Pathological effects of ECHINOSTOMA PARAENSEI infection associated with pyogenic abscesses caused by PSEUDOMONAS AERUGINOSA in swiss mice / Efectos patológicos de la infección por ECHINOSTOMA PARAENSEI asociada a abscesos piógenos causados ​​por PSEUDOMONAS AERUGINOSA en ratones suizos / Efeitos patológicos da infecção por ECHINOSTOMA PARAENSEI associada a abscessos piogênicos causados ​​por PSEUDOMONAS AERUGINOSA em camundongos suíços

The trematode Echinostoma paraensei is an intestinal parasite transmitted by ingestion of the infectious stage of metacercariae. For scientific purposes, its life cycle has been maintained in the laboratory, allowing analysis using various biological approaches. Different parasite isolates have revealed atypical patterns of migration and establishment in ectopic sites in Swiss-Webster mice. During the investigation of the biological life cycle of an E. paraensei isolate from the silvatic rodent Nectomys squamipes collected in the municipality of Rio Bonito (State of Rio de Janeiro), a bacterial coinfection with Pseudomonas aeruginosa was observed, which produced anatomopathological alterations, mainly in the liver, bile ducts, pancreas, and small intestine. The main macroscopic signs were the whitish suppurative pyogenic punctual lesions. The histological sections stained by hematoxylin-eosin showed an intense inflammatory reaction formed by mononuclear cells and macrophages surrounding the bile ducts, although the hepatic parenchyma still presented its normal aspect. Thus, pyogenic abscesses can be associated with E. paraensei infection depending on the strain and aggravating pathogenesis in the definitive host.

El trematodo Echinostoma paraensei es un parásito intestinal transmitido por ingestión de la etapa infecciosa de las metacercarias. Para fines científicos, su ciclo de vida se ha mantenido en el laboratorio, lo que permite el análisis mediante diversos enfoques biológicos. Diferentes aislamientos de parásitos han revelado patrones atípicos de migración y establecimiento en sitios ectópicos en ratones Swiss-Webster. Durante la investigación del ciclo biológico de un aislado de E. paraensei del roedor silvático Nectomys squamipes colectado en el municipio de Rio Bonito (Estado de Rio de Janeiro), se observó una coinfección bacteriana con Pseudomonas aeruginosa, que produjo alteraciones anatomopatológicas, principalmente en el hígado, los conductos biliares, el páncreas y el intestino delgado. Los principales signos macroscópicos fueron las lesiones puntuales piógenas blanquecinas supurativas. Los cortes histológicos teñidos con hematoxilina-eosina mostraron una intensa reacción inflamatoria formada por células mononucleares y macrófagos que rodeaban las vías biliares, aunque el parénquima hepático aún presentaba su aspecto normal. Por tanto, los abscesos piógenos pueden asociarse con la infección por E. paraensei dependiendo de la cepa y agravando la patogenia en el hospedador definitivo.

O trematódeo Echinostoma paraensei é um parasita intestinal transmitido pela ingestão da fase infecciosa das metacercárias. Para fins científicos, seu ciclo de vida foi mantido em laboratório, permitindo análises por meio de diversas abordagens biológicas. Diferentes isolados de parasitas revelaram padrões atípicos de migração e estabelecimento em sítios ectópicos em camundongos Swiss-Webster. Durante a investigação do ciclo de vida biológico de um isolado de E. paraensei do roedor silvático Nectomys squamipes coletado no município de Rio Bonito (Estado do Rio de Janeiro), foi observada uma coinfecção bacteriana por Pseudomonas aeruginosa, que produziu alterações anatomopatológicas, principalmente no fígado, dutos biliares, pâncreas e intestino delgado. Os principais sinais macroscópicos foram as lesões pontuais piogênicas supurativas esbranquiçadas. Os cortes histológicos corados pela hematoxilina-eosina mostraram intensa reação inflamatória formada por células mononucleares e macrófagos circundando as vias biliares, embora o parênquima hepático ainda apresentasse seu aspecto normal. Assim, abscessos piogênicos podem estar associados à infecção por E. paraensei dependendo da cepa e agravando a patogênese no hospedeiro definitivo.

