Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

First report of detection of Toxoplasma gondii DNA in oysters (Crassostrea sp.) in the state of Maranhão / Primeiro relato da detecção de DNA de Toxoplasma gondii em ostras (Crassostrea sp.) no Estado do Maranhão

Abstract The aim of this study was to report on detection of Toxoplasma gondii DNA in oysters (Crassostrea sp.) in the state of Maranhão. To conduct this study, 200 farmed oysters were acquired in the municipality of Raposa and 100 in Paço do Lumiar; and a further 100 oysters were taken from the natural stock in the municipality of Primeira Cruz. This total of 400 specimens sampled was divided into 80 pools composed of five animals each. The gills and visceral mass of each oyster were removed for DNA extraction (per pool of oysters), using a commercial kit. The nested PCR technique (with the primer SAG-1) was then used to investigate any presence of protozoa. This molecular technique demonstrated the presence of DNA of T. gondii in 2.5% of the pools of oysters (n = 2/80): these oysters were exclusively from farms. The results from this study allow the conclusion that oysters of the genus Crassostrea that are farmed in the state of Maranhão are capable of filtering oocysts of T. gondii and maintaining them in their tissues. They are therefore potential sources of contamination for humans and other animals.

Resumo: Objetivou-se com este estudo relatar a detecção do DNA de Toxoplasma gondii em ostras (Crassostrea sp.) no estado do Maranhão. Para a realização do estudo foram adquiridas 200 ostras de cultivo do município de Raposa, e 100 de Paço do Lumiar, além de 100 ostras extraídas de estoque natural do município de Primeira Cruz. Do total de 400 exemplares amostrados, formaram-se 80 pools em que cada pool foi constituído por cinco animais. De cada ostra foi procedida à retirada das brânquias e massa visceral, seguido da extração de DNA de cada pool de ostras, com a utilização de kit comercial. Posteriormente, realizou-se a pesquisa do protozoário por meio da técnica de nested PCR (primer SAG-1). Com a técnica molecular utilizada, foi diagnosticado o DNA do protozoário pesquisado em 2,5% (n=2/80) pools de ostras oriundas exclusivamente de cultivo. Com os resultados obtidos neste estudo, conclui-se que ostras do gênero Crassostrea sp., cultivadas no estado do Maranhão, são capazes de filtrar e manter nos seus tecidos oocistos de T. gondii, sendo, portanto, fontes potenciais de contaminação para seres humanos e outros animais.

Accumulation of oocysts of Cryptosporidium parvum in Biomphalaria glabrata (Pulmonata: Planorbidae) under experimental conditions

Abstract INTRODUCTION: Cryptosporidium oocysts are easily transported to various aquatic environments. The objective of this study was to evaluate B. glabrata mollusks exposed to food containing C. parvum oocysts. METHODS: Six experimental groups were used with B. glabrata either exposed or not to C. parvum oocysts. Microscopic and molecular diagnostics were conducted in water samples and tissues of B. glabrata. RESULTS: By light microscopy, C. parvum oocysts were identified in the water of the exposed groups. C. parvum DNA was not detected in water but was detected in tissue samples. CONCLUSIONS: Further studies should be conducted under natural conditions.

A new Eimeria (Apicomplexa: Eimeriidae), possessing mitra-shaped oocysts, from the Neotropical chelid turtle Batrachemys heliostemma (Testudines: Chelidae), and its comparison with Eimeria mitraria (Laveran & Mesnil 1902)

Eimeria jirkamoraveci sp. n. is described from faeces of two specimens of the toad-headed, side-necked turtle Batrachemys heliostemma collected at Iquitos in Peru. Oocysts are ovoid to almost spherical, 10.6 (8-12) Î 8.9 (7-10) mum, without micropyle, polar granule and oocyst residuum. One conically stretched end and three blunt conical tubercles at the opposite end of oocyst give it mitra-like appearance. Sporocysts are elongated, ellipsoidal, 7.2 (6-8) Î 4.1 (4-4.5) mum, with a small, knob-like Stieda body and sporocyst residuum composed of fine granules. To avoid possible conspecificity, the described new species is thoroughly compared with the most similar coccidium, E. mitraria, collected from its type host, Chinemys reevesii.

