Angiostrongylus Cantonensis-Conditioned Culture Medium Induces Myelin Basic Protein Alterations via Erk1/2 and NF-κB Activation in Rat RSC96 Schwann Cells.

Eating of excessive raw or undercooked environmental snails produces angiostrongyliasis demyelination caused by Angiostrongylus cantonensis (A. cantonensis). The aim of this study was to investigate the association between extracellular signal-regulated kinase (Erk)1/2-nuclear factor (NF)-κB pathway and myelin basic protein (MBP) expression in RSC96 Schwann cells treated with A. cantonensis-conditioned culture medium, which was prepared by culturing the third-stage (L3) nematode larvae in DMEM for 72 h. The supernatants were collected and filtered before use. Our results showed that MBP was produced in the RSC96 cells at 16 h to 48 h post-stimulation (PS). Phosphorylated (p)-NF-κB levels were significantly increased from 8 h to 48 h PS, as were the p-Erk1/2 levels at the same time points. Additionally, expression of p-NF-κB and MBP was significantly decreased by treatment with QNZ, an NF-κB inhibitor. Treatment with PD98059, an Erk kinase inhibitor, efficiently reduced p-Erk1/2, p-NF-κB and MBP expression in the Schwann cells. These results suggest that A. cantonensis-conditioned culture medium induced suppression of the Erk1/2-NF-κB signaling pathway leading to reduced MBP production in RSC96 Schwann cells. Thus, inhibiting this signaling intermediate involved in MBP expression may be a potential method for controlling inflammatory development of A. cantonensis-induced MBP changes in preceded demyelination.

Angiostrongylus cantonensis Eosinophilic Meningitis in an Infant, Tennessee, USA.

Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of eosinophilic meningoencephalitis worldwide. This parasite is endemic to Southeast Asia and the Pacific Islands, and its global distribution is increasing. We report A. cantonensis meningoencephalitis in a 12-month-old boy in Tennessee, USA, who had not traveled outside of southwestern Tennessee or northwestern Mississippi.

Autochthonous Case of Eosinophilic Meningitis Caused by Angiostrongylus cantonensis, France, 2016.

We report a case of a 54-year-old Moroccan woman living in France diagnosed with eosinophilic meningitis caused by Angiostrongylus cantonensis. Diagnosis was based on clinical symptoms and confirmed by testing of serum and cerebrospinal fluid samples. Physicians should consider the risk for A. cantonensis infection outside of endemic areas.

Angiostrongylus cantonensis Meningitis and Myelitis, Texas, USA.

Infection with Angiostrongylus cantonensis roundworms is endemic in Southeast Asia and the Pacific Basin. A. cantonensis meningitis and myelitis occurred in summer 2013 in a child with no history of travel outside of Texas, USA. Angiostrongyliasis is an emerging neurotropic helminthic disease in Texas and warrants increased awareness among healthcare providers.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis contributes to the immunosuppression of mice infected with Angiostrongylus cantonensis.

BACKGROUND: Immunosuppression has been described as a consequence of brain injury and infection by different mechanisms. Angiostrongylus cantonensis can cause injury to the central nervous system and eosinophilic meningitis to human. Both T cell and B cell immunity play an essential role in the resistance of the infection. However, whether brain injury caused by A. cantonensis infection can lead to immunosuppression is not clear. Therefore, the present study sought to observe the alteration of immune responses in mice infected with A. cantonensis. METHODS: Mice were infected with 20 third-stage A. cantonensis larvae. The messenger RNA (mRNA) expression of inflammatory mediators in brain tissues was observed by qRT-PCR. Cell surface markers including CD3, CD4, CD8, CD19, B220, 7-AAD, annexin-V, IgM, AA4.1, and CD23 were evaluated by using flow cytometry. The immune functions of T and B lymphocytes were detected upon stimulation by ConA and antibody responses to a nonself antigen OVA, respectively. Activation of the hypothalamic-pituitary-adrenal axis was evaluated by analyzing the concentration of plasma corticosterone and levels of mRNA for corticotropin-releasing hormone, tyrosine hydroxylase, and c-fos. RESULTS: A. cantonensis infection results in obvious immunosuppression evidenced as progressive spleen and thymus atrophy and significant decrease in the number of lymphocyte subsets including B cells, CD3+ T cells, CD4+ T cells, and CD8+ T cells, as well as reduced T cell proliferation at 21 days post-infection and antibody reaction to exogenous protein after infection. However, the sharp decrease of splenic and thymic cells was not due to cell apoptosis but to B cell genesis cessation and impairing thymocyte development. In addition, helminthicide treatment with albendazole on infected mice at 7 days post-infection could prevent immunosuppressive symptoms. Importantly, infected mice displayed hypothalamic-pituitary-adrenal axis activation, with peak responses occurring at 16 days post-infection, and glucocorticoid receptor antagonist could partially restore the infection-induced cessation of B cell genesis. CONCLUSIONS: Brain injury caused by A. cantonensis infection, like that of brain stroke and trauma, enhanced endogenous corticosteroid activity, resulting in peripheral immunosuppression.

