Drug resistance and genetic characteristics of one Eimeria tenella isolate from Xiantao, Hubei Province, China.

Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia.

Evaluation of immunoprotective effects of recombinant proteins and DNA vaccines derived from Eimeria tenella surface antigen 6 and 15 in vivo.

Coccidiosis is an intestinal parasitic disease that causes huge economic losses to the poultry industry globally. Eimeria tenella belonging to protozoon is the causative agent of cecal coccidiosis in chicken, and it causes enormous damage to poultry industry. The surface antigens (SAGs) of apicomplexan parasites have functions of attachment and invasion in host-parasite interaction. As a result of parasitic invasion, host immune response is triggered. However, the immunogenicity and potency of E. tenella surface antigen 6 and 15 (EtSAG 6 and 15), as vaccinal candidate antigen, remain largely unknown. Therefore, gene fragments of E. tenella EtSAG 6 and 15 were amplified and transformed to pET28a prokaryotic vector for recombinant protein expression. The pEGFP-N1 eukaryotic vectors with EtSAG 6 and 15 amplification fragments (pEGFP-N1-EtSAG 5 and 16) were transformed into 293 T cell line. The results of reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis revealed successful expressions of EtSAG 6 and 15 in Escherichia coli and 293 T cells. Subsequently, animal experiments of 49 cobb broilers were performed to evaluate immunoprotection of recombinant proteins and DNA vaccines derived from E. tenella EtSAG 5 and 16 with an immunizing dose of 100 µg, respectively. Chickens vaccinated with rEtSAG 6 protein, rEtSAG 15 protein, pEGFP-N1-EtSAG 6 plasmid, or pEGFP-N1-EtSAG 15 plasmid showed no significant increase in IFN-γor interleukin-4 (IL-4) level compared with control groups. Chickens vaccinated with protein rEtSAG 6, protein rEtSAG 15, pEGFP-N1-EtSAG 6 plasmid, or pEGFP-N1-EtSAG 15 exhibited higher weight gains, lower oocyst output, and lower mean lesion scores, compared with infection control group. Among the four immunized groups, plasmid EGFP-N1-EtSAG 6 (100 µg) group exhibited the highest anticoccidial index (ACI) value (150.20). Overall, plasmids EGFP-N1-EtSAG 6 and 15, as DNA vaccines, provided a more effective immunoprotection for chickens against E. tenella than protein rEtSAG 6 and protein rEtSAG 15 as subunit vaccines. EtSAG 6 and 15 are promising candidate antigen genes for developing coccidiosis vaccine.

A serine/threonine-specific protein kinase of Haemonchus contortus with a role in the development.

In the free-living nematode Caenorhabditis elegans, the serine/threonine-specific protein kinase, AKT, is known to play a key role in dauer formation, life-span, and stress-resistance through the insulin-like signaling pathway. Although the structure and function of AKT-coding genes of C. elegans are understood, this is not the case for homologous genes in parasitic nematodes. In the present study, we explored a C. elegans akt-1 gene homolog in the parasitic nematode Haemonchus contortus, investigated its transcript isoforms (Hc-akt-1a and Hc-akt-1b), and studied expression and function using both homologous and heterologous functional genomic tools. In C. elegans, we showed that the predicted promoter of Hc-akt-1 drives substantial expression in ASJ neurons of the N2 (wild-type) strain. In H. contortus (Haecon-5 stain), RNAi (soaking) led to a significantly decreased transcript abundance for both Hc-akt-1a and Hc-akt-1b, and reduced larval development in larval stages in vitro. Chemical inhibition was also shown to block larval development. Taken together, the evidence from this study points to a key functional role for Hc-akt-1 in H. contortus.

Prevalence of Eimeria parasites in the Hubei and Henan provinces of China.

