Worldwide Presence and Features of Flea-Borne Rickettsia asembonensis.

Rickettsia asembonensis, the most well-characterized rickettsia of the Rickettsia felis-like organisms (RFLO), is relatively unknown within the vector-borne diseases research community. The agent was initially identified in peri-domestic fleas from Asembo, Kenya in an area in which R. felis was associated with fever patients. Local fleas collected from domestic animals and within homes were predominately infected with R. asembonensis with < 10% infected with R. felis. Since the identification of R. asembonensis in Kenya, it has been reported in other locations within Africa, Asia, the Middle East, Europe, North America, and South America. With the description of R. asembonensis-like genotypes across the globe, a need exists to isolate these R. asembonensis genotypes in cell culture, conduct microscopic, and biological analysis, as well as whole genome sequencing to ascertain whether they are the same species. Additionally, interest has been building on the potential of R. asembonensis in infecting vertebrate hosts including humans, non-human primates, dogs, and other animals. The current knowledge of the presence, prevalence, and distribution of R. asembonensis worldwide, as well as its arthropod hosts and potential as a pathogen are discussed in this manuscript.

Introduction

Rickettsia asembonensis is a Gram negative, obligate intracellular bacteria of the order Rickettsiales and family Rickettsiaceae (1). Among Rickettsia spp. with validly published names, it is most closely related to R. felis (Table 1) (4–6, 8, 9, 11, 13–15, 17–19, 30). However, among incompletely characterize rickettsiae, R. asembonensis genetically groups with other R. felis-like organisms (RFLO). The RFLOs are genetically related to R. felis but consist of a unique group of rickettsiae that are associated with various arthropods including fleas, ticks, mites, and tsetse flies for which limited knowledge of their biology and pathogenicity is available (3, 16, 31). Unfortunately, the genetic information of the majority of RFLOs in the GenBank database is fragmentary. Of the RFLOs described, only R. asembonensis (32) and “Candidatus Rickettsia senegalensis” (3) have been cultured (from C. felis) and characterized.

Table 1

Worldwide distribution of Rickettsia asembonensis and closely related, incompletely characterized rickettsiae.

