Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

In vitro and ex vivo activity of Melaleuca alternifolia against protoscoleces of Echinococcus ortleppi.

Cystic echinococcosis is a zoonotic disease of difficult diagnosis and treatment. The use of protoscolicidal agents in procedures is of utmost importance for treatment success. This study was aimed at analysing the in vitro and ex vivo activity of Melaleuca alternifolia oil (tea tree oil - TTO), its nanoemulsion formulation (NE-TTO) and its major component (terpinen-4-ol) against Echinococcus ortleppi protoscoleces obtained from cattle. Concentrations of 2·5, 5 and 10 mg mL-1 of TTO, 10 mg mL-1 of NE-TTO and 1, 1·5 and 2 mg mL-1 of terpinen-4-ol were evaluated in vitro against protoscoleces at 5, 10, 15 and 30 min. TTO was also injected directly into hydatid cysts (ex vivo analysis, n = 20) and the viability of protoscoleces was evaluated at 5, 15 and 30 min. The results indicated protoscolicidal effect at all tested formulations and concentrations. Terpinen-4-ol (2 mg mL-1) activity was superior when compared with the highest concentration of TTO. NE-TTO reached a gradual protoscolicidal effect. TTO at 20 mg mL-1 showed 90% protoscolicidal action in hydatid cysts at 5 min. The results showed that TTO affects the viability of E. ortleppi protoscoleces, suggesting a new protoscolicidal option to the treatment of cystic equinococcosis.

Echinococcus granulosus sensu stricto, Echinococcus canadensis (G7), and Echinococcus ortleppi in fertile hydatid cysts isolated from cattle in Southern Brazil.

Echinococcosis is a cosmopolitan zoonotic infection that affects humans and animals. The aim of this study was to identify and characterize the fertile hydatid cysts from bovine viscera in order to verify different species and/or genotypes present in Southern Brazil. Firstly, cysts were collected from a slaughterhouse, which received animals from different regions of Rio Grande do Sul State (RS), considered an important area of occurrence of cystic echinococcosis. In total, 2396 cysts were analyzed by microscopy to verify the presence of protoscoleces. Protoscoleces were detected in 291 samples and were classified as fertile hydatid cysts. Total DNA was extracted from protoscoleces and amplified by polymerase chain reaction (PCR). Two hundred and fifty-one samples were identified by PCR and characterized as G5/G6/G7 genotypes, of which 40 belonged to Echinococcus granulosus sensu stricto (G1-G3). PCR was also performed, using G5-specific primers to identify 250 samples as Echinococcus ortleppi (G5). Only one sample was identified as Echinococcus canadensis (G7) by DNA sequencing using primers specific for the coxI gene. Phylogenetic analysis was also performed and identified three distinct groups E1 (G5), E2 (G7), and E3 (G1-G3), which were grouped according to similarity of their sequences. The study highlights the fact that E. granulosus sensu stricto, E. ortleppi, and E. canadensis (G7) were infecting cattle in RS, emphasizing the adaptation of different species of Echinococcus to this intermediate host.

Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil.

This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil / Echinococcus granulosus sensu lato e Taenia hydatigena em suínos no sul do Brasil

This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

Neste estudo, buscou-se identificar os agentes etiológicos de origem parasitária em cistos nas vísceras de suínos oriundos sa região centro/norte do sEstado do Rio Grande do Sul, Brasil. Cinquenta e oito cistos foram encontrados durante a inspeção veterinária em fígados de suínos abatidos entre janeiro de 2008 a 2012. As amostras foram submetidas às análises macroscópicas e moleculares. Reação em cadeia da polimerase (PCR), sequenciamento de DNA e alinhamento das sequências no BLAST (bases de dados do GenBank) foram utilizados na caracterização molecular das amostras. Das amostras analisadas, 10,3% (6/58) foram positivas para Echinococcus granulosus sensu lato e 56,9% (33/58) para Cysticercus tenuicollis. Evidenciou-se a ocorrência de larvas de E. granulosus sensu lato e Taenia hydatigena em rebanhos suínos na região centro/norte do RS. A presença de ambos os agentes é relevante devido às perdas econômicas para a indústria de carnes. Além disso, destaca-se que E. granulosus sensu lato possui importância zoonótica e pode contaminar os rebanhos suínos no sul do Brasil.

Influence of Toxoplasma gondii acute infection on cholinesterase activities of Wistar rats.

Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.

In vitro influence of temperature on the biological control activity of the fungus Duddingtonia flagrans against Haemonchus contortus in sheep.

