scholarly journals In vivo and in vitro models show unexpected degrees of virulence among Toxoplasma gondii type II and III isolates from sheep

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Mercedes Fernández-Escobar ◽  
Rafael Calero-Bernal ◽  
Javier Regidor-Cerrillo ◽  
Raquel Vallejo ◽  
Julio Benavides ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Toxoplasma Gondii ◽  
Small Ruminants ◽  
Parasite Burden ◽  
Target Cells ◽  
Post Inoculation ◽  
Type Ii ◽  
High Genetic Diversity

AbstractToxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiology, and variable clinical outcomes in animals and humans. In veterinary medicine, this apicomplexan parasite is considered one of the main infectious agents responsible for reproductive failure in small ruminants worldwide. The aim of this study was to phenotypically characterize 10 Spanish T. gondii isolates recently obtained from sheep in a normalized mouse model and in an ovine trophoblast cell line (AH-1) as infection target cells. The panel of isolates met selection criteria regarding such parameters as genetic diversity [types II (ToxoDB #1 and #3) and III (#2)], geographical location, and sample of origin (aborted foetal brain tissues or adult sheep myocardium). Evaluations of in vivo mortality, morbidity, parasite burden and histopathology were performed. Important variations between isolates were observed, although all isolates were classified as “nonvirulent” (< 30% cumulative mortality). The isolates TgShSp16 (#3) and TgShSp24 (#2) presented higher degrees of virulence. Significant differences were found in terms of in vitro invasion rates and tachyzoite yield at 72 h post-inoculation (hpi) between TgShSp1 and TgShSp24 isolates, which exhibited the lowest and highest rates, respectively. The study of the CS3, ROP18 and ROP5 loci allelic profiles revealed only type III alleles in ToxoDB #2 isolates and type II alleles in the #1 and #3 isolates included. We concluded that there are relevant intra- and inter-genotype virulence differences in Spanish T. gondii isolates, which could not be inferred by genetic characterization using currently described molecular markers.

scholarly journals Licarin-B Exhibits Activity Against the Toxoplasma gondii RH Strain by Damaging Mitochondria and Activating Autophagy

2021 ◽  
Vol 9 ◽  
Author(s):  
Jili Zhang ◽  
Hongfei Si ◽  
Kun Lv ◽  
Yanhua Qiu ◽  
Jichao Sun ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
High Efficiency ◽  
Quantitative Polymerase Chain Reaction ◽  
Parasite Burden ◽  
New Drugs ◽  
Ultrastructural Changes ◽  
Dependent Manner ◽  
Dapi Staining

Toxoplasma gondii is an obligate intracellular pathogen that infects warm-blooded animals and humans. However, side effects limit toxoplasmosis treatment, and new drugs with high efficiency and low toxicity need to be developed. Natural products found in plants have become a useful source of drugs for toxoplasmosis. In this study, twenty natural compounds were screened for anti-T. gondii activity by Giemsa staining or real-time fluorescence quantitative polymerase chain reaction (qPCR) in vitro. Among these, licarin-B from nutmeg exhibited excellent anti-T. gondii activity, inhibiting T. gondii invasion and proliferation in a dose-dependent manner, with an EC50 of 14.05 ± 3.96 μg/mL. In the in vivo, licarin-B treatment significantly reduced the parasite burden in tissues compared to no treatment, protected the 90% infected mice from to death at 50 mg/kg.bw. Flow cytometry analysis suggested a significant reduction in T. gondii survival after licarin-B treatment. Ultrastructural changes in T. gondii were observed by transmission electron microscopy (TEM), as licarin-B induced mitochondrial swelling and formation of cytoplasmic vacuoles, an autophagosome-like double-membrane structure and extensive clefts around the T. gondii nucleus. Furthermore, MitoTracker Red CMXRos, MDC, and DAPI staining showed that licarin-B promoted mitochondrial damage, autophagosome formation, and nuclear disintegration, which were consistent with the TEM observations. Together, these findings indicate that licarin-B is a promising anti-T. gondii agent that potentially functions by damaging mitochondria and activating autophagy, leading to T. gondii death.

scholarly journals Determination of lumefantrine as an effective drug against Toxoplasma gondii infection – in vitro and in vivo study

Parasitology ◽  
2020 ◽  
pp. 1-7
Author(s):  
Dawei Wang ◽  
Mengen Xing ◽  
Saeed El-Ashram ◽  
Yingying Ding ◽  
Xiao Zhang ◽  
...  
Keyword(s):  
Side Effects ◽  
Toxoplasma Gondii ◽  
Parasite Burden ◽  
Vero Cells ◽  
Negative Control ◽  
Protective Mechanism ◽  
High Dose ◽  
Mouse Tissues

