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Angiostrongylus cantonensis (AC), which parasitizes in the brain of the non-permissive host, such as mouse and human, is an etiologic agent of eosinophilic meningitis. Excretory-secretory (ES) products play an important role in the interaction between parasites and hosts' immune responses. Inflammatory macrophages are responsible for eosinophilic meningitis induced by AC, and the soluble antigens of Angiostrongylus cantonensis fourth stage larva (AC L4), a mimic of dead AC L4, aggravate eosinophilic meningitis in AC-infected mice model via promoting alternative activation of macrophages. In this study, we investigated the key molecules in the ES products of AC L4 on macrophages and observed the relationship between metabolic reprogramming and the PI3K-Akt pathway. First, a co-culture system of macrophage and AC L4 was established to define the role of AC L4 ES products on macrophage polarization. Then, AC L4 exosome and exosome-depleted excretory-secretory products (exofree) were separated from AC L4 ES products using differential centrifugation, and their distinct roles on macrophage polarization were confirmed using qPCR and ELISA experiments. Moreover, AC L4 exofree induced alternative activation of macrophages, which is partially associated with metabolic reprogramming by the PI3K-Akt pathway. Next, lectin blot and deglycosylation assay were done, suggesting the key role of N-linked glycoproteins in exofree. Then, glycoproteomic analysis of exofree and RNA-seq analysis of exofree-treated macrophage were performed. Bi-layer PPI network analysis based on these results identified macrophage-related protein Hexa as a key molecule in inducing alternative activation of macrophages. Our results indicate a great value for research of helminth-derived immunoregulatory molecules, which might contribute to drug development for immune-related diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wan, Sun, Tang, Wang, Wu and Sun.)

On Hawai'i Island, an increase in human neuroangiostrongyliasis cases has been primarily associated with the accidental ingestion of Angiostrongylus cantonensis L3 in snails or slugs, or potentially, from larvae left behind in the slug's slime or feces. We evaluated more than 40 different treatments in vitro for their ability to kill A. cantonensis larvae with the goal of identifying a safe and effective fruit and vegetable wash in order to reduce the risk of exposure. Our evaluation of treatment lethality was carried out in two phases; initially using motility as an indicator of larval survival after treatment, followed by the development and application of a propidium iodide staining assay to document larval mortality. Treatments tested included common household products, consumer vegetable washes and agricultural crop washes. We found minimal larvicidal efficacy among consumer-grade fruit and vegetable washes, nor among botanical extracts such as those from ginger or garlic, nor acid solutions such as vinegar. Alkaline solutions, on the other hand, as well as oxidizers such as bleach and chlorine dioxide, did show larvicidal potential. Surfactants, a frequent ingredient in detergents that lowers surface tension, had variable results, but dodecylbenzene sulfonic acid as a 70% w/w solution in 2-propanol was very effective, both in terms of the speed and the thoroughness with which it killed A. cantonensis L3 nematodes. Thus, our results suggest promising directions for future investigation.

Angiostrongylus cantonensis is one of the most widespread parasites causing central nervous system (CNS) diseases in mammals. Since the mitochondrion is an essential cell organelle responsible for both physiological and pathological processes, its dysfunction might lead to inflammation and multiple disorders. In this study we aimed to investigate the changes in mitochondrial dynamics that occur in the mouse brain upon infection with A. cantonensis, using molecular biology techniques such as polymerase chain reaction (PCR), western blot analysis, transmission electron microscopy (TEM), and different staining methods. Here, we show that mouse brain infected with A. cantonensis exhibits altered mitochondrial dynamics, including fission, fusion, and biogenesis. Additionally, we demonstrate that caspases and B-cell lymphoma 2 (BCL-2) were significantly upregulated in A. cantonensis-infected brain. These results are indicative of the occurrence of apoptosis during A. cantonensis infection, which was further confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These findings suggest the change in mitochondrial dynamics in A. cantonensis-infected brain, providing another point of view on the pathogenesis of meningoencephalitis caused by A. cantonensis infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In order to elucidate the infection pathways of third stage larvae (L3) of Angiostrongylus cantonensis, we performed experiments to assess: (i) the shedding of L3 from two species of experimental veronicellid slugs drowned in water and the ratio of emerged larvae, (ii) the transmission of viable L3 from drowned terrestrial gastropods to aquatic snails, and (iii) the transmission of viable L3 between terrestrial snails. Molluscs were experimentally infected by first stage larvae (L1) of A. cantonensis. Significantly more L3 larvae were released from Veronicella cubensis than from Veronicella sloanei. Numerous L3 were observed in the muscular foot, and also in the connective tissue between internal organs. Experimental exposure of P. maculata to L3 of A. cantonensis liberated from other gastropod species led to their infection and the infectivity of larvae after intermediesis was demonstrated by infection of laboratory rats (Rattus norvegicus). The transmission of L3 was observed in three out of four experiment replications and L3 were retrieved from 6 out of 24 Subulina octona snails. The infected synanthropic molluscs represent a key component in the epidemiology of human infections by A. cantonensis. Escape of L3 larvae from bodies of dead snails or slugs and their ability to infect further gastropod hosts (intermediesis) represents a public health risk. Thus, control of molluscs living in peri-domestic environment is an essential part of prevention of human infections.

Angiostrongylus cantonensis is the leading cause of eosinophilic meningitis worldwide, with life-threatening complications if not managed correctly. Previous in vitro studies have utilized change in motility patterns of adult female worms to assess the efficacy of anthelmintics qualitatively. However, it is the third stage larvae (L3) that are infectious to humans. With differential staining using propidium iodide penetration as the indicator of death, we can distinguish between dead and live larvae. This assay has enabled us to quantify the in vitro efficacy of nine clinically established anthelmintics on A. cantonensis L3. All drugs were tested at a 1 mm concentration. Piperazine and niclosamide were ineffective in inducing larval death; however, albendazole sulfoxide, pyrantel pamoate, diethylcarbamazine, levamisole and praziquantel were effective as compared to unexposed controls (P < 0.05). Ivermectin and moxidectin did not induce significant levels of mortality, but they considerably reduced larval motility almost immediately. This study indicates the need for further in vivo studies to determine the optimal dose and time frame for post-infection treatment with anthelmintics that demonstrated efficacy.

