New cassane-type alkaloids and diterpenoids from the pericarps of Caesalpinia bonduc.

The phytochemical investigation of the pericarps of Caesalpinia bonduc led to the isolation and identification of five new cassane-type alkaloids: caesalminines C - G (1-5) and six new diterpenoids: caesalbonducin K - P (6-11), along with seven known compounds (12-18). Compounds 1-5 were identified as a group of rare alkaloids possessing a tetracyclic cassane-type diterpenoid skeleton with a lactam D-ring instead of a typical furan or lactone moiety. The structures of 1-11 were elucidated on the basis of 1D and 2D NMR including HSQC, HMBC, COSY and NOESY, and other spectroscopic analyses. The cytotoxic activities of the isolated compounds were evaluated in the A431, A549 and U87MG cancer cell lines.

Functional analyses of Toxoplasma gondii dihydroorotase reveal a promising anti-parasitic target.

Toxoplasma gondii relies heavily on the de novo pyrimidine biosynthesis pathway for fueling the high uridine-5'-monophosphate (UMP) demand during parasite growth. The third step of de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (DHO), a metalloenzyme that catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate. Here, functional analyses of TgDHO reveal that tachyzoites lacking DHO are impaired in overall growth due to decreased levels of UMP, and the noticeably growth restriction could be partially rescued after supplementation with uracil or high concentrations of L-dihydroorotate in vitro. When pyrimidine salvage pathway is disrupted, both DHOH35A and DHOD284E mutant strains proliferated much slower than DHO-expressing parasites, suggesting an essential role of both TgDHO His35 and Asp284 residues in parasite growth. Additionally, DHO deletion causes the limitation of bradyzoite growth under the condition of uracil supplementation or uracil deprivation. During the infection in mice, the DHO-deficient parasites are avirulent, despite the generation of smaller tissue cysts. The results reveal that TgDHO contributes to parasite growth both in vitro and in vivo. The significantly differences between TgDHO and mammalian DHO reflect that DHO can be exploited to produce specific inhibitors targeting apicomplexan parasites. Moreover, potential DHO inhibitors exert beneficial effects on enzymatic activity of TgDHO and T. gondii growth in vitro. In conclusion, these data highlight the important role of TgDHO in parasite growth and reveal that it is a promising anti-parasitic target for future control of toxoplasmosis.

Development of a novel method for diagnosis of fasciolosis based on cathepsin L7 in ruminants.

Fasciolosis is a widely distributed zoonosis reported over 81 countries around the world. Good and early diagnostic method is critical in controlling this disease and prevention of injury to the liver and bile ducts. In this study, we identified a novel member (cathepsin L7) of cathepsin family from Fasciola spp.. Firstly, the biological character of CL7 was analyzed according to the information of cathepsin L family, and then rCL7 was expressed and purified, a new iELISA based on CL7 was developed. The results exhibited CL7 iELISA had 100% sensitivity 100% specificity in sheep (cut-off 1.329) and 100% sensitivity 93.75% specificity in cattle (cut-off 0.756). Moreover, anti-Fasciola CL7 antibodies could be detected in early Fasciola gigantica infected buffaloes, as early as 3 week-post-infection (WPI). In conclusion, it is suggested that CL7 with low cost, early detection, good specificity and sensitivity could be used as a candidate antigen for detection of ruminant fasciolosis.

ROP16 of Toxoplasma gondii Inhibits Innate Immunity by Triggering cGAS-STING Pathway Inactivity through the Polyubiquitination of STING.

cGAS-STING signaling is a major pathway in inducing type Ⅰ IFN, which plays a crucial role in the defense against T. gondii infection. In contrast, T. gondii develops multiple strategies to counteract the host defense, causing serious diseases in a wide range of hosts. Here, we demonstrate that T. gondii rhoptry protein 16 (ROP16) dampens type I interferon signaling via the inhibition of the cGAS (cyclic GMP-AMP synthase) pathway through the polyubiquitination of STING. Mechanistically, ROP16 interacts with STING through the SignalP domain and inhibits the K63-linked ubiquitination of STING in an NLS (nuclear localization signal)-domain-dependent manner. Consequently, knocking out the ROP16 in PRU tachyzoites promotes the STING-mediated production of type I IFNs and limits the replication of T. gondii. Together, these findings describe a distinct pathway where T. gondii exploits the ubiquitination of STING to evade host anti-parasite immunity, revealing new insights into the interaction between the host and parasites.

