Immunomodulatory effect of different extracts from Angiostrongylus cantonensis on airway inflammation in an allergic asthma model.

This study aimed to evaluate the effects of early-life exposure to different extracts of Angiostrongylus cantonensis (A. cantonensis) on airway inflammation in an allergic asthma model. The total soluble extract (TE) and the soluble extracts of the digestive (AcD), reproductive (AcR), and cuticle (AcC) systems of A. cantonensis were used for immunisation before ovalbumin (OVA)-sensitisation/challenge in an OVA-induced allergic asthma model. The initial hypothesis of the study was that some soluble extract of the systems (AcD, AcR, or AcC) could be more potent to the modulation of inflammation than the TE. Our data, however, shows that immunisation with the TE is more promising because it decreased the high influx of inflammatory cells on airways and promoted an increase of interferon-γ (IFN-ɣ) and interleukin-10 (IL-10) levels. Besides this, the immunisation with the TE also led to a reduction of goblet cells and mucus overproduction in the lung tissue of asthmatic mice. We believe that the extracts have a distinct capacity to modulate the immune system, due to the TE possessing a greater variability of molecules, which together leads to control of airway inflammation. In conclusion, this is the first study to reveal that the TE of A. cantonensis adult worms has a greater potential for developing a novel therapeutic for allergic asthma.

Prevalence and distribution of Angiostrongylus cantonensis (Nematoda, Angiostrongylidae) in Achatina fulica (Mollusca, Gastropoda) in Baixada Santista, São Paulo, Brazil.

INTRODUCTION: Angiostrongylus cantonensis is causes eosinophilic meningoencephalitis in humans. Worldwide expansion of this nematode is linked to the dispersion of their hosts. This study aimed to determine the prevalence of A. cantonensis infection in Achatina fulica in the nine municipalities that make up Baixada Santista, São Paulo, Brazil. METHODS: Angiostrongylus cantonensis larvae were analyzed using optical microscopy. We performed polymerase chain reaction and restriction fragment length polymorphism using restriction endonuclease ClaI, directed to the internal transcribed spacer region 2 of A. cantonensis larval DNA. RESULTS: Of the 540 snails analyzed, 117 (21.7%) were infected by A. cantonensis. For morphological and morphometric analyses, 60 larvae were used. Second-stage larvae were, on average, 358.2µm long and 26.4µm wide, while third-stage larvae were, on average, 450µm long and 21.12µm wide. The tails of the larvae ended in a fine tip. CONCLUSIONS: All municipalities comprising Baixada Santista had A. fulica that were naturally infected with A. cantonensis. All of the observed characteristics were typical of the species.

Prevalence and distribution of angiostrongylus cantonensis (nematoda, angiostrongylidae) in achatina fulica (mollusca, gastropoda) in baixada santista, São Paulo, Brazil

ABSTRACT INTRODUCTION Angiostrongylus cantonensis is causes eosinophilic meningoencephalitis in humans. Worldwide expansion of this nematode is linked to the dispersion of their hosts. This study aimed to determine the prevalence of A. cantonensis infection in Achatina fulica in the nine municipalities that make up Baixada Santista, São Paulo, Brazil. METHODS Angiostrongylus cantonensis larvae were analyzed using optical microscopy. We performed polymerase chain reaction and restriction fragment length polymorphism using restriction endonuclease ClaI, directed to the internal transcribed spacer region 2 of A. cantonensis larval DNA. RESULTS Of the 540 snails analyzed, 117 (21.7%) were infected by A. cantonensis. For morphological and morphometric analyses, 60 larvae were used. Second-stage larvae were, on average, 358.2µm long and 26.4µm wide, while third-stage larvae were, on average, 450µm long and 21.12µm wide. The tails of the larvae ended in a fine tip. CONCLUSIONS All municipalities comprising Baixada Santista had A. fulica that were naturally infected with A. cantonensis. All of the observed characteristics were typical of the species.

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.

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.

