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 S?o Vicente, state of S?o Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.     

Development of Angiostrongylus cantonensis in the lung of Achatina fulica (author's transl)

Twenty-four Achatina fulica from Pai-sha of the Pescardores Islands, where no Angiostrogylus cantonensis has been found in A. fulica(4), were inoculated with the first stage larvae of A cantonensis. One snail was killed every day after 24 days following inoculation. The lung of each snail was separated and examined for larvae under microscope. The same lung was then digested with pepsin. The larvae recovered after pepsin digestion were examined for more detailed morphology. This study confirmed that the first stage larvae of A. cantonensis passed through the respiratory pore to the lung of A. fulica and developed into infectious form (3rd stage larvae) in the lung. The second and third stage larvae were found in the lung on the 7th and 17th day respectively after inoculation. Worm capsules were found in the lung on the 12th day after inoculation and the size of capsule was in proportion to the duration of the infection.     

Low susceptibility of Achatina fulica from Brazil to infection with AngioIylus costaricensis and A. cantonensis

Introduction of Achatina fulica in Brazil has led to serious concerns about its role as vector for metaIylid worms: AngioIylus costaricensis and A. cantonensis. Experimental infection with both parasites was performed to evaluate the potential risk for their transmission by the giant African snail. Groups of 5 animals, both wild and bred at captivity were exposed at different inocula: 1, 5, and 10 x 10(3) L1 of A. costaricensis and A. cantonensis. In all groups, few snails got infected and parasitic burden was low. Two different ways of infection were tested: ingestion produced higher numbers of L3 than the inoculation through an artificial hole in the shell. We also report the parasitological examination of 6 batches of wild A. fulica from Florianópolis, state of Santa Catarina, Brazil: only 1 out of 244 animals were infected with metaIylid larvae. Taken together these data indicate that the giant African snail occurring in Southern Brazil is not a permissive host for both AngioIylus species and does not represent a significant risk for transmission of these parasites.     

Prevalence and intensity of Angiostrongylus cantonensis in freshwater snails in relation to some ecological and biological factors

The purpose of the study was to record different intermediate hosts of A. cantonensis and to determine the infection prevalence and intensity of this parasite in freshwater snails in relation to some ecological and biological factors. The study was conducted at Al-Salam irrigation Canal and Al-Abtal village (north Sinai) for one year, from March 2004 to February 2005. Thirteen species of freshwater snails of nine families were examined for A. cantonensis infection. Six species were found infected with A. cantonensis larvae. These species were L. carinatus, C. bulimoides, C. cyclostomoides, B. alexandrina, L. natalensis and M. tuberculta. The infection prevalence of A. cantonensis in the examined snails ranged from 0.63 to 2.24%. L. carinatus snail had the highest prevalence, mean abundance and mean intensity of A. cantonensis infection. Positive correlations were found between both prevalence and mean abundance of A. cantonensis and host size in L. carinatus and M. tuberculata. Negative correlations were detected between salinity and prevalence, mean abundance and mean intensity of larvae of A. cantonensis. The results demonstrated seasonal and spatial variation in the prevalence, mean abundance and mean intensity of infection among examined snails. In this study, A. cantonensis larvae were found in a wide range of freshwater snails and M. tuberculata snail was recorded as a new intermediate host for the first time. In conclusion, further investigations in other areas and controlled laboratory experiments of infection approaches are required to evaluate the possible threat of this parasite on humans.     

Experimental double infection of Biomphalaria glabrata snails with Angiostrongylus cantonensis and Schistosoma mansoni

Two groups of Biomphalaria glabrata snails primarily infected with Angiostrongylus contonensis were secondarily exposed to infection with Schistosoma mansoni. To investigate any anatagonistic effect of the first infection on a superimposed one and to compare to singly and non-infected snails, a series of experiments was undertaken in which snails were individually exposed, variously, to 1,000 and 2,000 first-stage larvae of A. cantonensis and then to 5 and 10 miracidia of S. mansoni 1 day and 3 weeks later. Snails became infected with S. mansoni in both groups of snails with double infections and shed cercariae after the same incubation period as in the singly infected groups. The number of snails shedding cercariae simultaneously was similar in single and double infection groups during the first two weeks of shedding, after which this number decreased somewhat in doubly infected groups. Snails with double infection showed higher cumulative mortality rates than in snail groups with single infection with either A. cantonensis or S. mansoni. Therefore, initial infection of B. glabrata with A. cantonensis produced no inhibitory or retarding effect on subsequent infection of snails with S. mansoni.     