Angiostrongylus cantonensis: a review of its distribution, molecular biology and clinical significance as a human pathogen.

Angiostrongylus cantonensis is a metastrongyloid nematode found widely in the Asia-Pacific region, and the aetiological agent of angiostrongyliasis; a disease characterized by eosinophilic meningitis. Rattus rats are definitive hosts of A. cantonensis, while intermediate hosts include terrestrial and aquatic molluscs. Humans are dead-end hosts that usually become infected upon ingestion of infected molluscs. A presumptive diagnosis is often made based on clinical features, a history of mollusc consumption, eosinophilic pleocytosis in cerebral spinal fluid, and advanced imaging such as computed tomography. Serological tests are available for angiostrongyliasis, though many tests are still under development. While there is no treatment consensus, therapy often includes a combination of anthelmintics and corticosteroids. Angiostrongyliasis is relatively rare, but is often associated with morbidity and sometimes mortality. Recent reports suggest the parasites' range is increasing, leading to fatalities in regions previously considered Angiostrongylus-free, and sometimes, delayed diagnosis in newly invaded regions. Increased awareness of angiostrongyliasis would facilitate rapid diagnosis and improved clinical outcomes. This paper summarizes knowledge on the parasites' life cycle, clinical aspects and epidemiology. The molecular biology of Angiostrongylus spp. is also discussed. Attention is paid to the significance of angiostrongyliasis in Australia, given the recent severe cases reported from the Sydney region.

Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.

This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

Temporal-spatial pathological changes in the brains of permissive and non-permissive hosts experimentally infected with Angiostrongylus cantonensis.

Human cerebral angiostrongyliasis becomes an emerging disease in many parts of the world. By postmortem examination, Angiostrongylus cantonensis have been reported to cause severe pathological changes in the central nervous system. The present study was designed to determine the temporal-spatial pathological changes through experimental infections and histopathological examination of permissive (SD rats) and non-permissive (ICR mice) hosts. After infecting SD rats with 25, 50, or 100 third-stage larvae (L3) and ICR mice with 25 L3, one animal from each group was sacrificed daily from day 1 to day 30 post-infection. Each rat brain was cut into six sections and mouse brain into five sections. These sections were stained with haematoxylin and eosin and examined microscopically. Eosinophilic meningitis was found to be the most commonly pathological change and occurred on day 17 post-infection in rats with 25 L3, day 9 in the 50- or 100-L3 groups, and day 12 in infected mice. Thickness of the meninges increased 9-24 folds in infected rats and 89 folds in an infected mouse on day 28. Encephalitis, congestion, perivascular cuffing, and haemorrhage were revealed in infected mice and rats with 100 L3. Fifth-stage larvae were frequently observed in the meninges but occasionally in the parenchyma. Significant correlations between meningitis and presence of larvae in the meninges were found in the three infected rat groups but not in the infected mice. The results indicate that the clinical course of A. cantonensis infection is not self-limited but becomes more severe with the intensity of infection.