Coccidiosis is an intestinal parasitic disease that causes huge economic losses in the poultry industry globally. Henan and Hubei, as important poultry production provinces in China, have great pressure for the prevention and control of chicken coccidiosis. In order to obtain information on the local prevalence of Eimeria species, we used an internal transcribed spacer 1 (ITS1) sequence of ribosomal DNA to identify the species from 318 fresh fecal samples. The fecal samples and the data relating to farm information were collected from 137 farms in Hubei and Henan provinces. As shown by genus-specific PCR results, the positivity rate of Eimeria was 97.17% (309/318), and the most common species were Eimeria mitis (66.67%), E. tenella (46.86%), and E. necatrix (41.51%). Then, we analyzed the correlation between the background information of each sample and the PCR identification results, which showed that indigenous farms in Henan province were at the greatest risk of harboring highly pathogenic Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. The results of this study showed that chicken coccidia was widespread, which provides important insights into the control of chicken coccidiosis in this region.

Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 µg) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-γ and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 µg) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

Serine/threonine protein phosphatase 1 (PP1) controls growth and reproduction in Schistosoma japonicum.

Schistosomiasis is a human parasitic disease caused by flatworms of the genus Schistosoma. Adult female schistosomes produce numerous eggs that are responsible for the pathogenesis and transmission of the disease, and the maturation of female gonads depends on the permanent pairing of females and males. Signaling protein kinases have been proven to control female gonad differentiation after pairing; however, little is known about the roles of protein phosphatases in the developmental and reproductive biology of schistosomes. Here we explored 3 genes encoding catalytic subunits of serine/threonine protein phosphatase 1 (PP1c) that were structurally and evolutionarily conserved in Schistosoma japonicum. In situ hybridization showed transcripts of 3 Sj-pp1c genes mainly localized in the reproductive organs and tissues. Triple knockdown of Sj-pp1c genes by RNA interference caused stunted growth and decreased pairing stability of worm pairs, as well as a remarkable reduction in cell proliferation activity and defects in reproductive maturation and fecundity. Transcriptomic analysis post-RNA interference suggested that Sj-pp1c genes are involved in controlling worm development and maturation mainly by regulating cell proliferation, eggshell synthesis, nutritional metabolism, cytoskeleton organization, and neural process. Our study provides the first insight into the fundamental contribution of Sj-PP1c to molecular mechanisms underlying the reproductive biology of schistosomes.-Zhao, L., Lu, Z., He, X., Mughal, M. N., Fang, R., Zhou, Y., Zhao, J., Gasser, R. B., Grevelding, C. G., Ye, Q., Hu, M. Serine/threonine protein phosphatase 1 (PP1) controls growth and reproduction in Schistosoma japonicum.

Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo.

Glycolysis was thought to be the major pathway of energy supply in both fast-replicating tachyzoites and slowly growing bradyzoites of Toxoplasma gondii. However, its biological significance has not been clearly verified. The genome of T. gondii encodes two lactate dehydrogenases (LDHs), which are differentially expressed in tachyzoites and bradyzoites. In this study, we knocked out the two LDH genes individually and in combination and found that neither gene was required for tachyzoite growth in vitro under standard growth conditions. However, during infection in mice, Δldh1 and Δldh1 Δldh2 mutants were unable to propagate and displayed significant virulence attenuation and cyst formation defects. LDH2 only played minor roles in these processes. To further elucidate the mechanisms underlying the critical requirement of LDH in vivo, we found that Δldh1 Δldh2 mutants replicated significantly more slowly than wild-type parasites when cultured under conditions with physiological levels of oxygen (3%). In addition, Δldh1 Δldh2 mutants were more susceptible to the oxidative phosphorylation inhibitor oligomycin A. Together these results suggest that lactate fermentation is critical for parasite growth under physiological conditions, likely because energy production from oxidative phosphorylation is insufficient when oxygen is limited and lactate fermentation becomes a key supplementation.

Micronemal protein 13 contributes to the optimal growth of Toxoplasma gondii under stress conditions.

