Angiostrongylosis in Cerdocyon thous (crab-eating fox) and Lycalopex gymnocercus (Pampas fox) in Southern Brazil.

The objective of this study was to identify species of Angiostrongylus spp. infecting wild carnivores in Southern Brazil, as well as to describe gross and histopathological findings associated with the infection. Necropsy was conducted in 16 wild carnivores parasitized by Angiostrongylus spp. Analysed lungs revealed multifocal dark-red areas of consolidation; in one case, multifocal firm white nodules spread in all pulmonary lobes were observed. In one animal, a focally extensive area of malacia associated with haemorrhage was noted in the encephalon. Histologically, multifocal granulomatous pneumonia or bronchopneumonia, associated with eggs and larvae in blood vessels, lung interstitium, alveoli, and sometimes in bronchi and bronchioles was observed. Adult nematodes were seen within blood vessels. The lesion observed in the brain was characterized as a focally extensive area of malacia associated with gitter cells, haemorrhage, thrombosis and a free intralesional larva. Through molecular techniques, seven positive samples of Angiostrongylus cantonensis were obtained, including the brain sample, and a positive sample of Angiostrongylus vasorum-like, all in Cerdocyon thous. The positive sample for A. vasorum showed 97% similarity with sequences deposited in GenBank, suggesting a new species or subspecies of Angiostrongylus sp. Infection of Lycalopex gymnocercus by Angiostrongylus spp. was confirmed by histological evaluation.

Angiostrongylus mackerrasae and A. cantonensis (Nematoda: Metastrongyloidea) belong to same genetic lineage: evidence from mitochondrial protein-coding genes.

Angiostrongylus mackerrasae is a parasitic nematode of rats found in Australia. When first reported, it was referred to as A. cantonensis. Recent molecular studies, including the mitochondrial genome, indicate that it is highly similar to A. cantonensis. These studies did not include A. malaysiensis, another member of the A. cantonensis species complex, for comparison. The present study examined the genetic distance and phylogenetic relationship between the component taxa (A. cantonensis, A. mackerrasae and A. malaysiensis) of the A. cantonensis species complex, based on the 12 protein-coding genes (PCGs) of their mitochondrial genome. Both the nucleotide and amino acid sequences were analysed. Angiostrongylus mackerrasae and A. cantonensis are members of the same genetic lineage and both are genetically distinct from A. malaysiensis. The genetic distance based on concatenated nucleotide sequences of 12 mt-PCGs between A. mackerrasae and A. cantonensis from Thailand is p = 1.73%, while that between the Thai and Chinese taxa of A. cantonensis is p = 3.52%; the genetic distance between A. mackerrasae and A. cantonensis from China is p = 3.70%. The results indicate that A. mackerrasae and A. cantonensis belong to the same genetic lineage, and that A. mackerrasae may be conspecific with A. cantonensis. It remains to be resolved whether A. mackerrasae is conspecific with A. cantonensis or undergoing incipient speciation.

Cryptic lineage diversity in the zoonotic pathogen Angiostrongylus cantonensis.

Delimitation of species is still a necessity among parasitic pathogens especially where morphological characters provide limited discernibility. Identification of cryptic lineages (independently evolving lineages that are morphologically similar) is critical as there could be lineage-specific traits that are of epidemiological importance. Angiostrongylus cantonensis is a zoonotic pathogen that can cause eosinophilic meningitis in humans. Recent reports of single marker sequence divergence hint at the potential for cryptic diversity in this lungworm. However, to definitively address if single marker divergence corresponds to independent evolving lineages, a multilocus approach is necessary. Using multilocus data, our goal was to determine if there were cryptic lineages within Thailand, a country plagued by several outbreaks and isolated cases of A. cantonensis infection. We analyzed the genetic structure of A. cantonensis samples collected from snails, Achatina fulica, across provinces in Thailand. Multilocus data (mitochondrial sequence data and 12 nuclear microsatellites) and individual based analyses were used to test for cryptic lineages. We found strong linkage disequilibrium patterns between mitochondrial haplotypes and nuclear-identified genetic clusters. There were clearly two divergent and independent clades. Moreover, within each clade, the data suggested additional substructure where individual provinces were likely to harbor unique genetic clusters. The results indicate there are at minimum two and possibly up to eight cryptic lineages within the assumed single species of A. cantonensis. Importantly, the two main clades do not show geographic affiliation and can be found in sympatry. With recent studies highlighting A. cantonensis strain diversity in pathogenicity and infectivity, it will be important to determine if these critical epidemiological traits are associated with specific lineages.