In Vitro Evaluation Reveals Effect and Mechanism of Artemether against Toxoplasma gondii.

Due to the limited effectiveness of existing drugs for the treatment of toxoplasmosis, there is a dire need for the discovery of new therapeutic options. Artemether is an important drug for malaria and several studies have indicated that it also exhibits anti-T. gondii activity. However, its specific effect and mechanisms are still not clear. To elucidate its specific role and potential mechanism, we first evaluated its cytotoxicity and anti-Toxoplasma effect on human foreskin fibroblast cells, and then analyzed its inhibitory activity during T. gondii invasion and intracellular proliferation. Finally, we examined its effect on mitochondrial membrane potential and reactive oxygen species (ROS) in T. gondii. The CC50 value of artemether was found to be 866.4 µM, and IC50 was 9.035 µM. It exhibited anti-T. gondii activity and inhibited the growth of T. gondii in a dose-dependent manner. We also found that the inhibition occurred primarily in intracellular proliferation, achieved by reducing the mitochondrial membrane integrity of T. gondii and stimulating ROS production. These findings suggest that the mechanism of artemether against T. gondii is related to a change in the mitochondrial membrane and the increase in ROS production, which may provide a theoretical basis for optimizing artemether derivatives and further improving their anti-Toxoplasma efficacy.

Mineralization Reduces the Toxicity and Improves Stability and Protective Immune Response Induced by Toxoplasma gondii.

Vaccination is an ideal strategy for the control and prevention of toxoplasmosis. However, the thermostability and effectiveness of vaccines limit their application. Here, calcium mineralization was used to fabricate Toxoplasma gondii tachyzoites as immunogenic core-shell particles with improved immune response and thermostability. In the current study, T. gondii RH particles coated with mineralized shells were fabricated by calcium mineralization. The mineralized shells could maintain the T. gondii tachyzoites structural integrity for at least 12 months and weaken the virulence. Immunization of mice with mineralized tachyzoites induced high levels of T. gondii-specific antibodies and cytokines. The immunized mice were protected with a 100% survival rate in acute and chronic infection, and brain cyst burdens were significantly reduced. This study reported for the first time the strategy of calcium mineralization on T. gondii and proved that mineralized tachyzoites could play an immune protective role, thus expanding the application of biomineralization in T. gondii vaccine delivery.

The determinants regulating Toxoplasma gondii bradyzoite development.

Toxoplasma gondii is an obligate intracellular zoonotic pathogen capable of infecting almost all cells of warm-blooded vertebrates. In intermediate hosts, this parasite reproduces asexually in two forms, the tachyzoite form during acute infection that proliferates rapidly and the bradyzoite form during chronic infection that grows slowly. Depending on the growth condition, the two forms can interconvert. The conversion of tachyzoites to bradyzoites is critical for T. gondii transmission, and the reactivation of persistent bradyzoites in intermediate hosts may lead to symptomatic toxoplasmosis. However, the mechanisms that control bradyzoite differentiation have not been well studied. Here, we review recent advances in the study of bradyzoite biology and stage conversion, aiming to highlight the determinants associated with bradyzoite development and provide insights to design better strategies for controlling toxoplasmosis.

Epidemiology of Fasciola spp. in the intermediate host in China: A potential risk for fasciolosis transmission.

Fasciolosis is a zoonotic disease as 600 million animals have been infected, and 180 million people are at risk of the infection in the world. Snail as the intermediate host of Fasciola is an essential and important factor in the transmission of fasciolosis, while its potential risk for transmission has not been studied. In this study, 3561 snails collected from large-scale regions of China were examined by nest-PCR method. A total of 345 snails were positive for Fasciola spp., with an overall prevalence of 9.7%. Prevalence in central and southern China, characterized by a subtropical monsoon climate, was relatively low (8.0%), while a high infection rate (36.9%) was found on the plateau area (altitude > 500 m). In combination with previous findings, the study showed a highly positive correlation between snails and animal infection in central and eastern China, thus indicating that infected snails could be an indispensable risk factor for fasciolosis transmission. Epidemiological surveillance of snails will help assess the risk of fasciolosis in humans or ruminants, which promotes future prevention of this zoonotic disease.

