. Some coccidial parasites ofmammals. Annals of Tropical Medicine and Parasitology62,252-9. LAINSON,R., LANDAU,I. & SHAW,J.J. (1971). On a new famiIy ofnon-pigmented parasites in the bIood ofreptiles: Garniidae fam.nov. (Coccidiida: Haemosporidiidea). Some speciesof the new genus, Garnia. International Journal of Parasitology 1, 241-50. LAINSON,R., SHAW,J. J. & LANDAU,I. (1975). Some bIood parasites ofthe BraziIian Iizards Plica umbra and Uranoscodonsupercüiosa (Iguanidae). Parasitology 70, 119-41. LANDAU, I. (1973). Diversité des mécanismes assurant Ia pérennité de I'infection chez Ies sporozoaires coccidiomorphes. Mémoires du Muséum National d'Histoire Naturelle A 77, 1-62. LANDAU,I. (1974). Hypotheses sur Ia phyIogénie desCoccidiomorphes de Vertébrés. Zeitschrift für Parasitenkunde 45,63-75. LE BAIL, O. & LANDAU,I. (1974). Description et cycIe biologique expérimental de Schellackia balli n.sp. (LankestereIIidae) parasite de Crapauds de Guyane. Annales de Parasitologie Humaine et Comparée49 (6), 663-668. LEVINE, N. D. (1973). In The Coccidia (ed. D. M. Hammond and P. L. Long). BaItimore: University Park Press. REICHENOW,E. (1919). Der Entwick1ungsgang des Hãmococcidien Karyolysus und Schellackia novo gen. SitzungsbuchBerlin GesellschaftNaturfreunde, Berlin, pp. 440-7. REICHENOW,E. (1921). Die Coccidien. In A. Prowazek, Handbuch derpathogenenProtozoen 3, Leif. 8,1136-277. ROGIER,E. & LANDAU,I. (1975). Description de Schellackiagvlvani n.sp. (LankestereIIidae), parasite de Lézards de GuadeIoupe. Bulletin du Muséum National d'Histoire Naturelle, 3, série,no. 284, Zoologie 194,91-7.
EXPLANATION OF THE PLATES PLATE 1 (F x 784; A-H x 1960) ScheUackialandauae Bp.nov., a haemogregarine of the lizard Polychrus marmoratus. GiemBa stained. (A-C) SporozoiteB in erythrocyteB and a lymphocyte, in the peripheral blood. (D) Extracellular Bporozoite in peripheral blood. (E) SporozoiteBin epithelial cellB of Btomach of Culex pipiens fatigan8, 14 daYB after blood-meal on infected lizard. (F) Low-power view of Bection of Bmall inteBtine of P. marmoratus, experimentally infected 30 daYBpreviouBly: parasiteB in almost every epithelial cell. (G) Developing microschizont in apical portion of epithelial cell of Bmall intestine: 23-day-old infection. (H) Almost mature microschizonts in smears from same intestine.
R. Parasitology, V 01. 72, Pare 3 Plate 1
R.LAINSON, J.J.SHAWAND R.D.WARD (Facing p. 242) . Parasitology,Parasito1ogy, V V 01. 01. 72,72, Pare Pare 3 3 PlatePlate 1 2
R.LAINSON, J.J.SHAWAND R.D.WARD (Facing p. 242) Parasitology, V 01. 72, Pare 3 Plate 3
R.LAINSON, J.J.SHAWAND R.D.WARD (Facing p. 242) Parasitology, V 01. 72, Pare 3 Plate 4.
R.LAINSON, J.J.SHAWAND R.D.WARD (Facing p. 242) Parasitology, V 01. 72, Pare 3 Plate 5
R.LAINSON, J.J.SHAWAND R.D.WARD (Facing p. 242) Plate 6. . Parasitology, V 01. 72, Pare 3
R.LAINSON, J.J.SHAWAND R.D.WARD (Facing p. 242) Schellackialandauae sp.nov. 243
PLATE 2 (x 1960) Schellackia landauaesp.nov., in the Iizard Palychrus marmoratus.Giemsa stained. (A, B) Smear of small intestine showing microschizonts with the micromerozoites budding off froII\ the periphery and a cytopIasmic network, respectiveIy. (C) Section of intestine showing a mature microschizont in apical part of epitheIiaI ceII. (D-F) Small macroschizonts, with Iimited number ofmacromerozoites, as seenin smears ofintestine. (G) Small, mature macroschizont, in section, within apicaI part of epitheIiaI cell of small intestine. (H-I) Large, immature and mature ruptured macroschizonts in smears. AlI these parasites from a 23-day-oId infection.
PLATE 3 (x 1960) Schellackia landauae sp. nov., in the Iizard Polychrua marmoratua. Giemsa stained. (A-J) Stages of asexuaI division within Iymphocytes and monocytes of the spIeen and Iiver (F), apparentIy by endodyogeny. (J) Group of freed tachyzoites: alI as seenin Bouin-fixed smearB. (K, L) Smear of intestine: young deveIoping microgametocytes. AlI parasites from a 30-day- oId infection. PLATE4 (x 1960) -Schellackia landauae sp.nov., in the lizard Polychrus marmoratUB.Giemsa stained. (A, B) Sections of small intestine showing nearly mature microgametocytes in apical part of epithelial cells. (O) Free microgametes as seen in air-dried smears of intestine: methanol- fixed and stained with Giemsa. (D) Section of intestine, with mature microgametocyte and macrogametocyte in same epithelial cell: apical portion of cell is uppermost. (E) Smear of intestine showing mature microgrametocyte: note large cytoplasmic residuum. The flagella have not stained following Bouin flxation.
PLATE5 (A-E x 1960; F x 784) Schellackia landauae sp.nov., in the lizard Polychrus marmoratus Giemsa stained. (A, B) Mature microgametocytes and macrogametocytes in epithelial cells of the small intestine, as seen in sections: apical part of cells uppermost. Thirty-day-old infection. (C) Section of lower part of epithelium of sma!l intestine showing macrogametocytes in basa! part of epithelial cells and developing and mature oocysts in the lamina propria, below. (D, E) Sections of mature oocysts in the lamina propria: note prominent residual body. (F) Low. power view of intestine, in section, showing large number of sporozoites from ruptured oocysts in the lamina propria: 45-day-old infection.
PLATE 6 (x 1960) Schellackia landauae sp.nov., in the lizard Polychrua marmoratus. Giemsa stained. (A) Sporo- zoites concentrated in the lamina propria of the small intestine, after the rupture of oocysts. 45-day-old infection: high-power view of previoUBphotograph. (B) Smear of same intestine, showing free and intracellular sporozoites: note variable number ofrefractile bodies (1 or 2). (C, D) Intracellular sporozoites, in diapause, within reticulo-endothelial cells of the lung (C) and liver (D) in old chronic infection.
Printed in Great Britain