A New Species of ThelohanellusKudo, 1933 (Myxozoa: Bivalvulida) Infecting Skeletal Muscle of Blacktail Shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in the Chattahoochee River Basin, Georgia.

Thelohanellus magnacysta n. sp. (Bivalvulida: Myxobolidae) infects the skeletal muscle of blacktail shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in Bull Creek, Chattahoochee River Basin, eastern Georgia. Although numerous members of ThelohanellusKudo, 1933 have overlapping myxospore dimensions with the new species, it differs from all nominal congeners by polar filament coil number and polar capsule width as well as by lacking a mucous envelope, iodinophilic vacuole, and sutural markings. With the use of novel primers for Myxozoa, a phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) suggests that the new species shares a recent common ancestor with a clade of cyprinid-infecting species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) and Thelohanellus. Consistent with other published research concerning the systematics of Thelohanellus, this result suggested that Thelohanellus and Myxobolus are polyphyletic and need revision. Histological sections of infected blacktail shiners confirmed that myxospores were only found within a plasmodium and only infected skeletal muscle and that plasmodia were encapsulated by a granuloma comprising varying degrees of acute granulomatous inflammation. The new species is the fourth of Thelohanellus reported from North America and the first reported from Cyprinella, as well as the first myxozoan described from the blacktail shiner.

Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy.

Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.

Are we There Yet? Understanding Interplanetary Microbial Hitchhikers using Molecular Methods.

Since the early time of space travel, planetary bodies undergoing chemical or biological evolution have been of particular interest for life detection missions. NASA's and ESA's Planetary Protection offices ensure responsible exploration of the solar system and aim at avoiding inadvertent contamination of celestial bodies with biomolecules or even living organisms. Life forms that have the potential to colonize foreign planetary bodies could be a threat to the integrity of science objectives of life detection missions. While standard requirements for assessing the cleanliness of spacecraft are still based on cultivation approaches, several molecular methods have been applied in the past to elucidate the full breadth of (micro)organisms that can be found on spacecraft and in cleanrooms, where the hardware is assembled. Here, we review molecular assays that have been applied in Planetary Protection research and list their significant advantages and disadvantages. By providing a comprehensive summary of the latest molecular methods yet to be applied in this research area, this article will not only aid in designing technological roadmaps for future Planetary Protection endeavors but also help other disciplines in environmental microbiology that deal with low biomass samples.

Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain.

Bacterial conjugation is a powerful tool used for DNA transfer from Escherichia coli into various bacteria including streptomycetes. In this methodology, spores are usually employed as recipient cells of the genetic information. However, some industrially important Streptomyces do not produce spores making difficult their genetic manipulation. In these strains, the use of mechanically fragmented mycelia allows DNA transfer with low efficiency. Streptomyces peucetius var. caesius is a non-sporulating bacteria which produces the antitumor compound doxorubicin. The use of aerial mycelia of this microorganism, failed to get intergeneric conjugation with E. coli. In the present work, by using young aerial mycelia of this microorganism and an excess of E. coli cells (~7×108cellsmL-1) in soybean-mannitol medium (MS) supplemented with 20mMMgCl2 resulted in a high number of exconjugant colonies (5×10-4) when compared to other reports from this genus (1.1×10-5 to 2.5×10-8). The effectiveness of these conditions was confirmed by isolating null mutants of two different glucokinases from S. peucetius var. caesius. The novelty in using young aerial mycelia as receptor cells, allowed an efficient conjugative process and opened the way for genetic manipulation of additional non-spore forming actinobacteria exhibiting natural resistance to be genetically manipulated.

Effects of postharvest oligochitosan treatment on fungal diseases and defence responses in Dongxue peach fruit.

