RESUMEN
BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.
Asunto(s)
Enfermedades de los Gatos , Toxoplasma , Toxoplasmosis Animal , Gatos , Animales , Antígenos de Protozoos , Sensibilidad y Especificidad , Reproducibilidad de los Resultados , Anticuerpos Antiprotozoarios , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Toxoplasmosis Animal/diagnóstico , Enfermedades de los Gatos/diagnósticoRESUMEN
BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.
Asunto(s)
Infecciones por Circoviridae , Circovirus , Enfermedades de los Porcinos , Sulfato de Amonio , Animales , Anticuerpos Antivirales , Proteínas de la Cápside , Infecciones por Circoviridae/diagnóstico , Infecciones por Circoviridae/veterinaria , Colodión , Oro Coloide/química , Inmunoensayo/veterinaria , Conejos , PorcinosRESUMEN
Zebrafish could serve as an alternative animal model for pathogenic bacteria in multiple infectious routes. Our previous study showed that immersion infection in zebrafish with Listeria monocytogenes did not cause lethality but induced transient expression of several immune response genes. We used an Affymetrix gene chip to examine the expression profiles of genes of zebrafish immersion-infected with L. monocytogenes. A total of 239 genes were up-regulated and 56 genes down-regulated compared with uninfected fish. Highest expression (>20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. By morpholino knockdown of mmp-9, we found that the morphants showed rapid death with much higher bacterial load after intravenous or intraventricular (brain ventricle) infection with L. monocytogenes. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 was also seen in transwell inserts with 8-µm pore polycarbonate membrane, as compared with the scrambled RNA. These findings suggest that Mmp-9 is a protective molecule against infection by L. monocytogenes by engaging in migration of zebrafish macrophages to the site of infection via a non-proteolytic role. Further work is required on the molecular mechanisms governing Mmp-9-driven macrophage migration in zebrafish.
Asunto(s)
Enfermedades de los Peces/genética , Listeria monocytogenes/fisiología , Listeriosis/veterinaria , Macrófagos/fisiología , Metaloproteinasa 9 de la Matriz/genética , Proteínas de Pez Cebra/genética , Pez Cebra , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Perfilación de la Expresión Génica , Listeriosis/genética , Listeriosis/inmunología , Listeriosis/microbiología , Longevidad , Metaloproteinasa 9 de la Matriz/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Pez Cebra/metabolismoRESUMEN
Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/µL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.
Asunto(s)
Sistemas CRISPR-Cas , Enfermedades de los Gatos , Enfermedades de los Perros , Toxoplasma , Toxoplasmosis Animal , Gatos , Animales , Perros , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Enfermedades de los Perros/parasitología , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Gatos/parasitología , Enfermedades de los Gatos/epidemiología , Enfermedades de los Gatos/diagnóstico , China/epidemiología , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/epidemiología , Toxoplasmosis Animal/diagnóstico , Ratones , Sensibilidad y Especificidad , Proteínas Asociadas a CRISPR/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Diagnóstico Molecular/métodos , Proteínas Bacterianas , EndodesoxirribonucleasasRESUMEN
BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.
Asunto(s)
Toxoplasma , Toxoplasmosis , Ratones , Animales , Perros , Humanos , Cromatografía de Afinidad/métodos , Sensibilidad y Especificidad , Inmunoensayo/métodos , Toxoplasmosis/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Antihelmínticos , Oro Coloide/análisis , Oro Coloide/químicaRESUMEN
BACKGROUND: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats. METHODS: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA. RESULTS: The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%). CONCLUSIONS: The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.
Asunto(s)
Toxoplasma , Toxoplasmosis Animal , Toxoplasmosis , Animales , Gatos , Ratones , Toxoplasma/genética , Linfocitos T CD8-positivos , Vacunas Atenuadas , Proteínas Protozoarias/genética , Citocinas , Ratones Endogámicos BALB C , Anticuerpos Antiprotozoarios , Toxoplasmosis Animal/prevención & controlRESUMEN
Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)|-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.
Asunto(s)
Toxoplasma , Toxoplasmosis , Animales , Apoptosis , Chlorocebus aethiops , Perfilación de la Expresión Génica , Humanos , Mamíferos/genética , Toxoplasma/genética , Toxoplasmosis/genética , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Células Vero , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
The immune system may aberrantly silence when against "altered self", which consequently may develop into malignancies. With the development of tumor immunology and molecular biology, the deepened understanding of the relationship between parasites and tumors shifts the attitude towards parasitic pathogens from elimination to utilization. In recent years, the antitumor impact implemented by protozoan parasites and the derived products has been confirmed. The immune system is activated and enhanced by some protozoan parasites, thereby inhibiting tumor growth, angiogenesis, and metastasis in many animal models. In this work, we reviewed the available information on the antitumor effect of parasitic infection or induced by parasitic antigen, as well as the involved immune mechanisms that modulate cancer progression. Despite the fact that clinical trials of the protozoan parasites against tumors are limited and the specific mechanisms of the effect on tumors are not totally clear, the use of genetically modified protozoan parasites and derived molecules combined with chemotherapy could be an important element for promoting antitumor treatment in the future.