Three new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the lesser seed-finch, Oryzoborus angolensis (Passeriformes: Emberizidae) from Brazil

Three new coccidian (Apicomplexa: Eimeriidae) species are reported from the lesser seed-finch, Oryzoborus angolensis from Brazil. Sporulated oocysts of Isospora curio n. sp. are spherical to subspherical; 24.6 Î 23.6 (22-26 Î 22-25) mum, shape-index (SI, length/width) of 1.04 (1.00-1.15). Oocyst wall is bilayerd, ~ 1.5 mum thick, smooth and colourless. Micropyle and oocyst residuum are absent. The sporocysts are ovoid, 13.2 Î 10.9 (15-17 Î 10-13) mum, SI = 1.56 (1.42-1.71), with a small Stieda body and residuum composed of numerous granules scattered among the sporozoites. Sporozoites are elongated and posses a smooth surface and two distinct refractile bodies. Oocysts of Isospora braziliensis n. sp. are spherical to subspherical, 17.8 Î 16.9 (16-19 Î 16-18) mum, with a shape-index of 1.06 (1.00-1.12) and a smooth, single-layered wall ~ 1 mum thick. A micropyle, oocyst residuum and polar granules are absent. Sporocysts are ellipsoid and slightly asymmetric, 13.2 Î 10.8 (12-14 Î 9-12) mum, SI = 1.48 (1.34-1.61). Each sporocyst contains a barely visible Stieda body and a residuum composed numerous of granules. Sporozoites are elongated and each of them contains two distinct refractile bodies. Oocysts of Isospora paranaensis n. sp. are subspherical to broadly ellipsoid 24.3 Î 19.8 (22-26 Î 18-22) mum, SI = 1.22 (1.15-1.38) with smooth single-layered wall ~ 1.5 mum thick. A micropyle and oocyst residuum are absent, but one distinct ellipsoid polar granule (2.5-3.5 Î 1.5-2.5 mum) is present. Sporocyst are ovoid, 15.7 Î 10.1 (14-18 Î 8-12) mum, SI = 1.46 (1.31-1.72), with distinct Stieda and sub-Stieda bodies. Each sporocyst contains a spherical sporocyst residuum, 4 mum in diameter. All described isosporan species represent a possible cause of acute coccidiosis for O. angolensis in captivity.

Cyclosporiasis in an infant.

This report describes cyclosporiasis in a seven month old infant who presented with incessant crying and refusal of feeds. The routine modified ZN stained smears showed the oocysts of Cyclospora when all other tests failed to reveal enteric pathogens. The need for the clinical laboratory to screen faeces samples for all possible pathogens in a given clinical situation needs to be emphasized.

Cryptosporidium and Giardia as determinants for selection of an appropriate source of drinking-water in southern Sri Lanka.

Four different water sources (irrigation canals, small reservoirs, shallow wells, and tubewells), used for domestic purposes, in an irrigated area in southern Sri Lanka, were tested for Giardia spp. cysts and Cryptosporidium spp. oocysts. Identification of these parasites in water sources is important as these are increasingly recognized as causative agents of waterborne diarrhoeal disease. All the four sources of water were contaminated with cysts and oocysts. The sources of surface-water contained a greater number of protozoa compared to tubewells and shallow wells (p < 0.05). The results indicate a reduction of high parasite loads by natural filtration as the water moves from canals to shallow wells through the soil profile. This could present an opportunity to reduce the burden of diarrhoeal disease due to protozoa by selecting an appropriate source of drinking-water and identifying those water sources that require treatment solutions.

Prevalence of cryptosporidiosis in HIV-infected patients in Kajang Hospital, Selangor.

A total of 66 fecal specimens obtained from patients infected with human immunodeficiency virus (HIV) from Kajang Hospital were screened for Cryptosporidium oocysts. The fecal specimens were concentrated using the formalin ethyl acetate concentration technique, stained with modified Ziehl-Neelsen and confirmed with immunofluorescence stain. It was established that 2 (3.0%) were positive for Cryptosporidium. The two cases involved a Chinese local man (with diarrhea) and an Indonesian foreigner (without diarrhea). A higher index of suspicion for clinical cryptosporidiosis in HIV patients, including those with chronic weight loss with or without diarrhea, is recommended. In addition, laboratory testing for Cryptosporidium in HIV-infected patients is highly recommended in order to have a better understanding of the epidemiology and management of the disease in Malaysia.

The Genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil: a review

A review is made of the recorded species of the coccidian genus Cyclospora and major events leading up to the discovery of C. cayetanensis, which is responsible for serious outbreaks of diarrhoea in man and is one of the aetiological agents of "traveller's diarrhoea". Humans appear to be the specific hosts, with the entire life-cycle in the intestine: to date there is no convincing evidence that the disease is a zoonosis. A description is given of oocysts and endogenous stages of C. schneideri n.sp., in the snake Anilius scytale scytale. Sporulation is exogenous and completed after about one week at 24-26°. Mature oocysts 19.8 Î 16.6 (15.1 Î 13.8-25.7 Î 20.1), shape-index 1.2 (1.0-1.3): no oocyst residuum or polar bodies. Oocyst wall a single colourless, smooth layer with no micropyle: it is rapidly deformed or broken. Sporocysts 13.6 Î 9.4 (11.3 Î 8.3-15.1 Î 9.9), shape-index 1.4 (1.2-1.5) with an inconspicuous Stieda body. Sporozoites 11-13 Î 2.5-3. Endogenous stages are intracytoplasmic in the epithelial cells of the small intestine and with the characters of the Eimeriorina.

Three new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the double-collared seed eater, Sporophila caerulescens (Passeriformes: Emberizidae), from Eastern Brazil

Three isosporan species are described from the double-collared seedeater, Sporophila caerulescens from Eastern Brazil. Isospora sporophilae n. sp. oocysts spherical to subspherical; oocyst wall bi-layered, smooth, inner layer colorless to pale yellowish, 21.6 Î 20.9 (19.20-23.20 Î 18.40-22.60) æm, shape-index 1.03 ± 0.02 (1-1.10), with no micropyle or oocyst residuum. Polar bodies splinter-like or comma-like. Sporocysts ovoidal, 15.2 Î 10.6 (17.40-12.80 Î 12.60-8.40) æm, shape-index 1.43 ± 0.14 (1.17-1.81), with knob-like Stieda body and residuum. Large crystalloid body in the center of the sporocyst. Isospora flausinoi n. sp. oocysts spherical to subspherical, oocyst wall bi-layered, smooth, colorless, 17.30 x 16.53 (14-20 Î 13.60-20) æm, shape-index 1.05 ± 0.04 (1-1.21). Micropyle and oocyst residuum absent; presence of a large polar body. Sporocyst piriform, 14.88 x 10.70 (11.80-18 Î 8-12.40) æm, shape-index 1.40 ± 0.18 (1.07-1.77), with smooth, thin, single-layered wall. Sporocyst with rounded Stieda body with no substieda body, and residuum composed of granular material. Isospora teixeirafilhoi n. sp. oocysts spherical to subspherical, oocyst wall bi-layered, smooth, colorless, 17.41 x 16.81 (15.60 19.40 Î 14.20-18.80) æm. Shape-index 1.04 ± 0.08 (1-1.12). Micropyle and oocyst residuum absent; presence of a small double-lobuled polar body. Sporocyst ovoid, 11.74 Î 8.12 (9-14.20 Î 6.20-9.40) æm. Shape-index 1.46 ± 0.23 (1.06-1.88). Sporocyst with knob-like Stieda body, no sub-Stieda body and residuum composed of granular material.

Patterns of shedding of cryptosporidial oocysts by ewes and lambs kept indoors

Este experimento foi realizado com o objetivo de determinar a variação na eliminação de oocistos de Cryptosporidium parvum nas fezes de cordeiros e ovelhas, mantidos confinados do nascimento a desmama, em uma criação localizada em Botucatu -SP. Um grupo de 20 ovelhas da raça Ile de France em final de gestação foi confinado em instalações com piso de concreto. O piso era lavado três vezes por semana e as fezes eram removidas diariamente. O nascimento dos cordeiros ocorreu em Agosto e Setembro / 2001. Amostras de fezes foram colhidas das ovelhas e dos cordeiros no dia do nascimento, 4, 8, 16, 32 e 64 dias pós-parto. As amostras foram processadas pela técnica de centrífugosedimentação em éter. Esfregaços foram confeccionados e corados com auramina O e pela técnica de ZiehI-Neelsen modificada. Do total de amostras de cordeiros e ovelhas, 26,7% e 31,9%, respectivamente (P>O,O5) apresentaram oocistos de Crypostosporidium. Quatro dos 20 cordeiros e duas das 20 ovelhas não apresentaram oocistos em nenhuma das amostras examinadas. O percentual mais alto de amostras positivas ocorreu nas amostras dos cordeiros com quatro dias de vida. Nas ovelhas o maior percentual de amostras positivas foi registrado quatro dias após o parto. Apesar da taxa relativamente elevada de animais que eliminaram oocistos nas fezes, a infecção por C. parvum foi subclínica nas ovelhas e nos cordeiros.

Técnicas de purificación y ruptura de quistes de Giardia spp / Purification and breaking techniques of cysts of Giardia spp

El objetivo de este trabajo fue optimizar y evaluar las técnicas de purificación, aislamiento y ruptura de quistes de Giardia spp a partir de heces formoladas para la obtención de ADN. La materia fecal filtrada fue sometida a 3 técnicas de purificación, utilizando soluciones de formol-éter, sacarosa y formol-éter más sacarosa. La solución de sacarosa permitió aislar los quistes con menos detritos. Los quistes purificados fueron tratados con 3 técnicas para la ruptura de los mismos: shock osmótico y calor, degradación química y shock térmico, acción enzimática y efecto mecánico. Solamente con la técnica de shock térmico, acción enzimática y efecto mecánico se observaron bandas fluorescentes en geles de agarosa. Los resultados de este trabajo permiten contar con una metodología de rutina, simple, que podría ser usada en los pasos previos a la técnica de PCR para la genotipificación de este parásito.

The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.

Padronização e avaliação de métodos moleculares para detecção de ocistos de Cryptosporidium spp (Apicomplexa: Cryptosporiidae) em amostras fecais: extração de DNA genômico e PCR (reação em cadeia pela polimerase) / Standardization and evaluation of molecular methods to detect oocysts of Cryptosporidium spp (Apicomplexa: Cryptosporiidae in faecal samples: extraction of genomic DNA and PCR (polymerase chain reaction)

O protozoário parasita Cryptosporidium parvum tem sido reconhecido como um importante patógeno emergente. Para estudos moleculares, a maioria das técnicas para extração do DNA requer o uso de kits importados para concentrar, romper a parede muito resistente do oocisto e purificar o DNA das matrizes das amostras. O objetivo do estudo foi desenvolver um método simples e rápido, baseado na reação em cadeia pela polimerase (PCR) para detectar Cryptosporidium em fezes preservadas. Oocistos foram concentrados das amostras fecais pela flutuação em gradiente de sacarose. Dos oocistos purificados foi extraído o DNA genômico através de incubação em tampão de lise contendo 70 mM -mercaptoetanol, digerido com proteinase K e extraído com fenol-clorofórmio-álcool isoamílico. A padronização foi iniciada com a PCR única para detectar Cryptosporidium spp usando um par de primer genérico (AWA). Para identificar C.parvum foi realizada a PCR única com o par de primer específico (LAX). Para aumentar a sensibilidade do método, foi atestada a nested-PCR, usando o primer externo XIA. Foram analisadas 39 amostras de DNA do bezerro padrão, 52 amostras de 17 pacientes e 45 amostras de 14 animais. Os resultados foram: 54,28 por cento de positividade na PCR AWA e, 71,42 por cento na nested-PCR XIA/AWA, 67,74 por cento na PCR LAX e 44,44 por cento na nested-PCR XIA/LAX das amostras do bezerro. A positividade geral nas amostras de pacientes e de animais foi: 34,48 por cento pela PCR and 54,83 por cento pela nested-PCR para Cryptosporidium spp e 16,00 por cento pela PCR e 50,00 por cento pela nested-PCR para C. parvum. O emprego do corante Vistra Green melhorou significativamente a visualização do gel. Os resultados levam a supor que este método simples e de baixo custo pode fornecer melhores resultados quando aplicado a amostras frescas, na rotina do laboratório.

Intestinal parasitic infections in Bekasi district, West Java, Indonesia and a comparison of the infection rates determined by different techniques for fecal examination.

This study was undertaken to determine the current status of intestinal parasitic infections among schoolchildren in West Java, Indonesia, and to compare the infection rates obtained by three different methods of fecal examination. A total of 285 fecal samples were collected from 131 males and 154 females at a junior high school. Samples were brought to the Department of Parasitology, Faculty of Medicine, University of Indonesia, and were examined for parasites by the Kato-Katz thick smear method (K-K). The residual samples were suspended in more than five volumes of 2% potassium dichromate solution and brought to the Department of Parasitology, Kobe University School of Medicine, Japan, where they were examined for parasites by the Army Medical School method (AMS III) and by the Sucrose Centrifugal Flotation method (SFL). The K-K revealed a total of two helminths with a prevalence of 10% (29/285). In contrast, nine species of parasites, 31% (89/285) positive, were obtained by AMS III, while 10 species, 22% (62/285) were found by SFL. Overall, 12 species of parasites were detected by the three methods: four species of nematoda (Trichuris trichiura, Ascaris lumbricoides, hookworm, and Enterobius vermicularis); five species of protozoa (Giardia intestinalis, Entamoeba histolytica-like cyst, E. coli, Cyclospora sp, Blastocystis hominis); two unidentified species of nematode eggs; and one unidentified species of mite egg.