Ultrastructure and cytopathology of a rickettsia-like organism causing systemic infection in the redclaw crayfish, Cherax quadricarinatus (Crustacea: decapoda), in Ecuador

A study of the ultrastructural characteristics of an intracellular bacterium infecting the redclaw crayfish, Cherax quadricarinatus, a pathogen referred to previously as a rickettsia-like organism (RLO), revealed the presence of different developmental stages. These included a rod-shaped and uniformly electron-dense elementary body (EB) and an intermediate body (IB). The length of the EB varied between 0.48 and 0.6 microm, and the diameter was 0.3 microm. The IB was 0.75 to 1.1 microm long by 0.36 to 0.44 microm in diameter. Although the EB of this bacterium has ultrastructural characteristics similar to those of Rickettsiella, no information is available regarding its genetic relationship to this genus, and the intracellular bacterium should continue to be referred to as a rickettsia-like organism. The hemocytes had different levels of infection, and the RLO proliferated inside these cells. The EB appeared to be free in the cytoplasm of infected hemocytes and other cells; however, this might be a fixation artifact. The EB was also contained in membrane-bound vacuoles along with the IB. RLO colonies were observed inside small granular cells. No large granular cells were observed in the sections examined; therefore, no data were obtained regarding infection of this type of hemocyte. The fixed phagocytes on the external side of the terminal hepatic arterioles had an activated interrupted layer containing RLO bacteria. Stem cells in the hematopoietic tissue were also infected, and some cells were apparently being released into circulation.     

Studies on rickettsia-like organism disease of the tropical marine pearl oyster I: the fine structure and morphogenesis of pinctada maxima pathogen rickettsia-like organism

The present paper reports for the first time the discovery of a rickettsia-like organism (RLO) in the cultured tropical marine pearl oyster Pinctada maxima with mass mortality in the Hainan Province of China. This organism parasitizes the cytoplasm of host cells and forms intracytoplasmic eosinophilic inclusions. These organisms are extremely pleiomorphic in shape and average 967 x 551 nm in size, as measured in cross sections of transmission electron micrographs. The organisms exhibit clearly recognizable ultrastructural characteristics of prokaryotic bacteria-like cells, including two trilaminar membranes, an increasing electron-dense periplasmic ribosome zone, and a thread-like DNA nucleoidal structure. In addition to the above prokaryotic characteristics, the following unique biological characteristics were confirmed by TEM: (i) These organisms are usually located in host cells in two ways, namely, free in the cell cytoplasm and involved within membrane-limited phagolysosomes; (ii) The organisms exist in two morphological cell types, namely a small cell variant (SCV) and a large cell variant (LCV). The most important morphological difference between two cell types is that the SCV is obviously ribosome-rich in the periphery of the body, which makes SCV more electron-dense in the cytoplasm and narrower in the central nucleoid area than the LCV; (iii) Two propagative modes of the organisms, transverse binary fission and budding, are observed in cytoplasm and phagolysosomes of host cells under TEM, in which the budding is more often seen in phagolysosomes. These characteristics indicate that the organism is a separate species in the family Rickettsiaceae and should be classified into the genus Rickettsia. On the basis of the existence of the two propagative modes and two cell types, and intracellular location, we propose a developmental cycle for this organism which includes a vegetative differentiation stage to develop LCV by transverse binary fisson and a budding differentiation stage to develop resistant SCV. Copyright 1999 Academic Press.     

Rickettsia-like organism associated with tremor disease and mortality of the Chinese mitten crab Eriocheir sinensis

We report the discovery of a rickettsia-like organism (RLO) in cultured freshwater Chinese mitten crab Eriocheir sinensis. The RLO caused tremor disease and was apparently responsible for a mass mortality (30 to 90%) in 2 provinces in southeast China. Moribund crabs were investigated from different districts during outbreaks in 1999 and 2000. With electron microscopy, 3 different pathogens were detected in moribund crabs: a rickettsia-like organism (RLO), virus-like particles (VLP) and a microsporidian-like protozoan (MLP). Based on the high prevalence, infection intensity and cytopathological signs, the RLO was considered to be the probable cause of the high mortality. Both VLP and MLP occurred at low prevalences and were considered secondary infections. The RLO was 0.22 to 0.35 microm in diameter, granular or clavate, bounded by a cell wall and membrane, and possessed no nucleus but a nucleoid was present. When dividing, RLOs occurred in irregular shapes, such as dumbbells, awls, and crescents. The RLOs exhibited a predilection for muscle and connective tissues and were probably transported to various tissues and organs by hemocytes.     

