A Note on Sporozoa of Undetermined Taxonomic Position in an Armadillo and a Heifer Calí

582 J. PROTOZOOL.19(4), 582-586 (1972).

R. LAINSON* The Wellcome Parasitology Unit, Instituto Evandro Chagas, Caixa Postal), Belém 66.000,Pard State,Brazil

SYNOPSIS. A sporozoan parasite of undetermined taxonomic position is described in the nine-banded armadillo, Dasypus novemcinctus, from British Honduras, Central America. Schizonts, giving rise to approximately 60 merozoites, are produced in the parenchyma cells of the liver and in the endothelial cells of the brain capillaries: rupture of the schizonts stimulates con- spicuous foci of inflammatory response. No sexual stages were observed. A similar parasite is recorded in the lung and liver of a heifer calí, Bos taurus, from England. The possible relationship of these parasites within the Sporozoa is discussed. lndex Key Words: Sporozoa of undetermined taxonomy; Coccidia; armadillo (Dasypus novemcinctus), from; British Honduras; calí (Bos taurus); lung; liver; brain.

Some years ago, in a report on Toxoplasma in the brains F. G. Clegg, of the Veterinary Investigation Centre of the of 2 captive armadillos (Dasypus novemcinctus) from British University of Nottingham, sent material to the Central Veteri- Honduras (9), I remarked on the abundance of foci of cellular nary Laboratory in Weybridge, Surrey, where Dr. L. M. Mark- reaction in the brain of another armadillo found dying in the son first noted the parasite which, till now, remains unidentified. same cage. In some instances there appeared to be the re- Tissues had been fixed in formalin-saline, and the sections mains of ruptured cysts in the center of the arcas of infiltra- studied here were stained by the Giemsa-colophonium method. tion, with "organisms resembling Toxoplasma scattered in the near vicinity." RESULTS In discussing the possibility that these might be the ruptured The Parasite in the Armadillo tissue-cystswhat appeared of T to oxoplasma, be "a different I mentioned protozoal the parasite complication undergoing of My úrst lmpres~lon was that .the anl~a! had mdeed dled of schizogony in the vis cera of the same armadillo. .." acute toxoplasmosls. Free parasltes, remmlscent of T oxoplasma, Over the past 6 years, repeated efforts have been made to were found sca.ttered abundantly. throughout th~ liver smear, rediscover this parasite in armadillos of the same species, in less freq~ently m that o~ the bram, and rarely m the ~pleen. Brazil, without success. There the matter might have rested, No parasltes were found m smears of the lung or the penpheral but in 1967 Professor P. C. C. Gamham showed me some sec- blood. tions of liver and lung from a heifer calf that had died on a ~ closer examin~tion of the liver sm~ar reve~led large, oval farm in England, and I was struck by certain similarities sch~onts from ~hlCh the T ~xoplasma-hke bodles wer~ ?learly between the parasite in this material and that from the arma- denved. In sectlons the schlzonts were seen to be wlthm the dillo. parenchyma cells (Figs. 12, 13). Their development, however, It was felt that a description of these 2 parasites should be was more r.eadily foll~wed in the smear, where the majority made, in view of the recent demonstration that Toxoplasma of the .orga~lsms were d1Slodge.d from the host c:lls.. "c itself is a coccidian (6, 7) and the new interest in the extra- Dur~g lts growth the schlzont usually mamtams a lemon- intestinal development of other gut-inhabiting coccidia (4). shaped or oval formo Early stages measured about 7.6 X 4.4 p.m, with the nucleus forming a well defined band across the MATERIALS AND METHODS cell (Figs. 1, 2). The cytoplasm stains a clear blue and is ..prominently vacuolated. With growth it appears to condense The arma.dlllo was one o~ s~veral .c~ptured m the general at the periphery, where it stains very deeply and gives the para- arca ~f Bak~~ Pot, Caro Thstnct, Bntls~ Honduras, an~ h~d site a somewhat cyst-like appearance. The chromatin begins been m captlvlty for -2 months before lt was fo~d dymg m to spread an increasingly broad band and ultimately forms the cage which it shared with 5 others. a diffuse network throughout the parasite (Figs. 3-5). At this The animal was killed and smears made from the lung, liver, stage the parasite mar reach 25.0 X 15.0 p.m, or more, without spleen, brain, and peripheral blood; these were air-dried, fixed any obvious division of the nucleus. There now seems to be a in methanol, and stained with Giemsa's stain. Tissues were rapid reorganization, during which bulky chromatin masses are fixed in Camoy's fluid and sections stained by the Giemsa- separated off; these ultimately give rise to some 40-60 merozoites colophonium method. (Figs. 6-9). Just before separation these merozoites frequently The heifer calf had died, apparently as a result of copper are. arranged. in rosette fashion,. with the nuclei towards the and manganese deficiency. Among the postmortem findings pen.phery (FlgS. 9, 12). ~erozoltes averaged 6.35 X 2.7 fm,

...havmg one end more pomted than the other. They dúfer were those of muscular dystrophy and úbrmous pleuntus. Mr. f h f T I . rom tose o oxop asma m t h elr poor stammg reactlon, ap- * .pearing as almost colorless crescents with a small vesicular I am mdebted to Mr. F. G. Clegg:, .Dr. L. M. 1.:Iarkson, and nucleus which usually contains a more densely staining granule Professor P. C. C. Garnham for proVlslon of materIal from the .. infected calí, and for pennission to make this comparison with mterpreted as a nucleolus or karyosome (FIg. 10). that from the annadillo; to Dr. Leonidas Braga Dias, Departa- Rupture of the merozoites from the parenchyma cells causes mento de Patologia, Universidade do P.ará, for advice. on histo- a variable degree of local inflammatory reaction (Fig. 14), pathology; and to Dr: J. J. Shaw for takm~ the photomlcrographs. within which the merozoites mar be engulfed by macrophages Dr. W. J. Hartley kmdly drew my attentlon to the paper by A.. . H. Corner et aI. and showed me histologic sections of their (FIg. 11) or actlvely enter them. Whether or not they undergo material. ' any further division process in these cells is uncertain. SPOROZOAOF UNDETERMINED TAXONOMY 583

schizonts (Fig. 17). The rupture of such capillaries and re- lease of parasiteswas clearly responsible for the areas of cellular reaction (Fig. 16). Morphologically, the products of the schizonts in the brain appeared similar to those in the liver and are consideredto belong to the sameparasite.

