Spotted Fever Group Rickettsiae in Ticks Collected from Wild Animals in Israel

Abstract

We report molecular evidence for the presence of spotted fever group rickettsiae (SFGR) in ticks collected from roe deer, addax, red foxes, and wild boars in Israel. Rickettsia aeschlimannii was detected in Hyalomma marginatum and Hyalomma detritum while Rickettsia massiliae was present in Rhipicephalus turanicus ticks. Furthermore, a novel uncultured SFGR was detected in Haemaphysalis adleri and Haemaphysalis parva ticks from golden jackals. The pathogenicity of the novel SFGR for humans is unknown; however, the presence of multiple SFGR agents should be considered when serological surveillance data from Israel are interpreted because of significant antigenic cross-reactivity among Rickettsia. The epidemiology and ecology of SFGR in Israel appear to be more complicated than was previously believed.

Introduction

Tick-borne spotted fever group (SFG) rickettsioses are caused by obligatory intracellular gram-negative bacteria of the genus Rickettsia. In Israel, Mediterranean spotted fever (MSF) caused by Rickettsia conorii subsp. israelensis is considered to be the primary cause of spotted fever group rickettsiosis associated with brown dog ticks, Rhipicephalus sanguineus Latreille.^1,2

To gauge the prevalence of SFGR in southern Israel, the hemolymph test showed the presence of rickettsiae in both Rh. sanguineus and Rhipicephalus turanicus collected from agricultural settlements in Israel.³ In the Negev region a correlation among the density of domestic animals, their ectoparasites (Rh. sanguineus, Rh. turanicus, and Hyalomma sp. ticks), and the incidence of spotted fever group rickettsiae was demonstrated.⁴ Serology was also a sensitive indicator for the presence and magnitude of human and canine exposure to ticks and to SFG rickettsiae (SFGR) based on the prevalence of immunoglobulin G (IgG)-antibodies to Rickettsia conorii in two rural villages in Israel.⁵ Recently Rickettsia massiliae and Rickettsia sibirica mongolotimonae were found in questing adult ticks collected from the vegetation in different parts of Israel.⁶ Furthermore, the presence of R. massiliae DNA sequences was detected in a Rh. sanguineus tick picked from the scalp of a pediatric patient in the north of Israel (Keysary A and others, unpublished data).

In Israel ticks such as Rh. sanguineus sensu lato, Rhipicephalus bursa Canestrini and Fanzago, Hyalomma marginatum Koch, and Haemaphysalis sulcata Canestrini and Fanzago have been found attached to humans.^7,8 Ixodid ticks, particularly Rh. sanguineus and Rh. turanicus Pomerantsev, are quite common on domestic and wild animals. Rhipicephalus (Boophilus) kohlsi has been found on the hilly land of the Mediterranean phytogeographic area, mainly on goats and sheep, whereas small numbers of this tick were also found on cattle, mules, horses, and camels.⁹ Adults of H. marginatum and Hyalomma detritum ticks occur on cattle and horses, although their larvae and nymphs can be found on rodents and birds.¹⁰ Previously, Haemaphysalis adleri was found on the golden jackal (Canis aureus), red fox (Vulpes vulpes), jungle cat (Felis chauss), and the desert cat (Felis lybica), whereas Haemaphysalis parva was found on dogs, golden jackal, gray wolf (Canis lupus), red fox, and hedgehog (Erinaceus europeus).^10,11

In contrast to studies of domestic animals and humans, there is a dearth of literature on the incidence of SFGR in populations of wild animals and their ticks in Israel. Only a single serological study showed high titers of SFG-rickettisal antibodies was detected in a substantial number of free-ranging jackals (Canis aureus syriacus) in Israel.¹²

We report here molecular evidence for the occurrence of two known human pathogenic species of SFGR, Rickettsia aeschlimannii in Hyalomma ticks, and Rickettsia massiliae in Rh. turanicus ticks collected from wild animals in Israel. In addition, we report the presence of a novel uncultivated SFGR found in Haemaphysalis ticks collected from golden jackals.