In this study, the effects of oligochitosan treatment on controlling postharvest diseases in Dongxue peach ( Prunus Persica L. Batsch, cv Dongxuemi) were examined and the possible underlying mechanisms were discussed. Results showed that the disease incidence and lesion area in peach fruit inoculated with Monilinia fructicola and Penicillium expansum were all remarkably reduced by oligochitosan treatment. Oligochitosan treatment inhibited spore germination and mycelial growth of the two fungi in vitro. Oligochitosan treatment also induced upregulation of the salicylic acid signalling pathway-related genes (NPR1, PR1 and phenylalanine ammonia lyase) and enhanced the levels of total phenolics, flavonoids and lignin in peach. Meanwhile, enzymatic activities of superoxide dismutase, catalase, polyphenoloxidase, ascorbate peroxidase and phenylalanine ammonia lyase also increased. These findings suggest that the effects of oligochitosan on the disease control of peach fruit may be associated with its direct antimicrobial effects as well as increasing antioxidant, phenylpropanoid metabolism and accumulating antifungal compounds by activating the salicylic acid-dependent pathway.

Myxobolus sp. and Henneguya sp. (Cnidaria: Myxobolidae) natural co-infection in the kidney of Piaractus mesopotamicus (Characiformes: Serrasalmidae).

This study evaluated the myxozoan infection and histopathology of the kidney of freshwater fish Piaractus mesopotamicus from intensive fish farming in Brazil. A total of 55 fish were examined for myxozoan infection. Infected organs were processed by usual histology and stained with hematoxylin-eosin (H&E) and Ziehl-Neelsen (ZN). From the total of 55 fish analyzed, 47 (85.45%) presented myxospores, being 9.09% (5/55) only with Myxobolus sp., 5.45% (3/55) only with Henneguya sp., and 70.91% (39/55) presenting both parasites. The presence of myxospores was associated with histological alterations in both stromal and renal parenchyma. Myxospores were found mostly in the peritubular interstitial tissue and in low intensity in the glomerulus which caused nuclear hypertrophy and loss of Bowman space. An increase in the glomerular tuft and a reduction in the lumen of the collector tubules were also observed, besides the high number of melanomacrophage cells in the glomerulus. This study reports for the first time detection of myxozoan mixed infection in one organ of pacu and discuss the possible transportation of myxospores in the circulating blood.

Myxosporidiosis in intensively-reared Piaractus mesopotamicus: Histopathological diagnosis by means of Ziehl-Neelsen staining / Mixosporidiose em Piaractus mesopotamicus de criação intensiva: diagnóstico histopatológico pela coloração Ziehl-Neelsen

Samples of different organs from intensively-reared Piaractus mesopotamicus were collected and processed using routine histological techniques in order to produce thin sections for staining with hematoxylin-eosin and with the Ziehl-Neelsen method. Through examination under an optical microscope, myxosporidians of the genera Henneguya sp. and Myxobolus sp. were identified, respectivelyin the gills and kidneys of P. mesopotamicus. Plasmodia with immature spores of Henneguya sp. were located along the secondary lamellae, with total length of 30.45±4.84µm and width of 3.52±0.33µm. Spores of Myxobolus sp. were located in the kidneys, with total length of 8.94±0.82µm and width of 5.59±0.39µm. Histopathological analysis of the gills showed plasmodia containing spores of Henneguya sp., at intralamellar and intravascular localities, at different stages of development. Spores of Myxobolus sp. were identified in the kidneys, in the peritubular region and in the interstices and glomerulus, surrounded by melanomacrophages. Focal hemorrhage was recorded in a few cases. Ziehl-Neelsen staining allowed to identify particular features of the spores and facilitated biometry and enabled classification in comparison with hematoxylin-eosin, thus demonstrating its usefulness for histopathological diagnosis of the parasitosis.