The Parasite in lhe Heifer Calf ,. Microscopically the lung had a generalized, interstitial pro- liferative pneumonia undoubtedly caused by the parasite. Con- nective tissue around the bronchioles, pleura, and blood vessels was increased considerably by a proliferation of fibroblasts and histiocytes (Fig. 18) and these changes always were associated with parasitized histiocytes. The reaction extended to the inter- lobular and alveolar septi, substantially thickening them (Figs. 19, 20)-here the parasites occupied histiocytes,and endothelial cells of the capillaries which sometimes were almost occluded (Figs. 20, 21). No parasites were seen in the alveolar cells themselves,and the lumena were free of any cellular exudate. In some lumena, however, there did arrear an exudate of an apparently serous-fibrinousnature. The smallest parasites (merozoites) averaged 6,0 X 3.2 p.m and were generally of the same form as described in the armadillo ( Fig. 23) .Again, they possesseda delicate, rounded nucleus with a distinct central nucleolus. Within the histiocyte or endothelial cell the parasite de- veloped into an oval body measuring 15.0 X 10.0 p.m, or more (Figs. 24, 25). Fromearly on there was a dense, heavily staining nucleolus in the center of the diffuse chromatin mass, the cytoplasm was highly vacuolated, and the organism strikingly resembled a young coccidial macrogametocyte (Fig. 25). With growth the chromatin apparently divided, although in sections the contours of individual nuclei were difficult to make out (Figs. 26-29). The developing schizont often had an irregular, ameboid shape (Fig. 29), such forms being more common in the alveolar septi, where the nature of the host tissue itself probably played a part in producing these shapes. With separation of the nuclear fiasses, some 60 merozoites were produced, often disposed in a rosette ( Figs. 29-31).Mature schizonts measured 20-30 X 10-15p.m. Figs. 1-11. [Line drawings of an unidentified sporozoan under- Schizogony in the liver appeared restricted to the Kupffer going schizogony in the tiver of an armadillo, Dasypus novem- cells (Fig. 22), and no evidence was found of development in cinctus. The parasites figured have been dislodged from host parenchyma cells. Liver smear. Giemsa's stain (G).] 1,2. Early parenchyma cells, as was so clearly the case in the parasite growth phase with singre, band-shaped nucleus. 3-5. arder forms from the armadillo. Rupture of merozoites from the schizonts with diffuse spread of chromatin. 6-9. Division of chromatin and produced minor cellular reaction. formation of merozoites. 10. Liberated merozoites. 11. Macro- Unfortunately, other tissues were not available for study phage with phagocytosed merozoites. and it is not known if development was taking place in other organs. The brain was the only other organ in which schizonts were found, although scanty merozoites were seen in smears of the DISCUSSION spleen. Considering the widely different hosts and geographic 10- The first examination of the brain was by histologic sec- calities it is probable that we are dealing with 2 different orga- tions, which contained very numerous foci of cellular infiltra- nisms and, even with so incomplete a description, there are tion. Such areas were sufficiently frequent to arrear in every individual features for each parasite which are quite striking. section (cut at 5 ,um, from a 10 mm piece of tissue), and Thus, the parasite of the armadillo had no nucleolus during individual sections sometimes had as many as 20 such reaction growth and division, whereas that of the calf possesseda very zones. heavy and distinct nucleolus throughout. The process of nu- As previously mentioned, my earlier impression was that clear division of the parasite in D. novemcinctus was relatively there were the remains of ruptured cysts in the center of these easy to follow in the smears. Notable features were the strange, toei, associated with scattered organisms resembling ToxoPlasma. band-like nucleus of the early stages (very reminiscent of a Examination of the brain smears, however, revealed large haemogregarine), the very diffuse disposition of the chromatin schizogonic masses within the capillaries, apparently in the en- in the growing body, and the delayed division which occurred dothelial cells (Fig. 15). What had appeared to be ruptured only when the parasite reached almost maximum size. Interpre- cysts in the sections were, in fact, capillaries occluded by tation of the division of the parasite in the calf was less easy, 584 SPOROZOAOF U NDETERMINEDTAXONOMY SPOROZOAOF UNDETERMINED TAXONOMY 585

raised the question as to whether or not the schizonts might belong to Toxoplasma. The morphology of both the schizonts and the merózoites differs,. however, from that described for Toxoplasma (5, 8). It is unfortunate that no stages of sexual developmentwere found for either the parasite in the armadillo or that in the calf. Schizogony of other intestinal coçcidia.-Although earlier workers have tended to stress the restriction of "intestinal coccidia" to the gut epithelium, there is increasing evidence that this is not always so. Thus, Lotze et aI. (11) described coccidial schizontsin the mesenteric lymph nades of experimentally infected sheep and goats; Shelton et aI. (13) reported on coccidian schizonts,merozoites, and gametocytesin macrophagesof granulomatous skin lesions of a dog; Box (1) described extraintestinal schizogony in canaries infected with Isospora; Dubey & Frenkel (4) discussed the whole subject of extraintestinal stages of coccidia, and experimentally produced such infections in cats with Isospora felis and Isospora rivolta. Finally, the present author has noted massive development of the sexual stages of an Eimeria species in the spleen, liver and intestinal musculature of crocodiles, as well as in its normal site in the lamina propria of the small intestine. Intestinal coccidia arrear rare (or poorly studied) in the Figs. 23-31. [Line drawings of an unidentified sporozoan 9-banded armadillo. Da Cunha & Torres (3) described very undergoing schizogony in the lung of a heifer calí (Bos taurus). FoS, Gc.] 23. Free merozoites liberated from an endothelial cell large schizonts in the intestine of Dasypus (Tatus) novem- of a capillary in the alveolar septum. 24, 25. Initial growth of cinctus, measuring up to 2,100 X 500 p.m. They named the parasites in endothelial cells. 26-28. Older schizonts with chro- parasite as Globidium tatusi, although Reichenow & Carini (12) matin apparently dividing. 29. Ameboid, older schizont with suggested that these enormous schizonts belonged to Eimeria nuclear material divided. 30, 31. Formation of merozoites. travassosi,normally found in other armadillos (Dasypus sex- cinctus and Muletia hybrida). Other armadillos of the genera in the absence of'smears. The presence of numerous karyo- Cabassusand Chaetophractusharbor Eimeria tatusi and Eimeria somes scattered among the chromatin mass

