Preventive effect of pidotimod on reactivated toxoplasmosis in mice.

As one of food-borne parasitic diseases, toxoplasmosis entails the risk of developing reactivation in immunocompromised patients. The synthetic dipeptide pidotimod is a potent immunostimulating agent that improves the immunodefenses in immunodepression. To investigate the efficacy of pidotimod as a preventive treatment, we used a murine model of reactivated toxoplasmosis with cyclophosphamide (CY)-induced immunosuppression. Pidotimod administration significantly restored the body weight and spleen organ index, increased survival time (from 70 to 90%), and decreased the parasitemia (from 80 to 35%) of CY-induced mice with reactivated toxoplasmosis. Cytokine profiles and CD4(+) T cells subpopulation analyses by Cytometric Bead Array and flow cytometry demonstrated that pidotimod treatment resulted in a significant upregulation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th1 cells (from 3.73 ± 0.39 to 5.88 ± 0.46%) after CY induction in infected mice. Additionally, histological findings and parasite DNA quantification revealed that mice administered with pidotimod had a remarkable reduction of parasite burden (two-log) and amelioration of histopathology in the brains. The in vitro studies showed that pidotimod significantly restored concanavalin A-induced splenocyte proliferation and pro-inflammatory cytokines in the supernatants of splenocyte culture. It could be concluded that the administration of pidotimod in immunocompromised mice significantly increases the Th1-biased immune response, prolongs survival time, and ameliorates the load of parasites in the blood. This is the first report of the preventive effect of pidotimod on reactivated toxoplasmosis.

[Kinetic distribution of cyst-forming Chinese isolate of Toxoplasma gondii in mice].

OBJECTIVE: To determine the kinetics of infection and cyst formation in CD1 mice following oral infection with cyst-forming Chinese isolate of Toxoplasma gondii TgCtwh1(genotype China 1, ToxoDB#9). METHODS: 50 CD1 female mice were obtained from specific pathogen-free (SPF) mouse colony in the Vital River Laboratories (VRL), Beijing. Mice were randomly divided into 10 groups each with 5 mice. All mice but control were peroral gavage infected with 50 cysts (1x10(4) bradyzoites) of TgCtwh1 isolate of T. gondii isolated from Wuhan, China. Cysts were isolated from the entire brain of mice infected with TgCtwh1 by density gradient centrifugation over Fycoll-paque plus. Animals were orally inoculated with cysts on day zero, and peripheral blood, lymph nodes, heart, liver, and brain of infected mice were collected on days 2, 4, 7, 10, 14, 21, 35, 50, and 72 post infection. Five mice were sacrificed by cervical dislocation under anesthesia at each time of collection, and the kinetic distribution was detected by fluorescence quantitative PCR and tissue inoculation into fresh mice. The cyst formation at various intervals after infection was also observed, as was the number of the cysts in brains and the cyst-forming rate. RESULTS: The body weight of the mice lessened (3.650 +/- 0.252)g post oral infection on day 7, and the weight was progressively decreased between day 10 [(1.730 +/- 0.017)g] and day 14 [(-0.390 +/- 0.554) g] after infection (P<0.05). In the brain tissue, cysts were first observed on day 21 post oral infection and the cyst-forming rate was 80%, and the average diameter of cysts was 20-40 microm. While on day 35 after infection, the cysts were formed in all infected mice(cyst-forming rate was 100%) and the average diameter was 50-60 microm. In chronic infection, DNA copies of parasites were first detected in blood, heart, liver and lymph node at 3.51 +/- 0.152, 4.100 +/- 0.198, 4.220 +/- 0.209 and 4.960 +/- 0.052 respectively on day 2, then in the brain on day 4 (3.800 +/- 0.154). During the early days of infection, the parasite burden in blood was progressively increased until days 7 (5.240 +/- 0.115) then gradually decreased and become undetectable on day 35. The burden of T. gondii in the heart and brain tissues increased significantly and reached their maximum on day 14 (5.640 +/- 0.214) and day 10 (5.790 +/- 0.060), respectively, and remained a stable level thereafter. Liver and lymph tissues reached their maximum on day 7 (5.310 +/- 0.038) and day 10 (6.200 +/- 0.152), then gradually decreased and become undetectable on day 50. CONCLUSION: The parasitemia in mice infected with T. gondii cyst-forming isolate lasts for 21 d at least, and cysts are detected in brain on day 21.

Evaluation of recombinant SjLAP and SjFBPA in detecting antibodies to Schistosoma japonicum.