Amostras de diferentes órgãos de Piaractus mesopotamicus mantidos em criação intensiva foram coletadas e processadas mediante as técnicas histológicas usuais para obtenção de cortes que foram corados com hematoxilina-eosina e pelo método de Ziehl-Neelsen. Ao exame em microscopia de luz foi possível identificar mixosporídeos dos gêneros Henneguya sp. e Myxobolus sp. em brânquia e rim de P. mesopotamicus respectivamente. Plasmódios com esporos imaturos de Henneguya sp. foram localizados ao longo das lamelas secundárias e mensurados (comprimento total 30,45±4,84µm e largura 3,52±0,33µm) e no rim esporos de Myxobolus sp. (comprimento total 8,94±0,82µm e largura 5,59±0,39µm). Na análise histopatológica das brânquias observaram-se plasmódios contendo esporos de Henneguya sp., com localização intralamelar e intravascular, em diferentes estágios de desenvolvimento. No rim identificaram-se esporos de Myxobolus sp., na região peritubular e no interstício e glomérulo, circundados por melanomacrófagos. Em poucos casos foi registrada hemorragia focal. O uso da coloração de Ziehl-Neelsen permitiu identificar particularidades dos esporos, facilitou sua biometria e classificação em comparação com a hematoxilina-eosina, demonstrando sua utilidade no diagnóstico histopatológico da referida parasitose.

Isolamento, identificação clássica e análise molecular de bactérias provenientes de coprólitos de sítios arqueológicos brasileiros / Isolation, identification classical and molecular analysis of bacteria from coprolites from archaeological sites in Brazil

A partir de amostras de coprólitos brasileiros datados entre 3490 mais ou menos 120 a 430 mais ou menos 70 anos, recuperados de escavações arqueológicas no Brasil, fomos capazes de isolar bactérias da família Bacillaceae. Objetivou-se com esse estudo testar e padronizar técnicas para detecção, identificação e caracterização molecular de microrganismos bacterianos nessas amostras. Os coprólitos foram inicialmente tratados com radiação ultravioleta, buscando eliminar contaminantes externos e posteriormente perfurados para retirada do material mais interno. Esse material foi cultivado em aerobiose em diferentes meios de cultura, originando o isolamento de bastonetes Gram positivos formadores de endósporos. Esses microrganismos foram submetidos a provas bioquímicas e fisiológicas, de sensibilidade a antimicrobianos, reação em cadeia da polimerase (PCR), clonagem e sequenciamento, buscando sua identificação e caracterização. Medidas para evitar a contaminação por microrganismos modernos foram tomadas, como a utilização de apenas controles negativos nas reações de PCR, duplicação dos experimentos, entre outras. Das 7 amostras de coprólitos utilizadas, apenas 5 originaram algum isolado, sendo estas datadas entre 3490 mais ou menos 120 a 430 mais ou menos 70 anos ou sem datação conhecida . O emprego de diferentes técnicas laboratoriais para identificação de microrganismos antigos ficou evidente. Essa diversificação aumenta as chances de identificação dos isolados, o que possibilitou concluir que se tratava de representantes da família Bacillaceae, do gênero Bacillus e correlatos.

Geoclimatic influences on invasive aspergillosis after hematopoietic stem cell transplantation.

BACKGROUND: Aspergillus species are ubiquitous. We hypothesized that climatic variables that affect airborne mold counts affect the incidence of invasive aspergillosis (IA). METHODS: Patients who received hematopoietic stem cell transplants (HSCTs) in geographically and climatically diverse regions (Seattle, WA, and Houston, TX) were examined. Cumulative incidence function, Kaplan-Meier analysis, and Cox proportional hazards regression were performed to examine the association between IA and season. Poisson regression analysis was performed to evaluate the seasonal patterns in IA rates and association with spore counts and climate. RESULTS: In Seattle, the 3-month incidence of IA was 4.6% (5.7% in allograft recipients and 0.8% in autograft recipients). During the 10-year study period, there was a decrease in the incidence of IA among allogeneic HSCT recipients, corresponding to decreased risks during the nonsummer months; receipt of HSCTs during the summer months was associated with an increased hazard for IA (hazard ratio, 1.87; 95% confidence interval, 1.25-2.81) after adjustment for other known risks. The person-month IA rate in Seattle was positively associated with environmental spore counts, which increased with high temperature and low precipitation. No seasonal effect on IA was observed in Houston, where total spore counts were lower and not variable by climate. CONCLUSIONS: Climatic variables differentially affect airborne spore counts and IA risk in geographically disparate centers.

