Detection and identification of spotted fever group Rickettsiae and Ehrlichiae in African ticks. (Research).

Rickettsia africae, a recently identified pathogen, was detected for the first time in Amblyomma ticks from Niger, Mali, Burundi, and Sudan, and iR. mongolotimonaei was identified for the first time in Africa. Rickettsiae of unknown pathogenicity and two new Ehrlichiae of the Ehrlichia canis group were identified in ticks from Mali and Niger.

***********

Spotted fever group Rickettsiae and Ehrlichiae are obligate intracellular gram-negative bacteria associated with arthropods, mainly ticks. While feeding, ticks can transmit these microorganisms to humans and animals (1). Two human tick-borne rickettsioses are known to occur in Africa (2). Mediterranean spotted fever, caused by Rickettsia conorii, is transmitted by the brown dog tick, Rhipicephalus sanguineus, which is well adapted to urban environments. R. conorii is prevalent in the Mediterranean area (Tunisia, Algeria, Morocco, Libya, and Egypt) and has also been isolated or detected in Kenya, Central Africa, Zimbabwe, and South Africa (2). Although African tick bite fever has been recognized since the beginning of the century as a rural disease usually contracted from ticks of cattle and game, it was regarded as synonymous with Mediterranean spotted fever, until the first human infection with R. africae was reported from Zimbabwe in 1992. Subsequently, numerous cases have been reported in tourists returning from southern Africa, where the cattle tick Amblyomma hebraeum is the vector (2,3). R. africae has also been recovered from A. variegatum ticks in Ethiopia and central Africa (2). In 1992, a survey for antibodies against Ehrlichia chaffeensis (the agent of human monocytic ehrlichiosis) in human sera from eight African countries indicated that human ehrlichioses might occur on the continent (4), and subsequently a case (diagnosed by serology only) was reported from Mali (5). Recently, new molecular methods have enabled the development of useful, sensitive, and rapid tools to detect and identify tick-borne pathogens in arthropods, including ticks (6). In this work, we tested ticks from Africa for rickettsial and ehrlichial DNA using polymerase chain reaction (PCR) and sequence analysis of amplified products.

Materials and Methods

Ticks were kept frozen at -20 [degrees] C (in Niger) or at -80 [degrees] C (in other countries) before being tested. DNA of each tick was extracted as described (7). Rickettsial and ehrlichial DNA was detected by PCR as described, using specific primers (Table).

The sequences of PCR products were obtained and analyzed with the corresponding sequences of rickettsial or ehrlichial species as described (7). Multiple alignment analysis was performed by using the ClustalW program version 1.8 in the DNA Data Bank of Japan (DDBJ; Mishima, Japan [http:/ /www.ddbj.nig.ac.jp/htmls/E-mail/clustalw-e.html]). All sequences used in the study are available in GenBank; the accession numbers of the new genotypes detected in this work are shown in the Table footnotes.

Results

Rickettsial DNA was detected in 24 (7.2%) of the 332 ticks examined (Table). R. africae was detected from A. variegatum from Mali (2/6), Niger (2/6), and Burundi (1/13), and from 1 of 16 A. lepidum from the Sudan. R. aeschlimmanii was detected in Hyalomma marginatum rufipes from Niger and Mali (8/24 and 3/20, respectively) and R. massiliae in 2/ 37 Rh. muhsamae from Mali. Further, three new ompA sequences (590 bp) were obtained from A. variegatum from Mali and Niger (Table). These were 99.3%-99.5% identical to those of R. africae. In the phylogenetic tree based on these ompA sequences, the three rickettsiae (named Rav1, RAv3, RAv9) were closely related to one another (95.7% bootstrap value) and branched with R. africae (86.1% bootstrap value) (data not shown). Partial sequences (316 bp) of the gltA gene of Rav1, RAv3, and RAv9 were also found to be closely related to those of R. africae (99% of similarity). Two new 16S rRNA ehrlichial genotypes were detected, including ERm58 (1,380 bp) in 7/37 Rh. muhsamae from Mali and EHt224 (1366 bp) in 1/5 H. truncatum from Niger. Both sequences were very similar (99.34% similarity), but different from those described for all the known ehrlichiae (i.e., 98.55% similarity with E. chaffeensis, 98.26% with E. canis and E. ewingii, and 97.75% with E. muris and Cowdria ruminantium). In a phylogenetic tree based on 16S rRNA gene sequences, ERm58 and EHt224 were found to be closely related and to belong to the E. canis group (data not shown). Enlarged gltA sequences of ERm58 (1,140 bp) and EHt224 (1,189 bp) were also obtained from the above ticks. Phylogenetic analyses of these sequences confirmed that ERm58 and EHt224 belonged to the E. canis group (data not shown).

