A Review of Neospora Caninum and Neospora-Like Infections in Animals

A Review of Neospora caninum and Neospora-like Infections in Animals

J. P. DUBEY

Zoonotic Diseases Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U. S. Department of Agriculture, BARC-East, Beltsville, Maryland 20705-2350, U. S. A.

Received 27 January 1992/ Accepted 15 March 1992

Key words: Neospora caninum, neosporosis, tachyzoites, tissue cysts, paralysis, abortion

ABSTRACT: Neospora caninum is a recently recognized protozoan parasite of animals. Until 1988, it was misdiagnosed as Toxoplasma gondii. Neospora caninum or Neospora-like parasites cause paralysis and death in dogs and neonatal mortality and abortion in cattle, sheep, goats and horses. Its life cycle is not known. Tachyzoites and tissue cysts are the only asexual stages known and the carnivorous definitive host is not known. Transplacental transmission is the only known natural route of infection.

INTRODUCTION AND HISTORY Neospora caninum is a recently recognized protozoon of dogs and other animals. Until 1988, it was misdiagnosed as Toxoplasma gondii. Neosporosis is not a new disease because in retrospective studies N. caninum was found in dogs in the United States that died in 1957 and 1958 (Dubey et al., 1988a; Dubey et al., 1990e). Historically, a neosporosis-like disease was first reported by Bjerkas et al. (1984). They identified a cyst-forming protozoon in a litter of six Boxer dogs in Norway. Five of these dogs developed neurologic disorders two to six months after birth. Toxoplasma gondii-like parasites were found in lesions in brain and muscles. However, T. gondii antibodies were not found in the sera of the dogs, nor was the parasite from dogs infective to mice. Dubey et al. (1988a) found a similar parasite in ten dogs in the United States, distinguished it from T. gondii, and named the parasite N. caninum. Dubey et al. (1988b) recovered live N. caninum in cell cultures and mice inoculated with tissues from naturally infected dogs and they induced

40 NEOSPOROSIS IN ANIMALS neosporosis in dogs experimentally. The development of an immunohistochemical test to specifically stain N. caninum in formalin-fixed tissues using anti-N. caninum serum raised in rabbits made it possible to diagnose Neospora infections in animals (Lindsay and Dubey, 1989a). Bjerkas and Dubey (1992) compared structure and antigenicity of the parasites in fixed tissues from dogs from Norway and U. S. A. and concluded that the parasite originally reported from Norwegian dogs was N. caninum or closely related to it. In this review the N. caninum-like parasites from livestock are considered Neospora although N. caninum has not been yet isolated and characterized from livestock.

STRUCTURE AND LIFE CYCLE The life cycle is unknown but the close similarities of the organism to T. gondii suggest that the parasite is a coccidian with a carnivore as the definitive host. Tachyzoites and tissue cysts are the only known stages. Tachyzoites are ovoid, lunate or globular 3 to 7 x 1 to 5 µm, depending on the stage of division (Figs. 1, 2). They divide into two zoites by endodyogeny. In infected animals, tachyzoites are found in neural cells (axons, Schwann cells, neurons, ependymal cells, retinal cells, astrocytes), macrophages, fibroblasts, vascular endothelial cells, myocytes, renal tubular epithelial cells, hepatocytes and other cells of the body (Cummings et al., 1988; Dubey et al., 1988a; Bjerkas and Presthus, 1989; Dubey and Lindsay, 1989b; Speer and Dubey, 1989). Host cells may contain as many as 100 tachyzoites. Tachyzoites are located within the host cell cytoplasm with or without a parasitophorous vacuole (PV). Few, many or no intravacular tubules may be present in the PV (Fig. 2). Why some tachyzoites are in a PV and others are without a PV is unknown. Neospora caninum tachyzoites have organelles typically found in T. gondii tachyzoites. Tachyzoites have a three layered plasmalemma, 22 subpellicular microtubules, two apical rings, a conoid, a polar ring, one to three mitochondria, up to 150 micronemes, eight to twelve rhoptries anterior to the nucleus and four to six rhoptries posterior to the nucleus, a Golgi complex, rough and smooth endoplasmic reticulum, a nucleus and a nucleolus (Bjerkas and Presthus, 1988; Speer and Dubey, 1989). Some of the micronemes may be perpendicular to the inner parasite membrane. The rhoptries contain solid electron dense material and are two to four times thicker than the diameter of the micronemes (Fig. 2). The descrepancy in the number of rhoptries reported to be up to 30 by Bjerkas and Presthus (1989) and up to 18 by Speer and Dubey (1989) may be due to difficulty in distinguishing rhoptries from dense granules (Bjerkas and Dubey, 1992). Micropores have not been seen in tachyzoites in animals (Speer and Dubey, 1989; Bjerkas and Presthus, 1989) but were found in tachyzoites grown in cell cultures (Speer and Dubey, 1989). Tissue cysts are often round to oval, up to 107 µm long and are found only in neural tissues (brain, spinal cord and retina). The cyst wall is smooth and up to 4 µm thick, depending upon how long the infection has existed (Figs. 3, 4, 5). In most tissue cysts the cyst wall is 1 to 2 µm thick. The cyst wall contains branched tubule-like structures (Bjerkas and Presthus, 1988). Septa are absent and there is no secondary cyst wall.