Angiostrongylus cantonensis causes severe neurological disorders in a wide range of warm-blooded animals, including several avian species. A laboratory isolate of A. cantonensis originating from French Polynesia, genotyped as clade 2, was used to assess the effect of experimental infection in chicken and Japanese quail. Low dose groups of birds were infected orally by 100 L3 larvae, high dose groups by 1500 L3 larvae and the birds in the third group were fed three infected snails, mimicking a natural infection. Clinical signs during the first week after infection, haematology, biochemistry, gross lesions and histology findings were used to assess the pathology of the infection. Some of the infected birds showed peripheral eosinophilia, while mild neurological signs were seen in others. No larvae were observed in serial sections of the central nervous system of infected birds 1 week after infection and no major gross lesions were observed during necropsy; histopathology did not reveal lesions directly attributable to A. cantonensis infection. Our results suggest that galliform birds are not highly susceptible to A. cantonensis infection and open a question of the importance of Galliformes in endemic areas as natural pest control, lowering the number of hosts carrying the infective larvae.

Background: Angiostrongylus cantonensis is an important food-borne zoonotic parasite. Humans are non-permissive hosts, and this parasite develops into fifth-stage larvae (L5) in the brain and subarachnoid cavity and then induces eosinophilic meningitis and eosinophilic meningoencephalitis. Excretory/secretory products (ESPs) are valuable targets for the investigation of host-parasite interactions. These products contain a wide range of molecules for penetrating defensive barriers and avoiding the immune response of the host. Endoplasmic reticulum (ER) stress has been found to be associated with a wide range of parasitic infections and inflammation. ER stress can increase cell survival via the activation of downstream signalling. However, the mechanisms of ER stress in A. cantonensis infection have not yet been clarified. This study was designed to investigate the molecular mechanisms of ER stress in astrocytes after treatment with the ESPs of A. cantonensis L5., Results: The results demonstrated that A. cantonensis infection activated astrocytes in the mouse hippocampus and induced the expression of ER stress-related molecules. Next, the data showed that the expression of ER stress-related molecules and the Ca 2+ concentration were significantly increased in activated astrocytes after treatment with the ESPs of L5 of A. cantonensis. Ultimately, we found that ESPs induced GRP78 expression via the Sonic hedgehog (Shh) signalling pathway., Conclusions: These findings suggest that in astrocytes, the ESPs of A. cantonensis L5 induce ER stress and that the Shh signalling pathway plays an important role in this process.

Angiostrongylus cantonensis is a parasitic nematode dwelling in the heart and pulmonary arteries of rats, which can cause angiostrongyliasis in human by accidental infections, manifested as eosinophilic meningitis or meningoencephalitis. Cysteine proteases are the major class of endopeptidases that are expressed at a high level in A. cantonensis, which suggests it may play key roles in pathogenesis of the disease. In this study, the biological properties of the cathepsin L-like peptidase (Ac-cathL) of A. cantonensis were investigated. The Ac-cathL gene was identified from the fourth stage cDNA library of A. cantonensis, and then cloned and characterized by bioinformatics analysis and heterologous expression. The open reading frame (ORF) of Ac-cathL (1068 bp) encodes a protein of 355 amino acids with an estimated molecular weight of 58.0 kDa. Sequence analysis and multiple sequence alignment demonstrated that Ac-cathL resembles members of cathepsin L family of other parasites and mammals. Stage-dependent mRNA expression analysis showed that Ac-cathL transcripts were expressed in all stages of A. cantonensis, with the highest expression in female stage. The recombinant Ac-cathL (rAc-cathL) expressed in Escherichia coli exhibited protease activity in acidic pH as demonstrated by gelatin zymography, as well as hydrolytic activity against natural substrates, including BSA, human IgG and human fibrinogen. Immunolocalization revealed that Ac-cathL is localized in tegument of the 18 days post infection stage and uterus of the female adult stage. Therefore, these results implied that the Ac-cathL plays important roles in host tissue migration, nutrition uptake and immune evasion., Competing Interests: Declaration of Competing Interest The authors certify that there is no conflict of interest with any individual/organization for the present work., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Snails are essential to complete the life cycle of the metastrongylid nematode Angiostrongylus cantonensis, the causative agent of infections in domestic and wild animals, mainly rodents, and also of neural angiostrongyliasis or eosinophilic meningitis in humans. There are many reports of mollusks that can act as intermediate hosts of this parasite, especially freshwater snails and the African giant Achatina fulica. The terrestrial gastropod Bulimulus tenuissimus is widely distributed in Brazil and other species of the same genus occur in Brazil and other countries, overlapping regions in which there are reports of the occurrence of A. cantonensis and angiostrongyliasis. In spite of this, there are no records in the literature of this species performing the role of intermediate host to A. cantonensis. The present study analyzed the experimental infection with first-stage larvae of A. cantonensis, under laboratory conditions, of B. tenuissimus, by using histology and electron microscopy techniques. Three weeks after exposure to L1 larvae, it was possible to recover L3 larvae in small numbers from the infected snails. Developing larvae were observed in the cephalopedal mass (foot), ovotestis, and mantle tissues, being located inside a granulomatous structure composed of hemocyte infiltration, but there was no calcium or collagen deposition in these structures in significant amounts. In the third week post exposure, it was possible observe a sheath around the developing larvae. The infected snails presented reduction in the fibrous muscular tissue in the foot region, loss of the acinar organization in the digestive gland, with increase of amorphous material inside the acini and loss of epithelial pattern of nuclear organization in the acinar cells. However, the ovotestis seemed unaffected by the infection, since there was a large number of developing oocytes and spermatozoa in different stages of formation. The digestion of infected snails allows us the third-stage recovery rate of 17.25%, at 14 days post exposure to the L1. These L3 recovered from B. tenuissimus were used to infect rats experimentally, and 43 days post infection first-stage (L1) larvae of A. cantonensis were recovered from fresh feces. The results presented constituted the first report of the role of B. tenuissimus as an experimental intermediate host to A. cantonensis and shed some light on a possible problem, since the overlapping distribution of B. tenuissimus and A. cantonensis in Brazil and other countries where different species of Bulimulus occur enables the establishment and maintenance of the life cycle of this parasite in nature, with wild rodents as reservoirs, acting as a source of infection to humans, causing neural angiostrongyliasis.