OBJECTIVE: To investigate the early response of immunoglobulin G (IgG) antibody responses to Schistosoma japonicum infection in mice by using the recombinant proteins, S. japonicum leucine aminopeptidase (rSjLAP) and S. japonicum fructose-1, 6-bisphosphate aldolase (rSjFBPA), and evaluate the potential of rSjLAP and rSjFBPA in diagnosis as well as in assessment of therapeutic efficacy in human schistosomiasis. METHODS: rSjLAP or rSjFBPA was induced from Escherichia coli BL21 strain transfected with the expression vectors, pET-28a-rSjFBPA/BL21 or pET-28a-rSjLAP/BL21 using isopropyl-beta-D-thiogalactoside (IPTG), and purified by Ni-NTA His Bind resin. 88 BALB/c female mice, inbred and 6 to 8 weeks old, were randomly divided into 4 groups. Groups A, B and C each made up of 21 mice and group D comprised 25 mice. Groups A, B and C were infected with 5, 15 and 25 S. japonicum cercariae respectively. As control, mice in group D were left uninfected. 3 mice from each of groups A, B and C were sacrificed and sera collected on days 3, 7, 10, 14, 20, 30, and 60 post infection. All the 25 mice in group D were sacrificed on the first day of the experiment for serum collection. rSjLAP and rSjFBPA were screened and used in ELISA to test the antibody response of the serum samples. Also, sera of 38 acute patients, 96 chronic patients with schistosomiasis japonica, 90 healthy donors and patients with other parasite infections including Clonorchis sinensis (33 cases), Paragonimus westermani (40) and hookworms (37) were tested using the recombinant protein-based ELISA. In addition, 36 sera each from the acute and chronic patients 12 months after treatment with praziquantel and 64 of the chronic patients in more than 2 years post-treatment of praziquantel were tested. The dosage of praziquantel for both acute and chronic patients was 60 mg/kg, 2 times/dx2 d. RESULTS: IgG antibody response was first detected at day 10 post infection by rSjLAP, rSjFBPA or the combined antigen assay. The mean absorbance (A450) on this day were 0.535 +/- 0.053, 0.595 +/- 0.033, 0.696 +/- 0.104 for group B; 0.548 +/- 0.060, 0.608 +/- 0.063, 0.621 +/- 0.090 for group C; and 0.415 +/- 0.038, 0.455 +/- 0.056, 0.498 +/- 0.077 for group A for rSjLAP, rSjFBPA and the combined assay respectively (P < 0.05). Early antibody level to both antigens was significantly higher in mice infected with 15 or 25 cercariae than those with 5 cercariae (P < 0.05). However, ELISA results in patients with confirmed schistosomiasis revealed positive rates of 97.4% (37/38) and 87.5% (84/96) for acute and chronic schistosomiasis with rSjLAP , 94.7% (36/38) and 88.5% (85/96) for acute and chronic schistosomiasis with rSjFBPA and 94.7% (36/38)and 85.4%(82/96) with both rSjLAP and rSjFBPA respectively. Statistical analysis showed no significant difference in the positive rate (P > 0.05). Also, rSjLAP and combined antigens showed a specificity of 96.7% (87/90) while that of rSjFBPA was 97.8% (88/90). There was a general decrease in the antibody titer of the patients after treatment. In 12 months after treatment it was 0.236 +/- 0.212 with rSjLAP, 0.287 +/- 0.191 with rSjFBPA, and 0.235 +/- 0.120 with both antigens respectively for acute cases; For chronic patients, it was 0.266 +/- 0.124, 0.261 +/- 0.143 and 0.265 +/- 0.140 in 12 months post-treatment, and 0.204 +/- 0.074, 0.176 +/- 0.074, and 0.176 +/- 0.073 in 2 years, respectively. For healthy control, it was 0.188 +/- 0.056, 0.173 +/- 0.45, and 0.184 +/- 0.051, respectively. No significant difference on antibody titer was found between treated patients and control (P > 0.05). The cross reaction with C. sinensis was 15.2% (5/33) for rSjLAP, 12.1% (4/33) for rSjFBPA and 9.2% (3/33) for combined antigens. With P. westermani, it was 15.0% (6/40), 12.5% (5/40) and 15.0% (6/40), respectively, and 8.1% (3/37) with hookworm infection. CONCLUSION: The study showed a satisfactory sensitivity and specificity of rSjLAP and rSjFBPA by ELISA which is promising for the immunological diagnosis of schistosomiasis.

Seroprevalence and genetic characterization of Toxoplasma gondii in cancer patients in Anhui Province, Eastern China.

BACKGROUND: Recent studies have indicated the predominance of Toxoplasma gondii genotype Chinese 1 in animals in China. However, little is known of the genetic features of the parasite in humans. This study aims to determine the prevalence of anti-T. gondii antibodies based on which the genetic character of the parasite was identified in cancer patients in China. METHODS: A total of 1014 serum samples with malignant neoplasms were collected from six tertiary-care hospitals (HAUCM, APH, HAMU, XAH, FHH and HBMC) from January, 2012 to August, 2013. Antibodies against T. gondii were examined by enzyme-linked immunosorbent assay (ELISA). Blood samples were subsequently used for PCR assay to detect T. gondii DNA (gra6). The DNA positive samples were subjected to genotyping using a multiplex multilocus nested PCR-RFLP at 10 loci, including sag1, sag2, sag3, btub, gra6, l358, c22-8, c29-2, pk1 and apico. Samples from the patients were anonymous and only data with regard to age and gender was available at sample collection. RESULTS: Overall, 8.38% (85/1014) of the examined patients showed positive antibodies against T. gondii. Among them, 61 (6.02%) were seropositive only for IgG, 16 (1.58%) were only for IgM, and 8 (0.79%) were found to be positive for both IgG and IgM. The seroprevalence of antibodies to Toxoplasma ranged from 5.8% to 11.0%, without regional difference (χ(2) = 4.764, P = 0.445). No significant differences of the positive rates of T. gondii infection were noted in genders (male, 8.96%; female, 7.45%) (χ(2) = 0.707, P = 0.400) and in ages (χ(2) = 1.172, P = 0.947). Of 1014 DNA samples, 36 (3.55%) were positive for T. gondii by nested PCR at gra6 locus and nine gave rise to complete genotyping results. All samples with achieved PCR-RFLP genotyping showed a common genetic character of type Chinese 1 (ToxoDB#9). CONCLUSION: Seroprevalence of toxoplasmosis in immunosuppressed individuals is rarely reported in China and we presented a positive rate of 8.38% in cancer patients. Toxoplasma genomic DNA genotyping demonstrated a common genetic character of Chinese 1, indicating a possible pathogenic origin of animals in human infection.