41 NEOSPOROSIS IN ANIMALS

Figures 1-5 Photographs of Neospora caninum. Figure 1.Tachyzoites (arrowheads) in section of liver of an experimentally infected rat. Note dividing tachyzoites (arrow) are larger in size than nondividing tachyzoites. H&E stain. x 750. Figure 2. Transmission electron micrograph of tachyzoites. Note six rhoptries (arrowheads), nucleus (N), a conoid (C), and parasitophorous vacuole (PV). x23,810. Figure 3. Tissue cyst in mouse brain 13 months after experimental infection. Unstained x 750. Note thick cystwall (opposing arrowheads). Figure 4. Tissue cyst in a neuron in spinal cord of a naturally infected bovine calf. Note thick cyst wall (arrowhead). The host cell nucleus is on left. H&E stain. x 750. Figure 5. Tissue cyst in the spinal cord of a bovine calf. Avidin-biotin complex immunohistochemcial reaction with anti-W. caninum serum. x 750.

42 NEOSPOROSIS IN ANIMALS

Bradyzoites are slender (6 to 8 x 1 to 1.8 µm) and contain the same organelles as are found in tachyzoites except that there are fewer rhoptries in bradyzoites and more PAS-positive (amylopectin) granules in the bradyzoites. The cyst wall stains variably with PAS and is argyrophilic. Tubular vesicular structures are present in between bradyzoites and bradyzoites may contain micropores (Bjerkas and Presthus, 1989). Bjerkas and Presthus (1989) found micropores in bradyzoites in tissue cysts from the Norwegian dogs whereas micropores were not seen in bradyzoites from dogs in U.S.A. However, it must be emphasized that bradyzoites from only one well fixed tissue cyst were examined by Speer and Dubey (1989). Fixation and the age of tissue cysts might also affect structure of organelles. These fine points can only be resolved when tissue cysts can be produced in large number in experimentally infected animals. Neospora or a Neospora-like parasite has been found in dogs, cattle, sheep, horses and goats in nature and the parasite can be transmitted to dogs, cats, mice, rats, gerbils, cattle and sheep experimentally (Dubey, 1990; Cudden et al., 1992). The only natural mode of infection identified so far is congenital (Dubey and Lindsay, 1989b, c). Transplacental infection was induced experimentally in dogs, cats, sheep, mice, and cattle (Dubey and Lindsay, 1989b, c; 1990a; Cole et al., 1991; Dubey et al., 1992b). Transplacental infection can occur repeatedly in the same animal. Up to three litters of congenitally infected pups were born from the same bitch (Bjerkas et al., 1984; Dubey et al., 1988b; Dubey et al., 1990e). Neospora infection in a cat before pregnancy led to congenital infection in a kitten (Dubey and Lindsay, 1989c). Neospora caninum tachyzoites were found in placenta and the uterus (Dubey and Lindsay, I989c). In experimental animals N. caninum is infective by the subcutaneous, intraperitoneal, intramuscular and oral routes. Bradyzoites in tissue cysts are resistant to acidic-pepsin solution indicating that carnivorism may be a part of the life cycle of N. caninum (Lindsay and Dubey, 1990c). Oocysts were not detected in feces of experimentally infected dogs and cats (Dubey et al., 1988b; Dubey and Lindsay, 1989a, b, c; Cudden et al., 1992). Neospora caninum tissue cysts can survive up to 14 days at 4OC but were rendered noninfective at –20OC for 1 day (Lindsay et al., 1991, 1992).

CULTIVAT ION Neospora caninum was initially cultivated in vitro in bovine monocytes (BM) and bovine cardio-pulmonary arterial (CPA) endothelial cells (Lindsay and Dubey, 1989b). Since that time N. caninum has been grown in Madin-Darby bovine kidney, fetal mouse brain and several other well established cell lines (Cole et al., 1990). More organisms are produced in BM than in CPA cells. Only tachyzoites have been identified in cultivated cells. Organisms from cell cultures are infective to animals (Dubey et al., 1988b; Lindsay and Dubey, 1989c, 1990c). Neospora caninum does not produce infection consistently in normal immunocompetent mice and rats. One way to infect rodents with N. caninum experimentally is to give 2 injections of 2 to 4 mg of

43 NEOSPOROSIS IN ANIMALS methylprednisolone acetate on seven days before and on the 0 day of the inoculation with ten or more N. caninum tachyzoites. Varying degrees of acute to chronic infection can be induced by varying the dose of N. caninum and corticosteroids given. Pneumonia and encephalomyelitis are the predominant signs of neosporosis in mice whereas rats develop hepatitis primarily (Lindsay and Dubey, 1989c, 1990b, c). The number of tissue cysts produced is variable and unpredictable. Tissue cysts in mice were much smaller (10-25 µm) than seen in naturally infected dogs (Lindsay et al., 1991). Neospora caninum can cause lethal infection in certain inbred mice not given corticosteroids.

HOST-PARASITE RELATIONSHIP Neospora caninum causes cell death by active multiplication of tachyzoites. It is capable of producing grossly visible lesions in a few days. It appears to be a primary pathogen because in naturally infected dogs with fatal neosporosis no other etiologic or modifying agents were identified (Dubey et al., 1988a). Neospora caninum can produce severe neuromuscular disease in dogs and probably other hosts. It can destroy a variety of neural cells including those of cranial and spinal nerves. The presence of large numbers of organisms may affect the conductivity of the affected cells. Tissue cysts are often not surrounded by a zone of host reaction. How long tissue cysts persist in the central nervous system is not known. Formation of granulomas around degenerating tissue cysts and bradyzoites suggest that some tissue cysts rupture and that the subsequent host reaction causes foci of inflammation (Dubey, 1990; Dubey et al., 1990c, 1992a). Administration of exogenous corticosteroids can exacerbate neosporosis (Dubey and Lindsay, 1990b; Dubey et al., 1990a)

TREATMENT Some of the drugs used for treatment of toxoplasmosis were tested against N. caninum. Lasalocid, monensin, piritrexim, pyrimethamine and trimethoprim prevented intracellular multiplication of N. caninum in infected cell cultures (Lindsay and Dubey, 1989c, d, 1990a). Sulfadiazine in drinking water prevented clinical neosporosis in experimentally infected mice (Lindsay and Dubey, 1990a). Treatment, however, was not effective if sulfadiazine was administered after clinical signs appeared.

NEOSPOROSIS IN DOGS Neospora caninum has been found in naturally infected dogs in the U.S.A. (Dubey et al., 1988a, b, 1990e; Braund et al., 1988; Cummings et al., 1988; Hay et al., 1990; Cudden et al., 1992; Hoskins et al., 1991); England (Dubey et al., 1990d; Trees et al., 1992; Mayhew et al., 1992); Norway (Bjerkas et al., 1984; Bjerkas and Presthus, 1988, 1989); Sweden (Hilali et al., 1986; Uggla et al., 1989a, b); Belgium (Poncelet et al., 1990a, b) Australia (Munday et al., 1990), Switzerland (Wolf et al., 1991), Canada (Cochrane and Dubey, 1992), Hungary (Sreter et al., 1992), South Africa (Jardine and Dubey, 1992), France, Germany, and Ireland (Dubey, unpublished). In dogs, the most severe disease is in young, congenitally infected pups. Young dogs develop an ascending paralysis with the hind limbs more severely affected than the front legs (Dubey et al., 1988a;