Estudo filogenético, biológico e morfológico de isolados de Angiostrongylus cantonensis provenientes de diferentes áreas geográficas do Brasil / Phylogenetic, biological and morphological analysis of angiostrongylus cantonensis isolates from different geographical areas of Brazil

Angiostrongylus cantonensis é responsável por causar meningoencefaliteeosinofílica em humanos e casos já foram registrados em diversas partes do mundoincluindo o Brasil (ES, PE e SP). Nesse estudo, relatamos a variabilidade genéticaentre isolados de A. cantonensis do Brasil utilizando sequências do genemitocondrial COI. Foram identificados três haplótipos brasileiros de A. cantonensis,baseados em oito haplótipos conhecidos (ac1-ac8). O haplótipo brasileiro ac5 ficouagrupado com isolados do Japão e o haplótipo brasileiro ac8 (isolados do RJ, SP,PA e PE) formaram um clado distinto. Foi relatado um novo haplótipo brasileiro,haplótipo ac9, o qual se encontra intimamente relacionado com os haplótipos daChina (ac6) e do Japão (ac7). Dois isolados brasileiros de A. cantonensis, Olinda eCaju (haplótipos ac8 e ac9, respectivamente) relatados no presente estudo, tiveramsua biologia e morfologia caracterizadas após infecção experimental. Foi observadadiferença significativa com maior carga parasitária recuperada nos isolados de Cajue um número significativamente maior de larvas L1 eliminadas nas fezes no início doperíodo patente. Entretanto, quando comparado o total de larvas eliminadas não foiverificada diferença significativa entre os dois isolados...

Angiostrongylus cantonensis is responsible for causing eosinophilicmeningoencephalitis in humans and cases have been recorded in various parts ofthe world including Brazil (ES, PE and SP). In this study, we report the geneticvariability among Brazilian isolates of A. cantonensis using sequences of themitochondrial COI gene. We identified three Brazilian haplotypes of A. cantonensis,based on eight known haplotypes (ac1-ac8). The Brazilian haplotype ac5, wasclustered with isolates from Japan and the Brazilian haplotype ac8 (isolates from RJ,SP, PA and PE) formed a distinct clade. It was reported a new Brazilian haplotype,haplotype ac9, which is closely related to haplotype from China (ac6) and Japan(ac7). Two Brazilian isolates of A. cantonensis, Olinda and Caju (haplotypes ac8 andac9, respectively) reported in this study, had their biology and morphologycharacterized after experimental infection. Significant differences were observed withhigher parasite load recovered in the isolates from Caju and a significantly greaternumber of L1 larvae eliminated in the feces at the beginning of the patent period.However, when compared to the total larvae eliminated there was no significantdifference between the two isolates. The isolates from Caju showed significantdifference in the proportion of female and male specimens (0,64:1), but it was notobserved for isolates from Olinda (1,16:1)...

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.

Detection of rat lungworm in intermediate, definitive, and paratenic hosts obtained from environmental sources.

Angiostrongylus cantonensis is the most common parasite causing human eosinophilic meningitis worldwide. The geographical distribution of this disease has changed dramatically in the last few decades. Various methods have been used to detect A. cantonensis in host animals around the world. A survey of mollusks collected on the island of Hawa'i in 2005 using PCR showed an infection rate of 24-78% depending on the mollusk species. In this study, samples from intermediate, definitive, and paratenic hosts were analyzed to further determine the presence of A. cantonensis in the United States. All samples were from Hawa'i, except for the apple snails (Pomacea maculata) that were collected in New Orleans, Louisiana. Angiostrongylus cantonensis was detected in the majority of species examined, including the apple snails from New Orleans and flatworms (planarians) from Hawa'i. Among the mollusks examined, the semi-slug Parmarion martensi had the highest parasite load, with an average larval burden of 445 larvae in 25 mg of tissue, as estimated by real-time PCR. In contrast, slime excreted from these highly infected mollusks contained no or very little A. cantonensis DNA. Analysis of definitive hosts (Rattus spp.) showed discrepancies between morphological and PCR-based identification; 54% of the rats were positive based on morphology, while 100% of tissue samples from these animals were positive by real-time PCR. This indicates that necropsies of rodents could underestimate the infection rates in definitive hosts of A. cantonensis.

Angiostrongylus cantonensis (Nematode: Metastrongiloidea): in vitro cultivation of infective third-stage larvae to fourth-stage larvae.

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.

Survey of Angiostrongylus cantonensis in rats and giant African land snails in Phitsanulok province, Thailand.

