RESUMO
Entamoebapolecki is a parasite of human and nonhuman primates, other mammals, and birds. Due to overlapping morphological features, cysts of E. polecki may be confused with those of other Entamoeba species commonly found in human fecal samples, including immature cysts of Entamoeba histolytica Although the presence of E. polecki in human Entamoeba-positive stool samples may be rare, its prevalence is likely underestimated due to such confusion. Here, we give examples of diagnostic approaches applied so far and summarize data on the molecular epidemiology of E. polecki, including host specificity and phylogeography. Moreover, we evaluate a novel diagnostic conventional PCR developed for the screening of fecal samples for E. polecki The assay was highly sensitive and specific when used on genomic DNA extracted directly from stool and Swedish wastewater samples. The PCR enabled the identification of all four subtypes (ST1 to ST4) of E. polecki by PCR product sequencing. Most (23/28) subtyped E. polecki-positive samples detected in patients in Sweden between 2002 and 2015 reflected colonization by ST4 and were seen in travelers/foreigners. Two and three human cases of ST2 and ST3, respectively, were also detected. Subtypes 1, 2, and 3 were detected in 3/21 wastewater samples, suggesting local endemicity of these E. polecki subtypes; interestingly, ST4 was not detected in wastewater. In conclusion, the current PCR assay enables simple and cost-effective screening of fecal and wastewater samples for E. polecki Human cases of E. polecki appear to involve primarily ST4, while E. polecki detected in wastewater may be primarily of animal origin.
Assuntos
Entamoeba/classificação , Entamoeba/isolamento & purificação , Entamebíase/epidemiologia , Entamebíase/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Águas Residuárias/parasitologia , Entamoeba/genética , Entamebíase/diagnóstico , Humanos , Epidemiologia Molecular , Prevalência , Sensibilidade e Especificidade , Suécia/epidemiologiaRESUMO
Cryptosporidium hominis is considered a strictly human-adapted species, and it is only occasionally diagnosed in animals. However, two variants, C. hominis monkey genotype and C. hominis Ik, were originally described in non-human hosts, monkeys and horses, respectively. During a Swedish national Cryptosporidium study, where all samples were analyzed at the small subunit rRNA and the 60â¯kDa (gp60) glycoprotein loci, we identified two patients infected with C. hominis monkey genotype (subtype IiA17) and two infected with C. hominis subtype IkA18G1. The isolates were further analyzed at the actin and the 70â¯kDa heat shock protein loci, and these analyses showed that these two subtype families are closely related to each other and to human-adapted C. hominis as well as to Cryptosporidium cuniculus. The two patients with C. hominis monkey genotype infection (a father and son) had visited a monkey farm in Thailand prior to infection, while the two cases with C. hominis Ik were unrelated, both probably infected in Sweden. This is the first time that a monkey genotype infection in humans has been related to contact with monkeys and where the gp60 subtype was identified. It is also the first time that human infection caused by C. hominis subtype Ik is described. Even though we were not able to detect any parasites in the animal samples, zoonotic transmission cannot be ruled out in any of these cases because both subtype families are regarded as animal adapted.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/genética , Actinas/genética , Adulto , Animais , Sequência de Bases , Criança , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , DNA de Protozoário/química , Fezes/parasitologia , Feminino , Genótipo , Proteínas de Choque Térmico HSP70/genética , Haplorrinos , Cavalos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Sialoglicoproteínas/genética , Suécia/epidemiologia , ViagemRESUMO
In order to improve genotyping and epidemiological analysis of Cryptosporidium spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different Cryptosporidium hominis patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encoding Cryptosporidium oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the Cryptosporidium parvum reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the C. hominis tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing Cryptosporidium directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of Cryptosporidium from human fecal samples, while alluding to the potential for a higher degree of genotyping within Cryptosporidium epidemiology.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/classificação , Cryptosporidium/genética , Fezes/parasitologia , Variação Genética , Genótipo , Cryptosporidium/isolamento & purificação , Genoma de Protozoário , Genômica , Humanos , Iowa , Carga Parasitária , Filogenia , Análise de Sequência de DNA , SinteniaRESUMO
Fourteen isolates of an unknown species identified as belonging to the genus Legionella by selective growth on BCYE agar were isolated from the biopurification systems of three different wood processing plants. The mip gene sequence of all 14 isolates was identical and a close match alignment revealed 86 % sequence similarity with Legionella pneumophila serogroup 8. The whole genome of isolate LEGN(T) was sequenced, and a phylogenetic tree based on the alignment of 16S rRNA, mip, rpoB, rnpB and the 23S-5S intergenic region clustered LEGN(T) with L. pneumophila ATCC 33152(T). Analysis of virulence factors showed that strain LEGN(T) carries the majority of known L. pneumophila virulence factors. An amoeba infection assay performed to assess the pathogenicity of strain LEGN(T) towards Acanthamoeba castellanii showed that it can establish a replication vacuole in A. castellanii but does not significantly affect replication of amoebae. Taken together, the results confirm that strain LEGN(T) represents a novel species of the genus Legionella, for which the name Legionella norrlandica sp. nov. is proposed. The type strain is LEGN(T) (â= ATCC BAA-2678(T)â= CCUG 65936(T)).
