Spotted fever group rickettsiae in ixodid ticks in Oromia, Ethiopia

Ticks and Tick-borne Diseases 6 (2015) 8–15

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Original article

Spotted fever group rickettsiae in ixodid ticks in Oromia, Ethiopia Bersissa Kumsa, Cristina Socolovschi, Didier Raoult, Philippe Parola ∗ Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, 13005 Marseille, France

a r t i c l e

i n f o

Article history: Received 16 May 2014 Received in revised form 19 August 2014 Accepted 22 August 2014 Available online 26 September 2014 Keywords: Ixodid ticks Domestic animals Spotted fever rickettsiae Oromia, Ethiopia

a b s t r a c t In Ethiopia, information on the transmission of human zoonotic pathogens through ixodid ticks remains scarce. To address the occurrence and molecular identity of spotted fever group rickettsiae using molecular tools, a total of 767 ixodid ticks belonging to thirteen different species were collected from domestic animals from September 2011 to March 2014. Rickettsia africae DNA was detected in 30.2% (16/53) Amblyommma variegatum, 28.6% (12/42) Am. gemma, 0.8% (1/119) Am. cohaerens, 18.2% (4/22) Amblyomma larvae, 6.7% (2/60) Amblyomma nymphs, 0.7% (1/139) Rhipicephalus (Boophilus) decoloratus and 25% (1/4) nymphs of Rh. (Bo.) decoloratus. A markedly low prevalence of R. africae was recorded in both Am. cohaerens and Rh. (Bo.) decoloratus (p < 0.0001) compared with that in Am. variegatum and Am. gemma. The prevalence of R. africae was markedly low in the western districts (Gachi and Abdela) (p < 0.0001); however, the prevalence of R. africae was relatively high in the central (Ada’a, Wolmara and Arsi) and eastern (Arero, Moyale and Yabelo) districts, where Am. variegatum and Am. gemma were predominantly associated with R. africae, respectively. R. aeschlimannii DNA was detected in 45.4% (5/11) Hyalomma marginatum rufipes and 2.2% (1/46) Hy. truncatum. Moreover, the first report of R. massiliae DNA in 1.9% (1/52) Rhipicephalus praetextatus ticks in Ethiopia is presented herein. Altogether, these results suggest that the transmission of spotted fever group rickettsiae through ixodid ticks is a potential risk for human health in different parts of Ethiopia. Clinicians in this country should consider these pathogens as a potential cause of febrile illness in patients. © 2014 Elsevier GmbH. All rights reserved.

Introduction Ticks are obligate hematophagous arthropods that act as reservoirs and vectors for a wide range of human and animal pathogens worldwide. Approximately 10% of the currently recognized tick species carry human and animal pathogens. Currently, ticks and mosquitoes are the major vectors for human and animal disease agents (Jongejan and Uilenberg, 2004; Parola and Raoult, 2001). Recent studies have indicated an increase in the spectrum of tickborne pathogens affecting humans and animals (Dantas-Torres et al., 2012; de la Fuente and Estrada-Pena, 2012; Nicholson et al., 2010). Ticks are common and widely distributed throughout Ethiopia (Mekonnen et al., 2001). Several species of ixodid ticks have been identified in Ethiopia and are considered to have more veterinary significance than medical importance (Kumsa et al., 2012). Most of

∗ Corresponding author at: URMITE, UMR CNRS 7278, IRD 198, INSERM U1095, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France. Tel.: +33 04 91 32 43 75; fax: +33 04 91 38 77 72. E-mail address: [email protected] (P. Parola). http://dx.doi.org/10.1016/j.ttbdis.2014.08.001 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

the studies on the role of ixodid ticks as vectors of human pathogens in Ethiopia were conducted before the discovery of well-advanced molecular tools during the 1950s and 60s (Burgdorfer et al., 1973; Philip et al., 1966). Indeed, the isolation of Rickettsia prowazekii, the causative agent of epidemic typhus, from Amblyomma ticks feeding on Ethiopian cattle remains a mystery and had never been confirmed in any other studies conducted in Ethiopia (Burgdorfer et al., 1973). Spotted fever group (SFG) rickettsiae are small, obligate intracellular, short rod, Gram-negative bacteria belonging to the genus Rickettsia, the family Rickettsiaceae and the order Rickettsiales (Parola et al., 2013). The genus Rickettsia comprises 3 main biogroups: the ‘spotted fever group’ (SFG), primarily transmitted through ixodid ticks, except R. felis and R. akari, which are vectored through fleas and mites, respectively; the ‘typhus group’ (TG), transmitted through fleas and lice; and the ‘scrub typhus group’ (STG), primarily vectored through chiggers (Parola, 2011; Parola et al., 2013). Currently, the genus Rickettsia comprises 31 species that cause diseases in vertebrate hosts, including humans, domestic animals, birds, and wildlife. Some ticks have also been implicated as SFG rickettsiae reservoirs, as these insects maintain rickettsiae both transstadially and transovarially.