RESUMEN
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.
TITLE: Un nouveau dispositif de bandelette à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) pour la détection rapide de Toxoplasma gondii dans le sang des chats et chiens errants. ABSTRACT: Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire qui provoque la toxoplasmose et menace la santé humaine et les animaux à sang chaud dans le monde entier. Des méthodes de diagnostic simples et applicables sont nécessaires de toute urgence pour guider le développement d'approches efficaces pour la prévention de la toxoplasmose. La plupart des outils de diagnostic moléculaire pour l'infection par T. gondii nécessitent des compétences techniques élevées, un équipement sophistiqué et un environnement de laboratoire contrôlé. Dans cette étude, nous avons développé un test par bandelettes à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) qui cible spécifiquement les 529 pb qui détectent une infection par T. gondii. Ce nouvel appareil portable est universel, rapide, convivial et garantit une sensibilité expérimentale ainsi qu'un faible risque de contamination par aérosol. Notre test LAMP-LFD a une limite de détection de 1 fg d'ADN de T. gondii et ne montre aucune réaction croisée avec d'autres pathogènes parasites, y compris Cryptosporidium parvum, Leishmania donovani et Plasmodium vivax. Nous avons validé le test en détectant T. gondii dans l'ADN extrait d'échantillons de sang prélevés sur 318 chats et chiens errants prélevés dans les villes de Deqing, Wenzhou, Yiwu, Lishui et Zhoushan dans la province du Zhejiang, dans l'est de la Chine. Le dispositif LAMP-LFD a détecté la prévalence de l'ADN de T. gondii chez respectivement 4,76 et 4,69% des chats et chiens errants. En conclusion, le test LAMP-LFD développé est efficace, minimise la contamination par les aérosols et convient donc à la détection de T. gondii dans les établissements médicaux simples et sur le terrain.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Toxoplasma , Animales , Gatos , China , Perros , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Toxoplasma/genéticaRESUMEN
Early diagnosis of trichinellosis is still difficult because of the lack of specific symptoms and limited window for serological detection. Here we established an assay based on tracing phosphate ions generated during loop-mediated isothermal amplification (LAMP) to detect Trichinella spiralis DNA in rat feces during its early stage of infection. By targeting a 1.6-kb repetitive element of Tri. spiralis, the assay was able to detect Tri. spiralis DNA in the feces of all infected rats as early as 1 day postinfection (dpi). The positive detection lasted to 7 dpi in the rats infected with 250 muscle larvae, and 21 dpi in the rats infected with 5,000 larvae. The assay was highly sensitive, and could detect 1.7 femtograms (fg) of Tri. spiralis DNA with high specificity, and with no cross reactivity with the DNA from Anisakis pegreffii, Gnathostoma spinigerum, Angiostrongylus cantonensis, Enterobius vermicularis, Schistosoma japonicum, and Trypanosoma evansi. Our present study provided a reliable technique for the early diagnosis of trichinellosis with the advantages of simplicity and speed, as well as high sensitivity and specificity.
Asunto(s)
ADN de Helmintos/aislamiento & purificación , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Fosfatos/aislamiento & purificación , Trichinella spiralis/aislamiento & purificación , Triquinelosis/parasitología , Animales , Heces/parasitología , Fosfatos/metabolismo , Plásmidos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Trichinella spiralis/genética , Trichinella spiralis/crecimiento & desarrollo , Triquinelosis/diagnósticoRESUMEN
Background: Convalescent plasma (CP) transfusion is considered to be the priority therapeutic option for COVID-19 inpatients when no specific drugs are available for emerging infections. An alternative, simple, and sensitive method is urgently needed for clinical use to detect neutralization activity of the CP to avoid the use of inconvenient micro-neutralization assay. Method: This study aims to explore optimal index in predicting the COVID-19 CP neutralization activity (neutralizing antibody titers, NAb titers) in an indirect ELISA format. Fifty-seven COVID-19-recovered patients plasma samples were subjected to anti-SARS-CoV-2 RBD, S1, and N protein IgG antibody by indirect ELISA. Results: ELISA-RBD exhibited high specificity (96.2%) and ELISA-N had high sensitivity (100%); while ELISA-S1 had low sensitivity (86.0%) and specificity (73.1%). Furthermore, ELISA-RBD IgG titers and pseudovirus-based NAb titers correlated significantly, with R2 of 0.2564 (P < 0.0001). Conclusion: ELISA-RBD could be a substitute for the neutralization assay in resource-limited situations to screen potential plasma donors for further plasma infusion therapy.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/sangre , COVID-19/terapia , Inmunización Pasiva/métodos , Plasma/inmunología , Animales , Anticuerpos Antivirales/uso terapéutico , Antivirales/uso terapéutico , Donantes de Sangre , China , Chlorocebus aethiops , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Inmunoglobulina G/sangre , SARS-CoV-2 , Sensibilidad y Especificidad , Células Vero , Sueroterapia para COVID-19RESUMEN
Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.
RESUMEN
Toxoplasma gondii causes infections in a wide range of intermediate hosts and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Dense granule protein 24 (GRA24) is a novel essential virulence factor that is transferred into the nucleus of host cells from the parasitophorous vacuole to regulate gene expression. In the present study, bioinformatic analysis showed that GRA24 had a high score for B-cell and T-cell epitopes compared with surface antigen 1 (SAG1), which has been studied as a promising vaccine candidate. As a DNA vaccine, pVAX1-GRA24 was injected intramuscularly into BALB/c mice and the induced immune response was evaluated. pVAX1-GRA24 induced high levels of a mixed Th1/Th2 cytokines at 6 weeks after immunization. Antibody determinations, cytokines [interferon gamma (IFN-γ), interleukin (IL)-12, IL-4, IL-10], antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, and cytotoxic T lymphocyte activity showed that mice immunized with pVAX1-GRA24 produced specific humoral and cellular immune responses. The expression levels of interferon regulatory factor 8 (IRF8), nuclear factor kappa B (NF-κB), and T-Box 21 (T-bet) were significantly higher in the pVAX1-GRA24 immunization group than in the control groups. Survival times were prolonged significantly (24.6 ± 5.5 days) in the mice immunized with pVAX1-GRA24 compared with the mice in the control groups, which died within 7 days of T. gondii challenge (p < 0.05). The results of the present study showed that pVAX1-GRA24 induced a T. gondii-specific immune response and thus represents a promising candidate vaccine to treat toxoplasmosis.
Asunto(s)
Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasmosis Animal/inmunología , Vacunas de ADN/inmunología , Factores de Virulencia/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Toxoplasma , Toxoplasmosis Animal/prevención & controlRESUMEN
Toxoplasma gondii causes serious public health problems, but there is no effective treatment strategy against it currently. DNA vaccines have shown promising findings in this regard. MYR1 is a new virulence factor identified in T. gondii that may have potential as a DNA vaccine candidate. We constructed a recombinant eukaryotic plasmid, pVAX1-MYR1, as a DNA vaccine, injected it intramuscularly into BALB/c mice, and evaluated its immunoprotective effects. pVAX1-MYR1 immunization induced a sequential Th1 and Th2 T-cell response, as indicated by high levels of Th1 and mixed Th1/Th2 cytokines at 2 and 6 weeks after immunization, respectively. These findings were corroborated by the antibody assays too. In addition, increased levels of antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, cytotoxic T lymphocyte activity and cytokine (IFN-γ, IL-12, and IL-10) production were also observed in the immunized mice. These findings showed that pVAX1-MYR1 stimulated humoral and cellular immune responses in the immunized mice. The increased production of IFN-γ and IL-12 was correlated with increased expression of the T-bet and p65 genes of the NF-κB pathway. However, no significant increase was observed in the level of IL-4. The survival of mice immunized with pVAX1-MYR1 was also significantly prolonged compared with the control group mice. Based on all the above findings, the current study proposes that pVAX1-MYR1 can induce a T. gondii-specific immune response and should therefore be considered as a promising vaccine candidate against toxoplasmosis. To the best of our knowledge, this is the first report to evaluate the immunoprotective value of an MYR1-based DNA vaccine against T. gondii.
RESUMEN
Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.
