'Candidatus Megaira polyxenophila' gen. nov., sp. nov.: considerations on evolutionary history, host range and shift of early divergent rickettsiae.

"Neglected Rickettsiaceae" (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as 'Candidatus Megaira polyxenophila' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica (Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (Alphaproteobacteria, Rickettsiales) as a sister group of the genus Rickettsia. In situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that 'Candidatus Megaira polyxenophila' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria.

Ultrastructure of the rickettsial endosymbiont "MIDORIKO" in the green alga Carteria cerasiformis as revealed by high-pressure freezing and freeze-substitution fixation.

Bacterial endosymbionts belonging to the family Rickettsiaceae were recently identified in the unicellular green alga Carteria cerasiformis, providing the first molecular evidence of rickettsial endosymbionts within photosynthetic eukaryotes. However, previous morphological studies using transmission electron microscopy (TEM) with conventional chemical fixation did not demonstrate whether the endosymbionts of C. cerasiformis have the diagnostic characteristics of the family Rickettsiaceae. In this study, we observed the rickettsial endosymbionts "MIDORIKO" within C. cerasiformis cells by TEM with high-pressure freezing and freeze-substitution fixation. The rickettsial endosymbionts resided directly in the C. cerasiformis cytoplasm without engulfing or encompassing membranes or vacuoles. The endosymbionts had a Gram-negative cell envelope composed of outer and inner bilayer membranes. The thicknesses of the outer and inner leaflets of the bacterial cell wall were almost identical. These morphological characteristics are consistent with those of the genus Rickettsia, but the cell wall structure differed from that of the genus Orientia within the family Rickettsiaceae.

"Candidatus Cryptoprodotis polytropus," a novel Rickettsia-like organism in the ciliated protist Pseudomicrothorax dubius (Ciliophora, Nassophorea).

Rickettsia-like organisms (RLO) are obligate, often highly fastidious, intracellular bacterial parasites associated with a variety of vertebrate and invertebrate hosts. Despite their importance as causative agents of severe mortality outbreaks in farmed aquatic species, little is known about their life cycle and their host range. The present work reports the characterization of "Candidatus Cryptoprodotis polytropus," a novel Rickettsia-like bacterium associated with the common ciliate species Pseudomicrothorax dubius by means of the "Full-Cycle rRNA Approach" and ultrastructural observations. The morphological description by in vivo and scanning electron microscopy and the 18S rRNA gene sequence of the host species is provided as well. Phylogenetic analysis based on the 16S rRNA gene supports the inclusion of "Candidatus Cryptoprodotis polytropus" within the family Rickettsiaceae (cl. Alphaproteobacteria) together with the genera Rickettsia and Orientia. Observations on natural ciliate populations account for the occasional nature of this likely parasitic association. The presence of a previously unknown RLO in ciliates sheds a new light on the possible role of protists as transient hosts, vectors or natural reservoir for some economically important pathogens.

Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould.

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.

Gene structure, activity and localization of a catalase from intracellular bacteria in Onchocerca volvulus.

Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O. volvulus adult lambda zapII cDNA library. Analysis of their nucleotide and encoded amino acid sequences revealed that they derive from intracellular bacteria, rather than the O. volvulus nuclear genome. The endobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene. The endobacterial CAT gene encodes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E. coli expression system. The purified 54 kDa protein has CAT activity over a broad pH range, with a specific activity of 103,000 +/- 3000 U mg(-1). The optical spectrum of the endobacterial CAT shows that it is a ferric haem-containing protein with a Soret band at 405 nm. To investigate the phylogeny of the intracellular bacterium in O. volvulus, a segment of the 16S rRNA gene was amplified from total genomic DNA by a polymerase chain reaction using universal eubacterial primers. A phylogenetic analysis of the O. volvulus-derived 16S rRNA sequence revealed that the endobacterium belongs to a distinct Wolbachia clade of the order Rickettsiales. Onchocercomata and biopsies containing different onchocercal species were immunohistochemically stained using polyclonal antibodies raised against the recombinant endobacterial CAT. CAT was detected in the endobacteria in the hypodermis of adult male and female O. volvulus, O. ochengi, O. gibsoni and O. fasciata. The endobacterial enzyme was also detected in onchocercal oocytes and all embryonic stages including intrauterine microfilariae as well as skin microfilariae. O. volvulus thus harbours Wolbachia-like endosymbionts which are transovarially transmitted and show particular affinity for the hypodermal tissues of the lateral chords.

