Ultrastructure of intracytoplasmic Rickettsia-like infection of the gills of the teleost Archosargus probatocephalus (Sparidae) in northeastern Brazil.

A histopathological survey was conducted to investigate the presence of microparasites in fish Archosargus probatocephalus in a river near Maceió, Brazil. Light microscope observations of fragments of gill showed the presence of small cysts containing numerous myxospores that were morphologically identified as Henneguya. Transmission electron microscopy observations further revealed several gill cells containing groups of prokaryotic cells within large cytoplasmic vacuoles. Each infected host cell displayed a single vacuole containing a variable number of Rickettsia-like cells (up to 11), some of which presented the dumbbell shape characteristic of binary fission. The Rickettsia-like cells were pleomorphic, without a nucleus and with chromatin dispersed in the cytoplasm. They had a thin electron-dense wall of Gram-negative type. The morphology of these prokaryotic was similar to those of the order Rickettsiales and was described as a Rickettsia-like organism. Histopathological evaluation showed that several vacuole membranes had a lysed appearance. Some had ruptured, thus allowing direct contact between the Rickettsia-like organism and the cytoplasm of the host cell. The rupturing of the branchial epithelium may have contributed towards reduction of the surface area of the gills, but it is not possible to say that this was the cause of the host's death.

First record of new rickettsia-like organism in the blue king crab Paralithodes platypus from the Sea of Okhotsk: Distribution, morphological evidence and genetic analysis.

An infection caused by a rickettsia-like organism (RLO) was detected in the blue king crab Paralithodes platypus from the eastern Sea of Okhotsk. The external signs of the disease are lethargy and an empty gastrointestinal tract. Dissection of infected individuals revealed that their hepatopancreas was light yellow in color. The causative agent of infection is Gram-negative rod-shaped bacterium targeted exclusively at hepatopancreas tissues. In the cytoplasm of infected cells, the bacteria are enclosed in parasite vacuoles or located immediately in cytosol. An ultrastructural analysis showed two main morphological types corresponding to the life cycle stages in the RLO: the vegetative stage of intermediate bodies, characterized by growth and division processes, and the infection stage of elementary bodies, which are spore-like non-dividing short rods surrounded by a multilayered membrane and having an osmiophilic inclusion body. At the terminal stage of infection, as a result of lysis of the infected cells, the RLO enters the lumen of the hepatopancreatic tubules which contributes to the spread of infection. According to genetic analysis based on 16S rRNA gene sequences, the RLO from P. platypus is most closely related to the Candidatus Hepatobacter penaei, NCBI #JX981946 (94.7% similarity) and NCBI #KY363553 (94.1% similarity). The high level of genetic differences (more than 5%) of the studied pathogen, along with the structural features, allows characterizing the RLO isolated from P. platypus as a new species of the genus Candidatus Hepatobacter paralithodi nov. sp., NCBI #MK928971.

Ultrastructure of intracytoplasmic Rickettsia-like infection of the gills of the teleost Archosargus probatocephalus (Sparidae) in northeastern Brazil / Ultraestrutura de Rickettsia intracitoplasmática infectando as brânquias do teleósteo Archosargus probatocephalus (Sparidae) no Nordeste do Brasil

Abstract A histopathological survey was conducted to investigate the presence of microparasites in fish Archosargus probatocephalus in a river near Maceió, Brazil. Light microscope observations of fragments of gill showed the presence of small cysts containing numerous myxospores that were morphologically identified as Henneguya. Transmission electron microscopy observations further revealed several gill cells containing groups of prokaryotic cells within large cytoplasmic vacuoles. Each infected host cell displayed a single vacuole containing a variable number of Rickettsia-like cells (up to 11), some of which presented the dumbbell shape characteristic of binary fission. The Rickettsia-like cells were pleomorphic, without a nucleus and with chromatin dispersed in the cytoplasm. They had a thin electron-dense wall of Gram-negative type. The morphology of these prokaryotic was similar to those of the order Rickettsiales and was described as a Rickettsia-like organism. Histopathological evaluation showed that several vacuole membranes had a lysed appearance. Some had ruptured, thus allowing direct contact between the Rickettsia-like organism and the cytoplasm of the host cell. The rupturing of the branchial epithelium may have contributed towards reduction of the surface area of the gills, but it is not possible to say that this was the cause of the host's death.