A global phosphoproteomics analysis of adult Fasciola gigantica by LC-MS/MS.

Protein phosphorylation plays key roles in a variety of essential cellular processes. Fasciola gigantica is a tropical liver fluke causing hepatobiliary disease fascioliasis, leading to human health threats and heavy economic losses. Although the genome and protein kinases of F. gigantica provided new insights to understand the molecular biology and etiology of this parasite, there is scant knowledge of protein phosphorylation events in F. gigantica. In this study, we characterized the global phosphoproteomics of adult F. gigantica by phosphopeptide enrichment-based LC-MS/MS, a high-throughput analysis to maximize the detection of a large repertoire of phosphoproteins and phosphosites. A total of 1030 phosphopeptides with 1244 phosphosites representing 635 F. gigantica phosphoproteins were identified. The phosphoproteins were involved in a wide variety of biological processes including cellular, metabolic, and single-organism processes. Meanwhile, these proteins were found predominantly in cellular components like membranes and organelles with molecular functions of binding (51.3%) and catalytic activity (40.6%). The KEGG annotation inferred that the most enriched pathways of the phosphoproteins included tight junction, spliceosome, and RNA transport (each one contains 15 identified proteins). Combining the reports in other protozoa and helminths, the phosphoproteins identified in this work play roles in metabolic regulation and signal transduction. To our knowledge, this work performed the first global phosphoproteomics analysis of adult F. gigantica, which provides valuable information for development of intervention strategies for fascioliasis.

Evaluation of Origanum vulgare Essential Oil and Its Active Ingredients as Potential Drugs for the Treatment of Toxoplasmosis.

Toxoplasma gondii is a serious hazard to public health and animal husbandry. Due to the current dilemma of treatment of toxoplasmosis, it is urgent to find new anti-T. gondii drugs to treat toxoplasmosis. In this study, the anti-T. gondii activity of Origanum vulgare essential oil (Ov EO) was firstly studied, and then, carvanol (Ca), the main ingredient of Ov EO was evaluated using the MTT assay on human foreskin fibroblast (HFF) cells in vitro. The cytotoxicity was evaluated using the MTT assay on HFF cells. The CC50 of Ov EO and Ca was 134.9 and 43.93 µg/ml, respectively. Both of them exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. For the inhibition effect, Ca was better than Ov EO at the same concentration, the IC50 of Ov EO and Ca was 16.08 and 7.688 µg/ml, respectively. In addition, treatment with Ca, was found to change the morphology of T. gondii tachyzoites and made their shapes curl up. These results showed that Ca was able to inhibit the proliferation of T. gondii by reducing invasion, which may be due to its detrimental effect on the mobility of tachyzoites. Our results indicated that Ca could be a potential new and effective drug for treating toxoplasmosis.

In Vitro Evaluation of Lavandula angustifolia Essential Oil on Anti-Toxoplasma Activity.

The current methods of treating toxoplasmosis have a number of side effects, and these therapies are only effective against the acute stage of the disease. Thus, development of new low toxicity and efficient anti-Toxoplasma drugs is extremely important. Natural products are important sources for screening new drugs; among them, essential oils (EOs) have efficacy in anti-bacterial, anti-inflammatory, anti-insect, and other aspects. In this study, 16 EOs were screened for their anti-T. gondii activity. Lavandula angustifolia essential oil (La EO)was found to have an anti-parasitic effect on T. gondii. The cytotoxicity of La EO was firstly evaluated using the MTT assay on human foreskin fibroblast (HFF) cells, and then the anti-T. gondii activity was evaluated by plaque assay. Finally, the invasion experiment and electron microscope observation were used to study the mechanism of La EO in anti-toxoplasma activity. The results indicated that the CC50 of La EO was 4.48 mg/ml and that La EO had activity against T. gondii and the inhibition was in a dose-dependent manner under safe concentrations. La EO was able to reduce T. gondii invasion, which may be due to its detrimental effect on changes of the morphology of tachyzoites. These findings indicated that La EO could be a potential drug for treating toxoplasmosis.