Toxoplasma gondii is a ubiquitous parasitic protozoan infecting humans and a wide variety of animals. Fast-replicating tachyzoites during acute infection and slowly growing bradyzoites during chronic infection are the two basic forms of T. gondii in intermediate hosts. Interconversion between the two contributes to the transmission and pathogenesis of this parasite. Secretory micronemal proteins are thought to mediate interactions with host cells and facilitate parasite invasion, therefore the majority of them are highly expressed in tachyzoites. Micronemal protein 13 (MIC13) is unique in that its expression is low in tachyzoites and is upregulated under bradyzoite-inducing conditions. Previous attempts to disrupt this gene were not successful, implying that it may play critical roles during parasite growth. However, in this study, MIC13 was successfully disrupted in type 1 strain RH and type 2 strain ME49 using CRISPR/Cas9-mediated gene disruption techniques. Consistent with its low expression in tachyzoites and increased expression under stress or bradyzoite-inducing conditions, MIC13-inactivated mutants displayed normal growth, host cell invasion, intracellular replication, and egress, as well as acute virulence at the tachyzoite stage. However, under stress conditions, such as high pH or oxygen limitation, MIC13-disrupted parasites showed significantly slower growth rates compared to the parental strains, suggesting that it is required for optimal parasite growth under bradyzoite-inducing or stress conditions. This is the first micronemal protein reported to have such expression pattern and function modes, which expands our understanding of the diverse functions of micronemal proteins.

The ABL kinase inhibitor imatinib causes phenotypic changes and lethality in adult Schistosoma japonicum.

Schistosomiasis caused by different species of schistosome parasites is one of the most debilitating helminthic diseases of humans worldwide. For decades, chemotherapy is the main method of controlling schistosomiasis. However, the fear of drug resistance has motivated the search for alternatives. It has been demonstrated that the ABL kinase inhibitor imatinib affected the development and survival of Schistosoma mansoni in vitro; however, there is still lack of information on whether imatinib also affects other schistosome species such as Schistosoma japonicum. In the present study, the anti-schistosomal potency of imatinib on adult S. japonicum was investigated in vitro, and the results showed that imatinib had a significant impact on various physiological processes of S. japonicum adult worms. Besides its negative effects on worm motility, pairing stability, and gonad development, imatinib caused pathological changes in the gastrodermis as well as the death of the parasite. Our findings suggest that imatinib is an intriguing candidate for further development as an option to fight S. japonicum.

A Novel Live Pichinde Virus-Based Vaccine Vector Induces Enhanced Humoral and Cellular Immunity after a Booster Dose.

UNLABELLED: Pichinde virus (PICV) is a bisegmented enveloped RNA virus that targets macrophages and dendritic cells (DCs) early in infection and induces strong innate and adaptive immunity in mice. We have developed a reverse genetics system to produce live recombinant PICV (strain P18) with a trisegmented RNA genome (rP18tri), which encodes all four PICV gene products and as many as two foreign genes. We have engineered the vector to express the green fluorescent protein (GFP) reporter gene (abbreviated as G in virus designations) and either the hemagglutination (HA [H]) or the nucleoprotein (NP [P]) gene of the influenza A/PR8 virus. The trisegmented viruses rP18tri-G/H and rP18tri-G/P showed slightly reduced growth in vitro and expressed HA and NP, respectively. Mice immunized with rP18tri-G/H were completely protected against lethal influenza virus challenge even 120 days after immunization. These rP18tri-based vectors could efficiently induce both neutralizing antibodies and antigen-specific T cell responses via different immunization routes. Interestingly, the immune responses were significantly increased upon a booster dose and remained at high levels even after three booster doses. In summary, we have developed a novel PICV-based live vaccine vector that can express foreign antigens to induce strong humoral and cell-mediated immunity and is ideal for a prime-and-boost vaccination strategy. IMPORTANCE: We have developed a novel Pichinde virus (PICV)-based live viral vector, rP18tri, that packages three RNA segments and encodes as many as two foreign genes. Using the influenza virus HA and NP genes as model antigens, we show that this rP18tri vector can induce strong humoral and cellular immunity via different immunization routes and can lead to protection in mice. Interestingly, a booster dose further enhances the immune responses, a feature that distinguishes this from other known live viral vectors. In summary, our study demonstrates a unique feature of this live rP18tri vector to be used as a novel vaccine platform for a prime-and-boost vaccination strategy.

In vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions.

UNLABELLED: Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. IMPORTANCE: Arenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China.

Several rhoptry proteins (ROPs) have been confirmed to be critical virulence factors of Toxoplasma gondii strains from North America and Europe. The two active kinases ROP17 and ROP18, and pseudokinase ROP5 were thought to be the key determinants of parasites' virulence in laboratory mice. Given the genetic diversity of Toxoplasma strains from different geographical regions, the virulence determinants in other strains, particularly the ones that are phylogenetically distant to the North American and European strains, are yet to be elucidated. In this study, we sought to examine the contribution of three known virulence factors to the virulence of a type I strain (T.gHB1) isolated from Central China. We deleted ROP17 and ROP18 individually, as well as in combination with GRA7 by the CRISPR-Cas9 system in this local isolate. Subsequent virulence tests in mice indicated that deletion of GRA7, ROP17, or ROP18 in T.gHB1showed similar attenuation in mice as the type I RH strain lacking the corresponding proteins. However, in contrast to the reported double knockouts in RH, double deletions of GRA7 plus ROP17 or GRA7 plus ROP18 in T.gHB1 did not show significant further virulence attenuation compared to the ROP17 or ROP18 single knockouts. These results indicated that GRA7, ROP18 and ROP17 may play different roles in virulence determination in genetically diverse strains of Toxoplasma.

Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3.

As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.

Anticoccidial activity of a botanical natural product based on eucalyptus, apigenin and eugenol against Eimeria tenella in broiler chickens.

BACKGROUND: Chicken coccidiosis is an intracellular parasitic disease that presents major challenges to the development of the commercial poultry industry. Perennial drug selective pressure has led to the multi-drug resistance of chicken coccidia, which makes the prevention and control of chicken coccidiosis extremely difficult. In recent years, natural plant products have attracted the attention of researchers due to their inherent advantages, such as the absence of veterinary drug residues. The development of these natural products provides a new direction for the prevention and treatment of chicken coccidiosis. METHODS: The anticoccidial effect of a natural plant product combination formulation (eucalyptus oil + apigenin + eugenol essential oil) was tested against Eimeria tenella in broilers. To search for the optimal concentration of the combination formulation, we screened 120 broilers in a chicken cage trial in which 100 broilers were infected with 5 × 104 sporulated Eimeria tenella oocysts; broilers receiving a decoquinate solution was set up as a chemical control. The optimal anticoccidial concentration was determined by calculating the anticoccidial index (ACI), and the suitable concentration was used as the recommended dose for a series of safety dose assessment tests, such as feed conversion ratio (FCR), hematological indices and serum biochemical indices, as well as liver and kidney sections, at onefold (low dose), threefold (medium dose) and sixfold (high dose) the recommended dose (RD). RESULTS: The results showed that this combination formulation of three plant natural products had a better anticoccidial effect than formulations containing two plant natural products or a single one, with an ACI of 169.3. The dose gradient anticoccidial test revealed that the high-dose formulation group had a better anticoccidial effect (ACI = 169.2) than the medium- and low-dose groups. The safety evaluation test showed that concentrations of the formulation at one-, three- and sixfold the RD were non-toxic to Arbor Acres broilers, indicating the high safety of the combination formulation. CONCLUSIONS: The combination formulation showed not only a moderate anticoccidial effect but also had a high safety profile for broilers. The results of this study indicate a new alternative for the prevention and control of coccidiosis in broilers.

Protective immunity induced by a DNA vaccine-encoding Toxoplasma gondii microneme protein 11 against acute toxoplasmosis in BALB/c mice.