Assuntos
Legionella/classificação , Filogenia , Madeira/microbiologia , Amoeba/microbiologia , DNA Bacteriano/genética , DNA Intergênico/genética , Genes Bacterianos , Legionella/genética , Legionella/isolamento & purificação , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Fatores de Virulência/genéticaRESUMO
The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), C. ubiquitum (n = 2), and one each of C. suis, C. viatorum, C. ditrichi, and Cryptosporidium horse genotype. One patient was co-infected with C. parvum and C. hominis. Subtyping was successful for all species/genotypes, except for C. ditrichi, and revealed large diversity, with 29 subtype families (including 4 novel ones: C. parvum IIr, IIs, IIt, and Cryptosporidium horse genotype Vic) and 81 different subtypes. The most common subtype families were IIa (n = 164) and IId (n = 118) for C. parvum and Ib (n = 26) and Ia (n = 12) for C. hominis. Infections caused by the zoonotic C. parvum subtype families IIa and IId dominated both in patients infected in Sweden and abroad, while most C. hominis cases were travel-related. Infections caused by non-hominis and non-parvum species were quite common (8%) and equally represented in cases infected in Sweden and abroad.
RESUMO
The genus Acanthamoeba represents free-living amoebae typically widespread in soil and water. It consists of more than 20 known species representing 15 genotypes of different pathogenicity and virulence. The aim of the study was the genotypic characterization of Acanthamoeba spp. isolated from human keratitis cases in Sweden. Thirteen amoeba isolates obtained from contact lens users with suspected Acanthamoeba keratitis (AK) were subjected to polymerase chain reaction amplification and subsequent sequencing of the SSU rRNA gene fragment. Sequence analysis identified 4 different genotypes in the studied material. The majority of samples (92%) represented sequences of T3, T4 and T11, all belonging to a cluster of related genotypes frequently described in AK cases. Similar to other reports, genotype T4 was the most common finding in our material (77% of samples). Interestingly, an uncommon genotype, T15, mostly reported from environmental sources, was found in a sample from a patient suffering from a protracted keratitis.
Assuntos
Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Animais , Lentes de Contato/parasitologia , Córnea/parasitologia , Genes de RNAr/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Especificidade da Espécie , SuéciaRESUMO
Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent beta-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.
Assuntos
Acanthamoeba/química , Celulase/metabolismo , Celulose/análise , Parasitologia/métodos , Esporos de Protozoários/química , Coloração e Rotulagem/métodos , Trichoderma/enzimologia , Acanthamoeba/isolamento & purificação , Animais , Histocitoquímica/métodos , Esporos de Protozoários/isolamento & purificaçãoRESUMO
Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoeba castellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.
