Duplex Reverse-Transcription Real-Time Polymerase Chain Reaction Assay Targeting 23S rRNA Single Nucleotide Polymorphisms for the Detection of Flea-Borne Rickettsioses.

Flea-borne spotted fever and flea-borne (murine) typhus are rickettsioses caused by Rickettsia felis and Rickettsia typhi, respectively, and typically present as undifferentiated febrile illnesses. The relative contribution of these agents to flea-borne rickettsioses in California is unclear. We have developed a duplex reverse transcription real-time polymerase chain reaction (RT-rtPCR) assay targeting R. felis- and R. typhi-specific 23S ribosomal RNA single nucleotide polymorphisms to better understand the respective roles of these agents in causing flea-borne rickettsioses in California. This assay was compared with an established duplex R. felis- and R. typhi-ompB rt-PCR assay and was shown to have 1,000-fold and 10-fold greater analytical sensitivity for the detection of R. felis and R. typhi, respectively. Retrospective testing of clinical specimens with both assays established R. typhi as the major etiologic agent of flea-borne rickettsioses in California.

First detection of Rickettsia felis and Ehrlichia canis in the common bed bug Cimex lectularius.

Bed bugs, common blood-feeding pests, have received limited attention regarding their potential involvement in emerging pathogen transmission. This study aimed to investigate the main vector-borne bacteria within bed bugs collected from Tunisian governorates and to genetically characterize the identified species. Molecular screening was conducted on field-collected bed bug samples, targeting zoonotic vector-borne bacteria from the Anaplasmataceae family, as well as the genera Rickettsia, Ehrlichia, Bartonella, and Borrelia. A total of 119 Cimex lectularius specimens were collected and grouped into 14 pools based on sampling Tunisian sites. Using genus-specific PCR assays, DNA of Rickettsia and Ehrlichia spp. was detected in a single pool. Sequencing and BLAST analysis of the obtained partial ompB and dsb sequences from positive samples revealed 100% similarity with those of Ehrlichia canis and Rickettsia felis available in GenBank. Obtained partial sequences showed phylogenetic similarity to R. felis and E. canis isolates found in dogs and ticks from American and European countries. To the best of our knowledge, this study is the first to investigate bed bugs in Tunisia and to report the worldwide identification of R. felis and E. canis DNA in the common bed bug, C. lectularius. These findings highlight the need for further research to explore the potential role of bed bugs in the epidemiology of these vector-borne bacteria.

Follow-up of a Rickettsia felis encephalitis: Some new insights into clinical and imaging features.

Rickettsia felis (R. felis) infection is a cause of unspecified encephalitis. However, the incidence has been underestimated due to the intracellular features of the pathogen and insufficient understanding of its clinical picture. This study reported a case of R. felis infection in a 26-year-old female who only manifested with certain neurological symptoms. With a lack of specific systemic inflammatory symptoms, the diagnosis was initially misdiagnosed as a brain glioma. However, a brain tissue biopsy showed prominent perivascular inflammatory infiltrations, which indicated inflammatory disease. Spinal fluid metagenomic next-generation sequencing (mNGS) was taken after ruling out other common infectious and autoimmune diseases. The results suggested R. felis infection, which was also supported by Weil-Felix reaction in the serum. After the diagnosis was corrected as R. felis encephalitis, the patient was successfully treated with doxycycline and had a good prognosis at the 1-year follow-up.

Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods.

INTRODUCTION: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. MATERIALS AND METHODS: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST>99). Information for the animal-hosts was available and statistically analyzed. RESULTS: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST>99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. CONCLUSIONS: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established.

Molecular detection of Bartonella henselae, Bartonella clarridgeiae and Rickettsia felis in cat and dog fleas in Tenerife, Canary Islands, Spain.

