scholarly journals Rickettsia felis from Cat Fleas: Isolation and Culture in a Tick-Derived Cell Line

2006 ◽  
Vol 72 (8) ◽  
pp. 5589-5595 ◽  
Author(s):  
Walairat Pornwiroon ◽  
Susan S. Pourciau ◽  
Lane D. Foil ◽  
Kevin R. Macaluso
Keyword(s):  
Cell Line ◽  
Citrate Synthase ◽  
Ixodes Scapularis ◽  
Spotted Fever ◽  
Protein A ◽  
Etiologic Agent ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Rickettsia Felis ◽  
Cat Fleas

ABSTRACT Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularis-derived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.

scholarly journals Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

2001 ◽  
Vol 67 (2) ◽  
pp. 546-552 ◽  
Author(s):  
Jason A. Simser ◽  
Ann T. Palmer ◽  
Ulrike G. Munderloh ◽  
Timothy J. Kurtti
Keyword(s):  
Cell Line ◽  
Citrate Synthase ◽  
Spotted Fever ◽  
Fluorescent Antibody ◽  
Rocky Mountain ◽  
Embryonic Cell ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Dermacentor Andersoni ◽  
Rocky Mountain Wood Tick

ABSTRACT An embryonic cell line (DAE100) of the Rocky Mountain wood tick,Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals.R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.

scholarly journals Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil

2011 ◽  
Vol 44 (3) ◽  
pp. 313-317 ◽  
Author(s):  
Roberta Santos Toledo ◽  
Katia Tamekuni ◽  
Mauro de Freitas Silva Filho ◽  
Valeska Bender Haydu ◽  
Richard Campos Pacheco ◽  
...  
Keyword(s):  
Citrate Synthase ◽  
Spotted Fever ◽  
Immunofluorescence Assay ◽  
Etiologic Agent ◽  
Spotted Fever Group ◽  
Rickettsia Species ◽  
Rickettsia Rickettsii ◽  
Serological Studies ◽  
Human Sera ◽  
Brazilian Spotted Fever

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF) and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens. Among the 34 sera analyzed, seven (20.6%) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species may be involved in transmission to humans.

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

2007 ◽  
Vol 73 (20) ◽  
pp. 6637-6643 ◽  
Author(s):  
Joyce M. Sakamoto ◽  
Abdu F. Azad
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Spotted Fever ◽  
Culture Conditions ◽  
Spotted Fever Group ◽  
Rickettsia Felis ◽  
Transitional Group ◽  
Mosquito Cell Lines

ABSTRACT 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.

Microscopy of Spotted Fever Rickettsia Movement through Tick Cells

1998 ◽  
Vol 4 (2) ◽  
pp. 115-121 ◽  
Author(s):  
Ulrike G. Munderloh ◽  
Stanley F. Hayes ◽  
Joel Cummings ◽  
Timothy J. Kurtti
Keyword(s):  
Host Cell ◽  
Ixodes Scapularis ◽  
Spotted Fever ◽  
Rhipicephalus Appendiculatus ◽  
Amblyomma Americanum ◽  
Host Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Spotted Fever Group ◽  
Symbiotic Association ◽  
Host Cell Membrane

Spotted fever group (SFG) rickettsiae are obligate intracellular prokaryotes that include tick-borne pathogens of vertebrates as well as nonpathogenic organisms living in symbiotic association with their tick hosts. We investigated the ability of SFG rickettsiae to move between and within host cells using tick cell culture and a SFG rickettsial isolate from a lone star tick (Amblyomma americanum) collected in Missouri. The isolate (MOAa), which is closely related to Rickettsia montana, grew in cell lines from the ticks Ixodes scapularis and Rhipicephalus appendiculatus. Transmission electron microscopy demonstrated that immediately following entry into tick cells, rickettsiae escaped from the host cell membrane, and intracellular rickettsiae came to lie in direct contact with host-cell cytoplasm. There was evidence of damage to the endoplasmic reticulum membrane which was broken down into vesicular structures. When rickettsiae exited host cells, host membrane stretched around them but was lost before re-entry. Use of a fluorescein-tagged monoclonal antibody to rickettsial outer membrane protein B and rhodamine-labeled phalloidin demonstrated association of actin tails with rickettsiae and suggested that SFG rickettsiae utilized host cytoskeletal components for movement. During early stages of infection, when cells harbored only one or a few organisms, “comet tails” of F-actin formed on one end of rickettsial cells, presumably pushing them ahead. Actin tails were not seen during later stages of infection when tick cells became completely filled with rickettsiae.

scholarly journals Zoonotic Rickettsia Species in Small Ruminant Ticks From Tunisia

2021 ◽  
Vol 8 ◽  
Author(s):  
Hanène Belkahia ◽  
Rachid Selmi ◽  
Sayed Zamiti ◽  
Monia Daaloul-Jedidi ◽  
Lilia Messadi ◽  
...  
Keyword(s):  
Small Ruminant ◽  
Citrate Synthase ◽  
Spotted Fever ◽  
Protein A ◽  
Rhipicephalus Sanguineus ◽  
Spotted Fever Group ◽  
Rickettsia Species ◽  
Rickettsia Monacensis ◽  
Significant Public Health ◽  
Pcr Targeting

Tick-borne rickettsioses present a significant public health threat among emerging tick-borne diseases. In Tunisia, little is known about tick-borne Rickettsia pathogens. Therefore, the aim of this study was to investigate the presence of Rickettsia species in small ruminant ticks from Tunisia. Adult ticks (n = 694) were collected from goats and sheep in northern Tunisia. Obtained ticks were identified as Rhipicephalus turanicus (n = 434) and Rhipicephalus sanguineus sensu lato (n = 260). Selected ticks (n = 666) were screened for the presence of Rickettsia spp. by PCR targeting a partial sequence of the ompB gene followed by sequence analysis. Rickettsial DNA was detected in 122 (18.3%) tested tick samples. The infection rates in Rh. turanicus and Rh. sanguineus s.l. ticks were 23.4 and 9.5%, respectively. The overall prevalence of rickettsial DNA was markedly higher in ticks collected from goats (23.2%) compared to those infesting sheep (7.9%). The detection of rickettsial DNA was significantly higher in ticks from the governorate of Beja (39.0%) than those from the governorate of Bizerte (13.9%). Two additional genes, the outer membrane protein A gene (ompA) and the citrate synthase gene (gltA), were also targeted for further characterization of the detected Rickettsia species. Genotyping and phylogenetic analysis based on partial sequences (n = 106) of the three different genes revealed that positive ticks are infected with different isolates of two Spotted Fever Group (SFG) Rickettsia, namely, Rickettsia massiliae and Rickettsia monacensis, closely related to those infecting camels and associated ticks from Tunisia, and humans and small ruminant ticks from neighboring countries like Italy, France, and Spain.

scholarly journals A streamlined method for transposon mutagenesis ofRickettsia parkeriyields numerous mutations that impact infection

2018 ◽  
Author(s):  
Rebecca L. Lamason ◽  
Natasha M. Kafai ◽  
Matthew D. Welch
Keyword(s):  
Host Cell ◽  
Virulence Factors ◽  
Vascular Endothelial Cells ◽  
Spotted Fever ◽  
Transposon Mutagenesis ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Loss Of Function ◽  
Rickettsia Parkeri ◽  
Obligate Intracellular

AbstractThe rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria,Rickettsiaare highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improvedmariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group speciesRickettsia parkeri. These mutants targeted genes implicated in a variety of pathways, including bacterial replication and metabolism, hypothetical proteins, the type IV secretion system, as well as factors with previously established roles in host cell interactions and pathogenesis. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

Application of the Numerical Characteristic of Formal Order Analysis of the Prokaryotic Genomes for Reclassification within the Genus Rickettsia

2016 ◽  
Vol 11 (2) ◽  
pp. 336-350 ◽  
Author(s):  
С.Н. Шпынов ◽  
S.N. Shpynov
Keyword(s):  
Spotted Fever ◽  
Numerical Characteristic ◽  
Spotted Fever Group ◽  
Rickettsia Felis ◽  
New Approach ◽  
Order Analysis ◽  
Ancestral Group ◽  
Prokaryotic Genomes ◽  
Information Chain

Genomes representing Rickettsiaceae family were analyzed using formal order analysis (FOA) of information chain in order to develop a new approach for the classification of prokaryotes. Average remoteness – the numerical characteristic of order was used to compare the genomes. FOA allows one to directly take into account arrangement of nucleotides in each sequence. The obtained results clarified the previously known classification. In addition Rickettsia felis group was discovered between the ancestral group and spotted fever group (SFG) and R. akari group located between the SFG and genus Orientia. Software used for the analysis of nucleotide sequences with FOA is freely available at http://foarlab.org.

scholarly journals Rickettsialpox — a rare but not extinct disease: review of the literature and new directions

2020 ◽  
Vol 10 (3) ◽  
pp. 477-485
Author(s):  
M. E. Eremeeva ◽  
K. Muniz-Rodriguez
Keyword(s):  
Genetic Diversity ◽  
New York ◽  
Spotted Fever ◽  
Research Priorities ◽  
Etiologic Agent ◽  
Spotted Fever Group ◽  
Public Health Importance ◽  
Sexually Transmitted ◽  
Worldwide Distribution ◽  
Clinical Triad

Rickettsialpox is an urban zoonosis caused by Rickettsia akari. To date R. akari is the only well-characterized mite-borne member of the spotted fever group. It is transmitted by the mouse mite, Liponyssoides sanguineus, commonly found on peridomestic rodents. While the disease was first discovered in New York City in 1946, a few years later a similar outbreak occurred in the Ukraine SSR. Numerous serosurveys and diagnosis of sporadic cases of rickettsialpox suggest its global distribution; however, the actual contemporary geography of rickettsialpox and its incidence are unknown. Rickettsialpox is characterized by the classic clinical triad found in rickettsioses of a black eschar, high fever, and rash but the latter is atypical as it is papulovesicular. Dermatological manifestations and the progression of rickettsialpox may mimic other infectious and noninfectious syndromes, including sexually transmitted diseases. The purpose of this review is to increase awareness of this unique disease through reanalysis of classic and contemporary clinical descriptions of rickettsialpox, evaluation of its worldwide distribution, and updates on the public health importance of the disease as well as the ecology and vector associations of R. akari. Our review data suggests that only limited genetic diversity exists among the available isolates of R. akari associated with previous outbreaks; additional effort is still required to define specific genetic markers permitting direct surveillance, accurate and reliable diagnosis, tracking and studying of the vector and host associations of contemporary isolates. The potential of R. akari to cross into other vector species emphasizes the necessity for detection of outbreaks of the disease in new regions of the world and in novel ecological settings. We describe existing gaps and limitations in our current understanding of the pathogenesis of rickettsialpox, the epidemiology of this disease and the genetic diversity of R. akari. We propose research priorities for what is needed to improve our understanding of this neglected rickettsial disease and its etiologic agent.

scholarly journals Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter ofTrypanosoma cruzi

2019 ◽  
Vol 30 (14) ◽  
pp. 1676-1690 ◽  
Author(s):  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Mayara S. Bertolini ◽  
Anibal E. Vercesi ◽  
Roberto Docampo
Keyword(s):  
Citrate Synthase ◽  
Etiologic Agent ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Amino Acid Residues ◽  
Cell Infection ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Low Glucose ◽  
Membrane Spanning

We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc and TcMCUd. The predicted structure of the proteins indicates that, as predicted for the TcMCU and TcMCUb paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca2+uptake, while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca2+uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca2+uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared with those in the mammalian complex.

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