Toxoplasma gondii is one of the most prevalent intracellular parasites and is threatening the health of both humans and animals, therefore causing incalculable economic losses worldwide. Vaccination is thought to be an efficient way of controlling toxoplasmosis. T. gondii microneme protein 11 (MIC11) is a soluble microneme protein which is presumably considered facilitating the early stage of cell invasion. To evaluate the protective efficacy of T. gondii MIC11, in the present study, a new DNA vaccine-encoding the α-chain of T. gondii MIC11 was constructed using the pcDNA3.1 vector. Expression of MIC11 from this vector was confirmed by indirect immunofluorescence assay following transfection into baby hamster kidney (BHK) cells. Intramuscular immunization of BALB/c mice with pcDNA/MIC11 was carried out to evaluate the immune responses by serum antibodies titers, lymphoproliferation assay, and cytokines assay. The protective efficacy was evaluated by survival rate in mice after challenging with highly virulent strain of T. gondii. The results demonstrated that this vaccination elicited significant humoral responses and T. gondii lysate antigen (TLA)-stimulated lymphoproliferation (p < 0.05). Compared to controls, the pcDNA/MIC11 immunized mice had high production of IFN-γ, IL-12, and IL-2 (p < 0.05), but not IL-4 (p > 0.05), indicating that a predominant Th1 type response was developed. The vaccination also increased the survival rate of immunized mice when they were challenged with a lethal dose of tachyzoites of T. gondii RH strain. These data suggest that T. gondii MIC11 is a reasonable vaccine candidate deserving further studies, and pcDNA/MIC11 is a potential strategy for the control of toxoplasmosis.

Detection of Mycoplasma wenyonii in cattle and transmission vectors by the loop-mediated isothermal amplification (LAMP) assay.

Mycoplasma wenyonii is a wall-less hemotrophic prokaryote with worldwide distribution. This paper describes the development of a LAMP method targeting 16S rRNA for specific detection of M. wenyonii in its vectors and cattle. The LAMP method is specific for M. wenyonii detection and more sensitive than PCR. A total of 330 blood samples from cattle were tested by LAMP and PCR detection, 71 (21.5 %) samples were positive by the LAMP, while only 62 (18.8 %) were positive by PCR. For detecting transmission vectors, 26 lice, 30 flies, and 26 mosquitoes were collected and 18 lice, 20 flies, and 21 mosquitoes were tested positive by LAMP and PCR. These results indicate that the LAMP assay is a simple and convenient diagnostic tool for M. wenyonii detection and can be used in epidemiological surveys.

Activation of NF-κB signaling by the dense granule protein GRA15 of a newly isolated type 1 Toxoplasma gondii strain.

BACKGROUND: It has been reported that the NF-κB pathway, an important component of host defense system against pathogens infections, can be differentially modulated by different Toxoplasma gondii strains, depending on the polymorphism of the GRA15 protein. The recently isolated Toxoplasma strain T.gHB1 is a type 1 (ToxoDB#10) strain but shows different virulence determination mechanisms compared to the classic type 1 strains like RH (ToxoDB#10). Therefore, it is worth investigating whether the T.gHB1 strain (ToxoDB#10) affects the host NF-κB signaling pathway. METHODS: The effects of T.gHB1 (ToxoDB#10) on host NF-κB pathway were investigated in HEK293T cells. The GRA15 gene product was analyzed by bioinformatics, and its effect on NF-κB activation was examined by Western blotting and nuclear translocation of p65. Different truncations of T.gHB1 GRA15 were constructed to map the critical domains for NF-κB activation. RESULTS: We demonstrated that the NF-κB pathway signaling pathway could be activated by the newly identified type 1 T.gHB1 strain (ToxoDB#10) of Toxoplasma, while the classic type 1 strain RH (ToxoDB#10) did not. T.gHB1 GRA15 possesses only one transmembrane region with an extended C terminal region, which is distinct from that of classic type 1 (ToxoDB#10) and type 2 (ToxoDB#1) strains. T.gHB1 GRA15 could clearly induce IκBα phosphorylation and p65 nuclear translocation. Dual luciferase assays in HEK293T cells revealed a requirement for 194-518 aa of T.gHB1 GRA15 to effectively activate NF-κB. CONCLUSIONS: The overall results indicated that the newly isolated type 1 isolate T.gHB1 (ToxoDB#10) had a unique GRA15, which could activate the host NF-κB signaling through inducing IκBα phosphorylation and p65 nuclear translocation. These results provide new insights for our understanding of the interaction between Toxoplasma parasites and its hosts.

