Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples.

Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries.

Early detection of Toxoplasma gondii-infected cats by interferon-gamma release assay.

Felines, the only definitive hosts that shed the environmentally-durable oocysts, are the key in the transmission of Toxoplasma gondii to all warm-blooded animals. They seroconvert as late as the third week and begin to shed oocysts as early as 3-8 days after being fed tissue cysts. Early detection of Toxoplasma-infected cats is crucial to evaluate Toxoplasma-contaminated environment and potential risks to public health. Moreover, it is fundamental for Toxoplasma infection control. Interferon-gamma release assay (IGRA) is a blood-based test assessing the presence of IFN-γ released by the T-lymphocytes directed against specific antigens, which is an ideal assay for early detection of Toxoplasma-infected cats. Here, cats were orally infected with the tissue cysts and blood was collected for toxoplasmic antigen stimulation, and the released IFN-γ was measured by ELISA. Results showed that Toxoplasma-infection was detected by IGRA as early as 4 days post-infection (dpi); while serum Toxoplasma IgM and IgG were detected by ELISA at 10 dpi and 14 dpi, respectively. Our findings demonstrated that IGRA-positive and ELISA-negative samples revealed an early Toxoplasma infection in cats, indicating a new strategy for the early diagnosis of Toxoplasma infection by combining IGRA and ELISA. Therefore, IGRA could emerge as a reliable diagnostic tool for the exploration of cat toxoplasmosis prevalence and its potential risks to public health.

Transfection of Eimeria mitis with yellow fluorescent protein as reporter and the endogenous development of the transgenic parasite.

BACKGROUND: Advancements have been made in the genetic manipulation of apicomplexan parasites. Both the in vitro transient and in vivo stable transfection of Eimeria tenella have been developed successfully. Herein, we report the transient and stable transfection of Eimeria mitis. METHODS AND FINDINGS: Sporozoites of E. mitis transfected with enhanced yellow fluorescent protein (EYFP) expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS) and then passaged 6 generations successively in chickens. The resulting population was analyzed by genome walking and Western blot. The endogenous development of the transgenic E. mitis was observed and its reproduction potential was tested. The stable transfection of E. mitis was developed. Genome walking confirmed the random integration of plasmid DNA into the genome; while Western blot analysis demonstrated the expression of foreign proteins. Constitutive expression of EYFP was observed in all stages of merogony, gametogony and sporogony. The peak of the transgenic oocyst output was delayed by 24 h and the total oocyst reproduction was reduced by 7-fold when compared to the parental strain. CONCLUSION: Stable transfection of E. mitis was successfully developed. The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E. mitis as a vaccine vector.

Toxoplasma gondii: effects of exogenous nitric oxide on egress of tachyzoites from infected macrophages.

Toxoplasma gondii is an obligate intracellular parasite that can infect any nucleated cells of warm-blood vertebrates. Invasion and egress by this protozoan parasite, both of which are crucial for its life cycle, are rapid events that are dependent upon parasite motility. A variety of chemicals and molecules have been utilized to induce Toxoplasma early egress from host cells. Here, we aimed to determine whether nitric oxide (NO) could induce egress of T. gondii tachyzoites from infected cells. Infected macrophages were collected from C57BL/6 mice and treated with different doses of sodium nitroferricyanide (III) dihydrate (SNP) which releases nitric oxide into cell culture medium. The pattern of parasite egress was analyzed by flow cytometry. The results showed that exogenous NO released by SNP could trigger egress of T. gondii tachyzoites from infected peritoneal macrophages which then underwent necrosis after parasite egress. Our findings provided a novel approach to study the interactions between host immune responses and T. gondii.

Identification of antigenic proteins of Toxoplasma gondii RH strain recognized by human immunoglobulin G using immunoproteomics.

Toxoplasma gondii, a ubiquitous intracellular protozoan, infects one third of the world human population. It is of great medical significance, especially for pregnant women and immune-compromised patients. Accurate and early detection of T. gondii infection is crucial in the management of this disease. To obtain potential diagnostic markers, immunoproteomics was employed to identify immunodominant proteins separated by 2-D immunobloting and probed with sera collected from Toxoplasma-positive pregnant women. MALDI-TOF MS and MS/MS analyses identified a total of 18 immunoreactive proteins that were recognized by Toxoplasma-positive sera, whereas none was reactive with the negative-control sera from healthy, Toxoplasma-negative volunteers. Pregnant women showed a diverse immunoreactivity pattern with each serum recognizing one to eight identified tachyzoite proteins. The identified proteins were localized in the membrane, cytoplasm and specific organelles of T. gondii, and are involved in host cell invasion, metabolism and cell structure. Among these 18 proteins, actin, catalase, GAPDH, and three hypothetical proteins had a broad reactivity with Toxoplasma-positive sera, indicating their potential as diagnostic markers for toxoplasmosis. Each of several combinations of the identified proteins offered 100% detection of Toxoplasma infections of all 28 Toxoplasma-positive women. The study findings suggest that Toxoplasma tachyzoites are highly immunogenic and highlights the heterogeneity of host responses to Toxoplasma infection and the importance of using combinations of immunogens as diagnostic antigens. The findings have significant implications to the development of diagnostic reagents with high sensitivity and specificity.

