Intermediate Hosts of Angiostrongylus cantonensis in Tenerife,Spain

The nematode Angiostrongylus cantonensis is the causative agent of human angiostrongyliasis, the main clinical manifestation of which is eosinophilic meningitis. Although this parasite has been found recently in its definitive rat host in Tenerife (Canary Islands, Spain), showing a widespread distribution over the north-east part of the island, there are no available data regarding which snail and/or slug species are acting as intermediate hosts on this island. Consequently, the objective of this work was to determine the possible role of three mollusc species, Plutonia lamarckii, Cornu aspersum and Theba pisana, as intermediate hosts of A. cantonensis in Tenerife. Between 2011 and 2014, 233 molluscs were collected from five biotopes where rats had been found previously to harbor either adult worms or antibodies against A. cantonensis, and the identification was carried out on the basis of morphological features and a LAMP technique. The prevalence of A. cantonensis larvae in the mollusc samples, based on morphological identification, was 19.3%, whereas 59 out of the 98 individuals (60.2%) analyzed by LAMP were positive. Positive results were obtained for the three mollusc species analyzed and two of the positive samples, both obtained from P. lamarckii, were confirmed as positive by 18S rRNA and ITS1 PCR. Sequence analysis of 18S rRNA PCR products showed 100% similarity with previously published A. cantonensis sequences. These results may be relevant from a public health point of view, since all the biotopes from which the samples were obtained were in inhabited areas or areas with human activity, but it is also important from the perspective of a possible transmission to other accidental hosts, such as dogs and horses, animals that are present in some of the areas analyzed.     

Invasive Snails and an Emerging Infectious Disease: Results from the First National Survey on Angiostrongylus cantonensis in China

Background

Eosinophilic meningitis (angiostrongyliasis) caused by Angiostrongylus cantonensis is emerging in mainland China. However, the distribution of A. cantonensis and its intermediate host snails, and the role of two invasive snail species in the emergence of angiostrongyliasis, are not well understood.

Methodology/Principal Findings

A national survey pertaining to A. cantonensis was carried out using a grid sampling approach (spatial resolution: 40×40 km). One village per grid cell was randomly selected from a 5% random sample of grid cells located in areas where the presence of the intermediate host snail Pomacea canaliculata had been predicted based on a degree-day model. Potential intermediate hosts of A. cantonensis were collected in the field, restaurants, markets and snail farms, and examined for infection. The infection prevalence among intermediate host snails was estimated, and the prevalence of A. cantonensis within P. canaliculata was displayed on a map, and predicted for non-sampled locations. It was confirmed that P. canaliculata and Achatina fulica were the predominant intermediate hosts of A. cantonensis in China, and these snails were found to be well established in 11 and six provinces, respectively. Infected snails of either species were found in seven provinces, closely matching the endemic area of A. cantonensis. Infected snails were also found in markets and restaurants. Two clusters of A. cantonensis–infected P. canaliculata were predicted in Fujian and Guangxi provinces.

Conclusions/Significance

The first national survey in China revealed a wide distribution of A. cantonensis and two invasive snail species, indicating that a considerable number of people are at risk of angiostrongyliasis. Health education, rigorous food inspection and surveillance are all needed to prevent recurrent angiostrongyliasis outbreaks.     

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%.     

PCR-Based Detection of Angiostrongylus cantonensis in Tissue and Mucus Secretions from Molluscan Hosts

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.     

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.     

Human Angiostrongyliasis Outbreak in Dali, China

Background

Several angiostrongyliasis outbreaks have been reported in recent years but the disease continues to be neglected in public health circles. We describe an outbreak in Dali, southwest China in order to highlight some key problems for the control of this helminth infection.

Methodology/Principal Findings

All available medical records of suspected angiostrongyliasis patients visiting hospitals in Dali in the period 1 October 2007–31 March 2008 were reviewed, and tentative diagnoses of varying strengths were reached according to given sets of criteria. Snails collected from local markets, restaurants and natural habitats were also screened for the presence of Angiostrongylus cantonensis. A total of 33 patients met criteria for infection, and 11 among them were classified as clinically confirmed. An additional eight patients were identified through a surveillance system put in operation in response to the outbreak. The epidemic lasted for 8 months with its peak in February 2008. Of the 33 patients, 97.0% complained of severe headache. 84.8% patients had high eosinophil cell counts either in the peripheral blood or in cerebrospinal fluid (CSF). Three-quarters of the patients were treated with a combination of albendazole and corticosteroids, resulting in significantly improved overall conditions. Twenty-two patients reported the consumption of raw or undercooked snails prior to the onset of the symptoms, and approximately 1.0% of the Pomacea canaliculata snails on sale were found to be infected with A. cantonensis. The snails were also found in certain habitats around Dali but no parasites were detected in these populations.

Conclusions/Significance

The import and sale of infected P. canaliculata is the likely trigger for this angiostrongyliasis outbreak. Awareness of angiostrongyliasis must be raised, and standardized diagnosis and treatment are needed in order to provide clinicians with a guide to address this disease. Health education campaigns could limit the risk, and a hospital-based surveillance system should be established in order to detect future outbreaks.     

Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.     

Comparative studies on the life history of Angiostrongylus mackerrasae Bhaibulaya, 1968 and Angiostrongylus cantonensis (Chen, 1935)

The life history of A. mackerrasae was found to differ from that of A. cantonensis as follows: (1) the moulting times of A. cantonensis in the definitive host occurred a few days earlier than those of A. mackerrasae; (2) the growth rate of A. cantonensis was more rapid than that of A. mackerrasae. However, there were no differences in the migratory pattern of the third-stage larvae of both species in experimentally-infected definitive hosts. It is concluded that Mackerras & Sandars (1955) described the life history of A. mackerrasae and not A. cantonensis.     

Comprehensive Review of Ocular Angiostrongyliasis with Special Reference to Optic Neuritis

Angiostrongyliasis, caused by Angiostrongylus cantonensis infection, is a food-borne parasitic disease. Its larvae evoke eosinophilic inflammation in the central nervous system, but can also cause pathological changes in the eyes. Among ocular angiostrongyliasis cases, the incidence of optic neuritis is low and only few sporadic reports exist. Some patients with optic neuritis developed obvious hypopsia or even vision loss, which would seriously influence the quality of life of patients. Prompt treatment of optic neuritis caused by A. cantonensis is the key factor for minimizing the incidence of serious complications of this disease. In this review, we first provide a comprehensive overview of ocular angiostrongyliasis, and then focus on the clinical features of optic neuritis caused by A. cantonensis.     

Release of Lungworm Larvae from Snails in the Environment: Potential for Alternative Transmission Pathways

Background

Gastropod-borne parasites may cause debilitating clinical conditions in animals and humans following the consumption of infected intermediate or paratenic hosts. However, the ingestion of fresh vegetables contaminated by snail mucus and/or water has also been proposed as a source of the infection for some zoonotic metastrongyloids (e.g., Angiostrongylus cantonensis). In the meantime, the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior are increasingly spreading among cat populations, along with their gastropod intermediate hosts. The aim of this study was to assess the potential of alternative transmission pathways for A. abstrusus and T. brevior L3 via the mucus of infected Helix aspersa snails and the water where gastropods died. In addition, the histological examination of snail specimens provided information on the larval localization and inflammatory reactions in the intermediate host.

Methodology/Principal Findings

Twenty-four specimens of H. aspersa received ~500 L1 of A. abstrusus and T. brevior, and were assigned to six study groups. Snails were subjected to different mechanical and chemical stimuli throughout 20 days in order to elicit the production of mucus. At the end of the study, gastropods were submerged in tap water and the sediment was observed for lungworm larvae for three consecutive days. Finally, snails were artificially digested and recovered larvae were counted and morphologically and molecularly identified. The anatomical localization of A. abstrusus and T. brevior larvae within snail tissues was investigated by histology. L3 were detected in the snail mucus (i.e., 37 A. abstrusus and 19 T. brevior) and in the sediment of submerged specimens (172 A. abstrusus and 39 T. brevior). Following the artificial digestion of H. aspersa snails, a mean number of 127.8 A. abstrusus and 60.3 T. brevior larvae were recovered. The number of snail sections positive for A. abstrusus was higher than those for T. brevior.

Conclusions

Results of this study indicate that A. abstrusus and T. brevior infective L3 are shed in the mucus of H. aspersa or in water where infected gastropods had died submerged. Both elimination pathways may represent alternative route(s) of environmental contamination and source of the infection for these nematodes under field conditions and may significantly affect the epidemiology of feline lungworms. Considering that snails may act as intermediate hosts for other metastrongyloid species, the environmental contamination by mucus-released larvae is discussed in a broader context.     

Acetylcholinesterase levels in Angiostrongylus cantonensis in relation to the immune response in rats

Beaver J. P. and Dobson C. 1978. Acetylcholinesterase levels in Angiostrongylus cantonensis in relation to the immune response in rats. International Journal for Parasitology8: 9–13. Angiostrongylus cantonensis larvae and adult nematodes synthesize three acetylcholinesterase (AChE) isozymes. The K_m of this isozyme complex changes with the development and migrations of the parasite in the rat host. The levels of parasite AChE changed as the development of A. cantonensis progressed; increasing quantities of AChE were found in young adult A. cantonensis from the brain of rats. After migration to the pulmonary arteries, the quantity of AChE in the parasite was reduced and continued to decline in the aging parasite. Anti-A. cantonensis antibody inhibited parasite AChE activity; this inhibition of the parasite AChE activity changed at stages during development of the parasite which suggested variation in parasite AChE isozyme levels. Haemagglutinating anti-A. cantonensis antibody appeared in the serum of infected rats when the parasites commenced to lay eggs and increased in titre thereafter until 103 days after infection.     

First record of a nematode Metastrongyloidea (Aelurostrongylus abstrusus larvae) in Achatina (Lissachatina) fulica (Mollusca, Achatinidae) in Brazil

Achatina (Lissachatina) fulica was introduced in Brazil in the 1980s for commercial purposes (“escargot” farming) and nowadays, mainly by human activity, it is widespread in at least 23 out of 26 Brazilian states and Brasília, including the Amazonian region and natural reserves, where besides a general nuisance for people it is a pest and also a public health concern, since it is one of the natural intermediate host of Angiostrongylus cantonensis, ethiological agent of the meningoencephalitis in Asia. As Brazil is experiencing the explosive phase of the invasion, the Laboratório de Malacologia do Instituto Oswaldo Cruz/Fiocruz has been receiving samples of these molluscs for identification and search for Angiostrongylus cantonensis and Angiostrongylus costaricensis larvae. While examining samples of A. fulica different nematode larvae were obtained, including Aelurostrongylus, whose different species are parasites of felids, dogs, primates, and badger. Morphological and morphometric analyses presented herein indicated the species Aelurostrongylus abstrusus, as well as the occurrence of other nematode larvae (Strongyluris-like) found in the interior of the pallial cavity of A. fulica. This is the first report in Brazil of the development of A. abstrusus infective larvae in A. fulica evidencing the veterinary importance of this mollusc in the transmission of A. abstrusus to domestic cats. Since the spread of A. fulica is pointed out in the literature as one of the main causative spread of the meningoencephalitis caused by A. cantonensis the authors emphasize the need of sanitary vigilance of snails and rats from vulnerable areas for A. cantonensis introduction as the port side areas.     

Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis

Background  

Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis.     

Messages in the mucus: Selection of land snail resting sites based on the presence of mucus from conspecifics and other species

Mucus is applied to substrates as snails move and when they rest. Beyond the use of mucus in movement and adhesion, the wide array of chemicals in mucus allows for supplemental use in communication. This communication is apparent in trail-following behavior. This study addresses the potential communication via mucus in selecting resting sites in a laboratory assay. In a series of six experiments, individuals of Cornu aspersum (which often rest on above-ground surfaces) were placed into lidded plastic containers with three or four sections swabbed with water (the control) or mucus, which included the test snail's own mucus, mucus from conspecifics from the same or a different container holding snails from the same lab colony, or from a different colony, and mucus from three additional snail species. The resting location was recorded on the following day. In two additional experiments, the facultative predator Rumina decollata was tested against conspecific and other snails' mucus. Individuals of C. aspersum preferentially rested in areas with the mucus of familiar conspecifics over mucus of unfamiliar conspecifics, a behavior that may contribute to homing and aggregation behaviors. There was evidence that juveniles may be less selective than adults. The mucus of only one of the three other species was avoided by individuals of C. aspersum. Unlike individuals of C. aspersum, individuals of the predator R. decollata avoided the mucus of conspecifics, which may be associated with less need for mate-finding in this self-fertilizing species. Individuals of R. decollata did not select among potential prey species.     

