Penetration Sites and Migratory Routes of Angiostrongylus costaricensis in the Experimental Intermediate Host (Sarasinula marginata)

The intermediate hosts of Angiostrongylus costaricensis are terrestrian molluscs, mostly of the family Veronicellidae. The present work aimed at clarifying more accurately the sites of penetration and the migratory routes of A. costaricensis in the tissue slugs and at verifying the pattern of the perilarval reaction at different times of infection. Slugs were individually infected with 5,000 L1, and killed from 30 min to 30 days after infection. From 30 min up to 2 hr after infection, L1 were found within the lumen of different segments of the digestive tube having their number diminished in more advanced times after exposition until complete disappearance. After 30 min of exposition, percutaneous infection occurred, simultaneously to oral infection. Perilarval reaction was observed from 2 hr of infection around larvae in fibromuscular layer, appearing later (after 6 hr) around larvae located in the viscera. A pre-granulomatous reaction was characterized by gradative concentration of amebocytes around larvae, evolving two well-organized granulomas. In this work we confirmed the simultaneous occurrence of oral and percutaneous infections. Perilarval reaction, when very well developed, defined typical granulomatous structure, including epithelioid cell transformation. The infection also caused a systemic mobilization of amebocytes and provoked amebocyte-endothelium interactions.

Angiostrongylus costaricensis and experimental infection of Sarasinula marginata II: elimination routes

Angiostrongylus costaricensis intermediate hosts are terrestrial mollusks mostly belonging to the Veronicellidae family. In the present investigation we focused on the mechanisms of larval expulsion from Sarasinula marginata infected with A. costaricensis. Twenty-five mollusks were individually infected with 5000 L1 and sacrificed at 30 min and 1, 2, 4, 6, and 8 h post-infection and at days 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28, and 30 post-infection; the mollusks were then fixed and stained. Diverse organs involved throughout the course of the migratory routes of larvae from oral penetration on were specified and the mechanisms of larval access to the fibromuscular layer through the kidney, rectum, and vascular system were defined. The elimination of L3, derived from oral and/or cutaneous infections, appears to depend on granulomas located close to the excretory ducts of mucous cells.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Alteration in the endogenous intestinal flora of swiss webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.

Angiostrongylus costaricensis: complete redescription of the migratory pathways based on experimental Sigmodon hispidus infection

Angiostrongylus costaricensis lives in the cecal and mesenteric arteries of its vertebrate hosts, and causes an inflammatory disease in humans. To investigate unknown aspects of the abdominal angiostrogyliasis pathogenesis, infected Sigmodon hispidus were sequentially studied in different times of infection. The study revealed that L3 goes alternatively through two migratory courses during its development into an adult worm: lymphatic/venous-arterial and venous portal pathways. The former is considered the principal one, because it is used by most of the larvae. Like other metastrongylides, A. costaricensis passes over the pulmonary circulation to migrate from the lymphatic system to the arterial circulation, where they circulate during some days before reaching their definitive habitat. The oviposition by mature females began on 15th day. Eggs and L1 were detected mainly in the intestine and stomach, surrounded by inflammatory reaction constituted by macrophages, monocytes, and eosinophils. They were also spread to the lungs, mesenteric lymph nodes, pancreas, spleen, and kidneys. The larvae (L1) exhibited the centripetal capacity to invade the lymphatic and venous vessels of the intestine and mesentery. Adult worms that developed in the venous intrahepatic pathway migrated downstream to reach the mesenteric veins and laid eggs that embolized in the portal hepatic vessels.

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

First report of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Achatina fulica (Mollusca: Gastropoda) from Southeast and South Brazil

The rat lungworm Angiostrongylus cantonensis is a worldwide-distributed zoonotic nematode that can cause human eosinophilic meningoencephalitis. Here, for the first time, we report the isolation of A. cantonensis from Achatina fulica from two Brazilian states: Rio de Janeiro (specifically the municipalities of Barra do Piraí, situated at the Paraiba River Valley region and São Gonçalo, situated at the edge of Guanabara Bay) and Santa Catarina (in municipality of Joinville). The lungworms were identified by comparing morphological and morphometrical data obtained from adult worms to values obtained from experimental infections of A. cantonensis from Pernambuco, Brazil, and Akita, Japan. Only a few minor morphological differences that were determined to represent intra-specific variation were observed. This report of A. cantonensis in South and Southeast Brazil, together with the recent report of the zoonosis and parasite-infected molluscs in Northeast Brazil, provide evidence of the wide distribution of A. cantonensis in the country. The need for efforts to better understand the role of A. fulica in the transmission of meningoencephalitis in Brazil and the surveillance of molluscs and rodents, particularly in ports, is emphasized.