í:- Figs. 12-17. [Unidentified sporozoan undergoing schizogony in the liver and brain of an armadillo, Dasypus novemcinctus. Figs. 12-14, liver sections, 5 ILm, Carnoy's fluid, Giemsa-colophonium (Gc).] 12. Almost mature schizont in a parenchyrna cell. Note rosette-like grouping of developing merozoites. X 850. 13. Mature schizont in a parenchyrna cell. X 1,200. 14. Cellular reaction following rupture of merozoites from a parenchyrna cell (arrow). X 415. 15. Mature schizont in endothelial cell of a brain capillary. Brain smear. G. X 850. 16. Focus of cellular reaction surrounding a ruptured capillary occluded by a mature schizont (arrow). Brain section, 5 ILm. Gc. X 415. 17. Higher magnification of part of the same capillary, packed with merozoites. X 850. Figs. 18-22. [Unidentified sporozoan undergoing schizogony in the lung and liver of a heifer calí (Bos taurus). Figs. 18-21, lung sections 5 lJ.In formalin-saline (FoS) Gc.] 18. Marked proliferation of connective tissue around a bronchiole. Parasites in histiocytes (arrow;). X '160. 19. Portion of i~terlobular septum of lung, thickened by connective-tissue proliferation. Numerous parasites in histiocytes. X 415. 20. Alveolar septum with thickening caused by cellular proliferation. A capillary is almost occluded by developing schizonts in the endothelial cells (arrow). X 630. 21. Alveolar septum, much thickened and containing developing and mature schizonts in endothelial cells (arrows). X 850. Developing and mature schizonts in Kupffer cells. 22. Liver section, 51Lm.FoS. Gc. X 850. 586 SPOROZOAOFU NDETERMINEDT AXONOMY