Assuntos
Amoeba/parasitologia , Toxoplasma/fisiologia , Toxoplasmose Animal/transmissão , Animais , Bioensaio , Gatos , Técnicas de Cocultura , Vetores de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Interações Hospedeiro-Parasita , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Microesferas , Oocistos/fisiologia , Reação em Cadeia da Polimerase , Toxoplasma/patogenicidade , Vacúolos/parasitologiaRESUMO
Free-living amebae (FLA) are known to occur worldwide in water-related biotopes, but only limited information is available on these organisms in developing countries and so far no information on their presence is available from Nicaragua. The aims of this study were to evaluate the prevalence of potentially pathogenic Acanthamoeba spp. and Naegleria spp. in different water sources to which the population of Le6n municipality is exposed. Since pathogenic amebae are thermotolerant, we were especially interested in the occurrence of FLA in geothermal areas. Water samples were collected from Le6n area in Nicaragua: 88 samples were from rivers and springs, 111 from wells, 74 from water taps and 21 from water tanks in urban and suburban Le6n and from three nearby geothermal areas of San Jacinto, Posoltega and Tipitapa. Amebae were identified using morphological and physiological criteria, immunohistochemical staining procedures and molecular methods. Indirect immunofluorescent test was performed on cysts and trophozoites fixed on microscopical slides and incubated for 30 min at room temperature in separate experiments with the following antibodies: rabbit-anti N fowleri/N lovanensis (Nf-Pab), mouse monoclonal antibody anti N. fowleri (Nf-5D12u), rabbit antibodies against Acanthamoeba spp. And fluorescent in situ hybridization (FISH) was performed using 18S rRNA-targeted fluorescent oligonucleotide probes. Probes: GSP for the detection of Acanthamoeba and NAEG1088 for the detection of Naegleria. Free-living amebae were recovered from approximately 43 % of the samples. Acanthamoeba spp was found in 21% of samples from León municipality and in 2% of samples from geothermal areas. Amoeboflagellates were found in 10 % of samples from Le6n and in 19% in geothermal areas. Fifty three percent of tested wells in the geothermal area contained thermotolerant amoeboflagellates. Naegleria spp. was identified in 24 out of 39 (61.5 %) of isolated amoeboflagellates. Twelve of them were assigned to N. lovanenesis while none of the isolates could be identified as N fowleri. However, the common presence of thermotolerant Naegleria in water, specially N. lovanensis, which is an indicator species for N. fowleri, suggests that also this pathogenic amoeba may pose a risk to public health in the area. On the other side, direct pathogenicity, free-living amebae are receiving increasing attention as reservoirs and potential vehicles for the transmission of bacteria in the environment. Thus the information provided in this study may serve as base-line for future studies on the role of free-living amebae e.g. in waterborne-disease outbreaks in the region. Among such potentially important enteropathgens are Vibrio cholerae, E. coli 0157, and Helicobacterpylori.
Assuntos
Acanthamoeba/isolamento & purificação , Água Doce/parasitologia , Naegleria/isolamento & purificação , Abastecimento de Água , Animais , NicaráguaRESUMO
Over a period of less than four weeks, 50 human cases of cryptosporidiosis were reported from a relatively small geographical area in Sweden. All cases were associated with visits to cattle spring pasture events at two farms (referred to as Farm A and B). Epidemiological and microbiological evidence show that contact with calves at the farms was the most likely source of Cryptosporidium infections. Gp60 sequences from human and calf isolates at Farm A were identical to each other, but differed from those at Farm B where, again, human and calf gp60 sequences were identical, proving that the two outbreaks had no common origin. As a direct consequence of these two outbreaks, and guided by knowledge gained from the outbreak investigations, the Swedish Board of Agriculture and all relevant farmer advisory organizations have updated their hygiene instructions for farm visits.
Assuntos
Doenças dos Bovinos/epidemiologia , Criptosporidiose/epidemiologia , Surtos de Doenças , Zoonoses/epidemiologia , Animais , Bovinos , Cryptosporidium/genética , Fazendas , Fezes/parasitologia , Humanos , Fatores de Risco , Estações do Ano , Suécia/epidemiologia , Zoonoses/parasitologiaRESUMO
Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2.
Assuntos
Cryptosporidium/classificação , Cryptosporidium/genética , Tipagem Molecular , Reação em Cadeia da Polimerase , Marcadores Genéticos , Variação Genética , Genoma de Protozoário , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Based on stool microscopy, an E. histolytica/E. dispar prevalence of 18.6% was found in León, Nicaragua about 10 years ago. Since then, new non-microscopic methods have been developed to discriminate between pathogenic E. histolytica and nonpathogenic E. dispar. The main objectives of the present study were to evaluate the true prevalence of E. histolytica among individuals with diarrhea and to assess the diagnostic procedures carried out at the health center level. METHODS: A descriptive study was carried out on patients with diarrhea. Parasite detection was performed by conventional microscopy on native preparations or concentrated and stained specimens, Triage Parasite Panel and by PCR for both E. histolytica and E. dispar. RESULTS: In 134 individuals with diarrhea, the prevalence of intestinal parasites was 69% as detected by direct stool examination. E. histolytica/E. dispar was found in eight (6%) of the samples, but the health centers reported 24%. In the Triage Parasite Panel only one case of E. histolytica/E. dispar was found. Analysis by PCR showed E. dispar in ten (7.5%) and E. histolytica in two cases (1.5%). The detection of intestinal coccidia and Dientamoeba fragilis required additional staining methods. CONCLUSIONS: PCR results showed that E. histolytica is a rare finding in patients with diarrhea. At the health centers, E. histolytica, E. histolytica/E. dispar were clearly overdiagnosed, with the consequence of overtreatment.