Angiostrongylus cantonensis infection in molluscs in the municipality of São Gonçalo, a metropolitan area of Rio de Janeiro, Brazil: role of the invasive species Achatina fulica in parasite transmission dynamics.

The aim of this study was to analyse the infection dynamics of Angiostrongylus cantonensisin its possible intermediate hosts over two years in an urban area in the state of Rio de Janeiro where the presence ofA. cantonensis had been previously recorded in molluscs. Four of the seven mollusc species found in the study were exotic.Bradybaena similaris was the most abundant, followed by Achatina fulica, Streptaxis sp., Subulina octona, Bulimulus tenuissimus, Sarasinula linguaeformis and Leptinaria unilamellata. Only A. fulica and B. similaris were parasitised by A. cantonensis and both presented co-infection with other helminths. The prevalence of A. cantonensis in A. fulica was more than 50% throughout the study. There was an inverse correlation between the population size ofA. fulica and the prevalence of A. cantonensis and abundance of the latter was negatively related to rainfall. The overall prevalence of A. cantonensis in B. similaris was 24.6%. A. fulica was the most important intermediary host of A. cantonensis in the studied area and B. similaris was secondary in importance for A. cantonensis transmission dynamics.

Interleukin 33 mediates type 2 immunity and inflammation in the central nervous system of mice infected with Angiostrongylus cantonensis.

Angiostrongylus cantonensis can induce central nervous system (CNS) injury and cause human eosinophilic meningitis. The CNS has been found to have high expression of interleukin 33 (IL-33), which promotes the pathogenesis of T-helper 2 (Th2)-related disease. Given the predominantly type 2 response induced by A. cantonensis-infected mice and human, it is likely that IL-33 may play a role in aiding this process. We report here that IL-33 protein and ST2L messenger RNA (mRNA) transcripts in the brains were upregulated during A. cantonensis infection and that both splenocytes and brain mononuclear cells became IL-33 responsive and produced interleukin 5 and interleukin 13. Furthermore, administration of IL-33 to A. cantonensis-infected mice enhanced ST2L expression and cytokine production. Interestingly, brain IL-33 protein and ST2L mRNA levels were elevated 14-21 days after infection in BALB/c mice, compared with C57BL/6 mice. Thus, our data indicate that IL-33 produced in the brain may function as an inflammatory mediator in eosinophilic meningitis induced by A. cantonensis.

Effects of infection by larvae of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) on the lipid metabolism of the experimental intermediate host Biomphalaria glabrata (Mollusca: Gastropoda).

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.

Molecular cloning, expression, and characterization of a putative activation-associated secreted protein from Angiostrongylus cantonensis.

Activation-associated secreted protein (ASP) had been found in many helminthes, which was associated with pathogenesis and stage transition. A complementary DNA (cDNA) sequence encoding a putative two-domain ASP was obtained from an Angiostrongylus cantonensis fourth-stage larvae cDNA library, which we designated as AgASP. The cDNA of AgASP contains an open reading frame encoding 424 amino acids, the first 19 residues being a putative secretion signal. The expression pattern of this protein was investigated by real-time polymerase chain reaction and Western blot. We found that this protein expressed most highly in the brain-stage larvae (Lbr) of this parasite and existed in the excretory/secretory products of this stage. Immunofluorescence showed it existed in the lumen of the Lbr. The recombinant protein can be recognized by the infection sera from mice (nonpermissive host), while it cannot be recognized by infection sera from rats (permissive host). The infiltration of neutrophils in infected nonpermissive host can be lessened by immunizing this host with this protein (immunized vs control group, 13.7 ± 10.2 vs 65.5 ± 19.2). These findings suggest that this protein plays a role in the pathogenesis of human angiostrongyliasis and is worthy of further study.

