In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Protective efficacy of recombinant Toxoplasma gondii dense granule protein 15 against toxoplasmosis in C57BL/6 mice.

Toxoplasma gondii is a pervasive protozoan parasite that is responsible for significant zoonoses. A wide array of vaccines using different effector molecules of T. gondii have been studied worldwide to control toxoplasmosis. None of the existing vaccines are sufficiently effective to confer protective immunity. Among the different Toxoplasma-derived effector molecules, T. gondii dense granule protein 15 from the type II strain (GRA15 (II)) was recently characterized as an immunomodulatory molecule that induced host immunity via NF-κB. Therefore, we assessed the immunostimulatory and protective efficacy of recombinant GRA15 (II) (rGRA15) against T. gondii infection in a C57BL/6 mouse model. We observed that rGRA15 treatment increased the production of IL-12p40 from mouse peritoneal macrophages in vitro. Immunization of mice with rGRA15 induced the production of anti-TgGRA15-specific IgG, IgG1 and IgG2c antibodies. The rGRA15-sensitized spleen cells from mice inoculated with the same antigen strongly promoted spleen cell proliferation and IFN-γ production. Immunization with rGRA15 significantly enhanced the survival rate of mice and dramatically decreased parasite burden in mice challenged with the Pru (type II) strain. These results suggested that rGRA15 triggered humoral and cellular immune responses to control infection. However, all of the immunized mice died when challenged with the GRA15-deficient Pru strain or the RH (type I) strain. These results suggest that GRA15 (II)-dependent immunity plays a crucial role in protection against challenge infection with the type II strain of T. gondii. This study is the first report to show GRA15 (II) as a recombinant vaccine antigen against Toxoplasma infection.

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice.

Introduction: NcSAG1 is one of most widely investigated antigens of Neospora caninum in various research fields. Such studies demonstrated the proficiency of NcSAG1 in the regulatory process of parasite adhesion and invasion of host cells. Accordingly, the contribution of NcSAG1 to the pathogenesis of neosporosis can undoubtedly be extrapolated, but direct evidence is lacking. Herein, we provide the first successful attempt at the gene disruption of NcSAG1 and novel data on the invasion and virulence potentials of N. caninum in vitro and in vivo. Methods: The disruption of the NcSAG1 gene was applied using the CRISPR/Cas9 system and confirmed by PCR, western blot and indirect fluorescent antibody tests as NcSAG1 knockout parasites (NcSAG1KO). Then, we investigated the role of NcSAG1 in the growth kinetics of the parasite in vitro. Results and discussion: The deletion of the NcSAG1 gene significantly decreased the infection rate and reduced the egress rate of the parasite. An in vivo study using nonpregnant female and male BALB/c mice revealed a significantly higher survival rate and lower body weight change in the group infected with the NcSAG1KO parasite than in the parental strain (Nc-1)-infected group. Regarding the vertical transmission model of BALB/c mice, the absence of the NcSAG1 gene significantly enhanced the survival of pups and greatly lowered the parasite burden in the brains of pups. In conclusion, our study suggested NcSAG1 as a key molecule in the pathogenesis of N. caninum.

Genetic characterization of Neospora caninum from aborted bovine fetuses in Hokkaido, Japan.

Neospora caninum is one of the main causes of bovine abortions worldwide, including Japan. Nothing is known about the N. caninum population substructures in Japan, and only one isolate from a pregnant sheep has been studied to date. This study describes, for the first time, the genetic characterization of isolates of N. caninum implicated in cattle abortions in Japan. Brains from five aborted fetuses were successfully genotyped based on multilocus microsatellite markers. Assigned genotypes showed high frequencies of mixed alleles in the sequenced markers MS7 and MS10, raising concerns about the subpopulation structures of N. caninum infecting animals in Japan. Clustering analysis of the genotypes, together with those from a previous dataset, showed that five of the six genotypes were distinct from other clusters. Meanwhile, the remaining genotype, together with the sheep isolate from Japan, was grouped with those from Mexico and Spain. These preliminary data may indicate a complex transmission pattern of N. caninum in Japan via clonal spreading by vertical and horizontal transmission and geographically related population substructuring.

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.

Neospora GRA6 possesses immune-stimulating activity and confers efficient protection against Neospora caninum infection in mice.

Vaccination has the potential to be the most cost-effective control measure for reducing the economic burden of neosporosis in cattle. In this study, the immune-stimulatory effect of recombinant Neospora caninum dense granule protein 6 (NcGRA6) was confirmed via its triggering of IL-12p40 production in murine macrophages. BALB/c mice were immunized with recombinant NcGRA6 fused with glutathione S-transferase (GST) protein with or without oligomannose-coated-liposomes (OMLs) as the potential adjuvant. Specific IgG1 antibody production was observed from 21 and 35 days after the first immunization in NcGRA6+GST- and NcGRA6+GST-OML-immunized mice, respectively. However, specific IgG2a was detected 1 week after the infection, and IgG2a levels of the NcGRA6+GST- group were higher than those of the NcGRA6+GST-OML-group. Moreover, spleen cell proliferation with concomitant interferon-gamma production was detected in mice immunized with NcGRA6+GST, indicating that a significant cellular immune response was induced. Mouse survival rates against N. caninum challenge infection were 91.7% for NcGRA6+GST and 83.3% for NcGRA6+GST-OML, which were significantly higher than those of control groups (GST-OML: 25%, phosphate-buffered saline: 16.7%). This indicates that naked NcGRA6+GST induced protective immunity. Thus, our findings highlight the immune-stimulating potential of NcGRA6 and the ability to induce protective immunity against N. caninum infection in mice.

