Rickettsia massiliae and its public health significance across Palearctic and Oriental regions: a scoping review

Abstract

Background

Arthropod including ticks, fleas, and lice have been found to be infected with Rickettsia massiliae, a pathogenic member of spotted fever group, causing rickettsiosis. This comprehensive scoping review summarize the known facts about its transmission, diagnostic-methods, phylogenetic position, human-case-reports, preventive-measures, and distribution in Palearctic and Oriental regions.

Methods

Three main steps were followed to compile this study: explanation of objective(s), identification of relevant literature, and retrieval of data determined by inclusion and exclusion criteria. Various databases, including Science Direct, Web of Science, PubMed, Scopus, Cochrane, and Google Scholar were screened for relevant literature. Our objectives were to collect data regarding transmission method(s) of R. massiliae, identification-assay(s), phylogenetic position, clinical-reports globally, and distribution via possible vectors & host animals surviving in Palearctic and Oriental regions. Descriptive analysis has been conducted to plot the frequency graphs of reported numbers in different countries and hosts.

Results

Findings presented that R. massiliae have been found across Palearctic and Oriental regions in 5 tick genera (Rhipicephalus, Hyalomma, Haemaphysalis, Ixodes, Amblyomma, and Dermacentor), 1 of louse (Haematopinus), 1 of sheep ked (Melophagus), and 1 of flea (Archaeopsylla), although their role as a vector(s) is still unknown. Dogs, sheep, cattle, and goats were recorded as epidemiologically important host animals for infected arthropods in 30, 23, 17, and 16 different studies, highlighting their role in its possible transmission. Additionally, Rh. sanguineus sensu lato (s.l) and Rh. turanicus were reported to infest the aforementioned animals, which is also being recognized as potential vector for its transmission. Highest number of R. massiliae reports (10) from different animals and vectors were recorded in China. Italy was recorded with highest (3) clinical cases of humans to-date. Moreover, transmission methods like transovarial via Rh. turanicus and horizontal/artificial-feeding via Rh. sanguineus s.l. and various diagnostic methods for R. massiliae have been documented.

Conclusions

R. massiliae has been widely documented throughout Palearctic and Oriental regions, with human cases reported in about six countries. Rh. sanguineus and Rh. turanicus stands out the potential vectors, while further investigation into the implications of diverse range of arthropods in epidemiology of this bacterium.

Graphical Abstract

Background

Rickettsiae are gram-negative bacteria that replicate entirely within cells and are named after Howard Ricketts, the American scientist who originally identified them as the cause of Rocky Mountain spotted fever in 1906 [1]. Rickettsial diseases are found globally, with several regions being endemic or hyper-endemic [2–4]. Rickettsiosis is an infectious disease caused by the obligate intracellular gram-negative bacteria, belonging to the order of rickettsiales, family Rickettsiaceae [5]. Rickettsiosis, caused by any other Rickettsia spp. except R. massiliae, is a significant contributor to morbidity and mortality in different regions of southeast Asian countries, and second most reported cause of non-malarial febrile illnesses, following dengue virus infection that is transmitted by mosquitoes in the region [6].

The family Rickettsiaceae includes the genera Rickettsia and Orientia. Rickettsia species are categorized according to differences in antigens and genomics. The taxonomy of this genus is currently growing and includes five distinct monophyletic groups: spotted fever group (SFG), transitional group, typhus group, R. canadensis group, and R. bellii group. Main disease-causing agents are prevalent worldwide and categorized mostly into SFG and Typhus groups [7–9]. Species in each of these groups vary in characteristics relating to their ecology, distribution, hosts, and pathogenicity [8]. All rickettsial species in these groups can potentially be propagated by ticks, fleas, lice, and mites in nature and can infect human beings [10–19]. The SFG rickettsial bacteria have a cosmopolitan distribution and are mainly propagated as well as transmitted to animals and humans via bites of approximately 198 infected arthropod vectors, which include species of ticks (n = 146), fleas (n = 24), mosquitoes (n = 15), mites (n = 6), lice (n = 4), keds (n = 2), and bugs (n = 1) [3, 4, 8, 13, 19]. Infected vectors transmit rickettsial bacteria through saliva while feeding on wounds or mucosal surfaces of host. The SFG species responsible for infectious illnesses are common in nature, with most human cases from the United States and Europe, with an estimated number of 66,133 human infections have been caused by SFG species worldwide from January 1906 to March 2021. The SFG rickettsiosis is becoming more widely recognized as a public health hazards globally, although its distribution and risk impact are not well defined [8, 13]. Although, insufficient information is available regarding the life cycles of majority of tick-borne (TB) Rickettsiae. Within natural vertebrate hosts, infections may lead to the development of rickettsemia, which enables noninfected ticks to get infected and maintains its natural life cycle [20]. During infection of SFG Rickettsia, propagation occurs via an infected arthropod vector, leading to the growth of Rickettsia in cells of endothelial layer. This process triggers inflammatory responses such as cytokine release and complement protein activation. Rickettsia growth and host immunological response cause the breakdown of blood vessels, leading to the distinctive maculopapular rash [21–23]. Although the bacteria mostly live in host's cytoplasm, certain bacteria leave via the luminal surface of vessel walls and enter into bloodstream [7].

