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Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species “Candidatus Trichorickettsia mobilis” (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split “Candidatus Trichorickettsia” into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed “Candidatus Trichorickettsia” hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture.

Ticks vector diverse pathogenic bacteria that are important to identify in public health and veterinary contexts. Technological advances in high throughput sequencing have given an unprecedented opportunity to comprehensively characterize bacterial associates of ticks, but recent studies have used different 16S rRNA variable regions and sequence read lengths with little consideration of whether they reveal the same bacterial diversity. We compare the effectiveness of bacterial surveys using three library preparations across nine 16S variable regions and a set of 12 tick specimens (Acari: Ixodidae). We identify the bacterial assemblages present in extractions from wild-collected Ixodes scapularis from two regions of Canada, and provide the first microbiome survey for Ixodes angustus. Four bacterial families accounted for most diversity, with Rickettsiaceae being replaced as most common by Enterobacteriaceae or Pseudomonadaceae in some I. scapularis, and Francisellaceae being most abundant in I. angustus. The commercially available Ion 16S kit, based on 6 amplicons representing 16S regions V2, V3, V4, V67, V8 and V9, gave the most comprehensive estimates of bacterial families, with the Ion V4 amplicon generally giving the highest estimated diversity. Sequencing of the V4 amplicon by the MR DNA commercial service also provided cost effective assays of tick microbiomes that were within the range of results from the Ion 16S kit. Subtraction of the number of reads found in an extraction control sample lowered estimates of the number of bacterial families by approximately half. Our study shows that diversity patterns obtained from 16S microbiome surveys depend on the amplicon and protocol used, demonstrating that more than one marker region is needed to provide reliable inferences.

Recently, several tick-borne pathogens were detected in reptile-associated ticks. However, studies on the microorganisms in reptile-associated ticks in Japan are limited. This molecular survey thus aimed to identify and characterize tick-borne pathogens (Rickettsiaceae and Anaplasmataceae) in reptile-associated ticks in Japan. In total, 77 Amblyomma nitidum and 104 Amblyomma geoemydae were collected from wild amphibious sea kraits (Laticauda semifasciata, Laticauda colubrina, and Laticauda laticaudata) and from yellow-margined box turtles (Cuora flavomarginata evelynae), respectively. Conventional polymerase chain reaction was performed using the DNA extracted from the ticks to detect the selected pathogens. Sequencing analysis of four Rickettsia genes (gltA, ompA, ompB, and sca4) led to the identification of a putative novel Rickettsia sp. and Rickettsia aeschlimannii-like rickettsia in A. nitidum and A. geoemydae, respectively. Sequencing analysis of gltA and groEL of Anaplasmataceae revealed that the Ehrlichia spp. in these ticks were novel and related to Candidatus Ehrlichia occidentalis. This is the first study on the microorganisms in A. nitidium and the first record of Rickettsia and Ehrlichia in A. geoemydae. Further studies are required to understand their pathogenicity to humans and animals and their life cycle in the wild.

Noctiluca scintillans is a bloom-forming dinoflagellate, which is widely distributed in the global coastal seas. Associated bacteria have been proven to be essential for the survival and growth of zooplanktons. However, the diversity and function of bacteria associated with Noctiluca scintillans are under studied and largely unknown. Here, we examined the diversity and function of bacteria associated with field-acquired and laboratory-maintained Noctiluca cells. Our results showed that the bacterial communities associated with the laboratory-maintained Noctiluca were dominated by Rhodobacterales, whereas those associated with the field-acquired Noctiluca varied over time. In addition, major Noctiluca-associated bacteria had low relative abundance in the ambient environment. We also observed that when field-acquired Noctiluca were cultivated with a mono-species food source, there was a shift in the associated bacterial communities. Metagenomic analysis showed that genes involved in DNA replication/repair and osmotic regulation were more abundant than other genes in the Noctiluca-associated bacterial community. Furthermore, the associated bacteria were able to degrade various complex carbohydrates and actively participate in the nitrogen cycle in their host cells. In addition, a draft genome of the Rickettsiaceae strain was recovered, and we showed that the genome did not contain genes encoding hexokinase and phosphoglucomutase, two key enzymes involved in glucose utilization. Instead, the primary energy sources of this bacteria were shown to be glutamate, glutamine and pyruvate, which might be obtained from the host. We suggest that in return, the Rickettsiaceae strain is likely to provide cofactors and amino acids to the host. This study highlights the spatial and temporal complexity of bacterial communities associated with Noctiluca, and provides valuable insights into the interaction between a host and its associated bacteria.

Rickettsial-like cells are reported from the body cavity of the Myanmar amber larval tick, Cornupalpatum burmanicum (Ixodida: Ixodidae). These cells are characterized and described in a new collective fossil genus erected for putative rickettsia in fossil ticks. The size and shape of the fossil cells resemble those of present day members of the Rickettsiaceae, many of which occur in the body cavity of present day ticks.