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Introduction: Angiostrongylus cantonensis is a serious public health problem and is widely distributed in the country. When the parasite infects the snails, it becomes deeply embedded in their interior and accidentally, it can infect people and other mammals. Objective: To establish the geographical distribution of A. cantonensis intermediate hosts (Achatina fulica) and definitive hosts (Rattus spp.) in Ecuador from 2014 to 2017. Materials and methods: We collected 2,908 A. fulica specimens in 16 provinces using the capture method per unit of effort for 30 minutes. We captured 211 hosts of which 20 were Rattus rattus and 191 R. norvegicus. The specimens were transported to the Instituto Nacional de Salud Pública e Investigación in Guayaquil where the larvae L3 were identified and counted in the tissues of the snail, which were then inoculated in laboratory rats to reproduce the life cycle. In the rodents, the brains, hearts, lung arteries and lungs were dissected, and the parasites were identified by taxonomic morphology. Results: Of the snails harvested, 441 were positive for A. cantonensis (15.2%) and a total of 6,166 L3 larvae were found; 77 (36.5%) specimens of Rattus spp., were infected with A. cantonensis and a total of 220 parasites (L4-L5 larvae and adult worms) were collected. Conclusions: We confirmed the presence of A. cantonensis in several provinces, which ratifies the endemic nature of this zoonosis in the national territory. Rattus spp. specimens constitute the definitive hosts of the parasite, which together with the presence of intermediate hosts, indicates the zoonotic potential of this parasitic infection.

MicroRNA plays a vital role in the regulation of host-parasite interaction. In recent years, genomic and transcriptomic resources have become increasingly available for many helminths, but only a limited number of reports in this area are on the regulatory effects of host microRNAs on parasitic nematodes. In this work, we screened increased expression of host microRNAs after nematode infection from miRNA-seq data and predicted target genes by combined bioinformatics analysis and transcriptional profiling. We elucidated regulatory effects of one host miRNA on nematode infection using miRNA inhibitor and adeno-associated virus (AAV)-based TuD miRNA inhibitor. Using AAV-based TuD miRNA inhibitor, we showed that stable blockade of mmu-miR-101b-3p could alleviate the pathological damages of Angiostrongylus cantonensis, a parasitic nematode. Data from a luciferase report assay showed that mmu-miR-101b-3p targeted the extracellular superoxide dismutase 3 (Acsod3). Increased Acsod3 expression in larvae and alleviated oxidative damages were seen in the groups receiving mmu-miR-101b-3p inhibitor treatment in vitro and AAV-based TuD miRNA inhibitor injection in vivo. Results of this study demonstrate that murine miR-101b-3p inhibits the expression of antioxidant enzyme in A. cantonensis to strengthen host oxidative responses to nematodes. This work expands our knowledge of interspecies regulation of nematode gene expression by of host miRNAs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The rat lungworm Angiostrongylus cantonensis causes human eosinophilic meningitis and it is endemic in Southeast Asia, but little is known about its distribution in Laos, Cambodia and Vietnam. We conducted a multi-country survey for A. cantonensis in these countries to estimate its prevalence in snails along the Mekong River and the east coast of Vietnam. We identified Angiostrongylus species by morphological and molecular analysis. We found A. cantonensis in the invasive snail, Pomacea spp. The wide accessibility of Pomacea snails, along with their infection by A. cantonensis, indicates that this snail species could be used in surveillance for preventing outbreaks of eosinophilic meningitis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Previous studies have shown that the Sonic hedgehog (Shh) signaling pathway may reduce cell apoptosis by inhibiting oxidative stress in A. cantonensis infection. In this study, we investigated the relationship between cytokine secretion and Shh pathway activation after treatment with excretory/secretory products (ESP) of fifth-stage larval A. cantonensis (L5)., Results: The results showed that IL-1β and IL-6 levels in mouse astrocytes were increased. Moreover, ESP stimulated the protein expression of Shh pathway molecules, including Shh, Ptch, Smo and Gli-1, and induced IL-1β and IL-6 secretion. The transcription factor nuclear factor-κB (NF-κB) plays an important role in inflammation, and it regulates the expression of proinflammatory genes, including cytokines and chemokines, such as IL-1β and TNF-α. After ESP treatment, NF-κB induced IL-1β and IL-6 secretion in astrocytes by activating the Shh signaling pathway., Conclusions: Overall, the data presented in this study showed that ESP of fifth-stage larval A. cantonensis stimulates astrocyte activation and cytokine generation through NF-κB and the Shh signaling pathway.

Angiostrongylus cantonensis (A. cantonensis), a parasitic nematode, is the important neurotropic pathogen which causes human angiostrongyliasis. It has a complex life-cycle and severe parasite-host interaction in contrast to free-living nematode. Establishment of a well-suited life-cycle and in vitro cultivation of A. cantonensis in the laboratory will be one of the key techniques to elucidate the mechanism of parasite-host interaction. However, the low survival and growth rate of worms is still to be the problem. We optimized the known life-cycle of A. cantonensis in the laboratory, showing that small in size, easy to breed, and high compatibility of Biomphalaria straminea precede the common snails as an intermediate host of A. cantonensis. Furthermore, the egg hatching rate in Ham's F-12 medium reached approximately 80% using the eggs of mature female adult worms. We also demonstrated that the survival of larvae could be sustained for more than 30 days by in vitro cultivation of L1 larvae in DMEM with mixed antibiotics (100 units/mL of penicillin G potassium, 50 μg/mL of streptomycin sulfate, and 0.5 μg/mL of amphotericin B) and L3, L4, and L5 larvae in Waymouth's medium with 20% fetal calf serum and mixed antibiotics. Infective L1 and L3 larvae kept high infective rate to the snail and rat after cultivation in these media, respectively. It will provide the basis for studying on genetic manipulations for functional genes, new drug screening, and the mechanism of parasite-host interaction of parasitic nematodes.

