Quantification of Toxoplasma gondii in the tissues of BALB/c mice after immunization with nanoliposomal excretory-secretory antigens using Real-Time PCR

Azadi, Yaghob; Ahmadpour, Ehsan; Hamishehkar, Hamed; Daryani, Ahmad; Spotin, Adel; Mahami-Oskouei, Mahmoud; Barać, Aleksandra; Rajabi, Saba; Alizadeh, Paria; Montazeri, Mahbobeh

doi:10.1016/j.cimid.2018.09.012

Cited by 6 publications

(3 citation statements)

References 30 publications

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“…For this purpose, we used the primers for the repeat element of T. gondii genome previously validated using TaqMan system for the assessment of amniotic fluids from patients (Kasper et al, 2009). Although the parasite load in tissue of mice infected with T. gondii has been performed previously (Lin et al, 2000;Jauregui et al, 2001;Kasper et al, 2009;Mesquita et al, 2010;Dadimoghaddam et al, 2014;Vargas-Villavicencio et al, 2016bAzadi et al, 2018), this is the first report of extensive validation in various mouse tissues, including placenta and fetal head, as well as maternal spleen, liver and brain. Furthermore, to increase the accuracy of the quantification of the parasite load, we propose the normalization of the parasite equivalents by the amount of tissue, which was also estimated by qPCR targeting the mice β-actin gene.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a murine model of congenital toxoplasmosis and validation of a qPCR assay to assess the parasite load in maternal and fetal tissues

Souza

Farani²,

Ferreira³

et al. 2023

Front. Microbiol.

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Toxoplasma gondii is the causative agent of toxoplasmosis, a disease that affects warm-blooded animals and one third of the human population worldwide. Pregnant women who have never been exposed to the parasite constitute an important risk group, as infection during pregnancy often leads to congenital toxoplasmosis, the most severe form of the disease. Current therapy for toxoplasmosis is the same as it was 50 years ago and has little or no effect when vertical transmission occurs. Therefore, it is urgent to develop new strategies to prevent mother-to-fetus transmission. The implementation of experimental animal models of congenital toxoplasmosis that reproduces the transmission rates and clinical signs in humans opens an avenue of possibilities to interfere in the progression of the disease. In addition, knowing the parasite load in maternal and fetal tissues after infection, which may be related to organ abnormalities and disease outcome, is another important step in designing a promising intervention strategy. Therefore, we implemented here a murine model of congenital toxoplasmosis with outbred Swiss Webster mice infected intravenously with tachyzoites of the ME49 strain of T. gondii that mimics the frequency of transmission of the parasite, as well as important clinical signs of human congenital toxoplasmosis, such as macrocephaly, in addition to providing a highly sensitive quantitative real-time PCR assay to assess parasite load in mouse tissues. As the disease is not restricted to humans, also affecting several domestic animals, including companion animals and livestock, they can also benefit from the model presented in this study.

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Section: Discussionmentioning

confidence: 99%

Establishment of a murine model of congenital toxoplasmosis and validation of a qPCR assay to assess the parasite load in maternal and fetal tissues

Souza

Farani²,

Ferreira³

et al. 2023

Front. Microbiol.

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show abstract

“…Some immunization studies with different types of nanoparticles (CaPNs, PLGA, and Chitosan) encapsulated with a variety of T. gondii recombinant proteins, namely MIC3, ROP8, and SAG1, showed similar results, with higher levels of specific IgA and IgG2a, leading to a Th1 response and consequently increased survival rate [96][97][98][99][100]. In addition to the use of parasite membrane proteins, the excretory secretory antigens (ESA) of T.gondii have also been used for vaccine development, since they have proved to play important roles in the immune escape and pathogenesis of the parasite, and the results showed increased levels of IFN-γ and IgG, as well as a reduction in the parasite load [101][102][103].…”