Abstract Toxoplasma gondii is an obligate intracellular protozoan parasite, which can infect almost all warm-blooded animals, including humans, leading to toxoplasmosis. Currently, the effective treatment for human toxoplasmosis is the combination of sulphadiazine and pyrimethamine. However, both drugs have serious side-effects and toxicity in the host. Therefore, there is an urgent need for the discovery of new anti-T. gondii drugs with high potency and less or no side-effects. Our findings suggest that lumefantrine exerts activity against T. gondii by inhibiting its proliferation in Vero cells in vitro without being toxic to Vero cells (P ≤ 0.01). Lumefantrine prolonged mice infected with T. gondii from death for 3 days at the concentration of 50 μg L−1 than negative control (phosphate-buffered saline treated only), and reduced the parasite burden in mouse tissues in vivo (P ≤ 0.01; P ≤ 0.05). In addition, a significant increase in interferon gamma (IFN-γ) production was observed in high-dose lumefantrine-treated mice (P ≤ 0.01), whereas interleukin 10 (IL-10) and IL-4 levels increased in low-dose lumefantrine-treated mice (P ≤ 0.01). The results demonstrated that lumefantrine may be a promising agent to treat toxoplasmosis, and more experiments on the protective mechanism of lumefantrine should be undertaken in further studies.

scholarly journals Spiroindolone That Inhibits PfATPase4 Is a Potent, Cidal Inhibitor of Toxoplasma gondii TachyzoitesIn VitroandIn Vivo

2013 ◽  
Vol 58 (3) ◽  
pp. 1789-1792 ◽  
Author(s):  
Ying Zhou ◽  
Alina Fomovska ◽  
Stephen Muench ◽  
Bo-Shiun Lai ◽  
Ernest Mui ◽  
...  
Keyword(s):  
Body Weight ◽  
Toxoplasma Gondii ◽  
Inhibitory Concentration ◽  
Parasite Burden ◽  
Content Type ◽  
Medicine Development ◽  
Lead Candidate ◽  
Oral Doses

ABSTRACTHere, we show that spiroindolone, an effective treatment for plasmodia, is also active againstToxoplasma gondiitachyzoites.In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1μM) and not toxic to human cells at ≥10μM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P= 0.002), measured 3 days after the last dose. This inhibition ofT. gondiitachyzoitesin vitroandin vivoindicates that spiroindolone is a promising lead candidate for further medicine development.

scholarly journals The Role of Type II Fatty Acid Synthesis Enzymes FabZ, ODSCI, and ODSCII in the Pathogenesis of Toxoplasma gondii Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao-Pei Xu ◽  
Hany M. Elsheikha ◽  
Wen-Ge Liu ◽  
Zhi-Wei Zhang ◽  
Li-Xiu Sun ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Toxoplasma Gondii ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Plaque Assay ◽  
Acid Synthesis ◽  
Host Cells ◽  
Type Ii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI, and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔFabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔODSCI and RHΔODSCII mutant strains no similar changes were detected. RHΔFabZ exhibited reduced virulence for mice compared with RHΔODSCI and RHΔODSCII, as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔODSCI and PRUΔODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii, while ODSCI and ODSCII do not contribute to these traits.

scholarly journals The Toxoplasma gondii-Shuttling Function of Dendritic Cells Is Linked to the Parasite Genotype

2009 ◽  
Vol 77 (4) ◽  
pp. 1679-1688 ◽  
Author(s):  
Henrik Lambert ◽  
Polya P. Vutova ◽  
William C. Adams ◽  
Karin Loré ◽  
Antonio Barragan
Keyword(s):  
Dendritic Cells ◽  
Toxoplasma Gondii ◽  
Phenotypic Traits ◽  
Type I ◽  
Type Ii ◽  
Parasite Genotype ◽  
Type Iii ◽  
Parasite Motility

ABSTRACT Following intestinal invasion, the processes leading to systemic dissemination of the obligate intracellular protozoan Toxoplasma gondii remain poorly understood. Recently, tachyzoites representative of type I, II and III T. gondii populations were shown to differ with respect to their ability to transmigrate across cellular barriers. In this process of active parasite motility, type I strains exhibit a migratory capacity superior to those of the type II and type III strains. Data also suggest that tachyzoites rely on migrating dendritic cells (DC) as shuttling leukocytes to disseminate in tissue, e.g., the brain, where cysts develop. In this study, T. gondii tachyzoites sampled from the three populations were allowed to infect primary human blood DC, murine intestinal DC, or in vitro-derived DC and were compared for different phenotypic traits. All three archetypical lineages of T. gondii induced a hypermigratory phenotype in DC shortly after infection in vitro. Type II (and III) strains induced higher migratory frequency and intensity in DC than type I strains did. Additionally, adoptive transfer of infected DC favored the dissemination of type II and type III parasites over that of type I parasites in syngeneic mice. Type II parasites exhibited stronger intracellular association with both CD11c+ DC and other leukocytes in vivo than did type I parasites. Altogether, these findings suggest that infected DC contribute to parasite propagation in a strain type-specific manner and that the parasite genotype (type II) most frequently associated with toxoplasmosis in humans efficiently exploits DC migration for parasite dissemination.