DNA vaccination with a gene encoding Toxoplasma gondii GRA6 induces partial protection against toxoplasmosis in BALB/c mice.

BACKGROUND: Infection with the protozoan Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Protection from acute toxoplasmosis is known to be mediated by CD8+ T cells, but the T. gondii antigens and host genes required for eliciting protective immunity have been poorly defined. The T. gondii dense granule protein 6 (GRA6), recently proved to be highly immunogenic and produces fully immune protection in T. gondii infected BALB/c mice with an H-2Ld gene. The CD8+ T cell response of H-2Ld mice infected by the T. gondii strain seemed to target entirely to a single GRA6 peptide HF10-H-2Ld complex. RESULTS: To determine whether a GRA6-based DNA vaccine can elicit protective immune responses to T. gondii in BALB/c mice, we constructed a eukaryotic expression vector pcDNA3.1-HisGRA6 and tested its immunogenicity in a mouse model. BALB/c mice were vaccinated intramuscularly with three doses of GRA6 DNA and then challenged with a lethal dose of T. gondii RH strain tachyzoites. All immunized mice developed high levels of serum anti-GRA6 IgG antibodies, and in vitro splenocyte proliferation was strongly enhanced in mice adjuvanted with levamisole (LMS). Immunization with pcDNA3.1-HisGRA6 with LMS resulted in 53.3% survival of challenged BALB/c mice as compared to 40% survival of BALB/c without LMS. Additionally, immunized Kunming mice without an allele of H-2Ld failed to survive. CONCLUSIONS: Our result supports the concept that the acquired immune response is MHC restricted. This study has a major implication for vaccine designs using a single antigen in a population with diverse MHC class I alleles.

[Researches on the effect of gas humidity on photoacoustic spectroscopy gas detection system].

Environmental influence is the important factor in a photoacoustic spectroscopy gas detection (PASGD) system when it is applied in the industrial field. The experiments show that the sensitivity of condenser microphone is affected mostly by the humidity of the gas under test, leading to the PASGD's result drift. The present paper puts forward a method to eliminate the influence of gas humidity. A speaker is fixed in the photoacoustic cell, whose amplitude is regarded as the sensitivity self-adaption characterization of sound sensor, used to correct the photoacoustic signal amplitude. Thus the problem of sensitivity changing in sound detection with condenser microphone is solved. Based on this method, a photoacoustic experimental setup, equipped with a diode laser, a resonant photoacoustic cell and a lock-in amplifier, was applied to compare the test for different humidity samples. The results show that this method is useful to eliminating the gas humidity influence and enhancing environmental adaptability of PASGD system.

3D shape measurement of objects with high dynamic range of surface reflectivity.

This paper presents a method that allows a conventional dual-camera structured light system to directly acquire the three-dimensional shape of the whole surface of an object with high dynamic range of surface reflectivity. To reduce the degradation in area-based correlation caused by specular highlights and diffused darkness, we first disregard these highly specular and dark pixels. Then, to solve this problem and further obtain unmatched area data, this binocular vision system was also used as two camera-projector monocular systems operated from different viewing angles at the same time to fill in missing data of the binocular reconstruction. This method involves producing measurable images by integrating such techniques as multiple exposures and high dynamic range imaging to ensure the capture of high-quality phase of each point. An image-segmentation technique was also introduced to distinguish which monocular system is suitable to reconstruct a certain lost point accurately. Our experiments demonstrate that these techniques extended the measurable areas on the high dynamic range of surface reflectivity such as specular objects or scenes with high contrast to the whole projector-illuminated field.

Evaluation of Toxoplasma gondii as a live vaccine vector in susceptible and resistant hosts.

BACKGROUND: Toxoplasma gondii has been shown to trigger strong cellular immune responses to heterologous antigens expressed by the parasite in the inbred mouse model. We studied the immune response induced by T. gondii as an effective vaccine vector in chickens and rabbits. RESULTS: T. gondii RH strain was engineered to express the yellow fluorescent protein (YFP) in the cytoplasm. A subcutaneous injection of the transgenic T. gondii YFP in chickens afforded partial protection against the infection of transgenic E. tenella YFP. T. gondii YFP induced low levels of antibodies to YFP in chickens, suggesting that YFP specific cellular immune response was probably responsible for the protective immunity against E. tenella YFP infection. The measurement of T-cell response and IFN-γ production further confirmed that YFP specific Th1 mediated immune response was induced by T. gondii YFP in immunized chickens. The transgenic T. gondii stimulated significantly higher YFP specific IgG titers in rabbits than in chickens, suggesting greater immunogenicity in a T. gondii susceptible species than in a resistant species. Priming with T. gondii YFP and boosting with the recombinant YFP can induce a strong anti-YFP antibody response in both animal species. CONCLUSIONS: Our findings suggest that T. gondii can be used as an effective vaccine vector and future research should focus on exploring avirulent no cyst-forming strains of T. gondii as a live vaccine vector in animals.

High-accuracy three-dimensional shape acquisition of a large-scale object from multiple uncalibrated camera views.

Acquiring a high-accuracy three-dimensional (3D) shape of a large-scale object from multiple uncalibrated camera views remains a big challenge, since a considerable number of images is required to cover the entire surface; the use of multiple images could, however, result in accumulative errors from each processed image. Here error propagation rules in the 3D reconstruction process have been deduced on the basis of the traditional dual-view reconstruction method. We propose a method that can control the accumulative errors by reducing the times of coordinate transformation with common-view-based dual-view reconstruction. This method involves constructing an image network composed of many image groups, each of which contains a common view. A baseline threshold method is introduced to construct a high-quality image network, and the sums or reprojection residual of all the common points is proposed to assess the validity of the solutions of the orientation. Experiments carried out with both synthetic and real images demonstrate that the proposed method can handle the accumulative error problem with robust and highly accurate results.

Regulation of subtilisin-like protease prC expression by nematode cuticle in the nematophagous fungus Clonostachys rosea.

Nematophagous fungi have been used as biological control agents against nematodes parasitic to plants and animals. These fungi can secret subtilisin-like extracellular serine proteases during the infection of nematodes. The expression of these subtilisin-like serine proteases is regulated by nitrogen sources, including nematode cuticle. However, the mechanisms underlying the nitrogen sources-induced expression of these serine proteases is not well understood. In this study, we investigated the effect of nitrogen sources on the expression of a subtilisin-like extracellular protease, prC, in the nematophagous fungus Clonostachys rosea. Disruption of prC attenuated infection of the fungus to nematodes, indicating that this gene functions as a virulence factor. The inhibition of basal expression of prC by the preferred nitrogen sources (glutamine, ammonia) occurred at the transcriptional level. In contrast, nematode cuticle induced the expression of prC at the post-transcriptional level. The inducible expression of prC by nematode cuticle was significantly suppressed by glutamine, ammonia and phenylmethylsulfonyl fluoride (an inhibitor of serine protease). Thus, the existence of active PrC, albeit at a low level in the medium, is probably essential for further induction of this gene by nematode cuticle. Moreover, the low molecule weight (< 3 kD) degradation products of nematode cuticle could significantly induce the expression of prC. Ammonia suppresses the virulence of C. rosea against nematodes, probably by inhibiting prC expression. Thus, the nematophagous fungi probably could not function well as biocontrol agents in fields fertilized with a large amount of ammonium salt.

[Theoretical study on radial resonance coupling of cylindrical photoacoustic cells].

Photoacoustic detection of trace concentrations of gases is one of the most sensitive techniques of infrared absorption spectroscopy. High-sensitivity photoacoustic detectors apply an acoustic resonator for the amplification of the weak photoacoustic signal. If the modulation frequency coincides with one of the resonance frequencies of the chamber, a standing acoustic wave is excited and the system works as an acoustic amplifier. The amplification of the resonator relies on the acting mode, quality factor, nature of microphone, and the coupling between electromagnetic radiation and the stand wave resonance mode. With different incidence orientation of the modulated IR laser relative to acoustic chamber, the sound pressure magnitude of resonance mode varies. The influence of different laser incidence orientation on the coupling coefficients of radial resonance mode of cylindrical photoacoustic cells was investigated by both theoretical deduction and numerical computation method. It is concluded that the coupling coefficients have two zeros and two maximums when the laser incidence angle varies from 0 to pi/2. When the incidence angle is 0 or tan(-1) (0.859 2 X 2R/L), the coupling coefficients are zeros and the radial resonance is invalid. When the incidence angle is tan(-1) (0.556 8 X 2R/L) or tan(-1) (2R/L), the coupling coefficients are the maximums and the radial resonance is the strongest. Here R is the radius and L the length of the cell. The results therein before give some theoretical guidelines for photoacoustic cell designing, optimizing, installing and adjusting, and for improvement of detection sensitivity in trace gas detectors through maximal excitement of radial modes in cylindrical acoustic cells.

