Atividade larvicida do extrato bruto enzimático do fungo Duddingtonia flagras sobre larvas de primeiro estádio de Angiostrongylus vasorum / Larvicidal activity of a crude enzyme extract of the fungus Duddingtonia flagrans on first-stage larvae of Angiostrongylus vasorum

INTRODUÇÃO: Angiostrongylus vasorum é um nematóide que parasita cães domésticos e eventualmente o homem. MÉTODOS: O objetivo deste trabalho foi observar a atividade predatória in vitro do extrato bruto enzimático do fungo Duddingtonia flagrans sobre larvas de primeiro estádio A. vasorum em condições laboratoriais no meio ágar-água 2 por cento. RESULTADOS: Ao final do experimento, os percentuais de redução das L1 de A. vasorum observados foram de: 53,5 por cento (24h) e 71,3 por cento (48h) CONCLUSÕES: O extrato bruto enzimático do fungo D. flagrans destruiu in vitro as L1, podendo ser utilizado como controle biológico desse nematóide.

INTRODUCTION: Angiostrongylus vasorum is a nematode parasite of domestic dogs and potentially of humans. METHODS: This study aimed to observe the predatory activity in vitro of a crude enzyme extract of the fungus Duddingtonia flagrans on first-stage larvae of A. vasorum in laboratory conditions on 2 percent water-agar. RESULTS: At the end of the experiment, the percentage reductions observed for A. vasorum L1 were 53.5 percent (24h) and 71.3 percent (48h). CONCLUSIONS: Crude enzyme extract of the fungus D. flagrans destroyed the L1 in vitro and can be used as a biological control for this nematode.

Susceptibility and morbidity between male and female Swiss mice infected with Angiostrongylus costaricensis: Susceptibilidade e morbidade entre camundongos Swiss machos e fêmeas infectados com Angiostrongylus costaricensis

The gender of vertebrate hosts may affect the outcome of parasitic infections. An experimental murine infection with Angiostrongylus costaricensis was followed with determinations of body weight, fecal larval elimination, number and length of adult worms, number of macroscopic intestinal lesions, and mortality. Groups of male and female Swiss mice were infected with 10 3rd-stage A. costaricensis larvae per animal. The results indicate there are no significant differences related to gender of the host, except for higher length of worms developed in male mice.

O sexo dos hospedeiros vertebrados pode influenciar no resultado de infecções parasitárias. A infecção experimental de camundongos com Angiostrongylus costaricensis foi acompanhada com observação do peso corporal, eliminação de larvas nas fezes, número e comprimento dos vermes adultos, número de lesões macroscópicas nos intestinos e mortalidade. Grupos de camundongos Swiss machos e fêmeas foram infectados cada um com 10 larvas de terceiro estágio de A. costaricensis. Os resultados indicam que não há diferenças significativas relacionados ao sexo dos hospedeiros, exceto pelo maior comprimento dos vermes nos hospedeiros machos.

Angiostrongylus costaricensis: complete redescription of the migratory pathways based on experimental Sigmodon hispidus infection

Angiostrongylus costaricensis lives in the cecal and mesenteric arteries of its vertebrate hosts, and causes an inflammatory disease in humans. To investigate unknown aspects of the abdominal angiostrogyliasis pathogenesis, infected Sigmodon hispidus were sequentially studied in different times of infection. The study revealed that L3 goes alternatively through two migratory courses during its development into an adult worm: lymphatic/venous-arterial and venous portal pathways. The former is considered the principal one, because it is used by most of the larvae. Like other metastrongylides, A. costaricensis passes over the pulmonary circulation to migrate from the lymphatic system to the arterial circulation, where they circulate during some days before reaching their definitive habitat. The oviposition by mature females began on 15th day. Eggs and L1 were detected mainly in the intestine and stomach, surrounded by inflammatory reaction constituted by macrophages, monocytes, and eosinophils. They were also spread to the lungs, mesenteric lymph nodes, pancreas, spleen, and kidneys. The larvae (L1) exhibited the centripetal capacity to invade the lymphatic and venous vessels of the intestine and mesentery. Adult worms that developed in the venous intrahepatic pathway migrated downstream to reach the mesenteric veins and laid eggs that embolized in the portal hepatic vessels.

Angiostrongylus costaricensis MORERA e CÉSPEDES 1971 no hospedeiro intermediário Sarasinula marginata SEMPER, 1885 (MOLLUSCA: SOLEOLIFERA): vias de infecçao, migraçao e eliminaçao larvar

Os hospedeiros intermediários do Angiostrongylus costaricensis são moluscos terrestres, principalmente da família Veronicellidae. No presente trabalho estudamos diferentes aspectos do ciclo biológico do A. costaricensis, na Sarasinula marginata: 1) as alterações seqüenciais durante o desenvolvimento larvar; 2) as vias migratórias, enfocando os sítios de penetração e eliminação; 3) as reações teciduais e celulares durante a penetração, maturação/migração e eliminação do parasita. Um total de 150 lesmas infectadas com 5.000 L1, foram sacrificadas e digeridas a partir do 1° até 30° e no 33°, 34°, 35°, 36°, 40°, 41° e 43° dia após infecção. As larvas foram estudadas em relação ao estágio de desenvolvimento e coradas para identificação das substâncias contidas no seu interior. Para o estudo dos itens 2 e 3 foram infectadas, individualmente, 25 lesmas com 5.000 L1, e sacrificadas com 30’, 1, 2, 4, 6, 8 horas e 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28 e 30 dias após infecção, fixadas e coradas.

Observou-se também que o tempo da muda de L2/L3 estendeu-se até o 43° dia e os lipídeos, ao contrário do glicogênio, parecem constituir-se na principal fonte de reserva energética das larvas. A partir de 30 minutos até 2 horas da infecção foram encontradas L1 na luz do trato digestivo, as quais diminuíram em número nos tempos posteriores, até completo desaparecimento. A infecção oral, ocorreu, concomitantemente, à infecção cutânea a partir de 30 minutos. A reação perilarvária foi observada a partir de 2 horas em larvas alojadas na camada fibromuscular, e no interior das vísceras 6 horas após a infecção. A reação pré-granulomatosa foi caracterizada pela concentração gradativa de amebócitos em torno das larvas, que culminou, 4 dias após a infecção, em granulomas bem organizados.

Neste trabalho, confirmamos a ocorrência simultânea das vias oral e cutânea de infecção do A. costaricensis no hospedeiro intermediário. Demonstramos ainda a forma e os sítios de penetração da L1, o envolvimento das diferentes vísceras na rota migratória do parasita, salientando a importância do reto e do rim, a ocorrência de larvas circulantes e a preferência larvar pelo tecido fibromuscular do molusco e a possibilidade de eliminação mecânica de L3 por ductos excretores. Caracterizamos também os padrões da reação amebocitária perilarval (pré-granuloma e granuloma) e sistêmica (hemocitose) e descrevemos os sítios de hemocitopoiese.

Angiostrongylus costaricensis MORERA e CÉSPEDES 1971 no hospedeiro intermediário Sarasinula marginata SEMPER, 1885 (MOLLUSCA: SOLEOLIFERA): vias de infecçao, migração e eliminação larvar / Angiostrongylus costaricensis MORERA CESPEDES 1971 and in intermediate host Sarasinula marginata Semper, 1885 (MOLLUSCA: SOLEOLIFERA): routes of infection, larval migration and elimination

Os hospedeiros intermediários do Angiostrongylus costaricensis são moluscos terrestres, principalmente da família Veronicellidae. No presente trabalho estudamos diferentes aspectos do ciclo biológico do A. costaricensis, na Sarasinula marginata: 1) as alterações seqüenciais durante o desenvolvimento larvar; 2) as vias migratórias, enfocando os sítios de penetração e eliminação; 3) as reações teciduais e celulares durante a penetração, maturação/migração e eliminação do parasita. Um total de 150 lesmas infectadas com 5.000 L1, foram sacrificadas e digeridas a partir do 1° até 30° e no 33°, 34°, 35°, 36°, 40°, 41° e 43° dia após infecção. As larvas foram estudadas em relação ao estágio de desenvolvimento e coradas para identificação das substâncias contidas no seu interior. Para o estudo dos itens 2 e 3 foram infectadas, individualmente, 25 lesmas com 5.000 L1, e sacrificadas com 30’, 1, 2, 4, 6, 8 horas e 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28 e 30 dias após infecção, fixadas e coradas. Observou-se também que o tempo da muda de L2/L3 estendeu-se até o 43° dia e os lipídeos, ao contrário do glicogênio, parecem constituir-se na principal fonte de reserva energética das larvas. A partir de 30 minutos até 2 horas da infecção foram encontradas L1 na luz do trato digestivo, as quais diminuíram em número nos tempos posteriores, até completo desaparecimento. A infecção oral, ocorreu, concomitantemente, à infecção cutânea a partir de 30 minutos. A reação perilarvária foi observada a partir de 2 horas em larvas alojadas na camada fibromuscular, e no interior das vísceras 6 horas após a infecção. A reação pré-granulomatosa foi caracterizada pela concentração gradativa de amebócitos em torno das larvas, que culminou, 4 dias após a infecção, em granulomas bem organizados. Neste trabalho, confirmamos a ocorrência simultânea das vias oral e cutânea de infecção do A. costaricensis no hospedeiro intermediário. Demonstramos ainda a forma e os sítios de penetração da L1, o envolvimento das diferentes vísceras na rota migratória do parasita, salientando a importância do reto e do rim, a ocorrência de larvas circulantes e a preferência larvar pelo tecido fibromuscular do molusco e a possibilidade de eliminação mecânica de L3 por ductos excretores. Caracterizamos também os padrões da reação amebocitária perilarval (pré-granuloma e granuloma) e sistêmica (hemocitose) e descrevemos os sítios de hemocitopoiese.

Transmission of Angiostrongylus cantonensis through the giant African snail Achatina fulica: an experimental study.

Observations on transmission of the rat lung worm, Angiostrongylus cantonensis, from rats to the snail intermediate host. Achatina fulica, in a vacant lot in Bangkok are described. The prevalence of A. cantonensis increased with snail age until 200 days of age when it attained a plateau of 50-60%. The overall prevalence was 53%. The worm burden slowly rose with age until 200 days of age beyond which it remained relatively constant. The highest mean worm burden of 5,478 was observed in the oldest age group. The parasite distribution in the snail population was highly aggregated both within each age class and in the overall population. Experiments on susceptibility of snails to laboratory infection revealed that worm recovery was dependent on dose of first stage larval infection but was independent of snail size in the range of 4-8 cm. The percent worm recovery of third stage larvae was negatively correlated with dose of infection, and no density-dependent effects of worm burden on worm size were observed.

Migratory effects on the body surface of Angiostrongylus cantonensis from subarachnoid space to pulmonary artery in rats.

Juvenile worms of Angiostrongylus cantonensis recovered from subarachnoid spaces and pulmonary arteries of rats, respectively, at 28 days post-infection have been compared with respect to their surface composition, antigenicity of surface proteins and morphological appearance. Quantitative and qualitative differences were shown between surface proteins of these two stages of worms. One major and 6 minor proteins appeared on brain stage worm's surface as assessed by surface-labelling and SDS-PAGE techniques. The same, but more predominant banding pattern, with one additional major protein of Mr 80,000 kDa presented on the lung stage worm's surface. Surface components from both stages were antigenic in permissive rat hosts but refractory in nonpermissive human hosts. The surface antigens are common to both stages within the rat. Observed by scanning electron microscopy, the surface appearance of brain stage worms is thickened, rough and irregular. Besides, particle clusters adhere randomly, without cluster adherence but transverse and longitudinal clefts were shown on the surface, before the outer layer was shed. The possible mechanisms of evasion from the host's immune attack with the surface-shedding phenomenon remain to be elucidated.

Studies on growth and distribution of Angiostrongylus cantonensis larvae in Ampullarium canaliculatus.

The intramolluscan development of Angiostrongylus cantonensis larvae in Ampullarium canaliculatus, a fresh-water migrant snail which multiplies in tremendous rate in Taiwan, was followed for 28 consecutive days in this study. The snails were infected with first stage larvae of A. cantonensis and groups of three snails were sacrificed daily to study various larval stages in different organs. A. cantonensis larvae were found in all snails examined. During the first four days, they were obtained only from the digestive tract. Mouth is thus suggested as the primary route of natural infection. The first molt was observed on the fifth day of infection, and the second molt, the 12th day. Molting occurred in almost all kinds of organs examined. Studies on tissue tropism showed that the predilection site of A. cantonensis in this freshwater snail was headfoot region. Relatively few larvae were found in visceral organs including liver, heart, and ovary. Most parasites, 62.1%, recovered on the 28th day of infection were third-stage larvae.

Angiostrongylus cantonensis infection in Ampullarius canaliculatus (Lamarck) in Kyushu, Japan.

Ampullarius canaliculatus is a large fresh-water snail of which the original habitat is said to be Argentina. Recently, the snail which grew wild has propagated in various parts of Japan, and is causing damage to aquatic plants by feeding on them. The present study was performed to find out whether the snail can be a suitable intermediate host for Angiostrongylus cantonensis. Natural infection with A. cantonensis was confirmed in the snails collected from 5 different places in 4 hamlets in Okinawa and in those from Ishigaki Island. All snails experimentally infected with first-stage larvae of the parasite showed second-stage and third-stage larvae developing at the time of examination later on. Thus, A. canaliculatus is a suitable intermediate host for A. cantonensis, and, if eaten raw, it can be a potential source of human infection with eosinophilic meningoencephalitis in the endemic areas.

Induction of protective immunity against infection by Angiostrongylus cantonensis in rats.

Attempts were made to induce acquired immunity against Angiostrongylus cantonensis infection by immunizing rats with somatic (L3S) and metabolic (L3ES) antigens from third-stage larvae and with somatic antigen from adult female worms (FACE) via either a combined intraperitoneal and oral (IP-OR-OR) or subcutaneous (SC-SC-SC) route. The most effective mean in inducing protection was the administration of L3S by the IP-OR-OR route, as judged from reduced mortality, prolonged survival periods and/or reduced worm burdens. Such a protocol reduced mortality to below 20% of the unimmunized controls. The worm burden of the L3S-immunized rats was only about 25% of the control value and the survival period was at least double. However, judging from gross morphology, the absence of stunted growth and unimpaired ficundity of the worms that developed into adult stage, it appears that those worms that survived had normal development. The latter observation is in contrast to that noted with FACE immunization which seemed to affect also the growth and development of worms that survived into adult stage.

Analysis of antigens from different developmental stages of Angiostrongylus cantonensis.

Protein profiles of somatic extracts from different developmental stages and excretory and secretory products of third-state larvae of Angiostrongylus cantonensis were analyzed and characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate under both reduced and unreduced conditions. Immunological identification of common and stage-specific antigens was determined by radioimmunoprecipitation using chronically infected rat serum and antisera from rats immunized with somatic extracts of third-stage larvae and adult female worms. The somatic extracts of first-and third-stage larvae and of adult worms were found to be highly complex, each consisting of more than 20 different peptides with molecular weights varying from higher than 130,000 to 8,200 daltons. Most proteins were composed of single polypeptide chains. Polypeptides with molecular weights of 80,000, 39,500 and 22,000 daltons were present in more than one developmental stages and were antigenically related. Those with molecular weight of 15,500 dalton, was present primarily in L3S and its possible usefulness in immunodiagnosis of infection that occurs in man was discussed.

The biology of Angiostrongylus cantonensis larvae in immune rats.

Rats acquired protective immunity to Angiostrongylus cantonensis after a single infection with 50 infective larvae. Immunity was stronger after subsequent infections. Fewer worms were recovered which were shorter and produced fewer progeny after secondary and tertiary infections. However, the growth of A. cantonensis during the early developmental stages in the brain and the ability of the young adult parasites to migrate to the lungs was not affected in the immune host.