Essential Functions of Calmodulin and Identification of Its Proximal Interacting Proteins in Tachyzoite-Stage Toxoplasma gondii via BioID Technology.

Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in cells, can regulate important biological processes, including parasite invasion and egress. Calmodulin (CaM) is a small, highly conserved, Ca2+-binding protein found in all eukaryotic cells. After binding to Ca2+, CaM can be activated to interact with various proteins. However, little is known about CaM's function and its interacting proteins in T. gondii. In this study, we successfully knocked down CaM in the T. gondii parent strain TATI using a tetracycline-off system with the Toxoplasma CaM promoter. The results indicated that CaM was required for tachyzoite proliferation, invasion, and egress and that CaM depletion resulted in apicoplast loss, thus threatening parasite survival in the next lytic cycle. In the tachyzoite stage, CaM loss caused significant anomalies in the parasite's basal constriction, motility, and parasite rosette-like arrangement in the parasitophorous vacuole (PV). These phenotypic defects caused by CaM depletion indicate the importance of CaM in T. gondii. Therefore, it is important to identify the CaM-interacting proteins in T. gondii. Applying BioID technology, more than 300 CaM's proximal interacting proteins were identified from T. gondii. These CaM partners were broadly distributed throughout the parasite. Furthermore, the protein interactome and transcriptome analyses indicated the potential role of CaM in ion binding, cation binding, metal ion binding, calcium ion binding, and oxidation-reduction. Our findings shed light on the CaM function and CaM-interactome in T. gondii and other eukaryotes. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion. The implementation efficiency of T. gondii biological processes is dependent on signal transmission. Ca2+, as a second messenger, is essential for the parasite's life cycle. Calmodulin, a ubiquitous Ca2+ receptor protein, is highly conserved and mediates numerous Ca2+-dependent events in eukaryotes. Few CaM functions or regulated partners have been characterized in T. gondii tachyzoites. Here, we reported the essential functions of calmodulin in T. gondii tachyzoite and the identification of its interacting partners using BioID technology, shedding light on the CaM function and CaM-interactome in Toxoplasma gondii and other eukaryotes.

Role of amylopectin synthesis in Toxoplasma gondii and its implication in vaccine development against toxoplasmosis.

Toxoplasma gondii is a ubiquitous pathogen infecting one-third of the global population. A significant fraction of toxoplasmosis cases is caused by reactivation of existing chronic infections. The encysted bradyzoites during chronic infection accumulate high levels of amylopectin that is barely present in fast-replicating tachyzoites. However, the physiological significance of amylopectin is not fully understood. Here, we identified a starch synthase (SS) that is required for amylopectin synthesis in T. gondii. Genetic ablation of SS abolished amylopectin production, reduced tachyzoite proliferation, and impaired the recrudescence of bradyzoites to tachyzoites. Disruption of the parasite Ca2+-dependent protein kinase 2 (CDPK2) was previously shown to cause massive amylopectin accumulation and bradyzoite death. Therefore, the Δcdpk2 mutant is thought to be a vaccine candidate. Notably, deleting SS in a Δcdpk2 mutant completely abolished starch accrual and restored cyst formation as well as virulence in mice. Together these results suggest that regulated amylopectin production is critical for the optimal growth, development and virulence of Toxoplasma. Not least, our data underscore a potential drawback of the Δcdpk2 mutant as a vaccine candidate as it may regain full virulence by mutating amylopectin synthesis genes like SS.

Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs.

BACKGROUND: Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits. METHODS: The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA). RESULTS: MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection). CONCLUSIONS: Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.