Asunto(s)
Gerbillinae/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Enfermedades de los Roedores/diagnóstico , Enfermedades de los Roedores/parasitología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/diagnóstico , Animales , Líquido Ascítico/parasitología , ADN Protozoario/análisis , ADN Protozoario/sangre , ADN Protozoario/aislamiento & purificación , Modelos Animales de Enfermedad , Hígado/parasitología , Pulmón/parasitología , Ratones , Sensibilidad y Especificidad , Organismos Libres de Patógenos Específicos , Bazo/parasitología , Toxoplasma/genética , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , VirulenciaRESUMEN
The immunochromatographic test (ICT) is a convenient and low-cost method that can rapidly obtain results (10â¯min) under normal conditions. In this study, we established an ICT assay with two monoclonal antibodies: TgSAG3-3A7 and TgSAG3-4D5 based on the conserved protein of TgSAG3 that can be expressed in all the infective stages of T. gondii. 20â¯nm gold particles were prepared and conjugated with TgSAG3-3A7 MAb at the concentration of 12.5⯵g/mL. TgSAG3-4D5 MAb were used as the capture antibody because of its higher affinity tested by ELISA. The detection limit of the ICT assay was 100â¯ng with visual detection under optimal conditions of analysis. Positive porcine serum of Cryptosporidium suis, Mycoplasma suis, Streptococcus suis, Salmonella choleraesuis, Cysticercus cellulosae, Isospora suis, and Trichinella spiralis were used to evaluate the specificity of this ICT and no cross-reactivity was observed. 310 porcine serum samples obtained from pig farms in Zhejiang Province, China were detected by this ICT and ELISA kit, 23 positive samples were found by the developed strip with the rate of 7.42% comparing with 22 positive samples detected by the commercially ELISA kit which the positive rate was 7.1%, the relative sensitivity and specificity of this ICT are 100% and 99.65%. Therefore, the ICT established in this study is proved effective, simple, specific and sensitive of T. gondii detection.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Glicoproteínas de Membrana/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Oro , Pruebas Inmunológicas/métodos , Límite de Detección , Sensibilidad y Especificidad , Porcinos , Toxoplasma/aislamiento & purificaciónRESUMEN
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.
Asunto(s)
Interferones/antagonistas & inhibidores , Interleucinas/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas de la Nucleocápside/fisiología , Virus de la Diarrea Epidémica Porcina/fisiología , Virus de la Diarrea Epidémica Porcina/patogenicidad , Transporte Activo de Núcleo Celular , Animales , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus , Genes Virales , Interacciones Huésped-Patógeno/inmunología , Interferones/biosíntesis , Interferones/genética , Interleucinas/biosíntesis , Interleucinas/genética , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Virus de la Diarrea Epidémica Porcina/genética , Regiones Promotoras Genéticas , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virologíaRESUMEN
Serologic tests are widely accepted and applied as means to detect anti- Toxoplasma gondii immunoglobulin G antibodies. In this study, recombinant matrix antigen (rMAG1) was induced by isopropyl-ß-d-thiogalactoside and purified by nickel-nitrilotriacetic acid purification system. We then developed and optimized an indirect enzyme-linked immunosorbent assay (ELISA) through checkerboard assays using serial dilutions of antigens and sera to assess the potential use of rMAG1 in serologic detection of T. gondii infection in dogs. Serum samples from 93 domestic dogs were analyzed by western blot and rMAG1-ELISA. The results were compared with those obtained from an ELISA with the soluble Toxoplasma lysate antigens (TLA). We found that although yielding an excellent agreement (96.7%) with western blot data (κ = 0.9659), rMAG1-ELISA produced higher sensitivity (93.9% vs. 87.8%) and specificity (98.3% vs. 96.7%) than TLA-ELISA. In addition, receiver operating characteristic analysis also revealed that rMAG1-ELISA is in more agreement with western blot (area under the curve [AUC] = 0.985) relative to TLA-ELISA (AUC = 0.955). These results indicated that the rMAG1-ELISA established in this study provides a promising and reliable tool for serologic detection of T. gondii infection in dogs.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Enfermedades de los Perros/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Proteínas Protozoarias/inmunología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/diagnóstico , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Western Blotting/veterinaria , Chlorocebus aethiops , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Enfermedades de los Perros/parasitología , Perros , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Toxoplasma/genética , Toxoplasma/inmunología , Células VeroRESUMEN
Echinococcus granulosus is the causative pathogen of cystic echinococcosis, a serious disease endangering human and animal health. In this study, an immunochromatographic strip was developed based on the recombinant protein Heat shock protein 70 (HSP70) for the serological detection of E. granulosus. The protocol completes within 20min requiring no specialized equipment or chemical reagents, while specificity tests confirmed no cross-reactivity with positive serum of Fasciola hepatica, Haemonchus contortus, Peste des petits ruminants virus (PPRV) and Foot-and-mouth disease virus (FMDV). The strips remained stable after storage at 4°C for up to 8 months. Both immunochromatographic strip and ELISA tests were applied to detect E. granulosus antibody in a total of 728 serum samples obtained from slaughter houses in Zhejiang Province. Our data revealed positive rates of 2.61 and 1.65% by immunochromatographic strip and ELISA methods, respectively. The immunochromatographic strip test developed in this study provides a simple, specific and rapid method of E. granulosus antibody detection and infected sheep monitoring.
Asunto(s)
Cromatografía de Afinidad/veterinaria , Equinococosis/veterinaria , Echinococcus granulosus/aislamiento & purificación , Oro Coloide/química , Proteínas HSP70 de Choque Térmico/inmunología , Tiras Reactivas , Enfermedades de las Ovejas/diagnóstico , Animales , Cromatografía de Afinidad/métodos , Equinococosis/sangre , Equinococosis/diagnóstico , Ovinos , Enfermedades de las Ovejas/parasitologíaRESUMEN
Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells.