Xun Suo

A Selective Review of Advances in Coccidiosis Research

Coccidiosis is a widespread and economically significant disease of livestock caused by protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and costs the animal agricultural industry many millions of dollars to control. In recent years, the modern tools of molecular biology, biochemistry, cell biology and immunology have been used to expand greatly our knowledge of these parasites and the disease they cause. Such studies are essential if we are to develop new means for the control of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with emphasis on recent research in poultry, are reviewed. Topics considered include taxonomy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst biogenesis, host cell invasion, immunobiology, diagnostics and control.

Stable transfection of Eimeria tenella: Constitutive expression of the YFP-YFP molecule throughout the life cycle

The obligate intracellular apicomplexan parasite Eimeria tenella, one of seven species of Eimeria that infect chickens, elicits protective cell-mediated immunity against challenge infection. For this reason, recombinant E. tenella parasites could be utilised as an effective vaccine vehicle for expressing foreign antigens and inducing immunity against heterologous intracellular microbes. A stable line of E. tenella expressing foreign genes is a prerequisite, and in this work an in vivo stable transfection system has been developed for this parasite using restriction enzyme-mediated integration (REMI). Two transgenic populations of E. tenella have been obtained that express YFP-YFP constitutively throughout the parasite life cycle. Southern blotting and plasmid rescue analyses show that the introduced exogenous DNA was integrated at random into the parasite genome. Although the life cycle of the transgenic populations was delayed by at least 12h and the output of oocysts was reduced 4-fold relative to the parental BJ strain of E. tenella, the transgenic parasites were sufficiently immunogenic to protect chickens against challenge with either transgenic or parental parasites. These results are encouraging for the development of transgenic E. tenella as a vaccine vector and for more detailed investigation of the biology of the genus Eimeria.

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

BackgroundInfection 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.ResultsTo 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.ConclusionsOur 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.

Transgenic Eimeria tenella Expressing Enhanced Yellow Fluorescent Protein Targeted to Different Cellular Compartments Stimulated Dichotomic Immune Responses in Chickens

Eimeria tenella, one of the seven species of chicken coccidia, elicits protective immunity against challenge infection with both homologous and heterologous strains. We endeavor to use recombinant E. tenella as a vaccine vehicle for expressing and delivering pathogen Ags and investigate immune responses against these foreign Ags. In this study, two lines of transgenic E. tenella expressing a model Ag, enhanced yellow fluorescent protein (EYFP), targeted to the micronemes and to the cytoplasm of the recombinant parasites were constructed to study the impact of Ag compartmentalization on immunogenicity. The MTT assay, intracellular cytokine staining, and real-time PCR were performed to detect the EYFP-specific proliferation and effector functions of splenic lymphocytes of immunized chickens. ELISA was used to measure anti-EYFP IgG and IgA responses. The results showed that both lines of transgenic parasites stimulated EYFP-specific lymphocyte proliferation and IFN-γ expression in CD4 and CD8 T cells, whereas a higher level of Ag-specific lymphocyte proliferation was elicited by the transgenic line expressing microneme-targeted EYFP. Furthermore, this line stimulated stronger IgA response than the one expressing cytoplasm-targeted EYFP after the second immunization. Our findings are encouraging for further investigation of the effect of Ag compartmentalization in transgenic Eimeria on immunogenicity and for the development of a eukaryotic vaccine vector using genetically modified Apicomplexa parasites.

An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin.

Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freunds Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.

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

BackgroundToxoplasma gondii has been shown to trigger strong cellular immune responses to heterologous antigens expressed by the parasite in the inbred mouse model [1]. We studied the immune response induced by T. gondii as an effective vaccine vector in chickens and rabbits.ResultsT. 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.ConclusionsOur 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.

Restriction enzyme-mediated transfection improved transfection efficiency in vitro in Apicomplexan parasite Eimeria tenella

Genetic manipulation of Apicomplexan parasite Eimeria tenella is only in its earliest stages. In the current study, transfection of E. tenella was conducted by electroporating sporozoites along with linear or circular plasmid DNA, and with or without restriction enzyme. Transfection system containing both linear DNA and restriction enzyme resulted in a transfection efficiency of 2.2x10(-3)in vitro, which is 200-fold higher than that using circular plasmid DNA alone. In another transfection strategy, PCR amplicons of expression cassette, instead of whole plasmid DNA, were subjected to transfection, and it was also found successful. These results suggest that linear DNA and restriction enzyme together in the transfection system greatly improve the transfection efficiency of E. tenella. The high transfection efficiency makes possible the establishment of stable transfection in vivo; and the success of PCR-based, restriction enzyme-mediated transfection will further simplify the transfection process for E. tenella and other Apicomplexan parasites.

Metabolomic Profiling of Mice Serum during Toxoplasmosis Progression Using Liquid Chromatography-Mass Spectrometry.

Better understanding of the molecular changes associated with disease is essential for identifying new routes to improved therapeutics and diagnostic tests. The aim of this study was to investigate the dynamic changes in the metabolic profile of mouse sera during T. gondii infection. We carried out untargeted metabolomic analysis of sera collected from female BALB/c mice experimentally infected with the T. gondii Pru strain (Genotype II). Serum samples were collected at 7, 14 and 21 day post infection (DPI) from infected and control mice and were subjected to liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS)-based global metabolomics analysis. Multivariate statistical analysis identified 79 differentially expressed metabolites in ESI+ mode and 74 in ESI− mode in sera of T. gondii-infected mice compared to the control mice. Further principal component analysis (PCA) and partial least squares-discrimination analysis (PLS-DA) identified 19 dysregulated metabolites (5 in ESI+ mode and 14 in ESI− mode) related to the metabolism of amino acids and energy metabolism. The potential utility of these metabolites as diagnostic biomarkers was validated through receiver operating characteristic (ROC) curve analysis. These findings provide putative metabolite biomarkers for future study and allow for hypothesis generation about the pathophysiology of toxoplasmosis.

Co-expression of reporter genes in the widespread pathogen Eimeria tenella using a double-cassette expression vector strategy

The double-cassette expression vector strategy is valuable for many studies, including comparative analysis of the function of promoters and expression of genes in different compartments. In this study, we report co-expression of enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP) in Eimeria tenella transfected with two double-cassette expression vectors, pMIC-EYFP/ACT-RFP and pMIC-EYFP/ACTss-RFP. The results showed that under regulation of the mic1 promoter, EYFP was expressed in sporulated oocysts but not in unsporulated ones, while under regulation of the actin promoter RFP was expressed in both forms. We found that the signal peptide of Toxoplasma gondii dense granule protein 8 (GRA8) located the RFP expression to the parasitophorous vacuoles of the parasites, the margins of the unsporulated oocysts and the cavities of the sporocysts. The feasibility of co-expression of exogenous proteins in E. tenella is important for the development of transgenic E. tenella as a novel vaccine vector.

Cryptic Eimeria genotypes are common across the southern but not northern hemisphere

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