Angiostrongylus cantonensis (A. cantonensis) is the most common infectious agent causing eosinophilic meningitis. As an important food-borne parasitic disease, angiostrongyliasis cantonensis is an emerging infectious disease which brings severe harm to central nerve system of human. Rat, one of the few permissive hosts of A. cantonensis known to date, plays an indispensable role in the worm's life cycle. However, the tolerance and adaptation of rat to A. cantonensis infection is rarely understood. In this study, we infected rats with different numbers the third stage larvae (L3) of A. cantonensis and explored their tolerance through analysis on survival curve, neurological function score, and detection of pathological damages in organs including the brain, lung, and heart of the animals. Results indicated that rats' survival condition worsens, and body weight dropped more significantly as more worms were used for infection. Death appeared in groups infected with 80 and more A. cantonesnsis per rat. Morris water maze revealed that the neurological function of rats damaged gradually with increasing infection number of A. cantonensis larvae. When the number of infected parasite exceeded 240 per animal, rats showed significant neurological impairments. Collection of A. cantonensis from rat lung after 35 days of infection implied an upper limit for worm entry, and the average length of worm was inversely proportional to the infection amount, while the ratio between female and male worms was positively related to the infection number. The degree of pulmonary and cardiac inflammation was proportional to the infection number of A. cantonensis. Meanwhile, there existed considerable amount of adult worms in rat's right atrium and right ventricle, leading to a right heart myocardial inflammation. The present study firstly reports the tolerance and adaptation of rat, a permissive host of A. cantonensis to its infection, which will not only provide accurate technical parameters for maintaining A. cantonensis life cycle under laboratory conditions but also help unveil the underlying mechanism of the distinct pathological outcomes in the permissive and non-permissive hosts with A. cantonensis infection.

Angiostrongyliasis caused by Angiostrongylus cantonensis (A. cantonensis) is an emerging food-borne parasitic disease, which refers basically to eosinophilic meningitis. Chitinase-like protein 3 (Chil3), a member of chitinase-like protein family which has chemotactic activity for eosinophils, is reported to be highly upregulated in brain of mouse infected with A. cantonensis. The mechanisms of high expression of Chil3 and the association between A. cantonensis and Chil3 are rarely reported. In order to understand the mechanism of high expression of Chil3 in A. cantonensis-infected mouse, we measured the level of Chil3 in RAW 264.7 and BV2 cell lines stimulated with soluble antigen of A. cantonensis by qPCR and ELISA. To explore the role of Chil3 in inflammation caused by A. cantonensis, we extracted and cultured brain mononuclear cells (BMNCs) and detected the eosinophil chemotactic activity of Chil3 using transwell assay and flow cytometer. Furthermore, we treated the infected mice by injection with rmChil3 and then counted the number of larvae in brains of infected mice and treated mice to examine the association between the worm and Chil3. Our results showed the soluble antigen from A. cantonensis could promote the Chil3 expression in macrophage and microglial cell lines induced by interleukin-13. In conclusion, we supposed that high expression of Chil3 enhanced by soluble antigens from A. cantonensis might be the reason of serious eosinophil infiltration in mouse brain after A. cantonensis infection.

Background: Angiostrongyliasis caused by the rat lungworm, Angiostrongylus cantonensis (A. cantonensis), has globally spread from the traditional epidemic areas. The small intestine is the site where the third-stage larvae (L3) of this worm entered the host body, and parasite proteases are involved in this process. Ac-cathB-2, a cathepsin B-like cysteine of A. cantonensis, was formerly isolated from the insoluble part of fragmentised Escherichia coli without activity. The unplanned low activity of prokaryotic expression proteins and difficulties in genetic modification hindered understanding the function of this protein., Methods: The recombinant Ac-cathB-2 was expressed and harvested from 293 T cells and the enzymatic property and the effects of processing on the activity of the recombinant protease were investigated in vitro. The expression of Ac-cathB-2 in response to external stimulation was assessed, and the function of this protease during host gut penetration was observed by using antiserum for inhibition., Results: Of the life-cycle stages studied, L3 expressed the highest level of Ac-cathB-2 gene and released the corresponding gene product from the body. The expression of this gene was rapidly upregulated after incubating L3 in small intestine homogenate of rat. Recombinant Ac-cathB-2 was harvested from 293 T cell culture medium. This protease was activated by pepsin-HCl and the enabled Ac-cathB-2 could subsequently digest laminin and fibronectin readily. Moreover, the small intestine isolated from rat was disrupted after incubating with the activated Ac-cathB-2, resulting in the detachment of epithelial cells. Antiserum treatment inhibited the hydrolytic ability of recombinant Ac-cathB-2 by 82.7 %, and also reduced the tissue penetration of activated L3 by 41.2 %. Additionally, pre-incubation of L3 with artificial gastric acid increased the number of penetrating larvae by 53.2 %, and this alteration could be partly blocked by antiserum treatment., Conclusion: We believe that Ac-cathB-2 from A. cantonensis might help the worm to penetrate the rat gut, because the protease was able to degrade the tissue components of host. Nevertheless, our results further indicated that host pepsin played a beneficial role in this process by cleaving Ac-cathB-2 for activation. Thus, Ac-cathB-2 may probably represent an important target for the control of A. cantonensis infection.