B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

In humans, spotted fever rickettsia induces symptoms that generally include fever, headache, myalgia, rash, local lymphadenopathy, and an eschar at the site of the tick bite, which might be useful in diagnosis (Parola et al., 2013). Rickettsia africae, the causative agent of ATBF, typically causes symptoms, including inoculation eschars, fever, regional lymphadenopathies and the frequent lack of cutaneous rash or pale vesicular eruptions, and the distribution of this pathogen is consistent with the geographical distribution of Amblyomma ticks (Parola et al., 2013). Rickettsia aeschlimannii is an emerging pathogen recently detected in human patients in Morocco, South Africa, Algeria, Tunisia and Greece, causing clinical symptoms resembling Mediterranean spotted fever caused by R. conorii (fever, generalized maculopapular rashes and an escar at the tick-bite site) (Parola et al., 2013). Rickettsia massiliae is a pathogenic SFG rickettsia associated with clinical symptoms, such as fever, eschar, night sweats, headache, maculopapular rash and necrotic skin lesions, in human patients in America, Europe and Africa (Parola et al., 2013). Previous studies on rickettsiae in Ethiopian ixodid ticks have documented the presence of Rickettsia spp. in Amblyomma variegatum, Amblyomma cohaerens and Rhipicephalus spp. in central Ethiopia (Philip et al., 1966) and Amblyomma spp. (Amblyomma gemma, Am. variegatum and Am. cohaerens) in the central and eastern regions of Ethiopia (Burgdorfer et al., 1973). R. aeschlimannii, the agent of SFG rickettsiosis, had been detected in Hyalomma marginatum rufipes and R. africae, the agent of African tick bite fever (ATBF), has been detected in Amblyomma lepidum and Am. variegatum ticks from eastern Ethiopia (Mura et al., 2008), and also R. africae had been detected in pools of Amblyomma and Rhipicephalus ticks (Pader et al., 2012) (summarized in Fig. 1). In Ethiopia, where the environment is suitable for ticks, 84% of the population is involved in agriculture and farmers and their family members interact with ectoparasite-infested animals on daily life activities. Moreover, the confirmatory diagnosis of fever and other diseases with unknown etiologies is not commonly practiced, reflecting the lack of health centers in many regions of the country. Therefore, information concerning ectoparasite-borne bacteria is extremely important. To update the knowledge on tick-borne rickettsiae in Ethiopia, the aim of the present study was to address the occurrence and molecular identity of Rickettsia species in ixodid ticks collected from domestic animals in nine districts in Oromia Regional State in Ethiopia.

9

Collection and identification of ticks Ticks were collected from September through November of 2011. A thorough visual examination of the body surfaces of each study animal was conducted to establish the presence or absence of ticks. All observed ticks attached to the skin of each animal were carefully removed using forceps or by hand to avoid any damage to the body, and the specimen were individually placed into small, pre-labeled plastic tubes containing 70% ethanol for subsequent identification as previously described (Kumsa et al., 2012, 2014a). All ticks from the same animal were placed into the same vial and transported to the Laboratory of the World Health Organization Collaborative Center for Rickettsial Diseases and Arthropod-borne Bacterial Diseases in Marseille, France. The morphological identification of ticks and molecular studies were performed from January 2012 through March 2014. All adult ticks were identified at the species level, and the larvae and nymphs were microscopically identified at the genus level using previously described morphological identification keys (Hoogstraal, 1956; Walker et al., 2000, 2003). The sex and stage of each tick was determined, and photographs of the dorsal and ventral body parts of each tick specimen were captured. The tick genera are abbreviated as previously described (Dantas-Torres, 2008). DNA extraction from ticks

Materials and methods

Prior to DNA extraction, each tick specimen was rinsed twice in sterile water for 15 min, dried on sterile filter paper, and longitudinally dissected into two equal halves. One half of each specimen was retained as reserve sample to avoid the risk of losing samples for any reason during or after DNA extraction (Kumsa et al., 2014a, 2014b). Genomic DNA was individually extracted from a total of 767 tick specimens using the QIAamp DNA tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In engorged tick DNA was extracted from small portion of the anterior part of one half of the ticks so as to minimize the PCR inhibitory effect of large amount of blood in the abdomen. The DNA from each tick specimen was eluted in 100 ␮l of Tris–EDTA (TE) buffer and stored at −20 ◦ C under sterile conditions to preclude contamination until the sample was used for PCR. To avoid crosscontamination among the samples during DNA extraction, the DNA extracting EZI Advanced XL Robot (Qiagen, Hilden, Germany) was thoroughly disinfected after each extraction according to the manufacturer’s recommendations. The second half of each tick specimen was stored at −80 ◦ C as a backup sample.