Summary Objectives To study the epidemiology, clinical features, and changing pattern of rickettsial infections on the western slopes of the hilly Central Province of Sri Lanka over 6 years. Methods All patients with rickettsial infections who presented to the Teaching Hospital, Peradeniya were studied prospectively from January 2002 to December 2007. An immunofluorescent antibody assay (IFA) was used to confirm the diagnosis. Results Of the 371 clinical cases of rickettsial infection, 122 underwent IFA to confirm the diagnosis. Species-specific IgG antibodies were positive in 105/122 (86.1%) cases: 43/105 (40.9%) to Rickettsia conorii and 6/105 (5.7%) to Orientia tsutsugamushi , with mixed antibody reactivity to more than one antigen in 56/105 (53.3%) cases, including Rickettsia typhi in 27/105 (25.7%). Among those with mixed IgG reactivity, IgM assays were used to detect pathogens responsible for acute infections. Finally, a total of 55 spotted fever group (SFG) infections, seven scrub typhus infections, and one case of murine typhus were identified. Of the 105 positive cases, 53 (50.5%) were male and 52 (49.5%) were female, and the mean age was 40 years (range 11–83 years). In the SFG patients, 13/55 (24%) had severe vasculitis with fern leaf type skin necrosis and 17/55 (31%) had arthritis. Three patients (5%) had an altered level of consciousness. A patient with scrub typhus had transient deafness. None of the 105 patients had an eschar. Conclusions It appears that SFG rickettsioses are on the rise in the hilly Central Province of Sri Lanka, whilst murine typhus and scrub typhus are present at a lower rate.

The vast majority of multicellular organisms coexist with bacterial symbionts that may play various roles during their life cycle. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) belongs to the smallest known insects whose size is comparable with some bacteria. Using 16S rRNA gene sequencing and Whole Genome Sequencing (WGS), we described microbiota diversity for this arthropod and its potential impact on their lifecycle. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX2363723 and SRX2363724. We found that small body size and limited lifespan do not lead to a significant reduction of bacterial symbionts diversity. At the same time, we show here a specific feature of microbiota composition in M. amalphitanum – the absence of the Rickettsiaceae family representatives that are known to cause sex-ratio distortion in arthropods and well represented in other populations of parasitoid wasps.

A survey of ectoparasites on feral pigs identified two commonly occurring ixodid tick species; Amblyomma triguttatum triguttatum and Ixodes australiensis. Molecular screening of A. t. triguttatum and I. australiensis for the presence of Rickettsia species detected the presence of rickettsiae belonging to the Spotted Fever Group (SFG) in 78.4% of screened A. t. triguttatum. None of the screened I. australiensis were positive for rickettsiae. Sequence analysis of the gltA and ompA loci of positive Rickettsia isolates were 100% homologous to the newly described species Rickettsia gravesii sp. nov. BWI-1. Serological screening of feral pigs detected antibodies to SFG Rickettsia in 50% of serum samples tested. These findings suggest that A. t. triguttatum is a vector/reservoir for R. gravesii sp. nov.

The spotted fever rickettsia, Rickettsia helvetica, is mostly reported to present a flu-like self-limiting disease. In the present case, however, where the presence of R. helvetica in the blood was verified by PCR together with serologic evidence of infection, the patient presented a different clinical picture with acute febrile illness, rash and long-lasting myasthenia.

Spotted Fever Group Rickettsia is important cause of emerging and re-emerging infectious disease in people and dogs. Importantly, dogs can serve as sentinels for disease in people. Sensitive and specific diagnostic tests that differentiate among species of infecting Rickettsia are needed. The objective of this study was to develop a sensitive and specific PCR that differentiates SFG Rickettsia infecting dog blood. Conventional and real-time PCR assays were developed using primers that targeted a small region of the ompA gene. Their sensitivity, determined by testing a cloned target sequence in the presence of host DNA, was 15-30 and 5 copies of DNA, respectively. Testing of Rickettsia cultures and analysis of Rickettsia gene sequences deposited in GenBank verified DNA could be amplified and used to differentiate species. DNA from the blood of infected dogs was also tested. Importantly, Rickettsia DNA was detected before seroconversion in some dogs. The species of infecting Rickettsia was also identified. We conclude these assays may assist in the timely diagnosis of infection with SFG Rickettsia. They may also facilitate the discovery of novel SFG Rickettsia infecting dogs, and in the investigation of dogs as sentinels for emerging rickettsioses.

Designation as a Category B biothreat agent has propelled Coxiella burnetii from a relatively obscure, underappreciated, "niche" microorganism on the periphery of bacteriology, to one of possibly great consequence if actually used in acts of bioterrorism. Advances in the study of this microorganism proceeded slowly, primarily because of the difficulty in studying this obligate intracellular pathogen that must be manipulated under biosafety level-3 conditions. The dogged determination of past and current C. burnetii researchers and the application of modern immunological and molecular techniques have more clearly defined the host and bacterial response to infection. This review is intended to provide a basic introduction to C. burnetii and Q fever, while emphasizing immunomodulatory properties, both positive and negative, of Q fever vaccines and C. burnetii infections.

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.

Resume La rickettsiologie ne recevra un nom qu’avec les travaux de Ricketts et von Prowazek en 1910, et ne cessera de s’alimenter d’especes et de pathologies nouvelles. En tant que premieres bacteries intracellulaires strictes decrites, la taxonomie des rickettsies rassemblait initialement sur la base de ce critere, un grand nombre de genres bacteriens ulterieurement reclasses avec l’avenement du sequencage et la decouverte d’horloges moleculaires telle que la sous-unite 16S de ARN ribosomique ou le cytochrome C. Pour l’identification des especes de Rickettsia, de nombreux criteres phenotypiques dont la morphologie, les tests de fixation du complement, de neutralisation de toxines, de serotypage et les profils proteiques ont longtemps ete utilises. Mais c’est la comparaison des sequences de genes, dont ompA , ompB et sca 4, qui ont permis d’identifier tres precisement les especes du genre Rickettsia et de proposer une classification phylogenique fiable. Cependant, la position phylogenique d’especes telles que Rickettsia helvetica, Rickettsia canadensis et Rickettsia bellii n’a pu etre determinee avec certitude. Aussi, l’analyse basee sur la concatenation de plusieurs genes, associee aux caracteres phenotypiques peut constituer une meilleure alternative.

Wolbachia bacteria are endosymbiotic partners of many animal species, in which they behave as either parasites (in arthropod hosts) or mutualists (in nematode hosts). What biochemistry and biology underpin these diverse lifestyles? The recent complete sequencing of genomes from Wolbachia that infect the arthropod Drosophila melanogaster and the nematode Brugia malayi, together with the partial genome sequencing of three Wolbachia strains found in drosophilids, enables this question to begin to be addressed. Parasitic arthropod Wolbachia are characterized by the presence of phages that carry ankyrin-repeat proteins; these proteins might be exported to the host cell to manipulate reproduction. In nematode Wolbachia, which lack these phages, several biochemical pathways can deliver essential metabolites to the nematode hosts. Nematode Wolbachia might also have a role in modulating the mammalian host immune system but the sequenced Wolbachia genomes lack the genes to synthesize lipopolysaccharide, raising questions about the nature of the inducing molecule. The Wolbachia surface protein might carry out this function.

Resume Nous presentons deux cas d'infections au cours desquelles la presence d'anticorps antiphospholipides (APL), anticardiolipine et anti-β2-GP1, s'est associee a la survenue d'evenements thrombotiques significatifs : un homme de 55 ans pour qui la serologie Coxiella burnetii (immunofluorescence indirecte) a retrouve la presence d'antigenes de phase 2 avec IgG a 1600 et IgM a 50 (titre significatif : IgG ≥ 200 et IgM ≥ 50), en faveur d'une fievre Q aigue ; et un homme de 20 ans avec une infection a CMV affirmee par la serologie (IgG : 44 U/ml, seuil significatif 6, IgM : 2,1 U/ml, seuil significatif 0,9). La presence transitoire d'anticorps antiphospholipides est frequente au cours des episodes infectieux, cependant elle s'associe rarement a des complications thrombotiques.

Human and animal parasitic filarial nematodes, which often are the cause of severe disease, harbor intracellular bacteria of the genus Wolbachia (Rickettsiaceae). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. In order to determine the possible role of Wolbachia in heartworm disease, dogs naturally infected with Dirofilaria immitis were studied for specific antibody response to Wolbachia surface protein (WSP). Antibody subclasses were analyzed to determine immune response polarization. Dogs that died from heartworm disease were necropsied, and various organs were studied by immunohistochemistry to determine whether Wolbachia-derived molecules were present in tissue from infected dogs. Humoral response to the WSP was present in all infected dogs and appeared to be predominantly of the Th1-type. Several organs, including lung, liver, and kidney, contained positive-staining cells for WSP, confirming that the canine host does come into contact with Wolbachia-derived molecules.

Livestock is considered to be the major "source" of human Q fever. The efficacy of two currently available vaccines (Coxevac, phase I, CEVA Santé Animale and Chlamyvax FQ, phase II, MERIAL) against Coxiella excretion was investigated in terms of risks to human health. Two months before mating, 17 goats were vaccinated subcutaneously against Coxiella burnetii with an inactivated phase I vaccine and 16 goats were vaccinated with an inactivated phase II Coxiella mixed with Chlamydophila abortus vaccine. Fourteen goats were left unvaccinated. At 84 days of gestation, the goats were subcutaneously challenged with 10(4) bacteria of C. burnetii strain CbC1. Phase I vaccine was effective and dramatically reduced both abortion and excretion of bacteria in the milk, vaginal mucus and feces. In contrast, the phase II vaccine did not affect the course of the disease or excretion.

