Canine Neosporosis: Clinical and Pathological ®Ndings and ®Rst Isolation of Neospora Caninum in Germany
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Parasitol Res (2000) 86: 1±7 Ó Springer-Verlag 2000 ORIGINAL PAPER Martin Peters á Frank Wagner á Gereon Schares Canine neosporosis: clinical and pathological ®ndings and ®rst isolation of Neospora caninum in Germany Received: 8 August 1999 / Accepted: 30 August 1999 Abstract Neosporosis was diagnosed in an 11-week-old species. In cattle, N. caninum has been found to be as- puppy of the breed Kleiner MuÈ nsterlaÈ nder with sociated with abortions. Clinical neosporosis in dogs is progressive hindlimb paresis. Pathohistological and most commonly observed in congenitally infected pup- immunohistological examinations revealed a dissemi- pies in the ®rst 6 months of life (Ruehlmann et al. 1995). nated infection with Neospora caninum. Parasitic stages Characteristically these animals show a progressively were demonstrated in the brain, spinal cord, retina, ascending paresis, usually starting at the pelvic limbs, muscles, thymus, heart, liver, kidney, stomach, adrenal caused by severe polymyositis, polyradiculitis, and dis- gland, and skin. Immunohistochemistry investigations seminated meningoencephalomyelitis. Generalized in- were carried out using polyclonal rabbit antisera devel- fection with multiple organ involvement in puppies has oped against N. caninum tachyzoites and the recombi- been reported (Dubey et al. 1988a; Barber et al. 1996). nant bradyzoite-speci®c antigen BAG-5 of Toxoplasma Some puppies suered and eventually died of myo- gondii, which is known to cross-react with N. caninum carditis caused by the parasite (Dubey et al. 1988a; Odin bradyzoites. BAG-5 antibodies recognized tissue cysts and Dubey 1993; Barber et al. 1996; WeissenboÈ ck et al. within the CNS and some protozoan stages that were 1997). Besides multifocal CNS involvement and not surrounded by a visible cyst wall. All parasite clus- polymyositis, pneumonia (Greig et al. 1995) or derma- ters in the retina and some in muscle tissue stained titis (Dubey et al. 1995; Fritz et al. 1997; Perl et al. 1998; positively with the BAG-5 antiserum. N. caninum was Poli et al. 1998) may be the predominant clinical feature isolated in cell culture and mice inoculated with brain in adult dogs. and spinal cord of the puppy. The new isolate is the ®rst Dogs are both de®nitive hosts (McAllister et al. reported in Germany and is designated NC-GER1. 1998) and intermediate hosts of N. caninum. Parasite stages in the intermediate host are the rapidly repli- cating tachyzoites and slowly replicating bradyzoites residing within tissue cysts. Intracellular multiplication Introduction of tachyzoites results in cell death with a subsequent in¯ammatory response (Dubey and Lindsay 1996). Neospora caninum is an apicomplexan parasite struc- In¯ammatory reactions do not seem to be caused by turally similar to Toxoplasma gondii. Natural infections intact tissue cysts as long as they do not rupture have been described worldwide in several mammalian (Mayhew et al. 1991). In dogs, tissue cysts have been described only within the CNS, including the retina (Dubey et al. 1990; Dubey and Lindsay 1996). Bradyzoites of N. caninum can be dierentiated from M. Peters (&) tachyzoites by immunohistological examination Institut fuÈ r Pathologie, TieraÈ rztliche Hochschule Hannover, (McAllister et al. 1996; Fuchs et al. 1998; Schares BuÈ nteweg 17, D-30559 Hannover, Germany et al. 1999). e-mail: [email protected]; Fax: +49-511-9538675 This paper reports on the clinical signs and patho- F. Wagner morphological lesions observed in a puppy infected with Klinik fuÈ r kleine Haustiere, TieraÈ rztliche Hochschule Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany N. caninum. The distribution of the parasite in several tissues, including the eye; the immunohistochemical G. Schares dierentiation between bradyzoites and tachyzoites in Bundesforschungsanstalt fuÈ r Viruskrankheiten der Tiere, Institut fuÈ r epidemiologische Diagnostik, Seestrasse 55, these tissues; and the ®rst isolation of the parasite in D-16868 Wusterhausen, Germany Germany are described. 2 antibody test (IFAT) and immunoblotting as described by Schares Materials and methods et al. (1998). Sera were examined for antibodies to T. gondii by immunoblotting (Schares et al. 1999). For PCR the CSF was cen- History trifuged; the pellet, digested with proteinase K; and the DNA, isolated using a DNA extraction kit (NucleoSpin C + T, Two of eight puppies of the fourth litter of a Kleiner MuÈ nster- Macherey & Nagel) according to the instructions of the manufac- laÈ nder bitch showed progressive hindlimb paresis. One male pup turer. Furthermore, DNA was extracted from tissues and examined developed clinical signs at 1 week of age and died at the age of for N. caninum DNA by PCR using primers Np6 and Np21 7 weeks. After postmortem examination, neosporosis was sus- (Yamage et al. 1996). pected. The second female pup showed stiness of the left hindlimb at 6 weeks of age and was treated with