In vitro infection of a cell line from Ictalurus nebulosus with Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiologic agent of salmonid rickettsial septicemia (SRS), affects several species of salmonids. Previous reports using the appearance of cytopathic effect (CPE) as the criterion for susceptibility, showed that Piscirickettsia salmonis (ATCC strain) can be grown in vitro in some cells lines derived from salmonid fish, but not in BB cells from brown bullhead (Ictalurus nebulosus) and BF-2 cells from bluegill (Lepomis macrochirus). In this study we describe growth of P. salmonis (ATCC strain VR 1361) in a cell line previously believed to be nonpermissive for this organism. CPE was first detected in chinook salmon embryo (CHSE-214) and epithelioma papulosum ciprini (EPC) cell lines at 6 d postinfection (dpi). In contrast, using BB cell line, CPE was first detected 45 dpi and the monolayer completed CPE by 78 dpi. Electron microscopic examination of BB cells 78 dpi revealed free, intracytoplasmic and extracellular localization of the agent. P. salmonis was also observed within membrane-bounded vacuoles in BB cells, similar to that described in CHSE 214 cells. Contrary to earlier reports, results from the present study show that the BB cell line, is susceptible to Piscirickettsia salmonis infection. The delayed onset of CPE in BB cells in comparison to other permissive cell lines suggests that BB cells are not ideal hosts for P. salmonis. Interestingly, however, these results demonstrate that P. salmonis can infect non-salmonid cell lines, and raises the possibility that non-salmonid fish may play a role in the persistence and transmission of SRS in the natural environment.

Fine needle aspiration diagnosis of intramuscular bacillary angiomatosis. A case report.

BACKGROUND: Bacillary angiomatosis (BA) is a localized infectious process that affects primarily patients with the acquired immunodeficiency syndrome. The microorganisms implicated in the pathogenesis of this disease belong to the Rickettsiaceae family. CASE: A 43-year-old, human immunodeficiency syndrome-positive male presented with diffuse swelling in the right deltoid area. A neoplastic process was considered in the differential diagnosis. Fine needle aspiration biopsy showed proliferation of blood vessels lined with plump endothelial cells, and the interstitial space was occupied by neutrophilic infiltrate, leukocytoclastic debris and clumps of characteristic amphophilic, granular material. Warthin-Starry stain demonstrated clusters of bacilli diagnostic of bacillary angiomatosis. CONCLUSION: The diagnosis of this entity, made by fine needle aspiration cytology (as the only diagnostic procedure), was instrumental in preventing further surgical manipulation and in initiating appropriate and immediate antibiotic therapy.

[Bacillary angiomatosis: report of 2 cases]. / Angiomatosis bacilar: a propósito de 2 casos.

BACKGROUND: Epithelioid bacillary angiomatosis (EBA) was studied as an infectious disease associated to immunosuppressive states, establishing the bases for performing differential diagnosis with other pathologic processes. METHODS: Two new cases of EBA in patients with human immunodeficiency virus infection (HIV) are presented. Diagnosis was performed by anatomopathologic study of the cutaneous lesions which had undergone biopsy. RESULTS: In one of the cases bacillary structures were observed under electron microscopy. This patient also presented Kaposi sarcoma (KS) with histologic study being therefore necessary to perform differential diagnosis between these pathologic processes. Both patients presented good response to treatment with erythromycin. CONCLUSIONS: 1) EBA is an infectious disease of good prognosis with antibiotic treatment which fundamentally affects severely immunosuppressed patients with human immunodeficiency virus infection. 2) Biopsy is the only differential diagnostic method for this disease with other processes with similar clinical appearance and different prognosis as in Kaposi sarcoma which may even coexist in these patients.

Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient.

Isolation of a Rochalimaea-like organism from a febrile patient infected with human immunodeficiency virus was confirmed. Analysis of 16S rRNA gene sequences, together with polymerase chain reaction and restriction endonuclease length polymorphism analysis of a portion of the citrate synthase gene, demonstrated that the agent is closely related to members of the genus Rochalimaea and that the isolate is genotypically identical to the presumptive etiologic agent of bacillary angiomatosis. However, the same genotypic analyses readily differentiated the new isolate from isolates of other recognized Rochalimaea species as well as other genera of bacteria previously suggested as putative etiologic agents of bacillary angiomatosis and related syndromes. We propose that the novel species be referred to as Rochalimaea henselae sp. now.

Purification of Cytoecetes phagocytophila, the causative agent of tick-borne fever, from infected ovine blood.