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine., (© Y. Long et al., published by EDP Sciences, 2015.)

Objective: To observe the effect of artificially construction of the life cycle of Angiostrongylus cantonensis in the laboratory condition, so as to provide the basis for the research of angiostrongyliasis., Methods: SD rats were infected orally with the third-stage larvae of A. cantonensis collected from infected Pomacea canaliculata. Six weeks after the infection, the first-stage larvae were isolated and counted from fresh feces of the rats and then were used to infect P. canaliculata. Three weeks later, the snails were dissected for counting the third-staged larvae of A. cantonensis., Results: The first-stage larvae were detected in the feces of the rats 6 weeks after the infection, and the third-staged larvae were successfully isolated after the infection of P. canaliculata., Conclusion: The animal model of the entire life cycle of A. cantonensis is successfully established in the laboratory with the infection of 50 larvae per rat.

In this study we have investigated the anatomic sites of expression and developmental expression patterns of two cathepsin B-like cysteine proteases (AC-cathB-1, -2) of Angiostrongylus cantonensis. The immunolocalization results revealed that native AC-cathBs were found present in the L1 and L3 larvae, female and male adults, and the AC-cathBs were localized mainly on the digestive tract of A. cantonensis and expressed at varied levels and in different patterns in the internal tissues according to their developmental stage. Consistent with the infective stage of L3 is a much more intense staining of AC-cathBs in the esophagus compared with the intestine. In contrast to L3, more abundant signals were located to the intestine of adults, suggesting that nutrition digestion likely to be the main function of the protease at this point. AC-cathBs fluorescent signals were present in excretory pore, excretory tube in lateral cords, and muscular esophagus of larvae, further supported the AC-cathB-1, -2 likely to be released by A. cantonensis as excretory/secretory products. Additionally, only the protein AC-cathB-2 was detected in the reproductive system, especially in the wall of vas deferens, uterus, and oviduct of the parasites, whether the AC-cathB-2 has some function in germ cells development and maturation need to be further characterized. Although the anatomic sites and expression patterns were different in larvae and adults and the corresponding function might not the same, AC-cathB-1 and -2 involved in the host-parasite interaction in addition to digestive function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Levels of calcium in the haemolymph and reserves in the shell of Biomphalaria glabrata experimentally infected by Angiostrongylus cantonensis were determined for the first time. At the same time, histochemical analyses of the digestive gland of infected and uninfected snails were performed to better understand the possible changes in metabolism of calcium in these organisms. After 1, 2 and 3 weeks of infection, the snails were dissected for collection of haemolymph and separation of tissues. The highest calcium concentrations in the haemolymph were found 2 weeks after infection, with a 39.61% increase in relation to the respective control group. However, there was a significant reduction in the concentration of this ion in the haemolymph of infected snails after 1 week of infection in relation to the uninfected specimens. In parallel, intense hypocalcification was shown in the shell of infected snails 1 and 2 weeks after infection, differing significantly in relation to the respective control groups. Morphological changes in the digestive gland of infected snails were also observed, confirming the role of this ion as an important element in the parasite encapsulation process.

Angiostrongylus cantonensis is the most common infectious agent causing eosinophilic meningitis and is present in Taiwan, Thailand and the Pacific islands. Clinical symptoms vary within different endemic regions, and their severity is probably dependent on the number of ingested parasites and the diversity among strains. The experimentally definitive host is the rat, and non-permissive hosts are certain mammals such as humans and mice. In this study, the partial gene sequences of two A. cantonensis strains isolated from five different regions in Taiwan were selected and molecularly analyzed. The internal transcribed spacer gene and cytochrome-c oxidase subunit I gene sequences of the Hualien (H) strain of A. cantonensis differed from those of the Pingtung (P) strain and the other three strains by 19% and 11%, respectively. We analyzed the infectivity, fecundity, and development of the H and P strain in rats and host pathogenicity in mice inoculated with both strains. The number of the emerged first-stage larvae, adult recovery, and average length of adults in Sprague-Dawley rats significantly differed between rats inoculated with the H and P strain. Young adult recovery, average length of young adults, eosinophil counts in the cerebrospinal fluid (CSF), glutathione peroxidase concentration, levels of reactive oxygen species as well as malondialdehyde concentration in the CSF, and the survival of mice significantly differed between BALB/c mice inoculated with the H and P strain. The H strain of A. cantonensis had lower infectivity, delayed fecundity, and poor development in rats, and caused milder pathology and lower mortality in mice than the P strain. These data clearly indicate that the H strain of A. cantonensis is a pathogenically distinct strain with lower infectivity to its definitive host, and causing mild pathogenic symptoms to its non-permissive host., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3β protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3β protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3β protein in patients with eosinophilic meningitis., Methods: In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3β protein in CSF were analyzed by western blot and an in-house 14-3-3β enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis., Results: The elevated 14-3-3β level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3β protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3β protein, it was found that the serum 14-3-3β level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3β protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3β level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3β level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0.798, p = 0.01) in the CSF of patients with eosinophilic meningitis (Spearman's correlation test)., Conclusions: The serum 14-3-3β concentrations may constitute a useful marker for blood brain barrier damage severity and follow up in patients with eosinophilic meningitis caused by A. cantonensis.