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.     

Development of brain injury in mice by Angiostrongylus cantonensis infection is associated with the induction of transcription factor NF-kappaB, nuclear protooncogenes, and protein tyrosine phosphorylation

Eosinophilic meningitis or meningoencephalitis caused by Angiostrongylus cantonensis is endemic to the Pacific area of Asia, especially Taiwan, Thailand, and Japan. Although eosinophilia is an important clinical manifestation of A. cantonensis infection, the role of eosinophils in the progress of the infection remains to be elucidated. In this experiment, we showed that A. cantonensis-caused eosinoplia and inflammation might lead to the induction of NF-kappaB and protooncogene expression via activation of the tyrosine phosphorylation signal pathway. After mice were infected daily with 30 third-stage larvae of A. cantonensis by oral adminstration for 6 weeks, no significant differences PKC-alpha, MEK-1, ERK-2, JNK, and p38 protein expression were found between the control and infected mice. However, the protein tyrosine phosphorylation levels, NF-kappaB, and iNOS protein products were significantly increased by 3.5-, 3.3-, and 6.3-fold, respectively, after 3 weeks of A. cantonensis infection. The same pattern was found for c-Myc, c-Jun, and c-Fos proteins, which were elevated by 3.2-, 2.3-, and 3.4-fold, respectively, compared to control animals after 3 weeks. The expression potency of these proteins started increasing in week 1, reaching maximal induction in week 3, and then declining in week 5 after A. cantonensis infection. Another consistent result was noted in the pathological observations, including eosinophilia, leukocyte infiltration, granulomatous reactions, and time responses in brain tissues of infected mice. These data suggest that the development of brain injury by eosinophlia of A. cantonensis infection is associated with NF-kappaB and/or nuclear protooncogenes expression, which is activated by the tyrosine phosphorylation pathway.     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.     

The effect of some biological and physical factors on infection of Biomphalaria glabrata with Angiostrongylus cantonensis.

Several biological and physical factors which may influence infection of Biomphalaria glabrata snails with the first stage larvae of Angiostrongylus cantonensis were studied. These factors were: the size of snails, the number of first stage larvae to which snails were exposed, the age of larvae, individual exposure compared with mass exposure of snails, the length of exposure period and the effect of temperature and light. The results showed that young snails, 2 mm in diameter, became infected with significantly smaller numbers of third stage larvae than larger snails (4, 8, 12 and 16 mm). No significant differences in the intensity of infection were evident between the larger size groups. The number of third stage larvae recovered from snails was directly related to the number of first stage larvae to which the snails were exposed. The mean percentage recovery per snail was more or less constant regardless of the infective dose. First stage larvae showed a slight reduction in their survival rate within 7 and 4 days, at 24 degrees C and 26 degrees C respectively, after which a sharp decrease in their survival rate occurred. However, the infectivity of larvae was progressively reduced from the second day at these two temperatures. The mean intensity of infection in snails was independent of whether the infection was by mass or individual exposure. The average number of first stage larvae entering a snail was greatest during the first half hour of exposure, this decreased considerably during the second half hour, and thereafter the number of larvae which entered a snail was low. It was concluded that 26 degrees C was the optimal temperature for infection and that the presence or absence of light had no effect on the infection.     