OBJECTIVE: To survey the Angiostrongylus cantonensis (A. cantonensis) or the rat lungworm in a rat, definitive host, and in a giant African land snail (Achatina fulica), the intermediate host, in Phitsanulok, Thailand. METHODS: Rats and giant African land snails were captured from Tha Pho sub-district, Phitsanulok, Thailand. Rats were killed and examined for adult A. cantonensis. The artificial digestion method following Baermann technique were used for isolation third stage larvae of A. cantonensis. RESULTS: Sixty-two rats were captured and they were identified as Rattus argentiventer, Rattus rattus (R. rattus), Bandicota savilei, and Bandicota indica but only one animal (R. rattus) of 62 rats (1.61%) was positive with adult worm of A. cantonensis. The third stage larvae of A. cantonensis were examined on 307 Angiostrongylus fulica snails. It was found that the overall infection rate was 12.38% (38 infected out of 307 Achatina snails). CONCLUSIONS: This study demonstrates that A. cantonensis is available in the natural hosts of Phitsanulok. This suggests that the transmissions of this parasite to human may occur in this region.

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.

Finding of Parastrongylus cantonensis (Chen, 1935) in Rattus rattus in Tenerife, Canary Islands (Spain).

Parastrongylus cantonensis is a parasite of murid rodents that can infect humans and cause health problems as eosinophilic meningitis. Although it is endemic in south Asia, the Pacific islands, Australia, USA, and a few Caribbean islands, it has been extended to new geographical regions. In the Canary Islands (Spain) a survey of helminths of Rattus rattus, Rattus norvegicus and Mus musculus domesticus was carried out. Furthermore, five species of molluscs were examined for nematode larvae to determine whether they are potential intermediate hosts of P. cantonensis. Nematodes were found in the lungs of 15% of 67 R. rattus examined in Tenerife, one of the four studied islands, with a prevalence of 20% in the highest focus of infection. Based on morphological and molecular analysis, with the complete internal transcribed spacer 2 (ITS-2) and a fragment of the small subunit ribosomal RNA (SSU rRNA) nucleotide sequences, nematodes were identified as P. cantonensis. Larval nematodes found from snails and slugs were identified as third-stage (L(3)) Metastrongyloidea, but the molecular study showed that they did not belong to P. cantonensis. This is the first finding of angiostrongyliasis in rats in the Canary Islands (Spain). New molecular data for this species and Parastrongylus dujardini are reported. The presence of P. cantonensis in Tenerife could be of importance from the public health point of view. Further studies are required in order to look for other potential foci of infections in the Canary Islands.

The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil.

The human cases of eosinophilic meningitis recently reported from Brazil have focused the attention of the public health agencies on the role the introduced snail Achatina fulica plays as hosts of the metastrongylid nematodes. Determining the potential of this snail to host and develop infective larval stages of metastrongylids in the wild and identify the species harbored by them is crucial for designing effective control measures. Here we assess if A. fulica may act as intermediate host of A. cantonensis at the peridomiciliary areas of a patient's house from state of Pernambuco (PE), who was diagnosed with eosinophilic meningitis and a history of ingesting raw molluscs. Larvae obtained from naturally infected A. fulica were orally administered to Rattus norvegicus. The worms were collected from the pulmonary artery and brain, and were morphologically characterized and compared to the Japan isolate of A. cantonensis. Adult worms and infective L(3) larvae (PE isolate) recovered from A. fulica specimens were also analyzed by polymerase chain reaction and restriction fragment length polymorphism of ITS2 region from rDNA and compared to A. cantonensis (ES isolate), A. vasorum (MG isolate) and A. costaricensis (RS isolate). The large size of the spicules (greater than those observed in other species of Angiostrongylus) and the pattern of the bursal rays agree with the original species description by Chen (1935). Furthermore, the morphology of the PE isolate was similar to that of Japan isolate. The PCR-RFLP profiles obtained were distinctive among species and no variation in patterns was detected among adult individuals from A. cantonensis isolates from PE and ES. The importance of A. fulica as an intermediate host of eosinophilic menigoencepahlitis in Brazil is emphasized.

Angiostrongylus cantonensis: morphological and behavioral investigation within the freshwater snail Pomacea canaliculata.

