RESUMO
Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.
Assuntos
Giardia lamblia , Encistamento de Parasitas , Proteínas de Protozoários , Giardia lamblia/enzimologia , Giardia lamblia/genética , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Encistamento de Parasitas/fisiologia , Encistamento de Parasitas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Processamento de Proteína Pós-TraducionalRESUMO
Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.
Assuntos
Giardia lamblia/genética , Encistamento de Parasitas/genética , Expressão Gênica , Giardia lamblia/fisiologia , RNA-SeqRESUMO
Giardia duodenalis is a flagellated protozoan that is responsible for many cases of diarrheal disease worldwide and is characterized by its great divergence from the model organisms commonly used in studies of basic cellular processes. The life cycle of Giardia involves an infectious cyst form and a proliferative and mobile trophozoite form. Each Giardia trophozoite has two nuclei and a complex microtubule cytoskeleton that consists of eight flagellar axonemes, basal bodies, the adhesive disc, the funis and the median body. Since the success of Giardia infecting other organisms depends on its ability to divide and proliferate efficiently, Giardia must coordinate its cell division to ensure the duplication and partitioning of both nuclei and the multiple cytoskeletal structures. The purpose of this review is to summarize current knowledge about cell division and its regulation in this protist.
Assuntos
Divisão Celular/fisiologia , Giardia lamblia/fisiologia , Trofozoítos/fisiologia , Divisão Celular/genética , Citoesqueleto/metabolismo , Giardia lamblia/genética , Microtúbulos/metabolismo , Mitose , Proteínas de Protozoários , Trofozoítos/genéticaRESUMO
BACKGROUND: Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. METHODS: To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites RESULTS: After incubating trophozoites with 5 µM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. CONCLUSIONS: These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese , Flagelos/fisiologia , Giardia lamblia/enzimologia , Giardia lamblia/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Protozoários/genética , Giardia lamblia/genética , Fosforilação , Proteínas de Protozoários/metabolismo , Trofozoítos/crescimento & desenvolvimento , Quinase 1 Polo-LikeRESUMO
BACKGROUND: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. METHODS: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. RESULTS: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. CONCLUSION: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.
Assuntos
Doenças do Cão/parasitologia , Giardia lamblia/genética , Giardíase/veterinária , Animais , Cães , Feminino , Genótipo , Giardia lamblia/classificação , Giardia lamblia/isolamento & purificação , Giardia lamblia/fisiologia , Giardíase/parasitologia , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Masculino , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. Giardia was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, Giardia is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain Giardia and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: In vitro maintenance and growth of Giardia trophozoites Basic Protocol 2: In vitro encystation of Giardia cysts Basic Protocol 3: In vivo infections using Giardia trophozoites.
Assuntos
Técnicas de Cultura de Células/métodos , Giardia lamblia/crescimento & desenvolvimento , Giardíase/parasitologia , Parasitologia/métodos , Preservação Biológica/métodos , Animais , Modelos Animais de Doenças , Giardia lamblia/genética , Giardia lamblia/fisiologia , Humanos , Estágios do Ciclo de Vida , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trofozoítos/genética , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/fisiologiaRESUMO
Giardia is an important cause of diarrhoea, and results in post-infectious and extra-intestinal complications. This chapter presents a state-of-the art of our understanding of how this parasite may cause such abnormalities, which appear to develop at least in part in Assemblage-dependent manner. Findings from prospective longitudinal cohort studies indicate that Giardia is one of the four most prevalent enteropathogens in early life, and represents a risk factor for stunting at 2 years of age. This may occur independently of diarrheal disease, in strong support of the pathophysiological significance of the intestinal abnormalities induced by this parasite. These include epithelial malabsorption and maldigestion, increased transit, mucus depletion, and disruptions of the commensal microbiota. Giardia increases epithelial permeability and facilitates the invasion of gut bacteria. Loss of intestinal barrier function is at the core of the acute and post-infectious complications associated with this infection. Recent findings demonstrate that the majority of the pathophysiological responses triggered by this parasite can be recapitulated by the effects of its membrane-bound and secreted cysteine proteases.