Studies on suspected clinical and experimental angiostrongyliasis: serological responses

Serum antibodies in suspected angiostrongyliasis patient were detected by ELISA. The antibody titre was 1:51,200 in the serum and 1:6,400 in CSF with preadult A. cantonensis antigen. Other tests like AGD and CIEP failed to show any positive reaction with both preadult and adult worm antigens. Experimental infection with 100 A. cantonensis larvae in albino rats indicated positive CIEP reaction in serum from the day 5 to 375 after infection. No precipitin line was seen on the other hand, in AGD during observation period. Different rat groups infected with larval doses of 100, 500, 2,000, and 5,000 showed positive CIEP reaction, on the 21st day of infection when preadult worms were seen in CNS. There was no CIEP reaction when a low dose of 15 larvae was used. Cerebral fluid of rats infected with heavy dose of 5,000 larvae showed positive CIEP reaction on the 21st day.     

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.     

Public health importance of Angiostrongylus cantonensis and its relatives

Species of Angiostrongylus are fairly common nematode parasites of a variety of small mammals, but some species - particularly A. cantonensis and A. costaricensis - are increasingly seen as human infections. The worms make use of a range of aquatic, amphibious or terrestrial gastropod molluscs as intermediate hosts, which become infected by ingestion of eggs shed in the faeces of infected mammals. Porotenic hosts, including crustaceans, amphibians and reptiles, are known for most Angiostrongylus species, and can also be a source of the infective third-stage larvae that initiate mammalian infections.     

The suitability of several aquatic snails as intermediate hosts for Angiostrongylus cantonensis.

Sixteen species of aquatic snails of four families were tested by quantitative technique under standardized conditions for their suitability as intermediate hosts for Angiostrongylus cantonensis. These species were the planorbid snails Biomphalaria glabrata, Biomphalaria alexandrina, Planorbis planorbis, Planorbis intermixtus, Bulinus truncatus, Bulinus contortus, Bulinus africanus, Bulinus tropicus and Helisoma sp.; the lymnaeid snails Lymnaea natalensis, Lymnaea tomentosa, Lymnaea stagnalis, and Stagnicola elodes; the physid snail Physa acuta (an Egyptian and a German strain) and the ampullariid snails Marisa cornuarietis and Lanistes carinatus. All these snail species proved to be susceptible to infection with A. cantonensis, and first stage larvae reached the infective third stage in all of them. However, the rate and intensity of infection varied with different species. B. glabrata was the most susceptible snail species with a 100% infection rate and an average percentage recovery of third stage larvae of 26.1. This was followed by S. elodes and B. africanus, with a 100% infection rate and an average percentage recovery of third stage larvae of 15.6 and 14.6 respectively. The rest of snail species proved to be less susceptible. For comparative evaluation of the suitability of the various snail species as intermediate hosts of A. cantonensis a "Capacity Index" was determined. This index should provide a useful method for the evaluation of the suitability of various snails as intermediate hosts of nematode parasites under standardized conditions in the laboratory.     

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.     

Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines

Mice infected orally with third-stage larvae of Heligmosomoides polygyrus were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hr-infected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36–72 hr before necropsy.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

Changes in worm burden, haematological and serological response in rats after single and multiple Angiostrongylus cantonensis infections.

Thirteen groups of rats were first sensitized with single or double doses of 5--30 third-stage larvae of Angiostrongylus cantonensis, followed by a challenge infection with 100 larvae at various periods after the primary infection. Seven other groups of rats receiving only the sensitizing infection served as the controls. In all the sensitized rats, a significantly (p less than 0.05) smaller mean number of adult worms was found established in the challenge infection as compared to the control. The frequency of the sensitizing dose and timing of the challenge infection appeared to influence the intensity of the host's response. There was no conclusive evidence to indicate that the immune response could retard the growth, development, or sex ratios of the worms established in subsequent infections. A positive haemagglutinating antibody response was first observed in some rats as early as four weeks post-infection with 100 larvae when the worms began migrating from the brain to the lungs. The antibody response and eosinophilia were most pronounced during the oviposition of the female worms and hatching of first-stage-larvae. Changes in white blood cell, lymphocyte, and neutrophil counts were also followed in some groups.     