Prevalence of parasites in amphipods Diporeia spp. from Lakes Michigan and Huron, USA

Amphipods of Diporeia spp. have declined considerably during the last decade in the Great Lakes. We examined the possibility that disease may be affecting these populations. A histological survey assessed the parasites in species of Diporeia within Lakes Huron and Michigan, USA, and the host response to some of them and to unknown factors. Amphipods were found to have an intranuclear inclusion body, and were hosts to a rickettsia-like organism, fungi, a haplosporidian, a microsporidian, epibiotic ciliates, a gregarine, a cestode, acanthocephalans and nodule formations. Epibiotic ciliates were most common (37% prevalence of infection), but a microsporidian (3.8%), a rickettsia-like organism (1.6%), fungi, including a yeast-like organism (1.3%), worms (1.3%), and a haplosporidian (0.7%) are likely associated with mortalities or detrimental effects on the host. The role these agents may have played in the decline of Diporeia spp. in the Great Lakes over the last decade is not clear. Interrelationships with the dynamics of various physical and biological factors such as high sedimentation, diminished food supplies, and virulent parasites could synergistically cause the decline in Diporeia spp. populations in Lakes Michigan and Huron.     

The role of cell death in the midgut epithelium in Filientomon takanawanum (Protura)

Midgut epithelium in Filientomon takanawanum is composed of epithelial cells and single, sporadic regenerative cells. In 80% of analyzed specimens midgut epithelial cells, as fat body and gonads, are infected with rickettsia-like microorganism. In non-infected specimens young and completely differentiated epithelial cells are distinguished among epithelial cells. Characteristic for midgut epithelial cells regionalization in organelles distribution is not observed. Autophagy is the sporadic process, but if the cytoplasm of epithelium cells possesses numerous spherites and sporadic autophagosomes, the apoptosis begins. Necrosis is observed sporadically.In the midgut epithelium cells of about 80% of analyzed specimens rickettsia-like microorganisms are observed. The more rickettsia-like microorganisms occur in the cytoplasm, the more autophagosomes are formed, and the process of apoptosis proceeds intensively.     

Infectivity, transmission and 16S rRNA sequencing of a rickettsia, Coxiella cheraxi sp. nov., from the freshwater crayfish Cherax quadricarinatus

A rickettsia-like organism isolated from infected, farm-reared Cherax quadricarinatus was cultured in the yolk sac of developing chicken eggs, but could not be cultured in 3 continuous cell lines, bluegill fry (BF-2), fathead minnow (FHM), and Spodoptera frugiperda (Sf-9). The organism was confirmed by fulfilling Koch's postulates as the aetiological agent of mortalities amongst C. quadricarinatus. When C. quadricarinatus was inoculated with the organism, mortality was 100% at 28 degrees C and 80% at an ambient temperature of 24 degrees C. Horizontal transmission with food and via the waterborne route was demonstrated, but mortalities were lower at 30 and 10% respectively over a 4 wk period. The 16S rRNA sequence of 1325 base pairs of the Gram-negative, obligate intracellular organism was 95.6% homologous to Coxiella burnetii. Of 18 species compared to this rickettsia, the next most closely related bacterium was Legionella pneumophila at 86.7%. The suggested classification of this organism is Order Rickettsiales, family Rickettsiaceae, tribe Rickettsieae, within the genus Coxiella. We suggest it should be named Coxiella cheraxi sp. nov.     

Characterisation of an emerging rickettsia-like organism in Tasmanian farmed Atlantic salmon Salmo salar

A rickettsia-like organism (RLO) was observed in farmed Atlantic salmon Salmo salar located in south-east Tasmania, Australia. Several assays such as immunoperoxidase, immunoelectron microscopy, polymerase chain reaction and nucleic acid sequencing, as well as phylogenetic analysis of rDNA sequences, were performed on infected fish tissues. Immunohistochemistry results suggested the presence of related antigenic determinants between the Tasmanian RLO and the type strain LF-89 of Piscirickettsia salmonis. However, sequence alignment demonstrated that the Tasmanian RLO contains a 19 bp deletion at the 3'-end of the internal transcribed spacer region of the rDNA operon, indicating a genetic divergence from P. salmonis isolates, which are exotic to Australia.     

