Angiostrongylus cantonensis: efficacy of albendazole-dexamethasone co-therapy against infection-induced plasminogen activators and eosinophilic meningitis

Angiostrongyliasis is one of the common causes leading to eosinophilic meningitis. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) may play a role in the pathogenesis. Administration of steroid drugs has been reported to possibly relieve the symptoms of eosinophilic meningitis. This study evaluates the curative effects of albendazole-dexamethasone co-therapy on eosinophilic meningitis in BALB/c mice. Assay indicators for the therapeutic effect include worm recovery, histopathological score of meningitis, tPA, uPA, total protein, and leukocyte counts. The results show that the albendazole-dexamethasone co-therapy significantly decreased (P<0.05) these factors after treatment on day 5 post-inoculation (PI), in contrast to treatment on 15 PI. Thus, the timing of medication is important and is closely related to the anthelmintic efficacy of a drug. At the same dosage and days post-infection, the earlier administration shows better results. This study showed that albendazole-dexamethasone co-therapy is an effective approach for the treatment of parasitic meningitis.     

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.     

Apoptosis in meningoencephalitis of Angiostrongylus cantonensis-infected mice

A hallmark of eosinophilic meningoencephalitis is infiltration of leukocytes into brain parenchyma and subarachnoid space infected by Angiostrongylus cantonensis. Apoptosis, a process that eliminates useless cells and counterbalances tissue homeostasis, is important for homeostasis of the immune system. In this study, we investigated the characteristics of cell death induced in BABL/c mice infected with A. cantonensis. We observed increased expression of the apoptotic proteins, caspase-3, caspase-8, caspase-9, and cytochrome c, and decreased expression of anti-apoptotic proteins, B-cell leukemia 2 and inhibitor of apoptosis protein 1. On immunohistochemistry, apoptotic proteins were localized within the leukocytes infiltrate. A terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay to detect DNA fragmentation confirmed these observations. The infiltration of leukocytes present in the brain parenchyma and subarachnoid space in vivo may also express these apoptotic regulatory molecules, which demonstrates the capacity of these cells to undergo apoptosis.     

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.     

Association of matrix metalloproteinase-9 and Purkinje cell degeneration in mouse cerebellum caused by Angiostrongylus cantonensis

Angiostrongylosis is a neurological disorder caused by invasion of the central nervous system by developing larvae of Angiostrongylus cantonensis. Purkinje cells in infected mouse cerebellums are small and irregular with degenerative atrophy or partial loss. Ultrastructural changes in degenerative cells included enlarged vacuolar structures and swollen mitochondria within the cytoplasm. The matrix metalloproteinase-9 mRNA which is low in normal cerebellums was expressed in A. cantonensis-infected mice cerebellum prior to Purkinje cell degeneration. Matrix metalloproteinase-9 protein level and enzyme activity increased when the Purkinje cells appeared degenerated. Using immunohistochemistry, matrix metalloproteinase-9 was localised within degenerative Purkinje cells. In addition, when the specific matrix metalloproteinase inhibitor, GM6001, was added, matrix metalloproteinase-9 enzyme activity was reduced by 41.6%. The numbers of degenerative Purkinje cells increased significantly upon establishment of infection but subsided upon inhibition. These results suggested that the expression of matrix metalloproteinase-9 may be associated with degeneration of Purkinje cells in mouse cerebellum infected by A. cantonensis.     

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.     

Angiostrongylus cantonensis: induction of urokinase-type PA and degradation of type IV collagen in rat lung granulomatous fibrosis

Granuloma formation and subsequent fibrosis around Angiostrongylus cantonensis larvae in the lungs were induced experimentally in Sprague-Dawley strain rats. Casein zymogram analysis demonstrated that urokinase-type plasminogen activator (uPA) activity was increased during lung inflammation and fibrosis. Granulomatous fibrosis, type IV collagen degradation and activation of uPA occur simultaneously. Furthermore, the present study demonstrated that collagen avidly binds uPA. Immunohistochemical observations showed localization of uPA within the infiltrating leucocytes. We propose that uPA may participate in A. cantonensis-induced granulomatous fibrosis.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Changes in the reproductive biology of Biomphalaria glabrata experimentally infected with the nematode Angiostrongylus cantonensis

This study showed for the first time changes in the reproductive biology of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis. The values of all the parameters analyzed (total number of eggs, number of egg masses, number of eggs/mass, number of eggs/snail, percentage of viable eggs and galactogen content in albumen gland) changed with progressive infection. The results indicate the occurrence of partial parasitic castration of B. glabrata by A. cantonensis larvae, probably in response to the depletion of energy reserves, with no injuries to the gonadal tissues.     

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.     

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.     

Detection of antibodies to Angiostrongylus cantonensis in serum and cerebrospinal fluid of patients with eosinophilic meningitis

Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student's t-test, P less than 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF.     

Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis.

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO₄) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15–36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15–34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x_L mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.     

Eosinophilic meningitis caused by Angiostrongylus cantonensis: an emergent disease in Brazil

Eosinophilic meningitis (EoM) is an acute disease that affects the central nervoussystem. It is primarily caused by infection with the nematode Angiostrongyluscantonensis. This infection was previously restricted to certain Asiancountries and the Pacific Islands, but it was first reported in Brazil in 2007. Sincethen, intermediate and definitive hosts infected with A. cantonensishave been identified within the urban areas of many states in Brazil,including those in the northern, northeastern, southeastern and southern regions. Thegoals of this review are to draw the attention of the medical community and healthcentres to the emergence of EoM in Brazil, to compile information about severalaspects of the human infection and mode of transmission and to provide a shortprotocol of procedures for the diagnosis of this disease.     

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

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.     

Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO₄) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15–36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15–34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x_L mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.     

Angiostrongylus cantonensis: experimental study on the susceptibility of apple snails, Pomacea canaliculata compared to Pila polita

Six groups (15 snails/group) of Pomacea canaliculata and Pila polita were infected orally with 0 (control), 200, 400, 800, 1600 and 3200 first-stage Angiostrongylus cantonensis larvae (L1). The respective mean+/-SD third stage larvae (L3) worm recovery 1-month post-infection (p.i.) for P. canaliculata was 0, 1.4+/-5.42 (0.7%), 0.13+/-0.35 (0.03%), 0.07+/-0.26 (0.009%), 0.07+/-0.26 (0.004%), 0, and for P. polita 0, 64.33+/-21.38 (32.25%), 115.36+/-36.82 (28.93%), 265.33+/-90.01 (33.27%), 471.33+/-92.98 (29.60%) and 849.00+/-243.23 (26.61%). The susceptibility of A. cantonensis in P. polita was dose-dependent (p<0.001). In the three groups (nine snails/group) of P. polita given 500 L1, we studied the distribution of L3 in the internal organs (i.e., foot, head+esophagus, kidney, albumin gland, mantle, intestine, digestive gland) and found the highest density after 1, 2 and 3 months p.i. in the mantle at 29.37%, 31.09% and 37.45%. The infection rate in P. canaliculata was too low to study distribution rates.     

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.     

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P < 0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2′-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.