Differential expression profiles of miRNAs and their putative targets in Schistosoma mansoni during its life cycle

BACKGROUND Schistosomiasis is a disease caused by Schistosoma. Due to its complex life cycle, evolutionary position and sexual dimorphism, schistosomes have several mechanisms of gene regulation. MicroRNAs (miRNAs) are short endogenous RNAs that regulate gene expression at the post-transcriptional level by targeting mRNA transcripts. OBJECTIVES Here, we tested 12 miRNAs and identified their putative targets using a computational approach. METHODS We performed the expression profiles of a set of miRNAs and their putative targets during the parasite's life cycle by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). FINDINGS Our results showed differential expression patterns of the mature miRNAs sma-miR-250; sma-miR-92a; sma-miR-new_4-3p; sma-miR-new_4-5p; sma-miR-new_5-5p; sma-miR-new_12-5p; sma-miR-new_13-3p and sma-miR-new_13-5p. Interestingly, many of the putative target genes are linked to oxidative phosphorylation and are up-regulated in adult-worms, which led us to suggest that miRNAs might play important roles in the post-transcriptional regulation of genes related to energetic metabolism inversion during parasite development. It is noteworthy that the expression of sma-miR-new_13-3p exhibited a negative correlation on SmNADH:ubiquinone oxidoreductase complex I. MAIN CONCLUSIONS Our analysis revealed putative miRNA genes related to important biological processes, such as transforming growth factor beta (TGF-β) signaling, proteasome regulation, glucose and lipid metabolism, immune system evasion and transcriptional regulation.

Morphological effects on helminth parasites caused by herbicide under experimental conditions / Efeitos morfológicos em helminto parasito causado por herbicida em condições experimentais

Abstract Helminth parasites have been studied as potential accumulators for different pollutants. Echinostoma paraensei is a foodborne trematode whose vertebrate host, the rodent Nectomys squamipes, is naturally exposed to environmental pesticides. However, little information exists regarding the pesticide's effects on helminths. This study investigated the morphological effects on the trematode, E. paraensei, after experimental Roundup® herbicide exposure, in concentrations below those recommended for agricultural use. After two hours of exposure, scanning electron microscopy (SEM) showed changes to the tegument, such as furrowing, shrinkage, peeling, spines loss on the peristomic collar, and histopathological evidence of altered cells in the cecum and acinus vitelline glands with vacuoles and structural changes to the muscular layers. Glycidic content was decreased, primarily in the connective tissue. As E. paraensei is an intestinal parasite of the semi-aquatic wild rodent, N. squamipes, it is predisposed to pesticide exposure resulting from agricultural practices. Therefore, we emphasize the need to evaluate its impact on helminth parasites, due to their pivotal role in regulating host populations.

Resumo Helmintos parasitos tem sido estudados como acumuladores potenciais para diferentes poluentes. O trematódeo E. paraensei tem como hospedeiro vertebrado o roedor Nectomys squamipes naturalmente exposto a pesticidas no meio ambiente. No entanto, pouca informação está disponível sobre os efeitos dos pesticidas em helmintos parasitos. O presente estudo investigou, em condições experimentais, os efeitos morfológicos no trematódeo E. paraensei após a exposição ao herbicida Roundup®, em concentrações abaixo das recomendadas para a utilização agrícola. A microscopia eletrônica de varredura (MEV) mostrou após duas horas de exposição, alterações no tegumento, como enrugamento, contração e descamação com perda de espinhos no colar peristômico e análise histopatológica evidenciou células do ceco alteradas, as glândulas vitelínicas com vacúolos e mudanças estruturais nas camadas musculares. Diminuição do conteúdo glicídico, principalmente no tecido conjuntivo, também foi observado. Considerando a predisposição à exposição a pesticidas agrícolas de N. squamipes infectado por E. paraensei, são necessários estudos para avaliar o impacto de tais resíduos frente aos helmintos e seus hospedeiros.

Characterisation of the vascular pathology in Sigmodon hispidus (Rodentia: Cricetidae) following experimental infection with Angiostrongylus costaricensis (Nematoda: Metastrongylidae)

BACKGROUND Angiostrongylus costaricensis is a nematode that causes human abdominal angiostrongyliasis, a disease found mainly in Latin American countries and particularly in Brazil and Costa Rica. Its life cycle involves exploitation of both invertebrate and vertebrate hosts. Its natural reservoir is a vertebrate host, the cotton rat Sigmodon hispidus. The adult worms live in the ileo-colic branches of the upper mesenteric artery of S. hispidus, causing periarteritis. However, there is a lack of data on the development of vasculitis in the course of infection. OBJECTIVE To describe the histopathology of vascular lesions in S. hispidus following infection with A. costaricensis. METHODS Twenty-one S. hispidus were euthanised at 30, 50, 90 and 114 days post-infection (dpi), and guts and mesentery (including the cecal artery) were collected. Tissues were fixed in Carson’s Millonig formalin, histologically processed for paraffin embedding, sectioned with a rotary microtome, and stained with hematoxylin-eosin, resorcin-fuchsin, Perls, Sirius Red (pH = 10.2), Congo Red, and Azan trichrome for brightfield microscopy analysis. FINDINGS At 30 and 50 dpi, live eggs and larvae were present inside the vasa vasorum of the cecal artery, leading to eosinophil infiltrates throughout the vessel adventitia and promoting centripetal vasculitis with disruption of the elastic layers. Disease severity increased at 90 and 114 dpi, when many worms had died and the intensity of the vascular lesions was greatest, with intimal alterations, thrombus formation, iron accumulation, and atherosclerosis. CONCLUSION In addition to abdominal angiostrongyliasis, our data suggest that this model could be very useful for autoimune vasculitis and atherosclerosis studies.

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.

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.

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

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.

Alteration in the endogenous intestinal flora of swiss webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.

Potentiality of Achatina fulica Bowdich, 1822 (Mollusca: Gastropoda) as intermediate host of the Angiostrongylus costaricensis Morera & Céspedes 1971

Samples of Achatina fulica were experimentally infected with Angiostrongylus costaricensis larvae, etiological agent of abdominal angiostrongyliasis, showing that A. fulica is susceptible to the parasite. Achatina fulica may be a risk to urbanization of abdominal angiostrongyliasis presumably due to its high proliferation, continuous dispersion and remarkable adaptation in several Brazilian towns.

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.

Intestinal fibrovascular nodules caused by Schistosoma mansoni infection in Calomys callosus Rengger, 1830 (Rodentia: Cricetidae): a model of concomitant fibrosis and angiogenesis

Human schistosomiasis develops extensive and dense fibrosis in portal space, together with congested new blood vessels. This study demonstrates that Calomys callosus infected with Schistosoma mansoni also develops fibrovascular lesions, which are found in intestinal subserosa. Animals were percutaneously infected with 70 cercariae and necropsied at 42, 45, 55, 80, 90 and 160 days after infection. Intestinal sections were stained for brightfield, polarization microscopy, confocal laser scanning, transmission and scanning electron microscopies. Immunohistological analysis was also performed and some nodules were aseptically collected for cell culture. Numerous intestinal nodules, appearing from 55 up to 160 days after infection, were localized at the interface between external muscular layer and intestinal serosa, consisting of fibrovascular tissue forming a shell about central granuloma(s). Intranodular new vessels were derived from the vasculature of the external vascular layer and were positive for laminin, chondroitin-sulfate, smooth muscle alpha-actin and FVIII-RA. Fibroblastic cells and extracellular matrix components (collagens I, III and VI, fibronectin and tenascin) comprised the stroma. Intermixed with the fibroblasts and vessels there were variable number of eosinophils, macrophages and haemorrhagic foci. In conclusion, the nodules constitute an excellent and accessible model to study fibrogenesis and angiogenesis, dependent on S. mansoni eggs. The fibrogenic activity is fibroblastic and not myofibroblastic-dependent. The angiogenesis is so prominent that causes haemorrhagic ascites

Penetration Sites and Migratory Routes of Angiostrongylus costaricensis in the Experimental Intermediate Host (Sarasinula marginata)

The intermediate hosts of Angiostrongylus costaricensis are terrestrian molluscs, mostly of the family Veronicellidae. The present work aimed at clarifying more accurately the sites of penetration and the migratory routes of A. costaricensis in the tissue slugs and at verifying the pattern of the perilarval reaction at different times of infection. Slugs were individually infected with 5,000 L1, and killed from 30 min to 30 days after infection. From 30 min up to 2 hr after infection, L1 were found within the lumen of different segments of the digestive tube having their number diminished in more advanced times after exposition until complete disappearance. After 30 min of exposition, percutaneous infection occurred, simultaneously to oral infection. Perilarval reaction was observed from 2 hr of infection around larvae in fibromuscular layer, appearing later (after 6 hr) around larvae located in the viscera. A pre-granulomatous reaction was characterized by gradative concentration of amebocytes around larvae, evolving two well-organized granulomas. In this work we confirmed the simultaneous occurrence of oral and percutaneous infections. Perilarval reaction, when very well developed, defined typical granulomatous structure, including epithelioid cell transformation. The infection also caused a systemic mobilization of amebocytes and provoked amebocyte-endothelium interactions

Effects of schistosomal mansoni infection on Calomys callosus coelom-associated lymphomyeloid tissue (milky spots)

Calomys callosus Rengger, 1830 (Rodentia: Cricetidae) is a mouse-like South American wild rodent, which is permissive to Schistosoma mansoni infection. In this paper we studied the effect of schistosomal infection in C. callosus mesenteric and omental milky spots (MS), subsidiary foci of coelom-associated lymphomyeloid tissue (CALT), during the acute, transitional (acute to chronic), and chronic phases of the infection. MS were morphologically analyzed by historical methods, using brightfield and confocal laser scanning microscopies. The MS of infected animals were mainly of lymphomyelocytic (42 to 90 days) and lymphoplasmacytic (160 days of infection) types and showed frequent presence of lymphoid follicles with germinal centers, plasmacytogenesis and plasmacytosis, mastocytosis, megakaryopoiesis, erythropoiesis and less pronounced eosinopoiesis. These results indicate that MS are a preferencial site of germinal-center-dependent and independent plasmacytogenesis, and a bone narrow-like organ, committed with various cellular lineages. The consequence of a C. callosus MS reactivity for schistosomal infection is still unknown and is under investigation.

Angiostrongylus costaricensis: via migratoria e histopatologia no modelo murino experimental / Angiostrongylus costaricensis: via migratoria and histopathology in a murine experimental model

A fim de esclarecer os aspectos do desenvolvimento e maturação dp Ansiostronsylus Costaricensis e a patogenia patologia da angiostrongiliase em hospedeiro vertebrado, estudamos o modelo do camundongo (Swiss Webster). Os animais foram infectados, oralmente, com L3 provenientes de lesmas Sarasinula sp. Após o sacrifício, conforme esquemas abaixo. os órgãos (cérebro, timo, coração, pulmão, fígado, pâncreas,baço, estomago, intestino, rins e aparelho reprodutor) foram fixados em Formalina-Millonia, incluídos em parafina e corados em HE. O estudo da penetração da L3 foi realizado em segmentos isolados de intestino delgado. Este material foi incluído em resina hidrofílica JB4, além de parafina. Quando necessário, as lâminas foram também coradas em Giemsa de Lennert, Reticulina e Resorcina-fucsina. Os tempos estudados após a infecção foram: Um,2,3,4,5,7,10,15,20,25,30,35,40 e 45 minutos(1000 L3; 1 animal/ponto) para a penetração. Seis, 12,24,48,72 horas, 4,5,6 dias (500 L3; 6 animais/pontos), 7,8,9,10,11,12,13, e 14 dias (500 L3; 2 animais /ponto) para observação de possíveis trajetórias para o habitat definitivo. Sete,10,14,18,21,24,28,31,35 e 40 diasa (8 L3; 6 animais/ponto) para os estudos histopatológico...