The cat flea Ctenocephalides felis is the main vector of Bartonella henselae and Bartonella clarridgeiae, the causative agents of cat-scratch disease (CSD) and the spotted-fever agent Rickettsia felis. In spite of their worldwide distribution, there are no data on the occurrence of CSD-causing Bartonella species or the prevalence of Rickettsia species in the Canary Islands, Spain. Therefore, the aim of our study was to screen cat and dog fleas for both pathogens. A total of 128 C. felis from cats and dogs were screened for Bartonella and Rickettsia by PCR. Bartonella henselae (2.3%) and B. clarridgeiae (3.9%) were found in fleas infesting cats, whereas R. felis was identified in both cat (36.6%) and dog (40.7%) fleas. Further, co-infections were observed. This work constitutes the first finding of CSD-causing Bartonella species and the first study on the prevalence of R. felis in fleas from domestic animals in the Canary Islands. These results indicate public health importance, as associated infections could be misdiagnosed in the Archipelago despite their clinical relevance. Establishing human and animal routine diagnosis procedures for these pathogens along with improving vector control in shelters is necessary in order to prevent the spread of the infections among animals.

Investigation of Ctenocephalides felis on domestic dogs and Rickettsia felis infection in the Democratic Republic of Sao Tome and Principe.

Rickettsia felis is an obligate intracellular Gram-negative bacterium which causes flea-borne spotted fever in humans. In the past decades, R. felis has been detected worldwide in Ctenocephalides felis fleas and various other arthropods. However, due to its shared symptoms with other common vector-borne diseases, human infection is prone to be underestimated or misdiagnosed, especially in the malaria-endemic areas including sub-Saharan Africa, where confirmatory laboratory diagnoses are not usually available. In this study, a 'One Health' approach was adopted to explore potential vector-borne and zoonotic pathogens in the Democratic Republic of Sao Tome and Principe (DRSTP), an island nation in the Gulf of Guinea. By collaborating with local veterinarians, 1,187 fleas were collected from 95 domestic dogs across the country and later identified as Ct. felis using taxonomic keys. A cytochrome oxidase gene-based phylogenetic analysis revealed that all collected fleas belonged to a single haplotype and were identical to isolates from Ivory Coast and Brazil that clustered into a clade of tropical distribution. Additional samples of 14 chigoe fleas (Tunga penetrans) were collected from the surrounding environment of the dogs' resting spots. Rickettsia felis infection in fleas was examined by molecular methods targeting the citrate synthase (gltA)- and outer membrane protein A (ompA)-coding genes as well as the R. felis-specific pRF plasmid. The bacterial DNA was detected in 21.01% (146/695) of cat fleas but none of the chigoe fleas. Microimmunofluorescence assay was then performed to assess pathogen exposure of the residents. Of 240 dried blood spots from participants with dog contacts, 8 (3.33%) exhibited R. felis antibodies. Our findings demonstrated the presence of R. felis in DRSTP. Further extensive epidemiological studies regarding its prevalence and its role in causing febrile illness while the nation is entering pre-elimination stage of malaria will be carried out.

Metagenomic analysis of human-biting cat fleas in urban northeastern United States of America reveals an emerging zoonotic pathogen.

An infestation of cat fleas in a research center led to the detection of two genotypes of Ctenocephalides felis biting humans in New Jersey, USA. The rarer flea genotype had an 83% incidence of Rickettsia asembonensis, a recently described bacterium closely related to R. felis, a known human pathogen. A metagenomics analysis developed in under a week recovered the entire R. asembonensis genome at high coverage and matched it to identical or almost identical (> 99% similarity) strains reported worldwide. Our study exposes the potential of cat fleas as vectors of human pathogens in crowded northeastern U.S, cities and suburbs where free-ranging cats are abundant. Furthermore, it demonstrates the power of metagenomics to glean large amounts of comparative data regarding both emerging vectors and their pathogens.

Molecular detection and identification of Rickettsia felis in Polygenis (Siphonaptera, Rhopalopsyllidae, Rhopalopsyllinae) associated with cricetid rodents in a rural area from central Argentina.