Recently, research for alternative methods to combat gastrointestinal parasites has increased, and the biological control activity of the fungus Duddingtonia flagrans stands out. In this study, the possible influence of temperature on the nematophagous activity of D. flagrans, after gastrointestinal passage, against Haemonchus contortus in sheep was analysed. Four female sheep, between 2 and 3 years of age and weighing between 40 and 50 kg, were used. Two sheep were parasitised with H. contortus, while two other sheep were dewormed. Before the collection of faeces, one of the dewormed animals received a dosage of 1 × 10(6) chlamydospores of D. flagrans, lyophilised in gelatin capsules, for three consecutive days. The faeces were collected with collector bags, mixed, and then separated as samples with (fungus; 800 eggs per gram (EPG) of faeces) or without fungus (control; 900 EPG). Each sample (five replicates) was maintained in a biochemical oxygen demand incubator under different temperatures (5, 10, 15, 20, 25, 30, or 35 °C) for 21 days, followed by determination of the larval recovery. Compared to the control group, the best temperature for fungal action was 30 °C, while no larvae were recovered at 5 °C. At 10 °C, fungal action was detected, yet there was no significant difference in the percent larval reduction between all temperatures, demonstrating that larval presence seems to be the main factor affecting the nematophagous action of D. flagrans. Temperature does not appear to be a limiting factor in the biological control activity of D. flagrans against H. contortus, but larval presence, which was not observed at 5 °C, is mandatory. At low temperatures, which are typically suboptimal conditions for fungal and larval development, the lyophilised D. flagrans reduced the number of H. contortus larvae, which demonstrates the biological control potential and the potential use of D. flagrans in the subtropics.

Predatory activity of the fungus Duddingtonia flagrans in equine strongyle infective larvae on natural pasture in the Southern Region of Brazil.

Biological control is an alternative method to reduce the population of parasites through natural predators. A promising option of biological control in the reduction of infective larvae on pasture is the use of nematophagous fungi. In this study, the efficacy of the nematophagous fungus Duddingtonia flagrans in controlling gastrointestinal nematode parasites in field-raised horses was tested. Ten foals with an average age of 12 months were divided in two groups: five males constituted the treated group and five females constituted the control group. Each group was introduced in a field of mixed pasture with approximately 5 ha. The treated group received the fungus D. flagrans at a concentration of 10(6) chlamydospores per kilogramme of animal body weight daily, mixed with horse food for 5 months. The control group did not receive the fungus. Samples were collected to perform eggs per gramme (EPG) counts weekly. Coproculture and collection of pasture were done monthly for larvae counting. No significant difference was observed in the EPG counting and in the number of larvae recovered from coprocultures, where cyathostomines, Strongylus and Trichostrongylus spp. were found after monthly larvae counting. No significant difference was observed in the EPG counts, and Trichostrongylus sp. was identified. The number of recovered larvae on pasture was significantly lower in the treated group in the last month of treatment, showing a reduction of 73.5% (p < 0.05). As such, the fungus was able to reduce the number of infective larvae in the pasture. Nevertheless, this did not reflect in a decrease of parasitic infection during the 5-month study period.

Duddingtonia flagrans: controle biológico de nematodeos de bovinos a campo / Duddingtonia flagrans: biological control of cattle nematodes in the field

O controle biológico é um método para diminuir uma população pela utilização de antagonista natural. No presente estudo, testou-se a eficácia do fungo nematófago Duddingtonia flagrans no controle de nematódeos parasitos gastrintestinais de bovinos criados à campo no município de Júlio de Castilhos. Foram utilizados 20 bezerros, distribuídos igualmente em duas áreas formadas por pastagem nativa. O grupo A foi tratado com o fungo D. flagrans, cultivado em sorgo, numa concentração de 1x10(6)clamidósporos kg-1 de peso animal, misturados em ração de manutenção, diariamente, durante oito meses. O grupo B serviu como controle e não recebeu fungo, apenas ração. Foram coletadas amostras para contagem de ovos por grama de fezes (OPG) semanalmente. Mensalmente, foram realizadas coproculturas para identificar as espécies de larvas de nematódeos, a pesagem dos animais e a coleta de pasto para contagem das larvas na pastagem. Dados de temperatura e índice pluviométrico foram registrados diariamente. O OPG foi reduzido no grupo tratado, em média 56,8, por cento nos últimos três meses de experimento, variando entre 40,4 e 67,1 por cento no grupo tratado (P<0,001). A coprocultura demonstrou que os principais nematódeos encontrados em ambos os grupos foram dos gêneros Cooperia e Haemonchus. A contagem de larvas na pastagem obteve um percentual de redução 77,1 por cento no grupo tratado ao final do experimento (P<0,01). Pôde-se concluir com este estudo que o papel do fungo Duddingtonia flagrans, é, sem dúvida, importante, principalmente, na diminuição do OPG e na redução significativa de larvas na pastagem. Portanto, este fungo nematófago é uma ferramenta biológica eficaz para ser empregado em um controle integrado de nematódeos de bovinos criados a campo.

Biological control is an alternative method to reduce parasite's population by the use of natural antagonist. In the present study, efficacy of nematophagous fungus Duddingtonia flagrans was tested to control gastrointestinal nematodes parasites of cattle livestock in the field. Twenty calves were used, distributed equally in two distinct plots formed by native pasture. Group A was treated with D. flagrans fungus, cultivated in sorghum (1x10(6) clamidospores kg-1 body weight) mixed with maintenance ration, each day, during eight months. Group B served as a control and did not receive the fungus. Samples for faecal egg count (FEC), were collected each week. There were monthly counts in faecal cultures to identify the species of nematodes larvae, animals weight, blood collection to determine red cell counts and collection of pasture to larvae counting. Temperature and rainfall data were registered daily. The FEC reduced around 56.8 percent in the last three months of the experiment, with a variation between 40.4 and 67.1 percent in the treated group (P<0.001). The faecal cultures demonstrated that the main nematodes found in both the groups were Cooperia and Haemonchus. Larvae counting in the pasture showed a reduction percentage around 77.1 percent in treated group at the end of experiment (P<0.01). It could be concluded with this study, that Duddingtonia flagrans has an important role in the reduction of FEC and significant reduction of larvae in the pasture. Therefore, this nematophagous fungus is efficient as a biological tool to be used in an integrated nematodes control of bovine raised in the field.

Echinococcosis in southern Brazil: efforts toward implementation of a control program in Santana do Livramento, Rio Grande do Sul.

This investigation aimed to design a strategy for echinococcosis control in Santana do Livramento county, an endemic area in state of Rio Grande do Sul (Brazil). Fecal samples from 65 dogs were obtained from urban, suburban and rural areas. Purging with Arecoline Bromhidrate (AB) was done to visualize Echinococcus granulosus, and Enzyme Linked Immunosorbent Assay (ELISA) was performed to detect parasite coproantigen. Samples were obtained at the beginning and at the end of treatment with Praziquantel. A third fecal sampling was also done in rural areas four months after the end of treatment. Each dog was treated immediately after the first purging and every 30 days for eight months. In urban and suburban areas no infected dogs were found. In rural areas, first evaluation showed 11.36% and 27.69% of infected dogs by AB and ELISA, respectively. No infected dogs were diagnosed in the second evaluation and in the third evaluation 36.84% and 47.37% infected dogs were identified by AB and ELISA, respectively. Medication program to combat dog infection resulted in successful interruption of parasite transmission, but the project failed to create awareness of the need for dog prophylaxis among rural populations as well as to establish a permanent control program in this municipality.

Echinococcosis in southern Brazil: efforts toward implementation of a control program in Santana do Livramento, Rio Grande do Sul

Este trabalho objetivou implementar um programa de controle da equinococose no município de Santana do Livramento, área endêmica no Estado do Rio Grande do Sul (Brasil). Amostras fecais de 65 cães foram coletadas em áreas urbanas, suburbanas e rurais. Para visualizar o Echinococcus granulosus foi realizada purgação com Bromhidrato de Arecolina (BA) e para identificar coproantígenos parasitários foi empregado um método imunoenzimático (ELISA). As amostras foram coletadas no começo e ao fim do tratamento com Praziquantel. Nas áreas rurais uma terceira coleta foi feita quatro meses após o fim do tratamento. Cada cão foi tratado no começo e a cada trinta dias durante 8 meses. Nas áreas urbanas e suburbanas nenhum cão parasitado foi identificado. Nas áreas rurais estavam parasitados 11,36 e 27,69 dos cães, por BA e ELISA, respectivamente. Na terceira avaliação, 36,84 e 47,37 dos cães estavam infectados, por BA e ELISA, respectivamente. Observou-se que um programa a base de drogas anti-parasitárias resultou em interrupção eficiente na transmissão parasitária, mas o projeto falhou em criar uma mentalidade consciente nos habitantes da zona rural relacionada a profilaxia, bem como em estabelecer um programa permanente de controle desta parasitose no município.