The R. massiliae (Mtu1; Latin name of Marseille, France, where organism was first isolated) is an obligatory gram-negative intracellular organism. It flourishes in tissue cultures of L929, Vero cells, and human embryonic lung fibroblast cells. Gimenez-stained infected cells contain tiny, intracellular, rod-shaped, or diplobacilli bacteria that are somewhat smaller (0.3–0.4 by 0.6–1 µmol) than other SFG Rickettsiae. Electron microscopy of R. massiliae revealed an exterior slime layer and trilaminar cell wall. Bacteria develop unrestrictedly in cytoplasm of cells without a membrane boundary and are absent in nuclei of cells [24, 25]. R. massiliae is one of the SFG bacteria that show cosmopolitan distribution globally and has been detected in many types of arthropods and is transmitted by ticks in genus Rhipicephalus. R. massiliae, a novel rickettsial bacteria, was first discovered in hemolymph of Rh. sanguineus ticks infesting dogs in France in 1992. Rh. sanguineus, commonly known as brown dog tick, is a globally widespread tick species with significant medical and veterinary importance due to its role in transmitting various pathogens to dogs and, occasionally humans [26–28]. It can be found on dogs in both urban and rural areas, though it occasionally infests other hosts and is highly adapted to living inside human residences. This tick remains active throughout year, not only in tropical and subtropical regions but also in certain temperate areas. The ability of Rh. sanguineus to complete up to four generations per year depends on factors such as climate and host availability. High temperatures can cause ticks to attach and feed on humans and rabbits more quickly, suggesting an increased risk of human parasitism and transmission of zoonotic agents like Rickettsia spp. in areas with warmer or longer summers [27]. The taxonomy and systematics of Rh. sanguineus group have long been unclear, but recent research has clarified many aspects [29]. Additionally, R. massiliae was officially classified as a new species by Beati and Raoult in 1993 after confirming its distinctiveness through various identification methods [24, 25, 30]. In 1996, a variant strain of R. massiliae designated Bar-29, was isolated in a Rh. sanguineus tick from Spain while Bar-29 was characterized by phenotypic and genotypic analyses. The new isolates were confirmed to have the same profiles as R. massiliae through various methods that included micro-immunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis. The profile of these isolates was distinct from other species of SFG and exhibited differentiable serotypes after identification via aforementioned assays [31]. R. massiliae Bar-29 has been detected in saliva of Rh. turanicus (within Rh. sanguineus group), providing evidence that bacteria could be transmitted through bite of this tick species [32]. Cardenosa et al. [33] concluded about pathogenicity of Bar-29 strain for humans. Additionally, it has been proved that R. massiliae has possibly transmitted by Rh. sanguineus s.l. tick species to dogs and to the patient’s family members which were documented positive with different levels of IgM and IgG for this pathogenic bacterium [34, 35].

Despite extensive literature on SFG Rickettsiae, a focused and comprehensive synthesis dedicated specifically to human pathogen, R. massiliae, has been lacking. This review fills that critical knowledge gap by providing a consolidated overview of key features of R. massiliae. The summarized information presented herein will aid in mitigation the impact of this emerging pathogen, and support the development of effective infection control strategies.

Methods

This scoping review began with the formation of a research team, including authors of this publication, who hold expertise in relevant topics. The investigators provided guidance on fundamental research question(s) to be explored, data search strategy, and criteria for inclusion and exclusion [36]. The research used a scoping review methodology in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping reviews (PRISMA-ScR), enabling a descriptive overview of the literature data as demonstrated in Fig. 1 [37, 38]. The study was conducted via a series of steps: (1) explanation of objective(s) and gap analysis; (2) identification and review of relevant literature; and (3) gathering of data determined by inclusion and exclusion criteria.

Fig. 1.

Method’s flow chart for article’s screening and processing

Focused objective(s) and gaps analysis

To obtain literature-based data regarding various possible transmission methods, phylogenetic position (ompA gene), diagnostic methods, zoonotic and pathogenic impacts, possible treatment, and control measures of R. massiliae in targeted region were the main objectives of current study. As no comprehensive review, presenting each aforementioned parameter(s) associated with R. massiliae, was not currently available in literature. Hence, we planned to overcome this scientific gap to minimize the risks associated with this zoonotic bacterium.

Literature data search strategy

To identify studies that align with our review objectives, a thorough computerized search strategy was devised for assembling all scientific papers that use English-language databases such as Science Direct, Web of Science, PubMed, Scopus, Cochrane, and Google Scholar to compile the appropriate scientific literature (Fig. 1). Various key terms, including SFG of Rickettsia and the detection of R. massiliae in various tick genera such as Rhipicephalus, Haemaphysalis, Dermacentor, Ixodes, Hyalomma, and Amblyomma were used in combinations in the aforementioned databases. Additionally, other terms were clinical cases of R. massiliae in humans, names of specific countries included in Palearctic & Oriental regions, microscopic, serological, and molecular detection of R. massiliae in cattle, buffaloes, sheep, goats, dogs, cats, foxes, jackals, wild boars, horses, donkeys, mules, hares, mice, mongooses, hedgehogs, sparrows, arthropods collected on vegetation, human beings, fleas, and louse were searched in different combinations in the aforementioned databases. Because of our main target, we intentionally employed the broader term "SFG Rickettsia" in our search strategy to ensure a comprehensive capture of literature. This approach mitigated the risk of omitting relevant studies that reported R. massiliae findings within general SFG surveillance or phylogenetic analyses but did not specify the species names including R. massiliae, in titles or keywords. Authors screened the obtained studies to determine if they contained individual data, removed duplicates, and excluded non-English literature after reviewing their titles and abstracts. Descriptive analysis has been conducted to plot the frequency graphs of the number of reports in different countries and different hosts.

Inclusion and exclusion criteria

Specifically, this review is intended to provide an overview of the studies that have clearly mentioned any of the aforementioned focused question(s). Each country (included in Palearctic & Oriental regions) were searched to obtain the total reports till 4 June 2025, addressing any of the aforementioned objectives. Additionally, duplicate, unrelated title & content-based, published in other than our targeted regions, and reporting other Rickettsia spp., except R. massiliae studies were excluded. Moreover, we searched and included all the relevant literature regarding human infectious reports throughout the world (Fig. 1).

Results and discussion

Transmission of R. massiliae

Ticks acts as a vector/carrier/reservoir/host for vertical and horizontal transmission of Rickettsiae. This zoonotic bacteria has been detected globally in various tick species including Rhipicephalus (turanicus, microplus, sanguineus, bursa, haemaphysaloides, decoloratus, secundus, pusillus), Hyalomma (anatolicum, hussaini, dromedarii, schulzei, marginatum, excavatum, scupense), Haemaphysalis (montgomeryi, longicornis, erinacei, sulcata, adleri, parva, cornupunctata, heinrichi, mageshimaensis, punctata), Ixodes (ricinus, Ixodes sp.), Amblyomma (testudinarium, Amblyomma sp.), and Dermacentor marginatus. Additionally, it has been identified in Haematopinus tuberculatus, Melophagus ovinus, and Archaeopsylla erinacei as presented in Tables 1 and 2 [8, 80, 84, 88]. Proven vectors and infected arthropods have been marked clearly in Table 1. Further details regarding variety of potentially possible infected arthropods, their associated host animals, and number of reports from each country, included in Palearctic and Oriental regions, are presented in Fig. 2A–B.

Table 1.

Published reports on Rickettsia massiliae detection in arthropods and the vertebrate animals on which arthropods were found, in Palearctic and Oriental regions

Country/year	Specimen type	Specimen sources	Serological (S)/molecular (M) evidences	Genetic marker (s)	Prevalence rate	References
Algeria/2001–2003	Rh. turanicus*	Cattle, goats, and hedgehog	M	gltA and ompA	Not known	[39]
	Rh. sanguineus*	Hedgehog
Algeria/2010–2013	Rh. sanguineus*	Dogs and cats	M (qPCR)	gltA	24.4%	[40]
Algeria/2014	Rh. bursa	Cattle	M (RT-PCR)	-	79.7%	[41]
Algeria/2014–015	Rh. sanguineus*	Wild boars	M (qPCR)	ompA	18.5%	[42]
Algeria/2012	Rh. sanguineus*	Cattle, sheep, dogs, boars, Mongoose, and jackals	M	ompA	40.4%	[43]
	Rh. bursa
China (northwest)	Rh. turanicus*	Sheep	M	17-kDa, ompA, gltA, rrs, sca1, and ompB	19.1%	[44]
China (Taklamakan desert)/2013–2014	Rh. turanicus*	Sheep	M	ompA, gltA, rrs, geneD, and ompB	3.3%	[45]
China/2015	Hy. anatolicum	Cattle	M	ompA	Not known	[46]
China	Hae. longicornis	Mice	M	ompA	Not known	[47]
China/2021–2022	Melophagus ovinus	Sheep	M	17-kDa, rrs, gltA, sca1, ompA, ompB, gene D	14.6%	[48]
China/2014–2016	Rh. turanicus*	Cattle and sheep	M	17-kDa, rrs, gltA, sca1, ompA, ompB, gene D	Not known	[49]
China/2017–2020	Rh. turanicus*	Pet dogs	M	gltA and OmpB	Not known	[50]
China/2016–2017	Rh. sanguineus*	Pet dogs	M	17 kDa, rrs, gltA, ompA, ompB, geneD	23.8%	[51]
China	Rh. sanguineus*	Dogs	M	gltA and ompB	2.8%	[52]
China	Hae. mageshimaensis	–	M	–	Not known	[53]
	Hae. longicornis
Cyprus/1999–2006	Rh. turanicus*	Dogs, foxes, hares	M	gltA and ompA	10.3%	[54]
	Rh. bursa	Goats		ompA	2.7%
	Rh. sanguineus*	Dogs			8.1%
Cyprus	Rh. sanguineus*	Dogs and cats	M	190-kDa	5.4%	[55]
Cyprus/2002–2006	Rh. bursa	Mouflons	M	ompA	6.4%	[56]
Denmark/2011	Ixodes spp.	Dogs	M	16S rRNA	5.0%	[57]
France (southern)/2007	Rh. sanguineus*	Human (because human was detected positive for Rickettsia serologically)	S (IgG and IgM) and M	ompA	20.0%	[35]
France/2009	Rh. sanguineus*	Hedgehog	M (qPCR)	gltA	91.7%	[58]
France/2010	Rh. sanguineus*	House sparrow (Passer domesticus), dogs, horses, cat, vegetation, and human	M	gltA and ompA	28.8%	[59]
France	Rh. sanguineus*	Vegetation	M	monoclonal antibodies	-	[60]
Georgia/2012–2016	Hy. marginatum	Cattle, dogs, and vegetation	M and Sequencing	gltA, ompA, ompB, and sca4	1.2%	[61]
	Rh. sanguineus*	Dogs			1.2%
Greece/2015	Rh. sanguineus*	Dogs	M	ompB	0.3%	[62]
	Rh. turanicus*				0.3%
Greece/2009	Rh. bursa	Humans	M	ompA	12.5%	[63]
	Rh. turanicus*				42.8%
Greece/2015–2016	Rh. bursa	Sheep	M	16S rRNA	–	[64]
Greece/1998–1999	Rh. turanicus*	Cattle, goats, and sheep	M	gltA and ompA	–	[65]
Greece/2014	Rh. sanguineus*	Dogs	M	Atp, RecA, gltA, DnaA, DnaK, ompA and OmpB	1.6%	[66]
Hungary/2011	De. marginatus	Shepherd dogs	RLB	–	7.7%	[67]
India/2017–2018	Hae. bispinosa	Cattle	M	ompA	Not known	[68]
India/2019–2020	Rh. sanguineus*	Shrews	M	23S rRNA	Not known	[69]
	Haematopinus tuberculatus	Buffaloes
Iran/2017–2018	Rh. turanicus*	Cattle, goats, sheep, and dogs	M	Rsp and gltA	24.6%	[70]
	Rh. sanguineous*	Cattle, goats, and sheep			24.6%
Iran/2018	Hy. marginatum	Goats and sheep	M	gltA	30.0%	[71]
	Hy. anatolicum
	Hy. dromedarii
	Hy. schulzei
	Rh. bursa
	Rh. turanicus*
Iran/2019	Rh. sanguineus*	Dogs	M	gltA, 17 kDa, and ompA	Not Known	[72]
Iran	Rh. sanguineus*	Sheep and goats	M	16S rRNA, gltA and ompA	Not Known	[73]
Iran/2019–2020	Rh. sanguineus*	Dogs	M	gltA	2.0%	[74]
Israel/2010	Rh. turanicus*	Vegetation	M	ompA	1.7%	[75]
	Rh. sanguineus*	Dogs			0.3%
	Hae. erinacei	Hedgehog			0.6%
Israel/2002–2008	Rh. turanicus*	Vegetation	M	ompA	24.4%	[76]
	Rh. sanguineus*				9.2%
Israel	Rh. sanguineus*	Vegetation	M	16S rRNA	Not known	[77]
Israel/2014	Rh. turanicus*	Vegetation	M	gltA, ompA, and ompB	Not known	[78]
	Rh. sanguineus*
Israel/2014	Rh. turanicus*	Roe deer	M	17 kDa, and ompA	53.7%	[79]
Italy	Rh. sanguineus*	Foxes	M	gltA	7.1%	[80]
Italy/2014–2016	Rh. turanicus*	Humans	M	23S rRNA, gltA, and ompA	3.2%	[81]
		Lepus europaeus (Hare)
	Rh. sanguineus*	Red fox
Italy/2012–2013	Rh. sanguineus*	Dogs	M	gltA, ompA, and ompB		[82]
Italy/2011	Rh. turanicus*	Vegetation	M	gltA, ompA, and ompB	27.3%	[83]
	Ix. ricinus				17.4%
Italy/2012–2013	Rh. sanguineus*	Humans	M	gltA, ompA, and ompB	Not known	[84]
	Rh. turanicus*
Italy/2007–2010	Ix. ricinus	Vegetation	M	gltA	6.2%	[85]
Italy/2007	Rh. sanguineus*	Dogs	M	gltA, ompA, and ompB	Not known	[86]
	Rh. turanicus*	Cattle and goats
Japan/1997–1999	Amblyomma sp.	Jackals	M	gltA and ompA	9.1%	[87]
Lebanon/2014	Rh. turanicus*	Ovine, bovine, and caprine	M	ompA	4.5%	[88]
Morocco/2002–2006	Rh. sanguineus*	Livestock and dogs	M	12S rDNA, gltA, and ompA	4.7%	[89]
Morocco/2018–2021	Rh. sanguineus*	Hedgehog	M	gltA, ompA, and ompB	5.6%	[90]
Morocco/2007	Rh. sanguineus*	Vegetation	M	ompA	26.7%	[91]
Morocco/2015–2020	Rh. sanguineus*	Foxes	M	gltA, ompA, and ompB	3.2%	[92]
Netherlands	Rh. sanguineus*	Sheep	M	16S rRNA	Not known	[93]
	Blood
Pakistan/2017–2021	Rh. haemaphysaloides	Wild boars	M	gltA and ompA	8.3%	[94]
	Rh. turanicus*
Pakistan	Hy. anatolicum	Cattle, buffaloes, Goats, and sheep	M	16S rRNA and ompA	0.2%	[95]
	Rh. microplus
Pakistan	Hae. sulcata	Sheep and goats	M	23S-5S ITS	42.6%	[96]
	Hy. anatolicum
	Rh. microplus
	Rh. turanicus*
	Rh. haemaphysaloides	Goats
Pakistan/2017	Hy. anatolicum	Cattle and buffaloes	M	23S-5S ITS	1.7%	[97]
	Hy. hussaini
Pakistan/2018–2019	Rh. microplus	Mules, horses, and donkeys	M	gltA, and ompA	2.4%	[98]
	Rh. haemaphysaloides
	Rh. turanicus*
	Hy. anatolicum
Pakistan/2023–2024	Rh. microplus	Cattle, sheep and goats	M	17 kDa, gltA, and ompA	Not known	[99]
	Rh. sanguineus*
Pakistan/2023	Rh. haemaphysaloides	Cattle	M	gltA, ompA and ompB	1.2%	[100]
	Rh. turanicus*
	Rh. microplus	Goats
	Hae. cornupunctata	Sheep
Pakistan/2020–2022	Hy. anatolicum	Goats and sheep	M	16S rRNA and gltA	20.2%	[101]
	Hy. excavatum
	Hy. scupense
	Hae. montgomeryi
	Rh. decoloratus
	Rh. haemaphysaloides
	Rh. microplus
Palestine/2014	Rh. turanicus*	Dogs and sheep	M	ompA, 16S rRNA and 17 kDa	10.1%	[102]
	Rh. sanguineus*				7.4%
	Hae. adleri				0.7%
	Hae. parva				0.7%
Palestine/2015	Rh. turanicus*	Sheep	M	16S rRNA	Not Known	[103]
Poland/2008–2009	Blood / serum	Humans	S	Specie specific antibodies	78.9%	[104]
Poland/2021	Rh. secundus	Vegetation	M	gltA and ompA	1.8%	[105]
Portugal/2021	Blood	Sheep	M	ompB and ompA	35.0%	[106]
	Rh. sanguineus*				62.0%
Portugal/2002–2004	Rh. turanicus*	Birds	M	gltA and ompA	Not Known	[107]
Portugal/2011–2012	Rh. sanguineus*	Vegetation	M	gltA	9.0%	[108]
Spain/1997–2003	Ixodes ricinus	Humans	M	gltA and ompA	15.0%	[109]
	Rh. turanicus*
	Rh. sanguineus*
	Rh. pusillus
Spain/2010–2016	Spleen	Dogs	M	ITS2 gene	1.6%	[110]
Spain	Blood	Red foxes	S	IgG-FITC antibody	50.3%	[111]
	Rh. sanguineus*		M	ompA (qPCR), multiplex PCR	78.1%
Spain/2008	Rh. pusillus	Vegetation	M	16S rRNA, ompB, atpA, dnaA, dnaK, and recA	Not Known	[112]
Spain/1997–2001	Rh. sanguineus*	Humans	M	gltA and ompA	Not Known	[113]
Spain/2001–2005	Rh. sanguineus*	Humans and dogs	M	gltA and ompA	2.5%)	[114]
Spain	Rh. sanguineus*	Dogs	M	16S rRNA and ITS2	Not Known	[115]
Spain/2014–2016	Rh. sanguineus*	Wild boars	M	16S rRNA and gltA	45.2%	[116]
Sri Lanka	Rh. sanguineus*	Dogs	M	ompA	6.5%	[117]
		Cows
Switzerland/1994–2001	Rh. sanguineus*	Dogs	M	16S rDNA, gltA and ompA	6.3%	[118]
Thailand/2008–2013	Amb. Testudinarium	Vegetations	M	17-kDa gene	Not known	[119]
Thailand/2015–2019	Hae. heinrichi	Burmese ferret-badger	M	ompA, ompB, and sca4	Not known	[120]
		Cattle
Tunisia/2009	Rh. sanguineus*	Sheep	M	23S-5S intergenic region and ompA	0.7%	[121]
		Dogs
Tunisia/2019–2020	Hy. marginatum	Cattle	M	gltA, ompA, and ompB	0.3%	[122]
	Rh. sanguineus*				10.67%
Tunisia	Rh. sanguineus*	Goats and sheep	M	gltA, ompA, and ompB	Not known	[123]
	Rh. turanicus*
Tunisia/2012–2014	Skin biopsies	Humans	M (qPCR)	23S-5S rRNA	63.7%	[124]
	Cutaneous swabs		RLB		40.6%
Tunisia	Rh. sanguineus*	Hedgehog	M	ompB	Not known	[125]
	Archaeopsylla erinaceid
Turkey/2016–2019	Rh. turanicus*	Dogs	M	16S rRNA, gltA, and ompB	1.8%	[126]
United Kingdom/2011	Hae. punctata	Vegetations	M	16S rRNA	2.0%	[127]

*Proven vectors, Rh Rhipicephalus, Hy Hyalomma, Hae Haemaphysalis, De Dermacentor, S serological, M molecular, RLB reverse line blot

year is representing the sampling time during specific study, while some studies were not having the exact sampling year

gltA Citrate synthase gene, ompA Outer membrane protein A, ompB Outer membrane protein B, 17-kDa 17-kD antigen gene, sca1 Surface cell antigen 1 gene, geneD/sca4 Surface cell antigen 4 gene, rRNA/rrs Ribosomal RNA gene, AtpA ATP synthase F1 alpha subunit gene, RecA Recombination protein gene, DnaA Chromosomal replication initiation protein gene, DnaK Heat-shock protein 70 gene/chaperone protein, ITS Intergenic space gene

Table 2.

Variety of vectors/carrier/reservoir/hosts and their reported countries

Reported vector/carrier/reservoir/hosts		Reported countries
Ticks	Species
	Rhipicephalus (turanicus, microplus, sanguineus, bursa, haemaphysaloides, decoloratus, secundus, pusillus)	Algeria, China, Cyprus, France, Greece, Georgia, Hungary, India, Iran, Italy, Israel, Lebanon, Morocco, Netherlands, Pakistan, Portugal, Palestine, Poland, Switzerland, Spain, Sri-Lanka, Turkey, Tunisia
	Hyalomma (anatolicum, hussaini, dromedarii, schulzei, marginatum, excavatum, scupense)	Algeria, China, Cyprus, Denmark, France, Greece, Georgia, Hungary, Iran, Pakistan, Tunisia.
	Haemaphysalis (montgomeryi, longicornis, erinacei, sulcata, adleri, parva, cornupunctata, heinrichi, mageshimaensis, punctata)	China, India, Israel, Pakistan, Palestine, Thailand, UK
	Ixodes (ricinus, Ixodes sp.)	Denmark, Italy, Spain,
	Dermacentor marginatus	Japan, Thailand,
		Hungary
Louse	Haematopinus tuberculatus	India
Sheep ked	Melophagus ovinus	China
Hedgehog flea	Archaeopsylla erinacei	Tunisia

Fig. 2.

A–B : A Number of Rickettsia massiliae reports from each country (included in Palearctic and Oriental regions), B Number of identification reports from variety of host animals

These tick species are widely distributed and harbor various pathogens, including R. massiliae, affecting animals and humans [27]. These findings may suggest the existence of R. massiliae in host animals because this bacterium can be transmitted from tick(s) to host animals during blood feeding [23, 35, 111, 113] (Fig. 3).

Fig. 3.

Putative mode of transmission of Rickettsia massiliae (not proven). Step 1: Biting of the tick to host animal for a blood meal and transfer of R. massiliae pathogen to the host, Step 2: Attachment of R. massiliae via adhesins (e.g., outer membrane protein B) to its host cells (vascular endothelial cells), Step 3: Engulfment of a bacterium by host cells, Step 4: The intracellular replication of R. massiliae by binary fission, Step 5: Destruction of vascular endothelial cells and release of R. massiliae into the blood vessels, that are ready to attach with other host cells (again infection starts), Step 6: Feeding of tick(s) for a blood meal from R. massiliae infected host animal, Step 7: Infected tick(s), which can bite another normal host (animal/human) and transfer the bacterium via saliva, hence the cycle starts back. Prepared via Biorender (Biorender (https://app.biorender.com) and according to Kim et al. [128]

Previous studies indicated that R. massiliae Bar-29 can be transmitted to vertebrate hosts via tick’s saliva during feeding. Furthermore, several other methods are also involved in the transmission of Rickettsia bacteria, which are discussed below.

Transovarial transmission is an efficient vertical transmission of rickettsial agents observed in many arthropod vectors, including ticks. It’s not a compulsory factor that such transmission will contribute to the amplification of TB pathogens, but only contribute to the maintenance of disease in surrounding environment [129]. More specifically, previous research has outlined the transovarial transmission of R. massiliae by identifying its presence in first-generation larvae of infected engorged Rh. turanicus ticks as well as by analyzing eggs from R. massiliae infected the same tick species by PCR. The observation that larvae and nymphs of second and third generation that fed on rabbits were positive for R. massiliae, suggests transovarial transmission. Infected female eggs, as well as larvae and nymphs throughout the developmental phases, exhibit a 100% transmission rate. Salivary transmission was detected in 80% of analyzed tick samples using PCR evaluation of saliva from the second generation of ticks from infected females [32].

This bacterium has been reported to be transmitted from ticks to blood via artificial feeding based on silicone membranes. In a previous study, R. massiliae infected Rh. sanguineus ticks were fed on bacteria-free bovine blood in an in vitro feeding system using a silicone membrane. Later, the blood samples were positive for the presence of R. massiliae via molecular techniques, and this was performed to validate that these ticks can act as vectors for R. massiliae [93].

Co-feeding/transsexual transmission of R. massiliae Bar-29 strain was evaluated by feeding R. massiliae-infected Rh. turanicus male ticks on a rabbit with non-infected Rh. sanguineus female ticks. After laying eggs, majority (69.2%) of female Rh. sanguineus were positive and confirmed via PCR for rickettsial gltA. Since, Rh. turanicus and Rh. sanguineus are closely related species; breeding between specimens of both species is conceivable (J. L. Camicas, personal communication) [32].

