Primers Designed for Amplification of Echinococcus multilocularis DNA Amplify the DNA of Encephalitozoon-Like Spores in the Polymerase Chain Reaction

ABSTRACT. Microsporidian spores were developed from cells which were grown in vitro from a human liver lesion which was due to larval Echinococcus multilocularis . The microsporidian spores developed in the same fashion as an Encephalitozoon cuniculi . The Encephalitozoon -like spores were completely separated on Percoll gradients. The separated spores contained DNA capable of amplification by two different primer sets designed for the polymerase chain reaction (PCR) of E. multilocularis DNA. However, the cell DNA from which microsporidium developed was thoroughly insensitive to the PCR using the E. multilocularis primer sets. The results strongly suggested that Encephalitozoon should be taken into consideration, when DNA isolated from larval E. multilocularis is analyzed.     

Encephalitozoon-Like Organisms in Patients with Alveolar Hydatid Disease: Cell Culture, Ultrastructure, Histoimmunochemical Localization and Seroprevalence

ABSTRACT. We found Encephalitozoon -like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis. The organisms developed in the same fashion as an Encephalitozoon cunculi. The spores that developed in parasitophorous vacuoles were 2.0–2.6 × 1.1–1.5 μm: each contained a single nucleus and 4–5 polar tubule coils, closely resembling E. cuniculi in its ultrastructure. Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients. We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05–1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml. Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ. The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD). Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test. The Percoll-separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting. Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals.     

Disseminated lethal Encephalitozoon cuniculi (genotype III) infections in cotton-top tamarins (Oedipomidas oedipus)--a case report

For the first time, Encephalitozoon (E.) cuniculi genotype III ('dog strain') was verified in two cotton-top tamarins (Oedipomidas oedipus) by light microscopy, immunohistochemistry, electron microscopy, PCR and sequencing. The animals had a disseminated lethal infection with this protist. In earlier reports, genotype III had been found only in domestic dogs, man, emperor tamarins (Saguinus imperator) and golden lion tamarins (Leontopithecus rosalia). This investigation establishes now that the 'dog strain' can occur in cotton-top tamarins too. This is further evidence for the zoonotic potential of E. cuniculi. Furthermore, free E. cuniculi spores were identified also in blood vessels of several tissues. These findings indicate that during a disseminated infection E. cuniculi spores can occur in peripheral blood, too. We propose that blood should also be included in the investigations for the detection of microsporidia, so that a possible disseminated course of an infection can be detected.     

Analysis of four human microsporidian isolates by MALDI-TOF mass spectrometry

Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000-8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.     

Infectivity of microsporidia spores stored in water at environmental temperatures

To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.     

Detection of Thelohania solenopsae (Microsporidia: Thelohaniidae) in Solenopsis invicta (Hymenoptera: Formicidae) by multiplex PCR

Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex PCR resulted in sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, seven additional species of microsporidia (including Vairimorpha invictae), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from Solenopsis xyloni, Solenopsis richteri, Solenopsis geminata, the invicta/richteri hybrid, and monogyne and polygyne S. invicta, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR amplification, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.     

Further molecular discrimination of Spanish strains of Echinococcus granulosus

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.     

EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall

Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.     

Encephalitozoonosis in pharmacologically immunosuppressed mice

Encephalitozoon cuniculi is a parasite that has been identified as a cause of opportunistic infections in immunocompromised individuals. This study was performed to evaluate E. cuniculi infection in pharmacologically immunosuppressed mice. Mice were immunosuppressed with cyclophosphamide (100mg/kg twice a week, IP) or cyclosporin (10mg/kg daily, IP) and inoculated with 10(7)E. cuniculi spores IP. The E. cuniculi spores were cultivated in MDCK cells. E. cuniculi identification was performed by light microscopy studies using Gram-Chromotrope, Hematoxylin-Eosin and Toluidine blue-fuchsin staining techniques, as well as by PCR at 15, 30 and 45 days post-inoculation (DPI). Cyclophosphamide-immunosuppressed mice have greatly reduced amounts of CD8(+), CD4(+) and CD3(+) T cells and CD19(+) B cells. The cells from these mice were analyzed by FACS and showed acute disseminated and fatal encephalitozoonosis. Mice treated with ciclosporin, which is both antiparasitic and immunosuppressive, have a milder, chronic, non-lethal infection and showed a significant reduction only in CD3(+) and CD4(+) T cell numbers. Our results support the role of CD8(+) T cells in controlling infection by E. cuniculi and show that preventive measures are essential for preventing this zoonosis in individuals undergoing chemotherapy for cancer or other immunosuppressive therapies.     

Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and 18 (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.     