Ym1, an eosinophilic chemotactic factor, participates in the brain inflammation induced by Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis is an emerging zoonotic pathogen that has caused hundreds of cases of human angiostrongyliasis worldwide. The larva in nonpermissive hosts cannot develop into an adult worm and can cause eosinophilic meningitis and ocular angiostrongyliasis. The mechanism of brain inflammation caused by the worm remains poorly defined. According to previous data of GeneChip, Ym1 in the brain of mice 21 days after infection with A. cantonensis was highly upregulated to over 7,300 times than the untreated mice. Ym1 is an eosinophilic chemotactic factor with the alternative names of chitinase-3-like protein 3, eosinophil chemotactic cytokine, and ECF-L. Ym1 displays chemotactic activity for T lymphocytes, bone marrow cells, and eosinophils and may favor inflammatory responses induced by parasitic infections and allergy. It has been reported that Ym1 is synthesized and secreted by activated macrophages during parasitic infection (Chang et al., J Biol Chem 276(20):17497-17506, 2001). In the brain, microglia are alternatively activated macrophage-derived cells which are the key immune cells in central nervous system inflammation. To explore the role of Ym1 in inflammation caused by A. cantonensis-infected mice, we examined the levels of Ym1 in the sera and cerebrospinal fluid (CSF) of the infected animals, followed by detection of the mRNA expression level of Ym1 in various organs including the brain, lung, liver, spleen, and kidney and of the cytokines IL-5 and IL-13 in the brain of the infected mice with or without intraperitoneal injection of minocycline (an inhibitor of microglial activation) by real-time reserve transcription PCR. Furthermore, immunolocalization of Ym1 in the brains of the infected mice was observed by using a fluorescence microscope. Our results showed that Ym1 was most highly expressed in the brains and CSF of the infected mice along with the process of inflammation. The antibody localized Ym1 to the microglia in the brain of the mice in both infection and minocycline + infection groups. And as in the brain, the mRNA level of Ym1 changed more obviously than IL-5 and IL-13. The study implies that Ym1 might serve as an alternative potential pathological marker which is detected not only in the sera and CSF but also in the brains of the infected mice and Ym1 secreted by microglia might be involved in eosinophilic meningitis and meningoencephalitis caused by A. cantonensis infection.

Severe form of radiculo - myelo - neuropathy with meningo - encephalitis secondary to Angiostrongylus cantonensis infection: unusual corpus callosal lesions and serial magnetic resonance imaging findings.

A 43-year-old man presented with the symptoms of recurrent lower abdominal pain, malaise and loss of appetite of 3-week duration, followed by acute onset of generalized paresthesias, fever and headache which progressed over few days to quadriparesis, altered sensorium, urinary and fecal incontinence. He had consumed raw tongue, liver, gall bladder and testicles of monitor lizard (Varanus bengalensis). Blood picture showed eosinophilia and cerebrospinal fluid (CSF) analysis revealed elevated protein and eosinophilia. Serum and CSF serology was positive for angiostrongyliasis. Magnetic resonance imaging showed focal hyperintense lesions in the corpus callosum and brainstem and an enhancing lesion in the cerebellum. Post-contrast T1-weighted axial images of spine showed evidence of cervical cord hyperintense lesions and root enhancement. Susceptibility weighted images/phase images showed unusual feature of multiple hemorrhagic lesions in the posterior fossa and supratentorial areas. Diffusion showed no restriction of corpus callosal lesions. Patient was treated with the high dose parenteral steroids with albendazole and at 6-month follow-up and had a remarkable recovery.

The use of albendazole and diammonium glycyrrhizinate in the treatment of eosinophilic meningitis in mice infected with Angiostrongylus cantonensis.

Angiostrongylus cantonensis (A. cantonensis) infection causes eosinophilic meningitis in humans. Eosinophilia and a Th2-type immune response are the crucial immune mechanisms for eosinophilic meningitis. CD4+CD25+ regulatory T cells (Treg) are involved in the pathogenesis of A. cantonensis. Diammonium glycyrrhizinate (DG) is a compound related to glycyrrhizin (GL), a triterpene glycoside extracted from liquorice root. We investigated the curative effects and probable mechanisms of therapy involving a combination of albendazole and DG in BALB/c mice infected with A. cantonensis, and compared these with therapy involving albendazole and dexamethasone. We analysed survival time, body weight, signs, eosinophil numbers, immunoglobulin E (IgE), interleukin-5 (IL-5), and eotaxin concentrations, numbers and Foxp3 expression of CD4+CD25+ Treg, worm recovery and histopathology. The present results demonstrated that the combination of albendazole and DG could increase survival time more efficiently and relieve neurological dysfunction; decrease weight loss, eosinophil numbers, concentrations of IgE, IL-5 and eotaxin, the number and expression of Foxp3 of CD4+CD25+ Treg; and improve worm recovery and histopathology changes in treated animals, compared with the combination of albendazole and dexamethasone. The observations presented here suggest that the albendazole and dexamethasone combination could be replaced by the combination of albendazole and DG.