Study areas and animals

Molecular detection of Rickettsia species

In this study, tick samples were collected from cattle, sheep, dogs and cats in the following districts in Oromia Regional State in Ethiopia: Arsi (7◦ 56 2.36 N, 39◦ 39 6.54 E), Wolmara (9◦ 6 17.87 N, 38◦ 28 8.50 E), Kimbibit (9◦ 24 39.95 N, 39◦ 21 14.75 E), Ada’a (9◦ 31 60.00 N, 38◦ 18 0.00 E), Bedele (8◦ 27 1.76 N, 36◦ 21 5.08 E), Gachi (9◦ 14 2.57 N, 35◦ 54 48.46 E), Arero (4◦ 43 33.28 N, 38◦ 45 46.78 E), Moyale (3◦ 31 60.00 N, 39◦ 3 0.00 E) and Yabelo (4◦ 53 41.91 N, 38◦ 6 0.59 E). The districts are located in six zones in the central, southwestern and southeast regions of the country, with various climates and agroecology. The livestock in the study areas are traditionally managed under extensive production systems (CSA, 2008). All the study animals were selected irrespective of sex and age. The animals were categorized into two age groups, young (up to one year) and adult (older than one year), according to a previous publication (Kumsa et al., 2012).

As a first step, all DNA samples were individually tested using a genus-specific qPCR system targeting the gltA gene (RKND03 system (Rolain et al., 2002): RKND03F-5 -GTG-AATGAA-AGA-TTA-CAC-TAT-TTA-T-3 , RKND03R- 5 -GTA-TCT-TAGCAA-TCA-TTC-TAA-TAG-C-3 and RKND03P- 6-FAM-CTA-TTATGC-TTG-CGG-CTG-TCG-GTT-C-TAMRA) in SFG rickettsiae and the Rpr274P gene in typhus group rickettsiae as previously described (Mediannikov et al., 2010a; Socolovschi et al., 2012). Sterile water was used as a negative control, and DNA from R. montanensis and R. typhi were used as positive controls for SFG and typhus group Rickettsia, respectively. All DNA samples positive for the gltA gene (RKND03 system) were further confirmed using species-specific genes for SFG Rickettsia mentioned below. The Amblyomma spp. (n = 37) and Rhipicephalus (Boophilus) decoloratus (n = 2) DNA samples positive for SFG Rickettsia were further tested using a previously described R. africae species-specific qPCR targeting the ITS gene (Mediannikov et al., 2012a). Sterile

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B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

Fig. 1. Map of Ethiopia showing the areas in which SFG rickettsiae has previously been reported.

water was used as a negative control, and DNA from R. africae was used as positive control. Hyalomma spp. DNA samples (n = 6) positive for SFG Rickettsia were further tested using a previously described species-specific qPCR targeting the Sca1 gene (a cell-surface antigen) of R. aeschlimannii with the same primers and probes used before (Socolovschi et al., 2012). Sterile water was used as a negative control, and DNA from R. aeschlimannii was used as positive control. Rhipicephalus sp. DNA sample (n = 1) positive for SFG Rickettsia was further tested using a previously described species-specific qPCR targeting hypothetical proteins of R. massiliae with the same primers and probes used before (Socolovschi et al., 2012). Sterile water was used as a negative control, and DNA from R. massiliae was used as positive control. Ethical statement Ethical approval for the collection of ticks from domestic animals was obtained from the animal research ethics board (Agreement # 14/160/550/2011) of the College of Veterinary Medicine and Agriculture at Addis Ababa University. All necessary oral permits were obtained for the field studies, including permission from each animal owner and the administration and agricultural office of each district. The collection of ticks from animals was not harmful or contrary to the welfare of the animals. Data analysis Microsoft Excel was used for data management. Data were stratified by districts for analysis and then descriptive statistics such as percentages and means, were employed to estimate and compare

the prevalence of Rickettsia DNA among different tick species in different districts. Statistical analysis was performed using EpiInfoTM 7 (CDC, 2008). The Mantel–Haenszel (MH) test in EpiInfoTM 7 was used to determine the relationships between the prevalence of Rickettsia DNA in different tick species and genera and districts of collection. A p-value of <0.05 was considered significant.

Results Ticks were collected from a total of 244 animals (206 cattle, 29 sheep, seven dogs and two cats) in nine districts: Abdela (37 cattle and fifteen sheep), Gachi (29 cattle and three sheep), Ada’a (nineteen cattle, seven dogs and two cats), Wolmara (seventeen cattle and one sheep), Kimbibit (23 cattle and ten sheep), Arsi (31 cattle), Arero (23 cattle), Moyale (eighteen cattle) and Yabelo (nine cattle). Overall, 329 male ticks, 314 female (260 non-engorged and 54 engorged) ticks, 98 nymphs and 26 larvae of ticks were collected. Greater proportion of the ticks was collected from cattle 89.9% (689/767) while lower proportion was collected from sheep 6.4% (49/767), from dogs 3% (23/767) and from cats 0.8% (6/767). From each infested animal from one up to ten ticks were collected. Further information on tick collection is presented on Table 1. A total of 767 ixodid ticks comprising thirteen different species (Table 2) were screened for both spotted fever and typhus group rickettsiae DNA using molecular methods. The overall prevalence of SFG rickettsiae was 6% (46/767) in all ticks collected from nine Ethiopian districts. In the study 6.5% (45/689) of the ticks from cattle and 16.7% (1/6) tick from one cat were positive for spotted fever group rickettsiae, however; spotted fever group rickettsiae was not detected in ticks taken from sheep (0/49) and dogs (0/23).

