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.     

Observations on the transmission of Angiostrongylus cantonensis from snail to rodent

A survey of Angiostrongylus cantonensis was carried out to investigate the mode of transmission from mollusc to rat in a fixed study area of Yoron Island from 1979 to 1982. Rattus rattus was found to be infected with a small number of worms in spite of heavy infection with third-stage larvae in Achatina fulica and an abundance of this snail in the area. Natural infection and/or susceptibility with A. cantonensis were confirmed in three small snail species. Bradybaena circulus, Fruticicola despecta and Luchuena reticulata. Young A. fulica was found to be infected with fewer third-stage larvae than mature A. fulica. It was concluded that molluscs which were infected with a small number of third-stage larvae of A. cantonensis play an important role in maintaining the life cycle of A. cantonensis. The percentage of rat stomachs containing mollusc tissue was relatively low, and the incidence and infection was low in rats. Infection with A. cantonensis did not occur very often in R. rattus in nature.     

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.     

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.     

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.     

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.     

Land molluscs as food of Malayan rodents and insectivores

Laboratory and field experiments demonstrate that, contrary to previously published stomach analysis data, rats selectively consume land molluscs. Rattus jalorensis and R. argentiventer prefer molluscs as food, while R. exulans and R. r. diardi are less inclined to do so. Macrochlamys resplendens , a land snail, is most favoured, with the slug Micropar-mariort malayanus next in preference. The heavily-shelled Achatina fulica is only occasionally eaten and Quantula striata is ignored. Examination of gastro-intestinal contents of wild rats caught from various habitats in Malaya show that when present in large numbers molluscs form an important part of the natural food of rats, yet laboratory evidence demonstrates that as many as 60% of mollusc-eating rats may fail to show evidence of shells on examination within 12 hours of feeding–an indication that stomach analyses alone may be of limited value.
The Malayan insectivores Echinosorex gymnurus and Suncus murinus often feed on snails and slugs, though they appear not to be suitable hosts for infection with the rat lungworm Angiostrongylus cantonensis.     

The correct identity of a louse sample (Phthiraptera: Menoponidae) from the roadside hawk, Rupornis magnirostris (Gmelin) (Falconiformes: Accipitridae)

A report of a louse sample identified as Colpocephalum cholibae Price & Beer by Oliveira et al. (2004), from the roadside hawk [Rupornis magnirostris (Gmelin)] in the state of S?o Paulo, Brazil, is regarded as a misidentification. A correction to the identity of the lice is given as Kurodaia (Kurodaia) fulvofasciata (Piaget). Key morphological differences between the genera Colpocephalum and Kurodaia are discussed, as well as possible reasons for the misidentification.     

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.     

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.     

Detection of antibodies to Angiostrongylus cantonensis in serum and cerebrospinal fluid of patients with eosinophilic meningitis

Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student's t-test, P less than 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF.     

Angiostrongylus cantonensis: role of eosinophils in the neurotoxic syndrome (Gordon-like phenomenon)

The role of eosinophils in the pathophysiology of Angiostrongylus cantonensis infections was investigated in nonpermissive (guinea pig) and permissive (rat) hosts. Neurological symptoms similar to the Gordon phenomenon (ataxia, tremor, paralysis) together with a loss of Purkinje cells in the cerebellum were observed after intracraneal injection of human eosinophil extracts or after infection with A. cantonensis, only in guinea pigs and not in rats. Blood eosinophilia as well as eosinophil numbers present in the cerebellum and in the cerebrospinal fluid were higher in guinea pigs than in rats, at all times after infection with A. cantonensis. Increased levels of cytotoxicity toward L3 larvae in vitro were obtained in the presence of guinea pig eosinophils and IgE antibodies, rather than with the corresponding rat effector system. The detection of one eosinophil granule component, the eosinophil peroxidase, in the cerebrospinal fluid from infected guinea pigs but not from rats suggested that in nonpermissive hosts, neurological disorders, similar to the previously described Gordon phenomenon, might be due to eosinophil neurotoxins released after interaction of eosinophils with the parasites.     

Melanoides tuberculata (Mollusca: Thiaridae) harboring renicolid cercariae (Trematoda: Renicolidae) in Brazil

Melanoides tuberculata , naturally infected by gymnocephalous cercariae, were found in aquatic collections from Belo Horizonte, Minas Gerais, Brazil. After morphological characterization, larvae were used for experimental infection of Poecilia reticulata. Metacercariae were obtained from the liver of these fish, which were also found to be naturally infected in the same locality. The morphology and biology of the developmental stages of trematodes we obtained were characteristic of Renicola sp. This is the first record of renicolid cercariae and metacercariae in Brazil.     

Spontaneous meningoencephalitis by Staphylococcus aureus in an infant golden‐headed lion tamarin (Leontopithecus chrysomelas)

Non‐human primates are susceptible to many bacteria, some of which bear zoonotic potential. We report the pathologic features of spontaneous fulminating meningoencephalitis by Staphylococcus aureus in a captive infant golden‐headed lion tamarin (Leontopithecus chrysomelas) from Brazil.     

First report of Lymnaea columella Say, 1817 (Pulmonata: Lymnaeidae) naturally infected with Fasciola hepatica (Linnaeus,1758) (Trematoda: Digenea) in Argentina

We report the first evidence of natural infection of Lymnaea columella with Fasciola hepatica in Argentina. A sample of 601 snails was collected in May 2003 in northeastern Corrientes, a province bounded on the north by Paraguay, on the east by Brazil and on the southeast by Uruguay. Among 500 examined snails, 44 (8.8%) were exclusively infected with F. hepatica. Parasite identification was based on morphological features of cercariae from snails, and of eggs and adult flukes from Wistar rats. We discuss the events suggesting that an enzootic transmission cycle of F. hepatica has been recently established in northeastern Corrientes.     

The B chromosome system of Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae) 1. Banding analysis

Karyotypic analyses of 366 specimens of the solitary wasp Trypoxylon (Trypargilum) albitarse collected from ten populations in the municipalities of Vi?osa and Porto Firme (Minas Gerais, Southeastern Brazil), revealed the presence of two morphological types of supernumerary (B) chromosomes. C-banding and fluorochrome banding suggest that the B chromosomes of T. albitarse may have originated from heterochromatin breaks within the standard (A) chromosome complement.     

EVOLUTION OF THE HELIORNITHIDAE: RECIPROCAL ILLUMINATION BY MORPHOLOGY, BIOGEOGRAPHY AND DNA HYBRIDIZATION (AVES: GRUIFORMES)

Abstract— Finfoots (Heliornithidae) were chosen to test the possibility that there has been a dramatic reversal in a suite of morphological characters, intimated by an earlier phylogenetic reconstruction of Gruiformes based on DNA hybridization. There are three nodes where unstudied finfoots could stem from the existing reconstruction. The resulting alternate trees have largely exclusive implications for morphological character suite polarity, biogeography and fossil identifications. A new DNA hybridization study that includes all relevant taxa was intended to form the basis for independent evaluation of the trees, but it produced results that conflict with the earlier DNA study. So, instead, DNA trees were evaluated by their reproducibility and consensus with most-parsimonious trees, biogeography, paleontology and traditional classifications, I concur with traditional classifications that finfoots are monophyletic, and that Limpkin (Gruiformes: Aramidae) is the sister of cranes (Gruiformes: Gruidae). Limpkin is not supported as the sister of the Sungrebe ( Heliornis fulica ) or as a member of the Heliornithidae, as reported in the earlier DNA study. It is alarming that the gross lack of consensus with traditional characters and concomitant implications for character suite polarity in this case went unquestioned.     

RAPD and mitochondrial DNA analysis of the soybean stalk weevil, Sternechus subsignatus (Coleoptera: Curculionidae)

Sternechus subsignatus Boheman (Curculionidae: Sternechini) is one of the primary Curculionidae species that reduces soybean yield in Brazil. Initially, outbreaks were reported in southern Brazil in 1973; but, more recent, outbreaks were reported in Bahia (summer 1997-1998) and Maranh?o (summer 2003-2004), two states in northeastern Brazil. A putative related species, S. pinguis (Fabricius), was first detected in Salta Province, Argentina. The objective of this study was to evaluate intraspecific molecular polymorphisms of geographically distinct Sternechus populations. Randomly amplified polymorphic DNA (RAPD) profiles and partial mitochondrial cytochrome B (CytB) gene sequences were used to determine whether individual soybean stalk weevils were one of two different species and to infer pest invasion pattern. Putative S. pinguis and S. subsignatus populations were collected in San Agustin (Cruz Alta, Tucumán Province, Argentina) and different sampling sites in the Brazilian states of Paraná, Bahia and Maranh?o. Polymorphic bands were obtained by RAPD and analyzed by Dice coefficients. Populations from southern Brazil were more closely related genetically to an Argentinean group than the populations sampled in northeastern Brazil. The Londrina Co., Brazil population displayed the highest intra-population genetic similarity. Most of the soybean stalk weevils collected from San Agustin, Tucumán, Argentina were divergent from those collected in Brazil. Sequencing and parsimony analysis of CytB did not differentiate specimens collected in Argentina and Brazil. Thus, our data show that soybean stalk weevil outbreaks and population increases in northeastern Brazil involved local genotypes.     

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.