Pathogenicity and human infection reports

To date, nine human cases of R. massiliae infection have been reported globally. First case of human infection was confirmed by molecular analysis of a 20-year frozen sample of a patient with an illness reminiscent of Mediterranean spotted fever (MSF) in Sicily (Italy) in 2006 [130]. The presence of R. massiliae was confirmed in serum samples of forest workers in Poland via indirect immunofluorescence assay (IFA), with subsequent molecular confirmation [104]. Serological techniques like direct IFA have been used to identify Rickettsiae in both humans and animals [35, 104, 111, 131, 132], however detecting these bacteria is challenging due to short durations of rickettsemia. Details regarding the nine human reports of R. massiliae are presented in Table 3.

Table 3.

Detail of human cases regarding Rickettsia massiliae

Country	Gender	Age (y)	Year of infection/sample collection	Sign and symptoms	Diagnostic assay	Medication	Effectiveness of medication	Study
Sicilia (Italy)	M	45	6th June 1985/identified in 2005 as a and R. massiliae after 20 years sample storage	Fever, rash, eschar, hepatomegaly	Cell culture and PCR from blood	Tetracycline	Cured	[130]
France	M	25	24th May 2007/10th June 2007	Fever, eschar, rash, acute vision loss	PCR (cutaneous biopsy, aqueous humor	Doxycycline, Ofloxacin and Glucocorticoid (IV)	Ophthalmic sequelae	[133]
Argentina	F	56	Un-known/1st July 2005	Fever, purpuric rash eschar, pleural effusion	PCR (cutaneous biopsy)	Doxycycline (12 days)	Cured	[134]
Sicilia (Italy)	M	13	10th May 2012/un-known	Headache, fever, cervical lymphadenitis, eschar, hepatomegaly	PCR (cutaneous biopsy)	Doxycycline	Cured	[135]
Romania	M	76	June 2011–June 2012a	Fever, rash, conjunctivitis, myalgia, eschar	Western blot with cross absorption	Doxycycline (7 days)	Cured	[136]
	F	49		Fever, rash, myalgia, eschar	Indirect immunofluorescence assay	Ibuprofen, Doxycycline, ofloxacin (14 days)	Cured
Greece	M	62	June 2013/July 2013	Fever, headache, myalgia, eschar, upper extremities rash	Indirect immunofluorescence assay	Ibuprofen, Doxycycline, ofloxacin (14 days)	Cured	[131]
Tunisia	Not known	Not known	2012–2014 from June to Octoberb	Fever, rash, eschar	PCR on skin biopsy	Not known	Not known	[124]
Italy	F	61	Not known	Erythematous lesion on her right eyelid and latero-cervical satellite lymphadenopathy, occipital headache with painful neck, lowgrade fever, general malaise, asthenia and generalized joint pain	Biopsy and qPCR for swab of eyelid samples	Not known	Not known	[132]

y year, M Male, F Female

^aPatients were examined during this time

^bStudy was conducted during this time, and no specific date of infection/sample collection is reported in both cases

The R. massiliae has been acknowledged as a newly identified etiological agent of MSF-like illnesses in humans and has an association with several tick species. The pathogenic role of R. massiliae was thought to be due to its sero-prevalence in humans. Some authors suggested that it may be the causal agent of certain cases of MSF [8]. To date, no case of R. massiliae infection has had a fatal outcome.

Phylogenetic position

Molecular approach based on genetic variance at the individual base-pair level gives a much more direct pathway for measuring the genetic diversity between and within species of Rickettsia genus. The gltA, ompA, ompB, 17-kDa, 190-kDa, 16S rRNA, 12S rDNA, 23S-5S ITS, ITS2, pgsA, Rsp, rrs, sca1, and sca4 genes are representative of different genomic functions and are believed to be active in most rickettsial species. Previous studies based on these molecular markers (Table 1) have been used for molecular detection and genetic analysis, providing sufficient and accurate information about evolutionary relationships of diverse Rickettsia species, specifically to characterize R. massiliae [52, 137, 138]. The R. massiliae group, specifically belonging to SFG Rickettsia and includes R. massiliae, R. rhipicephali, and R. aeschlimannii [139]. All about R. massiliae has been discussed earlier in this manuscript, however R. rhipicephali can infect ticks, and is generally considered a non-pathogenic agent especially for humans [140, 141] while R. aeschlimannii is recognized as an emerging human pathogen and is associated with ticks as a vector especially Hy. marginatum species [142], with cases reported in various regions of the world [143].

The importance of comparing results obtained for multiple identification markers to test the reliability of organismal phylogeny has recently been highlighted. The method of choice to establish bacterial phylogeny (including R. massiliae) is the comparison of different gene sequences [93, 111, 144–146]. Although R. massiliae is one of the earliest diverging species of the SFG, its genome is very similar in terms of sequence and gene order to the genomes of other SFG [147]. The sequences (based on ompA gene) of R. massiliae from other countries appears highly similar with only limited divergence (Fig. 4). This close homology may reflect the limited resolution of the single ompA gene fragment employed, as well as the overall genetic conservation of R. massiliae across various regions including Cyprus, Switzerland, Israel, Portugal, Spain, Tunisia, China, Senegal, India, Italy, and Pakistan [44–46, 70, 71, 94, 96–98, 122].

Fig. 4.

Constructed phylogenetic tree based on widely used genetic marker (ompA) sequences of Rickettsia massiliae using Minimum evolution method and p-distance model with 1000 bootstraps, representing that R. massiliae is making sister clade with R. rhipicephali and R. aeschlimannii. Three main groups including strain MTU5 (yellow), Bar-29 (green), and unclassified (no color) R. massiliae have been mentioned. R. akari (CP000847) was used as an outgroup (colour figure online)

Diagnostic methods

In general, diagnosis of rickettsiosis is difficult because of the manifestations with non-specific clinical symptoms, such as fever, severe headache, skin rash, and general malaise, which is often misdiagnosed as other febrile illnesses or viral diseases. Confirmation of a rickettsial infection requires direct molecular detection because serological testing to detect antibodies potentially leads to a false negative result if serum-based testing occurs too early before antibodies are generated [148–150]. Moreover, proper testing requires expensive equipment, reagents, and skilled laboratory technicians. These aspects can complicate the ability of countries with constrained resources to test for Rickettsia infections. Cases of febrile illnesses are often over-diagnosed as malaria and later proven otherwise by more sensitive and specific PCR assays. Distinguishing Rickettsia from other pathogenic agents early allows for timely treatment and informs any necessary public health measures [151].

