Characterization of immune cell infiltration in the placentome of water buffaloes (Bubalus bubalis) infected with neospora caninum during pregnancy.

Neospora caninum infection in cattle stimulates host immune responses, which may be responsible for placental damage leading to abortion. Susceptibility of water buffaloes (Bubalus bubalis) to neosporosis is not well understood, although vertical transmission and fetal death have been documented. The aim of this study was to characterize the immune response in the placentome of water buffalo following experimental infection in early gestation with the Nc-1 strain of N. caninum. Placentomes were examined by immunohistochemistry using antibodies specific for T-cell subsets, natural killer cells and CD79(αcy) cells. Placental inflammation was characterized by the infiltration of CD3(+) and CD4(+) T cells and T cells expressing the γδ T-cell receptor. The distribution of these cellular subsets in buffalo placentomes was similar to that previously described in cattle infected with N. caninum in early gestation, but the lesions were milder, which may explain the lower number of abortions observed in this species after infection.

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams.

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.

An outbreak of sodium fluoroacetate (1080) intoxication in selenium- and copper-deficient sheep in California.

Sodium fluoroacetate is an organofluorine compound toxic to mammals, insects, and birds, currently registered for use only in livestock protection collars as a predacide in some North American states, with restricted use in California. A flock of 445 lambs and ewes in California were moved into a native pasture on a municipal refuse disposal site. Within 24 hours, 14 ewes were found dead, and the remaining sheep were moved off the site. Both ewes and lambs exhibited disoriented running, followed by apparent blindness, weakness, ataxia, coma, and death. Over the next 4 days, 63 ewes and 80 lambs died with a peak at 3 days after grazing the suspect pasture (157/445, 35% mortality). Two dead 4-month-old lambs and 2 ewes were submitted to the California Animal Health and Food Safety laboratory for necropsy. Grossly, there were bilateral diffuse pulmonary congestion and edema, hydrothorax and hydropericardium with fibrin clots, and multifocally extensive areas of epicardial petechiae, ecchymoses, and pallor. In 1 ewe, there was regional caudodorsal pulmonary hemorrhage and intraluminal tracheal clotted blood. Microscopically in all cases, there was multifocal acute myocardial degeneration and necrosis with nonsuppurative pleocellular myocarditis. Sodium fluoroacetate was detected in kidney from a lamb and a ewe at 27.5 and 12.5 parts per billion, respectively. All sheep were selenium deficient, and concurrent copper deficiency was diagnosed in 3. The pathological and toxicological findings were consistent with 1080 poisoning, possibly exacerbated by micronutrient deficiency. This outbreak raised an alert about the use of restricted products with potential lethal effect in animals in California.

The Neospora caninum-Spain 7 isolate induces placental damage, fetal death and abortion in cattle when inoculated in early gestation.

The Nc-Spain 7 isolate of Neospora caninum, which was newly obtained from an asymptomatic congenitally infected calf, demonstrated a similar virulence as Nc-1 strain in mouse models. The aim of this study was to characterize the pathogenesis of Nc-Spain 7 isolate in cattle after experimental infection at 65 days of gestation. For this purpose, thirteen pregnant heifers were divided into three groups as follows: group A: 7 heifers inoculated with 1 × 10(8) tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1 × 10(8) tachyzoites of Nc 1 strain; and group C: 2 heifers received PBS. Serum samples were collected weekly and heparinized blood samples were collected three times (0, 28 and 42 days after inoculation) by jugular venipuncture. Placenta and fetal tissue samples were collected at time of necropsy. Specific antibody response in the dams was tested by IFAT, indirect ELISA, and rNcGRA7 and rNcSAG4 based-ELISA. Specific antibody response in fetal fluids was tested by IFAT. IFN-γ production was measured after in vitro culture of PBMC and the supernatant was assessed using a commercial kit (BOVIGAM). A significant increase in N. caninum antibody responses was detected in groups A and B by IFAT and by i-ELISA from day 14 after inoculation onwards. Besides, antibody response against rNCGra7 protein was also detected in all inoculated heifers by rNcGra7-based ELISA. Four fetuses from group A and one from group B were aborted between 3 and 5 weeks after infection. In the recovered fetuses, only 3 out of 4 fetal fluids from fetuses of group A and 1 out of 3 of group B were seropositive by IFAT, but all of them were positive by PCR. Transplacental transmission could be determined in all fetuses from groups A and B by PCR and/or IHC. Heifers of group C and their fetuses remained negative by all techniques. The results of this study demonstrate that the NC-Spain 7 isolate could be transmitted transplacentally, and produced fetal death and abortion in cattle.

Experimental inoculation of Neospora caninum in pregnant water buffalo.

The aim of this study was to characterize the pathogenesis of Neospora caninum in experimentally inoculated pregnant water buffalo (Bubalus bubalis). Twelve Mediterranean female water buffaloes ranging in age from 4 to 14 years old and seronegative to N. caninum by indirect fluorescent antibody test (IFAT) were involved. Ten females were intravenously inoculated with 10(8) tachyzoites of NC-1 strain at 70 (n=3) or 90 (n=7) days of pregnancy (dp). Two control animals were inoculated with placebo at 70 and 90 dp, respectively. Serum samples were obtained weekly following inoculation to the end of the experiment. Three animals inoculated at 70 dp were slaughtered at 28 days post inoculation (dpi), three animals inoculated at 90 dp were slaughtered at 28 dpi and the remaining four animals inoculated at 90 dp were slaughtered at 42 dpi. Fetal fluids from cavities and tissue samples were recovered for IFAT and histopathology, immunohistochemistry and PCR, respectively. Genomic DNA from fetal tissues was used for parasite DNA detection and microsatellite genotyping in order to confirm the NC-1 specific-infection. Dams developed specific antibodies one week after the inoculation and serological titers did not decrease significantly to the end of the experiment. No abortions were recorded during the experimental time; however, one fetus from a dam inoculated at 70 dp was not viable at necropsy. Specific antibodies were detected in only two fetuses from dams inoculated at 90 dp that were slaughtered at 42 dpi. No macroscopic changes in the placentas and organs of viable fetuses were observed. Nonsuppurative placentitis was a common microscopic observation in Neospora-inoculated specimens. Microscopic fetal lesions included nonsuppurative peribronchiolar interstitial pneumonia, epicarditis and myocarditis, interstitial nephritis, myositis and periportal hepatitis. Positive IHC results were obtained in two fetuses from dams inoculated at 70 dp and slaughtered at 28 dpi. N. caninum DNA was detected in placentas and fetuses from all inoculated animals. The pattern of amplified microsatellites from placental and fetal tissues resembled the NC-1 strain. Water buffaloes, like cattle, are susceptible to experimental inoculation with N. caninum at early pregnancy.