Tables 1 and 2 show the ages of the goats and the gestational stages at which abortion occurred, the occurrence and intensity of the histopathological lesions, the method of diagnosis of N. caninum infection, and the intensity of the cellular immunolabeling in the lesions. Fetuses 1–4 were the products of a single gestation (born to the same mother), as were fetuses 5 and 6, and fetuses 7 and 8. Fetuses 2, 3, 9, and 10, and the male goat 7, which were all negative for N. caninum, were used as negative controls. Goat 1 exhibited clinical neurological signs at birth, with moderate paresis, lack of coordination of the pelvic limbs, and difficulty in standing. These clinical signs had decreased a week after birth, and normal development proceeded until 12 months of age, when the animal was euthanized.
Necropsy and histopathological findings
The goats exhibited no macroscopic lesions. The microscopic findings in the fetuses were discrete to moderate perivascular mononuclear cuffs (fetuses 1, 4, 7, and 8), observed near the glioses. The glioses were focal or multifocal and were observed with decreasing frequency in the cerebral cortex (fetuses 5–7), rostral colliculus (fetuses 4–6), thalamus (fetuses 4, 7, and 8), caudal colliculus (fetuses 5 and 6), medulla oblongata and obex (fetuses 1 and 4) (Figure 1A), cerebellar peduncles (fetus 4), pons, and the cervical and lumbar spinal cord (fetus 8). Foci of necrosis surrounded by glial cells and inflammatory cells were also observed in fetus 4 (cranial colliculus, pons, and thalamus) and fetus 8 (thalamus and lumbar spinal cord). Discrete mononuclear meningitis was observed close to the cerebral cortex in fetuses 4–6. Neospora caninum cysts were observed in the thalamus (fetuses 4–7) and the cerebral cortex (fetuses 5–7), close to areas of inflammation (fetus 4) or not (fetuses 4–7). In fetus 7, a parasitic cyst was seen in the neuronal cytoplasm. In fetuses 5 and 6, there were rare foci of mineralization associated with necrosis.
Only two of the aborted fetuses showed lesions in the myocardium and skeletal striated muscle. These consisted of varying degrees of mononuclear inflammatory infiltration, and in one fetus, some tachyzoites were observed with immunohistochemistry in samples of the heart and skeletal muscles.
The microscopic lesions in the adult goats were glioses (goats 1–4) (Figure 1B), and perivascular mononuclear cuffs in the cerebral cortex (goats 1–3), obex (goats 1, 3, and 4), thalamus (goats 1 and 3), pons, cerebellum, caudal and rostral colliculi (goat 3), and cervical, thoracic, and lumbar portions of the spinal cord (goat 3). Goat 6 displayed discrete perivascular cuffs in the meninges. Multinucleate giant cells were seen associated with a focal inflammatory response in the cerebral cortex (goats 1 and 3), pons (goat 3), and obex (goat 4) (Figure 1C). Neospora caninum cysts were observed in the cerebral cortex (goats 2 and 3), rostral colliculus (goat 3), obex (goat 4), cervical, thoracic, and lumbar segments of the spinal cord (goat 3), the neuronal cytoplasm in the obex (goat 4) (Figure 1D), and the cervical spinal cord (goat 3). No lesions were observed in the male goat or fetuses used as negative controls.
Two adult goats (male goats 2 and 3) had focal lymphoplasmacytic myositis in their skeletal muscles (semitendinosus and semimembranosus), but these lesions could not be associated with the parasite.
Lectin histochemistry
The majority of cells within the areas of gliosis were positive for RCA1. Staining occurred in the thalamus (fetuses 4, 6–8 and goat 1) (Figure 2A), cerebral cortex (fetuses 1, 4 and 7 and goats 1 and 3), obex (fetus 1 and goat 4), cerebellum (fetus 4), pons (fetus 8 and goat 3), caudal colliculus (goat 3), and the cervical (goat 3), thoracic (goat 3), and lumbar segments (fetus 8) of the spinal cord. Staining was also seen in the cells of the perivascular cuffs in the cerebral cortex (fetuses 1, 4 and 7 and goats 1 and 3), cerebellum, and cerebellar peduncle (fetus 4), pons and caudal colliculus (goat 3) obex (goat 4), thalamus (fetuses 4 and 7), and the lumbar spinal cord (fetus 8). The multinucleate giant cells seen in the male goats also stained with RCA1.
Immunohistochemistry
Neospora caninum
Parasitic cysts and tachyzoites of N. caninum were immunolabeled in fetuses 1 and 4–7 and parasitic cysts in the adult male goats 2–4 (Figure 2B). The parasitic structures were negative for T. gondii.
GFAP
GFAP immunolabeling was observed in the cells within the glial foci in the cerebral cortex (fetuses 5 and 6), in the colliculi (fetus 4), and in an extensive area of gliosis in the cortex associated with a parasitic cyst (fetus 6), with characteristic astrocytosis (increased sizes and numbers of astrocytes) and astrogliosis (astrocyte hypertrophy: increased synthesis of intermediate filaments causing increased length and branching of the astrocytic processes). GFAP immunolabeling was also intense in the astrocytes adjacent to the glial foci in the cerebral cortex (fetus 1) and in the lumbar spinal cord (fetus 8). In the adult goats, GFAP immunolabeling occurred in the glial foci in the cerebral cortex (goats 1–3) and the thalamus (goat 1), and goats 1 and 2 displayed numerous and extremely dense astrocytic processes (glial scarring) (Figure 2C).
PCNA
PCNA labeling occurred in the macrophages of the perivascular cuffs in the cerebral cortex (fetus 1 and goats 1 and 3), thalamus (fetus 4), pons, caudal colliculus, and cerebellum (goat 3), and in the microglia of the glial foci in the cerebral cortex (fetus 1 and goats 1 and 3), thalamus (fetus 4 and goat 3), rostral colliculus, peduncle, and cerebellum (fetus 4), and cervical spinal cord (goat 3).
MHC-II
MHC-II immunolabeling occurred in the adult goats: in the cytoplasm of the endothelial cells of the meningeal blood vessels (goats 1 and 6) and the vessels of the cerebral parenchyma; in macrophages of the perivascular cuffs in the cerebral cortex (goats 1 and 3), obex (goat 4), pons, cervical spinal cord, cerebellum, thalamus, and caudal colliculus (goat 3). MHC-II immunolabeling was also seen in the glial foci in the cerebral cortex (goats 1 and 3) (Figure 2D), obex (goat 4), and the cervical spinal cord (goat 3). In fetuses 7 and 8, MHC-II labeling was observed in the glial foci, endothelia, and the perivascular cuffs.
CD3
Rare immunolabeled T lymphocytes were observed in the perivascular cuffs of the thalamic meninges in fetus 7, in the perivascular cuffs and foci of gliosis in the thalamus of fetus 4. In the adult goats, CD3 immunolabeling occurred in the perivascular cuffs in the meninges close to the cerebral cortex (goat 6), in the thalamic parenchyma (goat 1), and in the cerebral cortex (goats 2, 3, and 5).
CD79α
Rare immunolabeled B lymphocytes were observed in the perivascular cuffs and glial foci in the pons, cervical spinal cord, and thalamus of goat 3.
PCR and sequencing
Neospora caninum DNA was detected with PCR in the CNS samples of the fetuses (1, and 4–8) and goats (1–6) (Tables 1 and 2) and sequenced. The nucleotide sequences showed 99.9% homology with the corresponding sequence in N. caninum.