In vitro Anti-parasitic Activity of Pelargonium X. asperum Essential Oil Against Toxoplasma gondii.

Toxoplasmosis is a global zoonotic disease, and one-third of the human population is chronically infected by Toxoplasma gondii. Due to the limited effectiveness and prominent side effects of the existing drugs, there is a dire need for the discovery of new therapeutic options in the treatment of toxoplasmosis. In this study, five essential oils (EO) were screened for their anti-parasitic activity against T. gondii. The cytotoxicity of essential oils was evaluated using the MTT assay on human foreskin fibroblast cells. The CC50 values of Eucalyptus globulus EO, Cupressus sempervirens EO, Citrus aurantifolia EO, Melaleuca alternifolia EO, and Pelargonium X. asperum (Pa) EO were found to be 22.74, 7.25, 15.01, 6.26, and 4.77 mg/mL, respectively. Only PaEO exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. In addition, treatment with PaEO, was found to reduce the volume of T. gondii tachyzoites and make their membrane surfaces rough. These results showed that PaEO was able to inhibit the growth of T. gondii by reducing invasion, which may be due to its detrimental effect on the ability of tachyzoites to move. These findings suggest that PaEO could be a potential anti-T. gondii drug, which may facilitate the development of new and effective treatments against toxoplasmosis.

First report on the prevalence of Fasciola hepatica in the endangered Père David's deer (Elaphurus davidianus) in China.

BACKGROUND: Fasciola hepatica is an important zoonotic parasite that causes fasciolosis in a broad range of animals. No information is available about the prevalence of F. hepatica in Père David's deer (Elaphurus davidianus), an endangered species in the world. Therefore, the purpose of the study was to evaluate the prevalence of fasciolosis in Père David's deer in the Dafeng Elk National Natural Reserve, Jiangsu province, China. RESULTS: In this study, 142 fecal samples from Père David's deer were analyzed for F. hepatica by microscopy and nest-PCR. Only one sample was positive for F. hepatica according to microscopy examination, while 18 of 142 (12.68, 95%CI: 2.841-22.45%) samples were positive for F. hepatica according to nest-PCR results. CONCLUSIONS: This is the first report of prevalence of F. hepatica in Père David's deer. The prevalence data indicated that F. hepatica was also present in this endangered animal, which may cause a potential threat to this precious species.

Seroprevalence of Toxoplasma gondii infection in the endangered Père David's deer (Elaphurus davidianus) in China.

Père David's deer (Elaphurus davidianus) is an emblematic endangered species and regarded as a national treasure, toxoplasmosis is a serious zoonotic parasitic disease for wild animals. Little is known about the prevalence of antibodies to this parasite in Père David's deer. In this study, sera from 43 wild Père David's deer, from Dafeng nature reserve China were tested for antibodies to Toxoplasma gondii by MAT. The investigation showed that antibodies to toxoplasma were detected in 8 of 43 (18.60%, 95% CI 6.97-30.24) samples. Seroprevalence ranged from 15.00% to 21.74% between the different genders, but the difference was not significant according to SPSS analysis (P > 0.05). This report of seroprevalence of antibodies to T. gondii in Père David's deer provides basic data of T. gondii infection data, which is important for controlling and preventing toxoplasmosis in Père David's deer.

Prevalence and molecular characterization of Cryptosporidium spp. in Père David's deer (Elaphurus davidianus) in Jiangsu, China.

Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David's deer. In this study, 137 fecal samples from Père David's deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David's deer in this area.

Development of a nest-PCR for detection of Fasciola hepatica DNA in the intermediate snail host, Radix cucunorica, and the prevalence in northwestern China.

