Delin Zhang

Development of an immunochromatographic strip for the rapid detection of Toxoplasma gondii circulating antigens.

We developed a rapid immunochromatographic strip (ICS) procedure that can detect circulating antigens in the blood of animals during the acute stage of toxoplasmosis. The aim of this study was to evaluate this test using sera from field samples and from experimentally infected animals. The sensitivity and specificity of the ICS were compared with those of an enzyme-linked immunosorbent assay (ELISA). Both assays detected circulating antigens in the sera of animals experimentally infected with the Gansu Jingtai strain of Toxoplasma gondii, and the agreement between the two assays was 100%. In the infected animals, circulating antigens could be detected as early as the second day post-infection (PI) and in all animals by the fourth day. In the 381 field serum samples, the positive rates of the ICS and ELISA were 5.2% and 5.8%, respectively. In addition, there was no cross-reactivity of the antigens with Neospora caninum. The results presented here suggest that the ICS is a feasible, convenient, rapid and effective method to detect infection by T. gondii. This test could be a powerful supplement to the current diagnostic methods. Taken together, the results of this study encourage further research toward the production of commercial diagnostic tests for detecting T. gondii in animals.

Increased survival time in mice vaccinated with a branched lysine multiple antigenic peptide containing B- and T-cell epitopes from T. gondii antigens.

To develop a multiple antigenic peptide (MAP) vaccine against toxoplasmosis, tri-epitope MAP constructs were made in dimeric fashion. The constructs included one B-cell and two T-cell epitopes derived from Toxoplasma gondii antigens (SAG1, GRA4 and GRA1) situated in tandem through the GGG spacer sequence, with the latter positioned adjacent to a polylysine core. Immunization of BALB/c and Kunming mice with the MAP construct in Freunds adjuvant induced not only humoral immunes response but cellular responses. These responses were accompanied by significant levels of splenocyte proliferation and interferon gamma (IFN-γ) in vitro. After lethal challenge, vaccinated mice had increased survival time in comparison to unvaccinated controls. Our data demonstrate that a MAP construct could trigger strong humoral and cellular responses against T. gondii, and that this MAP is a vaccine candidate worth further development.

Serological survey of Toxoplasma gondii in Tibetan mastiffs (Canis lupus familiaris) and yaks (Bos grunniens) in Qinghai, China

BackgroundToxoplasma gondii is an amphixenosis which has extensive hosts. In recent years, the prevalence of T. gondii in China has been reported, while little is known on the survey of T. gondii infection in northwest China, especially in yaks (Bos grunniens) and Tibetan mastiffs (Canis lupus familiaris). The current study survey the infection of T. gondii in Tibetan mastiffs and yaks in Qinghai Province, China.MethodsThe indirect hemagglutination test (IHAT) was used to examine T. gondii antibodies in 1 795 serums, including 192 Tibetan mastiffs and 1603 yaks in Qinghai Province, China.ResultsIn this study, the seroprevalence of T. gondii infection was 8.52%. Twenty (10.42%) of 192 serums of Tibetan mastiffs and 133 (8.30%) of 1603 serums of yaks were seropositive. The seroprevalence of T.gondii infection in Tibetan mastiffs in breeding farm (1.08%) was lower than that in the field (19.19%), and the difference was statistically significant (P <0.05). The seroprevalence of antibodies to T.gondii in yaks ranged from 5.45% to 13.28% among the four different areas. The seroprevalence in different age groups were determined with apparent association.ConclusionsThe results indicated that T.gondii infection was prevalent in Tibetan mastiffs and yaks, which have implications for public health in this region. To our knowledge, this is the first seroprevalence survey of Tibetan mastiffs infected by T. gondii in The People’s Republic of China.

Analyzing and identifying novel B cell epitopes within Toxoplasma gondii GRA4

BackgroundThe identification of specific epitopes targeted by the host antibody response is important for understanding the natural response to infection and for the development of epitope-based marker vaccines and diagnostic tools for toxoplasmosis. In this study, Toxoplasma gondii GRA4 epitopes were identified using software-based prediction and a synthetic peptide technique.MethodsThe complete GRA4 gene sequence was obtained from T. gondii of the Gansu Jingtai strain of tachyzoites. The potential B cell epitopes of GRA4 was predicted using the PROTEAN subroutine in the DNASTAR software package. The peptides with good hydrophilicity, high accessibility, high flexibility and strong antigenicity were chemically synthesized and assessed by ELISA using pig sera from different time points after infection.ResultsThe potential B cell epitopes of GRA4 predicted by bioinformatics tools focused on six regions of GRA4, 52-77 aa, 93-112 aa, 127-157 aa, 178-201 aa, 223-252 aa and 314-333 aa. Eleven shorter peptides from the six regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the eleven peptides (amino acids 62-77, 233-252 and 314-333) tested were recognized by all sera.ConclusionsWe precisely located the T. gondii GRA4 epitopes using pig sera collected at different time points after infection. The identified epitopes may be useful for additional studies of epitope-based vaccines and diagnostic reagents.

Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1

BackgroundThe identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics.FindingsEleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully (amino acids 106–120, 166–180, 289–300 and 313–332).ConclusionsWe have precisely located the epitopes of T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents.

Identification of novel B cell epitopes within Toxoplasma gondii GRA1

Newly synthesized epitopes are one of the most promising antigens for the development of diagnostic kits and peptide vaccines. Very little is known about the B cell epitopes on GRA1 of Toxoplasma gondii, which are recognized by the humoral immune response in pigs. In this study, epitopes derived from GRA1 of T. gondii were identified using synthetic peptide techniques and bioinformatics. Three (PG10, PG13 and PG18) out of the eighteen peptides tested were recognized by all pig sera from different time points after infection, and the other peptides were recognized by select sera from various time points after infection. Our data indicate that many regions of GRA1, and in particular, the regions represented by the peptides PG10, PG13 and PG18, are involved in the pig antibody response. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope-based marker vaccines and diagnostic tools for toxoplasmosis.

Detection of Toxoplasma gondii oocysts in soils in Northwestern China using a new semi-nested PCR assay

BackgroundToxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China.ResultsThe one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 102T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0-1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%.ConclusionsThe present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.

Detection of Acute Toxoplasmosis in Pigs Using Loop-Mediated Isothermal Amplification and Quantitative PCR

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

An outbreak of lethal toxoplasmosis in pigs in the Gansu province of China.

In October 2004, a swine farm in Jinchang, Gansu Province, China, experienced an outbreak of toxoplasmosis. Most of the affected pigs had a rectal temperature greater than 40°C and gradually lost their appetite. Morbidity reached 57%, and mortality was approximately 2%. Analysis of blood samples from affected pigs using hemagglutination inhibition (HI) assay, immunoglobulin G—enzyme-linked immunosorbent assay (IgG-ELISA), and IgM-ELISA tests showed high titers of anti—Toxoplasma gondii antibody. Tachyzoites of T. gondii were found in body fluids of mice inoculated intraperitoneally with ground samples from the heart, liver, spleen, and brain of 2 sick pigs. In addition, the inoculation of 5 pigs with T. gondii tachyzoites caused death in 2 of the pigs. The origin of this outbreak was concluded to be food-borne T. gondii infection.

Evaluation of Protective Immune Responses Induced by Recombinant TrxLp and ENO2 Proteins against Toxoplasma gondii Infection in BALB/c Mice

Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all species of warm-blooded animals. As any chemical-based drugs could not act against the tissue cyst stage of T. gondii, vaccination may be one of the ideal control strategies. In the present study, two new vaccine candidates, named TgENO2 and TgTrxLp, were purified from Escherichia coli with pET-30a(+) expression system and then were injected into BALB/c mice to evaluate the protective efficacy against acute and chronic toxoplasmosis. The results showed that both the recombinant proteins, either alone or in combination, could elicit strong humoral and cellular immune responses with a higher level of IgG antibodies, IFN-γ, IL-2, CD4+, and CD8+ T cells as compared to those in mice from control groups. After acute challenge with tachyzoites of the GJS strain, mice immunized with rTgTrxLp (8 ± 2.77 d), rTgENO2 (7.4 ± 1.81 d), and rTgTrxLp + rTgENO2 (8.38 ± 4.57 d) proteins showed significantly longer survival time than those that received Freunds adjuvant (6.78 ± 2.08 d) and PBS (6.38 ± 4.65 d) (χ 2 = 9.687, df = 4, P = 0.046). The protective immunity of rTgTrxLp, rTgENO2, and rTgTrxLp + rTgENO2 proteins against chronic T. gondii infection showed 69.77%, 58.14%, and 20.93% brain cyst reduction as compared to mice that received PBS. The present study suggested that both TgENO2 and TgTrxLp were potential candidates for the development of multicomponent vaccines against toxoplasmosis.