An infection with Angiostrongylus cantonensis, the main causative agent for human eosinophilic encephalitis, can be acquired through the consumption of the freshwater snail Pomacea canaliculata. This snail also provides a suitable model to study the developmental morphology and behavior of A. cantonensis larvae, facilitated by the snail's distinct lung structure. We used microanatomy for studying the natural appearance and behavior of A. cantonensis larvae while developing within P. canaliculata. The distribution of refractile granules in the larval body and characteristic head structures changed during the developmental cycle. Two well-developed, rod-like structures with expanded knob-like tips at the anterior part were observed under the buccal cavity as early as the late second developmental stage. A "T"-shaped structure at the anterior end and its tenacity distinguished the outer sheath from that shed during the second molting. Early first-stage larvae obtained from fresh rat feces are free moving and characterized by a coiled tail, whereas a mellifluous "Q"-movement was the behavioral trait of third-stage A. cantonensis larvae outside the host tissue. In combination, the distribution of refractive granules, distinct head features, variations in sheaths, and behavioral characteristics can be utilized for differentiation of larval stages, and for distinguishing A. cantonensis larvae from those of other free-living nematodes.

[Morphology of III stage larvae of Angiostrongylus cantonensis in Pomacea canaliculata].

OBJECTIVE: To observe the morphologic characteristics of III stage larvae of Angiostrongylus cantonensis from Pomacea canaliculata. METHODS: P. canaliculata, the intermediate host snail of A. cantonensis, was infected with I stage larvae of A. cantonensis in laboratory. After 61 days, III stage larvae of A. cantonensis were harvested from snail's lungs and muscle of head-foot, followed by HE stain to observe morphological characteristics. RESULTS: The whole body of III stage larva was curling with obtuse head. Its pharyngeal canal extends from the buccal hole on the top of the head to the intestines at the pharyngeal intestine joint place, with apex cauda and clear anal tube. The tegument of the III stage larva was eosin-stained, with a transparent sheath outside of tegument. Some of the larvae cauda showed in circular cylinder, and some larvae presented ventral gland with two very short uterine which used to be the feature only showed in early IV stage larva. CONCLUSION: Morphologically characteristics of the III stage larvae is helpful to better understand the life-cycle and the control of A. cantonensis.

Light and electron microscopic studies on two nematodes, Angiostrongylus cantonensis and Trichuris muris, differing in their mode of nutrition.

The morphological characteristics of the adult heteroxenous blood nematode Angiostrongylus cantonensis and the adult monoxenous intestinal nematode Trichuris muris were compared with special regard to the ultrastructure of their digestive systems. The small circular mouth of A. cantonensis appears sucker like. The very narrow mouth of T. muris is surrounded by three lips covered by the cuticle that extends into the buccal space. In the buccal cavity of A. cantonensis, a single tooth occurs opposite to a cutting plate, while no teeth are present in T. muris. The lumen of the well-developed muscular pharynx of A. cantonensis shows a trifurcated star-like cross-section. The anterior segment of the bipartite pharynx presumably functions as a pump. The lumen of the bipartite pharynx and esophagus of T. muris exhibits a very narrow oval cross-section and possesses no musculature. It is composed of a long column of stichosome cells. The esophagus region is lined inside by bands of bacillary cells as well as outside by two longitudinal rows of funnel-like papillae. These structures may be involved in the uptake of nutrients by T. muris. The gland cells might excrete digestive exoenzymes, while the bacillary cells take up the predigested nutrients. The presence of many vesicles suggests a vesicular transport of the material into the pharynx. The intestinal epithelium of A. cantonensis is densely covered with short microvilli. The lumen itself is filled with red blood cells originating from host blood. The intestine of T. muris has a thick epithelium being placed on a basal lamina and shows long thin microvilli. The intestinal lumen is very narrow and free from particles or granules. This again suggests that T. muris lives on low molecular nutrients resorbed from the environment. The epithelium cells of the intestine of T. muris contain glycogen and electron light granules but are lacking mitochondria. This finding may indicate that the epithelium cells have an anaerobic energy metabolism. This statement fits with the fact that the habitat of the worm, the cecum, is largely anaerobic.

A fatal case of angiostrongyliasis in an 11-month-old infant.

An 11-month-old boy developed flaccid quadriparesis after two months in Fiji, and was transferred to Australia, where a diagnosis of postinfectious myelitis was made. Despite peripheral blood eosinophilia, eosinophils were not detected in the cerebrospinal fluid, and an infective aetiology was not identified. The patient died of progressive bulbar dysfunction. At autopsy, numerous nematodes, identified as Angiostrongylus cantonensis, were seen in vessels of the lungs, brain and spinal cord, associated with pulmonary abscesses and eosinophilic meningitis. A notable feature was the presence of adult nematodes in the lung.