Angiostrongylus cantonensis is an important zoonotic nematode. It is the causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. However, information of this parasite at the genomic level is very limited. In the present study, the transcriptomic profiles of the fifth-stage larvae (L5) of A. cantonensis were investigated by next-generation sequencing (NGS). In the NGS database established from the larvae isolated from the brain of Sprague-Dawley rats, 31,487 unique genes with a mean length of 617 nucleotides were assembled. These genes were found to have a 46.08% significant similarity to Caenorhabditis elegans by BLASTx. They were then compared with the expressed sequence tags of 18 other nematodes, and significant matches of 36.09-59.12% were found. Among these genes, 3,338 were found to participate in 124 Kyoto Encyclopedia of Genes and Genomes pathways. These pathways included 1,514 metabolisms, 846 genetic information processing, 358 environmental information processing, 264 cellular processes, and 91 organismal systems. Analysis of 30,816 sequences with the gene ontology database indicated that their annotations included 5,656 biological processes (3,364 cellular processes, 3,061 developmental processes, and 3,191 multicellular organismal processes), 7,218 molecular functions (4,597 binding and 3,084 catalytic activities), and 4,719 cellular components (4,459 cell parts and 4,466 cells). Moreover, stress-related genes (112 heat stress and 33 oxidation stress) and genes for proteases (159) were not uncommon. This study is the first NGS-based study to set up a transcriptomic database of A. cantonensis L5. The results provide new insights into the survival, development, and host-parasite interactions of this blood-feeding nematode.

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.

Ornithine decarboxylase antizyme (OAZ), a prominent regulator of cell proliferation, DNA/RNA transformation and tumorigenesis, can bind to ornithine decarboxylase (ODC) and facilitate its degradation. Expression of OAZ requires a unique ribosomal frame shift that is regulated by levels of polyamine in the cell. In this study, we cloned an OAZ gene with the +1 ribosomal frame-shift from a fourth-stage larvae cDNA library of Angiostrongylus cantonensis. We removed one nucleotide to express the gene without polyamine. The sequence analysis showed that the deleted-mutation ornithine decarboxylase antizyme (DM-AcOAZ) contained a conservative domain related to other species OAZ. Quantitative real-time PCR revealed that DM-AcOAZ was expressed in L3 and L4 stages and adult female worms. More notably the expression level is the highest in the adult female stage. Immunohistochemical studies indicated that DM-AcOAZ was specifically localized in the uterus, oocyte and intestine in adult female worms. MTT assays showed that in DM-AcOAZ transfected HeLa cells, cell proliferation is inhibited. In conclusion, DM-AcOAZ may be a female-enriched protein and may involved in the cell proliferation in A. cantonensis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Angiostrongylus cantonensis is a metastrongyloid nematode in the family Angiostrongylidae. It is the cause of angiostrongyliasis (rat lungworm disease), which manifests as eosinophilic meningitis. First described in 1935 from rats in China, A. cantonensis was placed in the genus Parastrongylus in 1986, but most workers have not adopted this treatment. The taxonomy of A. cantonensis and related worms is largely based on adult morphology, notably of the male bursa. However, identification of infective third stage larvae is more difficult. The natural life cycle involves rats as the definitive host and snails or slugs as the intermediate host. Human infection, as accidental hosts, results in worms maturing in the brain, but dying there instead of moving back into the bloodstream, as in rats, thereby leading to eosinophilic meningitis. The disease is an emerging infectious disease; Angiostrongylus cantonensis continues to be reported in new regions beyond its native range.

The freshwater apple snail Pomacea canaliculata was introduced to Taiwan then to mainland China in the early 1980s from Argentina, its native region, for the purpose of aquaculture. Because of the lack of natural enemies and its tolerance of a wide range of environmental conditions, both its abundance and distribution have dramatically increased and it has become a harmful species to local agriculture and other native species in many areas of China. Unfortunately, the snail also acts as an intermediate host of Angiostrongylus cantonensis, and has been implicated in transfer of the parasite to people, resulting in angiostrongyliasis manifested as eosinophilic meningitis. Efforts to prevent its further spread and population expansion were initiated many years ago, including the use of chemicals and biological control agents to control the snail.

The metastrongyloid nematode genus Angiostrongylus includes 18 species, two of which are relevant from a medical standpoint, Angiostrongylus costaricensis and Angiostrongylus cantonensis. The first was described from Costa Rica in 1971 and causes abdominal angiostrongyliasis in the Americas, including in Brazil. Angiostrongylus cantonensis, first described in 1935 from Canton, China, is the causative agent of eosinophilic meningitis. The natural definitive hosts are rodents, and molluscs are the intermediate hosts. Paratenic or carrier hosts include crabs, freshwater shrimp, amphibians, flatworms, and fish. Humans become infected accidentally by ingestion of intermediate or paratenic hosts and the parasite does not complete the life cycle as it does in rats. Worms in the brain cause eosinophilic meningitis. This zoonosis, widespread in Southeast Asia and the Pacific islands, has now been reported from other regions. In the Americas there are records from the United States, Cuba, Jamaica, Brazil, Ecuador, and Haiti. In Brazil seven human cases have been reported since 2007 from the southeastern and northeastern regions. Epidemiological studies found infected specimens of Rattus norvegicus and Rattus rattus as well as many species of molluscs, including the giant African land snail, Achatina fulica, from various regions of Brazil. The spread of angiostrongyliasis is currently a matter of concern in Brazil.

Experimental infection of Biomphalaria glabrata by Angiostrongylus cantonensis induces significant changes in the concentrations of triacylglycerol and cholesterol in the hemolymph and of neutral lipids in the digestive gonad-gland (DGG) complex of the host snail. In this study, snails were dissected after 1, 2, and 3 weeks of infection to collect the hemolymph and DGG and to measure the levels of cholesterol and triacylglycerol in the hemolymph and neutral lipid fractions in the tissues. The results show that infection by this nematode resulted in a significant decrease in the concentrations of both cholesterol and triacylglycerol in the hemolymph of B. glabrata during the parasite's initial ontogenic development period. This reduction indicates the possible use of these molecules by both parasite and host not only as energy substrates but also as structural factors required during development of the parasite's larval stages. In parallel, changes in the neutral lipid profile in the DGG and lipase activity of the infected snails were observed, indicating the importance of these molecules for successful infection.