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.

Between 1999 and 2002, 9349 sera and 517 aborted samples (422 foetuses and 95 placenta) were analysed from 675 sheep and 82 goat farms distributed all over the island of Sardinia. After abortion notification, sera collected at random from adult animals were examined to detect antibodies specific to Coxiella burnetii by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 255 (38%) sheep farms and in 39 (47%) goat herds whereas 40 ovine (10%) and 3 (6%) caprine foetuses were C. burnetii PCR-positive. Although C. burnetii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. Seroprevalence analysis indicates that C. burnetii distribution in sheep and goats is very high, but PCR results demonstrate that C. burnetii has a relatively low role in abortion, especially in goats.

Recent studies have suggested that intracellular Wolbachia bacteria are necessary for reproduction and survival of adult filarial worms. We now report results of in vitro studies of effects of antibacterial antibiotics (tetracycline, rifampicin, chloramphenicol, azithromycin, and doxycycline) on Brugia malayi infective larvae (L3) motility and molting. All of the antibiotics tested except chloramphenicol decreased L3 motility by 50% or more at 10 days, with minimal effective concentrations (MECs) of 20–100 μg/ml. Tetracyclines, rifampicin, and chloramphenicol inhibited L3 to L4 molting by 12 days in a concentration- and time-dependent manner, with MECs in the range of 1–20 μg/ml. These studies show that antibiotics active against Rickettsiaceae inhibit B. malayi L3 molting at low concentrations in vitro; higher concentrations kill the larvae. While it is possible that antibiotics directly affect filarial L3, we believe it is more likely that the effects seen are indirect effects related to bacterial killing. Index Descriptors and Abbreviations: Brugia malayi; Filarioidea; Rickettsiaceae; Wolbachia; antibiotics; NI-medium; NCTC-135 and IMDM, Iscove’s Modified Dulbecco’s Medium; FCS, fetal calf serum; Tet, tetracycline; Rif, rifampicin; Chlor, chloramphenicol; Azith, azithromycin; Doxy, doxycycline; IM, incomplete molting larvae; MEC, minimum effective concentration; PCR, polymerase chain reaction.

Coxiella burnetii infection in pregnant sheep typically causes abortion or the birth of weak lambs. Two C. burnetii-related abortions in a group of 34 pregnant ewes were reported at their first lambing in our research institute. The seroprevalence of C. burnetii infection and bacteria shedding were investigated using an ELISA and PCR, respectively, during the course of two subsequent pregnancies. None of the ewes examined seroconverted from negative to positive at the time of the second and the third parturition and most of the ewes that were seropositive at the abortion episode remained positive throughout the investigation. The two successive pregnancies resulted in the birth of healthy lambs without PCR evidence of Coxiella infection from placenta and vaginal swabs taken postpartum. PCR assay performed on vaginal swabs taken from all animals 1, 5 or 12 days after the second lambing were also negative for Coxiella. However, one ewe that had previously experienced C. burnetii shedding at the first lambing excreted the bacteria in the genital tract after the third parturition. The bacteria could not be detected by PCR in milk and faecal samples taken up to 12 days after both parturitions.

Objective To determine whether Coxiella burnetti is able to survive within free-living ameoba. Methods When C. burnetii was co-cultivated with the free-living amoeba species Acanthamoeba castellanii. Results Viable bacteria were detected for 18 days. Microscopic studies confirmed the presence of bacteria within vacuoles, some of which appeared to be spore-like forms. Conclusion These results indicate that free-living amoebae could provide an intracellular niche for the spore formation and survival of C. burnetii in the environment.

Colonization of dental pulp by blood-borne bacteria in the absence of previous inflammation has been hypothetized but has never been convincingly demonstrated. In order to provide convincing support for this hypothesis we attempted to detect Coxiella burnetii DNA in the dental pulp of bacteremic, intraperitoneally inoculated guinea-pigs by PCR amplification and direct sequencing of two molecular targets. Coxiella burnetii DNA was recovered from 20-50% of the animals depending on the molecular target, from 15-20 days after experimental challenge. These results demonstrated, for the first time, that dental pulp is contaminated by blood-borne bacteria and can be detected by molecular tools.

At the beginning of the 20th century, it was discovered at the Pasteur Institute in Tunis that epidemic typhus is transmitted by the human body louse. The complete genome sequence of its causative agent, Rickettsia prowazekii, was determined at Uppsala University in Sweden at the end of the century. In this mini-review, we discuss insights gained from the genome sequence of this fascinating and deadly organism.

Resume Au cours des infections aigues et chroniques a Coxiella burnetii, les anomalies immunologiques sont frequentes. Nous rapportons 13 cas de fievre Q aigue et discutons ces anomalies. Les mecanismes physiopathologiques ne sont qu'en partie elucides et font intervenir le germe lui-meme et le systeme immunitaire de l'hote. Le role des cytokines, notamment le TNF et l'interleukine 1, pourrait etre implique dans la pathogenie de la forme chronique et pourrait permettre de nouvelles approches therapeutiques au cours de cette infection.

Taylor, M. J., Bilo, K., Cross, H. F., Archer, J. P., and Underwood, A. P. 1999. 16S rDNA phylogeny and ultrastructural characterization ofWolbachiaintracellular bacteria of the filarial nematodesBrugia malayi, B. pahangi, andWuchereria bancrofti. Experimental Parasitology91, 356–361.

Resume Les rickettsies sont des bacteries de type Gram negatif, intracellulaires strictes, vehiculees par des arthropodes. Le genre Rickettsia est actuellement subdivise en deux groupes : le groupe typhus et le groupe boutonneux pourpre. La conjugaison des resultats obtenus par comparaison des sequences des genes codant la citrate synthase et la proteine rOmpA a permis d’obtenir une etude phylogenetique fiable pour l’ensemble des bacteries du genre Rickettsia . De plus, par PCR/sequencage il est maintenant possible d’identifier ces bacteries a partir de prelevements biologiques ou d’arthropodes vecteurs. L’identification moleculaire a ainsi permis d’objectiver la pathogenicite pour l’homme de certaines rickettsies, de decrire plusieurs nouvelles especes et de completer les donnees epidemiologiques dont on disposait.

rOmpA and rOmpB are immunodominant, surface-exposed proteins ofRickettsia rickettsii.Prior evidence suggests that adhesion ofR. rickettsiito the host cell is mediated by a rickettsial protein. Five monoclonal antibodies to rOmpA, five to rOmpB, and one to the rickettsial lipopolysaccharide (LPS) were tested for inhibition of rickettsial attachment. All the monoclonal antibodies to rOmpA inhibited adhesion of rickettsiae to the L-929 cells with some inhibition rates as high as 90%. In contrast, monoclonal antibodies to rOmpB and LPS did not block attachment. When Fab fragments of monoclonal antibodies against rOmpA and rOmpB were used, similar results were observed as for the intact monoclonals, non-adhesion and adhesion, respectively. Purified rOmpA showed a competitive inhibitive effect on the attachment ofR. rickettsiito host cells. Trypsin completely digested rOmpA but not rOmpB from the surface of intactR. rickettsii, resulting in loss of the ability of the rickettsiae to attach to the host cell. rOmpA appears to play an important role in the initial adhesion ofR. rickettsiito the host cell.

Resume L'osteomyelite chronique recurrente multifocale (OCRM) est une affection dont l'origine infecticuse est suspectee mais non demontree. Observation. Une enfant a presente un tableau caracteristique d'OCRM touchant successivement le perone, le radius, l'humerus gauche et le carpe droit. Une infection a Coxiella burnetii a pu etre affirmee lors de la poussee initiale. Deux recurrences sont survenues malgre une antibiotherapie adaptee avec un bilan infectieux negatif. Deux mois apres l'arret de l'antibiotherapie survenait un nouvel episode avec presence du germe dans le foyer osseux et reascension serologique. Conclusion. Coxiella burnetii peut etre a l'origine d'un tableau d'OCRM. Le mecanisme en cause est probablement celui d'une hypersensibilite retardee faisant de cette affection la premiere forme d'osteite reactionnelle.

Coxiella burnetii strains Nine Mile and Priscilla, considered to be associated with acute and chronic forms of Q fever, were investigated for variation in composition of their lipopolysaccharides. Though SDS-PAGE profiles of the lipopolysaccharides were distinct, chemical analyses showed only small differences in their overall composition. Further studies on lipid A-deprived O-polysaccharide fractions of both lipopolysaccharides, obtained by steric-exclusion chromatography, revealed noticeable differences in distribution and chemical composition of the O-polysaccharide chains. It is likely that C. burnetii strains are capable of synthesizing chemically distinct subclasses of O-specific polysaccharide molecules differing in their antigenic reactivities. The results provide suggestive evidence that virulence of C. burnetii may be modulated through lipopolysaccharide composition and structure.

The prevalence of antibodies to Rickettsiae and other tick-borne microrganisms in the sera of 181 forestry rangers from Friuli-Venezia-Giulia, Italy, was examined. Seven (3.9%) sera were positive for Rickettsia conorii and Rickettsia helvetica, as single or dual infections; four of these sera had been found previously to be positive for Borrelia burgdorferi. Antibodies to Coxiella burnetii were detected in five (2.8%) sera, four of which were also positive for B. burgdorferi. These findings indicate that patients in this north-eastern Italian region with fever subsequent to tick-bite should be investigated for Rickettsia and Coxiella infections.