44 NEOSPOROSIS IN ANIMALS

Wolf et al., 1991; Cudden et al., 1992). Other dysfunctions which occur include difficulty in swallowing, paralysis of the jaw, muscle flaccidity, and muscle atrophy (Hay et al., 1990; Mayhew et al., 1991; Cudden et al., 1992). Dogs with hind limb paralysis may be alert and survive for months. The disease may be localized or generalized and virtually all organs may be involved including the skin (Dubey et al., 1988a). Subclinically infected dams can transmit the parasite to their fetuses, and successive litters from the same dam may be born infected (Dubey et al., 1988b; Bjerkas and Presthus, 1989; Dubey et al., 1990e). Whether there is breed predisposition and differential sex susceptibility to neosporosis in dogs is not known, although most described cases were in Labrador retrievers and Basset hounds (Cummings et al., 1988; Dubey et al., 1988a, b; Hay et al., 1990; McGlennon et al., 1990). The most severe neosporosis was observed in four litters of German Shorthaired Pointers (GSHP) from one owner. In this outbreak, twenty-nine of thirty-nine GSHP dogs had hindlimb paralysis. Six dogs from two litters were necropsied and N. caninum was found in all 6 dogs (Dubey et al., 1990e). The cause of the hindlimb hyperextension is not known but is most likely due to a combination of upper motor neuron paralysis and myositis resulting in rapidly progressive fibrous contracture of the muscles and resulting in fixation of joints (Mayhew et al., 1991). For antemortem diagnosis, clinical signs may be helpful. Ascending paralysis in young dogs, particularly if several littermates are affected should arouse suspicion of neosporosis. Hematological valves are usually not altered. There may be increased levels of serum enzymes associated with necrosis of myocytes and liver. The detection of N. caninum antibodies in serum or of the parasite in biopsy tissue can aid diagnosis (Dubey et al., 1988b; Hay et al., 1990). However, anti-N. caninum antibodies can be found in clinically normal dogs (Lindsay et al. , 1990b). Neosporosis resembles toxoplasmosis clinically; however the parasite can be distinguished immunologically. An indirect fluorescent antibody test using cell cultured derived whole tachzyoites has been described for the diagnosing of canine neosporosis (Dubey et al., 1988b). The presence of IFA antibodies alone does not indicate clinical neosporosis (Lindsay et al., 1990b). Treatment with sulfadiazine and pyrimethamine and clindamycin therapy might be useful if diagnosis can be made early (Greene et al., 1985; McGlennon et al., 1990; Mayhew et al., 1991). Efficacies of trimethoprim, sulfadiazine, and pyrimethamine for treatment of dogs with paralysis is not known. None of these drugs were effective in dogs with stiff legs (Hay et al., 1990). Treatment was successful in dogs with early neosporosis-induced limb weakness. The combination of trimethoprim and sulfadiazine (Tribrissen, Coopers Pitman-Moore) at the standard combined dose of 15 mg/kg twice daily and pyrimethamine (Daraprim, Burrows Wellcome) at 1 mg/kg daily, all for four weeks reversed N. caninum associated paralysis in dogs (Mayhew et al., 1991; McGlennon et al., 1990). Treatment with clindamycin (13.5 mg/kg 3 times daily) proved useful in treating protozoal myositis in a six-year-old

45 NEOSPOROSIS IN ANIMALS

Weimaraner dog initially diagnosed as toxoplasmosis but retrospectively considered to be neosporosis (Greene et al., 1985; Dubey et al., 1990a).

NEOSPOROSIS IN CATTLE Neospora caninum-like parasites were first detected in aborted fetuses from dairy cattle from New Mexico (Thilsted and Dubey, 1989) and in a stillborn beef calf from Maryland (Dubey et al., 1990f). Retrospective search led to identification of N. caninum-like parasites in a calf from Australia (Hartley and Bridge, 1975; Dubey et al., 1990b), in calves in U.S.A. (Parish et al., 1987; Dubey et al., 1989; Shivprasad et al., 1989) and England (O’Toole and Jeffery, 1987; Dubey, 1989). Well planned extensive studies of causes of abortion in dairy cattle from California led to the discovery that a N. caninum-like parasite was a major cause of abortion in dairy cattle (Barr et al., 1991; Anderson et al., 1991). In one study of aborted fetuses submitted to the Veterinary Diagnostic Laboratory in Tulare, California, N. caninum-like parasites were identified in 19% of 468 bovine abortion during the 5-year period (Anderson et al., 1991). Since that time N. caninum associated abortions were diagnosed in several other states in U.S.A. including Wisconsin and South Dakota (Nietfeld et al., 1992). Neosporosis is thought to be a major cause of abortion in dairy cattle in New Zealand and Australia (Thorton et al., 1991). Dubey et al. (1992b) found clinical neosporosis in a four-week-old Hereford calf that was apparently healthy at birth. Little is known of the pathogenesis of N. caninum- associated lesions and neonatal mortality in cattle. The aborted fetuses were three to nine-month gestational age with a mean age of 5.6 months (Anderson et al., 1991; Barr et al., 1991a). Protozoa reactive to N. caninum antisera were found more in early gestational fetuses than in late gestational fetuses and tissue cysts were more common in early gestational fetuses than in the late gestational fetuses. However, N. caninum-like tachyzoites were found in four-month-old gestational fetuses. Paradoxically, tissue cysts predominated in stillborns or in calves necropsied before seven days of age (Barr et al., 199Ib; Dubey et al., 1989). Mummification has been an important clinical finding in outbreaks of N. caninum-associated abortions in cattle (Thompson et al., 1991; Nietfeld et al., 1992). The specific etiology of mummification is difficult to prove because fetal tissues are too autolysed for histologic examination or for bioassays. There were no specific gross lesions in fetuses or stillborn calves. One stillborn calf had an enlarged heart (Dubey et al., 1990f). In one study three of the four calves were recumbent from birth; the fourth calf became recumbent at three days of age (Parish et al., 1987; Dubey et al., 1989). The five-day-old Friesian calf reported by O’Toole and Jeffrey (1987) was unable to stand from birth and had bilateral areas of discoloration, especially of grey matter. In the calf from New South Wales, Australia there was marked unilateral reduction of ventral grey matter and focal cavitation (Hartley and Bridge, 1975). Microscopically, nonsuppurative encephalomyelitis characterized by necrosis and mononuclear cell