Rickettsial agents	Source	Country	Location	Sequence comparison with R. asembonensis NMRCii (%)						Year in	References
				rrs	gltA	om pA	om pB	sca4	17kDa	GenBank
Rickettsia felis URRWXCal2	Ctenocephalides felis	USA	California. El Labs at Soquel	99.5	98	92.5	94.7	95.7	97.3	1999	(2)
“Candidatus Rickettsia senegalensis”	Ctenocephalides felis	Senegal	Dakar	99.4	98	-	94	94.7	-	2013	(3)
Rickettsia sp. RF2125	Ctenocephalides canis	Thailand	Sangkhlaburi District, Kanchanaburi province	–	99.3	–	99.7	–	–	2002	(4)
Rickettsia sp. cf1and5	Ctenocephalides felis	USA	Greenville County, South Carolina	–	99.5	–	–	–	100	2005	(5)
Rickettsia sp. SE313	Echidnophaga gallinacea	Egypt	Mansoura, Zagazig	–	99.7	–	–	–	100	2005	(6)
Rickettsia sp. cf9	Ctenocephalides felis	USA	Not provided	–	–	–	99.9	99.8	–	2006	Reeves et al., (Unpubl.)
Rickettsia sp. FS27	Orchopeas horwadi	USA	not provided	–	99.7	–	–	–	99.7	2006	Reeves et al., (Unpubl.)
Rickettsia sp.	Ornithonyssus bacoti	Egypt	Ebshaway, El Quseir, Qara Oasis, Zagazig, Arab El Maamal	–	–	–	–	–	100	#	(7)
Uncultured R. sp. Clone Hf56–2	Archaeopsylla erinacei	Germany	Bavaria	–	–	–	100	–	–	2008	(8)
Uncultured R. sp. Clone ARV5606	Ctenocephalides felis	Peru	Iquitos	–	99.7	–	–	–	99.7	2009	(9)
Rickettsia sp. RF2125	Pulex irritans	Hungary	Various parts of the country, specific information not provided	–	>99.3	–	–	–	–	#	(10)
Rickettsia sp. R14	Ceratophylus fasciatus	India	Not provided	–	99.7	–	99.9	–	–	2010	Chahota et al., (Unpubl.)
R. endosymbiont of C. felis isolate F143	Ctenocephalides felis	Thailand	No specific information provided (45 Districts)	–	99.7	–	–	–	100	2011	(11)
R. endosymbiont of C. felis isolate F144	Ctenocephalides felis	Thailand	No specific information provided (45 Districts)	–	99.7	–	–	–	100	2011	(11)
Rickettsia sp. clone 4-G/G/JP-10-2	Ctenocephalides felis	Costa Rica	Limon (Guacimo)	–	99	–	–	–	–	2011	(12)
Rickettsia sp. 'Synosternus'	Synosternus pallidus	Senegal	Dielmo	–	100	–	100	–	–	2011	(13)
Rickettsia asembonensis F30	Ctenocephalides canis	Kenya	Nyanza	100	100	99.8	99.9	100	100	2011	(14)
Rickettsia asembonensis F82	Ctenocephalides felis	Kenya	Nyanza	100	–	99.9	–	100	100	2011	(14)
Uncultured R. sp. Clone HL2a	Ctenocephalides felis	Malaysia	Kuala Lumpar, Selangor	–	99.7	–	–	–	–	2013	(15)
Rickettsia sp. RFLO-18	Ctenocephalides felis	Thailand	Was not deposited in the GenBank	–	–	–	99.7	–	–	#	(16)
Rickettsia sp. J28p	Ctenocephalides felis	Peru	Not provided	–	99.7	–	–	–	–	2015	Palacios-Salvatiera et al., (Unpubl.)
Rickettsial strain from C. felis	Ctenocephalides felis	Ecuador	Pastaza	99.9	99.7	–	100	100	100	#	(17)
Rickettsia sp. Clone Xr	Xenopsylla ramesis	Israel	Negev	100	99.7	100	100	–	100	2014	(18)
Rickettsia sp. 9AL	Ctenocephalides felis	Colombia	Villeta	100	100	–	100	–	–	2014	(19)
Rickettsia sp. 0095	Macaca fascicularis	Malaysia	Not provided	–	100	–	99.9	–	–	2014	(20)
Rickettsia sp. Clone Mal	Homo sapiens	Malaysia	University Malaya Medical Center	–	99	–	99.9	–	–	2015	(21)
Uncultured Rickettsia sp. Isolate F1	Ctenocephalides felis	South Africa	Mpumalanga Province	100	–	–	–	–	–	2015	(22)
Rickettsia asembonensis 0-TP-1	Ctenocephalides felis	Costa Rica	Cahuita, La Virgen, Limon, Tulialba, Guapiles	–	99.7	–	–	–	–	2016	(23)
Rickettsia asembonensis 6-CP-4-3	Pulex simulans	Costa Rica	Cahuita, La Virgen, Limon, Tulialba, Guapiles	–	99.7	–	–	–	–	2016	(23)
Rickettsia asembonensis 6-CP-4-4	Ambylyomma ovale	Costa Rica	Cahuita, La Virgen, Limon, Tulialba, Guapiles	–	99.7	–	–	–	–	2016	(23)
Rickettsia asembonensis CF26B/US	Ctenocephalides felis	USA	Orange County, California	99.9	99.7	99.9	99.9	100	–	2016	(24)
Rickettsia asembonensis Tapes	Rhipicephalus sanguineus	Brazil	Tapes	–	99.6	–	–	–	100	2016	(25)
Rickettsia sp. Clone SP003-M	Ctenocephalides orientis	Malaysia	Kuala Lumpur, Perak, Johore, Kelantan, Pahang, Negeri Sembilan	–	99.2	–	–	–	–	2016	(26)
Rickettsia asembonensis DB32B	Rhipicephalus sanguineus	Malaysia	Kuala Lumpur, Selangor, Pahang	–	99.6	–	–	–	–	2017	(27)
Rickettsia asembonensis CF#68	Ctenocephalides felis	Brazil	Maranhao State	–	99.6	–	99.9	–	100	2017	(28)
Rickettsia asembonensis F30	Ctenocephalides felis	Uganda	Southwestern Uganda	–	100	–	99.6–100	–	–	#	(29)
Rickettsia asembonensis 7.2	Ctenocephalides felis	USA	Galveston, Texas	–	–	–	–	–	100	2018	Quade et al., (Unpubl.)
Rickettsia asembonensis VGD7	Ctenocephalides felis	Peru	Peruvian Amazon	–	99.8	99.8	100	99.8	100	2017	(30)

Rickettsia felis and Candidatus Rickettsia senegalensis are provided as reference rickettsiae that are closely related to but distinct from Rickettsia asembonensis.