Section: Recent Advances In the Use Of Nanoparticles For T Gondii Vac...mentioning

confidence: 99%

Nanoparticles as a Delivery System of Antigens for the Development of an Effective Vaccine against Toxoplasma gondii

et al. 2023

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Nanoparticles include particles ranging in size from nanometers to micrometers, whose physicochemical characteristics are optimized to make them appropriate delivery vehicles for drugs or immunogens important in the fight and/or prevention of infectious diseases. There has been a rise in the use of nanoparticles in preventive vaccine formulations as immunostimulatory adjuvants, and as vehicles for immunogen delivery to target immune cells. Toxoplasma is important worldwide, and may cause human toxoplasmosis. In immunocompetent hosts, infection is usually asymptomatic, but in immunocompromised patients it can cause serious neurological and ocular consequences, such as encephalitis and retinochoroiditis. Primary infection during pregnancy may cause abortion or congenital toxoplasmosis. Currently, there is no effective human vaccine against this disease. Evidence has emerged from several experimental studies testing nanovaccines showing them to be promising tools in the prevention of experimental toxoplasmosis. For the present study, a literature review was carried out on articles published over the last 10 years through the PubMed database, pertaining to in vivo experimental models of T. gondii infection where nanovaccines were tested and protection and immune responses evaluated. This review aims to highlight the way forward in the search for an effective vaccine for toxoplasmosis.

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“…As per the manufacturer's instructions, RT-PCR was performed in triplicate with three independent samples for each experimental group in an ABI Prism 7500 System (Applied Biosystems Inc., Foster, USA) with SYBR Green II (Takara Biotechnology, Dalian, Co., Ltd, China). The RT-PCR conditions were as follows: 94 °C for 5 s, followed by 40 cycles at 94 °C for 5 s and 60 °C for 30 s. The relative expression levels of genes were calculated from the quantification cycle (Cq) value and standardized by the 2 −ΔΔCq method [27]. All primers used in this study are listed in Additional file 1: Table S1.…”

Section: Quantitative Real-time Pcr (Qrt-pcr)mentioning

confidence: 99%

Functional characterization of acyl-CoA binding protein in Neospora caninum

Zhou

Zhang

et al. 2020

Parasites Vectors

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Background: Lipid metabolism is pivotal for the growth of apicomplexan parasites. Lipid synthesis requires bulk carbon skeleton acyl-CoAs, the transport of which depends on the acyl-CoA binding protein (ACBP). In Neospora caninum, the causative agent of neosporosis, the FASII pathway is required for growth and pathogenicity. However, little is known about the fatty acid transport mechanism in N. caninum. Methods:We have identified a cytosolic acyl-CoA binding protein, with highly conserved amino acid residues and a typical acyl-CoA binding domain in N. caninum. The recombinant NcACBP protein was expressed to verify the binding activities of NcACBP in vitro, and the heterologous expression of NcACBP in Δacbp yeast in vivo. Lipid extraction from ΔNcACBP or the wild-type of N. caninum was analyzed by GC-MS or TLC. Furthermore, transcriptome analysis was performed to compare the gene expression in different strains.Results: The NcACBP recombinant protein was able to specifically bind acyl-CoA esters in vitro. A yeast complementation assay showed that heterologous expression of NcACBP rescued the phenotypic defects in Δacbp yeast, indicating of the binding activity of NcACBP in vivo. The disruption of NcACBP did not perturb the parasite's growth but enhanced its pathogenicity in mice. The lipidomic analysis showed that disruption of NcACBP caused no obvious changes in the overall abundance and turnover of fatty acids while knockout resulted in the accumulation of triacylglycerol. Transcriptional analysis of ACBP-deficient parasites revealed differentially expressed genes involved in a wide range of biological processes such as lipid metabolism, posttranslational modification, and membrane biogenesis. Conclusions:Our study demonstrated that genetic ablation of NcACBP did not impair the survival and growth phenotype of N. caninum but enhanced its pathogenicity in mice. This deletion did not affect the overall fatty acid composition but modified the abundance of TAG. The loss of NcACBP resulted in global changes in the expression of multiple genes. This study provides a foundation for elucidating the molecular mechanism of lipid metabolism in N. caninum.

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Quantification of Toxoplasma gondii in the tissues of BALB/c mice after immunization with nanoliposomal excretory-secretory antigens using Real-Time PCR

Cited by 6 publications

References 30 publications

Establishment of a murine model of congenital toxoplasmosis and validation of a qPCR assay to assess the parasite load in maternal and fetal tissues

Establishment of a murine model of congenital toxoplasmosis and validation of a qPCR assay to assess the parasite load in maternal and fetal tissues

Nanoparticles as a Delivery System of Antigens for the Development of an Effective Vaccine against Toxoplasma gondii

Functional characterization of acyl-CoA binding protein in Neospora caninum

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