Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

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.

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.

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.

Mining host candidate regulators of schistosomiasis-induced liver fibrosis in response to artesunate therapy through transcriptomics approach.

BACKGROUND: Artesunate (ART) has been reported to have an antifibrotic effect in various organs. The underlying mechanism has not been systematically elucidated. We aimed to clarify the effect of ART on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in an experimentally infected rodent model and the potential underlying mechanisms. METHODS: The effect of ART on hepatic stellate cells (HSCs) was assessed using CCK-8 and Annexin V-FITC/PI staining assays. The experimental model of liver fibrosis was established in the Mongolian gerbil model infected with S. japonicum cercariae and then treated with 20 mg/kg or 40 mg/kg ART. The hydroxyproline (Hyp) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in liver tissue were measured and histopathological changes of liver tissues were observed. Whole-transcriptome RNA sequencing (RNA-seq) of the liver tissues was performed. Differentially expressed genes (DEGs) were identified using bioinformatic analysis and verified by quantitative PCR (qPCR) and western blot assay. RESULTS: ART significantly inhibited the proliferation and induce the apoptosis of HSCs in a dose-dependent manner. In vivo, Hyp content decreased significantly in the ART-H group compared to the model (MOD) group and GPX activity was significantly higher in the ART-H group than in the MOD group. Besides, ART treatment significantly reduced collagen production (p <0.05). A total of 158 DEGs and 44 differentially expressed miRNAs related to ART-induced anti-schistosomiasis liver fibrosis were identified. The qPCR and western blot results of selected DEGs were consistent with the sequencing results. These DEGs were implicated in key pathways such as immune and inflammatory response, integrin-mediated signaling and toll-like receptor signaling pathways. CONCLUSION: ART is effective against liver fibrosis using Mongolian gerbil model induced by S. japonicum infection. We identified host candidate regulators of schistosomiasis-induced liver fibrosis in response to ART through transcriptomics approach.

[A study of double antigen sandwich colloidal gold immunochromatography rapid detection for Mycobacterium tuberculosis antibody].

OBJECTIVE: To establish a simple, fast and accurate double antigen colloidal gold immunochromatographic technique for detecting Mycobacterium tuberculosis antibody from tuberculosis patients. METHODS: The fusion protein ESAT-6-16-38 was constructed by using gene cloning technique for the 6, 16 and 38 kDa early secreted antigenic target from Mycobacterium tuberculosis. The ESAT-6-16-38 fusion protein was marked to colloidal gold to establish the double antigen colloidal gold immunochromatographic assay. Serum samples from 163 patients with tuberculosis, including 57 sputum-positive cases, 64 sputum-negative cases, and 42 cases with extrapulmonary tuberculosis, were collected during 2007 and 2009 from the Disease Prevention and Control Center of Deqing County. In addition, 573 controls (224 healthy volunteers, 217 patients with acute pneumonia and bronchitis, 132 patients with paragonimiasis) were recruited for comparison. Mycobacterium tuberculosis specific antibodies were detected by using immunochromatographic and protein chip technique. Detection rate was compared with Chi-square test. RESULTS: Among the 163 tuberculosis patients, the positive rates of immunochromatographic detection and protein chip were 73.0% (120/163) and 72.4% (118/163) respectively; the difference was not statistically significant (χ()2 = 0.062, P > 0.05). Among the 573 controls, the negative rates of immunochromatographic detection and protein chip were 93.9% (538/573) and 92.0% (527/573) respectively; the difference was not statistically significant (χ()2 = 0.635, P > 0.05). There was no cross reaction in the paragonimiasis patients. The positive rate of the immunochromatographic assay was as high as 87.7% (50/57) in the sputum-positive patients. CONCLUSIONS: The double antigen immunochromatographic technique is an easy to operate, rapid, highly sensitive, specific, and reproducible method for the detection of Mycobacterium tuberculosis antibodies.

Early Detection of Trichinella spiralis DNA in Rat Feces Based on Tracing Phosphate Ions Generated During Loop-Mediated Isothermal Amplification.