PHYLOGENY OF ANGIOSTRONGYLUS CANTONENSIS IN THAILAND BASED ON CYTOCHROME C OXIDASE SUBUNIT I GENE SEQUENCE.

Angiostrongylus cantonensis is an emerging infectious agent causing eosinophilic meningitis or meningoencephalitis in humans with clinical manifestation of severe headache. Molecular genetic studies on classification and phylogeny of A. cantonensis in Thailand are limited. This study surveyed A. cantonensis larvae prevalence in natural intermediate hosts across Thailand and analyzed their phylogenetic relationships. A total of 14,032 freshwater and land snails were collected from 19 provinces of Thailand. None of Filopaludina sp, Pomacea sp, and Cyclophorus sp were infected with Angiostrongylus larvae, whereas Achatina fulica, Cryptozona siamensis, and Megaustenia siamensis collected from Kalasin, Kamphaeng Phet, Phetchabun, Phitsanulok, and Tak Provinces were infected, with C. siamensis being the common intermediate host. Based on morphology, larvae isolated from 11 samples of these naturally infected snails preliminarily were identified as A. cantonensis. Comparison of partial nucleotide sequences of cytochrome c oxidase subunit I gene revealed that four sequences are identical to A. cantonensis haplotype ac4 from Bangkok and the other seven to that of A. cantonensis isolate AC Thai, indicating two independent lineages of A. cantonensis in Thailand.

Levels of infection with the lungworm Angiostrongylus cantonensis in terrestrial snails from Thailand, with Cryptozona siamensis as a new intermediate host.

Angiostrongylus cantonensis is primarily considered an emerging infectious agent of eosinophilic meningitis or meningoencephalitis with a worldwide distribution. Rodents and snails are important invasive hosts for transmission and expansion of A. cantonensis. The objective of this study was to investigate infection levels of A. cantonensis in snails, the most important natural intermediate host. Our study location was Mueang Kamphaeng Phet district, Kamphaeng Phet Province, and was undertaken between October and December 2012. A total of 2228 freshwater and terrestrial snails were collected, comprising 1119 Filopaludina spp., 409 Pomacea caniculata, 275 Achatina fulica and 425 Cryptozona siamensis. Angiostrongylus larvae were isolated by artificial digestion methods following Baermann's techniques. A low prevalence and intensity of A. cantonensis were observed in A. fulica, while higher numbers were found in C. siamensis. None of the Filopaludina spp. and Pomacea caniculata were infected with A. cantonensis. Molecular characterization was performed by analysing the 264 bp of cytochrome c oxidase subunit I (COI). Three COI sequences of Angiostrongylus were identical to A. cantonensis with 91-99% identity. Cryptozona siamensis has not previously been recorded as an intermediate host for A. cantonensis in Thailand. The infection of A. cantonensis identified in the natural intermediate hosts is new and important information to assist in the prevention and control of human angiostrongyliasis.

A Case of Ocular Angiostrongyliasis with Molecular Identification of the Species in Vietnam.

A 23-year-old female residing in a village of Cao Bang Province, North Vietnam, visited the Hospital of Hanoi Medical University in July 2013. She felt dim eyes and a bulge-sticking pain in her left eye for some days before visiting the hospital. In the hospital, a clinical examination, an eye endoscopy, and an operation were carried out. A nematode specimen was collected from the eye of this patient. The body of this worm was thin and long and measured 22.0×0.3 mm. It was morphologically suggested as an immature female worm of Angiostrongylus cantonensis. By a molecular method using 18S rRNA gene, this nematode was confirmed as A. cantonensis. This is the first molecular study for identification of A. cantonensis in Vietnam.

Genetic differences in the rat lungworm, Angiostrongylus cantonensis (Nematoda: Angiostrongylidae), in Thailand.

This study surveyed the genetic differences among Angiostrongylus cantonensis (A. cantonensis) using the mitochondrial cytochrome b (cytb) gene. Partial cytb sequences were determined for 91 worms from eight locations in Thailand. Using morphological techniques, the nematodes were found to be A. cantonensis. Phylogenetic analysis found two main clades, which were subdivided into four subclades (clusters). Haplotype network analysis showed that 11 distinct cytb haplotypes were also present in four groups of A. cantonensis. There was no observable relationship between the genetic differentiation of gene flow and geographical distance. This low genetic variation and geographical distribution of A. cantonensis in each location indicates a founder effect, which may have resulted from multiple independent origins, and suggests that haplotypes migrated from endemic areas via human-related activities.

