Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 µg/ml of antigenic concentration.

Angiostrongylus costaricensis and experimental infection of Sarasinula marginata II: elimination routes

Angiostrongylus costaricensis intermediate hosts are terrestrial mollusks mostly belonging to the Veronicellidae family. In the present investigation we focused on the mechanisms of larval expulsion from Sarasinula marginata infected with A. costaricensis. Twenty-five mollusks were individually infected with 5000 L1 and sacrificed at 30 min and 1, 2, 4, 6, and 8 h post-infection and at days 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28, and 30 post-infection; the mollusks were then fixed and stained. Diverse organs involved throughout the course of the migratory routes of larvae from oral penetration on were specified and the mechanisms of larval access to the fibromuscular layer through the kidney, rectum, and vascular system were defined. The elimination of L3, derived from oral and/or cutaneous infections, appears to depend on granulomas located close to the excretory ducts of mucous cells.

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

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.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Low susceptibility of Achatina fulica from Brazil to infection with Angiostrongylus 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 ´ 10³ 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.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silicoana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and SmBAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

Estágios imaturos e bionomia de Cyclomia mopsaria Guenée (Lepidoptera, Geometridae)

Dados sobre estágios imaturos e bionomia de Cyclomia mopsaria Guenée, [1858] são apresentados. O material procede de São Carlos, São Paulo, Brasil. As larvas alimentaram-se de Erythroxylum microphyllum St.-Hilaire (Erythroxylaceae).

Desenvolvimento dos estágios imaturos de Palmistichus elaeisis Delvare & LaSalle (Hymenoptera, Eulophidae) em pupas de Lepidoptera

Palmistichus elaeisis Delvare & LaSalle, 1993 é um endoparasitóide coletado de pupas de Sabulodes sp. (Lepidoptera, Geometridae). Os estágios imaturos deste parasitóide foram estudados em laboratório (25 ± 1ºC; 70 ± 10% UR; fotofase 14 h) em pupas dos seguintes lepidópteros: Diatraea saccharalis (Fabricius, 1794) (Crambidae), Anticarsia gemmatalis Hübner, 1818, Heliothis virescens (Fabricius, 1781), Spodoptera frugiperda (J. E. Smith, 1797) (Noctuidae) e Thyrinteina arnobia (Stoll, 1782) (Geometridae). Observou-se que os ovos e as larvas de 1º ínstar são hialinas e himenopteriformes; as larvas dos 2º, 3º e 4º ínstares são esbranquiçadas e 12 segmentadas. A espécie de hospedeiro não influenciou o número de ínstares.

Desenvolvimento pré-imaginal de Eueides isabella dianasa (Hübner) (Lepidoptera, Nymphalidae, Heliconiinae) em folhas de Passiflora edulis L. (Passifloraceae)

Lagartas de Eueides isabella dianasa (Hübner, 1806) estão entre os insetos fitófagos que ocorrem em P. edulis. Para esse estudo, foram coletados ovos logo após a oviposição, sendo feitas observações diárias para o registro de eclosão, ecdises (confirmadas pelas cápsulas cefálicas), pupação e emergência de adultos. A viabilidade de ovos foi de 94,9%. O período embrionário observado (em dias ± erro padrão da média) foi de 4,25 ± 0,18. O período larval de 16,9 ± 0.34 dias apresentou cinco ínstares: 2,75 ± 0,2; 2,8 ± 0,2; 3,0 ± 0,1; 3,6 ± 0,2 e 4,6 ± 0,15. O período larval ativo foi de 15,83 ± 0,34 dias, seguido por um período prepupal e pupal de, respectivamente, de 7,42 ± 0,15 (período inativo dentro do desenvolvimento pós-embrionário de 8,4 ± 0,15). O desenvolvimento pós-embrionário foi de 24,25 ± 0,37. A duração do desenvolvimento pré-imaginal de 28,5 ± 0,4 pode ser considerada curta entre os lepidópteros. A duração relativa foi de 14,9% para o período embrionário e de 85,9% para o pós-embironário. O uso do tempo durante o período pós-embrionário foi de 11,3%; 11,7%; 12,4%; 14,8%; 18,9% e 30,6% respectivamente como L1, L2, L3, L4, L5 e pupa. O período larval representou 65,3% enquanto o período inativo (prepupa + pupa) 34,7%. A sobrevivência no final do desenvolvimento, considerando o período de ovo, foi de 27,5% e, considerando o período pós-embrionário, 30,0%, tendo sido mais evidente no quinto ínstar e no período pupal com 63,0% e 64,7%, respectivamente. Este é o primeiro registro de Eueides isabella dianasa no Estado de Alagoas, Brasil.

Levantamento das espécies de Simulium (Diptera, Simuliidae), Riacho dos Padres, município de Almirante Tamandaré, Paraná, Brasil

De janeiro a agosto de 2002, quinzenalmente, foi levada a efeito a coleta de simulídeos imaturos em substratos naturais e antrópicos no Riacho dos Padres. O trabalho visou proceder ao inventário das espécies e verificar as freqüências relativas. A área de propriedade particular localizada na Região Metropolitana de Curitiba constitui uma microempresa de agronegócio, com criação de peixes, coelhos, ovinos, bovinos, aves domésticas e, ainda, área de lazer com eqüinos para cavalgada de visitantes. A presença constante de animais hospedeiros e a ração fornecida aos peixes garantiram farta alimentação aos simulídeos, o que favoreceu a explosão desta praga. Em todos os substratos foram coletadas 24.021 pupas e um total de 197.812 larvas. A partir de pupas, foram identificadas com as seguintes freqüências: Simulium inaequale (55,24%), Simulium perflavum (16,81%), Simulium pertinax (13,93%), Simulium orbitale (8,03%), Simulium subnigrum (4,92%), Simulium distinctum (1,03%) e Simulium incrustatum (0,04%). Verificou-se também a quantidade de larvas que colonizaram todos os substratos.