A laboratory diagnostic paradigm shift in rickettsial infections is evolving. The latest and accurate diagnostic assay should be owned to expand the diagnostic window toward early infection intervals of R. massiliae. Further advancements in field of Rickettsia spp. diagnostics are necessary in order to enhance the identification of diseases and epidemiological surveillance, as well as to get a deeper understanding of the dynamics of transmission. There has been a growing number of reports on the application of next-generation sequencing (NGS) and metagenomics for diagnosing Rickettsia spp. infection. NGS is used to analyze microbial communities, while metagenomics is particularly effective in identifying bacteria that are located inside cells, challenging to cultivate, or may not exhibit their characteristic molecular target (such as partial DNA sequences) [152]. Moreover, the editing technique, clustered regularly interspaced short palindromic repeats (CRISPR) based analyses will also facilitate comprehensive research on challenging bacterial species, specifically in the field of diagnosis [153]. Although numerous diagnostic assays have been previously developed for the identification of R. massiliae and its infection.

This bacterium has been identified via Gimenez staining of tick’s hemolymph. For the said purpose, 11 Rh. turanicus ticks were tested, including 5 females and 6 males. The fumigated ticks were used to cut their body parts (legs), and a drop of hemolymph was obtained that was stained with Gimenez for the successful examination of R. massiliae which were later validated via PCR assay [32].

The R. massiliae (strain MTU5) can be cultivated and cultured in L929 cells that were growing on Eagle’s Minimum Essential Medium (EMEM) (Biowhittaker, Cambrex Biosciences), treated with 2 mmol/L glutamine and 4% fetal calf serum (GIBCO, Invitrogen) in culture flasks and incubated at 32 °C for their optimum growth and division of cells. Molecular confirmation is provided by shotgun sequencing followed by Southern blot [147]. Another study stated that its diagnosis relies on its culturing in Vero cell line (African green monkey kidney epithelial cells) and observation of growth using Gimenez staining [111, 146]. R. massiliae has been successfully isolated molecularly via IFA and PCR in cultured tick’s cells [145]. The shell vial culture technique has been used for accurate identification of R. massiliae in tick cells. Rickettsial agents were observed by applying the Gimenez stain on tick cells on the 16th day of their culture, which was later confirmed and verified (on day 18th of culture) through PCR by the amplification of gltA and ompA genes [32].

PCR and sequencing methods are sensitive and rapid tools that are used to detect and identify Rickettsiae in blood, tissue, or skin biopsy specimens globally. Most reliable method used for the detection of R. massiliae involved amplification of specific segments of R. massiliae DNA by PCR. This involves the initial detection of species by screening all DNA samples by a real-time PCR assay targeting fragments of different genes, including 17-kDa, htrA, ompA, gltA, rrs, sca1, 16S rRNA,12S rDNA, and ompB [31, 76, 89, 93, 111, 145, 146]. Additionally, the PCR-restriction fragment length polymorphisms (RFLP) method enables the rapid identification of bacteria in amplified PCR products. Different endonucleases like RsaI and PstI were used for RFLP analyses for the identification and confirmation of R. massiliae and specifically, the Mtu5 isolate/strain of R. massiliae [154].

The 23S-5S intergenic spacer gene can be molecularly amplified from the extracted DNA of ticks via PCR, followed by reverse line blot (RLB) hybridization for successful detection of R. massiliae with a sensitivity of 46.4% and specificity of 86.1%, using specific amino-oligonucleotide probes [67, 93, 121, 121].

The R. massiliae has been detected in serum, blood, spinal fluid, aqueous humor, and tissue specimens obtained from various animals by IFA, targeting IgG and IgM antibodies [34, 35, 104, 111, 131, 133]. Confirmation is subsequently provided by real-time PCR targeting the gltA gene [131]. The IFA was performed using human IgG against SFG Rickettsia provided in Rickettsia IFA IgG kit (Focus Diagnostics, Langenhagen Hannover, Germany) for the successful detection of R. massiliae [145]. R. massiliae Bar-29 has been successfully detected in saliva and feces of adult ticks through IFA, with successful amplification of gltA partial gene achieved by PCR [32].

Micro-immunofluorescence (MIF) is the ‘gold standard’ technique for the sero-diagnosis of MSF and is used as a reference technique in laboratories. This assay allows for simultaneous detection of multiple rickettsial agents in a single well because a single drop of diluted serum sample is reacted against many (usually nine) antigens simultaneously, which assist in differentiation of species [155]. This assay has been used for the detection of rickettsial agents, including R. massiliae, in Catalonian patients. Reactions to R. massiliae antigen can be very low but can be confirmed by specific reactions in western immunoblotting [33]. In a previous study, this bacterium has been detected using different assays, including IFA, MIF, SDS-PAGE, and western blot assay [31].

Treatment of R. massiliae infection

Given the challenge of diagnosing any rickettsial infection during acute phase and need of prompt treatment for quick recovery and complication avoidance, it is advised to use anti-rickettsial medications, preferably tetracycline, as a preliminary prescription when rickettsiosis is suspected [130, 132, 149]. Typical treatment involves doxycycline at a dosage of 200 mg per day for a period of 3 to 14 days, depending on patient's clinical condition. Chloramphenicol and more recent macrolides are likely suitable substitutes for doxycycline [131, 156]. Additionally, previous studies have reported that other antibiotics, including ofloxacin and glucocorticoids have been successfully applied for the treatment of R. massiliae infections in humans (Table 2) [133]. Furthermore, this pathogenic bacterium has a natural resistance to rifampicin that is similar to the resistance displayed by R. aeschlimannii [130]. Therapeutic strategy is the same as recommended for all TB rickettsiosis [132, 157]; prompt doxycycline treatment should be initiated in case of clinical suspicion.