46 NEOSPOROSIS IN ANIMALS infiltrations are the two most important lesions associated with naturally-acquired N. caninum-like infections in cattle (Barr et al., 1991a; Anderson et al., 1991). In an experimentaly infected cow, N. caninum was found associated with extensive necrosis of the cerebral white matter of the fetus without inflammation (Dubey et al., 1992c). In this calf, N. caninum was found only in the brain and spinal cord, indicating the predilection of N. caninum for the central nervous system in cattle. The heart is the next most parasitized organ although several tissues may be parasitized (Barr et al., 1990, 1991a). There is no antemortem diagnosis test for N. caninum- associated abortion in cattle. The identification of the parasite in fetal tissues is the only means of diagnosis. In routine hematoxylin and eosin stained sections it is difficult to locate N. caninum, especially tachyzoites. The immunohistochemical staining with specific N. caninum serum aids diagnosis. Unfortunately anti-N. caninum serum is not yet commercially available. Isolation of the parasite in cell cultures and mice can also aid diagnosis.

NEOSPOROSIS IN SHEEP Neosporosis-like disease was found in a one-week-old sheep from England (Hartley and Bridge, 1975; Dubey et al., 1990c). From birth the affected lamb was unable to stand but lay on its side exhibiting periodic running movements with its forelimbs. The lamb died and was necropsied. There was nonsuppurative encephalomyelitis and unilateral marked reduction in the gray matter of the ventral horn with focal cavitation; an identical lesion was also found in a calf (Hartley and Bridge, 1975). Numerous thick walled N. caninum-tissue cysts were found associated with neural lesions (Dubey et al., 1990c). Although there is no report of N. caninum-associated abortion in sheep, abortion has been induced in sheep experimentally infected with N. caninum (Dubey and Lindsay, 1990a). In experimentally induced aborted lambs, lesions of neosporosis were identical to those seen in ovine toxoplasmosis.

NEOSPOROSIS IN GOATS Neospora-like tissue cysts were found in 3 aborted pygmy goats in U.S.A. (Dubey et al., 1992a; Barr et al., 1992). Lesions were mainly confined to the central nervous system.

NEOSPOROSIS IN HORSES There is only one report of neosporosis in a horse (Dubey and Porter field, 1990). An eight-year-old mare aborted two months before term. Numerous N. caninum tachyzoites were seen in histologic sections of fetal lung. Toxoplasma gondii antibodies were not detected in mare’s serum.

NEOSPOROSIS IN CATS There is no report of natural N. caninum infection in cats. However, cats are susceptible to experimental N. caninum infection. Infection was severe in neonatally and perinatally infected cats (Dubey and Lindsay, 1990a, c). Neosporosis was more severe in lactating cats and in cats administered exogenous corticosteroids than in normal cats (Dubey and Lindsay, 1990c; Dubey et al., 1990a). Histologically, there was severe myositis and encephalitis in immunosuppressed cats (Dubey et al, 1990c).

