A Descriptive Study of Lectin Histochemistry of the Placenta in Cattle following Inoculation of Neospora caninum.

The aim of this study was to describe the lectin-binding pattern in the placentas of cows infected experimentally with Neospora caninum. Four cows were inoculated intravenously with 1 × 108 tachyzoites of the NC-1 strain of N. caninum at 150 ± 7 days of pregnancy. Two control cows were administered a placebo. An indirect fluorescence antibody test (IFAT) was performed on serum samples obtained before and after the inoculation. The cows were killed at 30 and 37 days post inoculation. Samples of placenta were taken for histopathology and lectin histochemistry. Fetal tissues and fluids were collected for histopathology and IFAT, respectively. All infected cows had high antibody titres. All fetuses had characteristic histopathological lesions, including non-suppurative meningoencephalitis, myocarditis, hepatitis and myositis, suggesting N. caninum infection. Only two infected fetuses developed specific antibodies. Mild non-suppurative inflammatory infiltrates were recorded in the placentae. Differences in the lectin-binding pattern were observed between infected animals and controls in the glycocalyx (CON-A and WGA) and apical cytoplasm (RCA-I and CON-A) of the trophoblastic cells; giant trophoblastic cells (CON-A and DBA); glycocalyx (PNA, WGA) and apical cytoplasm (CON-A, WGA, PNA, DBA and RCA-I) of endometrial cells; trophoblast of the interplacentomal region (WGA); endothelium (CON-A, SBA, RCA-1 and WGA); and finally, mesenchyme (CON-A, RCA-1, SBA, PNA and DBA). These findings indicate that there is a distinctive pattern of lectin binding in the placenta of cattle infected with N. caninum. The direct effect of the presence of the protozoa as well as the altered expression of cytokines could explain these changes in the maternofetal interface.

Neospora caninum tachyzoites inoculated by the conjunctival route are not vertically transmitted in pregnant cattle: a descriptive study.

The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5 × 10(8) tachyzoites, group 3 contained two cows inoculated with 2.5 × 10(8) tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG1/IgG2 ratios were ≤ 1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.

Humoral immune response in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival route.

The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.