Cytoecetes phagocytophila, the causative agent of tick-borne fever, was successfully separated by Percoll and Renografin density gradient centrifugation and by cellular affinity chromatography, from the peripheral blood leucocytes of experimentally infected sheep. After centrifugation on Renografin or Percoll density gradients, infectious particles of C. phagocytophila banded at buoyant densities which ranged between 1.063 to 1.140. Rickettsiae separated by wheat germ lectin cellular affinity chromatography retained their morphology but often lost their infectivity. Cell-free C. phagocytophila remained infective to susceptible sheep for 6 months when kept at -114 degrees C in sucrose phosphate buffer with 10 per cent dimethylsulphoxide as a cryopreservative.

Coxiella and Rickettsiella: comparison of ultrastructure with special reference to their envelope.

Ultrastructure of Rickettsiella phytoseiuli (R.p.) multiplying in female ticks Dermacentor reticulatus was compared with that of Coxiella burnetii (C.b.) in the same ticks and in mice. C.b. in ticks and mice were always represented by 2 main cell types: small dense round or rod-like cells (DC) and larger bacteria-like cells (BC). DC were surrounded with a characteristic five-layered 20 nm thick envelope. Under the envelope DC had a stack of parallel intracytoplasmic membranes with a periodicity 5-6 nm. R.p. in tick fat body and synganglion were also inside phagosomes and formed 6 sequentially developing cell forms: dense (elementary), intermediate, bacterial, giant, and crystal-forming in which small dark bodies (initial particles) condensed. Two of them--dense and bacterial--corresponded to DC and BC of C.b. The DC envelope structure of R.p. was strikingly similar to that of some C.b. DC in mouse. We confirmed the general morphologic similarities in the structure of C.b. and R.p. DC and that of C.b. BC and intermediate cells of R.p. The envelope structure of DC type was found in other gracilicute bacteria and is supposed to have no taxonomic value but to be a reflection of population heterogeneity.

Extracellular giant rickettsiae associated with bacteria in the gill of Crassostrea gigas (Mollusca, Bivalvia).

Extracellular giant rickettsiae closely associated with bacteria with a Gram-negative type wall are reported among the basal insertions of the cilia of the gill epithelium of the oyster Crassostrea gigas (Mollusca, Bivalvia) from the north of Spain. These rickettsiae are extremely pleomorphic cells with a maximum 9 microns length. The internal elements are ribosome-like particles and DNA strands are distributed in some light areas. Some rickettsial cells show one to two vacuole-like dense inclusions formed by curved concentric bands approximately 4 nm thick separated by an approximately 5-nm light space (myelin-like bodies). Numerous bacteria are closely associated with the rickettsiae in the surrounding zone. The cytopathic effects of this association seemed to give rise to the epithelial lysis and concomitant disappearance of the apical microvilli and cilia and the consequent destruction and disintegration of the gill epithelial cells, where these rickettsiae live in close proximity. This is believed to be the first description of extracellular giant pathogenic rickettsiae of these oyster species.

Endocytobionts in Dermacentor reticulatus ticks (Ixodidae): an electron microscope study.

Endocytobionts (ECBs) were detected in the ovaries of Dermacentor reticulatus. Their developmental cycle is directly related to the developmental stages of tick oocytes. Two basic forms of ECBs occur in tick cells, i.e. dense forms, occurring singly or in aggregates situated free in the cytoplasm of host cells, and the light forms which are larger, pleomorphic and always situated inside vacuoles of host cells. The light forms occur together with dense forms in all oocytes. The dense forms occur freely and independently in funicular cells of the oocyte, the epithelial cells of oviducts, and additionally in the cells of the Malpighian tubules. A probable function of ECBs in the tick host is discussed.

Comparative study on Rickettsia-like organisms in the midgut epithelial cells of different Glossina species.

The midgut epithelium of Glossina morsitans centralis, G. austeni, G. pallidipes, G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes, G. tachinoides and G. brevipalpis from ILRAD-bred colonies was examined, by electron microscopy, for the presence and distribution of Rickettsia-like organisms (RLOs). RLOs were present in the midgut epithelial cells of all non-teneral tsetse. In G.m. centralis, G. pallidipes and, to a much lesser extent, G. brevipalpis, RLOs were numerous and were present in all the specimens examined. RLOs were present in fewer numbers in the epithelial cells of tenerals of these three tsetse species. In contrast, RLOs occurred in very much lower numbers within the midgut cells of nonteneral G. austeni, G. p. palpalis, G. p. gambiensis, G.f. fuscipes and G. tachinoides; were not seen in every specimen, and were rarely observed in the midgut cells of teneral testse. The RLOs were typical rod-shaped bacteria with an inner and outer membrane, which occurred free within the host cell cytoplasm and appeared to cause no obvious pathology. The micro-organisms divided by binary fission and at least two distinct morphological forms plus a range of intermediate forms were seen in the midgut cells. A comparison of the presence and numbers of RLOs within the midgut cells and the midgut infection rates of both Trypanosoma congolense and T. b. brucei, both between Glossina species and also within the same stock of tsetse, clearly indicates that the ability of trypanosomes to establish and develop to mature infections is unlikely to be correlated solely with the presence of RLOs within the tsetse midgut.