47 NEOSPOROSIS IN ANIMALS

REFERENCES Anderson, M. L., Blanchard, P. C., Barr, B. C., Dubey, J. P. Hoffman, R. L., & Conrad, P. A. 1991. Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. J. Am. Vet. Med. Assoc. 198: 241-244. Barr, B. C., Anderson, M. L., Blanchard, P. C., Daft, B. M., Kinde, H., & Conrad, P. A. 1990. Bovine fetal encephalitis and myocarditis associated with protozoal infections. Vet. Pathol. 27: 354-361. Barr, B. C., Anderson, M. L., Dubey, J. P. & Conrad, P. A. 1991a. Neospora-like protozoal in factions associated with bovine abortions. Vet. Pathol. 28: 110-116. Barr, B. C., Anderson, M. L., Woods, L. W., Dubey, J. P. & Conrad, P. A. 1992. Neospora-like protozoal infections associated with abortion in goats. J. Vet. Diagn. Invest. In press. Barr, B. C., Conrad, P. A., Dubey, J. P. & Anderson, M. L. 1991b. Neospora-like encephalomylitis in a calf: Pathology, ultrastructure, and immunoreactivity. J. Vet. Diag. Invest. 3: 39-46. Bjerkas, I. & Dubey, J. P. 1992. Evidence that Neospora caninum is identical to the Toxoplasma-like parasite of Norwegian dogs. Acta. Vet. Scand. In press. Bjerkas, I. & Landsverk, T. 1986. Identification of Toxoplasma gondii and Encephalitozoon cuniculi by immunoperoxidase techniques and electron microscopy, in stored, formalin-fixed, paraffin-embedded tissue. Acta. Vet. Scand. 27: 11-22. Bjerkas, I., Mohn, S. F. & Presthus, J. 1984. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z. Parasitenk. 70: 271-274. Bjerkas, I. & Presthus, J. 1988. Immuno-histochemical and ultrastructural characteristics of a cyst-forming sporozoon associated with encephalomylitis and myositis in dogs. Acta. Path. Microbiol. Immunol. Scand. 95: 445-454. Bjerkas, I. & Presthus, J. 1989. The neuropathology in toxoplasmosis-like infection caused by a newly recognized cyst-forming sporozoon in dogs. Acta. Path. Microbiol. Immunol. Scand. 97: 459-468. Braund, K. G., Blagburn, B. L., Toivio-Kinnucan, M., Amling, K. A., & Pidgeon, G. L. 1988. Toxoplasma polymyositis of polyneuropathy - a new clinical variant in two mature dogs. J. Am. Anim. Hosp. Assoc. 24: 93-97. Cochrane, S. & Dubey, J. P. 1992. Fatal neosporosis in a Golden Retriever dog from Ontario, Canada. Unpublished. Cole, R. A., Lindsay, D. S., Blagburn, B. L. & Dubey, J. P. 1990. Neospora caninum (Protozoa: Apicomplexa): Comparison of in vitro development of NC-1 and NC-3 isolates in three cell lines and three primary cell cultures. Proceedings of Southeastern Society of Parasitologists, Boone, North Carolina, April 18-20. Abstract No. 34.

48 NEOSPOROSIS IN ANIMALS

Cole, R. A., Lindsay, D. S., Blagburn, B. L. & Dubey, J. P. 1991. Vertical transmission of Neospora caninum (Protozoa: Apicomplexa) in mice. Proceedings of Animal Disease Research Workers in Southern States, Southern Conference on Avian Diseases and Southern Conference on Animal Parasites, Auburn, Alabama, March 1991. Abstract No. 71. Cudden, P., Lin, D. S., Bowman, D. D., Lindsay, D. S., Miller, T. K., Duncan, I. D., de Lahunta, A., Cummings, J., Suter, M., Cooper, B., King, J. M. & Dubey, J. P. 1992. Neospora caninum infection in English Springer Spaniel littermates: Diagnostic evaluation and organism isolation. J. Vet. Int. Med. In press. Cummings, J. F, de Lahunta, A., Suter, M. M. & Jacobson, R. H. 1988. Canine protozoan polyradiculoneuritis. Acta. Neuropathol. 76: 46-54. Dubey, J. P. 1989. Congenital neosporosis in a calf. Vet. Rec. 125: 486. Dubey, J. P. 1990. Neospora caninum: A look at a new Toxoplasma-like parasite of dogs and other animals. Comp. Cont. Educt. Pract. Vet. 12: 653-663. Dubey, J. P, Acland, H. & Hamir, A. N. 1992a. Neospora caninum (Apicomplexa) in a stillborn goat. J. Parasitol. In press Dubey, J. P., Carpenter, J. L., Speer, C. A., Topper, M. J. & Uggla, A. 1988a. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192: 1269-1285. Dubey, J. P., Greene, C. E. & Lappin, M. R. 1990a. Toxoplasmosis and neosporosis. pp. 818-834. In: Infectious Diseases of the dog and cat, Greene, C. E. (Ed), W. B. Saunders, Philadelphia. Dubey, J. P., Hartley, W. J. & Lindsay, D. S. 1990b. Congenital Neospora caninum infection in a calf with spinal cord anomaly. J. Am. Vet. Med. Assoc. 197: 1043-1044. Dubey, J. P., Hartley, W. J., Lindsay, D. S. & Topper, M. J. 1990c. Fatal congenital Neospora caninum infection in a lamb. J. Parasitol. 76: 127-130. Dubey, J. P., Hattel, A. L., Lindsay, D. S. & Topper, M. J. 1988b. Neonatal Neospora caninum infection in dogs: Isolation of the causative agent and experimental transmission. J. Am. Vet. Med. Assoc. 193: 1259-1263. Dubey, J. P., Higgins, R. J., Smith, J. H. & O’Toole, T. D. 1990d. Neospora caninum encephalomyelitis in a British dog. Vet. Rec. 126: 193-194. Dubey, J. P., Janovitz, E. B., & Skowronek, A. J. 1992b. Clinical neosporosis in a four-week-old Hereford calf. Vet. Parasitol. In press. Dubey, J. P., Koestner, A. & Piper, R. C. 1990e. Repeated transplacental transmission of Neospora caninum in dogs. J. Am. Vet. Med. Assoc. 19: 857-860. Dubey, J. P., Leathers, C. W. & Lindsay, D. S. 1989. Neospora caninum-like protozoon associated with fatal myelitis in newborn calves. J. Parasitol. 75: 146-148. Dubey, J. P. & Lindsay, D. S. 1989a. Fatal Neospora caninum infection in kittens. J. Parasitol. 75: 148-151. Dubey, J. P. & Lindsay, D. S. 1989b. Transplacental transmission of Neospora caninum infection in dogs. Am. J. Vet. Res. 50: 1578-1579.