Resumo Um levantamento histopatológico foi realizado para pesquisar a presença de microparasitas, no peixe Archosargus probatocephalus, em um rio próximo a Maceió, Brasil. Observações ao microscópio óptico de fragmentos de brânquias mostraram a presença de pequenos cistos contendo numerosos mixósporos, identificados morfologicamente como Henneguya. Ocasionalmente, na microscopia eletrônica de transmissão, foram observados vários corpos citoplasmáticos de inclusão, grupo aparentemente de células procarióticas que vivem dentro de um grande vacúolo citoplasmático de algumas células branquiais. As células hospedeiras infectadas tinham um único vacúolo contendo um número variável de células do tipo Rickettsia, até 11, algumas das quais em forma do haltere, característica da fissão binária. Essas células eram pleomórficas sem núcleo, tendo a cromatina dispersa no citoplasma e possuíam uma parede densa de elétrons finos do tipo Gram-negativo. A morfologia dessas células procarióticas foi semelhante àquelas da ordem Rickettsiales e foram descritas como organismos tipo Rickettsiae. A histopatologia mostra várias membranas de vacúolos circundantes com aspetos lisados, enquanto outras apresentam rupturas que mostram contato direto do organismos tipo Rickettsiae com o citoplasma da célula hospedeira. A ruptura do epitélio branquial pode ter contribuído para a redução da superfície das brânquias, mas não é possível afirmar que foi a causa da morte do hospedeiro.

Identification of a Ribosomal Protein RpsB as a Surface-Exposed Protein and Adhesin of Rickettsia heilongjiangensis.

Rickettsia heilongjiangensis is an obligate intracellular bacterium that is responsible for far-eastern spotted fever. Surface-exposed proteins (SEPs) play important roles in its pathogenesis. Previous work identified a ribosomal protein RpsB as an SEP by biotin-avidin affinity, a seroreactive antigen, and a diagnostic candidate protein, indicating that it might play an important role in the pathogenesis of rickettsiae. However, in the absence of other evidence, its subcellular location of being surface-exposed was puzzling because ribosomal proteins are located in the cytoplasm. In the present study, the subcellular location of RpsB was analyzed with bioinformatics tools coupled with immunoelectron microscopy. The adhesion ability of RpsB was evaluated by protein microarray and cellular ELISA. Consequently, different bioinformatics tools gave different location predication results. Thus, RpsB was found in the cytoplasma and inner and outer membranes of R. heilongjiangensis by transmission electron microscopy. Protein microarray and cellular ELISA showed that RpsB binds to the host cell surface and its adhesion ability was even stronger than the known adhesin Adr1. In conclusion, RpsB was visually and directly shown for the time to be an SEP of rickettsiae and might be an important ligand and adhesin of rickettsiae. Its roles in pathogenesis warrant further study.

Ultrastructure of Presumed "Candidatus Rickettsia Andeanae" in Amblyomma maculatum (Acari: Ixodidae).

Amblyomma maculatum Koch, 1844 (also known as the Gulf Coast tick) is found in parts of the Americas, including the central and southern United States. Its primary importance is as the vector of Rickettsia parkeri, a spotted fever group rickettsia that causes an illness similar to, but milder than, Rocky Mountain spotted fever. A second spotted fever group rickettsia, "Candidatus Rickettsia andeanae," was detected in Gulf Coast ticks approximately 10 yr ago. However, the significance of this organism, including pathogenicity, has not yet been well-characterized. Here, we use transmission electron microscopy to describe bacteria within the tissues of A. maculatum ticks that were positive by polymerase chain reaction assay for "Ca. R. andeanae." In ultrathin sections of unfed A. maculatum adult females, we found evidence of bacteria with morphological features consistent with spotted fever group rickettsiae, including small size (≈0.3 by 0.9 µm), a halo zone (electron-lucent layer around the bacterium), and a trilaminar cell wall. In female ticks, bacteria were present in granular salivary glands and ducts, foregut, Malpighian tubules, nerve trunks, and reproductive tissue. These findings demonstrate evidence of "Ca. R. andeanae" in situ and contribute to our understanding of this novel rickettsia in A. maculatum.

Life cycle, growth characteristics and host cell response of Rickettsia helvetica in a Vero cell line.