scholarly journals Toxoplasma gondii Proteasome Subunit Alpha Type 1 with Chitosan: A Promising Alternative to Traditional Adjuvant

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 752
Author(s):  
Zhengqing Yu ◽  
Wenxi Ding ◽  
Muhammad Tahir Aleem ◽  
Junzhi Su ◽  
Junlong Liu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasite Burden ◽  
Effective Vaccine ◽  
Promising Alternative ◽  
Proteasome Subunit ◽  
Humoral Immune ◽  
Alpha Type

As an important zoonotic protozoan, Toxoplasma gondii (T. gondii) has spread around the world, leading to infections in one-third of the population. There is still no effective vaccine or medicine against T. gondii, and recombinant antigens entrapped within nanospheres have benefits over traditional vaccines. In the present study, we first expressed and purified T. gondii proteasome subunit alpha type 1 (TgPSA1), then encapsulated the recombinant TgPSA1 (rTgPSA1) in chitosan nanospheres (CS nanospheres, rTgPSA1/CS nanospheres) and incomplete Freund’s adjuvant (IFA, rTgPSA1/IFA emulsion). Antigens entrapped in CS nanospheres reached an encapsulation efficiency of 67.39%, and rTgPSA1/CS nanospheres showed a more stable release profile compared to rTgPSA1/IFA emulsion in vitro. In vivo, Th1-biased cellular and humoral immune responses were induced in mice and chickens immunized with rTgPSA1/CS nanospheres and rTgPSA1/IFA emulsion, accompanied by promoted production of antibodies, IFN-γ, IL-4, and IL-17, and modulated production of IL-10. Immunization with rTgPSA1/CS nanospheres and rTgPSA1/IFA emulsion conferred significant protection, with prolonged survival time in mice and significantly decreased parasite burden in chickens. Furthermore, our results also indicate that rTgPSA1/CS nanospheres could be used as a substitute for rTgPSA1/IFA emulsion, with the optimal administration route being intramuscular in mass vaccination. Collectively, the results of this study indicate that rTgPSA1/CS nanospheres represent a promising vaccine to protect animals against acute toxoplasmosis.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

scholarly journals Combined In Vitro and In Vivo Approaches to Propose a Putative Adverse Outcome Pathway for Acute Lung Inflammation Induced by Nanoparticles: A Study on Carbon Dots

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 180
Author(s):  
Maud Weiss ◽  
Jiahui Fan ◽  
Mickaël Claudel ◽  
Luc Lebeau ◽  
Françoise Pons ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Lung Inflammation ◽  
Adverse Outcome ◽  
Cellular Responses ◽  
Target Cells ◽  
Inflammasome Activation ◽  
Adverse Outcome Pathway ◽  
Acute Lung Inflammation

With the growth of nanotechnologies, concerns raised regarding the potential adverse effects of nanoparticles (NPs), especially on the respiratory tract. Adverse outcome pathways (AOP) have become recently the subject of intensive studies in order to get a better understanding of the mechanisms of NP toxicity, and hence hopefully predict the health risks associated with NP exposure. Herein, we propose a putative AOP for the lung toxicity of NPs using emerging nanomaterials called carbon dots (CDs), and in vivo and in vitro experimental approaches. We first investigated the effect of a single administration of CDs on mouse airways. We showed that CDs induce an acute lung inflammation and identified airway macrophages as target cells of CDs. Then, we studied the cellular responses induced by CDs in an in vitro model of macrophages. We observed that CDs are internalized by these cells (molecular initial event) and induce a series of key events, including loss of lysosomal integrity and mitochondrial disruption (organelle responses), as well as oxidative stress, inflammasome activation, inflammatory cytokine upregulation and macrophage death (cellular responses). All these effects triggering lung inflammation as tissular response may lead to acute lung injury.

scholarly journals Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 963
Author(s):  
Maria C. Holeva ◽  
Athanasios Sklavounos ◽  
Rajendran Rajeswaran ◽  
Mikhail M. Pooggin ◽  
Andreas E. Voloudakis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Topical Application ◽  
Plant Virus ◽  
Plant Protection ◽  
Post Inoculation ◽  
Tobacco Plants ◽  
Double Stranded Rna

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.

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