The aim of this study was to detect and identify the presence of Rickettsia in fleas associated with cricetid rodents from northeastern Buenos Aires Province, Argentina. Sixteen fleas belonging to three species of Polygenis were collected from 56 cricetid rodents and analyzed for the presence of Rickettsia performing the conventional polymerase chain reaction (PCR) technique. Only one specimen of Polygenis (Polygenis) axius axius collected from Oxymycterus rufus was positive for Rickettsia felis using the gltA gene, and to ompA gene. This is the first report of R. felis in a Rhopalopsyllidae flea from Argentina, and the first detection of this bacterium in P. (P.) a. axius. Since both, O. rufus and P. (P.) a. axius, are common in areas close to humans, and enzootic cycle of R. felis is not fully understood, the results herein obtained might be of epidemiological importance. Further studies are needed in order to analyze the capacity of the species of Polygenis to transmit R. felis.

The prevalence of Rickettsia felis DNA in fleas collected from cats and dogs in the UK.

In a large-scale survey in the UK, recruited veterinary practices were asked to inspect client-ownedcats and dogs, selected at random between April and June 2018, following a standardised flea inspection protocol. A total of 326 veterinary practices participated and 812 cats and 662 dogs were examined during the 3-month period. Fleas were collected, identified to species level and fleas of the same species collected from a single animal were pooled together and treated as a single sample. A total of 470 pooled flea samples were screened by PCR and DNA sequence analysis for a subset of Rickettsia species including R. felis and R. typhi. On analysis, 27 (5.7%) of the pooled flea samples were positive for R. felis DNA; these were predominantly in the cat flea, Ctenocephalides felis, but one dog flea, Ctenocephalides canis was also positive for this pathogen.

Rickettsia felis identified in two fatal cases of acute meningoencephalitis.

BACKGROUND: Rickettsia felis has recently emerged worldwide as a cause of human illness. Typically causing mild, undifferentiated fever, it has been implicated in several cases of non-fatal neurological disease in Mexico and Sweden. Its distribution and pathogenicity in Southeast Asia is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We retroactively tested cerebrospinal fluid (CSF) or sera from 64 adult patients admitted to hospital in North Sulawesi, Indonesia with acute neurological disease. Rickettsia felis DNA was identified in the CSF of two fatal cases of meningoencephalitis using multi-locus sequence typing semi-nested PCR followed by Sanger sequencing. DNA from both cases had 100% sequence homologies to the R. felis reference strain URRWXCal2 for the 17-kDa and ompB genes, and 99.91% to gltA. CONCLUSION/SIGNIFICANCE: The identification of R. felis in the CSF of two fatal cases of meningoencephalitis in Indonesia suggests the distribution and pathogenicity of this emerging vector-borne bacteria might be greater than generally recognized. Typically Rickettsia are susceptible to the tetracyclines and greater knowledge of R. felis endemicity in Indonesia should lead to better management of some acute neurological cases.

Molecular identification of vector-borne organisms in Ehrlichia seropositive Nicaraguan horses and first report of Rickettsia felis infection in the horse.

Certain vector-borne organisms serve as etiological agents of equine disease. After previously identifying a new Ehrlichia species in horses from Mérida, we aimed to determine the infection frequency and screen for a wide range of vector-borne organisms from 93 tick-exposed, Ehrlichia seropositive horses in this region. PCR assays were performed to identify infection by organisms within the following genera: Anaplasma, Babesia, Bartonella, Ehrlichia, Leishmania, Mycoplasma, Neorickettsia, Rickettsia and Theileria. Overall, 90/93 horses (96.8%) were infected with one or more vector-borne organisms. Ninety (96.8%) horses were infected with Theileria equi and 21 (26.8%) with Babesia caballi. Nine (9.7%) horses were infected with the novel Ehrlichia species previously designated H7, reported in horses from Nicaragua and Brazil. Two horses (2.2%) were infected with Rickettsia felis. Anaplasma, Bartonella, Leishmania, Mycoplasma, or Neorickettsia species DNA was not amplified from any horse. Ticks collected from horses infected with vector-borne organisms were identified as Amblyomma cajennense sensu lato and Dermacentor nitens. Horses in Mérida are infected by a range of vector-borne organisms, including B. caballi, T. equi, Ehrlichia species H7, and R. felis. To the authors' knowledge, this constitutes the first report of molecular detection of R. felis in horses.

Molecular Detection of Rickettsia felis and Rickettsia bellii in Mosquitoes.

To add to the limited information on Rickettsia in mosquitoes in China, we carried out a PCR survey on convenience samples of 3051 mosquitoes collected with hand nets in and around domestic dwellings in 25 provinces. Five species of mosquitoes were identified: Culex pipiens pallens (n = 1620), Aedes albopictus (806), Armigeres subalbatus (377), Anopheles sinensis (168), and Culex tritaeniorhynchus (80). A Rickettsia nested-PCR targeting the variable domain of gltA showed Rickettsia felis in four mosquito species of 16 provinces Cx. pipiens pallens (1.8%, 29/1620); Ae. albopictus (1.2%, 10/806); An. sinensis (1.2%, 2/168); and Ar. subalbatus (2.1%, 8/377). Rickettsia bellii was also widespread, occurring in 12 provinces and 2 species: Cx. pipiens pallens (4.3%, 69/1620) and An. sinensis (0.6%, 1/168). R. felis and R. bellii were found in almost similar numbers in female [1.5% (27/1809) and 1.2% (21/1809), respectively] as in male mosquitoes [1.8% (22/1242) and 4.0% (49/1242), respectively]. Our results indicated that mosquitoes in China are widely infected with R. felis, the agent of human flea-borne spotted fever, and that R. bellii can also occur outside of the Americas and its usual tick hosts.

Differentiation of Rickettsia felis and Rickettsia felis-Like Organisms via Restriction Fragment Length Polymorphism Analysis.

Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis) have been discovered-Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis-like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme NlaIV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of NlaIV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with NlaIV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis, and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae.

Molecular detection of Rickettsia felis in dogs, rodents and cat fleas in Zambia.

BACKGROUND: Flea-borne spotted fever is a zoonosis caused by Rickettsia felis, a Gram-negative obligate intracellular bacterium. The disease has a worldwide distribution including western and eastern sub-Saharan Africa where it is associated with febrile illness in humans. However, epidemiology and the public health risks it poses remain neglected especially in developing countries including Zambia. While Ctenocephalides felis (cat fleas) has been suggested to be the main vector, other arthropods including mosquitoes have been implicated in transmission and maintenance of the pathogen; however, their role in the epidemiological cycle remains to be elucidated. Thus, the aim of this study was to detect and characterize R. felis from animal hosts and blood-sucking arthropod vectors in Zambia. METHODS: Dog blood and rodent tissue samples as well as cat fleas and mosquitoes were collected from various areas in Zambia. DNA was extracted and screened by polymerase chain reaction (PCR) targeting genus Rickettsia and amplicons subjected to sequence analysis. Positive samples were further subjected to R. felis-specific real-time quantitative polymerase chain reactions. RESULTS: Rickettsia felis was detected in 4.7% (7/150) of dog blood samples and in 11.3% (12/106) of rodent tissue samples tested by PCR; this species was also detected in 3.7% (2/53) of cat fleas infesting dogs, co-infected with Rickettsia asembonensis. Furthermore, 37.7% (20/53) of cat flea samples tested positive for R. asembonensis, a member of spotted fever group rickettsiae of unknown pathogenicity. All the mosquitoes tested (n = 190 pools) were negative for Rickettsia spp. CONCLUSIONS: These observations suggest that R. felis is circulating among domestic dogs and cat fleas as well as rodents in Zambia, posing a potential public health risk to humans. This is because R. felis, a known human pathogen is present in hosts and vectors sharing habitat with humans.

Parallel Decline of Malaria and Rickettsia felis Infections in Senegal.

Rickettsia felis is a common emerging pathogen in sub-Saharan Africa. Comparing dynamics of morbidities due to malaria and R. felis infections in two Senegalese villages, we found a strong and significant correlation between them. Malaria morbidity is strongly decreasing because of the implementation of long-lasting insecticidal nets, so we hypothesize that the same measure may decrease the R. felis infections.

Rickettsial Infections among Cats and Cat Fleas in Riverside County, California.