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.

A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs.

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.

[Establishment and application of rapid dipstick of latex immunochromatographic assay for the diagnosis of schistosomiasis].

OBJECTIVE: To establish a simple and fast diagnostic assay for schistosomiasis. METHODS: Based on the immunochromatographic technique and the principle of indirect assay of ELISA, using soluble egg antigen (SEA) of Schistosoma japonicum and mouse anti-human monoclonal antibody labelled with red latex as color developing agents, a latex immunochromatographic assay (DLIA) was developed. Serum samples from 69 schistosomiasis patients were detected by DLIA. Tested were also 264 sera from healthy people, 15 sera from clonorchiasis patients, 8 sera from patients with angiostrongyliasis cantonensis, 11 sera from patients with intestinal nematode infection and 19 sera from paragonimiasis patients. ELISA was used as a parallel control. RESULTS: The sensitivity for detecting schistosomiasis antibodies with DLIA and ELISA was 94.2% (65/69) and 95.7% (66/69), respectively (chi2=0.15, P>0.05). The specificity in examining healthy persons was 97.4% (257/264) and 94.7% (250/264), respectively (chi2=2.43, P>0.05). No cross reaction was found with the sera of clonorchiasis, intestinal nematode infection and angiostrongyliasis. The cross reaction rate with paragonimiasis of the two assays was 42.1% (8/19) and 47.4% (9/19), respectively (chi2=0.11, P>0.05). CONCLUSION: DLIA is a simple, fast, sensitive and specific assay for the diagnosis of schistosomiasis.

Long Non-Coding RNA AC118344.1 Promotes Gastric Cancer Cell Proliferation, Invasion, and Metastasis via AKT2 and Its Downstream Molecules HK2 and MMP2.

BACKGROUND: Gastric cancer (GC) is a highly occurring cancer with poor prognosis. Reports indicate that long non-coding RNA (LncRNA) potentially regulates tumor progression. Herein, we aim to explore the effect of LncRNA AC118344.1 on the progression of gastric cancer. METHODS: Overexpression and knockout experiments were used to clarify the potential molecular signaling mechanisms induced by AC118344.1. CCK-8, transwell and in vivo metastasis assay were used to detect the function of AC118344.1 in AGS and SGC-7901 cells. Additionally, shRNA silencing techniques, qRT-PCR and Western blot assay were used to explore the relationship between AC118344.1, AKT2, and its downstream molecules. RESULTS: Upregulating the expression of AC118344.1 induces cell proliferation, invasion in vitro, and lung metastasis in vivo whereas downregulating the expression of AC118344.1 inhibits these effects. Besides, silencing the expression of AC118344.1 downregulated the expression of AKT2 in both the two cells. On the other hand, silencing the expression of AKT2 by shRNA was unable to downregulate the expression of AC118344.1 in both the gastric cancer cells. Also, AC118344.1 regulated AKT2 via its downstream molecules including HK2 and MMP2. CONCLUSION: AC118344.1 promotes gastric cancer cell proliferation and invasion and lung metastasis in nude mice by upregulating the expression of AKT2 and its downstream molecules (HK2 and MMP2). Therefore, our findings provide a novel mechanism of the AC118344.1-AKT2-HK2/MMP2 axis in regulating the development of gastric cancer cells.

GRA24-Based DNA Vaccine Prolongs Survival in Mice Challenged With a Virulent Toxoplasma gondii Strain.

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.

AKT2 phosphorylation of hexokinase 2 at T473 promotes tumorigenesis and metastasis in colon cancer cells via NF-κB, HIF1α, MMP2, and MMP9 upregulation.

It has been well-established that AKT2 plays an important role in the development and progression of colon cancer; however, its precise function remains unclear. In the present study, we found that AKT2 can interact with and phosphorylate hexokinase 2 (HK2), the rate-limiting enzyme in glycolysis. Moreover, threonine phosphorylation dramatically increases its catalytic activity and enhances glycolysis. Mechanistically, AKT2 phosphorylation of HK2 at T473 was found to increase hexokinase activity and lactic acid production. A mutation in the AKT2 phosphorylation site of HK2 substantially reduced the stimulating effects of AKT2 on glycolysis, cellular apoptosis, invasion, tumorigenesis, and metastasis. In addition, AKT2 regulated NF-κB, HIF1Α, MMP2, and MMP9 via the phosphorylation of HK2 at the T473 site. Taken together, AKT2 increases the invasion, tumorigenesis, and metastasis of colon cancer cells in vitro and promotes lung metastasis in nude mice in vivo through the phosphorylation of the T473 site of HK2 by upregulating NF-κB, HIF1α, MMP2, and MMP9. In conclusion, our findings highlight a novel mechanism for the AKT2-HK2-NF-κB/HIF1α/MMP2/MMP9 axis in the regulation of colon cancer progression. Moreover, our results suggest that both AKT2 and HK2 may be potential targets for the treatment of colon cancer.

The Virulence-Related MYR1 Protein of Toxoplasma gondii as a Novel DNA Vaccine Against Toxoplasmosis in Mice.

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.

Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections.

Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as 'test-of-cure' tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.

DNA detection of Trypanosoma evansi: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP).

Trypanosoma evansi (T. evansi) is the most widely spread pathogenic trypanosome in the world. The control of trypanosomiasis depends on accurate diagnosis and effective treatment. Focusing on the presence of T. evansi in Asia, we developed a detection assay based on tracing phosphate ions (Pi) generated during LAMP targeting the variant surface glycoprotein (VSG) gene of Rode Trypanozoon antigenic type 1.2 (RoTat 1.2 VSG). The diagnostic potential as well as the use of the assay as a test-of-cure method after berenil treatment, was assessed in mice at different time points of infection. In addition, 67 buffalo blood collected from Tongling county, Anhui province, as well as 42 cattle sera from the Shanghai area, were used to evaluate the diagnostic validity of the test. The detection limit of the novel LAMP assay was determined to be as low as 1 fg of T. evansi DNA, while the reaction time for the test was only 30min. Hence it outperforms both microscopy and PCR. In the test-of-cure assessment, successful berenil mediated cure could be confirmed within 48h after treatment. This offers a tremendous advantage over conventional antibody-based diagnostic tools in which successful cure only can be confirmed after months. In the cattle and buffalo screening, the LAMP was able to detect a false-negative determined sample, wrongly classified in a conventional microscopy and PCR screening. Finally, no cross-reactivity was observed with other zoonotic parasites, such as T. evansi type B, T. congolense, T. brucei, Schistosoma japonicum, Plasmodium falciparum, Leishmania donovani, Toxoplasma gondii and Angiostrongylus cantonensis. We conclude that the novel LAMP assay is sensitive, specific and convenient for field use, particularly in areas where infection incidence has become extremely low. The LAMP assay could be used as a tool for trypanosomiasis control and elimination strategies in areas where T. evansi Type A infections are causing a threat to livestock farming.

[Observation on the change of anti-S. japonicum antibody level in population migrated from outside embankment to new town].

OBJECTIVE: To detect the change of the anti-S. japonicum antibody level after people migrated from outside embankment to newly established town. METHODS: Three pilot spots were established for the investigation: one spot that both inhabitancy and cultivation disuse (A), one spot that only inhabitancy disuse but farming continued (B) and the third one served as control (C). DIGFA and ELISA were used to detect the antibody level in the populations from 2002 to 2005. RESULTS: The positive rate of anti-S. japonicum antibody declined significantly from 6.63% to 3.52% by DIGFA and from 7.26% to 3.71% by ELISA at spot A (chi2=5.2625, P<0.05; chi2=6.3296, P<0.05, respectively). There was no significant difference on the positive rate of antibody in spots B and C. The average A450 value of ELISA in the three spots was statistically analyzed by One-Way ANOVA. It was only in spot B that the average A450 value declined from 0.182 in 2003 to 0.147 in 2005 (P<0.01). CONCLUSION: The anti-S. japonicum antibody level in human population has decreased at certain degree after they migrated from outside embankment to new town.

Immuno-Efficacy of a T. gondii Secreted Protein with an Altered Thrombospondin Repeat (TgSPATR) As a Novel DNA Vaccine Candidate against Acute Toxoplasmosis in BALB/c Mice.

Toxoplasma gondii (T.gondii) is distributed worldwide and infects most species of warm-blooded animals, including humans. Toxoplasmosis has serious consequences, especially in people with an impaired or immature immune system. Thus, an effective vaccine is urgently required. Secretory microneme proteins are essential for the adhesion and invasion of T. gondii. The gene encoding the microneme protein, T. gondii secreted protein with an altered thrombospondin repeat (TgSPATR), we constructed a recombinant eukaryotic plasmid, pVAX1-TgSPATR, as a DNA vaccine, injected it intramuscularly into BALB/c mice and evaluated the induced immune response. Lymphocyte proliferation assays, cytokine (IFN-γ, IL-2, IL-4, IL-10), and antibody determinations showed that mice immunized with pVAX1-TgSPATR produced humoral and mixed Th1/Th2 type cellular immune responses. The survival times of mice immunized with pVAX1-TgSPATR were also significantly prolonged (15.7 ± 1.42 days) compared with control groups, which died within 7 days of challenge (p < 0.05). The current study indicated that pVAX1-TgSPATR induce a T. gondii specific immune response and might be a promising vaccine candidate against toxoplasmosis. To the best of our knowledge, this is the first report to evaluate the immunoprotective value of TgSPATR against T. gondii.

Genome-wide miRNAs expression profiles of Schistosoma japonicum schistosomula in response to artesunate.

AIM: miRNAs play a significant role in pharmacogenomics and are likely to be important in the molecular mechanism of atesunate (ART) effects on Schistosoma japonicum. METHODS: We sequenced the RNAs using an Illumina (Solexa) DNA sequencer and compared the relative expression levels of the miRNAs in 10-day-old schistosomula from ART and the parallel control group. RESULTS: We characterized 95 known miRNAs from S. japonicum schistosomula individuals, including 38 novel miRNA families. Among the detectable 134 miRNAs differentially expressed (>2.0-fold change, p < 0.01) after ART treatment in schistosomula, a total of seven known or novel 3p- or 5p- derived S. japonicum miRNAs were characterized. We propose that sja-miR-125b may regulate the expression of ART metabolizing enzymes, glutathione synthetase or heme-binding protein 2 to help S. japonicum resists or adapts to drug stress and also ART may significantly inhibit sexual maturation of female worms mediated by mir-71b/2 miRNA cluster. CONCLUSION: This was the first comprehensive miRNAs expression profile analysis of S. japonicum in response to ART, and provides an overview of the complex network of the mechanism of action of ART on S. japonicum.

Evaluation on the applied value of the dot immunogold filtration assay (DIGFA) for rapid detection of anti-Schistosoma japonicum antibody.

The dot immunogold filtration assay (DIGFA) is a rapid technique for the detection of anti-Schistosoma japonicum antibody. Its sensitivity with regard to sera obtained from patients with acute or chronic schistosomiasis was shown to be 100 and 96.9%, respectively. The specificity when using sera of people living in an area non-endemic for schistosomiasis japonica was 100%. Cross-reaction rates for paragonimiasis and clonorchiasis patients were 14.3% and 0%, respectively. Parallel serum tests of 1091 residents from an area endemic for S. japonicum by means of DIGFA, enzyme-linked immunosorbent assay and indirect haemagglutination test resulted in positive rates of 9.3%, 11.5% and 11.0%, respectively. Thus, there was a high level of agreement between the sets of results (P>0.05). In conclusion, DIGFA holds considerable promise for rapid and accurate diagnosis of S. japonicum, as it does not require any specific instruments and can be applied with ease. DIGFA has therefore several advantages over conventional diagnostic approaches and is useful not only for screening and sero-epidemiological surveys in the field, but also in clinical settings.

[Protective immunity of the recombinant Schistosoma japonicum specific very low density lipoprotein binding protein as a vaccine candidate].

OBJECTIVE: To investigate the protective immunity against Schistosoma japonicum in mice immunized with recombinant specific very low density lipoprotein binding protein (SVLBP) and its potential as vaccine candidate. METHODS: Recombinant SVLBP antigen was over-expressed under IPTG induction and purified by Ni-NTA affinity chromatography. C57BL/6 mice were immunized three times with purified reSVLBP complexed with Freund's adjuvant, at biweekly intervals. Then 35+/-1 cercariae of S. japonicum were given to each mouse by abdominal skin 10 days after the 3rd immunization. 45 days later, all mice were sacrificed to collect adult worms and count liver eggs. serum samples were collected before immunization and after challenge respectively, and were probed the antigen-specific antibodies using a panel of ELISAs. RESULTS: The worm burden and the egg deposition in liver tissue were reduced by 33.4% and 47.6% respectively in the immunized group, in comparison with the adjuvant control group (P<0.05). Higher titer (>1:6 400) of total IgG was observed after challenge infection. The vaccinated mice developed significantly higher levels of IgG2a, IgG2b, IgG1 than those of control mice. CONCLUSION: The recombinant tegumental SVLBP antigen could induce partial protection against S. japonicum infection. These data demonstrate the potential of SVLBP as a schistosome vaccine candidate.

Molecular identification of Anisakis and Hysterothylacium larvae in marine fishes from the East China Sea and the Pacific coast of central Japan.

Anisakiasis is a human disease caused by the accidental ingestion of larvae belonging to the family Anisakidae. Three fish species, the small yellow croaker Pseudosciaena polyactis, the mackerel Pneumatophorus japonicus and the hairtail Trichiurus haumela are important source for food products in the East China Sea. The prevalence and the identification of Anisakidae larvae in these fishes will benefit the prevention and control of anisakiasis. In this study, fish samples were obtained from fish markers in the East China Sea and the Pacific coast of central Japan during April 2011 and July 2013. For species identification, the PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis of the entire ITS region (ITS1, 5.8 S and ITS2) of nuclear ribosomal DNA (rDNA) was performed. In total, 2004 larvae were collected from 80 hairtail fish, 20 small yellow croaker, and 27 mackerel from the East China Sea and the Pacific coast of central Japan. High prevalence of Anisakidae larvae infection (116/122, 95.1%) was detected in the East China Sea. Seven species were identified belonging to the genera Anisakis (Nematoda: Anisakidae) and Hysterothylacium (Nematoda: Anisakidae). Anisakis pegreffii was the predominant species accounting for 84.8% of all larvae examined in East China Sea, while all Anisakidae larvae isolated from Japan were identified as Anisakis simplex sensu stricto (s.s.). In the East China Sea, A. simplex s.s. and Anisakis typica were 0.6% (4/619) and 1.5% (9/619) of the identified nematodes, respectively. Interestingly, one larva was identified as a recombinant genotype of A. simplex s.s. and A. pegreffii. In addition, four species of the genus Hysterothylacium, namely, Hysterothylacium amoyense (31/619, 5.0%), Hysterothylacium aduncum (10/619, 1.6%), Hysterothylacium fabri (21/619, 3.4%) and Hysterothylacium spp. (18/619, 2.9%) were also identified in the present study. This is a comprehensive epidemiological dataset for the family Anisakidae in the East China Sea. The identification of A. typica, recombinant genotype of A. simplex s.s. and A. pegreffii, H. amoyense and H. fabri is first reported in this area. The wide diversity and substantial geographical distributions of these nematodes will provide a foundation for future studies of Anisakidae family. The high prevalence of these nematodes in marine fishes off the East China Sea may pose considerable food safety problems, which is a potential cause of human anisakiasis.