The life cycle of the nematode Angiostrongylus cantonensis involves rats as the definitive host and slugs and snails as intermediate hosts. Humans can become infected upon ingestion of intermediate or paratenic (passive carrier) hosts containing stage L3 A. cantonensis larvae. Here, we report a quantitative PCR (qPCR) assay that provides a reliable, relative measure of parasite load in intermediate hosts. Quantification of the levels of infection of intermediate hosts is critical for determining A. cantonensis intensity on the Island of Hawaii. The identification of high intensity infection 'hotspots' will allow for more effective targeted rat and slug control measures. qPCR appears more efficient and sensitive than microscopy and provides a new tool for quantification of larvae from intermediate hosts, and potentially from other sources as well., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Three cysteine protease genes, cathepsin B-like enzyme gene 1, 2 (AC-cathB-1, AC-cathB-2) and hemoglobin-type cysteine protease gene (AC-hem) were isolated and described from Angiostrongylus cantonensis adult. The deduced amino acid sequence of Ac-cathB-1 and AC-cathB-2 contain all of the conserved regions of cathepsin B. AC-cathB-2 is similar to a host intrusion-related cysteine protease B from Parelaphostrongylus tenuis, and the AC-hem shares high similarity to legumain from Haemonchus contortus. AC-cathB-1 was expressed significantly higher in L1 as compared with AC-hem, the AC-cathB-2 followed; AC-cathB-2 transcripts in L3 were found increased rapidly and obviously abundant, suggesting that AC-cathB-1 and AC-cathB-2 may play an important role in intermediate and final host invasion, separately. The cysteine protease genes were more or less expressed in adult stage excepted for AC-cathB-2. As the AC-cathB-1 and AC-hem highly expressed in adult worms, suggesting AC-hem may activate AC-cathB-1 which involved in the host invasion and feeding process., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This study was conducted to gain an understanding of the Angiostrongylus cantonensis infection status of rodent definitive host, snail intermediate host, and local residents in Guangzhou, China. A total of 430 rats were captured and 23 rats, from two species, were infected, with an average infection rate of 5.35%. A total of 795 Achatina fulica snails and 734 Pomacea canaliculata snails were collected. The average infection rates of these two species were 13.96% (111 of 795) and 1.50% (11 of 734), respectively. As for the seroprevalence of different occupations, the rate among the "general group" was significantly lower than the "occupational group." From this survey, Guangzhou is implicated to be the natural focus of Angiostrongylus cantonensis. Rattus norvegicus and Achatina fulica play important roles in spreading this nematode in Guangzhou. Residents who live in Guangzhou, especially those working in certain industries such as agriculture, food-making, and aquaculture, face a higher risk of infection.

Infective larvae of Angiostrongylus cantonensis may take up and incorporate exogenous arachidonic acid into their lipid pool. By scintillation counting, uptake and incorporation were determined to be time dependent. Arachidonic acid was mainly incorporated into phospholipid (56.8%) and neutral lipid (22.4%) pools. In the neutral lipids, 64.0% was diglyceride and 36.0% triglyceride. Phosphatidylcholine was the predominant fatty acid in the phospholipid pool. In addition to the release of leukotriene B4, the parasite was found to generate radiolabelled CO2 after incubation with [U-14C]arachidonate. Moreover, enzymatic analysis of crude extracts revealed the presence of acyl-CoA dehydrogenase (short and long chain), thiolase, enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. These findings suggest that infective larvae of A. cantonensis not only take up and incorporate exogenous arachidonic acid into their lipid pool, but may also utilize the fatty acid through a functional β-oxidation pathway.

Background: Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails., Findings: We used a loop-mediated isothermal amplification (LAMP) assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1) in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis., Conclusions: LAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions.

Eosinophilic meningitis caused by Angiostrongylus cantonensis is an emerging infectious disease. It is the most common form of human angiostrongyliasis. The diagnosis is made by clinical criteria including the presence of cerebrospinal fluid eosinophils and a history of exposure to A. cantonensis larvae, e.g., from raw freshwater snails or contaminated vegetables. Among various treatment options, corticosteroid is the only effective treatment.

The human cases of eosinophilic meningitis recently reported from Brazil have focused the attention of the public health agencies on the role the introduced snail Achatina fulica plays as hosts of the metastrongylid nematodes. Determining the potential of this snail to host and develop infective larval stages of metastrongylids in the wild and identify the species harbored by them is crucial for designing effective control measures. Here we assess if A. fulica may act as intermediate host of A. cantonensis at the peridomiciliary areas of a patient's house from state of Pernambuco (PE), who was diagnosed with eosinophilic meningitis and a history of ingesting raw molluscs. Larvae obtained from naturally infected A. fulica were orally administered to Rattus norvegicus. The worms were collected from the pulmonary artery and brain, and were morphologically characterized and compared to the Japan isolate of A. cantonensis. Adult worms and infective L(3) larvae (PE isolate) recovered from A. fulica specimens were also analyzed by polymerase chain reaction and restriction fragment length polymorphism of ITS2 region from rDNA and compared to A. cantonensis (ES isolate), A. vasorum (MG isolate) and A. costaricensis (RS isolate). The large size of the spicules (greater than those observed in other species of Angiostrongylus) and the pattern of the bursal rays agree with the original species description by Chen (1935). Furthermore, the morphology of the PE isolate was similar to that of Japan isolate. The PCR-RFLP profiles obtained were distinctive among species and no variation in patterns was detected among adult individuals from A. cantonensis isolates from PE and ES. The importance of A. fulica as an intermediate host of eosinophilic menigoencepahlitis in Brazil is emphasized., (2010 Elsevier B.V. All rights reserved.)