Encephalitozoon cuniculi Isolated from the Urine of an AIDS Patient, which Differs from Canine and Murine Isolates

ABSTRACT. A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi . However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi , with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.     

Specific and sensitive detection of Nosema bombi (Microsporidia: Nosematidae) in bumble bees (Bombus spp.; Hymenoptera: Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10(7) down to 10 spores diluted in 100 microl of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of approximately 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.     

Class II photolyase in a microsporidian intracellular parasite

Photoreactivation is the repair of DNA damage induced by ultraviolet light radiation using the energy contained in visible-light photons. The process is carried out by a single enzyme, photolyase, which is part of a large and ancient photolyase/cryptochrome gene family. We have characterised a photolyase gene from the microsporidian parasite, Antonospora locustae (formerly Nosema locustae) and show that it encodes a functional photoreactivating enzyme and is expressed in the infectious spore stage of the parasite's life cycle. Sequence and phylogenetic analyses show that it belongs to the class II subfamily of cyclobutane pyrimidine dimer repair enzymes. No photolyase is present in the complete genome sequence of the distantly related microsporidian, Encephalitozoon cuniculi, and this class of photolyase has never yet been described in fungi, the closest relatives of Microsporidia, raising questions about the evolutionary origin of this enzyme. This is the second environmental stress enzyme to be found in A.locustae but absent in E.cuniculi, and in the other case (catalase), the gene is derived by lateral transfer from a bacterium. It appears that A.locustae spores deal with environmental stress differently from E.cuniculi, these results lead to the prediction that they are more robust to environmental damage.     

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.     

Chitinolytic activity in viable spores of Encephalitozoon species

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80 degrees C for 10 min or at 55 degrees C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.     

两种泥鳅中Sox基因的检出(英文)

性别决定基因ＳＲＹ都具有一个高度保守的基因序列──ＨＭＧ－ｂｏｘ,编码Ｓｏｘ蛋白,在胚胎发育过程中起重要作用。本文利用两种引物检测了泥鳅和大鳞副泥鳅的Ｓｏｘ基因。第一对引物特异扩增人类ＳＲＹ基因的保守区。在扩增泥鳅的基因组ＤＮＡ时可见长度分别为２００、５５０、９４０和１０００ｂｐ的四条扩增带。在大鳞副泥鳅中可以扩增出２００、５５０、９００ｂｐ三条带（Ｆｉｇ．１）。Ｓｏｕｔｈｅｒｎ杂交表明２００和５５０ｂｐ两条带可以和ＳＲＹ基因探针杂交（Ｆｉｇ．２）。第二对引物是兼并引物,可以特异扩增Ｓｏｘ基因的ＨＭＧ－ｂｏｘ区域。以基因组ＤＮＡ为模板可以在大鳞副泥鳅中扩增出２２０、５５０、７００和１５００ｂｐ四条带（Ｆｉｇ．３）;而在泥鳅中则为２２０、５３０、５７０和１５００ｂｐ四条带（Ｆｉｇ．４）。ＰＣＲ产物的长度差异被认为是由于部分Ｓｏｘ基因的ＨＭＧ－ｂｏｘ区域中存在着内含子。没有发现性别特异扩增带。以上结果说明哺乳动物与性决定有关的组分在脊椎动物中是广泛存在的。但这些组分在鱼类中是否与性决定有关,或仅是哺乳动物性决定因子的进化前体尚不得而知。无论如何广泛深入研究鱼类的Ｓｏｘ基因对于揭示性决定系统和因子的进化方式是十分     

Chlorine inactivation of spores of Encephalitozoon spp

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.     

Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia,Spain

In this survey we examined 87 domestic animal stool samples in order to detect the possible presence of microsporidia in animals in close contact with humans in Galicia (NW, Spain). The detection of Enterocytozoon bieneusi spores was confirmed in faecal samples from two dogs and one goat by polymerase chain reaction. None of the positive samples for microsporidia in the staining method were amplified with species-specific primers for Encephalitozoon intestinalis, E. hellem and E. cuniculi. Four rabbits faecal samples reacted with anti-E. cuniculi serum. Our results could indicate the importance of domestic animals as zoonotic reservoirs of microsporidial human infections.     

The immunoperoxidase test diagnosis of Encephalitozoon cuniculi in rabbits.

Sera collected from both naturally and artificially infected rabbits were found to show excellent correlation when examined for the presence of Encephalitozoon cuniculi antibodies using the immunoperoxidase and immunofluorescence tests. Out of 85 randomly selected rabbits, 21 were found to be serologically positive using both the tests. However, lesions which could be attributed to E. cuniculi infection were only demonstrated in 16.