A clinical case of presumed cerebellar medulloblastoma in a Japanese Black calf with increased neuron-specific enolase in cerebrospinal fluid.

A 2-day-old Japanese Black male calf that presented with opisthotonos with spastic extension of all four limbs and nystagmus was presented. Evaluation of cranial neurology revealed a horizontal slow nystagmus and absence of menace response in the left eye. Necropsy revealed a mass located between the posterior margin of the cerebrum and anterior margin of the cerebellum, and continuously with the cerebellar lesion. The brainstem was severely compressed by those lesions. Original structures of the cerebellum were mostly replaced by grayish-white and brownish tissues. Those lesions were diagnosed as presumed cerebellar medulloblastoma by histopathological and immunohistochemical examination. As neuron-specific enolase in the cerebrospinal fluid which is a biomarker for neuronal damage was increased compared with healthy calves.

Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis.

Abortion and reproductive failure caused by Neospora caninum infection has a dramatic negative economic impact on the cattle industry. To date, no definitive serodiagnostic tool for assessing N. caninum abortion has been reported. In this study, we evaluated the diagnostic performance of numerous N. caninum antigens in relation to abortion in cattle. Five recombinant proteins with potential as diagnostic antigens (NcGRA6, NcGRA7, NcGRA14, NcCyP, and NcSAG1) were compared by indirect enzyme-linked immunosorbent assay (iELISA) using sera from mice and cattle experimentally infected with N. caninum. The best-performing three antigens (NcSAG1, NcGRA7, and NcGRA6) were evaluated by IgG-iELISAs to assess their utility in diagnosing Neospora abortion using sera from confirmed N. caninum-aborted dams based on immunohistochemical assays (IHC). Additionally, all samples were tested using a commercial N. caninum antibody competitive ELISA (cELISA). The iELISAs against both NcSAG1 and NcGRA7 could efficiently distinguish IHC positive and negative samples compared with iELISAs against NcGRA6 and the cELISA. Furthermore, antibody levels against NcSAG1 and NcGRA7 were significantly higher in aborting cows comparing with infected but non-aborted dams in a herd experiencing a Neospora abortion outbreak. Tracking the dynamics of antibody levels during pregnancy revealed a marked increase in NcSAG1- and NcGRA7-specific antibodies at the last trimester of pregnancy. In contrast, no marked differences in antibody levels against either antigen were noted in neurologically symptomatic calves compared with non-symptomatic infected calves. Our data suggests NcSAG1 and NcGRA7 as indicators for Neospora abortion.

Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants further development.

Oligomannose-coated liposome-entrapped dense granule protein 7 induces protective immune response to Neospora caninum in cattle.

Neospora caninum is an intracellular protozoan parasite that causes abortion in cows. Vaccination is an important strategy for control of neosporosis, and a safe and effective vaccine suitable for cattle is required. Dense granule protein 7 of N. caninum (NcGRA7) is a secretory protein with high antigenicity in hosts. We demonstrated previously that NcGRA7 entrapped in liposomes coated with mannotriose (M3-NcGRA7) could induce a parasite-specific T-helper type 1 immune response and produce humoral antibodies that resulted in increased offspring survival and decreased infection in the brains of mice dams. In the present study, the efficacy of M3-NcGRA7 as a vaccine candidate against N. caninum has been evaluated in cattle (n=12). Cattle were immunized with M3-NcGRA7 containing 50 µg (n=4) or 200 µg NcGRA7 (n=4) subcutaneously twice with a 4-week interval and all cattle including the non-immunized controls (n=4) were inoculated with 10(7) tachyzoites of Nc-1 strain 27 days after the second immunization and euthanized at 85-87 days post infection (dpi). In immunized cattle, NcGRA7-specific antibody production and IFN-γ production in PBMC was induced before challenge. At 3 dpi, body temperature and concentration of serum IFN-γ tended to be higher in control cattle than in the immunized cattle. Furthermore, the parasite load in the brain significantly decreased in cattle immunized with 50 µg M3-NcGRA7 compared with controls. These results suggest that M3-NcGRA7 can induce protective immune responses to N. caninum tachyzoites in cattle, which could lead to practical application of safe and effective subunit vaccines.

Tissue distribution of Neospora caninum in experimentally infected cattle.

Histopathology and quantitative PCR (qPCR) were used to determine the tissue distribution of Neospora caninum in calves at 80 days postinfection. Our findings revealed that the most appropriate brain areas for researching N. caninum pathogenesis were the amygdala and hippocampus for qPCR and the corpus striatum and diencephalon for histopathology.

ELISAs based on rNcGRA7 and rNcSAG1 antigens as an indicator of Neospora caninum activation.

Bovine abortion caused by the Apicomplexa parasite Neospora caninum is a major economical problem in the livestock industry worldwide. However, the relationship between N. caninum infection and abortion is still unknown. Our study aimed to elucidate the relationship between parasite-specific antibody responses, parasite stages and abortion. In experimentally infected cattle, anti-NcGRA7 IgG1 antibody was predominantly detected during the acute infection stage, while the production of anti-NcSAG1 IgG1 antibody was observed during both acute and chronic stages. Furthermore, levels of anti-NcGRA7 IgG2 antibody were lower than those of anti-NcSAG1 IgG2 antibody. When tested on cattle with Neospora-associated abortion, positive rates of the anti-NcGRA7 IgG2 antibody were significantly lower than those of the anti-NcSAG1 antibody, although there was no difference in IgG1 antibody-positive rates between the two antigens. In addition, anti-NcGRA7 IgG2 antibodies were not detected in cattle for more than 30days after abortion. Our results suggested that anti-NcGRA7 and anti-NcSAG1 antibodies are suitable indicators for the activation stage of N. caninum infection and broad detection of the infection, respectively. In conclusion, the use of recombinant NcGRA7 and NcSAG1-based ELISAs will be useful for evaluating the abortion risk associated with N. caninum infection.

Enzyme-linked immunosorbent assays based on Neospora caninum dense granule protein 7 and profilin for estimating the stage of neosporosis.

Neospora caninum is an intracellular protozoan parasite that causes bovine and canine neosporosis, characterized by fetal abortion and neonatal mortality and by neuromuscular paralysis, respectively. Although many diagnostic methods to detect parasite-specific antibodies or parasite DNA have been reported, to date no effective serodiagnostic techniques for estimating pathological status have been described. Our study aimed to elucidate the relationship between the parasite-specific antibody response, parasite activation, and neurological symptoms caused by N. caninum infection by using a recombinant antigen-based enzyme-linked immunosorbent assay. Among experimentally infected mice, anti-N. caninum profilin (NcPF) antibody was only detected in neurologically symptomatic animals. Parasite numbers within the brains of the symptomatic mice were significantly higher than those in asymptomatic animals. In addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody-positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in the neurologically symptomatic dogs were significantly higher than those in the non-neurologically symptomatic animals. Our results suggested that the levels of anti-NcGRA7 and anti-NcPF antibodies reflect parasite activation and neurological symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have potential as suitable indicators for estimating the pathological status of neosporosis.

Host cholesterol synthesis contributes to growth of intracellular Toxoplasma gondii in macrophages.

The intracellular protozoan Toxoplasma gondii lacks the ability to synthesize sterol and scavenges cholesterol from the low-density lipoprotein receptor (LDLR) pathway of its host to facilitate replication. Sterol biosynthesis inhibitors, however, have a demonstrated anti-Toxoplasma effect. In this study, we examined the host mevalonate pathway as a novel source of cholesterol for T. gondii and its effects on parasite growth in macrophages. Parasite growth did not significantly change in the absence of LDLR or when LDL was exogenously supplemented. Lovastatin and compactin, both inhibitors of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the mevalonate pathway, significantly inhibited T. gondii growth in both wild-type and LDLR-knockout macrophages. Parasite growth was also suppressed by squalestatin, an inhibitor of squalene synthase, despite mevalonate producing isoprenoid intermediates in host cells. The present study demonstrates that lovastatin, compactin and squalestatin have anti-Toxoplasma activities and that the host cholesterol synthesis may contribute to parasite growth in macrophages.