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Abstract Background Clonorchiasis has significant socioeconomic importance in endemic areas; however, studies investigating the disease burden in specific sub-regions are lacking. This study aims to address the gap by quantifying the current disease burden caused by clonorchiasis in Guangdong province and assessing its distribution characteristics. Methods Comprehensive measures, including prevalence rates, disability-adjusted life years (DALYs), and direct medical costs, were used to assess the disease burden of clonorchiasis. To estimate the prevalence rate, the number of infections was divided by the examined population, based on the annual surveillance data on clonorchiasis cases during 2016–2021. The calculation of DALYs was based on the epidemiological parameters according to the definition issued by the World Health Organization. Cost data of clonorchiasis were utilized to quantify the direct medical costs. The distribution characteristics of disease burden were assessed through comparisons of groups of population defined by geographic area, time, and characteristics of people. Results In 2021, clonorchiasis posed a significant disease burden in Guangdong Province. The prevalence rate was found to be 4.25% [95% CI (4.02%, 4.49%)], with an associated burden of DALYs of 406,802.29 [95% CI (329,275.33, 49,215,163.78)] person-years. The per-case direct medical costs of patients with clonorchiasis were estimated to be CNY 7907.2 (SD = 5154.4). Notably, while the prevalence rate and DALYs showed a steady decrease from 2016 to 2020, there was a rising trend in 2021. Spatial clustering of clonorchiasis cases and DALYs was also observed, particularly along the Pearl River and Han River. This suggests a concentration of the disease in these regions. Furthermore, significant differences in prevalence rates were found among various demographic groups, including sex, age, occupation, and education level. Additionally, patients with longer hospital stays were more likely to incur higher direct medical costs. Conclusions The burden of clonorchiasis in Guangdong Province remains high, despite significant progress achieved through the implementation of the prevention and control programs. It is suggested that measures should be taken based on the distribution characteristics to maximize the effectiveness of prevention and control, with a primary focus on key populations and areas. Graphical Abstract

Abstract Background Aedes aegypti and Aedes albopictus are invasive mosquito species and significantly impact human health in southern China. Microbiota are confirmed to affect the development and immunity of mosquitoes. However, scientists have focused more on midgut microbiota of female mosquitoes and bacterial differences between female and male Aedes mosquitoes. The relationship between the midgut and entire body microbiota of Aedes is unclear. In this study, we collected mosquito samples reared under the same laboratory conditions and compared the microbial composition of midgut and entire bodies of Aedes aegypti and Aedes albopictus using 16S rRNA gene sequencing. Methods In this study, we collected mosquito samples reared under the same laboratory conditions and compared the microbial composition of midgut and entire bodies of Aedes aegypti and Aedes albopictus using 16S rRNA gene sequencing. Results A total of 341 OTUs were identified, showing that Proteobacteria was the dominant phylum and Methylobacterium the dominant genus in both Aedes aegypti and Aedes albopictus. The bacterial diversity and community structures of the entire bodies were similar between males and females in both Aedes aegypti and Aedes albopictus. Conversely, the bacterial compositions of male and female Aedes aegypti and Aedes albopictus were significantly different. NMDS analysis, UPGMA analysis, diversity indices and OTU distribution demonstrated that compositions and structures in midgut microbiota were similar but significantly different in the entire bodies of Aedes aegypti and Aedes albopictus. Functional prediction analysis showed that metabolism and environmental information processing were the dominant KEGG pathways at level 1. Our study showed that there were significantly different level 2 and 3 KEGG pathways in the midgut microbiota (16 level 2 and 24 level 3) and the entire bodies (33 level 2 and 248 level 3) between female Aedes albopictus and Aedes Aegypti. Conclusions Our findings that Aedes aegypti and Aedes albopictus reared in the same laboratory harbor a similar gut bacterial microbiome but different entire body microbiota imply that the gut microbiota of adult mosquitoes is environmentally determined regardless of the host genotype, but the entire body microbiota is more genetically determined. Our findings improved the understanding of the microbiota in the entire and partial tissues of Aedes mosquitoes. Graphical abstract

Abstract Background Potent granulomatous inflammation responses induced by schistosome eggs and resultant fibrosis are the primary causes of morbidity in schistosomiasis. Recombinant Sj16 (rSj16), a 16-kDa protein of Schistosoma japonicum produced in Escherichia coli, has been demonstrated to have novel immunoregulatory effects in vivo and in vitro. Thus, this study investigated the anti-inflammatory and anti-fibrotic effects of rSj16 treatment in S. japonicum-infected mice and demonstrated the immune modulation between the schistosome and the host. Methods Schistosoma japonicum infected mice were treated with the rSj16 protein and Sj16 peptide at different time points post-infection to assess their efficacy at the optimal time point. Sj16 peptide and/or Praziquantel (PZQ) treatments were initiated at week 5 post-infection to compare the therapeutic efficacy of each regimen. Hepatic granulomatous inflammation, fibrosis and cytokine production (pro-inflammatory, Th1, Th2, Th17 and regulatory cytokines IL-10) were detected. Moreover, M2 macrophages were measured to illuminate the mechanisms of Sj16. Results The rSj16 protein and Sj16 peptide had significant protective effects in S. japonicum-infected mice, as shown by decreased granuloma formation, areas of collagen deposition and inhibition of pro-inflammatory Th1, Th2 and Th17 cytokine production. These protective activities were more obvious when animals were treated with either the Sj16 protein or peptide at early stages post-infection. Interestingly, the combined treatment of PZQ and Sj16 was more effective and upregulated IL-10 production than administration of PZQ alone in infected mice. Furthermore, the Sj16 treatment alleviated the pathological effects associated with activated M2 macrophages. Conclusions This study demonstrates the anti-inflammatory and anti-fibrotic effects of rSj16 in schistosomiasis. Therefore, the combination of rSj16 with PZQ could be a viable and promising therapeutic strategy for schistosomiasis. In addition, this investigation provides additional information on schistosome-mediated immune modulation and host-parasite interactions.

Abstract Background Mass egg production is an important component of Aedes albopictus mosquito control programs, such as the sterile insect technique and incompatible insect technique, which requires the releases of large number of sterile males. Developing standard operating procedures and optimized cages for adult maintenance of Ae. albopictus can improve the mass rearing efficiency. Methods Three different sex ratios of females to males with a total number of 4,000 mosquitoes were tested by evaluating the insemination rate, egg production (total number of eggs per cage), female fecundity and egg hatch rate in small cage (30 × 30 × 30 cm). Blood meals with adenosine triphosphate (ATP, 0.05 g/ml), cage structures (Big cage A: 90 × 30 × 30 cm; Big cage B: 90 × 30 × 50 cm or 90 × 50 × 30 cm) and rearing densities (12,000, 16,000 and 20,000 mosquitoes, corresponding to 0.9 cm2/mosquito, 0.675 cm2/mosquito and 0.54 cm2/mosquito, respectively) were also tested and evaluated on the basis of egg production, female fecundity and egg hatch rate. An adult rearing unit holding 15 of Big cage A with optimal egg production was designed to produce 10 million eggs per rearing cycle in a 1.8 m2 space. Results Female to male ratios at 3:1 in small cages resulted in higher egg production but did not affect insemination rate, female fecundity and egg hatch rate. A concentration of 0.05 g/ml of ATP added to blood meals improved the blood-feeding frequency and thus increased the overall egg production per cage. Cage structures affected the egg production per cage, but not egg hatch rate. A medium rearing density at 0.675 cm2/mosquito (16,000 mosquitoes) resulted in higher egg production compared to both low and high densities. An adult rearing unit for Ae. albopictus on the basis of Big cage A has been developed with the capacity of producing 10 million eggs within 15 days. Conclusions Our results have indicated that the adult rearing methods and adult maintenance unit are recommended for Ae. albopictus mass rearing in support of the establishment of a medium-sized mosquito factory.