Angiostrongylus cantonensis (Nematode: Metastrongyloidea) in molluscs from harbour areas in Brazil

Angiostrongylus cantonensis is the most common aetiological agent of human eosinophilic meningoencephalitis. Following a report indicating the presence of this parasite in Brazil in 2007, the present study was undertaken to investigate the presence of A. cantonensis in the surrounding Brazilian port areas. In total, 30 ports were investigated and the following molluscs were identified: Achatina fulica, Belocaulus sp., Bradybaena similaris sp., Cyclodontina sp., Helix sp., Leptinaria sp., Melampus sp., Melanoides tuberculata, Phyllocaulis sp., Pomacea sp., Pseudoxychona sp., Rhinus sp., Sarasinula marginata, Streptaxis sp., Subulina octona, Succinea sp., Tomigerus sp., Wayampia sp. and specimens belonging to Limacidae and Orthalicinae. Digestion and sedimentation processes were performed and the sediments were examined. DNA was extracted from the obtained larvae and the internal transcribed spacer region 2 was analysed by polymerase chain reaction-restriction fragment length polymorphism after digestion with the endonuclease ClaI. Of the 30 ports investigated in this study, 11 contained molluscs infected with A. cantonensis larvae. The set of infected species consisted of S. octona, S. marginata, A. fulica and B. similaris. A total of 36.6% of the investigated ports were positive for A. cantonensis, indicating a wide distribution of this worm. It remains uncertain when and how A. cantonensis was introduced into South America.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

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.

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.

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

Eosinophilic meningitis (EoM) is an acute disease that affects the central nervous system. It is primarily caused by infection with the nematode Angiostrongylus cantonensis. This infection was previously restricted to certain Asian countries and the Pacific Islands, but it was first reported in Brazil in 2007. Since then, intermediate and definitive hosts infected with A. cantonensis have been identified within the urban areas of many states in Brazil, including those in the northern, northeastern, southeastern and southern regions. The goals of this review are to draw the attention of the medical community and health centres to the emergence of EoM in Brazil, to compile information about several aspects of the human infection and mode of transmission and to provide a short protocol of procedures for the diagnosis of this disease.

Brazilian Angiostrongylus cantonensis haplotypes, ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.

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 ofAngiostrongylus 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 similariswas the most abundant, followed byAchatina fulica, Streptaxissp., Subulina octona, Bulimulus tenuissimus, Sarasinula linguaeformisand Leptinaria unilamellata. Only A. fulicaand B. similariswere parasitised by A. cantonensis and both presented co-infection with other helminths. The prevalence of A. cantonensisin A. fulicawas more than 50% throughout the study. There was an inverse correlation between the population size ofA. fulicaand the prevalence of A. cantonensisand abundance of the latter was negatively related to rainfall. The overall prevalence of A. cantonensisin B. similariswas 24.6%. A. fulicawas the most important intermediary host of A. cantonensisin the studied area andB. similariswas secondary in importance for A. cantonensistransmission dynamics.

Seroprevalence of Angiostrongylus cantonensis in wild rodents from the Canary Islands

Background: Angiostrongylus cantonensis is a lungworm of rats (Muridae) that is the causative agent of human cerebral angiostrongyliasis. The life cycle of A. cantonensis involves rats and mollusks as the definitive and intermediate hosts, respectively. This study was designed to increase the knowledge about the occurrence and distribution of A. cantonensis in its definitive host in the Canary Islands, using parasitological and serological analysis in different areas and age groups.Methodology/Principal Findings: Between 2009 and 2010, 54 black rats (Rattus rattus) from Tenerife were captured from six human-inhabited areas and sera samples were obtained. The lung nematodes were identified by morphological and molecular tools as A. cantonensis. The 31-kDa glycoprotein antigen was purified from A. cantonensis adult worms by electrophoresis and electroelution. Of the 54 tested rodents, 30 showed IgG antibodies against A. cantonensis 31-kDa antigen by ELISA. Therefore, the overall seroprevalence was 55.6% (95% CI: 42.4-68). Seroprevalent rodents were found in all the 6 areas. This 31-kDa antigen was not recognized by some sera of rats infected by other helminth species (but not A. cantonensis). Seroprevalence of IgG antibodies against A. cantonensis and prevalence based on the presence of adult worms showed significant correlation (R2 = 0.954, p,0.05). Conclusions/Significance: The present results could indicate a high prevalence of A. cantonensisin Tenerife and suggest the inclusion of two new zones in the distribution area of the parasite. The commonness and wide distribution of A. cantonensis in rats implies the presence of intermediate hosts, indicating that humans may be at risk of getting infected.