Acinetobacter baumannii in Localised Cutaneous Mycobacteriosis in Falcons

Between May 2007 and April 2009, 29 falcons with identically localized, yellowish discolored cutaneous lesions in the thigh and lateral body wall region were presented at Abu Dhabi Falcon Hospital. Out of 18 falcons integrated in this study, 16 tested positive to Mycobacterium. avium complex. The 2 negative falcons tested positive in the Mycobacterium genus PCR. Moreover, 1 falcon tested positive to M. avium. paratuberculosis in tissue samples by PCR. In all cases, blood and fecal samples tested negative. In the acid-fast stain, all samples showed the for mycobacteriosis typical rods. Moreover, in 13 samples Acinetobacter baumannii was detected by PCR and proven by DNA sequencing. Clinical features included highly elevated WBCs, heterophilia, lymphocytopenia, monocytosis, severe anemia and weight loss. A. baumannii, a gram-negative bacillus with the ability to integrate foreign DNA, has emerged as one of the major multidrug resistant bacteria. In veterinary medicine, it has so far been detected in dogs, cats, horses and wild birds. To the authors' knowledge, this is the first report of an A. baumannii infection in falcons and of a veterinary Mycobacterium-Acinetobacter coinfection

Free-living Amoebae in Carbonate Precipitating Microhabitats of Karst Caves and a New Vahlkampfiid Amoeba, Allovahlkampfia spelaea gen. nov., sp. nov.

This is the first report on the diversity of small free-living amoebae (FLA) in carbonate precipitating habitats in karst caves. Of 11 samples from nine different habitats in four Slovenian karst caves ten samples were positive for FLA, four strains were successfully isolated and transferred to clonal monoxenic cultures, including Acanthamoeba castellanii genotype T4, Echinamoeba silvestris, Hartmannella vermiformis, and a new vahlkampfiid amoeba Allovahlkampfia spelaea gen. nov., sp. nov. The latter was isolated from a stromatolitic stalagmite, a typical biogenic speleothem. Echinamoeba silvestris was identified from an aerophytic algal community and Acanthamoeba and Hartmannella were isolated from a cave pool with floating calcite rafts. The grazing of FLA on bacteria may help in creating conditions that enhance carbonate precipitation

Pseudodidymium cryptomastigophorum gen. n., sp. n., a Hyperamoeba or a Slime Mould? A Combined Study on Morphology and 18S rDNA Sequence Data

Summary. The Hyperamoeba-like amoeboflagellate (Wi7/2-PE) has been isolated from a hydrotherapy pool inside a hospital at Wildbach/ Germany. Based on combined morphological and molecularbiological data we conclude that this isolate can neither be assigned to the genus Hyperamoeba nor to any of the myxogastrean slime moulds, which are the closest relatives of Hyperamoeba. We thus considered describing this isolate within a new genus, Pseudodidymium, as a new species, Pseudodidymium cryptomastigophorum. As observed by phase contrast microscopy the gross morphology and size of the three stages -trophozoite, cyst, and flagellate stage -is comparable to respective characters of Hyperamoeba as described b

Acanthamoeba castellanii : growth on human cell layers reactivates attenuated properties after prolonged axenic culture

The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba

Successful Treatment of Disseminated Acanthamoeba sp. Infection with Miltefosine

We report on an HIV-negative but immunocompromised patient with disseminated acanthamoebiasis, granulomatous amoebic encephalitis, and underlying miliary tuberculosis and tuberculous meningitis. The patient responded favorably to treatment with miltefosine, an alkylphosphocholine. The patient remained well with no signs of infection 2 years after treatment cessation

The identification of free-living environmental isolates of amoebae from Bulgaria

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named "6” (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)—all exhibiting a highly efficient proliferation in axenic cultures—were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCR

Molecular Identification of a Phage-infected Protochlamydia Strain Naturally Harboured by Non-Encysting Naegleria

A thermophilic strain of Naegleria clarki, isolated from a pond, has previously been investigated for its peculiarity to host a cytoplasmic symbiont, which causes a loss of the ability to form cysts. This endosymbiont, called Pcb, was itself infected by a phage, and exhibited chlamydia-like features resembling to another symbiont of Naegleria previously described as Protochlamydia naegleriophila. We report in this study, the results of amoeba host range and 16S rDNA molecular phylogeny of this strain, showing that Pcb is a new strain of the Naegleria endosymbiont chlamydial species Protochlamydia naegleriophila (Chlamydiae: Parachlamydiaceae)

Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota

Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays

BackgroundIn vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity.MethodsThe viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy.ResultsIt was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity.DiscussionOur findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests