RESUMO
Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.
Assuntos
Fezes/parasitologia , Doenças das Cabras/parasitologia , Hemoncose/veterinária , Haemonchus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Primers do DNA/genética , Doenças das Cabras/diagnóstico , Cabras , Hemoncose/diagnóstico , Hemoncose/parasitologia , Haemonchus/classificação , Haemonchus/genética , Sensibilidade e EspecificidadeRESUMO
Toxoplasmosis is a globally spread zoonosis. The pathogen Toxoplasma gondii can hijack cellular organelles of host for replication. Although a number of important cellular life events are controlled by cell organelles, very little is known of the transcriptional changes of host cellular organelles after infection with T. gondii. Herein, we performed RNA-sequencing (RNA-seq) and bioinformatics analyses to study the global organelle component changes. It was found that many transcripts of the mouse spleen cellular organelle components were altered by acute T. gondii infection with the RH strain (Type I). Most differentially expressed transcripts of mitochondrial components were downregulated, especially those involved in biosynthetic and metabolic processes. Moreover, mitochondria based apoptosis process was downregulated. In terms of cytoskeleton, most differentially expressed transcript of cytoskeleton components were also downregulated, including septin cytoskeleton, cytoskeleton organization, centrosome and myosin. For endolysosomal system, ion transporters were downregulated at mRNA level, whereas the cytolytic components were increased, such as granzymes, Rab27a and perforin1 (Prf1). The main transcripts of Golgi apparatus components involved in sialylation or vesicle-mediated transportation were downregulated, while immune related components were upregulated. For endoplasmic reticulum (ER), posttranslational modification, drug metabolism and material transportation related transcripts were downregulated. In addition, T. gondii antigen cross-presentation by MHC-I complex could be downregulated by the downregulation of CD76 and ubiquitination related transcripts. The present study, for the first time, described the transcriptional changes of the mouse spleen cellular organelles following acute T. gondii infection, which provides a foundation to study the interaction between T. gondii and host cells at the sub-cellular level.
Assuntos
Organelas/metabolismo , Baço/metabolismo , Toxoplasmose Animal/metabolismo , Animais , Apoptose , Biologia Computacional , Citoesqueleto/metabolismo , Regulação para Baixo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Metabolismo Energético , Expressão Gênica , Complexo de Golgi/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Organelas/parasitologia , Organelas/patologia , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Baço/parasitologia , Baço/patologia , Baço/ultraestrutura , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/patologia , Transcriptoma , Regulação para CimaRESUMO
Cryptosporidium is a widespread protozoan parasite that infects a large number of vertebrate animals, resulting in varying degrees of diarrhea or even death. As dairy cattle feces is an important source of Cryptosporidium spp. infection, development of a handy and accurate detection method via its oocysts in dairy cattle feces would be interesting and necessary. We herein developed a quick detecting method using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip to detect DNA of Cryptosporidium oocysts in dairy cattle feces. The DNA was released by boiled water with 0.1 % N-lauroylsarcosine sodium salt (LSS). The established method was proven to be of higher sensitivity than normal polymerase chain reaction (PCR) amplification with the lowest detection of 0.5 oocyst per reaction, and specificity with no cross reactivity to other common protozoan species in the intestine of dairy cattle. The diagnostic method established herein is simple, rapid, and cost-effective, and has potential for further development as a diagnostic kit for the diagnosis of cryptosporidiosis of dairy cattle.