Prevalence of schistosome infections within molluscan populations: observed patterns and theoretical predictions

The paper draws together a large and scattered body of empirical evidence concerning the prevalence of snail infection with schistosome parasites in field situations, the duration of the latent period of infection in snails (and its dependence on temperature), and the mortality rates of infected and uninfected snails in field and laboratory conditions. A review and synthesis of quantitative data on the population biology of schistosome infections within the molluscan host is attempted and observed patterns of infection are compared with predictions of a schistosomiasis model developed by May (1977) which incorporates differential snail mortality (between infected and uninfected snails) and latent periods of infection. It is suggested that the low levels of prevalence within snail populations in endemic areas of schistosomiasis are closely associated with high rates of infected snail mortality and the duration of the latent period of infection within the mollusc. In certain instances, the expected life-span of an infected snail may be less than the duration of the latent period of infection. Such patterns generate very low levels of parasite prevalence. A new age prevalence model for schistosome infections within snail populations is developed and its predictions compared with observed patterns. The implications of this study of observed and predicted patterns of snail infection within molluscan populations are discussed in relation to the overall transmission dynamics of schistosomiasis.     

Efficacy of albendazole combined with a marine fungal extract (m2-9) against Angiostrongylus cantonensis-induced meningitis in mice

The pathogenesis of angiostrongyliasis, resulting from Angiostrongylus cantonensis invasion of the human central nervous system, remains elusive. Anthelmintics are usually used to kill worms, although dead worms in the brain may cause severe inflammation which will lead to central nervous system damage. Therefore, combination therapy with anthelmintics and anti-inflammatory drugs in the treatment of human angiostrongyliasis needs further study. To evaluate the efficacy of albendazole combined with a marine fungal extract (m2-9) in A. cantonensis infection, BALB/c mice infected by the third-stage larvae of A. cantonensis were divided into three groups: mice treated with albendazole or m2-9 alone or in combination from day 5 post-inoculation (PI). Several efficacy parameters were recorded, including weight change, worm recovery, neurological function, behavioural analysis, eosinophil and leucocyte counts. The results showed that combination therapy increased body weight, reduced worm burden, improved learning ability, memory and action, decreased neurological dysfunction and leucocyte response in these mice. The combination of albendazole and m2-9 treatment significantly decreased leucocyte response and increased the frequency of rearing, compared to infected mice treated with either drug alone. Therefore, m2-9 is a natural product with potentially significant therapeutic value for angiostrongyliasis and is worthy of further study.     

Cross-reactions with Ascaris suum antigens of sera from mice infected with A. suum, Toxocara canis, and Angiostrongylus cantonensis

Ascaris suum larval excretory-secretory (AsES) antigen and larval (AsLA) as well as adult somatic antigen (AsAA) which were thought to be possibly helpful in the diagnosis of visceral larva migrans (VLM) due to A. suum infection were investigated in the present study. Serum taken from mice orally inoculated with approximately 250 embryonated eggs of A. suum or Toxocara canis, or 40 third-stage larvae of Angiostrongylus cantonensis were assessed by enzyme-linked immunosorbent assay (ELISA) using the AsES antigen, AsLA or AsAA at 1, 2, 3, 4, 6 and 8 weeks post infection (WPI). The titer of serum IgG from mice infected with A. suum increased from 1 WPI and a peak at 4 WPI was observed when it reached approximately three times the level of uninfected control mice. Thereafter, it decreased gradually but remained high as found from 6 to 8 WPI. No cross-reactions of heterologous serum IgG against AsES antigen was observed, whereas heterologous serum IgM exhibited significant cross-reactions to AsES antigen. Cross-reactivities to AsLA and AsAA by heterologous serum IgG as well as IgM antibodies were also observed in the trial. Altogether, the AsES antigen apparently seemed to be superior to the other two somatic antigens when used in the diagnosis of A. suum-induced VLM with serum IgG as tested by ELISA. Moreover, it was the first report to test the possibly antigenic cross-reactivity between A. suum and A. cantonensis.