Matrix Metalloproteinase-9 Leads to Claudin-5 Degradation via the NF-κB Pathway in BALB/c Mice with Eosinophilic Meningoencephalitis Caused by Angiostrongylus cantonensis

The epithelial barrier regulates the movement of ions, macromolecules, immune cells and pathogens. The objective of this study was to investigate the role of the matrix metalloproteinase (MMP)-9 in the degradation of tight junction protein during infection with rat nematode lungworm Angiostrongylus cantonensis. The results showed that phosphorylation of IκB and NF-κB was increased in mice with eosinophilic meningoencephalitis. Treatment with MG132 reduced the phosphorylation of NF-κB and the activity of MMP-9, indicating upregulation of MMP-9 through the NF-κB signaling pathway. Claudin-5 was reduced in the brain but elevated in the cerebrospinal fluid (CSF), implying that A. cantonensis infection caused tight junction breakdown and led to claudin-5 release into the CSF. Degradation of claudin-5 coincided with alteration of the blood-CSF barrier permeability and treatment with the MMP inhibitor GM6001 attenuated the degradation of claudin-5. These results suggested that degradation of claudin-5 was caused by MMP-9 in angiostrongyliasis meningoencephalitis. Claudin-5 could be used for the pathophysiologic evaluation of the blood-CSF barrier breakdown and tight junction disruption after infection with A. cantonensis.     

Comparative host reaction to Angiostrongylus malaysiensis (Nematoda) in three species of snails

Infection with Angiostrongylus malaysiensis was studied by means of tissue squash preparations and histological sections in three species of Malaysian aquatic snails: Lymnaea rubiginosa, Physastra sumatrana, and Melanoides tuberculata. Development to the third stage occurred normally in L. rubiginosa. The biphasic tissue reaction in this snail was similar to that described in Biomphalaria glabrata infected with A. cantonensis. However, the final architecture of the granuloma differed in the two hosts with the cells in the L. rubiginosa granuloma often appearing more vacuolated and less flattened. Nematode development in P. sumatrana was abnormal, with larvae beyond the early first stage occurring in only 21% of infected snails. The highly variable cellular reactions against larvae in P. sumatrana, even within the same snail, probably reflect the differing degrees of maturation attained by individual larvae. Except in one case, no infection of M. tuberculata occurred, presumably as a result of its impenetrable epithelia.     

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.     

Host-parasite relationship of Angiostrongylus cantonensis in Lymnaea palustris. III. Oxygen uptake of Lymnaea palustris.

Oxygen uptake rates by Lymnaea palustris infected with Angiostrongylus cantonensis and by noninfected controls were determined at 25, 30, and 35 C over 34-day periods. Significant decreases in oxygen uptake were noted from one day to the next for both infected and noninfected groups of snails. At 25 and 30 C groups of infected snails found to have high average larval recoveries frequently had significantly higher oxygen uptake rates than for corresponding groups of noninfected snails. At 35 C readings were less reliable due to an increase in mortality for both infected and noninfected snails. Respiratory rates were not significantly altered by increasing the temperature from 25 to 30 and 35 C.     

Eosinophilic meningitis outbreak related to religious practice

Three patients with eosinophilic meningitis (EoM) were investigated in two hospitals in Porto Alegre, Southern Brazil. These patients had a common exposure after the ingestion of raw mollusks in a religious ritual. Two of them had an uncommon presentation with intense lower distal extremities pain and small fiber neuropathy as defined by an electroneuromyography (ENMG) study. All three patients were positive for Angiostrongylus cantonensis serology and recovered after antihelminthic and anti-inflammatory treatment. Increased awareness of A. cantonensis infection is important to avoid new infections and to improved recognition and handling of cerebral angiostrongyliasis.     

Angiostrongylus cantonensis infection in molluscs in the municipality of S?o Gon?alo,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 cantonensis in 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 byAchatina 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. cantonensisin 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. similariswas 24.6%. A. fulica was the most important intermediary host of A. cantonensis in the studied area andB. similaris was secondary in importance for A. cantonensis transmission dynamics.