Material and Methods

One hundred eighty-one ticks were collected from 6 addax (Addax nasomaculatus), 7 red foxes (V. vulpes), 5 wild boars (Sus scrofa), and 3 golden jackals (Canis aureus); and from 4 roe deer (Capreolus capreolus) (descendant of European animals that were reintroduced into Israel in the 1980s and 1990s).¹³

Tick collections were performed randomly from animals that had been immobilized for different reasons, live-trapped or found dead. Fallow deer were sampled at Hai Bar Carmel in the north of Israel, addax at Hai Bar Yotvata in the Arava Rift valley in the south of Israel, and the rest from different sites (Table 1).

Table 1.

Prevalence of spotted fever group Rickettsia DNA in ticks picked from different hosts^*

Tick species	Host	Site of collection	Number of PCR-positive ticks/number of ticks tested	Sequence identification (NCBI GenBank accession no.)
Rhipicephalus turanicus	Roe deer	Carmel Mountains (northern Israel)	22/41	R. massiliae (GQ856265, GQ856267)
	Red fox	Ein Chemed Even Yehuda (central Israel)	2/17
	Boar	Granot (northern Israel)	1/5
Hyalomma marginatum	Roe deer	Carmel Mountains (northern Israel)	10/14	R. aeschlimannii (GQ856266, GQ856268)
Hyalomma dentritum	Addax	Yotveta Hai-Bar (southern Israel)	2/15
Hyalomma sp.	Boar	Ein Yakov (northern Israel)	1/4
	Red fox	Kfar Shimon (central Israel)	1/2
Haemaphysalis adleri	Golden jackal	Wadi Ara (central Israel)	1/5	“Candidatus Rickettsia goldwasserii” HM136923-HM136926
Haemaphysalis parva	Golden jackal	Wadi Ara (central Israel)	1/8	“Candidatus Rickettsia goldwasserii” HM136927-HM136930
Rhipicephalus sanguineus	Roe deer	Carmel Mountains (northern Israel)	0/40	Not applicable
Rhipicephalus (Boophilus) kohlsi	Roe deer	Carmel Mountains (northern Israel)	0/14
Haemaphysalis adleri	Red fox	Beth-Shemesh (central Israel)	0/6
	Boar	Golan Heights (northern Israel)	0/2
Haemaphysalis parva	Red fox	Beth-Shemesh (central Israel)	0/2
	Boar	Golan Heights (northern Israel)	0/6

^*PCR = polymerase chain reaction.

Ticks were identified to species using standard taxonomic keys^9,11,14–16 and comprised 40 Rh. sanguineus, 63 Rh. turanicus, 14 Rhipicephalus (Boophilus) kohlsi Hoogstraal and Kaiser, 14 Hyalomma marginatum Koch, 15 Hyalomma dentritum, 13 Haemaphysalis adleri Feldman-Muhsam, and 16 Haemaphysalis parva Neumann (Table 1). The six specimens of Hyalomma ticks were identified only to the genus.

The ticks were kept in 70% ethanol. DNA was extracted using the QIAamp Minikit (QIAGEN Inc., Valencia, CA), according to the manufacturer's instructions.

Tick extracts were first tested for rickettsial DNA by nested polymerase chain reaction (PCR) to amplify a fragment of 17 kDa protein antigen gene followed by restriction fragment length polymorphism analysis as described previously.¹⁷ Species identification of SFG Rickettsia was done by sequencing of 70–602 nucleotide fragments of the outer membrane protein A (OmpA) and 17 kDa protein gene fragments as described previously.¹⁸ Multiple locus sequence analysis included amplification and sequencing fragments of gltA, ompA, ompB, and sca4 as described previously.^18–20 New sequences generated during this study were submitted to NCBI GenBank under the following accession nos.: R. aeschlimannii - GQ856266 and GQ856268, R. massiliae - GQ856265 and GQ856267, and uncultured SFGR - HM136923-HM136930. Phylogenetic analysis was conducted using MEGA4.²¹

Results

DNA was extracted from 181 ticks collected from 25 wild animals of five species: roe deer, addax, red foxes, golden jackals, and wild boars (Table 1). DNA of SFGR was detected in 41 ticks (22.7% prevalence for the entire study).