Assuntos
Doenças dos Bovinos/diagnóstico , Criptosporidiose/diagnóstico , Cryptosporidium/genética , DNA de Protozoário/genética , Fezes/parasitologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Oocistos/citologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sarcosina/análogos & derivados , Sensibilidade e EspecificidadeRESUMO
Toxoplasmosis, caused by Toxoplasma gondii, is a parasitic zoonosis with worldwide distribution. The present study investigated the prevalence of T. gondii in dogs in Zhanjiang city, southern China, using both serological and molecular detection. A total of 364 serum samples and 432 liver tissue samples were collected from the slaughter house between December 2012 and January 2013 and were examined for T. gondii IgG antibody by ELISA and T. gondii DNA by semi-nested PCR based on B1 gene, respectively. The overall seroprevalence of T. gondii IgG antibody was 51.9%, and T. gondii DNA was detected in 37 of 432 (8.6%) liver tissue samples. These positive DNA samples were analyzed by PCR-RFLP at 3'- and 5'-SAG2. Only 8 samples gave the PCR-RFLP data, and they were all classified as type I, which may suggest that the T. gondii isolates from dogs in Zhanjiang city may represent type I or type I variant. This study revealed the high prevalence of T. gondii infection in dogs in Zhanjiang city, southern China. Integrated measures should be taken to prevent and control toxoplasmosis in dogs in this area for public health concern.
Assuntos
Doenças do Cão/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , China/epidemiologia , Doenças do Cão/epidemiologia , Cães , Feminino , Genótipo , Fígado/parasitologia , Masculino , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose Animal/sangue , Toxoplasmose Animal/epidemiologiaRESUMO
MicroRNA-36 (miR-36) is a recently discovered miRNA family which including at least eight members, and specifically existed in helminths compared with other miRNAs that widely exists in almost all kinds of lives. This paper reviews recent research advances about miR-36 to provide further fundamental information for helminth and miRNA study.
Assuntos
Helmintos/genética , MicroRNAs , AnimaisRESUMO
Toxoplasma gondii is a protozoan parasite infecting almost all warm-blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I-RH strain, Type II-PRU strain, Type II-TgQHO strain, and ToxoDB 9-TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI-TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.
Assuntos
Proteoma/análise , Proteômica/métodos , Proteínas de Protozoários/análise , Toxoplasma/química , Toxoplasma/genética , Genótipo , Estágios do Ciclo de Vida , Espectrometria de Massas , Redes e Vias Metabólicas , Proteoma/genética , Proteínas de Protozoários/genética , Espectrometria de Fluorescência , Eletroforese em Gel Diferencial BidimensionalRESUMO
BACKGROUND: The parasitic nematodes Ascaris lumbricoides and A. suum are of great public health and economic significance, and the two taxa were proposed to represent a single species. miRNAs are known with functions of gene regulations at post-transcriptional level. RESULTS: We herein compared the miRNA profiles of A. lumbricoides and A. suum female adults by Solexa deep sequencing combined with bioinformatics analysis and stem-loop real-time PCR. Using the A. suum genome as the reference genome, we obtained 171 and 494 miRNA candidates from A. lumbricoides and A. suum, respectively. Among which, 74 miRNAs were shared between the two taxa, 97 and 420 miRNAs were A. lumbricoides and A. suum specific. Target and function prediction revealed a significant set of targets which are related to ovarian message protein, vitellogenin and chondroitin proteoglycan of the two nematodes. Enrichment analysis revealed that the percentages of most predicted functions of the miRNA targets were similar, with some taxon specific or taxon enhanced functions, such as different target numbers, specific functions (NADH dehydrogenase and electron carrier functions), etc. CONCLUSIONS: This study characterized comparatively the miRNAs of adult A. lumbricoides and A. suum, and the findings provide additional evidence that A. lumbricoides and A. suum represent a single species. Due to the fast evolution nature of miRNAs and the different parasitic living conditions of humans and pigs, the phenomenon above might indicate a fast evolution of miRNAs of Ascaris in humans and pigs.
Assuntos
Ascaris lumbricoides/metabolismo , Ascaris suum/metabolismo , MicroRNAs/metabolismo , Transcriptoma , Animais , Ascaris lumbricoides/genética , Ascaris suum/genética , Feminino , Regulação da Expressão Gênica , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Currently, microRNAs (miRNAs) are known to regulate cellular processes such as apoptosis, differentiation, cell cycle, and immune functions, and their expression can be altered by distinct stress conditions, such as oxidative stress. In immune systems of fish, vitamin E (VE) has a defined role as an antioxidant. In order to understand the molecular mechanism of vitamin E defending from oxidative stress, three groups of juvenile Nile tilapia (Oreochromis niloticus) (initial weight 3.25 ± 0.02 g) were fed to satiation with 3 semi-purified diets containing VE (DL-α-tocopherol acetate) of 0, 50, and 2500 mg/kg supplementation, respectively, with the expressions of eight miRNAs (miR-21, miR-223, miR-146a, miR-125b, miR-181a, miR-16, miR-155 and miR-122) in the liver of tilapia subsequently detected after 8-week growth experiment. Results showed that VE-deficient (0 mg/kg supplementation) decreased the activity of superoxide dismutase (SOD), and decreased the expressions of miR-223, miR-146a, miR-16 and miR-122, while excessive supplementation of VE (2500 mg/kg) decreased SOD activity and increased the expressions of all the eight miRNAs. The targets of the eight miRNAs were further predicated with bioinformatic approach and the possible regulating mechanisms of VE via miRNAs were analyzed. The present study confirmed that the differences in dietary VE affected expression of hepatic miRNAs which may partly demonstrate the molecular mechanism of VE, and the new idea of introducing miRNAs into research will provide the basic data for researches of molecular nutrition.
Assuntos
Antioxidantes/administração & dosagem , Ciclídeos/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Estresse Oxidativo/efeitos dos fármacos , Vitamina E/administração & dosagem , Ração Animal/análise , Animais , Ciclídeos/metabolismo , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Fígado/enzimologia , Fígado/metabolismo , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Chlamydiaceae is a family of obligate intracellular pathogens with a worldwide distribution in many animal species, including humans. No information exists on the prevalence of Chlamydia felis infections in cats and dogs in Lanzhou, the geographical center of China. The aim of this study was to carry out a census of cats and dogs in Lanzhou and document the seroprevalence of C. felis exposure in these companion animals. RESULTS: In this study, blood samples were collected from 485 animals (221 cats and 264 pet dogs) in Lanzhou between November 2010 and July 2011 to identify antibodies against C. felis. Thirteen of 221 (5.9%) cats and 32 of 264 (12.1%) pet dogs were positive for C. felis infection using indirect hemagglutination at a cutoff of 1:16. The seroprevalence in household and stray cats was 3.9% and 14.3%, respectively, and the difference was statistically significant (P < 0.05). Among different age groups, the seroprevalence in cats varied from 1.9 to 7.9%, and that in dogs ranged from 9.6 to 20.4%; however, the differences were not statistically significant (P > 0.05). CONCLUSIONS: This is the first report of the seroprevalence of C. felis exposure in cats and dogs in Lanzhou, northwestern China. Our results indicate that the presence of C. felis exposure in cats and dogs may pose a potential threat to human health.
Assuntos
Doenças do Gato/microbiologia , Infecções por Chlamydia/veterinária , Chlamydia , Doenças do Cão/microbiologia , Fatores Etários , Animais , Doenças do Gato/epidemiologia , Gatos , China/epidemiologia , Infecções por Chlamydia/epidemiologia , Doenças do Cão/epidemiologia , Cães , Feminino , Testes de Hemaglutinação/veterinária , Masculino , Animais de Estimação/microbiologia , Estudos Soroepidemiológicos , Fatores SexuaisRESUMO
Ascaris nematodes, which cause ascariasis in humans and pigs, are among the most important nematodes from both health and economic perspectives. microRNA (miRNA) is now recognized as key regulator of gene expression at posttranscription level. The public availability of the genome and transcripts of Ascaris suum provides powerful resources for the research of miRNA profiles of the parasite. Therefore, we investigated and compared the miRNA profiles of male and female adult A. suum using Solexa deep sequencing combined with bioinformatic analysis and stem-loop reverse transcription polymerase chain reaction. Deep sequencing of small RNAs yielded 11.71 and 11.72 million raw reads from male and female adults of A. suum, respectively. Analysis showed that the noncoding RNA of the two genders, including tRNA, rRNA, snRNA, and snoRNA, were similar. By mapping to the A. suum genome, we obtained 494 and 505 miRNA candidates from the female and male parasite, respectively, and 87 and 82 of miRNA candidates were consistent with A. suum miRNAs deposited in the miRBase database. Among the miRNA candidates, 154 were shared by the two genders, and 340 and 351 were female and male specific with their target numbers ranged from one to thousands, respectively. Functional prediction revealed a set of elongation factors, heat shock proteins, and growth factors from the targets of gender-specific miRNAs, which were essential for the development of the parasite. Moreover, major sperm protein and nematode sperm cell motility protein were found in targets of the male-specific miRNAs. Ovarian message protein was found in targets of the female-specific miRNAs. Enrichment analysis revealed significant differences among Gene Ontology terms of miRNA targets of the two genders, such as electron carrier and biological adhesion process. The regulating functions of gender-specific miRNAs was therefore not only related to the fundamental functions of cells but also were essential to the germ development of the parasite. The present study provides a framework for further research of Ascaris miRNAs, and consequently leads to the development of potential nucleotide vaccines against Ascaris of human and animal health significance.
Assuntos
Ascaris suum/genética , MicroRNAs/análise , MicroRNAs/genética , Animais , Biologia Computacional , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Análise de Sequência de DNA , SexoRESUMO
Chlamydia spp. are obligate intracellular gram-negative bacteria that cause a wide range of significant diseases in humans and animals worldwide, resulting in significant economic losses. Chlamydial infection in cattle has been reported in many countries including China. However, there has been no survey of chlamydial infection of dairy cattle in Guangzhou, southern China. The objective of the present investigation was to examine the chlamydial seroprevalence in dairy cattle in Guangzhou, subtropical southern China by using an indirect hemagglutination assay (IHA). The overall seroprevalence of chlamydial infection in dairy cattle was 7.25% (29/400). Greater than or equal to eight-yr-old dairy cattle had the highest seroprevalence (10.34%), followed by those that were ≥ 6 years old or < 7 years old dairy cattle (10.20%), although there were no statistically significant differences among different groups (P > 0.05). Dairy cattle with 5 pregnancies had the highest seroprevalence (10.81%). These results indicate that chlamydial infection was present in dairy cattle in Guangzhou, subtropical southern China, and integrated strategies and measures should be executed to control and prevent chlamydial infection and disease outbreak in the study region.
RESUMO
Interleukin-15 (IL-15) is a pro-inflammatory cytokine and also a chemotactic factor for a variety of immune cells, which participates in and regulates the inflammatory response and immune response to organisms. Up to now, the molecular structure and receptor of IL-15 have been widely researched, with important advances in its signal transduction way. However, its application in parasitology is still a relatively new topic. This article summarizes the latest research development of IL-15 in anti-parasitic infection, prospects its mechanism and application.
Assuntos
Interleucina-15/imunologia , Interleucina-15/farmacologia , Interleucina-15/uso terapêutico , Animais , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Helmintíase/tratamento farmacológico , Helmintíase/imunologia , Helmintos/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Doenças Parasitárias/tratamento farmacológico , Doenças Parasitárias/imunologia , Infecções por Protozoários/tratamento farmacológico , Infecções por Protozoários/imunologiaRESUMO
Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.
Assuntos
Angiostrongylus cantonensis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Caramujos/parasitologia , Angiostrongylus cantonensis/patogenicidade , Animais , Primers do DNA/química , DNA de Helmintos/química , DNA Ribossômico/química , Larva/parasitologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Infecções por Strongylida/diagnóstico , Infecções por Strongylida/prevenção & controleRESUMO
Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1×10(3) tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P<0.05) increased survival time (12.7±1.2days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3±0.9days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.
Assuntos
Perforina/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias , Toxoplasma/imunologia , Toxoplasmose Animal/prevenção & controle , Vacinas de DNA , Animais , Anticorpos Antiprotozoários/sangue , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Imunoglobulina G/sangue , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , Perforina/genética , Proteínas de Protozoários/genética , Vacinas Protozoárias/genética , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/imunologia , Toxoplasma/genética , Toxoplasmose Animal/imunologia , TransfecçãoRESUMO
Ascaris suum is an important parasite of pigs that causes tremendous economic losses globally to agriculture and animal husbandry annually. RNA interference (RNAi) technology has been described as a successful and useful approach for the elucidation of gene function in parasitic nematodes. In the present study, RNAi was used to silence the expression of a gene encoding enolase in A. suum by soaking infective larvae in double-stranded RNA derived from an EST (representing As-enol-1) selected from an A. suum infective larvae-specific cDNA library. The mRNA levels of RNAi-treated larvae were examined by Reverse-Transcription PCR (RT-PCR) analysis. The survival of RNAi-treated larvae was compared with larvae treated with dsRNA-free culture medium. The effect of enolase depletion on the development of A. suum larvae was assessed by infecting BALB/c mice with RNAi-treated larvae. The results showed that enolase gene expression was silenced completely and the survival rate of the RNAi-treated nematodes was reduced by 20.11% (P<0.01) after soaking for 72 h. Although no significant difference was detected in the numbers of larvae recovered from the liver and lungs of infected mice 4 days post infection, RNAi knockdown of the A. suum enolase mRNA led to significant shorter larvae, indicating that loss of enolase expression may cause delays in larval development.
Assuntos
Ascaris suum/enzimologia , Ascaris suum/genética , Regulação Enzimológica da Expressão Gênica/genética , Fosfopiruvato Hidratase/genética , Interferência de RNA , Animais , Ascaríase/parasitologia , Ascaris suum/crescimento & desenvolvimento , Feminino , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Fígado/parasitologia , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , SuínosRESUMO
Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.
Assuntos
Angiostrongylus cantonensis/genética , MicroRNAs/isolamento & purificação , Animais , Biologia Computacional , Feminino , Expressão Gênica , Masculino , MicroRNAs/genética , RNA de Helmintos/genética , RNA de Helmintos/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Fatores SexuaisRESUMO
BACKGROUND: Clonorchis sinensis is a zoonotic parasite causing clonorchiasis-associated human disease such as biliary calculi, cholecystitis, liver cirrhosis, and it is currently classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs) are non-coding, regulating small RNA molecules which are essential for the complex life cycles of parasites and are involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing and bioinformatic predictions with stem-loop real-time PCR analysis. RESULTS: Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belonged to 284 families. There was strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil was the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and end of conserved miRNAs. There was no significant "seed region" at the first and ninth positions which were commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis were still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There were two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping stringent conserved seed regions with high active innovation in other places of miRNAs mainly in the middle and the end, which were perfect for the parasite to perform its complex life style and for host changes. CONCLUSIONS: The present study represented the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as miRNA studies of other related species such as Opisthorchis viverrini and Opisthorchis felineus of human and animal health significance.
Assuntos
Clonorchis sinensis/genética , Saúde , MicroRNAs/genética , RNA de Helmintos/genética , Animais , Sequência de Bases , Viés , Sequência Conservada/genética , Evolução Molecular , Humanos , MicroRNAs/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Toxoplasma gondii is a worldwide prevalent pathogen that infects most of the warm-blood vertebrates. To investigate the regulation network of splenic miRNAs altered by acute infection with T. gondii, we herein investigated the changes of miRNA profile in mouse spleen via next generation sequencing and bioinformatics analysis. A total of 379 miRNAs was identified, 131 miRNAs of them were differentially expressed (including 97 upregulated and 34 downregulated miRNAs). 48 differentially expressed miRNAs had validated targets in the miRWalk2.0 database. Gene Ontology (GO) enrichment analysis revealed that the validated targets of differently expressed miRNAs were significantly enriched in gene transcription regulation. It suggested that T. gondii can modulate host gene expression through targeting to trans-regulation factors. The genes involved in apoptosis or anti-apoptosis were both targeted by differentially expressed miRNAs. The change of power balance between the miRNAs targeting host apoptosis genes and those regulating host anti-apoptosis genes contributes to the fate of host apoptosis process. Twelve pathways were significantly enriched in KEGG analysis with most of them being cancer related, including pathways in cancer, pancreatic cancer, colorectal cancer, axon guidance, MAPK signaling pathway, focal adhesion, chronic myeloid leukemia, renal cell carcinoma, prostate cancer, glioma, regulation of actin cytoskeleton, and Wnt signaling pathway. Our study showed a changed miRNA regulation network in mouse spleen infected by T. gondii. These findings will be helpful for better understanding of miRNA regulation network in host-T. gondii interaction, revealing the relationship among T. gondii infection, gene regulation, apoptosis and cancer process alterations in infected spleen.
Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Baço/parasitologia , Toxoplasmose/genética , Animais , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Análise de Sequência de RNA/métodosRESUMO
Eurytrema pancreaticum is one of the most common trematodes living in the pancreatic and bile ducts of ruminants and also occasionally infects humans, causing eurytremiasis. In spite of its economic and medical importance, very little is known about the genomic resources of this parasite. Herein, we performed de novo sequencing, assembly and characterization of the transcriptome of adult E. pancreaticum. Approximately 36.4 million high-quality clean reads were obtained, and the length of the transcript contigs ranged from 66 to 19,968 nt with mean length of 479 nt and N50 length of 1094 nt, and then 23,573 unigenes were assembled. Of these unigenes, 15,353 (65.1%) were annotated by blast searches against the NCBI non-redundant protein database. Among these, 15,267 (64.8%), 2732 (11.6%) and 10,354 (43.9%) of the unigenes had significant similarity with proteins in the NR, NT and Swiss-Prot databases, respectively. 5510 (23.4%) and 4567 (19.4%) unigenes were assigned to GO and COG, respectively. 8886 (37.7%) unigenes were identified and mapped onto 254 pathways in the KEGG Pathway database. Furthermore, we found that 105 (1.18%) unigenes were related to pancreatic secretion and 61 (0.7%) to pancreatic cancer. The present study represents the first transcriptome of any members of the family Dicrocoeliidae, which has little genomic information available in the public databases. The novel transcriptome of E. pancreaticum should provide a useful resource for designing new strategies against pancreatic flukes and other trematodes of human and animal health significance.
Assuntos
Dicrocoeliidae/genética , Transcriptoma , Animais , Bases de Dados de Proteínas , Dicrocoeliidae/patogenicidade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Repetições de Microssatélites , Anotação de Sequência Molecular , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/parasitologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Schistosoma turkestanicum is a parasite of considerable veterinary importance as an agent of animal schistosomiasis in many countries, including China. The S. turkestanicum cercariae can also infect humans, causing cercarial dermatitis in many countries and regions of the world. In spite of its significance as a pathogen of animals and humans, there is little transcriptomic and genomic data in the public databases. METHODS: Herein, we performed the transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females of S. turkestanicum and de novo transcriptome assembly. RESULTS: Approximately 81.1 (female) and 80.5 (male) million high-quality clean reads were obtained and then 29,526 (female) and 41,346 (male) unigenes were assembled. A total of 34,624 unigenes were produced from S. turkestanicum females and males, with an average length of 878 nucleotides (nt) and N50 of 1480 nt. Of these unigenes, 25,158 (72.7 %) were annotated by blast searches against the NCBI non-redundant protein database. Among these, 21,995 (63.5 %), 22,189 (64.1 %) and 13,754 (39.7 %) of the unigenes had significant similarity in the NCBI non-redundant protein (NR), non-redundant nucleotide (NT) and Swiss-Prot databases, respectively. In addition, 3150 unigenes were identified to be expressed specifically in females and 1014 unigenes were identified to be expressed specifically in males. Interestingly, several pathways associated with gonadal development and sex maintenance were found, including the Wnt signaling pathway (103; 2 %) and progesterone-mediated oocyte maturation (77; 1.5 %). CONCLUSIONS: The present study characterized and compared the transcriptomes of adult female and male blood fluke, S. turkestanicum. These results will not only serve as valuable resources for future functional genomics studies to understand the molecular aspects of S. turkestanicum, but also will provide essential information for ongoing whole genome sequencing efforts on this pathogenic blood fluke.