Assuntos
Entamoeba/citologia , Entamoeba/genética , Entamebíase/diagnóstico , Entamebíase/parasitologia , Animais , Erros de Diagnóstico , Entamoeba/isolamento & purificação , Humanos , Microscopia , Nicarágua , Reação em Cadeia da Polimerase , TriagemRESUMO
Giardia lamblia is a protozoan parasite infecting the upper mammalian small intestine. Infection relies upon the ability of the parasite to attach to the intestine via a unique cytoskeletal organelle, the ventral disk. We determined the composition and structure of the disk throughout the life cycle of the parasite and identified a new disk protein, SALP-1. SALP-1 is an immunodominant protein related to striated fiber-assemblin (SFA). The disk is disassembled during encystation and stored as four fragments in the immobile cyst. Serial Analysis of Gene Expression (SAGE) showed that the mRNA levels of the disk proteins decreased in encystation but two-dimensional protein gels showed that the protein levels were more constant. The parasite emerges without a functional disk but the four disk fragments are quickly reassembled into two new disks on the dividing, early excysting form. Thus, disk proteins are stored within the cyst, ready to be used in the rapid steps of excystation.
Assuntos
Giardia lamblia/crescimento & desenvolvimento , Morfogênese , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Giardia lamblia/citologia , Giardia lamblia/genética , Giardia lamblia/metabolismo , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/análise , RNA de Protozoário/análise , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
In humans, the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low, and most of the confirmed cases have been observed in immunocompromised patients. Both anthroponotic and zoonotic transmission routes have been suggested. Here, we report a case of suspected zoonotic transmission of C. felis from a cat to a human. The cat developed diarrhea several months before such symptoms were displayed by its owner, a 37-year-old immunocompetent woman. The presence of identical C. felis SSU rRNA, HSP70, and COWP gene sequences was verified in both hosts. In conclusion, it is highly probable that the cat was the initial source of infection and not the opposite. Our results show that Cryptosporidium infection can be transmitted from pets to humans and that molecular analysis is needed to confirm the identity of the oocysts.
RESUMO
We have developed a novel strategy for the purification of Cryptosporidium oocysts from clinical samples using IMS and PCR amplification of target DNA to facilitate uniform coverage genome sequencing and de novo assembly. Our procedure could also be used for other microbial pathogens from clinical specimens.
Assuntos
Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Genoma de Protozoário , Separação Imunomagnética , Reação em Cadeia da Polimerase , Criptosporidiose/parasitologia , DNA de Protozoário/análise , Fezes/parasitologia , Humanos , OocistosRESUMO
BACKGROUND: The basis for correctly assessing the burden of parasitic infections and the effects of interventions relies on a somewhat shaky foundation as long as we do not know how reliable the reported laboratory findings are. Thus virtual microscopy, successfully introduced as a histopathology tool, has been adapted for medical parasitology. METHODOLOGY/PRINCIPAL FINDINGS: Specimens containing parasites in tissues, stools, and blood have been digitized and made accessible as a "webmicroscope for parasitology" (WMP) on the Internet (http://www.webmicroscope.net/parasitology).These digitized specimens can be viewed ("navigated" both in the x-axis and the y-axis) at the desired magnification by an unrestricted number of individuals simultaneously. For virtual microscopy of specimens containing stool parasites, it was necessary to develop the technique further in order to enable navigation in the z plane (i.e., "focusing"). Specimens were therefore scanned and photographed in two or more focal planes. The resulting digitized specimens consist of stacks of laterally "stiched" individual images covering the entire area of the sample photographed at high magnification. The digitized image information (approximately 10 GB uncompressed data per specimen) is accessible at data transfer speeds from 2 to 10 Mb/s via a network of five image servers located in different parts of Europe. Image streaming and rapid data transfer to an ordinary personal computer makes web-based virtual microscopy similar to conventional microscopy. CONCLUSION/SIGNIFICANCE: The potential of this novel technique in the field of medical parasitology to share identical parasitological specimens means that we can provide a "gold standard", which can overcome several problems encountered in quality control of diagnostic parasitology. Thus, the WMP may have an impact on the reliability of data, which constitute the basis for our understanding of the vast problem of neglected tropical diseases. The WMP can be used also in the absence of a fast Internet communication. An ordinary PC, or even a laptop, may function as a local image server, e.g., in health centers in tropical endemic areas.