PacC in the nematophagous fungus Clonostachys rosea controls virulence to nematodes.

Nematophagous fungi are commonly used as biological control agents of plant and animal parasitic nematodes. However, relatively little is known of the environmental attributes conferring pathogenicity in these fungi. In this report, we investigated the role of PacC-mediated pH response in the pathogenesis of the nematophagous fungus Clonostachys rosea. We identified a pacC orthologue from this fungus and found that its transcript was elevated in C. rosea during the early stage of its infection of nematode. Disruption of pacC resulted in slowed growth at alkaline pH, altered filamentation, reduced conidiation and attenuated virulence to nematodes. The expression of an extracellular serine protease PrC, a putative virulence factor, was downregulated in the pacC mutants. The PrC transcript levels were significantly higher under alkaline growth conditions than under acidic growth conditions. Promoter activity analysis and electrophoretic mobility shift assay indicated that the regulation of PrC by pH via the PacC pathway occurred at the transcriptional level. In conclusion, PacC functions as a positive regulator of virulence to nematodes in C. rosea.

The molecular mechanism of endoplasmic reticulum stress-induced apoptosis in PC-12 neuronal cells: the protective effect of insulin-like growth factor I.

Endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative diseases. Although CCAAT/enhancer-binding protein homologous protein (CHOP) has been shown to play a critical role in ER stress, the precise apoptosis cascade downstream of CHOP is unknown. In this report, we investigated the mechanism of ER stress-mediated apoptosis as well as the action of IGF-I in PC-12 neuronal cells. Our results demonstrated that tribbles-related protein 3 (TRB3), which is a target gene of CHOP, was responsible for tunicamycin (an ER stress inducer)-induced apoptosis. TRB3 could promote dephosphorylation of Akt in PC-12 cells. IGF-I inhibited ER stress-induced apoptosis by restoring the phosphorylation level of Akt. Both wortmannin (a phosphatidylinositide 3-kinase inhibitor) and SB 212090 (a p38 MAPK inhibitor) suppressed the protective effect of IGF-I on ER stress-induced apoptosis. Interestingly, IGF-I attenuated ER stress-mediated expression of TRB3 but not CHOP. This action of IGF-I was abolished by SB 212090 but not by wortmannin. Immunoprecipitation analysis revealed that IGF-I promoted the phosphorylation of CHOP by activating p38 MAPK, probably leading to a decrease in the transcriptional activity of CHOP. The dephosphorylation of Akt resulted in increased expression of a proapoptotic protein, p53 up-regulated modulator of apoptosis (PUMA), in a forkhead box O3a-dependent manner. Knockdown of PUMA by short hairpin RNA attenuated ER stress-mediated apoptosis. Thus, our current study indicates that both TRB3 and PUMA are critical molecules in ER stress-induced apoptosis. IGF-I effectively protects PC-12 neuronal cells against ER stress-induced apoptosis through the phosphatidylinositide 3-kinase/Akt and p38 MAPK pathways.

[Effect of reducing caloric intake on mice transplanted with S180 ascitic cancer].

OBJECTIVE: To examine the effect of different reduced caloric intake on mice transplanted with S180 ascitic tumor. METHODS: The institute for cancer research (ICR) mice were randomly divided into control group, 3.0 standard feed (SF) group, 2.0 SF group and 1.3 SF group. The mice in control group were fed enough (about 5 g/d) dietary intake, while the amounts of dietary intake in the latter three groups were scaled down in the proportion of 65%, which were 3.0 g, 2.0 g and 1.3 g standard feed respectively. Meanwhile the essential vitamins were added to the latter three groups to keep the amount of intake the same as that of the control's. RESULTS: For most of the mice, the caloric intake obviously prolonged the mean survival days and improved the life quality was 7.14 kcal/d, and the fasting blood glucose level was 2-3 mmol/L. CONCLUSION: Properly reduced caloric intake and keeping lower blood glucose level is beneficial to prolonging the survival time of mice transplanted with S180 ascitic cancer.