Seed dormancy release of Halenia elliptica in response to stratification temperature, duration and soil moisture content.

BACKGROUND: Although the effect of cold stratification on seed dormancy release has been extensively studied for many species, knowledge of the role of stratifying temperature, soil moisture content and duration of stratification on seed dormancy release at the population level is limited. Here, we aimed to determine the response of seed dormancy release to these factors in six populations of Halenia elliptica. RESULTS: Seed dormancy release was more responsive to low than high temperatures, and no dormancy break occurred at 8 °C. Seed germination percentage increased first and then remained unchanged as stratifying soil moisture content increased from 0 to 24%. Seed dormancy release of populations from low altitude was more sensitive to increased stratifying temperature and decreased soil moisture content than those from high altitudes. CONCLUSIONS: Temperature and soil moisture changes resulting from global warming could affect seed dormancy release and consequently seedling establishment. Thus, incorporating data on seed dormancy release involving temperature, soil moisture content and stratification duration is beneficial for predicting plant species regeneration, migration and coexistence in a scenario of climate change.

Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection.

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice.

Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice.

In this study, we generated a tkl1 deletion mutant in the Toxoplasma gondii type 1 RH (RHΔtkl1) strain and tested the protective efficacies of vaccination using RHΔtkl1 tachyzoites against acute, chronic, and congenital T. gondii infections in Kunming mice. Mice vaccinated with RHΔtkl1 mounted a strong humoral and cellular response as shown by elevated levels of anti-T. gondii-specific IgG, IL-2, IL-12, IFN-γ, and IL-10. All RHΔtkl1-vaccinated mice survived a lethal challenge with 1 × 103 tachyzoites of type 1 RH or ToxoDB#9 (PYS or TgC7) strain as well as 100 cysts or oocysts of Prugniuad strain. All mock-vaccinated plus infected mice have died. Vaccination also protected against cyst- or oocyst-caused chronic infection, reduced vertical transmission caused by oocysts, increased litter size, and maintained body weight of pups born to dams challenged with 10 oocysts on day 5 of gestation. In contrast, all mock-vaccinated plus oocysts-infected dams had aborted, and no fetus has survived. Vaccinated dams remained healthy postinfection, and their brain cyst burden was significantly reduced compared with mock-vaccinated dams infected with oocysts. In vivo depletion of CD4+ T cells, CD8+ T cells, and B cells revealed that CD8+ T cells are involved in the protection of mice against T. gondii infection. Additionally, adoptive transfer of CD8+ T cells from RHΔtkl1-vaccinated mice significantly enhanced the survival of naive mice infected with the pathogenic strain. Together, these data reaffirm the importance of CD8+ T cell responses in future vaccine design for toxoplasmosis and present T. gondii tkl1 gene as a promising vaccine candidate.

Efficacy of antiretroviral compounds against Toxoplasma gondii in vitro.

The obligate intracellular parasite Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. Although infection with this parasite is generally benign, severe illness may occur in infected individuals if their immunity becomes less competent, such as in human immunodeficiency virus (HIV)-infected patients. In this study, the inhibitory activity of 44 commonly used antiretroviral compounds was determined against T. gondii in vitro. Of the 44 tested antiretroviral compounds, 14 showed potency against T. gondii at IC50 concentrations (concentration inhibiting T. gondii tachyzoite growth by 50%) ranging from 1.18 ± 2.21 µM (nelfinavir) to 18.89 ± 1.87 µM (trovirdine). Of the 14 potent antiretroviral compounds, 7 are HIV-1 protease inhibitors. This study also investigated whether co-administration of these 14 antiretroviral compounds interferes with the anti-T. gondii activity of existing anti-T. gondii drugs, namely sulfadiazine and pyrimethamine. The results showed no significant interaction between any of the 14 tested antiretroviral compounds and pyrimethamine or sulfadiazine. These results warrant investigation of whether administration of the lead antiretroviral drugs with highly potent anti-T. gondii activity to HIV patients may help to limit the occurrence of toxoplasmic encephalitis.

Serological evidence of hepatitis E virus and influenza A virus infection in farmed wild boars in China.

Hepatitis E virus (HEV) and influenza A virus (IAV) are two important pathogens which can infect humans and various animals causing public health problems. In this study, the seroprevalence and risk factors associated with HEV and IAV infection in farmed wild boars were investigated in China. A total of 758 serum samples were collected from farmed wild boars between 2015 and 2016, and antibodies against HEV and IAV were examined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits. The overall prevalence of anti-HEV antibodies was 24.54% (186/758, 95% CI 21.48-27.60) in farmed wild boars. There were statistically significant differences in the HEV seroprevalence in farmed wild boars of different ages (<22 days: 8.33%; 22-66 days: 18.89%; >66 days: 26.36%) (P < 0.05) and different genders (50.00% in male and 23.49% in female) (P < 0.01). However, there was no statistically significant difference in the HEV seroprevalence in farmed wild boars of different regions and different years. The overall IAV seroprevalence was 5.80% (44/758, 95% CI 4.14-7.46), and there was no statistically significant difference in the IAV seroprevalence in farmed wild boars of different ages and genders, collected from different regions and different years. Our results indicate that HEV and IAV infections in farmed wild boars may pose a potential risk for human infection. To our knowledge, this is the first report of HEV and IAV seroprevalence in farmed wild boars in China, which provides baseline data for further studies and for control of HEV and IAV infection in farmed wild boars.

Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System.

Infection with the apicomplexan protozoan parasite Toxoplasma gondii is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of T. gondii pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 GRA genes (GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38, and GRA40) in the virulent T. gondii RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 GRA genes. Functional characterization of single ΔGRA mutants was achieved in vitro using cell-based plaque assay and egress assay, and in vivo in BALB/c mice. Targeted deletion of these 17 GRA genes had no significant effect neither on the in vitro growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17 GRA genes suggest that GRAs may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth in vitro or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations.

Live Attenuated Pru:Δcdpk2 Strain of Toxoplasma gondii Protects Against Acute, Chronic, and Congenital Toxoplasmosis.

Background: The threat of Toxoplasma gondii infection in immunocompromised individuals and pregnant women necessitates the development of a safe and effective vaccine. Here, we examined the immune protection conferred by a live attenuated strain of T. gondii. Methods: We tested the efficacy of intraperitoneal vaccination using 500 Ca2+-dependent protein kinase 2 (cdpk2)-deficient tachyzoites of T. gondii Pru strain against acute, chronic, and congenital toxoplasmosis in mice. The kinetics of antibody response, cytokines, and other quantifiable correlates of protection against T. gondii infection were determined. Results: Vaccination with Pru:Δcdpk2 induced a high level of anti-T. gondii immunoglobulin G titer, type 1 T-helper (Th1) response at 28 days postvaccination, and a mixed Th1/type 2 T-helper response at 70 days postvaccination. All vaccinated mice survived a heterologous challenge with 1000 tachyzoites of RH or ToxoDB#9 (PYS or TgC7) strains. Also, vaccination protected against homologous infection with 20 T. gondii Pru cysts, and improved pregnancy outcome by reducing parasite cyst load in the brain, maintaining litter size and body weight of pups born to vaccinated dams challenged with 10 Pru cysts compared to pups born to unvaccinated dams. Conclusions: The use of T. gondii Pru:Δcdpk2 mutant strain represents a promising approach to protection against acute, chronic, and congenital toxoplasmosis in mice.

Seroprevalence and risk factors of hepatitis E virus infection in cancer patients in eastern China.

OBJECTIVES: Hepatitis E virus (HEV) is a single-stranded RNA virus infecting a variety of animals and humans. Little is known of HEV infection among cancer patients in China. This study provides new epidemiological data for the prevalence of HEV co-infection in cancer patients, indicating that HEV infection is common in this group. METHODS: A total of 950 cancer patients and 950 control volunteers from Shandong Province, eastern China, were recruited to participate in this investigation. Blood samples from patients and controls were examined for anti-HEV IgG and IgM antibodies using ELISAs. RESULTS: The overall seroprevalence of IgG and IgM antibodies to HEV was significantly higher in cancer patients (247/950, 26.0%) than in controls (123/950, 13.0%) (p<0.001). In cancer patients, 22.7% of serum samples were anti-HEV IgG-positive, and in control subjects, 11.0% were anti-HEV IgG-positive (p<0.001). For IgM antibodies, seroprevalence was 4.2% in cancer patients (n=40) and 2.1% in controls (n=20) (p=0.009). The seroprevalence of HEV was higher in cancer patients who had dogs in their home (31.7%) when compared to other cancer patients. The highest seroprevalence of HEV infection was found in leukemia patients (32.3%), followed by liver cancer patients (31.1%); the lowest HEV seroprevalence was detected in patients with gastric cancer (18.9%). CONCLUSIONS: This investigation revealed that the seroprevalence of HEV was significantly higher in cancer patients than in controls in eastern China. Therefore, cancer patients are a high-risk population and should be kept away from sources of HEV infection.

Toxoplasma gondii Infection in Farmed Wild Boars (Sus scrofa) in Three Cities of Northeast China.

The apicomplexan protozoan parasite Toxoplasma gondii is a widely distributed etiological agent of foodborne illness. This parasite can cause production losses in livestock and serious disease in humans through consumption of contaminated meat. Pig meat is the most likely source of human infection, and wild boars may play a role in the transmission of T. gondii by serving as a reservoir host. This study aimed to investigate the seroprevalence of antibodies to T. gondii among farmed wild boars in China. In an 11-month survey, a total of 882 serum samples were obtained from farmed wild boars from three cities (Jilin City, Siping City, and Baishan City) in Jilin province, Northeast China and were tested for antibodies specific for T. gondii. Using modified agglutination test and a cutoff titer of 1:25, the prevalence of T. gondii infection in the examined samples was 10.0% (88 of 882). The highest seroprevalence was observed in animals from Jilin city (15.3%, 43/281) and followed by Siping (11.4%, 30/263) and Baishan (4.4%, 15/338). Logistic regression analysis revealed a significant correlation between the investigated geographic region and T. gondii infection. In addition, prevalence was higher in females compared to males, and the highest prevalence was detected in piglets. These findings indicate that farmed wild boars may become a source of foodborne toxoplasmosis, posing a food safety threat to the public health in the investigated areas. Implementation of effective measures to control T. gondii infection in farmed wild boars in China may be warranted.