B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

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Table 1 Summary of species of ticks collected from different domestic animal in 9 districts in Oromia, Ethiopia. Tick spp.

District

Animal spp. (number)

No. of ticks collected (m = male, f = female)

No. of each tick spp. collected from one animal

No. of engorged ticks

Amblyomma cohaerens (n = 119)

Abdela

Cattle (19) Sheep (8) Cattle (24) Sheep (1) Cattle (2) Cattle (9) Cattle (2) Cattle (1) Cattle (1) Cattle (8) Cattle (11) Cattle (5) Cattle (1) Cattle (1) Cattle (3) Sheep (2) Cattle (9) Cattle (6) Cattle (19) Cattle (2) Cattle (1) Cattle (8) Sheep (2) Cattle (5) Cattle (4) Cattle (2) Cattle (1) Cattle (13) Sheep (12) Cattle (11) Sheep (2) Cattle (6) Cat (1) Cattle (1) Cattle (10) Cattle (1) Cattle (1) Cattle (1) Cattle (14) Cattle (12) Sheep (1) Cattle (8) Cattle (15) Cattle (15) Sheep (1) Cattle (24) Cattle (10) Cattle (3) Cattle (1) Cattle (2) Cattle (2) Cattle (3) Cattle (6) Cattle (4) Cattle (11) Cattle (6) Cattle (1) Cattle (7) Cattle (16) Sheep (9) Cattle (6) Cattle (1) Cattle (3) Sheep (1) Cattle (5) Cattle (2) Sheep (1) Cattle (4) Cattle (6) Cattle (1) Cattle (1)

39 (23m, 16f) 10 (6m, 4f) 49 (28m, 21f) 2 (1m, 1f) 2 (2m) 12 (8m, 4f) 3 (2m, 1f) 2 (1m, 1f) 1 (1m) 13 (7m, 6f) 20 (10m, 10f) 8 (4m, 4f) 1 (1f) 1 (1f) 3 (3m) 2 (2m) 13 (10m, 3f) 7 (6m, 1f) 25 (20m, 5f) 2 (2m) 1 (1m) 8 2 5 4 2 1 13 13 11 2 6 1 1 10 1 1 1 14 (14f) 14 (2m, 12f) 1 (1f) 11 (3m, 8f) 31 (16m, 15f) 20 (7m, 13f) 3 (3f) 31 (6m, 25f) 10 (10f) 3 (3f) 1 (1f) 2 2 3 6 4 11 6 1 9 (4m, 5f) 23 (12m, 11f)) 12 (5m, 7f) 8 (5m, 3f) 1(1m) 3 (2m, 1f) 1 (1f) 8 (5m, 3f) 4 (2m, 2f) 1 (1f) 7 (4m, 3f) 10 (6m, 4f) 1 (1f) 1 (1f)

1–3 1–2 1–3 2 1 1–2 1–2 2 1 1–2 1–2 1–2 1 1 1 1 1–2 1–2 1–2 1 1 1 1 1 1 1 1 1 1–2 1 1 1 1 1 1 1 1 1 1 1–2 1 1–2 1–3 1–2 3 1–2 1 1 1 1 1 1 1 1 1 1 1 1–2 1–3 1–2 1–2 1 1 1 1–2 2 1 1–2 1–2 1 1

4 female

Gachi

Amblyomma variegatum (n = 119)

Arsi Arero Moyale Yabelo Ada’a Arero Moyale Yabelo Arero Moyale Gachi

Amblyomma spp. larva (n = 22)

Ada’a Wolmara Arsi Arero Yabelo Abdela

Amblyomma spp. nymph (n = 60)

Gachi Ada’a Wolmara Yabelo Abdela

Amblyomma gemma (n = 28)

Amblyomma lepidum (n = 2)

Gachi Ada’a

Rhpicephalus (Boophilus) decoloratus (n = 139)

Wolmara Arsi Arero Moyale Yabelo Abdela Gachi Ada’a Wolmara Kimbibit

Rhipicephalus (Boophilus)decoloratus larva (n = 4) Rhipicephalus(Boophilus)decoloratus nymph (n = 31)

Rhipicephalus praetextatus (n = 52)

Rhipicephalus eversti evetsi (n = 37)

Arsi Arero Moyale Yabelo Ada’a Kimbibit Abdela Gachi Ada’a Wolmara Kimbibit Arero Ada’a Kimbibit Arsi Abdela Gachi Ada’a Wolmara Kimbibit Arsi Arero Moyale

4 female

1 female

2 female 2 female 1 female

1 female 2 female

3 female 1 female

2 female 1 female 2 female 8 female

1 female

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B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

Table 1 (Continued) Tick spp.