Neospora caninum Dense Granule Protein 7 Regulates the Pathogenesis of Neosporosis by Modulating Host Immune Response.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii Neosporosis caused by N. caninum is considered one of the main causes of abortion in cattle and nervous-system dysfunction in dogs, and identification of the virulence factors of this parasite is important for the development of control measures. Here, we used a luciferase reporter assay to screen the dense granule proteins genes of N. caninum, and we found that NcGRA6, NcGRA7, and NcGRA14 are involved in the activation of the NF-κB, calcium/calcineurin, and cAMP/PKA signals. To analyze the functions of these proteins and Neospora cyclophilin, we successfully knocked out their genes in the Nc1 strain using plasmids containing the CRISPR/Cas9 components. Among the deficient lines, the NcGRA7-deficient parasites showed reduced virulence in mice. An RNA sequencing analysis of infected macrophage cultures showed that NcGRA7 mainly regulates the host cytokine and chemokine production. The levels of gamma interferon in the ascites fluid, CXCL10 expression in the peritoneal cells, and CCL2 expression in the spleen were lower 5 days after infection with the NcGRA7-deficient parasite than after infection with the parental strain. The parasite burden and the degree of necrosis in the brains of mice infected with the NcGRA7-deficient parasite were also lower than in those of the parental strain. Collectively, our data suggest that both the NcGRA7-dependent activation of the inflammatory response and the parasite burden are important in Neospora virulence.IMPORTANCENeospora caninum invades and replicates in a broad range of host species and cells within those hosts. The effector proteins exported by Neospora induce its pathogenesis by modulating the host immunity. We show that most of the transcriptomic effects in N. caninum-infected cells depend upon the activity of NcGRA7. A deficiency in NcGRA7 reduced the virulence of the parasite in mice. This study demonstrates the importance of NcGRA7 in the pathogenesis of neosporosis.

Effects of Extracts from Thai Piperaceae Plants against Infection with Toxoplasma gondii.

Herbal medicines and natural herb extracts are widely used as alternative treatments for various parasitic diseases, and such extracts may also have potential to decrease the side effects of the standard regimen drugs used to treat toxoplasmosis (sulfadiazine-pyrimethamine combination). We evaluated how effective the Thai piperaceae plants Piper betle, P. nigrum and P. sarmentosum are against Toxoplasma gondii infection in vitro and in vivo. Individually, we extracted the piperaceae plants with ethanol, passed them through a rotary evaporator and then lyophilized them to obtain crude extracts for each one. The in vitro study indicated that the P. betle extract was the most effective extract at inhibiting parasite growth in HFF cells (IC50 on RH-GFP: 23.2 µg/mL, IC50 on PLK-GFP: 21.4 µg/mL). Furthermore, treatment of experimental mice with the P. betle extract for 7 days after infection with 1,000 tachyzoites of the T. gondii PLK strain increased their survival (survival rates: 100% in 400 mg/kg-treated, 83.3% in 100 mg/kg-treated, 33.3% in 25 mg/kg-treated, 33.3% in untreated mice). Furthermore, treatment with 400 mg/kg of the P. betle extract resulted in 100% mouse survival following infection with 100,000 tachyzoites. The present study shows that P. betle extract has the potential to act as a medical plant for the treatment of toxoplasmosis.

Development of genome-wide PCR-based markers from insertion, deletion and single nucleotide polymorphisms for closely related Japanese rice cultivars and identification of QTLs for the appearance of cooked rice and polished rice.

Appearance of rice grain is an important property, affecting its acceptance by consumers. Moreover, appearance is a complex characteristic involving many components, including glossiness and whiteness. The genetic bases for the glossiness of cooked rice and the whiteness of polished rice (WPR) were determined using 133 recombinant inbred lines (RILs) derived from a cross between two closely related cultivars from Hokkaido, Joiku462, with high glossiness and whiteness, and Yukihikari, an ancestor of Joiku462 with low glossiness and whiteness. Analyses identified 167 genome-wide InDel markers, five cleaved amplified polymorphic sequences (CAPS) and eight derived CAPS markers differentiating the parental lines. The glossiness area (GLA) and glossiness strength (GLS) of cooked rice and WPR were determined for RILs in two locations, Pippu and Sapporo, Hokkaido. Four QTLs were detected. qGLA10 and qGLS9 were detected on chromosomes 10 and 9, respectively, with both being significant at both geographic locations. qWPR1 on chromosome 1 was significant at Pippu, and qWPR4 on chromosome 4 was significant at Sapporo. The Joiku462 alleles at all QTLs increased each trait. The PCR-based markers flanking these four QTLs may be useful for improvement of GLA, GLS and WPR.