trimethoprim and sulfa- diazine (Tribrissen 20, Essex) for 12 days without clinical im- Isolation of N. caninum provement. During this period a serum sample was taken for serology. After a week without therapy the pup had become re- For isolation of protozoan parasites, brain and spinal cord tissues cumbent. At the age of 11 weeks, physical, neurological, and were treated separately. All solutions (buer, medium) used for ophthalmological examinations were done. Routine blood counts isolation were supplemented with penicillin (1,000 IU/ml) and and blood biochemistry were performed. A second serum sample streptomycin (1,000 lg/ml) to prevent bacterial growth. Tissues was taken from the pup and the bitch was bled for serological were homogenized in 100 ml PBS, ®ltered through sterile gauze, examination. A cerebrospinal ¯uid (CSF) sample was taken and centrifuged (800 g). The brain sediment and spinal cord sedi- atlantooccipitally for dierential cell counts, serology, and poly- ment were resuspended in 100 and 50 ml RPMI supplemented with merase chain reaction (PCR). The pup was euthanized. 0.5% (w/v) trypsin (Sigma, Deisenhofen), respectively, and then incubated at 37 °C. After 30 min the homogenate was centrifuged three times (800 g). Pathological and immunohistochemical examination The resulting sediments were aliquoted and inoculated both onto 1-day-old Vero-cell monolayers (25 cm2) and intraperitone- Immediately after euthanasia a complete postmortem examination ally into mice. Cell cultures were examined microscopically every was carried out. Half of the longitudinally divided brain as well as day for the presence of parasite stages. Five and three Vero-cell the major parts of the spinal cord were removed for parasite iso- cultures were infected using aliquots of brain and spinal cord ho- lation. Native samples of the brain, spinal cord, heart, lung, liver, mogenate, respectively. Further aliquots of brain homogenate were spleen, kidney, and skeletal muscles were removed for Neospora inoculated into four BALB/c J-nu mice (nude mice; 3 months old), caninum PCR. Another set of tissue samples was ®xed in 4% buf- into two AKR mice (3 months old), and into four BALB/c mice fered formol saline and processed using routine techniques. Sec- (1 month old). Aliquots of spinal cord homogenate were inoculated tions were cut at 5 lm and stained with hematoxylin and eosin into three BALB/c J-nu mice (2±3 months old), into two AKR mice (H&E) for microscope examination. Sections were deparanized in (3 months old), and into two BALB/c mice (1 month old). At 9 h xylene for immunohistochemical examinations. After incubation in after inoculation, AKR and BALB/c mice were treated with a isopropanol and 96% (v/v) ethanol, endogenous peroxidase was subcutaneous injection of 2.5 mg prednisolone-21-acetate (Al- quenched with 0.5% (v/v) hydrogen peroxide in methanol for brecht, Aulendorf). Mice were examined daily for the development 30 min. After further rehydration in 75% (v/v) and 50% (v/v) of disease. Sick mice were euthanized, and their peritoneal exudate ethanol the slides were rinsed in phosphate-buered saline (PBS) was examined microscopically for the presence of parasites. Posi- and then incubated in 0.05% (v/v) pronase E (Merck, Darmstadt) tive peritoneal exudate was washed once in a 20-fold volume of for 20 min at 37 °C. RPMI and inoculated onto a 1-day-old Vero-cell monolayer After another rinse in TRIS-PBS, sections were blocked in (25 cm2). TRIS-PBS containing 20% (v/v) normal goat serum and were in- cubated (45 min, 37 °C) with a rabbit antiserum (1:500 in PBS) developed against N. caninum (NC-1) tachyzoites (Schares et al. 1997). This antiserum detects bradyzoites and tachyzoites of Results N. caninum. To obtain exclusive immunolabeling of bradyzoites of N. caninum we used (45 min, 37 °C) a polyclonal rabbit antiserum (1:14,000 in PBS) developed against a BAG-5 fusion protein of Clinical ®ndings Toxoplasma gondii, which does not cross-react with tachyzoites. This antiserum does strongly cross-react with N. caninum brady- Clinical examination of the pup revealed paresis and zoites (McAllister et al. 1996). Furthermore, a mouse monoclonal hyperextension of the left hind leg along with a marked antibody (74.1.8.) against T. gondii BAG-5 antigen developed by atrophy and hardening of the thigh muscles of both Weiss et al. (1992) was used on a few representative sections of the CNS and skeletal muscle (1:25 in PBS). Antibodies were detected hindlimbs. The left tarsal joint could not be ¯exed using commercial kits (Vectastain Elite ABC Kit, Vector Labora- mechanically. The dog was not capable of getting up tories) as described by the manufacturer. without help but could stand after receiving passive For color development, 3,3¢-diaminobenzidine tetrahydro- support. Urination and defecation were under control. chloride (0.05 mg/ml in 0.05 M TRIS-PBS, pH 7.6) and 0.03% (v/ v) hydroxide peroxide were used. The enzyme reaction was stopped The proprioceptive positioning reaction was normal in after 5 min by thorough rinsing in tap water for 10 min.