Human Angiostrongylus cantonensis: an update.

Angiostrongylus cantonensis was first discovered in 1935 and has become an important emerging pathogen causing human angiostrongyliasis. Major outbreaks of human angiostrongyliasis have been reported in endemic regions. Thousands of cases of human angiostrongyliasis have been documented worldwide. A. cantonensis has spread from its traditional endemic regions of the Pacific islands and Southeast Asia to the American continent including the USA, Caribbean islands and Brazil. Humans acquire A. cantonensis by consumption of raw or undercooked intermediate snail hosts or paratenic hosts. The main clinical manifestations of human angiostrongyliasis are eosinophilic meningitis and ocular angiostrongyliasis. The treatment of this disease includes supportive treatment, corticosteroid therapy, and combined therapy with corticosteroids and anthelminthics. The most effective method for prevention is to persuade people not to eat raw or undercooked intermediate and paratenic hosts.

Differences of larval development and pathological changes in permissive and nonpermissive rodent hosts for Angiostrongylus cantonensis infection.

Angiostrongylus cantonensis is a neurotrophic and pulmonary parasite which causes severe neuropathological damages by invading and developing in the central nervous system (CNS). Nonpermissive host with A. cantonensis infection appeared to have more serious neurologic symptoms, and there is still not much knowledge about the host-parasite interrelationship in different hosts. We investigated and compared the larval size, recovery rate, distribution, and the severity of pathologic injuries in the CNS of both permissive host (e.g., rats) and nonpermissive hosts (e.g., mice). In present study, mice infected with A. cantonensis showed higher worm recovery rate in late-stage infection and smaller size of intracranial larvae as compared to the infected rats. Intracranial larvae mainly aggregated on cerebral surface of infected rats but on surface of cerebellum and brainstem in mice. Hemorrhage and tissue edema on brain surface caused by worm migration appeared earlier and severer in infected mice than in rats. Neuropathological examination revealed that injuries induced by A. cantonensis in brain parenchyma included hemorrhage, vascular dilatation, focal necrosis with neuronal loss, and infiltration of inflammatory cells. In the comparison of these pathological changes in rats and mice, infected mice suffered more serious injuries and provoked more intense inflammatory response as compared to infected rats. All these morphological evidences indicate that larval development was retardant in the CNS of nonpermissive host, and nonpermissive host experienced more serious pathological injuries than permissive host. It implies that the difference in innate immune response to parasite infection attribute to host specificity.

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis.

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood-brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-ß. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay.

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.

An outbreak of angiostrongyliasis in Guanging, People's Republic of China: migrants vulnerable to an emerging disease.

Angiostrongyliasis has been frequently reported from the People's Republic of China during the last decade. An outbreak of angiostrongyliasis among migrant laborers in Guangning, Guangdong Province is described here. A questionnaire was developed to collect epidemiological and clinical information about 17 migrant laborers from the Bai ethnic group in Dali, Yunnan Province. Serum samples were collected and tested by enzyme-linked immunosorbent assay. Rats and mollusks from the same area where patients had collected Pomacea canaliculata were examined for presence of Angiostrongylus cantonensis. All 17 Bai migrant laborers consumed P. canaliculata and six had meningitis 3-19 days after consumption of P. canaliculata. Headache, myalgia and fatigue were the most common symptoms. Blood samples from 5 patients were positive for antibodies to A. cantonensis. The places where the migrant laborers collected P. canaliculata were identified as endemic areas for A. cantonensis. This outbreak highlights the vulnerability of migrants to angiostrongyliasis.