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Volume 7, Number 6—December 2001

Research

Detection and Identification of Spotted Fever Group Rickettsiae and Ehrlichiae in African Ticks

Philippe Parola*, Hisashi Inokuma*, Jean-Louis Camicas†, Philippe Brouqui*, and Didier Raoult*Comments to Author 
Author affiliations: *Université de la Méditerranée, CNRS UMR 6020, Marseille, France;; †Centre IRD, Montpellier, France

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Table

Detection and identification of spotted fever group rickettsiae and ehrlichiae from African ticks by polymerase chain reaction (PCR)a,b

Tick species Animal Location No. pos. ticks/ total examined Gene sequence Identification GenBank accession no. for new genotypes
Amblyomma variegatum Cattle Mali 6/6 (rickettsiae) ompA Rickettsia africae (2/6) -
RAv1 (2/6) genotype AF311959
RAv3 (2/6) genotype AF311960
gltA R. africae (2/6) -
RAv1 (2/6) genotype AF311962
RAv3 (2/6) genotype AF311963
Niger 6/6 (rickettsiae) ompA R. africae (2/6) -
Rav9 (1/6) genotype AF311961
RAv3 (3/6) genotype AF311960
gltA R. africae (2/6) -
RAv9 (1/6) genotype AF311964
RAv3 (3/6) genotype AF311963
Burundi 1/13 (rickettsiae) ompA + gltA R. africae -
A. lepidum Cattle Sudan 1/16 (rickettsiae) ompA + gltA R. africae -
Hyalomma impeltatum Cattle Niger 0/42 - - -
Mauritania 0/42 - - -
H. dromedarii Cattle Niger 0/7 - - -
H. impressum Cattle Niger 0/8 - - -
H. marginatum rufipes Cattle Niger 8/24 (rickettsiae) ompA + gltA R. aeschlimmanii -
Mali 3/20 (rickettsiae) ompA + gltA R. aeschlimmanii -
H. truncatum Cattle Niger 1/5 (rickettsiae ompA + gltA R. mongolotimonae -
1/5 (ehrlichiae) 16S rRNA Eht224 genotype AF311968
gene Eht224 genotype AF311966
Mali 0/5 gltA - -
Sudan 0/5 ompA + gltA - -
ompA + gltA
Rhipicephalus muhsamae Cattle Mali 2/37 (rickettsiae) ompA + gltA R. massiliae- -
7/37 (ehrlichae) 16S RNA gene Erm58 genotype AF311967
gltA Erm58 genotype AF311965
R. evertsi evertsi Cattle Sudan 0/10 - - -
R. sanguineus Dogs Mali 0/24 - - -
Sudan 0/62 - - -

a A convenience sample of ticks was obtained as part of other, ongoing studies, as summarized above. In October 1997, 42 Hyalomma impeltatum were collected from cattle at Kiffa (16°37'N, 11°24'O) in Mauritania. In Mali in February 1998, 6 Amblyomma variegatum, 37 R. muhsamae, 20 H. marginatum rufipes, and 5 H. truncatum were collected from cattle in Bamako (12°39' N, 8°00'W) and Bougouni (11°25' N, 7°29' W) and 24 R. sanguineus from dogs in Bamako. In 1999, 6 A. variegatum, 42 H. impeltatum, 7 H. dromedarii, 8 H. impressum, 24 H. marginatum rufipes, and 5 H. truncatum were collected from cattle at Niamey (13°30' N, 2°07' E) in the Republic of Niger; 5 H. truncatum, 10 Rh. evertsi evertsi, and 16 A. lepidum were collected from cattle and 62 Rh. sanguineus were collected from dogs in Khartoum (15°31' N, 32°47' E) in the Sudan; 13 A. variegatum were collected from cattle in Bujumbura (3°22' S, 29°21' E) in Burundi. All ticks were adults attached on mammals.
bPrimers include Rr190.70p and Rr190.701n, which amplify a fragment of 629-632 bp of ompA encoding for a 190-kD protein (7, and RpCS.877p-RpCS.1273r, which amplify a 396-bp fragment of the citrate synthase gene, gltA (7). Ehrlichial DNA was detected with EHR16SR-EHR16SD primers, which amplify a 345-bp fragment of the 16S rRNA gene of all the known ehrlichiae (8). To amplify the main part of the 16S rRNA gene, tick DNA samples that were found to be positive with the above primers were amplified with the EHR16SR and EHR16SD primers and the universal primers fD1 and rp2 (7). The positive DNA samples were also used in PCR reactions to amplify the citrate synthase gene, gltA, of ehrlichiae. A sest of primers, EHR-CS133F (5'-GGW-TTY-ATG-TCY-ACT-GCT-GC-3') and EHR-CS778R (5'-GCN-CCM-CCA-TGM-GCT-GG-3'), which amplify a fragment of about 650 bp of the citrate synthase gene of tick-borne ehrlichiae, were used for the screening PCR. Two other primer sets, Chaff-M4F (5'-AAT-TAT-GRT-YAA-ARA-RGC-AG-3')/EHR-CS778R and F1b(5'-GAT-CAT-GAR-CAR-AAT-GCT-TC-3')/Chaff1233R (5'-ACC-AGT-ATA-YAA-YTG-ACG-3') were used to amplify the main part of the citrate synthase gene sequence in tick DNA samples that were found to be positive from the screening PCR (Inokuma, et al., unpub data).
The sequences of the newly rickettsial genotypes found in this study have been deposited in GenBank under the following accession numbers (ompA and gltA, respectively): RAv1, AF311959 and AF311962; RAv3, AF311960 and AF311963; RAv9, AF311961 and AF311964. The sequences of the newly ehrlichical genotypes found in this study have been deposited in GenBank under the following accession numbers (16S rRNA gene and gltA, respectively): Erm58, AF311967 and AF311965; Eht224, AF311968, and AF311966.

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