Aspartic proteases are a family of proteinases with catalytic aspartate residues in the active site. These enzymes have been reported to initialize the degradation of host hemoglobin in blood-feeding helminths. After identifying an expressed sequence tag representing an aspartic protease from an Angiostrongylus cantonensis young adult dataset, this sequence was found to encode a protein with a predicted molecular mass of 46 kDa. It also showed good homologies to aspartic proteases from Caenorhabditis elegans (50.7% identity), Haemonchus contortus (43.0% identity), Necator americanus (41.5% identity), Strongyloides stercoralis (35.9% identity), and Burgia malayi (29.6% identity). This putative aspartic protease was determined to be expressed in the infective larvae, young adults, and adult worms of A. cantonensis by quantitative real-time PCR. Among male worms, the expression level was determined to increase by 223.0 + or - 24.2 fold in young adults relative to the infective larvae and then decreased to 7.1 + or - 0.2 fold in adult worms. In female worms, the expression level was observed to increase by 118.5 + or - 10.1 fold in young adults and by 277.5 + or - 29.2 fold in the adults, when compared with infective larvae. These findings not only indicate that the expression level of aspartic protease gene in A. cantonensis changes with development but also has a sexual difference in individual developmental stages in the final host., (Copyright 2009 Elsevier B.V. All rights reserved.)

Prevalences and intensity of infection with Angiostrongylus cantonensis third stage larvae were examined in mollusks to determine whether they are potential intermediate hosts in eight provinces, northeast Thailand. Mollusk samples were collected from 24 reservoirs (3 reservoirs/province) in close to human cases during the previous year. Six out of 24 localities and 9 (3 new record species) out of 27 species were found with the infection. The highest intensity in infected species was found to be only one or two snails, whereas the majority had very low or no infection. The highest density was found in Pila pesmei and the lowest in Pila polita. The edible snails, P. polita, P. pesmei, and Hemiplecta distincta have the potential to transmit A. cantonensis to man. The varying density levels of larvae in infected snails may reflect observed variation in symptoms of people who traditionally eat a raw snail dish.

An infection with Angiostrongylus cantonensis, the main causative agent for human eosinophilic encephalitis, can be acquired through the consumption of the freshwater snail Pomacea canaliculata. This snail also provides a suitable model to study the developmental morphology and behavior of A. cantonensis larvae, facilitated by the snail's distinct lung structure. We used microanatomy for studying the natural appearance and behavior of A. cantonensis larvae while developing within P. canaliculata. The distribution of refractile granules in the larval body and characteristic head structures changed during the developmental cycle. Two well-developed, rod-like structures with expanded knob-like tips at the anterior part were observed under the buccal cavity as early as the late second developmental stage. A "T"-shaped structure at the anterior end and its tenacity distinguished the outer sheath from that shed during the second molting. Early first-stage larvae obtained from fresh rat feces are free moving and characterized by a coiled tail, whereas a mellifluous "Q"-movement was the behavioral trait of third-stage A. cantonensis larvae outside the host tissue. In combination, the distribution of refractive granules, distinct head features, variations in sheaths, and behavioral characteristics can be utilized for differentiation of larval stages, and for distinguishing A. cantonensis larvae from those of other free-living nematodes.

Objective: To construct the life cycle of Angiostrongylus cantonensis (A.cantonensis) in laboratory condition., Methods: SD rats were infected orally with the third-stage larvae of A.cantonensis collected from Jiangmen, Guangdong province. Six weeks after infection, the first-stage larvae were isolated from fresh feces of the rats by using Baermann funnel to infect 25 second-generation white jade snails raised in laboratory at the daily dose of 300 000 for 3 consecutive days. Three weeks later, the snails were dissected for counting the third-staged larvae of A.cantonensis, and those positive for A.cantonensis infection were fed directly to 10 fasting rats. The serum samples of the rats were then collected 2 weeks later for examination of specific antibodies using ELISA. The feces of the infected rats were examined microscopically after 6 weeks, and the brain, heart and lungs of the infected rats were dissected to observe the larvae at 3, 5, and 8 weeks, respectively., Results: The 3-stage larvae of A.cantonensis were found in the second-generation snails 3 weeks after infection. The positivity rate of serum specific antibodies was 100% in the 10 rats 2 weeks after feeding of the infected snails. The 1-stage larvae were detected in the feces of the rats 6 weeks after infection, and the fourth-stage larvae were found in the brain of the rats at 3 weeks, while adult worm and eggs were found in the heart and lungs of the infected rats at 5 and 8 weeks., Conclusion: The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.

We have developed a molecular method using PCR-direct sequencing to identify the infective 3rd juvenile stage of Angiostrongylus cantonensis, a nematode parasite of rodents that can accidentally infect humans and cause eosinophilic meningitis. We demonstrate that the 5' end of the small subunit (SSU) ribosomal (r) RNA gene is a suitable marker to identify A. cantonensis and distinguish it from other closely related Angiostrongylus species. When the SSU rRNA marker was employed on nematode populations extracted from the black slug Laevicaulis altae collected in 2 test sites in the Philippines, the infective 3rd juvenile stage A. cantonensis was detected without difficulty. The phylogenetic position of other non-angiostrongylid nematodes isolated was also determined. The molecular technique developed in this study provides a rapid and accurate method for the identification of A. cantonensis when morphological identification proves difficult or inadequate.

Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.

Objective: To compare the efficiency of three methods, lung-microscopy, tissue homogenate and enzyme digestion in the detection of Angiostrongylus cantonensis larvae from the lungs of snails., Methods: 60 Pomacea canalicular infected by the first-stage larvae of A. cantonensis were divided into 2 groups and the lung of each snail from the two groups was separated from the soft body. All the lungs were examined under microscope and larval nodes were counted. Each lung from one group was ground and that from the other was artificially digested by enzyme, the number of larvae in each lung was recorded. The efficiency of three methods was compared. Enzyme digestion was also used to estimate number of larvae in lung and in other body parts., Results: By using enzyme digestion as the standard method, the detection rate of lung-microscopy, tissue homogenate and enzyme digestion was 96.7%, 93.4% and 100% respectively (chi2=2.069, P>0.05), while the lung-microscopy was significantly faster (Z=4.782, P<0.01). The number of larvae in snail lung was positively correlated with that in other part (r=0.847, P<0.01)., Conclusions: The lung-microscopy in larvae detection is similarly efficient to the other two methods but faster, which is therefore more suitable for snail screening in the field.