Comer and his fellow workers used the term "cyst" be- Thus, the parasites of the English calí and the Canadian cattle cause "the situation of this disease appears analogous to that are so similar that it is tempting to regard them as the garoe. described in chronic toxoplasmosis..." From their photographs A closer examination of the life cycles of the existing bovine and slides of their material that I have examined, however, coccidia would seem important to see if extraintestinal develop- the aggregations of parasites are clearly stages in schizogony, ment is a normal part of these parasites' development or if and the parasite bears a striking resemblanceto that I have just under certain conditions (e.g. stress) they mar forsake their described in the calí. This similarity is particularly noticeable usual site of development in the intestine, invade other tissues, in the young forros, which have the garoe dark-staining dots and undergo a violent schizogonywhich mar produce a disease in the chromatin masses(karyosomes?) and the rosette formation akin to acute toxoplasmosis. of the developing merozoites. AIlowing for the wide varia- tion seen for both parasites, the size of the schizonts and mero- REFERENCES zoites is similar. Finally, the localization of the dividing para- 1. B?x, E. D: 1970. AtoxoPlasma associated with an isosporan sites in the endothelial cells of the alveolar capillaries, often oocyst m cananes. J. Pr.otozool. 17,391-6. I d. h f th f .. h 2. Comer, A. H., Mltchell, D., Meads, E. B. & Taylor, P. A. occ u mg tem, ... IS most remmlscent o e . m ectlon m t e 1963. Dalmeny disease. An infection of cattle presumed to be calí. causedby an unidentified protozoon. Cano VetoJ. 4, 252-64. Comer et aI. went to great lengths in attempts to isolate the .3; Da Cunha, ~. & Torres, C. 1924. Sur .un n°u,veau qlo- organism in inoculated cows and calves, goats, guinea pigs, mice, bldlum, CI. '{'atusl ,cunha et Torres, 1923, parasrte de I annadlllo. b . em ryonatmg eggs,an d even. m ce11 cu Itures o f b ovme. emb ryoruc.C. 4. R. Dubey,SocoBlol J. P.Paris, & Frenkel, 90, 242. J. K. 1972. Extra-intestinal stages kidney, but alI attempts failed. Finally, complement-fixation of Isospora felis and I. rivolta (Protozoa: Eimeriidae) in cats. and Sabin-Feldmantests for Toxoplasma gave negative reactions J. Protozool. 19, 89-92. ... with gera from many of the infected animaIs and the authors 5. -& -.1972. Cyst-mduced toxoplasmosls m cats. I d d h h d . d b ' h .J. Protozool. 19, 155-77. conc u e t at t e. lse~sewas cause. y some.ot er. organISmo 6. Frenkel, J. K., Dubey, J. P. & Miller, N. L. 1970. Toxo- Unfortunately, m splte of exhauStlve conSlderatlon of other plasma gondii in cats: recaI stages identified as coccidian oocysts. pathogens, no mention was made of the presence or absence Science 167., 893-6. ... of intestinal coccidia in the infected cattle. The schizonts were 7. Hutch!sc;>n,':V. M., Dunachle, J. F., Sllm, J...C. &. Work, K. f d h " th h t th I gth f th . t t . I t t " 1970. Coccldlan-hke nature of Toxoplasma gond!!. Brlt. Med. J. oun, owever, roug ou e en o e m es ma rac. 1, 142-4.

S h' . f KI . 11 Th h .8. -, -, Work, K. & Siim, J. C. 1971. The life c zzogonlc DIeve opment o a ossle a sp.- e sc lzonts cycle of the coccidian parasite Toxoplasma gondii in the domestic describedhere in the armadillo and the calí could possibly have cato Trans. Roy. SocoTrop. Med. Hyg. 65, 380-99. belonged to. a Klossiella or related parasite. K. cobayae of the 9. Lainson, R. 1964. Toxoplasma infection of the annadillo guinea pig, for example, undergoes gametogony and schizogony (Dasypus novemcinctus) in Briti~h Honduras, with particular the end th I. II f th kidn I I . Ab d t reference to the rupture of cysts m the central nervous system. in ..o ela I ... "e s o e ey gomerul.. ~.an Trans. Roy. Soco Trop. Med. Hyg. 58, 287. schIzogornc stages mar algo be found, however, m sImIlar 10. Levine, N. D. & Ivens, V. 1970. The Coccidian Parasites situations in other organs of the body. Lack óf kidney material of Ruminants. Univ. of Illinois Press,Urbana. . from the infected animaIs has made it impossible to investigate 11. Lotze, J. C., Shalkop, W. T., Leek, R. G. & Behin, R. .bl t .. th th ' . t 1964. Coccidial schizonts in mesenteric Iymph nades of sheep a pOSSI e connec Ion WI IS parasI e. and goats. J. Parasito 50, 205-8. In conclusion, only the acquisition of further, more complete 12. Reichenow,E. & Carini, A. 1937. über Eimeria travassosi material will enable the correct classification of these interesting und die Gattung Clobidi.um. Arch. Protistenk: 88, 374-86.

P resent extraintestinal 13. ~11.eltc;>n, G. C,! Kmtner.. L. D..& Mackmtosh, D. O. 1968. organisms. I reei it most..~ likel that they re ..A coccldla-hke organlsm assoclated wrth subcutaneous granulomata development of coccIdIa, possIbly of already recognIzed specIes. in a dog. J. Am. VetoMed. Ass. 152, 263-7.