49 NEOSPOROSIS IN ANIMALS

Dubey, J. P. & Lindsay, D. S. 1989c. Transplacental Neospora caninum infection in cats. J. Parasitol. 75: 765-771. Dubey, J. P. & Lindsay, D. S. 1990a. Neospora caninum induced abortion in sheep. J. Vet. Diagn. Invest. 2: 230-233. Dubey, J. P. & Lindsay, D. S. 1990b. Neosporosis in dogs. Vet. Parasitol. 36: 147-151. Dubey, J. P., Lindsay, D. S. & Lipscomb, T. P. 1990a. Neosporosis in cats. Vet. Pathol. 27: 335-339. Dubey, J. P, Lindsay, D. S., Anderson, M. L., Davis, S. W. & Shen, S. K. 1992c. Induced transplacental transmission of Neospora caninum in cattle. J. Am. Vet. Med. Assoc. Submitted. Dubey, J. P., Miller, S., Lindsay, D. S. & Topper, M. J. 1990f. Neospora caninum-associated myocarditis and encephalitis in an aborted calf. J. Vet. Diag. Invest. 2: 66-69. Dubey, J. P. & Porterfield, M. L. 1990. Neospora caninum (Apicomplexa) in an aborted equine fetus. J. Parasitol. 76: 732-734. Greene, C. E., Cook, J. R. & Mahaffey, E. A. 1985. Clindamycin for treatment of Toxoplasma polymyositis in a dog. J. Am. Vet. Med. Assoc. 187: 631-634. Hartley, W. J. & Bridge, P. S. 1975. A case of suspected congenital Toxoplasma encephalomyelitis in a lamb associated with a spinal cord anomaly. Brit. Vet. J. 131: 380-384. Hay, W. H, Shell, L. G., Lindsay, D. S. & Dubey, J. P. 1990. Diagnosis and treatment of Neospora caninum infection in a dog. J. Am. Vet. Med. Assoc. 197: 87-89. Hilali, M., Lindberg, R., Waller, T. & Wallin, B. 1986. Enigmatic cyst-forming sporozoon in the spinal cord of a dog. Acta. Vet. Scand. 272: 623-625. Hoskins, J. D., Bunge, M. M., Dubey, J. P. & Duncan, D. E. 1991. Disseminated infection with Neospora caninum in a ten-year-old dog. Cornell Vet. 81: 329-334. Jardine, J. E. & Dubey, J. P. 1992. Canine neosporosis in South Africa. To be published. Lindsay, D. S., Blagburn, B. L. & Dubey, J. P. 1990a. Infections of mice with Neospora caninum (Protozoa: Apicomplexa) does not protect against challenge with Toxoplasma gondii. Infect. Immun. 58: 2699-2700. Lindsay, D. S., Blagburn, B. L. & Dubey, J. P. 1992. Examination of factors affecting the survival of Neospora caninum tissue cysts in mouse brains. J. Parasitol. 77: 70-72. Lindsay, D. S. & Dubey, J. P. 1989a. Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am. J. Vet. Res. 50: 1981-1983. Lindsay, D. S. & Dubey, J. P. 1989b. In vitro development of Neospora caninum (Protozoa: Apicomplexa) from dogs. J. Parasitol. 75: 163-165. Lindsay, D. S. & Dubey, J. P. 1989c. Neospora caninum (Protozoa: Apicomplexa) infections in mice. J. Parasitol. 75: 772-779. Lindsay, D. S. & Dubey, J. P. 1989d. Evaluation of anticoccidial drugs to inhibit development of Neospora caninum in cell cultures. J. Parasitol. 75: 990-992.