Fasciolosis, a foodborne zoonotic disease, caused by Fasciola species which is considered an important problem for human health and livestock husbandry development. Snails are intermediate hosts of F. hepatica, the epidemiological surveillance of snails can evaluate the transmission risk of this disease in human and livestock. In this study, we developed a nest-polymerase chain reaction (nest-PCR) to detect the DNA of F. hepatica in Radix cucunorica, a prevalent intermediate host of this parasite in northwestern China. The nest-PCR was used to amplify a 208 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica with two pairs of primers. The method was able to detect up to 0.16 fg genomic DNA in a 25 µL PCR reaction system even effected with high concentrations of snail DNA, and no cross reaction was observed from the genomic DNA of Paramphistomum cervi, Clonorchis sinensis, Orientobilharzia turkestanicum, Metorchis orientalis, Dicrocoelium chinensis. To evaluate the transmission risk of this disease, 409 snail samples collected from different areas of Gansu province were used to detect and analyze the transmission risk of F. hepatica in this area. Of 409 snail samples, the overall prevalence is 43.76%. The prevalence was 92.75% in Gannan Tibetan Autonomous Prefecture, while no snail was positive for F. hepatica in Linxia Hui Autonomous Prefecture. The nest-PCR was firstly used to detect the infection of F. hepatica in snail. It is a novel, useful and convenient method with high sensitivity and specificity. This study is the first report about the epidemiological surveillance of snail infection by F. hepatica in northwestern China, which will help to evaluate the transmission risk of F. hepatica in northwestern China.

Evaluation of protective immunity induced by recombinant calcium-dependent protein kinase 1 (TgCDPK1) protein against acute toxoplasmosis in mice.

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8- and CD3+CD8+CD4- T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ±â€¯2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis.

Seroprevalence and risk assessment of Toxoplasma gondii in Java sparrows (Lonchura oryzivora) in China.

BACKGROUND: Toxoplasma gondii, an intracellular zoonotic parasite, infects all mammalian and birds. Understanding the prevalence of Toxoplasma in bird is important for evaluating the transmission of this parasite. No information about the seroprevalence of T. gondii in Java sparrows (Lonchura oryzivora) is available. RESULTS: In this study, from 2014 to 2015, 350 serum samples from Java sparrows were collected in Beijing and Shangqiu, Henan province, and the antibodies against T. gondii were evaluated with MAT. The seroprevalence in Java sparrows was 34.29% (CI95% 29.31-39.26). A phenomenon of seropositivity tended to increase with age were observed, but the difference is not significant. The prevalence was significant different in gender and color, which could be risk factors. CONCLUSIONS: This study firstly reported T. gondii seroprevalence in Java sparrows, which extended the host range of T. gondii. Java sparrows may pose significant transmission medium, accelerating the spread of T. gondii diffusion.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) interacting with buffalo serum of different infection periods by shotgun LC-MS/MS.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is an important zoonotic disease in the world. It affects livestock, especially for sheep and cattle, causing major economic loss due to morbidity and mortality. Although the excretory and secretory products (ESPs) of F. hepatica have been relatively well studied, little is known about the interaction between the ESP and host, and the mechanism of the key proteins involved in interaction. In this study, buffaloes were infected by Fasciola gigantica, and infection serum was collected at three different periods (42dpi, 70dpi, and 98dpi). The interaction proteins were pulled down with three different period serum by Co-IP assay, respectively, and then identified by LC-MS/MS analysis. A number of proteins were identified; some of them related to the biological function of the parasite, while most of them the functions were unknown. For the annotated proteins, 13, 5, and 7 proteins were pulled down by the infected serum in 42dpi, 70dpi, and 98dpi, respectively, and 18 proteins could be detected in all three periods. Among them, 13 belong to the cathepsin family, 4 proteins related to glutathione S-transferase, and 3 proteins are calcium-binding protein; other proteins related to catalytic activity and cellular process. This study could provide new insights into the central role played by ESPs in the protection of F. gigantica from the host immune response. At the same time, our research provided material for further studies about the interaction between F. gigantica and host.

The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells.

BACKGROUND: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats. METHODS: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0. RESULTS: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-ß) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway. CONCLUSIONS: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.