District

Animal spp. (number)

No. of ticks collected (m = male, f = female)

No. of each tick spp. collected from one animal

Rhipicephalus pulchullus (n = 118)

Ada’a

Rhipicephalus sanguienus (n = 14) Rhipicephalus spp. nymph (n = 9)

Arero Moyale Yabelo Ada’a Ada’a

Cattle (2) Dog (1) Cattle (22) Cattle (18) Cattle (8) Dog (7) Cattle (1) Dog (3) Cattle (2) Cattle (2) Cattle (1) Cattle (1) Cattle (1) Cattle (2) Cattle (4) Cattle (13) Cattle (3) Cattle (1) Cattle (10) Dog (1) Cat (2) Dog (1) 244 animals

2 (1m, 1f) 2 (1m, 1f)) 52 (30m, 22f) 38 (19m, 19f) 24 (14m, 10f) 14 (9m, 5f) 1 4 2 2 1 (1m) 2 (1m, 1f)) 1 (1m) 2 (2f)) 5 (4m, 1f)) 23 (13m, 10f) 6 (3m, 3f) 2 (1m, 1f) 15 (11m, 4f) 1 (1m) 5 (1m, 4f) 2 (2f) 329m, 314f, 26 larvae, 98 nymphs

1 2 1–4 1–4 1–5 1–6 1 1–2 1 1 1 2 1 1 1–2 1–2 2 2 1–2 1 1–4 2 1–10 ticks

Haemaphysalis leachi (n = 6)

Arero Moyale Ada’a Wolmara Kimbibit Arero Moyale Ada’a Wolmara Kimbibit Arsi Ada’a

Haemaphysalis spinulosa (n = 2) 767 ticks Total

Ada’a 9 districts

Hyalomma marignatum rufipes (n = 11)

Hyalomma truncatum (n = 46)

R. africae DNA was detected in 30.2% (16/53) Am. variegatum, 28.6% (12/42) Am. gemma, 0.8% (1/119) Am. cohaerens, 18.2% (4/22) Amblyomma larvae, 6.7% (4/60) Amblyomma nymphs, 0.7% (1/139) Rh. (Bo.) decoloratus and 25% (1/4) nymphs of Rh. (Bo.) decoloratus (Table 2). The overall prevalence of R. africae was significantly higher in both Am. variegatum and Am. gemma than in the other tick species (Am. cohaerens and Rh (Bo.) decoloratus) (Mantel–Haenszel (MH), p < 0.0001). In the southwestern districts (Gachi and Abdela), the overall prevalence of R. africae was markedly low compared with the central and southeastern districts (MH, p < 0.0001). In the central districts (Ada’a, Wolmara and Arsi), Am. variegatum was predominantly associated with R. africae, whereas Am. gemma was predominantly associated with this species in the southeastern districts (Arero, Moyale and Yabelo) (Fig. 2). This study is the first to report R. africae in Am. gemma, Rh (Bo). decoloratus, in larval and nymphal ticks in Ethiopia. Statistically significant variation was not found (MH, p < 0.07) in the prevalence of R. africae between 3/54 (5.5%) engorged and 4/260 (1.5%) non engorged female ticks. R. aeschlimannii DNA was detected in 45.4% (5/11) Hy. m. rufipes and 2.2% (1/46) Hyalomma truncatum (Table 2). The prevalence of R. aeschlimannii was significantly higher in Hy. m. rufipes than in Hy. truncatum ticks (MH, p = 0.001). R. massiliae DNA was detected in 1.9% (1/52) Rhipicephalus praetextatus ticks (Table 2). Spotted fever group rickettsia DNA was not detected in Haemaphysalis (Hae. leachi (n = 6) and Hae. spinulosa (n = 2) ticks collected from domestic animals. Similarly, SFG rickettsiae were not detected in Am. lepidum, Rhipicephalus evertsi evertsi, Rhipicephalus pulchellus and Rhipicephalus sanguineus ticks (Table 2). We did not detect any typhus group rickettsia DNA in the 767 ixodid ticks collected from domestic animals in Oromia. Discussion The overall prevalence of pathogenic SFG rickettsiae was detected in 6% (46/767) of ixodid ticks belonging to four genera (Amblyomma, Rhipicephalus and Hyalomma) collected from animals in nine districts, highlighting the importance of hard ticks for human health in Ethiopia. Our molecular strategy was based on

No. of engorged ticks

4 female 6 female 3 female

1 female

2 female 54 females

recent scientific studies, and the results were confirmed through the amplification of two different target genes for each positive result using positive and negative controls as previously described (Mediannikov et al., 2012a; Socolovschi et al., 2012). The absence of spotted fever group rickettsiae in ticks collected from sheep and dogs most probably reflect the very lower percentage of sheep 11.9% (29/244) and dogs 2.9% (7/244) as compared to the very higher percentage of ticks from cattle as well as the greater number of cattle 84.4%(206/244) studied. Future studies with representative number of domestic animals and their ticks are necessary to address the comparative importance of animal species and their ticks in Ethiopia. The detection of R. africae, the agent of African tick bite fever, in Am. variegatum confirms the results of a previous report associating R. africae with this Ethiopian tick species (Mura et al., 2008). Similarly, this spotted fever group rickettsia, erroneously considered as R. conorii, the agent of Mediterranean spotted fever, was reported in Am. variegatum ticks from Ethiopia approximately 41 years ago (Burgdorfer et al., 1973; Philip et al., 1966). This erroneous consideration of spotted fever group rickettsia as R. conorii, vectored by Rhipicephalus spp., at that time in Ethiopia was due to the fact that new techniques such as species sensitive and specific serology, shell vial assay and molecular methods that enabled the isolation, identification and characterization of most of these bacteria including R. africae, vectored principally by Amblyomma spp., were developed only during the last 20 years after the 1990 (Raoult et al., 2001). In consistent with our observation R. africae has been detected in Am. variegatum ticks collected from cattle in several African countries including in (7/26) in Kenya (Mutai et al., 2013), in (22/39) Uganda and 22/141 Nigeria (Lorusso et al., 2013), in 4–8% in Nigeria (Reye et al., 2012), in Senegal (Mediannikov et al., 2010b), in 11/12 in Sudan (Morita et al., 2004) and in 6/6 Mali, 6/6 Niger and 1/13 Burundi (Parola et al., 2001). Similarly, in support of our finding R. africae was detected in A. gemma ticks collected from cattle in Kenya (Mutai et al., 2013). Results of our study suggest that A. gemma is potentially an important vector for R. africae in addition to the already known Am. variegatum in Ethiopia. The identification of R. africae in Am. gemma and Rh. (Bo.) decoloratus in the present study expands the current knowledge concerning tick species that

Table 2 Prevalence of SFG rickettsiae in ixodid ticks using species-specific gene qPCR in 9 districts in Oromia, Ethiopia. Zones

IluAba Bora

District

Abdela

Gachi

West Showa

North Showa

Arsi

Borana

Ada’a

Wolmara

Kimbibit

Arsi

Arero

Moyale

Yabelo

Total

– 1/1 (100%) – 4/13 (30.8%) 2/4 (50%) 1/7 (14.3%) 8/25 (32%) 0/11 1/2 (50%) 0/4 1/17 (3.2%) 9/42 (26.4%)

– – – 5/7 (71.4%) ½ (50%) 0/1 6/10 (60%) 0/31 – 0/11 0/42 6/52 (11.5%)

– – – – – – – 0/23 0/2 0/6 0/31 0/31

0/2 – – 7/25 (28%) – 1/10 (10%) 8/37 (21.6%) 1/31 (3.2%) – – 1/31 (3.2%) 9/68 (13.2%)

0/12 6/13 (46.1%) 0/1 0/2 – 0/1 6/29 (20.7%) 0/10 – 0/1 0/11 6/40 (15%)

0/3 4/20 (20%) 0/1 – – 0/1 4/25 (16%) 0/3 – – 0/3 4/28 (14.3%)

0/2 (0%) 1/8 (12.5%) – 0/1 0/1 1/1 (100%) 2/13(15.4%) 0/1 – – 0/1 2/14 (14.3%)

1/119 (0.8%) 12/42 (28.6%) 0/2 16/53 (30.2%) 4/22 (18.2.%) 4/60 (6.7%) 37/298 (12.4%) 1/139 (0.7%) ¼ (25%) 0/31 2/174 (1.1%) 39/472 (8.3%)

1/9 (11.1%) 0/8 0/4 0/14 0/5 1/40 (2.5%)

– 0/5 – – – 0/5

0/35 0/7 – – – 0/42

0/8 0/10 – – – 0/18

– 0/1 0/52 – 0/2 0/55

– 0/1 0/38 – 0/2 0/41

– – 0/24 – – 0/24

1/52 (1.9%) 0/37 0/118 0/14 0/9 1/230 (0.4%)

1/1 (100%) 0/23 1/24 (4.2%)

0/2 0/6 0/8

1/1 (100%) 0/2 1/3 (33.3%)

– 1/15 (6.7%) 1/15 (6.7%)

½ (50%) – ½ (50%)

2/5 (40%) – 2/5 (40%)

– – –

5/11 (45.4%) 1/46 (2.2%) 6/57 (10.5%)

0/6 0/2 0/8 9.6% (11/114)

– – – 9.2% (6/65)

– – – 1.3% (1/76)

– – – 9.9% (10/101)

– – – 7.2% (7/97)

– – – 8.1% (6/74)

– – – 5.3% (2/38)

0/6 0/2 0/8 6% (46/767)