Conclusion

This study has shown for the first time that R. africae, the agent of African tick bite fever, is present in West Africa (Mali and Niger), the Sudan, and Burundi. It also indicates a potential role for A. variegatum and A. lepidum as vectors of R. africae in these areas. Recently, we also documented cases in tourists returning from numerous countries, including those in West and East Africa (9) (Figure). Our results support the hypothesis that the geographic distribution of African tick bite fever parallels that of the distribution of Amblyomma spp., as ticks are known to be vectors and also reservoirs of tick-borne rickettsiae (2). In Africa, although the principal vector of R. africae appeared to be A. hebraeum, which is prevalent in southern Africa, A. variegatum (which is widely distributed throughout sub-Saharan Africa) appears as a potential vector. Amblyomma are known to readily feed on people in Africa and are commonly infected with rickettsiae (up to 100%). Thus, African tick bite fever may have a high prevalence throughout the continent. Studies have shown sero-prevalences of 30%-80% for spotted fever group rickettsiae in sub-Saharan Africa (2), although it is unclear what proportion of those infections might be due to R. africae infection.

[FIGURE OMITTED]

In this study, we report for the first time the presence of "R. mongolotimonae" in Africa (Niger). This pathogen was first isolated from an H. asiaticum collected in Inner Mongolia, China. Later, the same agent was isolated from the blood and skin of a febrile woman from Marseille in 1996 (2), which demonstrated its pathogenicity for humans. Subsequently, we have recognized four more cases in southern France (10, and unpub, data). Results of this study suggest that R. mongolotimonae may be associated with Hyalomma sp. ticks throughout the world. In this work, we also detected two rickettsiae of unknown pathogenicity, namely R. aeschlimannii and R. massiliae. Although this is the first recognition of these rickettsiae in Mali and Niger, the epidemiologic importance of this finding has yet to be determined. Finally, we detected three new spotted fever group genotypes closely related to R. africae. Until further studies clarify the position of these organisms, we suggest they may be considered variant strains of R. africae.

Although previous reports, based on the results of sero-surveys, have indicated that human ehrlichioses occur in Africa, firm evidence is still absent. Because of the serologic cross-reactivity between ehrlichiae, serosurvey results have to be interpreted carefully. We detected two new ehrlichial genotypes, that is, Erin58 in Rh. muhsamae from Mali and Eht224 in H. truncatum from Niger. Both belong to the E. canis group, which includes E. chaffeensis, C. ruminantium, E. muris, E. ewingii, and a new isolate detected in Japanese ticks (11). In this group, as within each group of ehrlichiae, members share homologous surface antigens and thus cross-react extensively in serologic assays (12). Erm58 and Eht224 may also be organisms responsible for such serologic cross-reactions, including in serosurveys and case reports of human ehrlichioses in Africa. In 1997, new ehrlichial genotypes were also detected in Namibia and Zimbabwe, and a number of ehrlichiae in Africa may be responsible for serologic cross-reactions in serosurveys of humans and animals for currently recognized pathogenic ehrlichiae (13,14). The pathogenicity of the Erm58 and Eht224 recognized in our study has yet to be determined, and further studies to characterize the human ehrlichioses in Africa are indicated. Moreover, it remains to be demonstrated whether H. truncaturn and Rh. muhsamae ticks act as vectors or reservoirs of the new ehrlichiae, since ticks also could have been infected while feeding on bacteremic mammals.

