Detection of “<Emphasis Type="Italic">Candidatus</Emphasis> Rickettsia sp. strain Argentina”and <Emphasis Type="Italic">Rickettsia bellii</Emphasis> in <Emphasis Type="Italic">Amblyomma</Emphasis> ticks (Acari: Ixodidae) from Northern Argentina

Ixodid ticks were collected from vegetation and from humans, wild and domestic mammals in a rural area in the semi-arid Argentine Chaco in late spring 2006 to evaluate their potential role as vectors of Spotted Fever Group (SFG) rickettsiae. A total of 233 adult ticks, identified as Amblyomma parvum, Amblyomma tigrinum and Amblyomma pseudoconcolor, was examined for Rickettsia spp. We identified an SFG rickettsia of unknown pathogenicity, “Candidatus Rickettsia sp. strain Argentina”, in A. parvum and A. pseudoconcolor by PCR assays targeting gltA, ompA, ompB and 17-kDa outer membrane antigen rickettsial genes. Rickettsia bellii was detected in a host-seeking male of A. tigrinum. Amblyomma parvum is widespread in the study area and is a potential threat to human health.     

Molecular detection of various rickettsiae in mites (acari: trombiculidae) in southern Jeolla Province, Korea

This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.     

Proposal to create subspecies of <Emphasis Type="Italic">Rickettsia conorii</Emphasis> based on multi-locus sequence typing and an emended description of <Emphasis Type="Italic">Rickettsia conorii</Emphasis>

Background  

Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.     

Species-specific BALB/c mouse antibodies to rickettsiae studied by Western blotting

Abstract BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Spotted fever rickettsioses in southern and eastern Europe

Mediterranean spotted fever due to Rickettsia conorii conorii was thought, for many years, to be the only tick-borne rickettsial disease prevalent in southern and eastern Europe. However, in recent years, six more species or subspecies within the spotted fever group of the genus Rickettsia have been described as emerging pathogens in this part of the world. Tick-borne agents include Rickettsia conorii israelensis, Rickettsia conorii caspia, Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia sibirica mongolitimonae and Rickettsia massiliae. Many Rickettsia of unknown pathogenicity have also been detected from ticks and could represent potential emerging pathogens to be discovered in the future. Furthermore, a new spotted fever rickettsia, Rickettsia felis, was found to be associated with cat fleas and is an emerging human pathogen. Finally, the mite-transmitted Rickettsia akari, the agent of rickettsialpox, is also known to be prevalent in Europe. We present here an overview of these rickettsioses, focusing on emerging diseases.     

Infection by <Emphasis Type="Italic">Rickettsia bellii</Emphasis> and <Emphasis Type="Italic">Candidatus “Rickettsia amblyommii”</Emphasis> in <Emphasis Type="Italic">Amblyomma neumanni</Emphasis> Ticks from Argentina

The tick species, Amblyomma neumanni (Acari: Ixodidae) is the most frequent tick parasitizing humans in northwestern Argentina. The present study evaluated the rickettsial infection among 55 A. neumanni adult free-living ticks collected in Dean Funes, Córdoba Province. Ticks were individually processed by the hemolymph test with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction (PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated and established in Vero cell culture from two ticks (ticks 4 and 13), which previously showed to contain Rickettsia-like organisms by the hemolymph test. These two Rickettsia isolates were designated as An4 and An13. Molecular characterization (partial DNA sequences of two to three rickettsial genes were determined) of these two isolates and phylogenetic analyses identified them as Rickettsia bellii (isolate An4) and Candidatus “Rickettsia amblyommii” (isolate An13). After testing all A. neumanni ticks by PCR, the prevalence of Candidatus R. amblyommii and R. bellii was 23.6% (13/55) and 3.6% (2/55), respectively. These two rickettsiae have been considered of unknown pathogenicity and appropriate studies to test their pathogenicity to humans or animals need to be conducted. This is the first report of Rickettsia in ticks from Argentina, and also in the species A. neumanni. The results reinforce previous findings that R. bellii (and probably Candidatus R. amblyommii) are widespread among some Neotropical Amblyomma species, suggesting that these ticks gained these bacterial agents from a common ancestor and/or by recent horizontal transmission of rickettsiae between ticks.     

Isolation of a rickettsial pathogen from a non-hematophagous arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.     

Restriction of the growth of a nonpathogenic spotted fever group rickettsia

The growth kinetics of pathogenic and nonpathogenic rickettsiae were compared to elucidate the mechanism responsible for the pathogenicity of rickettsiae. Vero and HeLa cells derived from mammals were inoculated with a nonpathogenic species of spotted fever group rickettsia, Rickettsia montanensis, before being infected with the pathogenic species Rickettsia japonica. The mammalian cells became persistently infected with R. montanensis and produced low levels of rickettsiae. On the other hand, superinfection of the R. montanensis-infected cells with R. japonica resulted in increased yields of R. montanensis accompanied by R. japonica growth. Both rickettsiae also grew well in the R. japonica-infected cells subjected to superinfection with R. montanensis. Western blotting with an antibody to the autophagy-related protein LC3B found that autophagy was induced in the cells infected with R. montanensis alone. On the contrary, autophagy was restricted in the cells that were co-infected with R. japonica. Electron microscopy of the cells infected with R. montanensis alone demonstrated rickettsia particles being digested in intracytoplasmic vacuoles. Conversely, many freely growing rickettsiae were detected in the co-infected cells.     

Intracytoplasmic Localization of Antigenic Heat-Stable 120- to 130-Kilodalton Proteins (PS120) Common to Spotted Fever Group Rickettsiae Demonstrated by Immunoelectron Microscopy

Immunoelectron microscopy demonstrated antigenic heat-stable 120- to 130-kilodalton proteins (PS120) of spotted fever group (SFG) rickettsiae with antiserum against recombinant PS120 of Rickettsia japonica. In the case of R. japonica, a major part of the protein was shown to be localized outside the electron-lucent nucleoid-like region in the cytoplasm of the organisms. The other SFG rickettsiae represented a similar localization of the PS120 antigens cross-reactive to that of R. japonica. On the other hand, a typhus group rickettsia demonstrated no antigens cross-reactive to the PS120 of SFG rickettsiae.     

Propagation of arthropod-borne Rickettsia spp. in two mosquito cell lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.     

Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools.

The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. I. Inhibition of Growth of Rickettsia tsutsugamushi,Karp Strain,in Cultured Murine Embryonic Cells and Macrophages by Recombinant Tumor Necrosis Factor-Alpha

Recombinant murine tumor necrosis factor-alpha (TNF-α) inhibited intracellular growth of Rickettsia tsutsugamushi, Karp strain, in the mouse embryo cell line C3H/10T1/2 clone 8 at doses of 100 to 10 U/ml. The growth inhibitory effect of TNF-α was also evident when peritoneal exudate macrophages or bone marrow-derived macrophages were used as the host cell for rickettsial growth. Interferon-gamma (IFN-γ), at doses up to 1,000 U/ml, did not affect the growth of this strain of rickettsiae in the mouse embryo cell line but, as expected, profoundly inhibited rickettsial growth in peritoneal exudate macrophages and bone marrow-derived macrophages. The effect of TNF-α on rickettsial growth in the mouse embryo cell line was not reproducibly enhanced by IFN-γ. Treatment of the cell line with TNF-α delayed rickettsial cytopathic effects, but the rickettsiae ultimately grew to high numbers in the cells and caused cell death. These findings show that, at least in our system, R. tsutsugamushi is resistant to IFN-γ-mediated antirickettsial effects in cells other than macrophages. The results of this study support the suggestion that TNF-α may inhibit rickettsial growth in cells other than macrophages.     

Tick-Borne Rickettsioses,Neglected Emerging Diseases in Rural Senegal

Background

Rickettsioses are one of the most important causes of systemic febrile illness among travelers from developed countries, but little is known about their incidence in indigenous populations, especially in West Africa.

Methodology/Principal Findings

Overall seroprevalence evaluated by immunofluorescence using six rickettsial antigens (spotted fever and typhus group) in rural populations of two villages of the Sine-Saloum region of Senegal was found to be 21.4% and 51% for spotted fever group rickettsiae for Dielmo and Ndiop villages, respectively. We investigated the role of tick-borne rickettsiae as the cause of acute non-malarial febrile diseases in the same villages. The incidence of rickettsial DNA in 204 blood samples from 134 (62M and 72F) febrile patients negative for malaria was studied. DNA extracted from whole blood was tested by two qPCR systems. Rickettsial DNA was found in nine patients, eight with Rickettsia felis (separately reported). For the first time in West Africa, Rickettsia conorii was diagnosed in one patient. We also tested 2,767 Ixodid ticks collected in two regions of Senegal (Niakhar and Sine-Saloum) from domestic animals (cows, sheep, goats, donkeys and horses) by qPCR and identified five different pathogenic rickettsiae. We found the following: Rickettsia aeschlimannii in Hyalomma marginatum rufipes (51.3% and 44.8% in Niakhar and Sine-Saloum region, respectively), in Hyalomma truncatum (6% and 6.8%) and in Rhipicephalus evertsi evertsi (0.5%, only in Niakhar); R. c. conorii in Rh. e. evertsi (0.4%, only in Sine-Saloum); Rickettsia massiliae in Rhipicephalus guilhoni (22.4%, only in Niakhar); Rickettsia sibirica mongolitimonae in Hyalomma truncatum (13.5%, only in Sine-Saloum); and Rickettsia africae in Rhipicephalus evertsi evertsi (0.7% and 0.4% in Niakhar and Sine-Saloum region, respectively) as well as in Rhipicephalus annulatus (20%, only in Sine-Saloum). We isolated two rickettsial strains from H. truncatum: R. s. mongolitimonae and R. aeschlimannii.

Conclusions/Significance

We believe that together with our previous data on the high prevalence of R. africae in Amblyomma ticks and R. felis infection in patients, the presented results on the distribution of pathogenic rickettsiae in ticks and the first R. conorii case in West Africa show that the rural population of Senegal is at risk for other tick-borne rickettsioses, which are significant causes of febrile disease in this area.     

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free‐ranging crab‐eating foxes (Cerdocyon thous)

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free‐ranging crab‐eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty‐nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector‐borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.     

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.     

Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich^T) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.