B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

1. Prevalence of R. africae in Amblyomma spp. and Rh (Bo). decoloratus Am. cohaerens 0/49 1/51 (2%) – – Am. gemma – – Am. lepdium – 0/5 Am. variegatum 0/10 1/5 (20%) Amblyomma larva 0/26 1/13 (7.7%) Amblyomma nymph 0/85 3/74 (4%) Overall Amblyomma spp. 0/14 0/15 Rh (Bo). decoloratus – – Rh (Bo). decoloratus larva 0/3 0/6 Rh (Bo). decoloratus nymph 0/17 0/21 Overall Rh (Bo). decoloratus spp. 0/102 3/95 (3.1%) Overall tick spp. 2. Prevalence of R. massiliae in Rhipicephalus spp. – – Rh. praetextaatus 0/1 0/4 Rh. e.evertsi – – Rh. pulchellus Rh. sanguineus – – Rhipicephalus spp. nymph – – Overall Rhipicephalus spp. 0/1 0/4 3. Prevalence of R. aeschlimannii in Hyalomma spp. Hy. m. rufipes – – – – Hy. truncatum – – Overall Hyalomma spp. 4. Prevalence of SFG rickettsia in Haemaphysalis spp. – – Hae. leachi – – Hae. spinulosa Overall Haemaphysalis spp. Overall prevalence of SFG rickettsiae in ticks 0/103 3% (3/99)

East Showa

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B. Kumsa et al. / Ticks and Tick-borne Diseases 6 (2015) 8–15

Fig. 2. Map of Ethiopia showing the geographical distribution of SFG rickettsiae and their host ticks in the present study.

host R. africae in Ethiopia. The absence of significant variations in the prevalence of R. africae between engorged and none engorged female ticks most probably reflects the avoiding of lager portion of abdomen of engorged ticks containing large blood. We observed a strong geographic correlation between the high prevalence of R. africae (in Am. variegatum in the central districts and Am. gemma in the southeastern districts) and the distribution of different Amblyomma species across Oromia, consistent with the previously established geographical distribution of Am. variegatum as the predominant Amblyomma spp. in central Ethiopia (Kumsa et al., 2012; Mekonnen et al., 2001) and Am. gemma as the predominant Amblyomma spp. in the southeastern and eastern regions of Oromia, Ethiopia (Regassa, 2001). Am. cohaerens is the predominant tick species in western Ethiopia, where the climate is humid and wet for most of the year, whereas Am. variegatum is the predominant tick species in areas with high rainfall in central and other regions of Ethiopia (Mekonnen et al., 2001), and Am. gemma is restricted to the semi-arid regions east of Rift Valley in Ethiopia (Regassa, 2001). The high prevalence of R. africae in Am. gemma has also been reported in Kenya (Mutai et al., 2013). The markedly low prevalence of 1.9% (3/159) R. africae in western Oromia (Gachi and Abdela districts), where Am. cohaerens is the predominant Amblyomma spp. (Kumsa et al., 2012), compared with that in the central (Ada’a, Wolmara and Arsi) (21.6%, 22/102) and southeastern (Arero, Moyale and Yabelo) (17.9%, 12/67) districts is consistent with previous reports of low levels of SFG rickettsiae in Am. cohaerens compared with Am. variegatum and Am. gemma ticks in Ethiopia (Burgdorfer et al., 1973; Philip et al., 1966). In contrast, the absence of R. africae DNA in 109 Am. cohaerens has been recently reported in southwestern Ethiopia (Mediannikov et al., 2013). This inconsistency likely reflects differences in the number of Amblyomma ticks tested and the geographical locations examined in the two studies. In the present study, we examined a total

of 298 Amblyomma ticks comprising four different species in nine districts in Oromia; however, in the previous study (Mediannikov et al., 2013), only 109 Amblyomma ticks representing Am. cohaerens in a single district in southwest Ethiopia were studied. The low prevalence of R. africae in 1.1% Rh. (Bo.) decoloratus compared with the prevalence of this species in Amblyomma ticks (12.4%) is consistent with the argument that the rate of R. africae infection in Rhipicephalus ticks is typically low, reflecting concomitant co-feeding with Amlyomma ticks (Parola et al., 2013). In addition, Rh. (Bo.) decoloratus is infected only when cohabiting with Amblyomma ticks, regarded as the primary reservoir of R. africae (Macaluso et al., 2003; Mediannikov et al., 2013). R. africae has also been reported in Rh (Bo.) decoloratus ticks in Liberia (Mediannikov et al., 2012b) and Nigeria (Ogo et al., 2012). The prevalence of R. aeschlimannii in 45.4% of Hy. m. rufipes ticks confirms a previous report in eastern Ethiopia (Mura et al., 2008) and previous studies in other countries, reporting a prevalence of 44.8% in Senegal (Mediannikov et al., 2010a), 60% in Corsica (Matsumoto et al., 2004), 33.3% in Niger and 15% in Mali (Parola et al., 2001) and Camargue in southern France (Socolovschi et al., 2012). R. aeschlimannii has been detected in Hy. truncatum in Senegal (Mediannikov et al., 2010a), Sudan (Morita et al., 2004) and Kenya (Mutai et al., 2013). The significantly lower prevalence of R. aeschlimannii in Hy. truncatum suggests that Hy. m. rufipes is the principal vector of this bacterium, as previously suggested (Demoncheaux et al., 2012; Parola et al., 2013). Interestingly, this study is the first to report R. massiliae in 1.9% (1/52) Rh. praetextatus in Ethiopia. Consistent with this observation, R. massiliae has been previously detected in several Rhipicephalus spp. in other African countries, including 8.1% (2/37) in Rh. muhsamae in Mali (Parola et al., 2001), 8.2% (5/61) in Rh. senegalensis in Guinea (Mediannikov et al., 2012b) and in 22.4% (11/49) in Rh. guilhoni in Senegal (Mediannikov et al., 2010a). Rh. praetextatus is one of