Adalia bipunctata, the two spot ladybird, carries a vertically transmitted bacterial agent which kills male progeny during embryogenesis. Some matrilines of A. bipunctata give rise to strongly female-biased sex ratios. 16S rDNA sequence analysis revealed a bacterium of the genus Rickettsia associated with this trait, a conclusion which is corroborated here. Using light microscopy, an association between a bacterium located in A. bipunctata hemocyte cytoplasm and matrilines which show the sex ratio trait was found. This element was not found in hemocytes taken from females from normal sex ratio lines, nor in hemocytes taken from males. The association is confirmed by study of the inheritance of the sex ratio trait. Only daughters of sex ratio crosses that bear this cytoplasmic bacterium also show the sex ratio trait, with other daughters being normal with respect to sex ratio. Transmission electron microscopy of hemocytes revealed a walled bacterium, bearing features of members of the genus Rickettsia, free in the cytoplasm of hemocytes taken from infected lines, but not in those taken from uninfected lines.

Cytoplasmic incompatibility (CI) in Drosophila is related to the presence of Wolbachia, an intracelluar microorganism found in many species of insects. In order to study the intracellular localization of Wolbachia in eggs and embryos, we have purified the bacteria from fly embryos and subsequently generated a monoclonal antibody (Mab Wol-1) specific for Wolbachia. Indirect immunofluorescence staining using Wol-1 reveals that during mitosis, Wolbachia are localized near spindle poles and centrosomes. Double label immunofluorescence experiments using anti-tubulin and anti-Wolbachia antibodies show that Wolbachia co-localize with centrosomal microtubules throughout the cell cycle. Direct interactions between the bacteria and centrosome-organized microtubules are implied from seven observations: (1) throughout the mitotic cycle, the position and movement of Wolbachia precisely mimic the behavior of the centrosome and apparently associates with centrosome-organized microtubules; (2) Wolbachia segregate equally to each spindle pole during mitosis; (3) Wolbachia do not associate with spindle microtubules during mitosis; (4) Wolbachia located in the egg cortex localize to the domains of cytoplasm organized by microtubules during blastoderm formation; (5) polar body nuclei that lack centrosomes but contain associated microtubules do not contain Wolbachia; (6) Wolbachia no longer associate with yolk nuclei, following differentiation and loss of centrosomes; (7) during pole cell formation, Wolbachia co-localize with the centrosome on the apical side of the nucleus as pole cells form. Quantitative data indicates that no Wolbachia growth occurs during the preblastoderm period even though rapid nuclear, and subsequent cellular, proliferation takes place during this same period. This indicates that Wolbachia are under strict growth regulation by the host suggesting that host factors play a role in regulating growth of Wolbachia in the egg. Further cellular and molecular studies of the extensive, global interactions between host and symbiont observed in this egg should provide important new insights into the evolution of host/symbiosis and the cell biology of cytoplasmic incompatibility.

Rickettsiae are Gram-negative bacteria which multiply only inside host cells and need arthropods either as reservoirs or as vectors. Using the polymerase chain reaction and an automated laser fluorescent DNA sequencer, we amplified and sequenced the 16S rRNA (rDNA) of all available bacteria of the genus Rickettsia. R. tsutsugamushi remained close to the other bacteria of the genus Rickettsia using this technique, contrary to previous conclusions based on the study of the Sta-58 protein antigen. We found that R. canada was not included in the typhus group, as is currently recognized, but was grouped with the rickettsiae of the spotted fever group (SFG). All the SFG rickettsiae tested were grouped in the same cluster (R. conorii, Indian tick typhus rickettsia, Astrakhan fever rickettsia, Israeli tick typhus rickettsia, HA-91, R. sibirica, R. parkeri, "R. africae", "R. slovaca", R. rickettsii, Thai tick typhus rickettsia, R. japonica, R. massiliae, R. rhipicephali, R. montana, two recent isolates GS and Bar 29, R. australis, R. akari, R. bellii and R. helvetica). The recently described ELB bacterium, the agent of the Californian murine typhus, and AB bacterium, a bacterium associated with male killing in the ladybird beetle, were found in this cluster. The sequences of R. conorii Moroccan strain/Indian tick typhus rickettsia, R. massiliae/GS and R. sibirica/HA-91 were identical. All the rickettsiae had a unique ancestor with bacteria also isolated in arthropods (Ehrlichia, Cowdria, Anaplasma, Wolbachia pipientis), eventually pathogenic for mammals and implicated in parthenogenesis and cytoplasmic incompatibility. We conclude that a unique bacterium started a stable association with arthropod ancestors and generated the observed diversity of the currently isolated members of the Rickettsiales.

Strains of Coxiella burnetii phase I and II whole cells (WC-I and WC-II) or whole cell fractions were assessed for their potential to induce long-lasting protection in endotoxin-non-responder C3H/HeJ or CD-1 mice against Rift Valley fever (RVF) virus challenge. Among the whole cell fractions, only the chloroform-methanol residue (CMR), administered as a single dose (100 micrograms per mouse) 24 h before viral challenge, effectively protected 100% of the mice from RVF virus; the CMR of the Ohio strain of C. burnetii was not protective. Most of the RVF virus-infected mice treated with other C. burnetii cell fractions died, although their times to death varied. Lipopolysaccharide (LPS) associated with CMR preparations used in these studies, did not protect against RVF virus challenge. A single dose of 100 micrograms of CMR given 24 h before viral challenge completely eradicated 4-5 logs of RVF virus in the serum, liver, spleen, and central nervous system. Compared to several other immunomodulators, CMR was an equally effective antiviral agent. Efficacy of CMR of both Henzerling and Ohio strains disappeared or was marginal when treatment was initiated 2-3 days before RVF viral challenge, even when a second or a third dose of CMR was administered after challenge. A single dose of liposome-encapsulated CMR to RVF virus-infected mice extended the range of therapeutic efficacy of this biologically active component of C. burnetii to 4 days before infection.

Baculovirus recombinants that express the Rickettsia rickettsii rOmpA protein were constructed. Monoclonal antibodies (mAbs) against the rOmpA protein reacted with recombinant-infected Spodoptera frugiperda (Sf9) cells in indirect immunofluorescence assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of infected Sf9 cell lysates with a mAb against rOmpA showed that the recombinant-expressed rOmpA protein migrated slightly below rOmpA extracted from R. rickettsii. Guinea-pigs immunized with lysates of recombinant-infected Sf9 cells developed antibodies reactive with R. rickettsii and were protected against challenge, indicating that the baculovirus-expressed rOmpA protein could be useful in subunit vaccines and for studies of the immune response to R. rickettsii infection.

cited By 23; International audience; The rRNA-encoding genes (rDNAs) have been cloned and characterized from Wolbachia pipientis (Wp), the gonadial bacteria-like parasite of the mosquito Culex pipiens (Cp) and the moth Ephestia cautella (Ec). In Wp from both insect species the rDNAs are organized in a way which appears to be very unusual. The rRNAs are encoded by two unlinked transcription units, each present in a single copy per genome. One contains the 16S rDNA only, while the other is an operon encoding both the 23S and 5S rDNAs. Each transcription unit contains two putative upstream promoters, and downstream a Rho-independent terminator. The 16S rDNA, as well as the 23S-5S rRNA operon are not linked to any tRNA-encoding sequence and lack the antitermination boxes which are usually present immediately downstream from eubacterial promoters of rDNAs. Wp infecting Ec and Cp are highly similar taking as criteria the rDNAs and their flanking sequences. However, it clearly appears that each insect species harbours a different and specific Wp strain, or even subspecies. Phylogenetic relationships deduced from the complete sequences of their rDNAs undoubtedly confirm that Wp from Cp and Ec belong to the α-group of Proteobacteria, and are closely related to the Rickettsia. © 1995.

Summary The bacterial genus Rickettsia is traditionally divided into three biotypes, the spotted fever group (SFG), the typhus group (TG), and the scrub typhus group (STG) based on vector host and antigenic cross-reactivity. DNA sequence data were gathered from the 16S ribosomal RNA gene of several SFG and TG species. Comparative sequence analysis shows that: i) all species of Rickettsia are closely related, exhibiting 0.3–2.6% sequence divergence; ii) although there are identifiable clusters corresponding to the SFG and TG, species of Rickettsia fall into more than two distinct phylogenetic groups; iii) the tick-borne species Rickettsia bellii and Rickettsia canada diverged prior to the schism between the spotted fever and typhus groups; iv) the newly described AB bacterium is clearly a member of Rickettsia , but its phylogenetic placement within the genus is problematic; v) the mite-borne Rickettsia akari , the tick-borne Rickettsia australis and the recently described flea-borne ELB agent form a loose cluster that cannot be definitively associated with either the TG or the traditional SFG cluster. This latter Glade may represent a unique group(s) distinct from the main cluster of spotted fever and typhus group species. The divergence of Rickettsia was an ancient event within the α-subclass of the proteobacteria. The sequence divergence between Rickettsia and Ehrlichia , the closest known genus to Rickettsia , is nearly equal to the sequence divergence between Rickettsia and all other α-subclass proteobacterial taxa included in the analysis. When Rickettsia was compared to a representative group of the α-subclass, twenty-eight nucleotide sites were identified which uniquely characterize the 16S rRNA sequences of all species of Rickettsia . The approximate time of divergence between the various species of Rickettsia , estimated from the bacterial 16S rRNA molecular clock, coincides with the approximate divergence time of the hard body ticks which are the arthropod hosts of many Rickettsia . Thus, the possibility of coevolution between these intracellular bacteria and their tick hosts exists.

The firA and lpxA genes, as well as an ORF coding for a putative 16-kDa protein of unknown function, have been identified and characterized in the obligate intracellular bacterium, Rickettsia rickettsii . This is the first description of these genes, which code for enzymes involved in the biosynthesis of lipid A, in a species outside of the Enterobacteriaceae. The deduced amino acid (aa) sequences of FirA, ORF16 and LpxA of R. rickettsii , when compared to their Escherichia coli analogs, exhibited 35, 44 and 41% aa identity, respectively. In addition, the order of genes in R. rickettsii , firA ORF16- lpxA , was identical to that found in E. coli ; however, the spacing between the rickettsial genes was greater. Interestingly, the R. rickettsii FirA and LpxA deduced proteins retain an unusual hexapeptide repeat pattern found in E. coli and Salmonella typhimurium FirA/Ssc and E. coli LpxA, as well as other acyltransferases, providing additional support for the importance of this structure.

The diagnosis of Q fever is based on the detection of serum antibodies to Coxiella burnetii antigens. The isolation of the aetiologic agent is not recommended as a routine procedure due to difficulties in growing these bacteria and their infectivity. Since it has been well documented that infected animals and humans excrete bacteria in urine and semen, we have developed a dot-ELISA test and indirect immunofluorescence assay (IFA) for diagnosing the infection. The minimum concentration of bacteria detectable in urine with dot-ELISA was estimated as 1 × 10 6 cells ml −1 . IFA was less useful because single C. burnetii cells are hardly visible. In semen C. burnetii bacteria were detected equally well with both IFA and dot-ELISA test.

Any hitherto available technique used to detect the presence of Wolbachia, a rickettsia-like endosymbiont of Drosophila simulans, i.e., crossing with tester strains, electron microscope observations, DAPI-staining of embryo, or amplification of specific sequences by PCR are notably time-consuming techniques. Staining sperm cysts with the fluorochrome DAPI is here shown to be a fast and reliable technique to detect the symbiont and evaluate the level of infection of individual males. Wolbachia cells are abundant in sperm cysts and make up a mass which is eliminated in the waste bag during spermatogenesis. Such a mass was absent in all those strains known to be aposymbiotic. In infected strains, tetracycline treatment led to the disappearance of the symbionts within sperm cysts. In aging males, a decrease in the proportion of infected cysts were observed. There was however no apparent change in the number of symbionts in the cysts which remained infected. The reduction of reproductive incompatibility, which is observed in older males, may be explained by this process, i.e., a decrease in the frequency of infected cysts.

Human vascular endothelial, Vero and human embryonic lung cells infected with rickettsiae for 24 h or 48 h were labelled for polymerized actin with NBD-phallacidin. Between 20 and 68% of the intracellular Rickettsia conorii had an actin tail of between 0.33 and 15 μm, with the longest tails being observed in Vero cells. In the case of R. typhi less than 1% of the organisms had actin tails and these were considerably shorter than those of R. conorii . These findings provide new information concerning the different cytopathic effects observed with the two rickettsial species.

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.

A novel spotted fever group rickettsia has recently been isolated from Amblyomma hebraeum ticks in Zimbabwe. In a survey of 172 goat sera collected throughout Zimbabwe the highest prevalence of antibodies reactive with this rickettsia was in the south of the country, the area where A hebraeum is most commonly found. Nine goats were infected using male and female A hebraeum taken from a tick line shown to be infected with the novel rickettsia. By week 3 after infection, seroconversion occurred in all nine goats but no clinical signs of disease could be detected. A leucocytosis due to a mature neutrophilia one to two weeks after infection was the only abnormality. Rickettsaemia was detected only on day 3 after exposure to infected ticks. Immunosuppression failed to induce recrudescence of the rickettsaemia.

Cytoplasmic incompatibility is known to occur between strains of both Drosophila simulans and D. melanogaster. Incompatibility is associated with the infection of Drosophila with microorganismal endosymbionts. This paper reports survey work conducted on strains of D. simulans and D. melanogaster from diverse geographical locations finding that infected populations are relatively rare and scattered in their distribution. The distribution of infected populations of D. simulans appears to be at odds with deterministic models predicting the rapid spread of the infection through uninfected populations. Examination of isofemale lines from four localities in California where populations appear to be polymorphic for the infection failed to find evidence for consistent assortative mating preferences between infected and uninfected populations that may explain the basis for the observed polymorphism.

Antigenic diversity in five stocks of the tick-borne rickettsia Cowdria ruminandum, the causal agent of heartwater disease of ruminants, was studied by cross-immunity trials in goats and sheep. Complete absence of cross-protection was found only between the Kumm and Kwanyanga stocks, and in all other combinations there were various degrees of cross-immunity. Immunological strain differences were more pronounced in goats than in sheep.

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.