Characterisation and preliminary functional analysis of N-acetyltransferase 13 from Schistosoma japonicum.

BACKGROUND: N-acetyltransferase 13 (NAT13) is a probable catalytic component of the ARD1A-NARG1 complex possessing alpha (N-terminal) acetyltransferase activity. RESULTS: In this study, a full-length complementary DNA (cDNA) encoding Schistosoma japonicum NAT13 (SjNAT13) was isolated from schistosome cDNAs. The 621 bp open reading frame of SjNAT13 encodes a polypeptide of 206 amino acids. Real-time PCR analysis revealed SjNAT13 expression in all tested developmental stages. Transcript levels were highest in cercariae and 21-day-old worms, and higher in male adult worms than female adult worms. The rSjNAT13 protein induced high levels of anti-rSjNAT13 IgG antibodies. In two independent immunoprotection trials, rSjNAT13 induced 24.23% and 24.47% reductions in the numbers of eggs in liver. RNA interference (RNAi) results showed that small interfering RNA (siRNA) Sj-514 significantly reduced SjNAT13 transcript levels in worms and decreased egg production in vitro. CONCLUSIONS: Thus, rSjNAT13 might play an important role in the development and reproduction of schistosomes.

Two Molecular Plasma-Based Diagnostic Methods to Evaluate Early Infection of Schistosoma japonicum and Schistosomiasis Japonica.

The prevalence and infectious intensity of schistosomiasis japonica has decreased significantly in China in the past few decades. However, more accurate and sensitive diagnostic methods are urgently required for the further control, surveillance, and final elimination of the disease. In this study, we assessed the diagnostic efficacy of a real-time fluorescence quantitative PCR (qPCR) method and recombinase polymerase amplification (RPA) combined with a lateral-flow dipstick (LFD) assay for detecting early infections of Schistosoma japonicum and different infection intensities. The sensitivity of the qPCR at 40 days post-infection (dpi) was 100% (8/8) in mice infected with 40 cercariae, which was higher than in mice infected with 10 cercariae (90%, 9/10) or five cercariae (77.8%, 7/9). The results of the RPA-LFD assays were similar, with sensitivities of 55.6% (5/9), 80% (8/10), and 100% (8/8) in mice infected with 5, 10, and 40 cercariae, respectively. In goats, both the qPCR and RPA-LFD assays showed 100% (8/8) sensitivity at 56 dpi. In the early detection of S. japonicum infection in mice and goats with qPCR, the first peak in positivity appeared at 3-4 dpi, when the positivity rate exceeded 40%, even in the low infection, intensity mice. In the RPA-LFD assays, positive results first peaked at 4-5 dpi in the mice, and the positivity rate was 37.5% in the goats at 1 dpi. In conclusion, neither of the molecular methods produced exceptional results for the early diagnosis of S. japonicum infection. However, they were useful methods for the regular diagnosis of schistosomiasis in mice and goats.

Diagnostic Efficacy of Plasma-Based Real-Time PCR for Schistosomiasis Japonica in Mice before and after Treatment with Praziquantel.

The prevalence of schistosomiasis japonica in China is now characterized by a low epidemic rate and low-intensity infections. Some diagnostic methods with high sensitivity and specificity are urgently needed to better monitor this disease in the current situation. In this study, the detection efficacy of a real-time fluorescent quantitative PCR (qPCR) assay was assessed for schistosomiasis japonica in mice, and before and after treatment with praziquantel (PZQ). Our results showed that the sensitivity of the qPCR was 99.3% (152/153, 95% CI: 96.41-99.98%) and its specificity was 100% (77/77, 95% CI: 95.32-100%) in mice infected with different numbers of Schistosoma japonicum. After the oral administration of PZQ, mice infected with 10 cercariae or 40 cercariae were all Schistosoma japonicum-negative 6 weeks after treatment. However, the negativity rates on a soluble egg antigen (SEA)-based enzyme-linked immunosorbent assay (ELISA) were only 34.8% (8/23, 10 cercariae group) and 6.7% (1/15, 40 cercariae group) at the sixth week after PZQ treatment. These results demonstrated that the qPCR method had good sensitivity and specificity, and suggested that its sensitivity correlated with the infection intensity in mice. Moreover, this method had better potential utility for evaluating the treatment efficacy of PZQ in schistosome-infected mice than SEA-based ELISA.

Characterization of aspartyl aminopeptidase from Schistosoma japonicum.