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the predominant tick species observed on ruminants in the central, north and eastern regions of Ethiopia (Mekonnen et al., 2001). Although published reports are not available on human hard tick infestations in Ethiopia, Am. variegatum, Am. cohaerens and Rh. praetextatus have been reported to feed on humans (Dantas-Torres et al., 2012; Jongejan and Uilenberg, 2004; Parola and Raoult, 2001). The significance of the present study is further strengthened through recent reports of SFG rickettsiae in humans in Ethiopia, including R. africae in a French man who stayed for 2 months in western Ethiopia (Stephany et al., 2009) and 2.9% (3/102) SFG Rickettsia DNA in dried thin blood smears prepared to test for malaria in children with febrile illnesses at Soddo Christian Hospital in Wolaitta Soddo in southern Ethiopia (Aarsland et al., 2012). The absence of any typhus group rickettsiae in ixodid ticks in this study is consistent with previous studies that did not detect this bacterium in hard ticks in Ethiopia (Mediannikov et al., 2012a; Mura et al., 2008; Pader et al., 2012), suggesting that other hematophagous arthropods might play a role in the transmission of TG rickettsiae. In conclusion, the findings presented herein provide additional clear information on the geographic distribution and SFG rickettsia species detected in different ixodid ticks in various regions of Ethiopia. These results suggest that physicians managing patients with fever of unknown etiology in Ethiopia and those who care for travelers from Ethiopia should consider the presence of several SFG rickettsiae as potential causative agents. In addition, future studies are needed to address the isolation and culture of SFG rickettsiae from ticks collected from animals and vegetation (questing ticks), and in blood samples from human, domestic and other reservoir animals and determine the public health significance of this bacterium in Ethiopia. Conflict of interest The authors declare that no competing interests exist. Acknowledgments The authors would like to thank the domestic animal owners in the different districts of Oromia for allowing the collection of ticks from their animals. We would also like to thank the laboratory technicians from URMITE, Marseille, particularly Veronique Brice and Annick Bernard, for technical support. References Aarsland, S.J., Castellanos-Gonzalez, A., Lockamy, K.P., Mulu-Droppers, R., Mulu, M., White, A.C., Cabada, M.M., 2012. Treatable bacterial infections are underrecognized causes of fever in Ethiopian children. Am. J. Trop. Med. Hyg. 87, 128–133. Burgdorfer, W., Ormsbee, R.A., Schmidt, M.L., Hoogstraal, H., 1973. A search for the epidemic typhus agent in Ethiopian ticks. Bull. World Health Org. 48, 563–569. CDC, 2008. Centers for Disease Control and Prevention 1600 Clifton Rd. Atlanta, GA 30333, USA 800-CDC-INFO (800-232-4636) TTY: (888)., pp. 232–6348. CSA, 2008. Addis Ababa, Ethiopia, 2007/08. Dantas-Torres, F., 2008. Towards the standardization of the abbreviations of genus names of ticks (Acari: Parasitiformes: Ixodida). Vet. Parasitol. 154, 94–97. Dantas-Torres, F., Chomel, B.B., Otranto, D., 2012. Ticks and tick-borne diseases: a one health perspective. Trends Parasitol. 28, 437–446. de la Fuente, J., Estrada-Pena, A., 2012. Ticks and tick-borne pathogens on the rise. Ticks. Tick. Borne. Dis. 3, 115–116. Demoncheaux, J.P., Socolovschi, C., Davoust, B., Haddad, S., Raoult, D., Parola, P., 2012. First detection of Rickettsia aeschlimannii in Hyalomma dromedarii ticks from Tunisia. Ticks Tick Borne Dis. 3, 398–402. Hoogstraal, H., 1956. African Ixodidae. 1. Ticks of the Sudan (with special reference to equatorial province and with preliminary reviews of the genera Boophilus, Margaropus and Hyalomma). Department of the Navy, Bureau of the Medicine and Surgery, United States Government Printing Office, Washington, DC. Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology (Suppl. 129), S3–S14. Kumsa, B., Beyecha, K., Geloye, M., 2012. Ectoparasites of sheep in three agroecological zones in central Oromia, Ethiopia. Onderstepoort J. Vet. Res. 79, E1–E7.

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