Protective immunity against Toxoplasma gondii induced by DNA immunization with the gene encoding a novel vaccine candidate: calcium-dependent protein kinase 3.

BACKGROUND: Toxoplasma gondii can infect almost all warm-blood animals including human beings. The plant-like calcium-dependent protein kinases (CDPKs) harbored by T. gondii are involved in gliding motility, cell invasion, egress and some other developmental processes, and so have been implicated as important virulence factors. METHODS: In the present study, we constructed a DNA vaccine expressing T. gondii CDPK3 (TgCDPK3) and evaluated its protective efficacy against T. gondii infection in Kunming mice. The gene sequence encoding TgCDPK3 was inserted into the eukaryotic expression vector pVAX I, and mice were immunized with pVAX-CDPK3 intramuscularly. RESULTS: The results showed that mice immunized with pVAX-CDPK3 developed a high level of specific antibodies and a strong lymphoproliferative response. The significantly increased levels of IFN-γ, IL-2, IL-12 (p70) and IL-23 and high ratio of IgG2a to IgG1 antibody titers indicated that a Th1 type response was elicited after immunization with pVAX-CDPK3. Furthermore, the percentage of CD4+ T cells in mice vaccinated with pVAX-CDPK3 was significantly increased. After lethal challenge with the tachyzoites of the virulent T. gondii RH strain, the mice immunized with pVAX-CDPK3 prolonged the survival time from 10 days to 24 days (13.5 ± 4.89) compared to untreated mice or those received PBS or pVAX I which died within 7 days (P < 0.05). In chronic infection model (10 cysts of the T. gondii PRU strain), the numbers of brain cysts of the mice immunized with pVAX-CDPK3 reduced significantly when compared with those in control groups (P < 0.05), and the rate of reduction could reach to about 50%. CONCLUSIONS: TgCDPK3 can generate protective immunity against acute and chronic T. gondii infection in Kunming mice and is a promising vaccine candidate for further development of an effective vaccine against T. gondii.

Experimental Study on the Effect of CO2 Injection Pressure on the Migration Characteristics and Extraction Effects of Replacement CH4.

To study the effect of CO2 injection pressure on gas migration characteristics and coalbed methane (CBM) extraction, a platform for the experimental replacement of CH4 with CO2 was used to conduct experiments on the replacement of CH4 under different CO2 injection pressures and analyze the gas transport characteristics and CH4 extraction during the experiment. The results reveal that the rate of gas migration out of the coal seam accelerates with increasing gas injection pressure, as determined by comparisons of the migration rates between adjacent monitoring points. The change trend of the CH4 desorption rate under different gas injection pressures is divided into slow decline, sharp decline, and stability stages, and the maximum value of the effective diffusion coefficient increases from 2.3 × 10-5 to 3.4 × 10-5 and 4.6 × 10-5 cm2/s as the gas injection pressure increases from 0.6 to 0.8 and 1.0 MPa. Similarly, the change pattern of coal seam permeability can be divided into slow decline, sharp decline, and stability stages. After the gas injection pressure was increased from 0.6 to 0.8 and 1.0 MPa, the CH4 desorption volume increased from 90.2 to 94.1 and 97.8 L, whereas the coal seam CO2 sequestration volume increased from 269.2 to 274.2 and 322.8 L, respectively. In contrast, the CH4 extraction efficiency increased from 76.9 to 80.2 and 82.9%, respectively. The research results have important reference value and practical significance for optimizing the CO2 injection pressure and improving the CBM extraction.

Evaluation of immune responses in mice after DNA immunization with putative Toxoplasma gondii calcium-dependent protein kinase 5.

Toxoplasma gondii can cause serious public health problems and economic losses worldwide. Calcium-dependent protein kinases (CDPKs) are key mediators of T. gondii signaling pathways and are implicated as important virulence factors. In the present study, we cloned a novel T. gondii CDPK gene, named TgCDPK5, and constructed the eukaryotic expression vector pVAX-CDPK5. Then, we evaluated the immune protection induced by pVAX-CDPK5 in Kunming mice. After injection of pVAX-CDPK5 intramuscularly, immune responses, determined with lymphoproliferative assays and cytokine and antibody measurements, were monitored, and mouse survival times and brain cyst formation were evaluated following challenges with the T. gondii RH strain (genotype I) and the PRU strain (genotype II). pVAX-CDPK5 effectively induced immune responses with increased specific antibodies, a predominance of IgG2a production, and a strong lymphocyte proliferative response. The levels of gamma interferon (IFN-γ), interleukin 2 (IL-2), and IL-12(p70) and the percentages of CD3(+) CD4(+) and CD3(+) CD8(+) cells in mice vaccinated with pVAX-CDPK5 were significantly increased. However, IL-4 and IL-10 were not produced in the vaccinated mice. These results demonstrate that pVAX-CDPK5 can elicit strong humoral and cellular Th1 immune responses. The survival time of immunized mice challenged with the T. gondii RH strain (8.67 ± 4.34 days) was slightly, but not significantly, longer than that in the control groups within 7 days (P > 0.05). The numbers of brain cysts in the mice in the pVAX-CDPK5 group were reduced by ∼40% compared with those in the control groups (P < 0.05), which provides a foundation for the further development of effective subunit vaccines against T. gondii.

First report of Toxoplasma gondii seroprevalence in pet parrots in China.

Toxoplasmosis, caused by the obligate intracellular protozoan parasite Toxoplasma gondii, has become a serious public health problem worldwide. T. gondii can infect almost all warm-blooded animals, including parrots. However, little is known of T. gondii infection in parrots in China. Antibodies against T. gondii in 311 parrots including 202 Budgerigars (Melopsittacus undulatus), 26 Lovebirds (Agapornis sp.), 22 Cockatiels (Nymphicus hollandicus), and 61 Alexandrine Parakeets (Psittacula eupatria) in the cities of Beijing and Weifang in north China were tested using the modified agglutination test (MAT). Twenty-six (8.36%) out of 311 serum samples were positive for T. gondii at the cutoff of 1:5. Among the four species, a higher seroprevalence of T. gondii was found in Cockatiels (13.64%, 95% confidence interval [CI] 0.00-27.98), although the difference was not statistically significant (p=0.61). Seropositivity rates against T. gondii in male parrots (10.43%, 95% CI 5.74-15.12) were not statistically different from that in female parrots (6.08%, 95% CI 2.23-9.93, p=0.17). The seropositivity of T. gondii in parrots from Weifang and Beijing was 11.11% (95% CI 6.13-16.09) and 5.70% (95% CI 2.08-9.31), respectively. The seroprevalence varied in parrots of different age groups, ranging from 5.71% (95% CI 1.27-10.15) to 13.00% (95% CI 6.41-19.69), however, the difference among age groups was not statistically significant (p=0.12). The seroprevalence of T. gondii infection in parrots in summer (11.63%, 95% CI 6.84-16.42) was significantly higher than in spring (4.32%, 95% CI 0.94-7.70, p=0.02). The results of the present survey indicated that parrots in China are exposed to T. gondii. To our knowledge, this is the first report of T. gondii seroprevalence in parrots in China.