Central nervous system (CNS) is the main site for encystment of Neospora caninum in different animal species. In this tissue, glial cells (astrocytes and microglia) modulate responses to aggression in order to preserve home-ostasis and neuronal function. Previous data showed that when primary cultures of glial cells are infected with N. caninum, they develop gliosis and the immune response is characterized by the release of TNF and IL-10, followed by the control of parasite proliferation. In order to elucidate this control, three enzymatic systems involved in parasite-versus-host interactions were observed on a model of neuron/glia co/cultures obtained from rat brains. Indoleamine 2,3-dioxygenase (IDO), induced nitric oxide synthase (iNOS) responsible for the catabolism of tryptophan and arginine, respectively, and cycloxigenase (COX) were studied comparing their modulation by respective inhibitors with the number of tachyzoites or the immune response measured by the release of IL-10 and TNF. Cells were treated with the inhibitors of iNOS (1.5 mM L-NAME), IDO (1 mM 1-methyl tryptophan), COX-1 (1 mu M indomethacin) and COX-2 (1 mu M nimesulide) before infection with tachyzoites of N. caninum (1:1 cell: parasite). After 72 h of infection, immunocytochemistry showed astrogliosis and a significant increase in the number and length of neurites, compared with uninfected co-cultures, while an increase of IL-10 and TNF was verified. N. caninum did not change iNOS activity, but the inhibition of the basal levels of this enzyme stimulated parasite proliferation. Additionally, a significant increase of about 40% was verified in the IDO activity, whose inhibition caused 1.2-fold increase in parasitic growth. For COX-2 activity, infection of cultures stimulated a significant increase in release of PGE(2) and its inhibition by nimesulide allowed the parasitic growth. These data indicate that iNOS, IDO and COX-2 control the proliferation of N. caninum in this in vitro model. On the other hand, the release of IL-10 by glia besides modulating the inflammation also allow the continuity of parasitism.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-β antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-β, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-β did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-β, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-β cytokines participate in parasite proliferation control mechanisms.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly Th1-type; however, glial cells in infected primary cultures release IL-10 and IL-6 but tiol IFN-gamma. This suggests a protective response of glia to avoid inflammatory damage of nervous tissues. This study investigated the effects of pro-inflammatory cytokines in primary cell co-cultures of rat astrocytes and microglia infected with N. caninum. Cells were treated with IFN-gamma or TNF-alpha and 24 h after, infected with parasite tachyzoites. Lactate dehydrogenase (LDH) activity, measured to indicate cell viability, was increased following TNF-alpha treatment. Infected cultures also increase LDII activity 72 h post-infection. Treatment with 50, 150 and 320 IU/ml of IFN-gamma orTNF-A reduced tachyzoitcs in cultures by 41.5, 54.9 and 61.5%, and by 30.2, 38.8 and 31.7%, respectively. These results suggest that IFN-gamma may act synergistically with the parasite in its immune evasion mechanisms and both cytokines can reduce parasite proliferation, but they may also be toxic to infected glial cells.

Neospora caninum infection provokes neurological disorders, recurrent abortion and death in dogs and cattle. Dogs are both intermediate and definitive host of N. caninum. Thus, the development of sensitive and specific immunoassays to diagnose canine neosporosis is essential to control this disease. This work investigated serum anti-neosporal IgG and IgE antibodies in 140 dogs represented by 30 healthy animals (group I), 11 dogs showing acute N. caninum infection (group II), 50 urban dogs with serological evidence of canine neosporosis in indirect fluorescent antibody test (IFAT) (group III) and 49 urban dogs without clinical and laboratory evidences of neosporosis (group IV). Enzyme-linked immunosorbent assay (ELISA) and western immunoblotting, both using a soluble N. caninum tachyzoite antigen (SNA), investigated these two isotypes of antibodies, while a Urea-ELISA measured the avidity of the IgG antibodies. Anti-Toxoplasma gondii IgG antibodies were also investigated in the animals. Anti-neosporal IgG was found in all animals from groups II and III, whereas 32.7% (16/49) of dogs from group IV were reactive. IgG antibodies of low avidity were demonstrated in dogs from group II (median 35.3%), while animals from groups III and IV had IgG antibodies of high avidity (medians of 61.5% and 61.7% respectively). IgE antibodies were found in four (13.3%) and five (16.6%) dogs from groups III and IV respectively. Dogs presenting acute infection (group II) or chronic infection (group III) had IgG antibodies to several neosporal antigens, mainly of 29-30 and 35 kDa, while 13 of 16 dogs from group IV recognized antigens from 14 to 170 kDa. Antibodies to T. gondii were detected in 36 of 50 (72%) sera from group III and 25 of 49 (51%) sera from group IV. We concluded that IgG-ELISA and Urea-ELISA with SNA may substitute for IFAT in both laboratory routine and epidemiological studies of canine neosporosis.

The proteinogram of six 12 month-old Alpine goats, intensively raised and naturally infected by gastrointestinal parasites, was evaluated. Blood and feces samples of each animal were monthly collected. Total serum protein and their fractions were determined by agarose gel eletrophoresis, using Tris buffer, pH 9.2. The identified protein fractions were albumin, alfa-globulin, beta1-globulin, beta2-globulin and gama-globulin, whose average and standard deviation (g/dl) were, respectively: 2.35±0.39, 0.69±0.36, 0.70±0.08, 0.48±0.08 and 1.52±0.41. It was not observed significative correlation (P>0.05), according to the Spearman non-parametric test, either between the Strongyloides eggs count per gram of feces or the Haemonchus spp. larval count per gram of feces and the fraction electrophorectly variable.

The proteinogram of six 12 month-old Alpine goats, intensively raised and naturally infected by gastrointestinal parasites, was evaluated Blood and feces samples of each animal were monthly collected. Total serum protein and their fractions were determined by agarose gel eletrophoresis, using Tris buffer, pH 9.2. The identified protein fractions were albumin, alfa-globulin, beta1-globulin, beta2-globulin and gama-globulin, whose average and standard deviation (g/dl) were, respectively: 2.35 +/- 0.39, 0.69 +/- 0.36, 0.70 +/- 0.08, 0.48 +/- 40.08 and 1.52 +/- 0.41. It was not observed significative correlation (P > 0.05), according to the Spearman non-parametric test, either between the Strongyloides eggs count per gram of feces or the Haemonchus spp. larval count per gram of feces and the fraction electrophorectly variable.