Presently, few studies have investigated the role of domestic cats (Felis catus) in the recrudescence of flea-borne rickettsioses in California and the southern United States. In this study, we aimed to investigate the presence of Rickettsia typhi or Rickettisa felis in domestic cats (F. catus) and the fleas (primarily Ctenocephalides felis, the cat flea) associated with these cats in Riverside County, California. Thirty cats and 64 pools of fleas collected from these cats were investigated for rickettsial infections. Three cats and 17 flea pools (from 10 cats) tested positive for rickettsial infections. polymerase chain reaction and DNA sequencing indicated that one of the cats was positive for R. felis infections, whereas two were positive for Candidatus Rickettsia senegalensis infection. In addition, 12 of the flea pools were positive for R. felis, whereas five were positive for Ca. R. senegalensis. By contrast, no cats or their associated fleas tested positive for R. typhi. Finally, eight sera from these cats contained spotted fever group Rickettsia (SFGR) antibodies. The detection of R. felis and SFGR antibodies and the lack of R. typhi and TGR antibodies support R. felis as the main rickettsial species infecting cat fleas. The detection of Ca. R. senegalensis in both fleas and cats also provides additional evidence that cats and their associated fleas are infected with other R. felis-like organisms highlighting the potential risk for human infections with R. felis or R. felis-like organisms.

Serum cytokine responses in Rickettsia felis infected febrile children, Ghana.

The intracellular pathogen Rickettsia felis causes flea-borne spotted fever and is increasingly recognized as an emerging cause of febrile illness in Africa, where co-infection with Plasmodium falciparum is common. Rickettsiae invade endothelial cells. Little is known, however, about the early immune responses to infection. In this study, we characterize for the first time the cytokine profile in the acute phase of illness caused by R. felis infection, as well as in plasmodial co-infection, using serum from 23 febrile children < 15 years of age and 20 age-matched healthy controls from Ghana. Levels of IL-8 (interleukin-8), IP-10 (interferon-γ-induced protein-10), MCP-1 (monocyte chemotactic protein-1), MIP-1α (macrophage inflammatory protein-1α) and VEGF (vascular endothelial growth factor) were significantly elevated in R. felis mono-infection; however, IL-8 and VEGF elevation was not observed in plasmodial co-infections. These results have important implications in understanding the early immune responses to R. felis and suggest a complex interplay in co-infections.

A 2015 outbreak of flea-borne rickettsiosis in San Gabriel Valley, Los Angeles County, California.

Although flea-borne rickettsiosis is endemic in Los Angeles County, outbreaks are rare. In the spring of 2015 three human cases of flea-borne rickettsiosis among residents of a mobile home community (MHC) prompted an investigation. Fleas were ubiquitous in common areas due to presence of flea-infested opossums and overabundant outdoor cats and dogs. The MHC was summarily abated in June 2015, and within five months, flea control and removal of animals significantly reduced the flea population. Two additional epidemiologically-linked human cases of flea-borne rickettsiosis detected at the MHC were suspected to have occurred before control efforts began. Molecular testing of 106 individual and 85 pooled cat fleas, blood and ear tissue samples from three opossums and thirteen feral cats using PCR amplification and DNA sequencing detected rickettsial DNA in 18.8% of the fleas. Seventeen percent of these cat fleas tested positive for R. felis-specific DNA compared to under two (<2) percent for Candidatus R. senegalensis-specific DNA. In addition, serological testing of 13 cats using a group-specific IgG-ELISA detected antibodies against typhus group rickettsiae and spotted fever group rickettsiae in six (46.2%) and one (7.7%) cat, respectively. These results indicate that cats and their fleas may have played an active role in the epidemiology of the typhus group and/or spotted fever group rickettsial disease(s) in this outbreak.

Bartonella henselae and Rickettsia felis Detected in Cat Fleas (Ctenocephalides felis) Derived from Eastern Austrian Cats.

Cats and cat fleas (Ctenocephalides felis) are vectors of the zoonotic bacterial pathogens Bartonella henselae and Rickettsia felis, which are the causative agents of "cat scratch disease" and "cat flea typhus," respectively. In the surroundings of Vienna (Austria), we identified 11 (10.5%; n = 105) B. henselae-positive fleas originating from 8 cats (20.5%; n = 39). One flea was positive for R. felis. There should be high levels of awareness among veterinarians and animal keepers as to the handling of cats, especially if free roaming, stray, or feral.