桑萎缩病的类菌原体病原物及其超微病变特征

对感染桑萎缩病的桑叶及嫩梢进行超薄切片的电镜观察发现,其韧皮部组织,筛管及伴胞内有多型性类菌原体。菌体为圆形及椭圆形,大小约为50～160nm,双层膜,厚度约为8～10nm,内含物中具有核质样的纤维状物质,而在健株叶梢中未观察到任何病原体．随着病害的发展,可观察到细胞成份的降解。第一,在感病植株叶肉细胞内存在部分细胞核的降解、核膜破裂或核质流失甚至核仁分散消失。第二,叶绿体内有不同程度的淀粉粒和嗜锇颗粒累积,部分叶绿体外膜破裂,基质流失,基粒降解。第三,线粒体及粗面内质网在数量上有所增加,部分线粒体嵴已降解。     

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.     

Rickettsial and mollicute infections in hepatopancreatic cells of cultured Pacific white shrimp (Penaeus vannamei)

Infections by multiple species of bacteria occurred in hepatopancreatic epithelial cells of cultured Pacific white shrimp (Penaeus vannamei). Grossly, hepatopancreases of moribund shrimp were pale white. Light microscopically, hepatopancreatic tubules appeared atrophied and were associated with granulomas. Examination by scanning and transmission electron microscopy revealed heavy cytoplasmic infections by three forms of microorganisms: (1) a rickettsia-like bacterium, (2) a helical form of a mollicute-like bacterium, and (3) a filamentous mollicute-like bacterium. The rod-shaped rickettsia (900 nm long by 300 nm wide) appeared to be free in the cytoplasm and had both a plasma membrane and a cell wall. Neither form of mollicute possessed a cell wall. The helical mollicute was blunt at its wide end (about 260 nm in diameter) where it contained electron-lucent bodies. Helical turns along its tapered axis resembled those of a spiroplasma (the only helical form of mycoplasma in the class Mollicutes) or a spirochete. The helical bacterium did not possess periplasmic flagella characteristic of spirochetes, which lends support to its being a type of spiroplasma. The filamentous mollicute consisted of masses of short, branched filaments 60 nm wide with intermittent spherical dilations and terminal blebs on the branches. The presumed mollicutes have not been reported previously in crustaceans. Each bacterium, or concurrent infections of the bacteria, are pathogenic to cultured shrimp, could impact culture operations and thus deserve more study.     

Evidence that the novel microorganism 'Z' may belong to a new genus in the family Chlamydiaceae

Abstract The purpose of this study was to investigate possible phylogenetic relationships of a new microorganism called 'Z'. The organism was previously shown to be similar to chlamydia in its growth cycle and some of its metabolic requirements, but different in others and in its major outer membrane protein. In this study we report the sequencing of 'Z's 16S ribosomal DNA and comparison of the sequence with that of other microorganisms, including chlamydia and rickettsiae. While chlamydial species have 95.5% sequence identity among themselves, 'Z' had 83% identity with them, and 73% identity with certain rickettsia-like organisms. Based on the sequence analyses and taking into account physiologic considerations, we believe that 'Z' may belong to a novel genus in the family Chlamydiaceae.     

Ultrastructure and distribution of intracellular rickettsia-like microorganisms in various organs of the laboratory-reared adult tickArgas (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 ticksArgas (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.     

Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon

A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.     

Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.     

Fine Structure of Cryptococcus neoformans Grown In Vitro as Observed by Freeze-Etching

Cryptococcus neoformans grown on culture media was observed by the freeze-etching technique. In the capsule, short fibrils were seen when freezeetched. This organism was unique in the appearance of the cell wall, which showed two strata. The outer one was dense with particles of about 20 nm in diameter, whereas the inner one was sparse in particles. The appearance of the cell membrane of this organism differed distinctly depending on the culture media. When grown on glycerol medium, the cell membrane possessed, as do other yeasts, clear but somewhat longer and curved invaginations. The membrane of cells grown on nonglycerol medium exhibited, however, only a few invaginations of irregular shape. Instead, characteristically of this organism, the cell membrane showed round depressions of 40 to 200 nm in diameter which were the surface view of the paramural bodies. In cross-fractured cells, both types of paramural bodies were found. Some of them contained a single vesicle of about 50 nm in diameter. These seem to play a role in secreting the cytoplasmic vesicles. Data suggesting the existence of multivesicular bodies in the cytoplasm and multivesicular lomasomes were also obtained. Some of the baglike paramural bodies showed multilayered membrane. These are thought to be plasmalemmasomes. This organism was similar to other yeasts reported in other respects.     