Assuntos
Giardíase/complicações , Giardíase/parasitologia , Enteropatias/etiologia , Giardia lamblia/fisiologia , Giardíase/patologia , Humanos , Enteropatias/parasitologiaRESUMO
Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3ß) with similarity to the mammalian topoisomerase IIIß. We found that TOP3ß was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3ß can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3ß can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3ß increased expression of cwp1-3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3ß. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3ß gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3ß may be functionally conserved, and involved in inducing Giardia cyst formation.
Assuntos
DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Perfilação da Expressão Gênica/métodos , Giardia lamblia/fisiologia , Domínio Catalítico , Parede Celular/metabolismo , DNA Topoisomerases Tipo I/química , Regulação da Expressão Gênica , Giardia lamblia/enzimologia , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transativadores/metabolismo , Regulação para CimaRESUMO
The objectives of this study were to explore risk factors associated with Giardia and Cryptosporidium infections in dogs and cats in Chiang Mai, Thailand, to describe the seasonal distributions of Giardia and Cryptosporidium prevalence, and to determine the potential for zoonotic transmission through genetic characterization of isolates. Fecal samples from 301 dogs and 66 cats were collected between August 2009 and February 2010. The presence of Giardia cysts and Cryptosporidium oocysts was determined using zinc sulfate centrifugal flotation and immunofluorescent assay (IFA). Genotype/species were determined by DNA sequence analyses of PCR products from Giardia glutamate dehydrogenase (gdh), beta-giardin (bg), and triosephosphateisomerase (tpi) and Cryptosporidium heat shock protein 70KDa (hsp70) and small subunit-rRNA (SSU-rRNA) genes. Information related to specific risk factors was collected from owners of each animal using a questionnaire. The risk factor data were analyzed for associations with Giardia and Cryptosporidium infections using logistic regression. The overall estimated prevalence of Giardia and Cryptosporidium in dogs was 25.2% and 7.6%, respectively and in cats, 27.3% and 12.1%, respectively. The estimated prevalence of Giardia infection in dogs in the rainy season (31.7%) was significantly higher than in the drier, winter season (17.2%) (pâ¯<â¯0.01). The estimated prevalence of Cryptosporidium infection in dogs and of Giardia and Cryptosporidium infections in cats was not associated with season (pâ¯>â¯0.05). Multivariable analysis indicated that Giardia cysts were more likely to be detected in fecal samples of dogs that resided in high-density environments, drank untreated water, were shedding Cryptosporidium oocysts, were having acute diarrhea or a history of chronic diarrhea, and were collected in the rainy season. All 19 Giardia PCR positive samples typed as G. duodenalis canine adapted genotypes (assemblages C or D). In cats, of six Giardia PCR positive samples, five typed as dog assemblages and one typed as assemblage AI. Of ten dogs with Cryptosporidium PCR positive samples, eight typed as C. canis, one as C. parvum (a zoonotic species) and one had both C. canis and C. parvum. Of three Cryptosporidium PCR positive samples in cats, one typed as C. felis and two typed as C. parvum. The presence of zoonotic G. duodenalis assemblage AI in a cat, and C. parvum in feces of dogs and cats suggests a potential role for a reservoir for zoonotic transmission. Whether or not these presences were from exposure to other animal or human hosts or environment are needed to be confirmed.