Impacto dos nutrientes N e K e de açúcares solúveis sobre populações de Diabrotica speciosa (Germar) (Coleoptera, Chrysomelidae) e Agrotis ipsilon (Hüfnagel) (Lepidoptera, Noctuidae) na cultura da batata, Solanum tuberosum L. (Solanaceae)

Foi estudada a ocorrência de Diabrotica speciosa (Germar, 1824) (Coleoptera, Chrysomelidae) e de Agrotis ipsilon (Hüfnagel, 1767) (Lepidoptera, Noctuidae) em plantas de batata, cultivares Achat e Monalisa, influenciadas por dosagens de nitrogênio e potássio, e teor mínimo de açúcares solúveis. Os seguintes parâmetros foram avaliados: concentração de nutrientes minerais e açúcar em folha verde, folha senescente, folha em abcisão, haste, tubérculo e planta total usando extratos de infusão em etanol 80%. A maior infestação por larvas de D. speciosa foi na cultivar Monalisa a 150 kg.ha-1 de N + K com 27,03% a P<0,05. Foi observado que o efeito da dosagem de N + K no incremento da concentração de açúcares solúveis aumentou os danos provocados por A. ipsilon nos tubérculos e hastes. A infestação por essas espécies aumentou para 58,82% na cultivar Monalisa, quando a dosagem de nitrogênio foi elevada de 0 para 150 kg.ha-1, na ausência de potássio. Por outro lado, alta dosagem de potássio reduziu os danos causados por A. ipsilon na cultivar Monalisa. Entretanto, não influiu no armazenamento de açúcar solúvel. Os resultados indicaram que na cultivar Achat os teores de açúcares solúveis foram reduzidos, provavelmente sensibilizados pela elevação da dosagem de adubação potássica, diferindo da cultivar Monalisa cuja influência verificada foi pela dosagem do nitrogênio.

Controle integrado de espécies de Simulium (Diptera, Simuliidae) por Bacillus thuringiensis e manejos mecânicos no riacho e nos vertedouros de tanques de piscicultura, Almirante Tamandaré, Paraná, Brasil

No Riacho dos Padres na Chácara Evíssima, numa microempresa de agronegócio com criação de peixes e animais domésticos, área de lazer e salão de eventos, localizada na Região Metropolitana de Curitiba, os simulídeos em grande proliferação causaram sério impacto com as picadas a tal ponto que dificultaram o convívio habitual. De janeiro a agosto de 2002, coletou-se 24.021 pupas por meio das quais foram identificadas as espécies: Simulium inaequale, Simulium perflavum, Simulium pertinax com 85,98% e Simulium orbitale, Simulium subnigrum, Simulium distinctum e Simulium incrustatum com somente 14,02% de freqüência. O combate a estas populações iniciado em 28 de fevereiro terminou em 08 de agosto de 2002. O controle integrado consistiu na aplicação de biopesticida Bacillus thuringiensis var. israelensis (Bti) e em manejos mecânicos não só removendo os imaturos (ovos, larvas e pupas) por meio de escovação de vertedouros, mas também pela retirada de substratos naturais e antrópicos do leito. A densidade larvária média inicial, antes da aplicação de Bti no leito do riacho (75 m) e no último vertedouro, verificada em substratos antrópicos fitilhos (área 13.500 cm²), foi de 6,7 e 11,5 larvas/cm², respectivamente. Nestes habitats, o controle integrado com Bti de 1,77 a 2,09 mg/litro do ingrediente ativo, suplementado com manejos mecânicos, monitorado em substratos fitilhos (área 20.250 cm²), resultou em redução larvar de 72,61% a 99,97% e de 74,91% a 99,45%, respectivamente, nas vazões de 0,39 a 0,45 e de 0,38 a 0,43 m³/min. No primeiro vertedouro (área 5.110 cm²), porém, só com manejos mecânicos, atingiu-se a redução larvária de 53,81% a 99,59%. O controle efetuado reduziu as picadas hematófagas ao nível esperado, surtindo o efeito positivo almejado.

Composição e preferência por microhábitat de imaturos de Odonata (Insecta) em um trecho de baixada do Rio Ubatiba, Maricá-RJ, Brasil

No período de maio de 1999 a maio de 2000 foram efetuadas coletas de imaturos de Odonata (Insecta), em sete diferentes substratos, ao longo de 50 metros de um trecho do Rio Ubatiba, Maricá, Rio de Janeiro, Brasil. Um total de 1.279 larvas de Odonata foi coligido e identificado em 27 espécies de cinco famílias. Acanthagrion lancea (Selys, 1876), Hetaerina auripennis (Burmeister, 1839), Micrathyria hesperis Ris, 1911 e Telebasis filiola (Perty, 1834) foram as espécies mais numerosas. O substrato com o maior número de indivíduos foi "vegetação em áreas de remanso". Grande parte das espécies estudadas demonstrou preferência por algum dos substratos. Dythemis multipunctata Kirby,1894, Erythrodiplax sp., M. hesperis, T. filiola, A. lancea, Erythemis sp., Coryphaeschna adnexa (Hagen, 1861) e H. auripennis demonstraram preferência por substratos orgânicos, enquanto que Brechmorhoga sp., B. praeCOX (Hagen, 1869) e Progomphus complicatus (Selys, 1854), por inorgânicos.