DNA of R. massilliae was detected in 25 of 63 Rh. turanicus ticks, collected from roe deer, foxes, and boars. Nucleotide sequences were identical among all ompA amplicons obtained from Rh. turanicus and had 99% nucleotide identity to the homologous ompA fragment of both R. massiliae Mtu5 and Bar29 and 100% similarity to Mtu1 strain.

No ompA sequence differences were observed in DNA from R. aeschlimannii detected in 14 Hyalomma ticks out of 35 collected from roe deer, addax, boars and foxes. DNA of a novel SFGR was found in two Haemaphysalis species ticks: H. adleri (in 1 of 5 ticks) and H. parva (in 1 of 8 ticks) from golden jackals (Table 1). Homologous rickettsial fragments of gltA, ompA, ompB, and sca4 amplified from these two different ticks were found to be identical. The 381 bp gltA fragment sequenced had 99% sequence similarity and at least 1 to 3 unique single-nucleotide polymorphisms (SNP) compared with gltA fragment of Rickettsia honei, Rickettsia africae, Rickettsia slovaca, and Rickettsia japonica. The 575 bp ompA fragment had < 96% sequence similarity to ompA of R. honei and R. slovaca, R. africae and Rickettsia sibirica mongolotimonae, corresponding to 18–21 SNP. The 1,765 bp sca4 fragment had < 97% sequence similarity to the homologous fragments of R. slovaca and other SFGR encompassing multiple SNPs and several unique insertion/deletion (INDEL). The 4,899 bp ompB fragment had ≤ 97% sequence similarity to the nearest rickettsial relative. Phylogenetic analysis of the four concatenated gene fragments, gltA-ompA-sca4-ompB indicated that the nucleotide sequences are those of a SFGR belonging to a novel phylogenetic lineage that appears to be most related to “Candidatus Rickettsia siciliensis” that was recently found in Rh. turanicus (Figure 1).²² “Candidatus Rickettsia siciliensis” and the SFGR from H. adleri and H. parva ticks share 99%, 95%, 98%, and 96% nucleotide sequence similarity in their homologous fragments of gltA (HM014438), ompA (HM014439), sca4 (HM014440), and ompB (HM014441), respectively.

Figure 1.

Phylogenetic position of “Candidatus Rickettsia goldwasserii.” Phylogenetic position was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.78594827 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches; only bootstrap values of ≥ 80 are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method as base substitutions per site. All positions containing gaps, missing data and primer sequences were eliminated from the dataset. Fragments of the four genes were concatenated (gltA-ompA-sca4-ompB), and a total of 2,028 positions were analyzed. Phylogenetic analyses were conducted with MEGA4.²¹

Rickettsial DNA was not detected in Rh. sanguineus and Rh. (B.) kohlsi collected on roe deer or H. adleri from boar and red fox (Table 1).

Discussion

We detected R. aeschlimannii in Hyalomma sp. ticks and R. massiliae in Rh. turanicus ticks collected from wild animals and captive bred wildlife in Israel (Table 1). We also characterized a previously unknown SFGR in H. adleri and H. parva from jackals.

