Sarcocystosis with involvement of the central nervous system in lambs

J Vet Diagn Invest 5:291-296 (1993) Sarcocystosis with involvement of the central nervous system in lambs Scott D. Fitzgerald, Evan B. Janovitz, Kevin R. Kazacos, J. P. Dubey, Duane A. Murphy An outbreak of neurologic disease in a flock of 5-month- taining large rounded metrocytes and early bradyzoites, and old Rambouillet lambs on a sheep farm in west-central In- were associated with some degree of myositis with degendiana was investigated. Over a 4-week period during the eration of muscle fibers and lymphocytic perimysial infiltrasummer of 1990, 32 of 48 lambs in 1 pen were affected, and tion. 22 of these lambs died. Approximately 300 sheep were on Multifocal, nonsuppurative meningoencephalomyelitis was the farm. The affected lambs were confined to a single pen evident in the cerebrum, cerebellum, brain stem, and cerin a large open-sided pole barn and were the youngest group vital, thoracic, and lumbar spinal cord of all 3 lambs (Fig. of spring lambs on the farm. 1). Inflammation was characterized by glial nodules, multi- Clinical signs described by the owner and referring vet- focal necrosis with gliosis and mononuclear cell infiltration, erinarian included anorexia, weight loss, incoordination, stiff and lymphocytic infiltration around blood vessels and in the limbs, trembling, and generalized weakness. The condition meninges (Fig. 2). An immature schizont was found in the progressed rapidly in fatal cases, and death generally occurred pons of 1 of these lambs (Fig. 3). The density of neuroglia within 7 days of the onset of clinical signs. around this schizont was increased, but the schizont was not Three clinically affected lambs (2 live and 1 dead) were surrounded by microglia. Immunohistochemical examinasubmitted to the Animal Disease Diagnostic Laboratory, tion of paraffin-embedded sections of samples of brain from Purdue University, West Lafayette, Indiana, for diagnostic all 3 lambs using antisera to Toxoplasma gondii, Neospora evaluation. Both live lambs were thin, weak, and ataxic. All caninum, and Sarcocystis spp. was negative. 11 The FA test 3 lambs had bronchopneumonia of varying severity. One for pseudorabies was negative, and no viral agent was isolamb had moderate subcutaneous edema of the neck and lated. inguinal region and mild serous effusions in the pleural, peri- The small intestine of 1 lamb was infected by coccidia and cardial, and peritoneal cavities. Tissue samples from all 3 cryptosporidia. After the diagnosis of intestinal coccidiosis lambs were routinely processed for histopathologic exami- was conveyed, the referring veterinarian directed the owner nation. Samples of pneumonic lung were inoculated on blood to add amprolium to the lambs feed. Following treatment, and MacConkey s agar in an attempt to culture for aerobic affected lambs improved clinically, no more died, and no bacteria. Selected areas of the pneumonic lungs were ex- additional lambs developed clinical signs. amined for bovine respiratory syncytial virus (BRSV), in- Approximately 6 weeks later, a team of diagnostic perfectious bovine rhinotracheitis virus (IBR), and sonnel visited the farm for an on-site evaluation, to collect Parainfluenza-3 virus (P13) by direct immunofluorescence convalescent sera from affected and nonaffected lambs, to (FA) and were inoculated onto bovine turbinate cells for virus collect fecal samples from dogs, and to choose a convalescent isolation. A portion of the brain from 1 of the live lambs lamb for further diagnostic evaluation. was examined for pseudorabies virus by FA and was also Hay was found stacked in 1 comer of the pole barn, and inoculated onto cell cultures. grain was stored in uncovered wooden bins at the opposite Pneumonic processes were suppurative, compatible with side. At least 10 Great Pyrenees dogs were present to guard bacterial infection; however, no aerobic bacteria were iso- the sheep from predators. These dogs had free access to all lated from lungs. In 2 of the lambs, there was also hyperplasia locations on the farm and generally remained with the sheep of peribronchiolar lymphoid tissue, suggestive of mycoplas- at all times. One pup that was 8 months old at the time of mosis. The lungs of all 3 lambs were negative for IBR, BRSV, the visit had been raised with the affected lambs, spending and PI3 by FA, but PI3 was isolated from 1 lamb. most of its time in the same pen. According to the owner, Striated muscles (diaphragm, tongue, esophagus, triceps, at least 20 free-roaming cats had access to the pens and feed adductor, rectus femoris and psoas muscles, and the myo- storage areas, but these were subsequently removed. cardium) in all 3 lambs contained numerous sarcocysts (up The owner indicated that sheep occasionally died on the to 65/cm 2 in the cardiac muscle of 1 of the lambs) 50-250 woods or pasture, and carcasses were available for con- µm in diameter. Many of the sarcocysts were immature, con- sumption by his dogs. It was possible that while in the pen, the pup had access to lambs tails removed at docking or to parts of a carcass brought in by another dog. From the Animal Disease Diagnostic Laboratory and the De- Rectal fecal samples were collected from 4 of the Great partment of Veterinary Pathobiology, School of Veterinary Medi- Pyrenees dogs, including an &year-old lactating female, an tine, Purdue University, West Lafayette, IN 47907 (Fitzgerald, Janoadult male, a 1.5-year-old female, and the &month-old male vitz, Kazacos, Murphy), and the Zoonotic Parasites Laboratory, Livestock and Poultry Sciences Institute, ARS, USDA, BARC-East, that had been raised with the affected lambs. Four samples Beltsville, MD 20705 (Dubey). Current address (Fitzgerald): Animal of dried feces (presumably from the dogs) were recovered Health Diagnostic Laboratory, College of Veterinary Medicine, from the ground near the hay stored in the barn. Fecal sam- Michigan State University, East Lansing, MI 48824. ples were hydrated and mixed with concentrated sucrose so- Received for publication July 18, 1991. lution (specific gravity = 1.30), filtered, processed by cen- 291

292 Brief communications Figure 1. Photomicrograph of caudate nucleus from a clinically affected lamb with sarcocystosis. Note glial nodules randomly scattered in gray matter. trifugal flotation, and examined microscopically for parasite the g-year-old dog low numbers of Sarcocystis sporocysts. eggs, larvae, and cysts. Sporocysts from the 3 dogs were measured to the nearest 0.5 Three of the 4 dogs were positive for helminth eggs and µm and compared morphologically. Sporocysts from the 2 Sarcocystis sporocysts in the feces. The helminth eggs found younger dogs (13 from each dog) were 14-15 x 9-10.5 µm included Trichuris, Ancylostoma, Taenia, and Toxocara; (X = 14.5 x 9.6 µm). These measurements and other mor- Toxocara was found in the pup only. The pup was shedding phologic characteristics indicate the sarcocysts were Sarcohigh numbers, the 1.5-year-old dog moderate numbers, and cystis tenella. 5 Figure 2. Photomicrograph of cerebral cortex from a clinically affected lamb with sarcocystosis. This glial nodule was at the junction of the gray and subcortical white matter. In the adjacent inner layer of cerebral cortex, several neuroglia were pyknotic.

Brief communications 293 Figure 3. Photomicrograph of pons from a clinically affected lamb with sarcocystosis. Note the immature schizont (second generation) with merozoites toward the periphery of the central nuclear mass. Approximately 1 week after this farm visit, a lamb that had recovered from clinical signs was euthanized and examined at necropsy. The bronchopneumonia in this convalescent lamb was more severe than that in the other 3 lambs. A retrosternal lymph node was abscessed. Pasteurella haemolytica and Mycoplasma ovipneumoniae were isolated from the lung. Haemonchus contortus and other trichostrongyles were in the abomasum of the convalescent lamb. Histologically, meningoencephalomyelitis was similar to but less extensive than in the other 3 lambs. Four mature sarcocysts were found in randomly selected sections of the brain and spinal cord: 1 in the cerebrum, 1 in the brain stem, and 2 in the spinal cord (Fig. 4). Mature sarcocysts were not intimately associated with glial nodules but were in areas of the CNS with inflammation. Numerous mature sarcocysts containing a myriad of 2- x 5-µm bradyzoites were in skeletal muscles (Fig. 5), but only a few were in the myocardium. Sections of skeletal muscle with high numbers of mature Figure 4. Photomicrograph of spinal cord from a convalescent lamb with sarcocystosis. This mature sarcocyst containing bradyzoites was at junction of gray and white matter.

294 Brief communications Figure 5. Photomicrograph of rectus femoris muscle from a convalescent lamb with sarcocystosis. Note the high density of mature sarcocysts in muscle fibers. sarcocysts from the convalescent lamb were postfixed in 3% glutaraldehyde and routinely processed for transmission electron microscopy. The protozoa1 cysts within the skeletal muscle contained numerous mature bradyzoites. Bradyzoites had a single, prominent nucleus and, in the anterior half of the cytoplasm, numerous micronemes but no rhoptries. No septa were present within the cyst. The cyst wall was thick (approximately 3 µm) and had numerous, tightly packed, evenly spaced, villous protrusions arranged in parallel, which interdigitated with intracellular membranes (sarcoplasmic reticulum) of the host myocyte (Fig. 6). The villous protrusions had plaques at their apices and contained no microtubules. These features were consistent with the cyst structure of S. tenella. 5 The diagnosis of sarcocystosis was based on the presence of developing sarcocysts within inflammatory lesions in skeletal muscle and myocardium. In the clinically affected lambs, sarcocysts containing metrocytes and early bradyzoites were indicative of infection 5-6 weeks prior to necropsy. 4 Although only 1 immature sarcocyst was found in the central nervous system (CNS) of any clinically affected lamb, the nature of the inflammatory response (nonsuppurative to granulomatous) was compatible with Sarcocystis as an etiology. Furthermore, the mature sarcocysts in the brain of the convalescent lamb indicated that there had been infection of the CNS. Definitive diagnosis of Sarcocystis-induced CNS lesions, i.e., presence of schizonts in CNS endothelium in association with nonsuppurative inflammation, is difficult because this stage is transient and precedes clinical signs. 4 The bronchopneumonia was probably not directly caused by Sarcocystis; no organisms were found in the lung and the nature of the inflammation was typical of infection with the organisms isolated from the convalescent lamb. Differential diagnoses for ovine meningoencephalitis include listeriosis, pseudorabies and other viral encephalitides, toxoplasmosis and neosporosis, and parelaphostrongylosis. Although brains were not cultured for bacteria, listeriosis was considered unlikely because there was a lack of suppurative inflammation in the brain stem of these lambs. Pseudorabies was not identified by either FA or virus isolation, and neither primary neural degeneration nor viral inclusions were seen histologically in any CNS sections. Furthermore, pseudorabies in sheep tends to have a rapidly fatal clinical course. 1 In cerebral toxoplasmosis, necrosis is usually more extensive than was seen in these lambs with sarcocystosis. Because Toxoplasma gondii cysts are located within parasitophorous vacuoles whereas Sarcocystis cysts are not, 6 electron microscopy may differentiate these protozoa; however, sections of brain were not examined by electron microscopy because so few sarcocysts were seen. Immunocytochemical methods can also differentiate these protozoa; 7 the lack of positive staining in the brains of these lambs is consistent with the life cycle of Sarcocystis spp., which transiently infect the CNS before becoming encysted in striated muscle. Parelaphostrongylus tenuis, a nematode that infects the meninges of white-tailed deer, may cause myelitis in aberrant hosts such as sheep. Lesions of parelaphostrongylosis mostly involve the white matter of the spinal cord and are characterized by necrotic tracts with varying degrees of histiocytic infiltration, gliosis, demyelination, and cavitation. 8 These lesions were not seen in the spinal cords of lambs, nor were any nematode larvae found histologically. At least 4 species of Sarcocystis have been described in sheep, which serve as intermediate hosts. 5 Carnivores, primarily canids and felids, are the usual definitive hosts for these organisms. Canids serve as definitive hosts for S. tenella and S. arieticanis, and felids for S. gigantea and S. medusiformis. These species of Sarcocystis may be identified by

Brief communications 295 Figure 6. Transmission of electron micrograph of a sarcocyst within the sarcoplasm (A) of a muscle fiber from a convalescent lamb with sarcocystosis. Note the evenly spaced, villous protrusions arranged in parallel (B) of the cyst wall, consistent with Sarcocystis tenella, and the bradyzoites (C) within the cyst. 6,500 x. ultrastructural features of their cyst wall, in particular the presence and type of villous protrusions. 5 The sarcocysts in the skeletal muscle of the lambs in this report had ultrastructural features consistent with S. tenella. In a recent survey, S. tenella was the most prevalent (84% of sheep surveyed) species of Sarcocyst present within muscles of sheep slaughtered in the USA. 3 In sheep experimentally infected with S. tenella, clinical signs and lesions were associated with the endothelial schizonts rather than with the encysted sarcocysts in striated muscle. 2,10 Neurologic disease and lesions, including multifocal gliosis and perivascular mononuclear cell infiltration with mature sarcocysts in the neuropil, were similar to what was found in the lambs in this report. This is the first report of a flock outbreak of sarcocystosis in which large numbers of lambs had clinical CNS disease and high mortality. The close association of lambs and dogs on this farm was considered responsible for the epizootic. It was not determined conclusively why the lambs in only 1 pen were affected. These lambs may have been particularly susceptible because they were the youngest lambs on the farm. Alternatively and more likely, affected lambs were probably exposed to larger numbers of sporocysts than were unaffected sheep because the pup that was raised with theaffected lambs was shedding high numbers of sporocysts and disease due to Sarcocystis has been shown to be dose related. 5 Methods of controlling sarcocystosis in sheep have been previously summarized. 5 Although exclusion of dogs from the premises would have been effective in preventing further contamination of the environment with sporocysts, the sheep farmer preferred using dogs for raising his sheep and protecting them from predators. Strict removal of dead lambs and sheep was recommended to prevent dogs from ingesting ovine muscle tissues. There is no treatment currently available for dogs shedding sporocysts, and these stages can last for several months in the environment. Strict sanitation concerning dog feces and administration of amprolium to future lambs were recommended to prevent further epizootics. The apparent beneficial effect of amprolium was attributed to prevention of further infection and inhibition of development of schizonts in the lambs. Amprolium is effective in reducing mortalities and decreasing the severity of clinical disease in sheep when administered before, or concurrent with, sporocyst ingestion. 9 Amprolium interferes with schizogony but probably has no effect on mature sarcocysts; 5 sarcocysts were numerous in the convalescent lamb despite several weeks of amprolium therapy. Acknowledgements. We thank Janeice Samman for preparation of histopathology specimens, Sam Royer for technical assistance with photography, Deborah Van Horn for preparation and photography of electron microscopy specimens, Mary Woodruff for virology, and Dr. Terry Bowersock for bacteriology. This report is published as journal paper no. 13070, Purdue Agricultural Experiment Station, West Lafayette, IN 47907. References 1. Clark LK, Molitor TW, Gunther R, et al.: 1984, Pathogenicity of a modified-live pseudorabies vaccine virus in lambs. J Am Vet Med Assoc 185:1535-1537.

296 Brief communications 2. Dubey JP: 1988, Lesions in sheep inoculated with Sarcocystis tenella sporocysts from canine feces. Vet Parasitol 26:237-252. 3. Dubey JP, Lindsay DS, Speer CA, et al.: 1988, Sarcocystis arieticanis and other Sarcocystis species in sheep in the United States. J Parasitol 74: 1033-1038. 4. Dubey JP, Speer CA, Callis G, Blixt JA: 1982, Development of the sheep-canid cycle of Sarcocystis tenella. Can J Zool 60: 2464-2477. 5. Dubey JP, Speer CA, Fayer R: Sarcocystosis of animals and man. CRC Press, Boca Raton, FL. 6. Dubey JP, Speer CA, Munday BL, Lipscomb TP: 1989, Ovine sporozoan encephalomyelitis linked to Sarcocystis infection. Vet Parasitol 34: 159-163. 7. Jeffrey M, O Toole D, Smith T, Bridges AW: 1988, Immunocytochemistry of ovine sporozoan encephalitis and encephalomyelitis. J Comp Pathol 98:213-224. 8. Jortner BS, Troutt HF, Collins T, Scarratt K: 1985, Lesions of spinal cord parelaphostrongylosis in sheep. Sequential changes following intramedullary larval migration, Vet Pathol 22:137-140. 9. Leek RG, Fayer R: 1980, Amprolium for prophylaxis of ovine Sarcocystis. J Parasitol 66: 100-106. 10. Leek RG, Payer R, Johnson AJ: 1977, Sheep experimentally infected with Sarcocystis from dogs. I. Disease in young lambs. J Parasitol 63:642-650. 11. Lindsay DS, Dubey JP: 1989, Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am J Vet Res 50: 1981-1983. J Vet Diagn Invest 5:296-300 (1993) Intramuscular Sarcocystis in two beluga whales and an Atlantic white-sided dolphin from the St. Lawrence estuary, Quebec, Canada Sylvain De Guise, André Lagacé, Christiane Girard, Pierre Béland A small isolated population of around 500 beluga whales (Delphinapterus leuces) live in permanence in the St. Lawrence estuary. 17 They have been found to be highly contaminated, mostly with organochlorines and heavy metals, and to show many pathological lesions. 3,14,15 Sarcocystis is a protozoan parasite with an obligatory 2-host life cycle. The intermediate host, usually a herbivore, is infected by ingesting sporocysts from the feces of an infected definitive host, usually a carnivore. The definitive host becomes infected by ingesting the encysted form of the parasite present in the musculature of the intermediate host 8 Sarcocystis has been found in many animal species, and in a few marine mammals including pinnipeds 4,5,18 and cetaceans, 1,6,7,19,20 but to the authors knowledge, it has never been reported in belugas and Atlantic white-sided dolphins. The present paper reports Sarcocystis in 2 beluga whales and an Atlantic white-sided dolphin (Lagenorhyncus acutus) from the St. Lawrence, and comments on the possible life cycle. During a study on the pathology of marine mammals in the St. Lawrence river and estuary, stranded carcasses were recuperated and carried to the necropsy room of the Veterinary School of the Université de Montréal, whether fresh (belugas) or frozen (dolphins, porpoises, and seals). After a complete gross examination, tissues were fixed in 10% buffered formalin, embedded in paraffin, cut into 5-7-µm sections, and routinely stained with hematoxylin, phloxin, and saffron (HPS) for histopathology. Formalin-fixed tissues selected for electron microscopy were fixed with 5% glutaraldehyde, postfixed in 1% osmium tetroxide, and stained en bloc with uranyl acetate. The small blocks were then embedded in epon, cut to 80 nm, and stained with lead citrate. The sections were examined with a Philips 201 transmission electron microscope. From the Faculte de Medecine Veterinaire, Universite de Montreal, 3200 Sicotte, C.P. 5000, St-Hyacinthe, Quebec J2S 7C6, Canada (De Guise, Lagact, Girard), and the Institut National d Écotoxicologie du St-Laurent, 3872 Parc Lafontaine Montreal, Quebec H2L 3M6, Canada (De Guise, Béland). Received for publication April 5, 1992. Figure 1. Microphotograph of a thin-walled sarcocyst in the striated muscle of an adult beluga whale (Delphinapterus leucas) (DL- 5-88). Note the thin septa (arrow) dividing the sarcocyst into compartments filled with bradyzoites and the central area where few bradyzoites can be seen. Hematoxylin, phloxin, and saffron. Bar = 50 µm.