Other flea-borne rickettsiae include, besides the aforementioned R. felis and “Ca. R. senegalensis,” Rickettsia typhi, a member of the typhus group of rickettsiae (TGR). R. typhi is the causative agent of murine typhus, a febrile disease that is found throughout the world. R. typhi is vectored by various flea species-especially X. cheopis, but also other Xenopsylla species such as X. astia and X. brazilliensis (33, 34), Synosternus pallidus, and rarely, but importantly, Ctenocephalides felis the common cat flea that readily parasitizes cats, opossums, and other domestic, peri-domestic, and wild animals. C. felis is believed to be capable of hosting R. typhi and to vector murine typhus in areas outside the traditional range of rat fleas and rats (35, 36).

R. felis, R. asembonensis, and “Ca. R. senegalensis” fall within the spotted fever group rickettsiae (SFGR) that genetically clusters within the transitional group of rickettsiae (37). R. felis is associated with flea-borne spotted fever (38, 39) and the pathogenicity of R. asembonensis and “Ca. R. senegalensis” is currently unknown. These three agents have worldwide distribution, are often sympatric and most often found parasitizing cat and dog fleas (3, 4, 14, 38, 40, 41).

“Candidatus R. senegalensis” was first described in C. felis fleas from Senegal (3) and an agent believed to be “Ca. R. senegalensis”-like (Rickettsia sp. RF31) had been detected previously in C. felis near the Thailand-Myanmar border (4). A very close genetic relationship (99.9% based on gltA gene sequence) between Rickettsia sp. RF31 and the latter is notable (3). “Ca. R. senegalensis” is distinct from, but can be sympatric with, R. felis and R. asembonensis (40). It has worldwide distribution but is not reported as often as R. felis or R. asembonensis. Reports of its molecular presence in cat tissues suggests it may be able to infect vertebrate animals (41).

History of Rickettsia asembonensis

Incompletely characterized rickettsiae with various identities most closely related to R. asembonensis populated the literature in the early 2000s (Table 1). These agents were detected by molecular techniques [i.e., PCR, nested PCR (nPCR), and/or quantitative real-time PCR (qPCR)] and then characterized by sequencing different size fragments of one or more commonly used gene targets (rrs, gltA, ompA, ompB, sca4, or the 17 kDa antigen gene). The first agent, referred to as Rickettsia sp. RF2125, was detected in Ctenocephalides canis in western Thailand near the Myanmar border (4). The agent was characterized by the sequence of a 1,171 bp fragment of the gltA that showed the rickettsial agent to be unique but most closely related to R. felis (4). The sequence of a 790 bp fragment of ompB (JX183538) from the original Rickettsia sp. RF2125 DNA preparation was obtained at that same time as the gltA but was not reported in the original article (4). It was reported in 2013 (14). We believe that RF2125 may have been the first detection of R. asembonensis or a very similar agent. Additional reports of R. asembonensis or an agent closely related to it continued to occur worldwide (Figure 1) shortly thereafter including: Rickettsia sp. cf1 and 5, USA (5); Rickettsia sp. SE313, Egypt (6); Rickettsia sp. Hf56-2, Germany (8); Rickettsia sp. ARV5606, Peru (9); and Rickettsia sp. Synosternus, Senegal (13). These partially characterized agents were described prior to our complete characterization of R. asembonensis (1). These agents are summarized along with R. asembonensis to include their distribution, vector hosts, and genetic characterization (see Table 1).

Figure 1

Worldwide mapof the locations of Rickettsia asembonensis, genetically similar rickettsiae, and associated vertebrate and invertebrate hosts. Inset maps are for points in (A): Costa Rica; (B): Egypt and Israel; and (C): Malaysia. This map was created using ArcGIS® software by Esri. ArcGIS® and ArcMap™ which are the intellectual property of Esri and are used herein under license. Copyright © Esri.