Rickettsia helvetica, a spotted fever rickettsia and emerging pathogen with Ixodes ricinus ticks as the main vector, is an agent of human disease and may cause febrile illness as well as meningitis. In three parallel series the isolated standard type of R. helvetica, obtained from a PCR-positive I. ricinus tick, was high-passaged and propagated in a Vero cell line. By using quantitative real-time PCR, the generation time from inoculation to stationary phase of growth was calculated to 20-22 h. In the static cultivation system the stationary phase was observed from the seventh day after inoculation, and there was no observed degradation of R. helvetica DNA during the 14 days studied. Microscopy showed that the organisms invaded the host cells rapidly and were primarily found free in the cytoplasm and only occasionally located in the nucleus. Four days after inoculation some of the host cells were broken and many indifferent stages of cytoplasmic organic decomposition were seen. However the R. helvetica organism did not show any morphologic alterations and the number of organisms was stable after the replication peak which may indicate that R. helvetica is adapted to growth in a Vero cell line and/or that the phase of degradation occurs later than the 14 days studied. The findings differ from what has been reported for other rickettsiae of the spotted fever group and may be of importance for invasiveness and virulence of R. helvetica.

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.

Ultrastructural study of the life cycle of Rickettsia slovaca, wild and standard type, cultivated in L929 and Vero cell lines.

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.

Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia.

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk(T) (=CSUR R3(T) =ATCC VR-1596(T)).

Molecular detection and characterization of spotted fever group rickettsiae in Taiwan.

Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.

Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

Ultrastructural description of new Rickettsia-like organisms in the commercial abalone Haliotis tuberculata (Gastropoda: Haliotidae) from the NW of Spain.

Rickettsia-like organisms (RLOs) were found in the commercially farmed abalone Haliotis tuberculata in the northwestern region of the Atlantic Coast of Spain and are described from light and transmission electron microscopy observations. The RLOs measured approximately 1.6 x 0.9 microm and were found in intracytoplasmic, spherical to ellipsoidal vacuoles (up to 8 microm) in the epithelial cells of the digestive diverticulae. The morphological ultrastructure of these organisms was typically prokaryotic, including a plasmalemma and a thin Gram-negative type cell wall. Several ultrastructural changes were observed in the epithelial cells of the host containing the RLOs. The nuclei became pycnotic and several basophilic dense inclusions appeared in the cytoplasm. In addition, the host cell appeared lysed and was ruptured in advanced stages of infection. It was impossible to ascertain whether the RLOs are responsible for this disease, as a haplosporidian infection was also present. We can only conclude that the presence of RLOs simultaneously with a haplosporidian parasite may contribute to the mortality of the abalone host.

Ultrastructural and molecular identification of a new Rickettsia endosymbiont in the springtail Onychiurus sinensis (Hexapoda, Collembola).

In this paper we provide microscopic and molecular evidence for the presence of an endosymbiontic bacterium in male and female gonads of the soil arthropod Onychiurus sinensis. The sequence of the gene encoding for the 16S rRNA shows that the bacterium is a member of the genus Rickettsia, and some anomalies presumably associated with the presence of these microorganisms have been detected. Although the Rickettsia found in O. sinensis has the smallest genetic divergence with Rickettsia bellii, the phylogenetic analysis fails to find support for a sister-group relationship between these two species, rather suggesting that most Rickettsia species/strains isolated in various arthropods have rapidly evolved and diversified in what appears to be a sudden burst of evolution.

The first finding of a Rickettsia bacterium associated with parthenogenesis induction among insects.

The larval endoparasitoid, Neochrysocharis formosa (Westwood), is an important natural enemy of the leafminer Liriomyza trifolii (Burgess) in Japan. The thelytokous strain of N. formosa mostly produces female progeny. Male progeny were produced by females treated with tetracycline, suggesting that microorganisms induce thelytoky in N. formosa. The result of nucleotide sequencing of the 16S rRNA gene indicated that the parasitoid is infected with a Rickettsia bacterium, which appears to be causative of the thelytoky. Although Rickettsia-bellii-like bacteria have been found to be associated with various reproductive disorders, this is the first finding of a parthenogenesis-inducing Rickettsia among insects.

Ultrastructural analysis of rickettsia - like organisms in the oyster Crassostrea rizophorae from the northeastern atlantic coast of Brazil

In this report, we describe the ultrastructural morphology of rickettsia–like organisms (RLOs) present in gill epithelial tissue of the oyster, Crassostrea rizophorae, from the estuarine region of the Parnaíba river, on the northeastern Atlantic coast of Brazil. Numerous rod-shaped RLOs formed microcolonies that were located in intracytoplasmic vacuoles up to 85 micrometers in diameter. These RLOs, which measured about 2 micrometers x 0.6 micrometers, had ultrastructural characteristics of prokaryotes that included a plasma membrane and a thin, Gram-negative type cell wall. Some nondividing RLOs had a transversal constriction indicative of binary fission. The disappearance of the apical cilia with concomitant lysis of the gill epithelial cells close to the parasitized cells suggested that the RLOs may be associated with mortality in these oysters. Numerous free RLOs were seen following disruption of the vacuoles during host cell necrosis and degeneration. This is the first description of a Rickettsiales-like organism in the Atlantic fauna of Brazil.