Although this study detected for the first time certain richettsiae and ehrlichiae in African countries, systematic sampling was not done, and results cannot address their prevalence and distribution. However, this work provides a starting point for epidemiologic studies there.

Table. Detection and identification of spotted fever group
Rickettsiae and Ehrlichiae from African ticks by polymerase chain
reaction (PCR) (a,b)

                                           No. pos. ticks/total
Tick species         Animal    Location          examined

Amblyomma            Cattle      Mali       6/6 (rickettsiae)
variegatum                      Niger       6/6 (rickettsiae)
                               Burundi      1/13 (rickettsiae)
A. lepidum           Cattle     Sudan       1/16 (rickettsiae)
Hyalomma             Cattle     Niger              0/42
impeltatum                    Mauritania           0/42
H. dromedarii        Cattle     Niger              0/7
H. impressum         Cattle     Niger              0/8
H. marginatum        Cattle     Niger       8/24 (rickettsiae)
rufipes                          Mali       3/20 (rickettsiae)
H. truncatum         Cattle     Niger        1/5 (rickettsiae
                                             1/5 (ehrlichiae)
                                 Mali              0/5
                                Sudan              0/5
Rhipicephalus        Cattle      Mali       2/37 (rickettsiae)
muhsamae                                     7/37 (ehrlichae)
R. evertsi evertsi   Cattle     Sudan              0/10
R. sanguineus        Dogs        Mali              0/24
                                Sudan              0/62

                         Gene
Tick species           sequence       Identification

Amblyomma                ompA       Rickettsia africae (2/6)
variegatum                            RAv1 (2/6) genotype
                                      RAv3 (2/6) genotype
                         gltA           R. africae (2/6)
                                      RAv1 (2/6) genotype
                                      RAv3 (2/6) genotype
                         ompA           R. africae (2/6)
                                      Rav9 (1/6) genotype
                                      RAv3 (3/6) genotype
                         gltA           R. africae (2/6)
                                      RAv9 (1/6) genotype
                                      RAv3 (3/6) genotype
                     ompA + gltA        R. africae
A. lepidum           ompA + gltA        R. africae
Hyalomma                  --                  --
impeltatum                --                  --
H. dromedarii             --                  --
H. impressum              --                  --
H. marginatum        ompA + gltA      R. aeschlimannii
rufipes              ompA + gltA      R. aeschlimannii
H. truncatum         ompA + gltA      R. mongolotimonae
                       16S rRNA       Eht224 genotype
                         gene         Eht224 genotype
                         gltA                 --
                     ompA + gltA              --
                     ompA + gltA
Rhipicephalus        ompA + gltA        R. massiliae
muhsamae               16S RNA        Erm58 genotype
                         gene
                         gltA         Erm58 genotype
R. evertsi evertsi        --                  --
R. sanguineus             --                  --
                          --                  --
                              GenBank accession
Tick species                     no. for new
                                  genotypes
Amblyomma
variegatum                           --
                                  AF311959
                                  AF311960
                                     --
                                  AF311962
                                  AF311963
                                     --
                                  AF311961
                                  AF311960
                                     --
                                  AF311964
                                  AF311963
A. lepidum                           --
Hyalomma                             --
impeltatum                           --
H. dromedarii                        --
H. impressum                         --
H. marginatum                        --
rufipes                              --
H. truncatum                         --
                                     --
                                   AF311968
                                   AF311966
                                     --
                                     --
Rhipicephalus
muhsamae                             --
                                   AF311967
R. evertsi evertsi                 AF311965
R. sanguineus                        --
                                     --
                                     --

(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 [degrees] 37'N,
11 [degrees] 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 [degrees] 39' N,
8 [degrees] 00'W) and Bougouni (11 [degrees] 25' N, 7 [degrees] 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 [degrees] 30' N, 2 [degrees] 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 [degrees] 31' N, 32 [degrees] 47' E) in the Sudan; 13 A. variegatum
were collected from cattle in Bujumbura (3 [degrees] 22' S, 29 [degrees]
21' E) in Burundi. All ticks were adults attached on mammals.

(b) Primers 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).

Acknowledgments

We thank Djibo Garba, Issa Baradji, Salif Diagana, Jean-Bosco Ndihokubwayo, Bernard Davoust, Michael Cino, and Patricia Amargier for their help in collecting the ticks. We are grateful to Patrick Kelly for reviewing the manuscript.

This study was funded partly by French Ministry of Research and Technology ("Programme de recherche fondamentale en microbiologie et maladies infectieuses et parasitaires 2000"). During part of the field work, Dr. Parola was supported by Universite de la Mediterranee.

Dr. Philippe Parola obtained both MD and PhD degrees at the Faculty of Medecine of Marseille, France, and is now a postdoctoral fellow at the Harvard School of Public Health, Boston, Massachusetts. His research interests include medical entomology and tick-borne diseases.

References

(1.) Parola P, Raoult D. Ticks and tick-borne bacterial human diseases, an emerging infectious threat. [published erratum appears in Clin Infect Dis 2001;33:749]. Clin Infect Dis 2001;32:897-8.

(2.) Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 1997;10:694-719.

(3.) Fournier PE, Beytout J, Raoult D. Related tick-transmitted infections in Transvaal: consider Rickettsia africae. Emerg Infect Dis 1999;5:178-81.

(4.) Brouqui P, Le Cam C, Kelly PJ, Laurens R, Tounkara A, Sawadogo S, et al. Serologic evidence for human ehrlichiosis in Africa. Eur J Epidemiol 1994:10:695-8.

(5.) Uhaa IJ, MacLean JD, Greene CR, Fishbein DB. A case of human ehrlichiosis acquired in Mali: clinical and laboratory findings. Am J Trop Med Hyg 1992;46:161-4.

(6.) Sparagano OA, Allsopp MT, Mank RA, Rijpkema SG, Figueroa JV, Jongejan F. Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp Appl Acarol 1999;23:929-60.

(7.) Rydkina E, Roux V, Fetisova N, Rudakov N, Gafarova M, Tarasevich I, et al. New Rickettsiae in ticks collected in territories of the former Soviet Union. Emerg Infect Dis 1999;5:811-4.

(8.) Parola P, Roux V, Camicas JL, Brouqui P, Raoult D. Detection of ehrlichiae in African ticks by polymerase chain reaction. Trans R Soc Trop Med Hyg 2000;94:707-8.

(9.) Raoult D, Fournier PE, Fenollar F, Jensenius M, Prioe T, de Pina JJ, et al. Rickettsia africae, a tick-borne pathogen in travelers to sub-Saharan Africa. N Engl J Med 2001;344:1504-10.

(10.) Fournier PE, Tissot-Dupont H, Gallais H, Raoult D. Rickettsia mongolotimonae: a rare pathogen in France. Emerg Infect Dis 2000;6:290-2.

(11.) Inokuma H, Parola P, Raoult D, Brouqui P. Molecular survey of Ehrlichia infection in ticks from animals in Yamagushi prefecture, Japan. Vet Parasitol 2001;99:335-9.

(12.) Rikihisa Y. Ehrlichiae of veterinary importance. In: Raoult D, Brouqui P, editors. Rickettsiae and rickettsial diseases at the turn of the third millennium. Paris: Elsevier; 1999. p. 393-405.

(13.) Allsopp M, Visser ES, du Plessis JL, Vogel SW, Allsopp BA. Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences. Vet Parasitol 1997;71:283-300.

(14.) Savadye DT, Kelly PJ, Mahan SM. Evidence to show that an agent that cross-reacts serologically with Cowdria ruminantium in Zimbabwe is transmitted by ticks. Exp Appl Acarol 1998;22:111-22.

Philippe Parola, * Hisashi Inokuma, * Jean-Louis Camicas, ([dagger]) Philippe Brouqui, * and Didier Raoult *

* Unite des Rickettsies, CNRS UMR 6020, Marseille, France; and ([dagger]) Centre IRD, Montpellier, France

Address for correspondence: Didier Raoult, Unite des Rickettsies, Faculte de Medecine, Universite de la Mediterranee, CNRS UMR 6020, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France; fax: [phone]; e-mail: [e-mail].