The tegument of schistosomes is the interface between the worm and the host environment. Some molecules distributed on the tegument participate in host-parasite interactions. Aspartyl aminopeptidase (AAP), identified on the tegument of Schistosoma japonicum (S. japonicum), facilitate protein turnover by acting in concert with other aminopeptidases. In this study, the gene encoding S. japonicum aspartyl aminopeptidase (SjAAP) was cloned, expressed and characterized. Quantitative real-time PCR analysis showed that SjAAP was expressed in all studied developmental stages. The transcript level was higher in 8, 14, 21, and 28 days old worms than the other detected stages. Moreover, the level of expression in 42-day-old male worms was significantly higher than that in females. The recombinant SjAAP (rSjAAP) was expressed as both supernatant and inclusion bodies in Escherichia coli BL21 cells. The enzymatic activity of rSjAAP was 4.45 U/mg. The Km and Vmax values for H-Asp-pNA hydrolysis were discovered to be 5.93 mM and 0.018 mM·min-1. Immunofluorescence analysis revealed that SjAAP is primarily distributed on the tegument and parenchyma of schistosomes. Western blot showed that rSjAAP possessed good immunogenicity. Although specific antibodies were produced in BALB/c mice vaccinated with rSjAAP emulsified with ISA 206 adjuvant, no significant reduction of worm burden and number of eggs in the liver was observed. Therefore, rSjAAP may not be suitable to act as a potential vaccine candidate against schistosomiasis japonica in mice. However, this study provides some foundation for further exploration of the biological function of this molecule.

Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats.

Although the prevalence of schistosomiasis japonica has declined gradually in China, more accurate and sensitive diagnostic methods are urgently needed for the prevention and control of this disease. Molecular diagnostic methods are advantageous in terms of sensitivity and specificity, but they are time-consuming and require expensive instruments and skilled personnel, which limits their application in low-resource settings. In this study, an isothermal DNA amplification assay and recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) were set up. It was used to detect S. japonicum infections in experimental mice and domestic goats by amplifying a specific DNA fragment of S. japonicum. The lower limit of detection for the LFD-RPA assay was evaluated using dilutions of plasmid containing the target sequence. Cross-reactivity was evaluated using genomic DNA from eight other parasites. The effectiveness of the LFD-RPA assay was verified by assessing 36 positive plasma samples and 36 negative plasma samples from mice. The LFD-RPA assay and real-time PCR were also used to assess 48 schistosomiasis japonica-positive plasma samples and 53 negative plasma samples from goats. The LFD-RPA assay could detect 2.6 femtogram (fg) of S. japonicum target DNA (~39 fg genomic DNA of S. japonicum), only 10-fold less sensitive than real-time PCR assay. There was no cross-reactivity with DNA from the other eight parasites, such as Haemonchus contortus and Spirometra. The whole amplification process could be completed within 15 min at 39°C, and the results can be observed easily using the LFD. The sensitivity and specificity of the LFD-RPA assay were 97.22% (35/36, 95% CI, 85.47%-99.93%) and 100% (36/36, 95% CI, 90.26%-100%) in mice, and 93.75% (45/48, 95% CI, 82.80%-98.69%) and 100% (53/53, 95% CI, 93.28%-100%) in goats. By comparison, the sensitivity and specificity of real-time PCR were 100% (36/36, 95% CI, 90.26%-100%) and 100% (36/36, 95% CI, 90.26%-100%) for mice, and 97.92% (47/48, 95% CI, 88.93%-99.95%) and 100% (53/53, 95% CI, 93.28%-100%) for goats. The LFD-RPA assay exhibits high sensitivity and specificity for the diagnosis of schistosomiasis japonica, and it is an alternative method for diagnosis schistosomiasis japonica in low resource setting.

Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat.

BACKGROUND: Schistosomiasis japonica is an infectious disease caused by Schistosoma japonicum that seriously endangers human health. Domestic animals have important roles in disease transmission and goats are considered a primary reservoir host and source of infection. The prevalence and intensity of schistosomiasis infections have significantly decreased in China, and a more sensitive, specific detection method is urgently needed. The aim of this study was to develop a real-time PCR assay for accurate detection of S. japonicum infection in goats. METHODS: A real-time PCR method for detecting schistosomiasis japonica in goats was developed by amplification of a specific S. japonicum DNA fragment, and validated using a total of 94 negative and 159 positive plasma and serum samples collected in our previous study of S. japonicum infection. Both plasma and serum samples were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA). In addition, 120 goat plasma samples from an S. japonicum-endemic area (Wangjiang) and 33 from a non-endemic region (Weihai) were collected and evaluated using our method. RESULTS: The sensitivity and specificity of the real-time PCR for detecting infected samples were 98.74% (157/159, 95% CI: 95.53-99.85%) and 100% (94/94, 95% CI: 96.15-100%), respectively. For the ELISA, sensitivity and specificity were 98.11% (156/159, 95% CI: 94.59-99.61%) and 90.43% (85/94, 95% CI: 82.60-95.53%), respectively. Further, we found positivity rates for S. japonicum infection in Wangjiang and Weihai of 8.33% (10/120, 95% CI: 4.07-14.79%) and 0% (0/33, 95% CI: 0-10.58%), respectively. CONCLUSIONS: The results of this study indicate that our real-time PCR method exhibits higher sensitivity and specificity than ELISA and is a useful method for detection of S. japonicum infection in goats.