Control/preventive measures

Additionally, there is a need to mitigate the potential vectors/reservoirs of R. massiliae using chemical acaricides, plant extracts, fungi, or vaccines [158–163]. It has been documented comprehensively that rickettsial infection rate has significantly increased from 33.3% in 2005 to 50.8% in 2015 (seasonally, especially from July to October) in Germany due to increased incidence of Ixodes ticks [164]. Early removal of ticks from their respective host animals (after attachment) could help in minimizing the risks of rickettsiosis. This is based on the detection of R. massiliae DNA as early as 8 h after the ticks were applied to host animals. These findings proved the early transmission of TB rickettsial DNA to host animals [93]. To prevent rickettsiosis, common risk environments should be avoided when traveling in endemic regions. These also include avoiding contact with rats, dogs, and domestic cattle, as well as bush plants that may be contaminated with ticks or mites. Specifically Rh. sanguineus s.l (competent proven vector) should be effectively eliminated via applications of different acaricides on its potential vectors (mainly dogs) as well as environments. The reinfestations after acaricides applications should be also minimized for a certain period of time [26, 165]. Unfortunately, resistance against various acaricides by this tick species has been reported globally, resulting in failure of its effusive control [166], although the mechanisms regarding development of resistance in this tick specie are poorly understood and demands further comprehensive mechanistic research. Additionally, molecular examination of human blood is also mandatory when relevant signs/symptoms are present, to check the presence of R. massiliae, so that control strategies could be developed to minimize the risks of human infections. For this purpose, recent advances in technology, such as next generation of sequencing and CRISPR-based gene editing, will facilitate further research on these obligate intracellular bacteria because these have been applied to identify many bacterial species, including those that pose challenges in terms of cultivation and manipulation, which presents a potential opportunity for the advancement of tools in Rickettsia species [153]. Moreover, the recombinase polymerase amplification (RPA) method in combination with a lateral flow (LF) test is recommended as a suitable approach for developing a straightforward, fast, highly sensitive, and specific method to detect Rickettsia species. This method would be particularly useful in areas with limited resources. The RPA technique involves three proteins: a recombinase, a polymerase, and a single-strand binding protein. It is capable of amplification within a time frame of 5–20 min at a constant temperature of approximately 37 °C. Furthermore, it has a detection limit of less than 10 copies per reaction. The amplified products are marked using a biotin-labelled reverse primer and a probe labelled with carboxyfluorescein (FAM) that is easily observable using the LF test [167].

Limitations and future recommendations

Some unresolved questions regarding the confirmation of primary vertebrate host, reservoir hosts, or carriers for R. massiliae bacterium remained poorly defined because of the ambiguous role of vertebrate animals in its transmission and tick infestations. So future studies are recommended to comprehensively perform the xenodiagnoses based research and controlled experimental infections to move beyond the serosurveys for quantification of reservoir competence via amplification of R. massiliae bacterium. It is an important gap in literature, and more laboratory studies are needed to explore the vector capacity (transmission capability) of other arthropods, just like Rh. turanicus and Rh. sanguineus as proven vectors.

Moving forward, robust species-level confirmation via molecular methods in both typical and atypical SFG presentations is essential to definitively map the pathogenicity and true public health burden of this emerging agent. Specifically, the full spectrum of clinical signs from infection R. massiliae in people and animals should be documented and variations (if possible) in subspecies of R. massiliae should be addressed.

Similarly, future research outlining the concrete research approaches, including comparative genomics of humans vs tick-derived strains of R. massiliae, systematic febrile-illness surveillance, and refined animal models, should be performed to transform these uncertainties into a targeted research roadmap. We recommend that future research integrate detailed clinical reporting with whole-genome sequencing of patient-derived isolates should be performed, which is essential to determine if phylogenetic clades or specific virulence gene profiles correlate with disease severity. We believe that these approaches will resolve the unknown spectrum of R. massiliae pathogenicity (from endosymbiont-like relationships to genuine virulence) and will fulfil the existing knowledge gaps.

Conclusions

Ticks associated rickettsial bacterium are growing day by day due to their variety of vectors and capacity to feed on various host animals. In Palearctic and Oriental regions, our study highlighted the geographical distribution of R. massiliae and its localization in all the infected arthropods as well as host animals, to recognize their existence and to know about their potential threats to the living community. Due to its potential threat for human beings, further investigations are required to minimize its zoonotic impacts. As most of the reported host animals are companions to humans beings and can be infested by variety of tick species belonging to Rhipicephalus genus, which increase the chances of their transmission to human beings, hence, R. massiliae is a human concern currently. Therefore, further comprehensive research studies are needed to mitigate the known vectors of R. massiliae in genus Rhipicephalus, so that negative impacts of R. massiliae could be minimized.

Abbreviations

SFG

Spotted Fever Group

MSF

Mediterranean Spotted Fever

IFA

Immunofluorescence assay

NGS

Next-generation sequencing

CRISPR

Clustered regularly interspaced short palindromic repeats

LF

Lateral flow

RPA

Recombinase polymerase amplification

EMEM

Eagle’s minimum essential medium

RFLP

Restriction fragment length polymorphisms

RLB

Reverse line blot

MIF

Micro-immunofluorescence

PRISMA

Preferred reporting items for systematic reviews and meta-analyses

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel

Author contributions

M.K.O., CY, J.L., S.S., T.A.K., A.Z.K.: Conceptualization, Literature based data-collection, Writing–original draft, reviewing & editing draft. Q.R., M.R., G.G.: Supervision of the overall investigations, Conceptualization, reviewing & editing draft. All authors equally contributed to the formulation of the whole manuscript.

Funding

This study was financially supported by the National Parasitic Resources Center (NPRC-2019-194-30), National Natural Science Foundation of China (32373037), The Natural Science Foundation of Gansu Province, China (25JRPA1090), National Key Research and Development Program of China (2024YFD1800100), The Science Fund for Creative Research Groups (22JR5RA024) of Gansu Province, Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-LVRI, NBCITS (CARS-37).

Data availability

Not applicable. As we have presented all the included literature data in the manuscript.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

None of the authors of this review paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper.