A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens / O efeito da fonte de água e suplementação de solo na contaminação parasitária em hortas orgânicas

Abstract The objective of this study was to determine factors associated with vegetable contamination with zoonotic protozoan. Samples of water, soil and vegetables were collected from July/2014 to May/2016, totaling 83 samples, 21 properties of Londrina region, Paraná, Brazil. DNA amplification of Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis in the samples was conducted using polymerase chain reaction (PCR). The PCR results were positive for T. gondii in 12.9% (8/62), Cryptosporidium spp. in 11.3% (7/62) and G. intestinalis in 25.8% (16/62) of the samples. DNA sequencing identified C. parvum in five samples and G. intestinalis Assemblage E in three. The statistical associations demonstrated greater probability of positive samples for T. gondii and for at least one of the three protozoa when the source of irrigation water was the river; a greater chance of positive samples for Cryptosporidium spp. when deer were present on the property; and a smaller chance of positive samples for at least one of the three etiologic agents when soil was supplemented with limestone. The results expose some critical contamination points, providing support for training farmers on good management practices during the production process.

Resumo O trabalho teve como objetivo determinar os fatores associados à contaminação de vegetais por protozoários zoonóticos. Amostras de água, solo e vegetais foram coletadas de julho/2014 a maio/2016, totalizando 83 amostras de 21 propriedades da região de Londrina, Paraná, Brasil. A amplificação de fragmentos de DNA de T. gondii, Cryptosporidium spp. e Giardia intestinalis foi realizada por meio da reação em cadeia da polimerase (PCR). Os resultados da PCR foram positivos para T. gondii em 12,9% (8/62), Cryptosporidium spp. em 11,3% (7/62) e G. intestinalis. em 25,8% (16/62) das amostras. O sequenciamento de DNA identificou C. parvum em cinco amostras e G. intestinalis, Assemblage E em três amostras. As associações estatísticas evidenciaram maior probabilidade de amostras serem positivas para T. gondii ou para pelo menos um dos três protozoários quando a fonte de água de irrigação era o rio; uma maior chance de amostras positivas para Cryptosporidium spp. quando havia cervos na propriedade; e uma menor chance das amostras serem positivas para pelo menos um dos três agentes etiológicos quando o solo era suplementado com calcário. Os resultados expõem alguns pontos críticos de contaminação, fornecendo suporte para capacitar os agricultores em boas práticas de gestão durante o processo de produção.

Ocorrência e identificação de Cryptosporidium e Giardia em amostras de água superficial destinada ao abastecimento público do estado de São Paulo / Occurrence and identification of Cryptosporidium and Giardia from surface water catchment in Sao Paulo, Brazil

Estudos de revisão sobre surtos associados à transmissão hídrica revelaram que os protozoários parasitas Cryptosporidium parvum e Giardia duodenalis (sinonímia: G. lamblia e G. intestinalis) são os principais responsáveis pelo maior número de casos registrados em todo o mundo. A contaminação das águas superficiais que servem ao abastecimento público por estes protozoários representa risco à saúde humana e animal, pois ambos parasitas apresentam resistência à cloração, processo convencional utilizado para desinfecção em Estações de Tratamento de Água (ETAs).Em vista desta lacuna, o presente estudo propõe identificar espécies e genótipos de Cryptosporidium e Giardia a partir de 128 amostras de águas superficiais de 11 mananciais do estado de São Paulo, de acordo com o Método 1623.1 (USEPA, 2012). Para identificar estes parasitas, foi realizada a recuperação dos (oo) cistos a partir de lâminas raspadas, seguindo o protocolo adaptado da USEPA, em seguida, utilizou-se o PCR em tempo real para identificar os genes 18S rRNA para Cryptosporidium e SSU para Giardia. Os resultados mostraram que a frequência de ocorrência desses protozoários nos pontos de captação foi de 29,7% para Giardia e 30,4% para Cryptosporidium. Os cistos estavam presentes em 10 dos 11 pontos de captação com frequências que variaram de 17 a 100%, e concentrações que variaram de

Review studies on waterborne outbreaks have been showing that Cryptosporidium parvum and Giardia duodenalis (synonym: G. lamblia and G. intestinalis) are the primarily responsible for the highest number of the cases recorded worldwide. Contamination of surface waters catchments by these protozoa is a risk factor to human health because both parasites are resistant to chlorination, which is a conventional process used for disinfection in water treatment plants (WTP). The present study aimed to identify species of Cryptosporidium and Giardia recovered from surface water catchment samples from 11 municipalities from the State of São Paulo, totalizing 128 samples. Quantification of both parasites was carried out according to method 1623.1 (USEPA, 2012). In order to identify parasites, the recovering of (oo)cysts from slides followed USEPA´s protocol by scraping slides, then Real Time PCR using the 18S rRNA genes for Cryptosporidium and SSU for Giardia were carried out. Results showed that the frequency of occurrence of these protozoa at the catchment points was 29,7% for Giardia and 30,4% for Cryptosporidium. Cysts were present in 10 of 11 catchments points with frequencies varying from 17 to 100% with concentrations ranging from

Evidence of heterozygosity and recombinant alleles in single cysts of Giardia duodenalis / Evidência de heterozigose e alelos recombinantes em cistos individualizados de Giardia duodenalis

Abstract Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.

Resumo A espécie Giardia duodenalis é dividida em oito grupos (nomeados de A a H). Isolados do grupo A são divididos em quatro subgrupos (AI, AII, AIII and AIV). Enquanto isolados do subgrupo AII são detectados quase exclusivamente em hospedeiros humanos, isolados do subgrupo B são encontrados em uma grande variedade de hospedeiros entre animais e humanos. Neste trabalho, foi constatado que, dentre diversos cistos individualizados de G. duodenalis provenientes de fezes de origem humana, um cisto continha os alelos AII e B e um número inesperado de variantes de alelos codificadores de beta giardina e GLORF-C4. Ainda, um dos alelos beta giardina desse cisto possuía fragmentos AII intercalando um fragmento B, indicando que esse alelo pode ser um recombinante entre alelos AII e B. Os resultados aqui apresentados são inéditos e colocam em dúvida o conceito atual de que os diferentes grupos de G. duodenalis representam espécies distintas com diferentes graus de especificidade por hospedeiros.

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.

Genotipos de Giardia duodenalis en muestras de niños de las guarderías del Instituto Colombiano de Bienestar Familiar y de perros en Ibagué, Colombia / Giardia duodenalis genotypes found in the Instituto Colombiano de Bienestar Familiar day care centers and dogs in Ibagué, Colombia

Introducción. Se han descrito ocho genotipos de Giardia duodenalis, del A al H. Los genotipos A y B se han aislado de humanos y de una gran variedad de mamíferos; sin embargo, los genotipos del C al H han mostrado mayor especificidad de huésped. Objetivo. Identificar los genotipos de G. duodenalis a partir de quistes obtenidos en heces de niños de las guarderías del Instituto Colombiano de Bienestar Familiar (ICBF) y de perros en Ibagué, mediante PCR-RFLP de los genes de la beta giardina y la glutamato deshidrogenasa. Materiales y métodos. Los quistes de las muestras positivas para G. duodenalis fueron sometidos a concentración; se extrajo su ADN y se efectuó el análisis de PCR-RFLP de los genes de la beta giardina y de la glutamato deshidrogenasa. Como control positivo se utilizó la cepa MHOM/CO/04/G40 procedente del Grupo de Parasitología del Instituto Nacional de Salud. Resultados. De las muestras tomadas de niños, 11/23 (48 %) correspondieron al genotipo A y, 12/23 (52 %), al genotipo B. Cuatro muestras de perros presentaron los genotipos C y D, específicos de este huésped. Conclusiones. En los niños solamente se encontraron los genotipos asociados a infecciones humanas (AII, BIII y BIV) y en los perros, los genotipos específicos para este huésped (C y D). Debido al reducido tamaño de las muestras analizadas provenientes de perros, y dado que estos no estuvieron en contacto con los niños de las guarderías del ICBF, no fue posible determinar una interacción entre el ciclo de transmisión de los humanos y el de los animales.