Rickettsia aeschlimannii was first described from H. marginatum marginatum ticks from Morocco in 1997²³ and was subsequently detected in 2002 in a patient returning from Morocco.²⁴ Subsequently, the presence of R. aeschlimannii has been demonstrated in H. marginatum marginatum ticks with a distribution from Portugal and northern Spain to Kazakhstan and from Mediterranean countries to South Africa.^25,26 The prevalence of R. aeschlimannii in H. marginatum ticks tested ranges from 1.8% to 64% in different studies.^23,27–29 Rickettsia aeschlimannii has also been detected in Hyalomma aegyptum (L.) in Algeria,³⁰ Haemaphysalis inermis Birula in Spain,²⁷ Hyalomma marginatum rufipes Koch in Egypt, Ethiopia, and Chad,^28,31 Hyalomma anatolicum excavatum Koch from the Greek Island of Cephalonia,³² and Hyalomma dromedarii Koch and Hyalomma impeltatum Schulze and Schlottke from Egypt.³¹ Furthermore, five other human biting tick species including Haemaphysalis punctata Canestrini and Fanzago, Ixodes ricinus (L.), Rh. bursa, and Rh. sanguineus collected from Spanish patients were shown to contain DNA of R. aeschlimannii.³³ The association of Rhipicephalus complex ticks with R. aeschlimannii is probably not surprising, because it was also detected in Rhipicephalus apendiculatus Neumann ticks collected from a patient suffering from R. aeschlimannii infection.³⁴

Rickettsia massiliae has been detected in Rh. sanguineus, Rhipicephalus sulcatus Neumann, Rhipicephalus lunulatus Neumann, Rhipicephalus muhsamae Morel and Vassiliades, Rhipicephalus senegalensis Koch, Rh. bursa, and Rh. turanicus.^{23,28,35–37} Rickettsia massiliae has been detected in Rhipicephalus ticks in Europe, Africa, and in South and North America.^37–39 There are several confirmed clinical cases caused by R. massiliae reported in the peer-reviewed literature.^37–39 Furthermore, it is believed that R. massiliae is responsible for cases of SFG rickettsioses resistant to rifampin in Catalonia, Spain,³⁹ which corresponds closely to the observation that R. massiliae is naturally resistant to this antibiotic.⁴⁰

The SFGR detected in H. adleri and H. parva has unique genetic characteristics that meet the minimum current requirement for identification as a new species based on the proposed molecular similarity criteria: ≤ 99.9%, ≤ 98.8%, ≤ 99.2%, and ≤ 99.3% for the gltA, ompA and ompB, and sca4, respectively, to its closest SFGR relative.^27,41 We cannot propose a formal species description because only two specimens from different tick species were analyzed and a rickettsial isolate was not established to complete its characterization. However, we can assign Candidatus status to this yet uncultivated SFGR Rickettsia and name it “Candidatus Rickettsia goldwasserii” in recognition of Dr. Robert A. Goldwasser for his work on rickettsiae and rickettsial diseases in Israel and his important contributions to the development of indirect fluorescent antibody assays for rickettsiae.^42,43 Further detailed study will be necessary to establish the prevalence and distribution of this SFGR in Haemaphysalis ticks from Israel, its primary vector and reservoir, and its ability to cause human and animal rickettsioses.

In Israel, ixodid ticks, which have been found on humans include 13 various species of Hyalomma, the sheep tick, Rh. bursa and H. sulcata as documented by Feldman-Muhsam.⁸ Cwilich and Hadani found H. excavatum, H. detritum, H. marginatum, Rh. sanguineus, Rh. turanicus, and Rh. bursa on humans.⁷ To the best of our knowledge, there is no published document showing that H. parva and H. adleri infest humans.

Our results provide evidence for the presence of R. aeschlimannii and confirm the evidence for the presence of R. massiliae in Israel.⁶ Further surveillance will be needed to characterize tick and animal reservoir for each SFGR reported.

Serological diagnosis of spotted fever infections cannot distinguish between those caused by R. massiliae, R. aeschlimannii, and R. conorii or potentially other SFGR because of the strong cross-reaction among the spotted fever group rickettsiae. Definitive diagnosis of the specific etiological SFGR agent causing rickettsioses in Israel requires molecular techniques conducted on whole blood and skin biopsy samples collected during the acute stage of illness and before antibiotic administration.