Abstract Background Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research. Results Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. Conclusions The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

Abstract Background Standardized larval rearing units for mosquito production are essential for the establishment of a mass-rearing facility. Two larval rearing units, developed respectively by the Guangzhou Wolbaki Biotech Co. Ltd. (Wolbaki) and Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (FAO/IAEA-IPCL), are tested to assess their potential uses to mass-rear the larval stages of Aedes albopictus in support of the establishment of a medium-scale mosquito facility for the application of mosquito genetic control strategies. Methods The triple Wolbachia-infected Ae. albopictus strain (HC strain) was used in this study. The effects of larval densities of two larval rearing trays (corresponding to 2.4, 3.0 and 3.6 larvae/cm2) and tray size/position (top, middle and bottom layers) on the pupae production and larval survival were assessed when trays were stacked within the larval rearing units. The male pupae production, female pupae contamination after sex separation, and male mating competitiveness were also studied by using both larval rearing units in their entirety. Results The optimal larval rearing density for Wolbaki-tray (Wol-tray) was 6,600 larvae (equal to 3.0 larvae/cm2) and 18,000 larvae (3.6 larvae/cm2) for the FAO/IAEA-IPCL tray (IAEA-tray). No significant difference in pupae production was observed when trays were stacked within top, middle or bottom layers for both units. At thirty-four hours after the first pupation, the average male pupae production was (0.89 × 105) for the Wol-unit and (3.16 × 105) for the IAEA-unit. No significant difference was observed in female pupae contamination between these two units. The HC males showed equal male mating competitiveness to wild type males for mating with wild type females in large cages, regardless of whether they were reared in the Wol-unit or IAEA-unit. Conclusions The current study has indicated that both the Wol-unit and IAEA-unit are suitable for larvae mass-rearing for Ae. albopictus. However, the IAEA-unit, with higher male production and less space required compared to the Wol-unit, is recommended to be used in support of the establishment of a medium-sized mosquito facility.

Abstract Background Schistosomiasis mansoni is one of the most important, but often neglected, tropical diseases transmitted by snails of the genus Biomphalaria. Control of the intermediate host snail plays a crucial role in preventing the spread of schistosomiasis. However, there is only one molluscicide, niclosamide, recommended by the World Health Organization. Niclosamide has been used for several decades but is toxic to non-target organisms. Therefore, it is necessary to optimize the scaffold of niclosamide and develop novel molluscicides with enhanced potency and decreased toxicity to non-target organisms. Methods In this study, a candidate compound was analyzed by nuclear magnetic resonance and mass spectrometry. The molluscicidal potential against Biomphalaria species and cercaricidal potential against S. mansoni were evaluated using the immersion method. Furthermore, the preliminary mechanism was studied through cellular enzyme tests and electron microscopy. Results 5-chloro-2-[(2-chloro-4-nitrophenyl)carbamoyl]phenyl-4-methoxybenzoate (salicylanilidate), a novel salicylanilide ester derivative, was derived from niclosamide. The 50% lethal concentration to B. glabrata, B. straminea and B. pfeifferi was 0.261 mg/l, 0.172 mg/l and 0.241 mg/l, respectively. The effective dose required to completely kill S. mansoni cercariae was 0.625 mg/l for salicylanilidate and 0.125 mg/l for niclosamide. However, salicylanilidate was approximately 100-fold less toxic to the fish Danio rerio than niclosamide. Furthermore, salicylanilidate reduced the enzymatic activities of nitric oxide synthase (NOS), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) in the snail, demonstrating that it could affect neurohypophysis transmission and energy metabolism. Severe swelling in the tentacle and deformation of cilia in the tentacle and mantle were observed through scanning electron microscopy. The results of transmission electron microscopy showed that salicylanilidate could damage critical organelles in hepatopancreas tissues, including degeneration of the endoplasmic reticulum and vacuolization in mitochondria. In addition, transcriptional levels of superoxide dismutase (SOD), acid phosphatase (ACP) and NOS in the hepatopancreas were significantly downregulated as shown by real-time quantitative polymerase chain reaction (RT-PCR). These results indicated that the hepatopancreas is a primary target organ of salicylanilidate. Conclusions Salicylanilidate not only had deleterious effects on Biomphalaria species and S. mansoni cercariae but also showed very low toxicity to D. rerio, suggesting that it has broad potential applications.