Resistance of polysaccharide coatings to proteins, hematopoietic cells, and marine organisms.

The interaction of covalently coupled hyaluronic acid, alginic acid, and pectic acid with proteins, cells (hematopoietic KG1a and Jurkat cells), and marine organisms (algal zoospores and barnacle cypris larvae) is compared. In contrast to cells and proteins for which such polysaccharide coatings are known for their antiadhesive properties, marine algal spores and barnacle cyprids were able to colonize the surfaces. Of the three polysaccharides, hyaluronic acid showed the lowest settlement of both Ulva zoopores and barnacles. Photoelectron spectroscopy reveals that the polysaccharide coatings tend to bind bivalent ions, such as calcium, from salt water. Such pretreatment with a high salinity medium significantly changes the protein and hematopoietic cell resistance of the surfaces. Complexation of bivalent ions is therefore considered as one reason for the decreased resistance of polysaccharide coatings when applied in the marine environment.

Efficiency of Artemia cysts removal as a model invasive spore using a continuous microwave system with heat recovery.

A continuous microwave system to treat ballast water inoculated with Artemia salina cysts as a model invasive spore was tested for its efficacy in inactivating the cysts present. The system was tested at two different flow rates (1 and 2 L x min(-1)) and two different power levels (2.5 and 4.5 kW). Temperature profiles indicate that the system could deliver heating loads in excess of 100 degrees C in a uniform and near-instantaneous manner when using a heat recovery system. Except for a power and flow rate combination of 2.5 kW and 2 L x min(-1), complete inactivation of the cysts was observed at all combinations at holding times below 100 s. The microwave treatment was better or equal to the control treatment in inactivating the cysts. Use of heat exchangers increased the power conversion efficiency and the overall efficiency of the treatment system. Cost economics analysis indicates that in the present form of development microwave treatment costs are higher than the existing ballast water treatment methods. Overall, tests results indicated that microwave treatment of ballast water is a promising method that can be used in conjunction with other methods to form an efficient treatment system that can prevent introduction of potentially invasive spore forming species in non-native waters.

Cytological diagnosis of rhinosporidiosis with skeletal involvement--a case report.

A young Hindu male presented with painful swelling of left lower thigh for 6 months. The provisional diagnosis both clinically and radiologically was osteosarcoma. FNAC and biopsy proved the lesion to be a case of rhinosporidiosis. The present case is reported due to rare incidence of skeletal rhinosporidiosis.

Molecular detection and phylogenetic placement of a microsporidian from English sole (Pleuronectes vetulus) affected by X-cell pseudotumors.

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.

Cytologic detection of microsporidia spores in bile. A comparison of stains.

OBJECTIVE: To compare stains in preparations of bile in a patient with AIDS and microsporidial cholangitis. STUDY DESIGN: Bile was obtained from a 30-year-old male with AIDS and symptoms of cholangitis. Comparative staining of the specimen was performed using a formalin-fixed preparation stained with Chromotrope 2R stain and with alcohol-fixed preparations stained with Gram and Giemsa stain and Diff-Quik. An alcohol-fixed ThinPrep slide was stained with Papanicolaou stain. RESULTS: Diagnostic microsporidia spores were detected under oil immersion using Papanicolaou, Chromotrope 2R, Giemsa and Gram stain. The Diff-Quik-stained preparation also revealed microsporidia but with suboptimal morphology. CONCLUSION: Detection of microsporidia in bile can be achieved using several different stains routinely available to cytologists, most optimally with alcohol-fixed Papanicolaou- or Giemsa-stained preparations or with Chromotrope 2R stain, which is available in parasitology laboratories. These findings should be applicable to fluids from other body sites with this emerging pathogen in AIDS.

Onicomicosis: técnicas diagnósticas / Onychomycosis: diagnostic techniques

El objetivo de nuestro estudio es determinar el rendimiento de diferentes técnicas diagnósticas de onicomicosis, la correlación entre ellas y entre las diversas formas clínicas de onicomicosis. Se realizó un estudio prospectivo de 124 muestras de uñas de pacientes con el diagnóstico clínico de onicomicosis examinados en el Hospital Clínico de la Universidad de Chile, entre diciembre de 1995 y septiembre de 1996. Pacientes que recibieron tratamientos antimicóticos orales tres meses antes o tópicos una semana previa al estudio, fueron excluidos. A las muestras ungueales se les realizó estudio micológico directo, cultivo y examen histopatológico. Al 25 por ciento de las muestras se les realizó microscopia electrónica de barrido. Los resultados demostraron que la edad promedio fue de 44 años. Las manifestaciones clínicas en más de un 90 por ciento de los casos fueron cambios de color, de grosor y, en menor porcentaje, onicolisis. Las formas clínicas subungueal distal y distrófica total fueron las más frecuentes. Un 81,5 por ciento de las muestras presentaron un examen directo positivo, un 39,5 por ciento un cultivo positivo y más del 50 por ciento un examen histopatológico y de microscopia electrónica positivo. Existió una correlación estadística significativa entre las formas clínicas más frecuentes de onicomicosis y el examen micológico directo

Pathology and morphology of Ichthyophonus hoferi in naturally infected fishes off the Swedish west coast.

The pathology and morphology of Ichthyophonus hoferi was studied in naturally infected Atlantic herring Clupea harengus, in sprat Sprattus sprattus, and in flounder Pleuronectes flesus from the west coast of Sweden. The pathogen was found in all organs examined, with the intensity of infection varying in different organs of the different fish species. Two main phases in the life of infecting parasites were identified, 'active' and 'passive', the latter being able to switch to active. The active phase of the infection in herring was usually accompanied by a lean and slender appearance of the body, a drastic decrease in intestinal fat, emaciation of the somatic muscles, swelling of the visceral organs, poor quality of flesh texture and a distinctive off-odour. The most characteristic macroscopic sign of ichthyophonosis in herring and flounder was the occurrence of creamy white nodules on the heart. The infection causes a chronic systemic granulomatous inflammation. The nature of the granulomatous inflammation was host- and tissue-dependent. The pathogenicity of the parasite in its active form and the side effects of host defence cells were also reflected in dramatic tissue damage and loss of structure and function of the infected organs. Three kinds of spores were identified: 'un-developing spore', 'developing spore' and 'plasmodio-spore'. The formation and spread of 'plasmodia', from plasmodio-spores, as a secondary infection agent is documented. Transmission electron microscopy revealed I. hoferi to be multinucleated, containing different organelles and structures. These included a cell wall, an undulating cell membrane, a thin paramural endoplasm, an endoplasmic reticulum, polymorphic but usually spherical mitochondria with short tubulo-vesicular cristae, dictyosomes with plate-like cristernae, large electron-dense lipid droplets and electron-lucid vacuoles, probably containing glycogen.

Identification of a human isolate of Encephalitozoon cuniculi type I from Italy.

A microsporidial strain, obtained from a person with AIDS living in Italy was isolated and cultivated on RK13 (rabbit kidney) cell monolayers. Identification at the species level was performed by immunological and molecular methods. Western blot analysis showed that the human isolate and the Encephalitozoon cuniculi reference strain had similar banding patterns. The small subunit rRNA sequence analysis confirmed the identification of the isolate as E. cuniculi, which is a widespread microsporidian species infecting a wide range of natural hosts, including humans. Moreover, based on the sequence of the rDNA internal transcribed spacer region, this isolate was classified as E. cuniculi type I (rabbit strain), previously reported in six persons with AIDS living in Switzerland. These results provide further information on the geographical distribution of E. cuniculi types.