Growth of typhus group and spotted fever group rickettsiae in insect cells.

To analyze the host dependency of rickettsial growth, NIAS-AeAl-2 insect cells (AeAl2) derived from mosquito were first used in this study. It was demonstrated that typhus group rickettsiae (TGR) grew well in AeAl2 cells, but spotted fever group rickettsiae (SFGR) failed. To elucidate the inhibitory process of the growth of SFGR in AeAl2 cells, the adherence and invasion were first analyzed. SFGR possessed abilities to adhere to and invade AeAl2 cells as well as TGR in contrast to their inability of the growth in the cells. Morphologically, generation of microvilli could not be observed on AeAl2 cells inoculated with either group of rickettsiae. On the contrary, Vero cells inoculated with rickettsiae generated a great number of microvilli that adhered to rickettsiae and engulfed them into the cells. The roles of rickettsial major outer membrane protein A and B (rOmpA and rOmpB) were later investigated using E. coli expressing either rOmpA or rOmpB on their surface. Bacteria expressing either one of the major outer membrane proteins of rickettsiae as well as bacteria not expressing these proteins showed adherence to and invasion of AeAl2 cells. Thus, it is yet to be elucidated whether these major outer membrane proteins have any roles in these steps.

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.

Ultrastructure of a Japanese Rickettsial strain genetically identified as Rickettsia helvetica which was originally found in Europe.

A rickettsial strain IO-1 has been isolated from a tick, Ixodes ovatus, in Japan and genetically identified as Rickettsia helvetica, a member of the spotted fever group rickettsiae. Ultrastructural observations were made on the microorganism. The ultrastructure of R. helvetica IO-1 appeared to be generally the same as that previously shown for other rickettsiae of the spotted fever and typhus groups. The rickettsiae were primarily found free in the cytoplasm of L929 cultured cells. Occasionally, the rickettsiae may also invade the host cell nucleus; however, the frequency of the nuclear localization was very low.

Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where Brazilian spotted fever is endemic.

Owing to the potential role of the tick Amblyomma cooperi in the enzootic cycle of Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection by Rickettsia species in A. cooperi ticks collected from an area in Brazil where BSF is endemic. Among a total of 40 A. cooperi adult ticks collected in an area of BSF endemicity in the state of São Paulo, PCR analysis detected DNA of Rickettsia bellii in 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individual A. cooperi ticks resulted in two isolates of R. bellii and one isolate genotypically characterized as an SFG rickettsia. The two R. bellii isolates were established in Vero cell cultures in the laboratory and were confirmed to be R. bellii by molecular analysis of the gltA and 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica. These results do not support the role of A. cooperi in the ecology of R. rickettsii in the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.

Ultrastructural and genetic evidence of a reptilian tick, Aponomma hydrosauri, as a host of Rickettsia honei in Australia: possible transovarial transmission.

In 1993, a novel rickettsia was isolated from the blood of inhabitants of Flinders Island, Australia, with acute febrile illnesses. This rickettsia was found to be a new species of spotted fever group (SFG) rickettsia, eventually named Rickettsia honei. The suspected ectoparasite vector of this rickettsia has yet to be identified. The purpose of this study was to evaluate the presence of this rickettsial species in a suspected tick vector, Aponomma hydrosauri, by DNA sequencing and electron microscopy (EM). Ticks collected from an Australian blue-tongued lizard on Flinders Island and a copperhead snake in Tasmania were demonstrated to be infected with R. honei by PCR, DNA sequencing, and EM. Rickettsiae were found in ultrathin sections of salivary glands, malpighian tubules, and midgut epithelial cells. In a previous study with a R. honei-infected tick from Flinders Island, rickettsiae were found in the nuclei of midgut epithelial cells, and EM also revealed the presence of rickettsiae in the cytosol of oocytes and immature eggs, suggesting transovarial transmission. These results implicate A. hydrosauri as a possible host of R. honei on Flinders Island and Tasmania and also provide evidence favoring transovarial maintenance of R. honei.