The first detection of the insertion sequence ISW1 in the intracellular reproductive parasite Wolbachia.

Wolbachia are maternally inherited intracellular rickettsia-like bacteria known to infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis, and feminization. We report on a novel insertion sequence (IS), ISW1, which was identified in the region downstream of groEL of a Wolbachia strain, wTai. The 573-bp-long ISW1 sequence is the first IS element observed in this organism, displays significant similarity to IS200, and lacks terminal inverted repeats. There were more than 20 copies of ISW1 on the chromosome of wTai. Sequence analysis of nine distinct ISW1 copies and their flanking regions showed that the copies were identical and suggested that ISW1 has no preference for its insertion sites. Possible roles of ISW1 in the adaptation of Wolbachia to intracellular environments and in various reproductive alterations caused by this bacterium are discussed.     

Autometallographic silver-enhancement of colloidal gold particles used to label phagocytic cells.

The present paper demonstrates that colloidal gold silver-enhanced by autometallography (AMG) can be used to label phagocytic cells for light microscopic detection. Cultured macrophages were exposed to 0.5 microliters 6 nm colloidal gold particles for 24 or 48 h. Other cultures were exposed to 25 microliters of the same solution for 1 to 14 days. The staining was found to be stable also when new unmarked cells were applied. The colloidal gold had no adverse effect on the cells. The presented technique might also prove valuable for estimation of the total number of phagocytes in a culture or in an organism by applying labelled cells to culture or organism, and to ascertain the fate of a population of marked cells.     

A Note on the Detection of Penicillin Acylase Activity in Escherichia coli by the Reaction of Ampicillin with Biuret Reagent

Ampicillin reacts with biuret reagent in a novel manner to give an absorption peak at 410 nm. Treatment of ampicillin with penicillin acylase from Escherichia coli ATCC 9637 or whole cells of the same organism results in loss of biuret-reacting properties. The reaction could possibly form the basis of a screening test for penicillin acylase.     

Carotenoid pigments and the enhanced resistance of Pseudomonas methanolica to the action of ultraviolet radiation

The correlation was studied between the elevated resistance of Pseudomonas methanolica, a facultative methylotroph, to the bactericidal action of UV (254 nm) and the content of a pink pigment in this organism. Absorption spectra of cell extracts containing the pigment were recorded. The ethanol and acetone extracts had two absorption maxima at 420-430 and 500-510 nm, respectively, which was typical of carotenoids. The culture with the pigment and its variant without the pigment had nearly identical survival rates. Therefore, the elevated resistance of P. methanolica cells seems to stem from the activity of DNA reparation cellular systems rather than from the presence of carotenoid pigments in the cells.     

Role of the parasporal body in causing toxicity of Bacillus thuringiensis toward Aedes aegypti larvae

A unique type of microorganism has been found causing an unusual disease in larvae of the clover cutworm, Scotogramma trifolii (Lepidoptera; Noctuidae). The organism contains DNA and reproduces exclusively by self-assembly forming enveloped reniform/bacilliform particles which measure 170 × 450 nm in negatively stained preparations. During initial stages of development, the organism apparently reproduces primarily within vesicles in the cytoplasm of a variety of cell types including hemocytes, and epidermal, fat body, and tracheal matrix cells. Most reproduction, however, occurs in vesicles that circulate in the hemolymph. These vesicles, most of which are derived from host cells, measure 2–10 μm in diameter, are highly refractile, reach populations as high as 10⁸/ml of hemolymph, and are diagnostic for the disease. The pathology caused by this organism, its shape and ultrastructure, and reproduction within vesicles indicate it is either a peculiar type of rickettsia, possibly related to those of the genus Rickettsiella, or a new type of invertebrate virus. Among its unusual features are its ability to induce the formation of reproductive vesicles from host cell components, and its apparent control of de novo ribosome and membrane synthesis within these vesicles as it develops. The possible relationship of this organism to baculoviruses and rickettsia is discussed.