R. asembonensis was initially described as an unknown Rickettsia sp. detected in various flea species (i.e., C. felis, C. canis, Echidnophaga gallinacean, X. cheopis, and Pulex irritans) collected from various domestic animals (i.e., dogs, cats, and rodents) and houses (by light traps) in Asembo, Kisumu, in western Kenya during an epidemiologic surveillance study (14). This study was conducted concurrently with a fever study in which the presence of R. felis was identified in 7.2% of febrile patients (42). The initial molecular characterization of the R. asembonensis agent was accomplished utilizing a multilocus sequence typing (MLST) algorithm (43). Prevalence of this new agent (~91.7%) in collected fleas was found to be distinctly different from that of R. felis (8.3%) (14).

Subsequently, additional fleas collected from the same hosts and locations within the livestock-owning compounds in Asembo were processed for rickettsial culture. The new agent, Rickettsia asembonensis NMRCii, was successfully cultured from a pool of five individual flea triturate cultures isolated from C. canis and C. felis fleas obtained from domestic dogs. The cultures were initially grown in S2 and subsequently in C6/36 cell lines at 25°C (32), but not in Vero and L929 cell lines or embryonated chicken eggs incubated at 37°C (1).

The culture of R. asembonensis NMRCii was analyzed by microscopy, including Diff-Quik/acridine orange staining and transmission electron microscopy (32). The R. asembonensis were observed in the Drosophila S2 and Aedes albopictus C6/36 cells lines as early as 3 days post-infection, and could be observed at multiple time points throughout the average culture time of 40–45 days (32). Rickettsiae were observed both intra- and extracellularly at time points ranging from 15 to 30 days throughout the course of the continuous culture (32). The new agent was observed by acridine orange staining in singlets, doublets, and during heavy parasitization of host cells, in long chains (32). Transmission electron microscopy of the R. asembonensis revealed multiple free rickettsiae (round to elongated morphology) in the cytoplasm of the host cells, with normal rickettsial size [diameter 0.375–0.5 μm (round morphology), length 0.5–0.625 μm, width/diameter 0.25–0.375 μm (elongated morphology)]. A cell wall membrane, defined periplasmic space, and cytoplasmic membrane were observed, as well as the electron lucent “halo” (rickettsial slime layer) (32). Intranuclear localization/growth of the agent was not detected by acridine orange or by transmission electron microscopy (32).

Genetic characterization of the cultured R. asembonensis NMRCii by MLST using rickettsial genes rrs, gltA, ompA, ompB, and sca4; plasmid analysis; and whole genome sequencing confirmed that the new agent was indeed a unique Rickettsia species (1, 44). R. asembonensis NMRCii was shown to have an estimated genome size of 1.40 Mb, possessed a 21,692 bp circular plasmid and had a G+C content of 32.2%. The R. asembonensis plasmid, pRAS01, was discovered to be unique as it only shared 89% homology with that of R. africae ESF5 and only 84% homology with that of R. felis. The R. asembonensis genome has 1,147 predicted protein-coding genes, 33 tRNA genes, and three rrn operons. These characteristics are similar with those found within the genome of R. felis (NC_007109), which is 1.49 Mb in size and contains 1,400 protein-coding genes, 33 tRNA genes, and three rrn operons. Of the R. felis proteins, 1,157 (83%) have homologs in R. asembonensis (1, 44).

The sequences of R. asembonensis NMRCii, were 100% identical to those previously described for “Ca. R. asemboensis” isolates F30 and F82 for the following genes: rrs, gltA, sca4, and the 17kD antigen gene. For the ompA and ompB genes, the R. asembonensis NMRCii shared 99.86 and 99.98% similarity respectively, with the “Ca. R. asemboensis” isolates F30 and F82. The differences observed were as a result of nucleotide substitutions in two positions for the ompA gene and in one position for the ompB gene. A molecular phylogenetic analysis using 4,130 bp sequence of the variable gene-ompB open reading frame was conducted and the phylogenetic relationship between Rickettsia asembonensis NMRCii with R. felis, Rickettsia sp. PU01-02 (“Ca. R. senegalensis”) and other recognized Rickettsia species was determined (Figure 2).

Figure 2

Molecular phylogenetic analysis using ompB open reading frame (4,130 bp). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Evolutionary analyses were conducted with MEGA7.

Rickettsia asembonensis NMRCii was deposited in two separate culture collections (= DSM 100172T and = CDC CRIRC RAS001T) and the name officially changed (according to the rules of the International Journal of Systematics and Evolutionary Biology) from “Candidatus Rickettsia asemboensis” to Rickettsia asembonensis (1).