Introduction: Eight Giardia duodenalis genotypes (A-H) have been described to date. Genotypes A and B have been isolated from humans and a wide range of mammals; however, genotypes C-H have shown greater host specificity. Objective: Identifying G. duodenalis genotypes from cysts in faeces obtained from children attending the Instituto Colombiano de Bienestar Familiar (ICBF) day care centres and from dogs in Ibagué by PCR-RFLP targeting both the b -giardin and glutamate dehydrogenase genes. Materials and methods: Cysts from G. duodenalis positive samples were concentrated, DNA was extracted and the b -giardin and glutamate dehydrogenase genes were analysed by PCR-RFLP. The MHOM/CO/04/G40 strain was used as positive control (this was obtained from the Grupo de Parasitología at the Instituto Nacional de Salud ). Results: Of the total human samples, 11/23 (48%) were genotyped as A and 12/23 (52%) as B; PCR-RFLP revealed that four canine samples were genotypes C and D, these being host-specific. Conclusions: Only genotypes associated with human infection (AII, BIII and BIV) were found in the children and host-specific genotypes were observed in canines (C and D). No interaction could be established between animal and human transmission cycles due to the small canine sample size and as the former did not come into contact with children attending ICBF day-care centres.

Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/microl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63degrees C by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1alpha) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

Evaluation of alpha-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The alpha/beta-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant alpha-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of alpha-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the alpha-tubulin gene from G. lamblia. PCR-RFLP analysis of this alpha-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that alpha-tubulin can be used as a molecular probe to detect G. lamblia.

Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

First genotyping of Giardia lamblia from human and animal feces in Argentina, South America

The purpose of this study was to investigate the genotypes of Giardia lamblia from human and animal feces and their epidemiological and clinical characteristics in Argentina, South America. Seventy isolates, 60 from humans (adults and children), eight from dogs and two from cows were processed by polymerase chain reaction-restriction fragment length polymorphism. Data corresponding to demographic, socio-cultural and environmental variables and presence/absence of signs/symptoms were collected. The triosephosphate isomerase gene was amplified from 43 (71.66 percent) of the 60 human fecal samples. Among these, 3/43 (6.98 percent) were genotype AII and 40/43 (93.02 percent) were genotype B. Assemblage AII was detected in three children who lived together in a shantytown and they were oligosymptomatic and none had diarrhea. This genotype was not found in animals. Genotype B showed a high prevalence in both adults and children. It was also found in polysymptomatic people, many of whom presented diarrhea. It was also found only in one dog. The present study represents the first contribution to the knowledge of G. lamblia genotypes in Argentina.

Comparación de métodos de lisis y extracción de ADN de trofozoítos de Giardia lamblia / Comparison of lysis methods and DNA extraction of Giardia lamblia trophozoites

Se evaluó la eficiencia de procedimientos de lisis y tratamientos de extracción de ADN de trofozoítos de Giardia lamblia respecto a la eficiencia de ruptura, cantidad y pureza de ADN, además de los tiempos de procesamiento y costos. Se testearon cinco métodos de lisis (agua destilada y calor; agua destilada, calor y proteinasa K; buffer de lisis D; buffer de lisis E y un kit comercial) y tres métodos de purificación de ADN (fenol:cloroformo: isoamílico; Chelex 100 y un kit comercial). Los datos obtenidos se analizaron estadísticamente. La combinación de buffer de lisis E y Chelex fue un método simple y económico, que produjo alto rendimiento de ADN con baja pureza. Ella técnica comercial fue un método simple, más costoso que produjo bajas cantidades de ADN con un nivel de pureza apropiado para estudios moleculares.

Giardia y giardiasis / Giardia and giardiasis

Giardia lamblia es un protozoario parásito que habita el intestino delgado de los seres humanos y de muchos otros vertebrados y es una de las más comunes causas de diarrea en todo el mundo. Durante su ciclo de vida Giardia sufre significativos cambios bioquímicos y morfológicos que le permiten sobrevivir en ambientes y condiciones que de otro modo lo destruirían. Para sobrevivir fuera del intestino del hospedador, los trofozoítos de Giardia se diferencian a quistes, los que se caracterizan por poseer una rígida pared glicoproteica externa que les permiten sobrevivir inclusive frente a la acción de los desinfectantes más comunes. Otro de los mecanismos de adaptación de este parásito es la variación de los antígenos de superficie que le permite a los trofozoítos evadir la respuesta inmune del huésped y generar infecciones tanto agudas como crónicas o recurrentes en individuos infectados. Durante los últimos años se han producido importantes avances en el conocimiento de las bases moleculares de los procesos de enquistamiento y variación antigénica en Giardia que pronostican el pronto hallazgo de nuevos agentes quimioterapéuticos y/o inmunoprofilácticos contra este importante parásito intestinal.

In vivo determination of the gap2 gene promoter activity in Giardia lamblia

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.

Mobile genetic elements in protozoan parasites.

Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids-Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtleomeric in location while one is chromosome-internal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.