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. cantonensis: 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.

Population structure of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Thailand based on PCR-RAPD markers.

Angiostrongylus cantonensis is the causative agent of angiostrongyliasis, which is widely distributed throughout the world. It can specifically infect many species of intermediate and definitive hosts. This study examined the genetic differentiation and population structure using the RAPD-PCR method of parasites obtained from 8 different geographical areas of Thailand. Based on 8 primers, high levels of genetic diversity and low levels of gene flow among populations were found. Using genetic distance and neighbor-joining dendrogram methods, A. cantonensis in Thailand could be divided into two groups with statistically significant genetic differentiation of the two populations. However, genotypic variations and haplotype relationships need to be further elucidated using other markers.

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 66kDa 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%.

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.

The genetic basis of adaptive evolution in parasitic environment from the Angiostrongylus cantonensis genome.

Angiostrongylus cantonensis (rat lungworm) is the etiological agent of angiostrongyliasis, mainly causing eosinophilic meningitis or meningoencephalitis in human. Although the biology of A. cantonensis is relatively well known, little is understood about the mechanisms of the parasite's development and survival in definitive hosts, or its adaptation to a broad range of snail intermediate hosts. Here, we generate a high-quality assembly of a well-defined laboratory strain of A. cantonensis from Guangzhou, China, by using Illumina and PacBio sequencing technologies. We undertake comparative analyses with representative helminth genomes and explore transcriptomic data throughout key developmental life-cycles of the parasite. We find that part of retrotransposons and gene families undergo multiple waves of expansions. These include extracellular superoxide dismutase (EC-SOD) and astacin-like proteases which are considered to be associated with invasion and survival of the parasite. Furthermore, these paralogs from different sub-clades based on phylogeny, have different expression patterns in the molluscan and rodent stages, suggesting divergent functions under the different parasitic environment. We also find five candidate convergent signatures in the EC-SOD proteins from flukes and one sub-clade of A. cantonensis. Additionally, genes encoding proteolytic enzymes, involved in host hemoglobin digestion, exhibit expansion in A. cantonensis as well as two other blood-feeding nematodes. Overall, we find several potential adaptive evolutionary signatures in A. cantonensis, and also in some other helminths with similar traits. The genome and transcriptomes provide a useful resource for detailed studies of A. cantonensis-host adaptation and an in-depth understanding of the global-spread of angiostrongyliasis.

The small subunit (SSU) ribosomal (r) RNA gene as a genetic marker for identifying infective 3rd juvenile stage Angiostrongylus cantonensis.

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

Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails.

Angiostrongylus cantonensis is a zoonotic nematode parasite causing human eosinophilic meningitis (or meningoencephalitis) worldwide. A closely related species, Angiostrongylus malaysiensis, might also be a human pathogen. Larvae were obtained from land snails in Lao PDR, Cambodia, Myanmar and Thailand. We sequenced two nuclear gene regions (nuclear ribosomal ITS2 and SSU rRNA) and a portion of one mitochondrial gene (COI) from these larvae. Angiostrongylus cantonensis and A. malaysiensis were identified. This is the first report of the molecular identification of the two Angiostrongylus species in Lao PDR, Cambodia and Myanmar. The regional distributions of the two species broadly overlap. Phylogenetic relationships were inferred including data from Angiostrongylus species deposited in public databases. All the gene regions we sequenced have potential value in distinguishing between species of Angiostrongylus. The COI gene exhibited the greatest intraspecific variation in the study region (five haplotypes in A. cantonensis and four in A. malaysiensis) and might be suitable for more detailed phylogeographic studies.

Complete mitochondrial genome reveals genetic diversity of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae).

Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. Earlier work on its mitochondrial genome was based on long polymerase chain reaction method. To date, only the mitogenome of the isolates from China has been studied. We report here the complete mitogenome of the Thailand isolate based on next generation sequencing and compare the genetic diversity with other isolates. The mitogenome of the Thailand isolate (13,519bp) is longer than those of the China isolates (13,497-13,502bp). Five protein-coding genes (atp6, cox1, cox2, cob, nad2) show variations in length among the isolates. The stop codon of the Thailand isolate differs from the China and Taiwan isolates in 4 genes (atp6, cob, nad2, nad6). Additionally, the Thailand isolate has 4 incomplete T stop codon compared to 3 in the China and Taiwan isolates. The control region is longer in the Thailand isolate (258bp) than the China (230-236bp) and Taiwan (237bp) isolates. The intergenic sequence between nad4 and cox1 genes in the Thailand isolate lacks 2bp (indels) at the 5'-end of the sequence as well as differs at 7 other sites compared to the China and Taiwan isolates. In the Thailand isolate, 18 tRNAs lack the entire TΨC-arm, compared to 17 in the China isolate and 16 in the Taiwan isolate. Phylogenetic analyses based on 36 mt-genes, 12 PCGs, 2 rRNA genes, 22 tRNA genes and control region all indicate closer genetic affinity between the China and Taiwan isolates compared to the Thailand isolate. Based on 36 mt-genes, the inter-isolate genetic distance varies from p=3.2% between China and Taiwan isolates to p=11.6% between Thailand and China isolates. The mitogenome will be useful for population, phylogenetics and phylogeography studies.

Neuro-angiostrongyliasis: unresolved issues.

Angiostrongylus cantonensis, the rat lungworm, probably evolved with its hosts, members of the genus Rattus and closely related species, in south-east Asia. Since its first discovery in rats in China and in a case of human infection in Taiwan, the parasite has been found to infect humans and other mammals across a wide and ever-increasing territory, which now encompasses much of south-east Asia, Melanesia, Polynesia and eastern Australia. It has also established a foothold in Africa, India, the Caribbean and south-eastern USA. This dispersal has been a direct result of human activity, and in some cases has been linked with the spread of the African giant land snail, Achatina fulica. However, this snail is not critical to the extension of the parasite's range, as numerous other indigenous molluscan species serve as adequate intermediate hosts; the importance of Achatina to the life cycle may have been over-emphasized. In Australia, the parasite is established along parts of the east coast, and the presence of an indigenous close relative, Angiostrongylus mackerrasae, suggests a long association of the parasite with its local rat hosts, a situation analogous to that of Angiostrongylus malaysiensis in south-east Asia. These three Angiostrongylus species share virtually the same life cycle, but only A. cantonensis has been confirmed to be a human pathogen.

Experimental infection of five subspecies of Oncomelania snails with Angiostrongylus cantonensis.

Five subspecies of Oncomelania snails, Oncomelania hupensis nosophora, O.h. hupensis, O.h.chiui, O.h.formosana and O.h.quadrasi, were experimentally exposed to the first stage larvae of Angiostrongylus cantonesis. The presence of third stage larvae was observed in all of the five subspecies of Oncomelania snails 20 days after infection. Infection rates of the third stage larvae of the parasite in Oncomelania snails were 38.0-40.0%. There were no differences in preferences among Oncomelania snails. The third stage larvae in Oncomelania snails almost distributed in kidney and intestine region, and most of the larvae were active and free in tissues. The distribution pattern of the larvae in Oncomelania snails was quite different from that in Achatina fulica and Ampullarium sp. These third stage larvae were ingested by rats, and developed to adults. These data suggest that Oncomelania snails may play important role when A. cantonensis will spread, and indicate the possibility of human infection with A. cantonensis.

Identification of Angiostrongylus cantonensis and other nematodes using the SSU rDNA in Achatina fulica populations of Metro Manila.

Angiostrongylus cantonensis is a parasitic nematode that causes eosinophilic meningitis in humans. Accidental infection occurs by consumption of contaminated intermediates, such as the giant African land snail, Achatina fulica. This study surveyed the presence of A. cantonensis juveniles in A. fulica populations from 12 sites in Metropolitan Manila, Philippines using the SSU rDNA. Fourteen distinct sequences from 226 nematodes were obtained; of these, two matched A. cantonensis and Ancylostoma caninum, respectively, with 100% identity. Exact identities of the remaining twelve sequences could not be determined due to low percent similarities. Of the sequenced nematodes, A. cantonensis occurred with the highest frequency (139 out of 226). Most of these (131 out of 139) were collected in just one area in Quezon City. Nematode infection of A. fulica in this area and two others from Makati and another area in Quezon City, respectively, were highest, combining for 95% of the total infection. Ancylostoma caninum, on the other hand, was detected in four different sites. A. caninum is a canine parasite, and this is the first report of the nematode in A. fulica. These results cause public health concerns as both A. cantonensis and A. caninum are zoonotic to humans.

Biology, systematics, life cycle, and distribution of Angiostrongylus cantonensis, the cause of rat lungworm disease.

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

Angiostrongylus cantonensis and rat lungworm disease in Brazil.

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