Rickettsiae in gill epithelial cells of the hard clam, Mercenaria mercenaria.

Rickettsiae are found in the gill epithelium of the hard clam, Mercenaria mercenaria. The procaryotes occur free in the cytoplasm of the epithelial cells at the tip of the filament and in the more proximal cells that support the lateral J cilia. The fine structure of the organisms, showing rippled cell walls, is typical of the rickettsiae. The increasing size of the inclusion representing late phase growth often culminates in lysis of the host cell. Masses (Gram-negative, Feulgen-positive) in ova, similar to those observed in the gill epithelium, suggest that transovarian transmission may occur.

Ultrastructural changes in salivary glands of tsetse, Glossina morsitans morsitans, infected with virus and rickettsia-like organisms.

Electron microscope observations on enlarged hypertrophied salivary glands dissected from adult laboratory-reared male Glossina morsitans morsitans show a concurrent infection of the salivary gland tissue with rod-shaped virus particles and intracellular rickettsia-like organisms. The latter are found intracellular in the epithelium and in the gland lumen enclosed within lytic zones. The virus particles are found within the degenerating cytoplasm, nuclei, and lumen of the cell where they are especially numerous. Stratified epithelium and gland enlargement are a prominent feature of the infection. These observations suggest that biological associations between salivary gland tissue and diverse microbes may be more common than formerly recognized. The microbes appear to cause damage to salivary gland cells, causing hyperplasia which assumes pathologic proportions.

Ultrastructure and distribution of intracellular rickettsia-like microorganisms in various organs of the laboratory-reared adult tick Argas (Persicargas) arboreus (Ixodoidea: Argasidae).

Rod (RS) and coccoid (CS) rickettsia-like microorganisms were found in single and group forms in organs of the laboratory-reared adult ticks Argas (Persicarges) arboreus. RS are distributed in most organs but are mainly concentrated in the salivary glands, mid-gut, and testes. CS single forms were concentrated in the rectal sac, while the group forms were limited to Malpighian tubules and haemocytes of both sexes. The primary oocytes were heavily infected with both forms of CS. No RS or CS were detected in the muscles. Despite the structural differences between RS and CS, they are suggested to be different morphotypes of the same organism.

Feline anaemia associated with Ehrlichia-like bodies in three domestic short-haired cats.

Three domestic short-haired cats with a history of anorexia and loss of body condition had high rectal temperatures, and a normocytic, normochromic anaemia. Two of them were also dyspnoeic, and thoracic radiographs revealed a diffuse, unstructured increase in radio-opacity involving all the lung lobes. Examination of Giemsa-stained blood smears and culture of blood monocytes revealed purplish-staining intracytoplasmic inclusions, in monocytes and lymphocytes, which occurred either singly or in aggregates. Electron micrographs of a buffy coat smear from one of the cats revealed round intracytoplasmic inclusions, with electron dense and lucid areas morphometrically similar to those found in other members of the genus Ehrlichia. An attempt to culture chlamydia from one of the cats was unsuccessful. The cats were treated successfully, one with tetracycline hydrochloride and the other two with imidocarb dipropionate.

Experimental infection with Rickettsiella phytoseiuli in adult female Dermacentor reticulatus (Ixodidae): an electron microscopy study.

Rickettsiella phytoseiuli naturally occurring in Phytoseiulus persimilis mites was cultivated in adult female Dermacentor reticulatus ticks. It demonstrates all six known developmental stages: dense, intermediate, bacterial, giant, crystal-forming and small dark particles. These stages of rickettsiae were found in salivary glands, Malpighian tubules, synganglion, ovaries, tracheal complex, haemolymph, fat body and alimentary tract. Rickettsiella phytoseiuli did not infect the Gené's organ. It multiplied in female ticks in a manner similar to that in the typical host mite, P. persimilis.