Assuntos
Microscopia/métodos , Parasitos/citologia , Doenças Parasitárias/patologia , Parasitologia/educação , Parasitologia/métodos , Interface Usuário-Computador , Animais , Humanos , Internet , Camundongos , Microscopia/normas , Doenças Parasitárias/parasitologia , Parasitologia/normas , Controle de QualidadeRESUMO
Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.
Assuntos
Acanthamoeba/isolamento & purificação , Celulase/metabolismo , Celulose/metabolismo , Trichoderma/enzimologia , Animais , Sítios de Ligação , Celulase/genética , Celulase/imunologia , Dimerização , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The protective effect of anti-Giardia antibodies in mother's milk on the acquisition of Giardia infection in their children during the first 2 y of life was analysed as part of a prospective study on infant diarrhoea in a group of 307 mothers and children in Leòn, Nicaragua. Among 24 children acquiring infection within the first 6 months, 23 were born to mothers lacking antibodies. These children also developed more severe diarrhoea. A significant difference between children born to mothers with and without antibodies with respect to the age at which the first Giardia infection was acquired was demonstrated by survival analysis and log rank test (p = 0.036). In conclusion, children born to non-immune mothers are at significantly higher risk of acquiring Giardia infection and developing giardiasis with more severe symptoms compared with children of immune mothers.
Assuntos
Anticorpos Anti-Idiotípicos/sangue , Giardia lamblia/imunologia , Giardíase/prevenção & controle , Imunidade Materno-Adquirida/imunologia , Leite Humano/imunologia , Animais , Aleitamento Materno , Pré-Escolar , Países em Desenvolvimento , Diarreia Infantil/epidemiologia , Diarreia Infantil/etiologia , Feminino , Giardíase/epidemiologia , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Nicarágua/epidemiologia , Estudos Prospectivos , Medição de Risco , Estudos de Amostragem , Sensibilidade e EspecificidadeRESUMO
Transmission routes of Helicobacter pylori remain poorly understood. The finding of bacterial DNA in water suggests the involvement of environmental factors. Here we report successful co-cultivation of H. pylori with Acanthamoeba castellanii, which circumvents the requirement of this bacterium for precise microaerobic conditions and a large supply of nutrients in order to grow. H. pylori was able to propagate and remain viable for several weeks in the presence of amoebae under experimental conditions. Intact, metabolically active bacteria could be demonstrated in vacuoles. The putative dependence of H. pylori on free-living amoebae in nature could be important with respect to transmission and prevalence, as shown for some other pathogenic bacteria.
Assuntos
Amoeba/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Acanthamoeba/microbiologia , Amoeba/crescimento & desenvolvimento , Animais , Divisão Celular , Meios de Cultivo Condicionados , Imunofluorescência , Vida Livre de Germes , Helicobacter pylori/metabolismo , Hibridização in Situ Fluorescente , Vacúolos/microbiologiaRESUMO
Free-living amoebae and Acanthamoebae are known to harbour a range of opportunistic microbial pathogens such as Legionellae, sequestering them from antimicrobial agents as well as environmental stresses. Less is known however of the interaction between the thermotolerant free-living amoebae and Legionellae. In the current study, such phenomena were investigated between an environmental and clinical thermotolerant Acanthamoebae isolate and 6 Legionellae; L. anisa, L. birminghamiensis, L. bozemanii, L. dumoffii, L. erythra and L. pneumophila. All Legionellae could be located within either Acanthamoeba isolate, with L. erythra, and L. pneumophila found located within vacuoles. At concentrations exceeding 2 mg/l, free chlorine was a better disinfectant than combined chlorine against Acanthamoebae-bound Legionellae, though thermal treatment was the most effective of the treatment types investigated. While the interaction with free-living Acanthamoebae increased the resistance of Legionellae to thermal treatment, it increased the sensitivity of Legionellae to free and combined chlorine. Interaction with biofilms did not affect the sensitivity of sessile and intracellular Legionellae to disinfection, caused in part by the thin coverage of biofilm on coupon surfaces. Acanthamoebae cysts remained viable after treatment with 100 mg/l chlorine (free and combined) for 10 min, as well as 80 degrees C, implying that conventional hyper-disinfection may be insufficient for long-term control of Acanthamoebae-bound Legionellae in water distribution systems.