Arthropods Associated With Rickettsia asembonensis

R. asembonensis DNA has been detected in various arthropods, but most commonly in fleas (Table 1). It has been identified in fleas from three families namely the Pulicidae, Ceratophyllidae and Coptopsyllidae. In the cosmopolitan Pulicidae family it has been associated with seven genera: Ctenocephalides (C. felis, C. canis, and C. orientis); Xenopsylla (X. cheopis, X. ramesis, and X. gerbilli); Archaeopsylla (A. erinacei); Echidnophaga (E. gallinacea); Pulex (P. irritans); and Synosternus (S. pallidus). In the family Ceratophyllidae, R. asembonensis has been detected in three genera: Ceratopsyllus (C. fasciatus); Orchopeas (O. howardi); and Nosopsyllus (N. laeviceps) and in one genus in the family Coptopsyllidae: Coptopsylla (C. lamellifer) (45).

High prevalence rates of R. asembonensis have been reported in C. felis and C. canis (sympatric species), S. pallidus, X. ramesis, and X. gerbilli with up to 95, 95, 91.4, 100, and 33.3% of the fleas positive for R. asembonensis, respectively (13, 14, 18, 40, 46). Similar results in Costa Rica and Brazil confirm the high prevalence of R. asembonensis in C. felis (23, 28). In addition, R. asembonensis has been associated with other fleas, usually in much lower prevalence than in the aforementioned fleas. These include E. gallinacea, P. irritans, C. lamellifer, X. hirtipes, and N. laeviceps. Often these fleas are positive for R. asembonensis in the same areas as fleas highly infected with R. asembonensis (14, 46). The presence of the R. asembonensis in minimally infected flea species may be due to co-feeding and not that these fleas are reservoir hosts for R. asembonensis. Other arthropods in which evidence of R. asembonensis has been found include the tropical rat mites (Ornithonysus bacoti) in Egypt (7) and ticks (Amblyomma ovale and Rhipicephalus sanguineus) (23, 25–27).

Pathogenicity

In limited laboratory studies no marked cytopathic effects were observed in S2 and C6/36 cells, beyond lysis of overly parasitized host cells (32). Additionally, no growth was observed in embyronated chicken eggs (1). Moreover, in two febrile studies conducted in Kenya no molecular evidence of this agent in patients' blood was seen whereas R. felis DNA was detected in 3.7 and 7.2% of fever patients' blood (42, 47). However, there is molecular evidence of R. asembonensis in a patient from Malaysia with fever, myalgia, arthralgia, mild headache, conjunctival suffusion, and the presence of petechiae noted on his limbs. Molecular analysis (gltA and ompB sequences) of the patient's blood identified R. sp. RF2125 (21). In addition, in the blood from a healthy free range domestic dog from Mnisi community situated in the northeastern corner of the Bushbuckridge Municipal Area, Mpumalanga Province, South Africa R. asembonensis was detected by NGS (22). Lastly, 12 of 50 healthy monkeys from Peninsular Malaysia had molecular evidence (100% gltA sequence similarity) of R. sp. RF2125/”Ca. R. asemboensis” (20). Thus, from the mixed results presented, the question of pathogenicity for humans and other animals is not yet resolved and requires more investigation.

Future Research Direction

R. asembonensis-genotypes have been described in various biting and non-biting arthropods. Apart from R. asembonensis NMRCii that has been isolated in cell culture and whose full genome sequence is available in the GenBank Database, many of the others are just molecular isolates derived from arthropods with very limited sequence data for comparison. Functional and structural analysis of R. asembonensis is needed to ascertain differences and/or similarities between it and other rickettsial species. Moreover, research concerning the known/potential hosts of R. asembonensis, its current/potential arthropod vectors (both common and non-common), and its potential for interference with other rickettisal flea-borne pathogens (R. felis and R. typhi), as well as non-rickettsial pathogens such as Yersinia pestis, will be crucial to fully defining its pathogenicity and probability as a public health concern/nuisance across the world.

Author Contributions

All authors contributed to the conception and design of the review. AM wrote the first draft of the manuscript. JJ, AL-F, HS, CF, and AR wrote revisions of the manuscript. All authors contributed to the manuscript's final version, and read and approved the submitted version.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.