50 NEOSPOROSIS IN ANIMALS

Lindsay, D. S. & Dubey, J. P. 1990a. Effects of sulfadiazine and amprolium on Neospora caninum (Protozoa : Apicomplexa) infections in mice. J. Parasito1. 76: 177-179. Lindsay, D. S. & Dubey, J. P. 1990b. Neospora caninum (Protozoa: Apicomplexa) infections in rats. Canad, J. Zool. 68: 1595-1599. Lindsay, D. S. & Dubey, J. P. 1990c. Infections in mice with tachyzoites and bradyzoites of Neospora caninum (Protozoa : Apicomplexa). J. Parasitol. 76: 410-413. Lindsay, D. S., Dubey, J. P. & Blagburn, B. L. 1991. Characterization of a Neospora caninum (Protozoa: Apicomplexa) isolate (NC-3) in mice. Alabama Acad. Sci. 62: 1-8. Lindsay, D. S., Dubey, J. P., Upton, S. J. & Ridley, R. K. 1990b. Serological prevalence of Neospora caninum and Toxoplasma gondii in dogs from Kansas. J. Helminthol. Soc. Wash. 57: 86-88. Mayhew, I. G., Smith, K. C., Dubey, J. P., Gatwald, L. K. & McGlennon, N. J. 1991. Treatment of encephalomyelitis due to Neospora caninum in a litter of puppies. J. Small Anim. Pract. 32: 609-612. McGlennon, N. J., Jefferies, A. R. & Casas, C. 1990. Polyradiculoneuritis polymyositis due to a toxoplasma-like protozoan: Diagnosis and treatment. J. Small Anim. Pract. 31: 102-104. Munday, B. L., Dubey, J. P. & Mason, R. W. 1990. Neospora caninum infection in dogs. Aust. Vet. J. 67: 76. Nietfeld, J. C., Dubey, J. P., Anderson, M. L. & Libal, M. C. 1992. Neospora-like protozoan infections as a major cause of bovine abortion in upper midwest United States. J. Vet. Diagn. Invest. In press. O’Toole, J. M. & Jeffrey, M. 1987. Congenital sporozoan encephalomyelitis in a calf. Vet. Rec. 121: 563-566. Parish, S. M., Maag-Miller, L., Basser, T. E., Weidner, J. P., McElwain, T., Knowles, D. P. & Leathers, C. W. 1987. Myelitis associated with protozoal infection in newborn calves. J. Am. Vet. Med. Assoc. 191: 1599-1600. Poncelet, L., Bjerkas, I., Charlier, G., Coignoul, F., Losson, B. & Balligand. 1990a. Confirmation de la presence de Neospora caninum en Belgique. Ann. Med. Vet. 134: 501-503. Poncelet, L., Coignoul, P., Fontaine, J. & Balligand, M. 1990b. Infestation de chiots par Neospora caninum en Belgique et en France? Ann. Med. Vet. 134: 167-171. Shivaprasad, H. L., Ely, R. & Dubey, J. P. 1989. A Neospora-like protozoon found in an aborted bovine placenta. Vet. Parasitol. 34: 145-148. Speer, C. A. & Dubey, J. P. 1989. Ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum. J. Protozool. 36: 458-463. Sreter, P., Sebestyen, P., & Dubey, J. P. 1992. Neosporosis in a dog in Hungary. Unpublished. Thilsted, J. P. & Dubey, J. P. 1989. Neosporosis-like abortions in a herd of dairy cattle. J. Vet. Diag. Invest. 1: 205-209.

51 NEOSPOROSIS IN ANIMALS

Thorton, R. N., Thompson, E. J. & Dubey, J. P. 1991. Neospora protozoal abortion in New Zealand cattle. N. Z. Vet. J. In press. Trees, A. J., Tennant, B., Kelly, D. F., Guy, F. & Dubey, J. P. 1992. Neospora caninum - a newly identified protozoan pathogen. Trans. Roy. Soc. Trop. Med. Hyg. Abstract. Unpublished. Uggla, A., Dubey, J. P., Funkquist, B. & Segall, T. 1989a. Fatal Neospora caninum-infektion hos riesenschnauzer. Svensk. Veterinärtidning. 41: 271-274. Uggla, A., Dubey, J. P., Lundmark, G. & Olsen, P. 1989b. Encephalomyelitis and myositis in a Boxer puppy due to a Neospora-like infection. Vet. Parasitol. 32: 255-260. Wolf, M., Cachin, M., Vandevelde, M., Tipold, A. & Dubey, J. P. 1991. Zur Klinischen Diagnostik des Protozoaren Myositissyndroms (Neospora caninum) des Welpen. Tierarztliche Praxis 19: 302-306.