Assuntos
Doenças do Gato/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium/fisiologia , Doenças do Cão/epidemiologia , Giardia lamblia/fisiologia , Giardíase/veterinária , Animais , Doenças do Gato/parasitologia , Gatos , Criptosporidiose/parasitologia , Doenças do Cão/parasitologia , Cães , Feminino , Giardíase/epidemiologia , Giardíase/parasitologia , Masculino , Fatores de Risco , Estações do Ano , Tailândia/epidemiologiaRESUMO
Entamoeba histolytica and Giardia duodenalis are widespread intestinal protozoan parasites which both spread via cysts that have to be ingested to infect a new host. Their environment, the small intestine for G. duodenalis and the colon for E. histolytica, contains only very limited amounts of oxygen, so both parasites generate energy by fermentation and substrate level phosphorylation rather than by oxidative phosphorylation. They both contain reducing agents able to reduce and activate nitroimidazole drugs such as metronidazole which is the gold standard drug to treat Entamoeba or Giardia infections. Although metronidazole works well in the majority of cases, it has a number of drawbacks. In animal models, the drug has carcinogenic activity, and concerns about a possible teratogenic activity remain. In addition, the treatment of G. duodenalis infections is hampered by emerging metronidazole resistance. Plant-derived drugs play a dominant role in human medicine, therefore we tested the activity of 14 isolated plant compounds belonging to seven different classes in vitro against both parasites. The tests were performed in a new setting in microtiter plates under anaerobic conditions. The compound with the highest activity was methylgerambullin, a sulphur-containing amide found in Glycosmis species of the family Rutaceae with an EC50 of 14.5⯵M (6.08⯵g/ml) after 24â¯h treatment for E. histolytica and 14.6⯵M (6.14⯵g/ml) for G. duodenalis. The compound was successfully synthesised in the laboratory which opens the door for the generation of new derivatives with higher activity.
Assuntos
Acrilamidas/farmacologia , Amidas/farmacologia , Antiprotozoários/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Giardia lamblia/efeitos dos fármacos , Extratos Vegetais/farmacologia , Rutaceae/química , Compostos de Enxofre/farmacologia , Acrilamidas/síntese química , Acrilamidas/química , Amidas/síntese química , Amidas/química , Antiprotozoários/síntese química , Antiprotozoários/química , Entamoeba histolytica/fisiologia , Entamebíase/tratamento farmacológico , Entamebíase/parasitologia , Giardia lamblia/fisiologia , Giardíase/tratamento farmacológico , Giardíase/parasitologia , Humanos , Extratos Vegetais/química , Compostos de Enxofre/síntese química , Compostos de Enxofre/químicaRESUMO
Giardia lamblia is usually cultured axenically in TYI-S-33, a complex medium which does not permit survival and growth of mammalian cells. Likewise, medium commonly used to maintain and grow mammalian cells does not support healthy trophozoite survival for more than a few hours. The inability to coculture trophozoites and epithelial cells under optimal conditions limits studies of their interactions as well as interpretation of results. Trophozoites of the WB isolate but not the GS isolate were repeatedly adapted to grow stably in long-term cocultures with Caco2, Cos7, and mouse tumor rectal (RIT) cell lines using hybridoma-screened Dulbecco's modified Eagle's medium and 10% fetal calf serum. Giardia did not grow in spent cell culture medium or when separated by a permeable membrane using transwell methodology. Giardia chronically cocultured with specific cell lines became adapted (conditioned). These Giardia cocultures grew better than nonconditioned trophozoites, and the cell lines differed in their ability to support trophozoite growth in the order of RIT > Cos7 > Caco2. Trophozoites conditioned on one cell line and then grown in the presence of a heterologous cell line changed their growth rate to that seen in conditioned Giardia from the heterologous cell line. Trophozoite survival required intimate contact with cells, suggesting that trophozoites obtain an essential nutrient or growth factor from mammalian cells. This may explain why Giardia trophozoites adhere to the small intestinal epithelium during human and animal infections. This coculture system will be useful to understand the complex interactions between the host cells and parasite.
Assuntos
Giardia lamblia/fisiologia , Animais , Células COS , Células CACO-2 , Chlorocebus aethiops , Técnicas de Cocultura , Humanos , Neoplasias RetaisRESUMO
The interaction between human protozoan parasites and the immune cells of bivalves, that can accumulate them, is poorly described. The purpose of this study is to consider the mechanisms of action of some of these protozoa on zebra mussel haemocytes, by evaluating their cytotoxic potential. Haemocytes were exposed to Toxoplasma gondii, Giardia duodenalis or Cryptosporidium parvum (oo)cysts. The results showed a cytotoxic potency of the two largest protozoa on haemocytes and suggested the formation of haemocyte aggregates. Thus, this study reveals the first signs of a haemocyte:protozoan interaction.
Assuntos
Dreissena/parasitologia , Hemócitos/patologia , Parasitos/fisiologia , Animais , Sobrevivência Celular , Cryptosporidium parvum/fisiologia , Dreissena/imunologia , Giardia lamblia/fisiologia , Interações Hospedeiro-Parasita , Oocistos/fisiologia , Toxoplasma/fisiologiaRESUMO
Giardia duodenalis is a ubiquitous flagellated protozoan parasite known to cause giardiasis throughout the world. Potential transmission vehicles for this zoonotic parasite are both water and food sources. As such consumption of water contaminated by feces, or food sources washed in contaminated water containing parasite cysts, may result in outbreaks. This creates local public health risks which can potentially cause widespread infection and long-term post-infection sequelae. This paper provides an up-to-date overview of G. duodenalis assemblages, sub-assemblages, hosts and locations identified. It also summarizes knowledge of potential infection/transmission routes covering water, food, person-to-person infection and zoonotic transmission from livestock and companion animals. Public health implications focused within the UK, based on epidemiological data, are discussed and recommendations for essential Giardia developments are highlighted.
Assuntos
Giardia lamblia/fisiologia , Giardíase , Animais , Giardíase/diagnóstico , Giardíase/epidemiologia , Giardíase/transmissão , Giardíase/veterinária , Humanos , Saúde Pública , Fatores de Risco , Reino UnidoRESUMO
The risk of exposure to zoonotic factors among veterinarians comprises still underestimated problem. Many etiological factors of infectious diseases are so far poorly known, including the way of their transmission from environment to humans and their impact for health. The main aim of the study was to determine the risk of two selected zoonosis infections caused by Giardia duodenalis and Cryptosporidium spp. among occupational group of veterinarians in Poland. Two hundred ninety seven samples of stool were tested for the presence of Giardia cysts and Cryptosporidium oocysts using Direct Fluorescent Assay (DFA). There were no positive results for Cryptosporidium. The presence of Giardia cysts was found in two samples of faeces (0.67%), confirmed by polymerase chain reaction and sequencing. The risk with regard to the parasites Giardia duodenalis and Cryptosporidium spp. seems to be low among the group of veterinarians.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Giardia lamblia/isolamento & purificação , Giardíase/parasitologia , Médicos Veterinários/estatística & dados numéricos , Adulto , Cryptosporidium/genética , Cryptosporidium/fisiologia , Fezes/parasitologia , Feminino , Giardia lamblia/genética , Giardia lamblia/fisiologia , Giardíase/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Oocistos/classificação , Oocistos/genética , Polônia/epidemiologiaRESUMO
Fifteen Mongolian gerbils were inoculated with 10 × 106 viable trophozoites of Giardia intestinalis. Their faeces were examined daily by flotation method and the number of shed cysts was counted. Two animals (male and female) were euthanised at 4- to 5-day intervals (9, 14, 18 days post-infection (DPI)). The remaining nine gerbils were sacrificed and dissected at the end of the experiment (23 DPI). Their small intestinal tissues were processed for examination using histological sectioning and scanning electron microscopy and their complete blood count (CBC) was examined. The highest number of trophozoites at the total was observed in the duodenum in gerbils sacrificed on 14 DPI. Number of shed cysts was positively correlated with number of trophozoites rinsed from the intestine. Infected gerbils had lower body weight gain in comparison with control group and in three male gerbils; diarrhoea occurred during infection. Cyst shedding was negatively correlated with values of mean corpuscular haemoglobin concentration. Females showed another pattern in cyst shedding than males. This information needs to be taken into account while planning the experiments.
Assuntos
Gerbillinae , Giardia lamblia/fisiologia , Giardíase/parasitologia , Animais , Modelos Animais de Doenças , Duodeno/parasitologia , Duodeno/patologia , Fezes/parasitologia , Feminino , Gerbillinae/parasitologia , Giardia lamblia/crescimento & desenvolvimento , Giardíase/patologia , Humanos , Masculino , Microscopia Eletrônica de Varredura , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/fisiologiaRESUMO
Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.
TITLE: Estimation de la viabilité et infectiosité des stades (kystes et oocystes) de Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii transmis par la nourriture et l'eau : une revue des méthodes. ABSTRACT: Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii sont des parasites protozoaires qui ont été soulignés comme agents pathogènes émergents dans les aliments par l'Organisation des Nations Unies pour l'alimentation et l'agriculture et l'Organisation Mondiale de la Santé. Selon l'Autorité Européenne de Sécurité des Aliments, 4786 épidémies d'origine alimentaire et hydrique ont été enregistrées en Europe en 2016, dont 0.4% ont été attribuées à des parasites, incluant Cryptosporidium, Giardia et Trichinella. Jusqu'en 2016, aucune méthode standardisée n'était disponible pour détecter les kystes de Giardia et les oocystes de Cryptosporidium et Toxoplasma dans les aliments. Aucune réglementation n'est donc proposée concernant ces dangers. Cependant, compte tenu de leur faible dose infectieuse, l'ingestion d'une quantité d'aliments faiblement contaminés peut entraîner une infection de l'homme. Pour évaluer le risque lié aux protozoaires dans les aliments, des efforts doivent être faits dans l'évaluation de l'exposition pour estimer la contamination le long de la chaîne alimentaire, depuis la matière première jusqu'aux consommateurs. Cette évaluation nécessite de déterminer : (i) la prévalence de parasites infectieux dans les aliments, (ii) l'efficacité des mesures de maîtrise pour éliminer cette contamination. Pour mener une telle évaluation, des méthodes capables d'identifier des parasites viables (vivants) et infectieux sont requises. Cette revue décrit les méthodes actuellement disponibles permettant d'évaluer l'infectiosité et la viabilité des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. et T. gondii, et leur potentiel pour être appliquées dans l'évaluation de l'exposition pour déterminer la présence de parasites infectieux et/ou caractériser l'efficacité des mesures de maîtrise. Les avantages et limites de chaque méthode sont présentés et une stratégie d'analyses est proposée pour évaluer l'exposition à ces protozoaires.
Assuntos
Cryptosporidium/fisiologia , Parasitologia de Alimentos/métodos , Giardia lamblia/fisiologia , Toxoplasma/fisiologia , Água/parasitologia , Animais , Bioensaio/métodos , Células Cultivadas/parasitologia , Criptosporidiose/parasitologia , Criptosporidiose/prevenção & controle , Cryptosporidium/patogenicidade , Técnicas Genéticas , Giardia lamblia/patogenicidade , Giardíase/parasitologia , Giardíase/prevenção & controle , Humanos , Oocistos/patogenicidade , Oocistos/fisiologia , Medição de Risco , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Toxoplasmose/prevenção & controleRESUMO
At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium parvum/fisiologia , Giardia lamblia/fisiologia , Giardíase/diagnóstico , Técnicas Analíticas Microfluídicas , Oocistos/fisiologia , Animais , Células Cultivadas , Diagnóstico Diferencial , Impedância Elétrica , Citometria de Fluxo , Humanos , Sensibilidade e Especificidade , Análise de Célula Única , Especificidade da EspécieRESUMO
Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-γ (gEF1Bγ), and structural proteins, e.g. α-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1Bγ and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/fisiologia , Oxidiazóis/farmacologia , Proteoma/efeitos dos fármacos , Ligação Proteica , Proteômica , Proteínas de Protozoários/análiseRESUMO
Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.