Abstract Background Schistosomiasis, also generally known as snail fever, is a parasitic disease caused by trematode flatworms of the genus Schistosoma. In Hong Kong and mainland China, the freshwater snail Biomphalaria straminea has been introduced and has the potential to transmit intestinal schistosomiasis caused by S. mansoni, a parasite of man which has a wide distribution in Africa and parts of the New World, especially Brazil. The first identification of B. straminea in Hong Kong dates back to the 1970s, and its geographical distribution, phylogenetic relationships, and infection status have not been updated for more than 30 years. Thus, this study aims to reveal the distribution and current infection status of B. straminea in contemporary Hong Kong. Methods Snails were collected from different parts of Hong Kong from July 2016 to January 2017. Both anatomical and molecular methods were applied to identify B. straminea. Cytochrome c oxidase subunit 1 (cox1), internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and 16S ribosomal DNA (rDNA) were sequenced from individual snails and analyzed. To detect the presence of S. mansoni, both biopsy and PCR analyses were carried out. Results Using both anatomical and molecular analyses, this study demonstrated the existence of black- and red-coloured shell B. straminea in different districts in the New Territories in Hong Kong, including places close to the mainland China border. None of the B. straminea (n = 87) investigated were found to be infected with S. mansoni when tested by biopsy and PCR. The Hong Kong B. straminea are genetically indistinguishable, based on the chosen molecular markers (cox1, ITS1-5.8S-ITS2, and 16S rDNA), and are similar to those obtained in mainland China and South America. Conclusion Biomphalaria straminea is now well established in freshwater habitats in Hong Kong. No evidence of infection with S. mansoni has been found. Surveillance should be continued to monitor and better understand this schistosomiasis intermediate host in mainland China and Hong Kong.

Abstract Background Schistosomiasis is considered second only to malaria as the most devastating parasitic disease in tropical countries. Schistosome cercariae invade the host by penetrating the skin and migrate though the lungs and portal circulation to their final destination in the hepatic portal system and eventually the mesenteric veins. Previous studies have shown that the cytotoxic pathways that target schistosomulum in the lung-stage involve nitric oxide (NO) produced by macrophages. By contrast, skin-stage schistosomulas can evade clearance, indicating that they might be freed from macrophage NO-mediated cytotoxicity to achieve immune evasion; however, the critical molecules and mechanisms involved remain unknown. Methods Recombinant SjCa8 (rSjCa8), an 8-kDa calcium-binding protein that is stage-specifically expressed in cercaria and early skin-stage schistosomulas of Schistosoma japonicum, was incubated with mouse RAW264.7 macrophages. Effects on macrophage proliferation were determined using Cell Counting Kit-8. Next, transwell assay was carried out to further investigate the role of rSjCa8 in macrophage migration. The effects of rSjCa8 on macrophage apoptosis were evaluated using confocal microscopy and flow cytometry. Additional impacts of rSjCa8 on NO release by lipopolysaccharide (LPS)-stimulated macrophages as well as the underlying mechanisms were explored using fluorescent probe, nitric oxide signaling pathway microarray, quantitative real-time PCR, mutagenesis, and neutralizing antibody approaches. Results rSjCa8 exhibited a striking inhibitory effect on macrophage migration, but did not markedly increase cell proliferation or apoptosis. Additionally, rSjCa8 potently inhibited NO release by LPS-stimulated macrophages in a dose- and time-dependent manner, and the inhibitory mechanism was closely associated with intracellular Ca2+ levels, the up-regulation of catalase expression, and the down-regulation of the expression of 47 genes, including Myc, Gadd45a, Txnip, Fas, Sod2, Nos2, and Hmgb1. Vaccination with rSjCa8 increased NO concentration in the challenging skin area of infected mice and reduced the number of migrated schistosomula after skin penetration by cercariae. Conclusions Our findings indicate that SjCa8 might be a novel molecule that plays a critical role in immune evasion by S. japonicum cercaria during the process of skin penetration. The inhibitory impacts of rSjCa8 on macrophage migration and [Ca2+]i-dependent NO release suggest it might represent a novel vaccine candidate and chemotherapeutic target for the prevention and treatment of schistosomiasis.

Abstract Background Angiostrongylus cantonensis (A. cantonensis) infection can result in increased risk of eosinophilic meningitis. Accumulation of eosinophils and inflammation can result in the A. cantonensis infection playing an important role in brain tissue injury during this pathological process. However, underlying mechanisms regarding the transcriptomic responses during brain tissue injury caused by A. cantonensis infection are yet to be elucidated. This study is aimed at identifying some genomic and transcriptomic factors influencing the accumulation of eosinophils and inflammation in the mouse brain infected with A. cantonensis. Methods An infected mouse model was prepared based on our laboratory experimental process, and then the mouse brain RNA Libraries were constructed for deep Sequencing with Illumina Genome Analyzer. The raw data was processed with a bioinformatics’ pipeline including Refseq genes expression analysis using cufflinks, annotation and classification of RNAs, lncRNA prediction as well as analysis of co-expression network. The analysis of Refseq data provides the measure of the presence and prevalence of transcripts from known and previously unknown genes. Results This study showed that Cys-Cys (CC) type chemokines such as CCL2, CCL8, CCL1, CCL24, CCL11, CCL7, CCL12 and CCL5 were elevated significantly at the late phase of infection. The up-regulation of CCL2 indicated that the worm of A. cantonensis had migrated into the mouse brain at an early infection phase. CCL2 could be induced in the brain injury during migration and CCL2 might play a major role in the neuropathic pain caused by A. cantonensis infection. The up-regulated expression of IL-4, IL-5, IL-10, and IL-13 showed Th2 cell predominance in immunopathological reactions at late infection phase in response to infection by A. cantonensis. These different cytokines can modulate and inhibit each other and function as a network with the specific potential to drive brain eosinophilic inflammation. The increase of ATF-3 expression at 21 dpi suggested the injury of neuronal cells at late phase of infection. 1217 new potential lncRNA were candidates of interest for further research. Conclusions These cytokine networks play an important role in the development of central nervous system inflammation caused by A. cantonensis infection.

Abstract Background Schistosomiasis japonicum remains a considerable economic and public health concern in China, the Philippines and Indonesia. Currently available measures to control the unique intermediate host Oncomelania hupensis are frequently associated with severe side effects. Previous studies have demonstrated that linalool-rich extracts from various plants exhibited promising biological activities including cytotoxic, anti-microbial and anti-parasitic properties. Methods We identified the components of leaf extracts from Cinnamomum camphora by gas chromatography coupled to mass spectrometry (GC-MS) and investigated molluscicidal and larvicidal effects of linalool against O. hupensis and Schistosoma japonicium. The ultrastructural alterations in gills, salivary gland, stomach and hepatopancreas of snails were observed under the light microscope and transmission electron microscope, and lesions to tegument of cercaria were examined under a light microscope and fluorescence microscope. We then evaluated the effects of linalool on skin penetration and migration of schistosomula and adult survival by measurement of worm burden and egg counts in Balb/C mice infected with linalool-treated cercariae. Results In the present work, 44 components were identified from the leaf extracts of C. camphora, of which linalool was the most abundant constituent. Linalool exhibited the striking molluscicidal and larvicidal effects with LC50 = 0.25 mg/L for O. hupensis and LC50 = 0.07 mg/L for cercaria of S. japonicium. After exposure to linalool, damage to the gills and hepatopancreas of the snails, and to the tegument and body-tail joint of cercariae was apparent. In addition, linalool markedly reduced the recovered schistosomulum from mouse skin after challenge infection, and therefore decreased the worm burden in infected animals, but not fecundity of female adults of the parasite. Conclusions Our findings indicated that linalool might be a novel chemotherapeutic agent against S. japonicium and the snail intermediate host.