Vet. Path. 11: 1-12 (1974) An Unidentified Sporozoan Encephalomyelitis in Sheep W. J. HARTLEY and W. F. BLAKEMORE Department of Veterinary Medicine, The University of Sydney, Camden; and Wellcome Laboratory for Comparative Neurology, School of Veterinary Medicine, Cambridge Abstract. Severe encephalomyelitis with myelomalacia in two young sheep was associated with an unidentified sporozoan parasite that was passing through a schizogonic cycle in astrocytes. Mature merozoites were 5-7 urn in length, had a conoid, subpellicular microtubules, paired organelles and many micronemes. Their morphology is compared with that of Toxoplasma and other related sporozoans. The apparent absence of gametogony suggests that the schizogonic cycle in the central nervous system could be an aberrant dead-end cycle. There are many well-recognized bacterial and viral infections that cause encephalomyelitis in sheep [7]. A protozoan parasite suspected of being Toxoplasma gondii has also been seen in isolated cases of ovine encephalomyelitis [5, 12, 16]. COLE et al. [I] described toxoplasmosis in sheep characterized by respiratory and neurological signs, and KOESTNER and COLE [9] described the neural lesions. Isolated cysts, morphologically resem bling Sarcocystis are occasionally seen in ovine brain but are not associated with any host reaction [4, 17]. The present paper reports a severe encephalomyelitis and myelomalacia in two sheep, which was associated with an unidentified protozoan parasite. Field Observations Case I. A 6-month-old Suffolk ram lamb from Tasmania had been artificially reared and had gradually become unthrifty with diarrhoea. It then developed posterior incoordination followed by general incoordination, apparent blindness, periodic head jerking, and leg biting. The only significant finding on autopsy was a heavy infestation with Cysticercus tenuicollis.
2 HARTLEYIBLAKEMORE Case II. A 5-month-old stud Dorset Horn ewe lamb from New South Wales had shown progressive posterior incoordination over a period of 3 weeks and finally complete flaccid paralysis of its hind quarters. On the same property at least six other sheep between 2 months and 3 years of age had shown signs of incoordination during the preceding 8 months, and all had made a gradual recovery. Materials and Methods The brain and cord from both of these sheep were fixed in 10% formalin. For histological examination blocks were taken at six representative levels of brain and four of cord from each sheep. These were processed by the usual methods and embedded in paraffin wax. Sections were routinely stained with haematoxylin and eosin (HE), and selected sections of cord were stained by one or more of the following techniques; Luxolfast blue, periodic acid-schiff (PAS), haematoxylin, Giemsa, and Gram. Six years later small portions of formalin-fixed cord from both cases were taken for electron microscopy. These were osmicated in osmium tetroxide, dehydrated in ethanol and embedded in araldite. Thin sections were stained with uranyl acetate and lead citrate and examined at 50 kv in an Hitachi HS electron microscope. Serum samples were taken from 20 healthy lambs in contact with the sheep in case I and from six previously affected and six unaffected sheep in contact with the sheep in case II. These sera were examined for Toxoplasma dye test antibodies. Results Light Microscopy Brain. In both cases there was a universal moderate to severe nonsuppurative subacute meningoencephalitis affecting grey and white matter and characterized by disseminated microglial nodules and perivascular cuffing. In the mid- and posterior brain stem, but apparently not elsewhere, there were scattered clumps of an intracytoplasmic protozoan parasite in various stages of development. In case II there was one large cyst in the mid-brain stem morphologically indistinguishable from Sarcocystis tenella. Cord. At all levels there were widespread focal lesions of malacia affecting the white matter. In case I these lesions were recent and were associated with only a few gitter cells and mild perivascular cuffing (fig. 1), whereas in case II Fig. 1. Case 1. Spinal cord. A large area of recent malacia in the peripheral white matter with early gliosis and perivascular cuffing. Giemsa. Fig. 2. Case II. Spinal cord. A large area of relatively old standingmalacia of peripheral white matter with extensive replacement gliosis. HE.
Sporozoan Encep ha lomye litis 3
4 HARTLEy/BLAKEMORE the malacic areas were largely replaced by gitter cells in association with pronounced perivascular cuffing and gliosis (fig. 2). In both cases there was a moderate diffuse gliosis and perivascular cuffing of grey matter. Many clumps of intracellular parasites were present, in both grey and white matter, together with scattered free forms. Dorsal root ganglia and spinal nerves were unaffected. Parasite. The parasite was identified in many cells, particularly in the white matter of the cord, where it was undergoing an apparent developmental cycle. The earliest stage recognized was a distinct intracytoplasmic circular body, about 10 urn in diameter that, in HE-stained sections, was pale bluepurple with one or two small dark purple inclusions. As the body increased in size the cytoplasm became darker and the purple inclusions more numerous (fig. 3). At a later stage the purple inclusions became bigger and much less dense. Finally the intracytoplasmic body, which was now about 40 urn in diameter, consisted of 25 or more distinct elongated parasites about 5 flm in length, having an eosinophilic cytoplasm and a fairly dense purple nucleus. Not infrequently these parasites occurred also in rosette formation. One host cell contained an intracytoplasmic clump of parasites that were only 2 urn long. The parasites did not stain more distinctly with Giemsa, and they did not take up the Gram stain. An occasional clump of mature parasites was associated with PAS-positive granules. Electron Microscopy The developmental forms of the parasite were identical in both cases and were mainly in the cytoplasm of hypertrophic astrocytes, sometimes apparently free in the parenchyma and occasionally in macrophages. The blue bodies seen under the light microscope consisted of an intracytoplasmic mass, bounded by a double membrane, containing mitochondria, micronemes, chromatin-like condensations, and many ribosomes. At a later stage there was focal condensation of chromatin-like material to form circular membrane-bound structures (nuclei precursors) (fig.4). Still later there was invagination of the surrounding outer cell membrane to take in a nucleus and other cytoplasmic elements to form a colony of immature daughter parasites (fig. 5). Further development of the colony was associated, in some instances at least, with the peripheral migration of the contained parasites to form a rosette within the cytoplasm of the astrocyte. At this stage the double outer cell
Sporozoan Encepha lomye litis 5 a b d e ---_... / Fig. 3. Cases I and II. Spi nal cord. Various stages in the intracy top lasmic developm ent of the parasite. HE.
4 5 Fig. 4. Early schizont within a n astrocyte. Fig. 5. Later schizont with invaginati on of the outer memb rane to form dau ght er cells.
6 Fig. 6. Maturing pa rasite s in rosett e forma tion with one still a tta ched to a cen tral residual body. Fig. 7. Mature schizo nt con ta ining mero zoites. 7
8 HARTLEy /BLAKEMORE a b Fig. 8. Merozoites. C = conoid ; Er = endo plasmic reticulum ; Mn micronemes ; Mt = subpellicular micro tubules ; N = nucleus; PO = paired orga nelles; PP = posterior pore. a Immatur e merozoite fro m a rosette. b Mature merozoite. membran e of the par asite was complete except at its posterior end, where its cytopl asm was co ntinuous with the rem aining undifferentiated cytoplasm at the centre of the rosette. The par asites themselves conta ined a deve loping co no id, micronemes, mitochondria, man y free ribosomes, and posteriorly large aggregate s of spira lly arranged rough endoplasmic reticulum. At a slightly later sta ge of development the outer cell membrane of the par asites was complete, except in one instance in which the parasite was still connected posteriorly to what was prob ab ly a residual body (fig. 6). The latter was membrane-bound and co ntained numerous various-sized vesicles, mit ochondria a nd ribo somes. T he maturing parasites now had subpellicular microtubules, more microneme s, less rou gh endo plas mic reticulum, occasional large vesicles, but no evidence of paired or gan elles (fig. 8a).
Spo rozoan Encepha lomye litis 9 Fig, 9. Parts of mature merozoites. C = conoid; Me = mit ochon dri a; Mn = micronernes ; Mv = microvi lli ; PO = paired or ganelles ; N = nucleus. T he next stage was a co lony of mature parasite s (fig. 7) ind iscriminately a rra nged within the cyto plasm of th e host cell. T he intracytop lasmic mat ure individu al pa rasite was S- 7 1lm lo ng a nd 2-3 11 m wide (fig. 8b). T he a nterior end was always d istend ed as co mpa red with th e poster ior extremity, and the nucleus was situa ted posteri orly. Th ere was a well-developed striated pellicle with ap prox ima te ly 24 subpellicular micro tubules. T here was a lso a distinct spira l cono id, a posterior pore, at least two sets of pair ed or ga nelles, lar ge mit ochondria, free rib osom es and some ro ugh endo plasmic ret iculum (fig. 8, 9). Th ere were also numer ou s micronernes lar gely co nfined to t he anterior half of th e parasite. Most of th ese struct ures were of uni form moderate electro n de nsity but so me, pa rticularly in the imm atu re merozo ites (fig. 8a), had distinct co nce ntric ba nd s of low a nd high electron den sity. No micr opore and no Golgi a ppa ra tus co uld be pos itive ly ide nt ified. T he cytop lasm co nta ining these mature parasites co n ta ined nume ro us elonga ted micro villi (fig. 9). At no stage of development was th ere evide nce of a definite parasito ph orus vac uo le.
10 HARTLEy/BLAKEMORE Serology Two of the 20 sera taken from lambs in contact with the sheep in case I had low positive Toxoplasma dye test titres of I :64. None of six sheep previously showing neurologic signs had titres, and only one of six others in contact with the sheep in case II had a low positive titre of I :64. Discussion The morphologic observations on this ovine parasite indicate that it goes through a complete schizogonic cycle in astrocytes with the production of numerous merozoites. It can thus be ascribed to the class Sporozoa. The mature merozoites have many of the ultrastructural characteristics of the trophozoites of Toxoplasma [15]; however, they differ somewhat from the merozoites produced by Toxoplasma in the cat small intestine [14]. As schizogony with oocyst formation has, to date, been found only in Felidae [8, II] and only in the small intestine and bile ductules [3,6], it is likely that the schizogonic cycle we saw in the central nervous system (CNS) of sheep was not caused by Toxoplasma. This view is confirmed by the very low incidence of antibodies to Toxoplasma in affected and unaffected in-contact sheep. Our merozoites also have many of the ultrastructural features of the merozoites of several other sporozoans including Coccidia, Besnoitia and Sarcosporidia, The presence of a presumed S. tenella cyst in the brain of one of our sheep is worthy of comment because of their rarity in the CNS. Recent experimental work from Germany [13] has indicated that S. tenella derived from sheep oesophagus undergoes a cycle similar to that of Toxoplasma in the intestine of cats. To date, however, no detailed account of the morphology of the intestinal schizogonic cycle has been published. In view of the unusual location of the schizogony and since there was minimal evidence of a gametogonic cycle - one possible clump of microgametocytes - it seems likely that our cases represent an aberrant dead-end cycle of a known ovine sporozoan, rather than infection by a new species. Because of the close similarity of the late schizogonic stages of our parasite with the trophozoite and early cystozoite stages of Toxoplasma it is probable that these two parasites have been confused. The first account of a field case of ovine Toxoplasma encephalomyelitis [12] describes a sporadic case in an adult sheep in the United States. In that case the inflammatory lesions were far more severe in the cervical and thoracic cord than in the brain and were associated with 'cyst-like structures with the typical morphology of
Sporozoan Encephalomyelitis 11 Toxoplasma', including some in rosette formation. As that case has many features in common with ours, it is conceivable that all three sheep were affected by the same parasite. CORNER et al. [2] reported a severe disease outbreak in dairy cows in Canada that was associated with disseminated infection by a Toxoplasma-like organism parasitizing vascular endothelium; however, the authors were not able to confirm infection by this parasite by serological and biological methods. LAINSON [10] has reported two intracellular protozoan parasites, one from the nine banded armadillo from British Honduras, and the other from a calf in England. He considered that both were undergoing schizogony in a variety of ti ssues but not gametogony. Several years ago, one of us (W.]. H.) had the opportunity to examine histological sections derived from the animals in both the Canadian and English cases. A comparison between these bovine cases and our ovine cases indicated that all developmental forms of the parasites, no matter in which tissue they occurred, had a closely similar morphology. Whether they represent one species of sporozoan or a number of related ones is unknown. Acknowledgements The authors thank Mr. B. L. MUNDAY and Miss J. C. KATER, respectively, of the Tasmanian and New South Wales Departments of Agriculture for referring these cases; Mr. K. MCWILLIAM who prepared the histological sections; and Dr. J. K. FRENKEL of the University of Kansas Medical Center and Prof. E. SCHOLTYSECK of the University of Bonn for their assistance. Dr. BLAKEMORE was supported in part by the National Fund for Research into Crippling Diseases and by the Wellcome Trust. References 1 COLE, C. R.; SANGER, Y. L.; FARRELL, R. L., and KORDER, J. D.: The present status of toxoplasmosis in veterinary medicine. Nth amer. Yet. 35: 265-270 (1954). 2 CORNER, A.H.; MITCHELL, D.; MEADS, E.B., and TAYLOR, P.A.: Dalmeny disease. An infection ofcattle presumed to be caused by an unidentified protozoan. Canad. vet. J. 4: 252-264 (1963). 3 DUBEY, J. P. and FRENKEL, J. K.: Cyst induced toxoplasmosis in cats. J. Protozool. /9: 155-177 (1972). 4 HARTLEY, W. J.: Unpubl. observations.
12 HARTLEy/BLAKEMORE 5 HARTLEY, W. J. and KATER, J. c.: Observations on diseases of the central nervous system of sheep in New Zealand. New Zeald vet.j.10: 128-142 (1962). 6 HUTCHISON, W. M. and DUNACHIE, J. F.: The life cycle of the coccidian parasite Toxoplasma gondii in the domestic cat. Trans. roy. Soc. trop. Med. Hyg. 65: 380-399 (1971). 7 INNES, J. R. M. and SAUNDERS, L. Z.: Comparative neuropathology, pp. 345, 508, 514 (Academic Press, New York 1962). 8 JEWELL, M. L.; FRENKEL, J. K.; JOHNSON, K. M., and RUIZ, A.: Development of Toxoplasma oocysts in neotropical Felidae. AmeLJ. trop. Med. Hyg. 21: 512-517 (1972). 9 KOESTNER, A. and COLE, C. R.: Neuropathology of ovine and bovine toxoplasmosis. AmeLJ. vet. Res. 22: 53-66 (1961). 10 LAINSON, R.: A note on Sporozoa of undetermined taxonomic position in an armadillo (Dasypus novemcinctus edentata ) and a heifer calf (Bos taurus). J. Protozool. 19: 582-586 (1973). II MILLER, N. L.; FRENKEL, J. K., and DUBEY, J. P.: Oral infections with Toxoplasma cysts and oocysts in felines, other mammals and in birds. J. Parasit. 58: 928-937 (1972). 12 OLAFSON, P. and MONLUX, W.S.: Toxoplasma infection in animals. Cornell Vet. 32: 176-190 (1942). 13 ROMMEL, M. VON und HEYDERN, A. 0.: Beitrage zum Lebenszyklus der Sarkosporidien. I. Die Sporozyste von S. tenella in den Fazes der Katze. Berl. Munch. tierarztl, Wschr, 85: 101-105 (1972). 14 SHEFFIELD, H.G.: Schizogony in Toxoplasma gondii: an electron microscope study. Proc. helm. Soc., Wash. 37: 237-242 (1970). 15 SHEFFIELD, H.G. and MELTON, M.L.: The fine structure and reproduction of Toxoplasma gondii. J. Parasit. 54: 209-226 (1968). 16 WICKHAM, N. and CARRE, H.R.: Toxoplasmosis in domestic animals in Australia. Austr. vetj. 26: 1-3 (1950). 17 ZLOTNIK, I.: Toxoplasma in sheep. Lancet ii: 295 (1959). Request reprints from: W.J. HARTLEY, Department of Veterinary Medicine, The University of Sydney, Camden, 2570 NSW (Australia)