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# First molecular detection of Rickettsia africanae in ticks from the Union of the Comoros

Amina Yssouf, Cristina Socolovschi, Tahar Kernif, Sarah Temmam, Erwan Lagadec, Pablo Tortosa, Philippe Parola

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Amina Yssouf, Cristina Socolovschi, Tahar Kernif, Sarah Temmam, Erwan Lagadec, et al. First molecular detection of Rickettsia africanae in ticks from the Union of the Comoros. Parasites & Vectors, 2014, 7 (1), pp.444. 10.1186/1756-3305-7-444. hal-01274564

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Distributed under a Creative Commons Attribution 4.0 International License
First molecular detection of *Rickettsia africana* in
ticks from the Union of the Comoros

Amina Yssouf¹, Cristina Socolovschi¹, Tahar Kernif¹, Sarah Temmam¹, Erwan Lagadec², Pablo Tortosa²,³ and Philippe Parola¹*

**Abstract**

**Background:** *Rickettsia africana* is the agent of African tick bite fever, a disease transmitted by ticks in sub-Saharan Africa. In Union of the Comoros, a recent study reported the presence of a *Rickettsia africana* vector but no information has been provided on the circulation of the pathogenic agent in this country.

**Methods:** To evaluate the possible circulation of *Rickettsia* spp. in Comorian cattle, genomic DNA was extracted from 512 ticks collected either in the Union of the Comoros or from animals imported from Tanzania and subsequently tested for *Rickettsia* infection by quantitative PCR.

**Results:** *Rickettsia africana* was detected in 90% (60/67) of *Amblyomma variegatum*, 1% (1/92) of *Rhipicephalus appendiculatus* and 2.7% (8/296) of *Rhipicephalus (Boophilus) microplus* ticks collected in the Union of the Comoros, as well as in 77.14% (27/35) of *Amblyomma variegatum* ticks collected from imported cattle. Partial sequences of both bacterial *gltA* and *ompA* genes were used in a phylogenetic analysis revealing the presence of several haplotypes, all included within the *Rickettsia africana* clade.

**Conclusions:** Our study reports the first evidence of *Rickettsia africana* in ticks collected from the Union of the Comoros. The data show a significant difference of infection rate of *Rickettsia africana* infected ticks between the Islands, with maximum rates measured in Grande Comore Island, sheltering the main entry port for live animal importation from Tanzania. The high infection levels reported herein indicate the need for an in-depth assessment of the burden of rickettsioses in the Union of the Comoros, especially among those at risk of infection, such as cattle herders.

**Keywords:** Cattle ticks, *Rickettsia africana*, *Amblyomma variegatum*, *Rhipicephalus*, Comoros

**Background**

Tick-borne rickettsioses are considered among the oldest known vector-borne zoonotic diseases; they are caused by obligate intracellular Gram-negative bacteria belonging to the spotted fever group (FSG) of the genus *Rickettsia* [1]. Many species of this genus are considered to be vertically transmitted symbionts of invertebrates, suggesting that the arthropod vectors act as reservoirs or amplifiers of rickettsiae in the wild [2-4]. In sub-Saharan Africa, several rickettsial strains have been isolated and detected from

ticks and vertebrate animals [5], among which *Rickettsia africana*, the etiological agent of African tick-bite fever (ATBF), is the most common [3,6,7]. The main tick-vectors of *Rickettsia africana* are *Amblyomma hebraeum* in southern Africa and *Amblyomma variegatum* in West, Central and Eastern Africa, as well as in the eastern Caribbean [7-11]. In the Indian Ocean, *Rickettsia africana* has been previously detected in *Amblyomma variegatum* in La Reunion and Madagascar [4,12] but never in arthropod and human samples from other islands of the region, including the Comorian Islands.

The Union of the Comoros is composed of three vol-
canic islands: Grande Comore (the youngest and most
elevated island), Anjouan and Moheli. The archipelago is
located in the western Indian Ocean, at the northern en-
trance of the Mozambique Channel between Madagascar

<footnote>* Correspondence: philippe.parola@univ-amu.fr

¹Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, WHO Collaborative Center for Rickettsioses and Other Arthropod-borne Bacterial Diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
and the East African coast, and is characterized by a
warm and humid tropical climate. Cattle are imported as
a food source, mainly from Madagascar. In 2000, a free
trade agreement was signed between Comoros and
Tanzania, facilitating the reciprocal travel and flow of
cattle. Consequently, the export of cattle from Tanzania
to Comoros, and particularly to Grande Comore Island,
has steadily increased [13]. The likelihood of an increase
in the number of pathogens introduced through cattle-
associated ticks has also risen.

Recently, an entomological survey carried out on all three
islands of the Union of the Comoros showed the presence
of *Amblyomma variegatum*, *Rhipicephalus appendiculatus*
and *Rhipicephalus microplus* on cattle [13]. Thus, the aim
of this study was to detect and determine the prevalence of
*Rickettsia* species that infect ticks on autochthonous cattle
and on cattle imported from Tanzania and to evaluate the
role of cattle importation in the introduction and of the hu-
man pathogen, *Rickettsia*, throughout the country.

**Methods**

Study sites and tick sampling

The present study used adult ticks that were previously
collected to describe cattle tick diversity and distribution
in the country. Briefly, the ticks were collected from ani-
mals on the three islands of the Union of the Comoros,
including 16 of the 17 districts of the country. Adult
ticks were collected on cows and goats from the three
islands during the 2010 rainy season. For each district,
between one and five animals were sampled and identi-
fied, and the number of collected ticks was recorded for
each site of collection. Following the same protocol,
ticks were also collected from cattle imported from
Tanzania that were held in the quarantine enclosure lo-
cated in the harbor vicinity, or in any of the three other
quarantine enclosures located in the capital, Moroni
(Grande Comore). All ticks were immediately stored in
70% ethanol until morphological and molecular analyses.
Tick species were determined morphologically using
standard identification keys [14].

DNA extraction and PCR detection of *Rickettsia*

Each tick was sliced longitudinally with a disposable
scalpel, and each half was crushed in a buffered solution
(G2) with proteinase K (Qiagen Hilden, Germany) and
incubated at 56°C overnight. Total DNA from half of
each tick was extracted in 50 µl of eluate using the EZ1
DNA Tissue kit (Qiagen, Hilden, Germany). Rickettsial
DNA detection was performed by quantitative PCR
using the Eurogentec MasterMix Probe PCR kit (Qiagen,
Hilden, Germany) following the manufacturer's instruc-
tions with a final volume of 10 µl in each reaction as
previously described [15]. Each DNA sample was tested
by quantitative (q) real-time PCR using a CFX 96 Real

Time System (BIO-RAD, Singapore). The presence of
SFG *Rickettsia* from was determined with a Taqman
probe (Eurogentec, Seraing, Germany) and RKND03F
and RKND03R specific primers targeting the citrate syn-
thase A (*gltA*) encoding gene [16,17]. Positive samples
were subsequently screened with a previously described
*Rickettsia africana*-specific qPCR method. Samples with
fewer than 35 cycle thresholds (Ct) were considered posi-
tive [18].

In order to generate sequence data allowing phylogen-
etic analyses of infecting *Rickettsia*, *gltA* and *ompA* were
amplified and subsequently sequenced from a subset of
randomly selected *Rickettsia*-positive tick samples. A frag-
ment of *gltA* gene was amplified using the Rp CS.409d and
CS.1258n primers, previously reported to amplify a 750-bp
fragment from all known *Rickettsia* species [6], and *ompA*
gene was targeted by using the primers 190.70,190.180,
and 190.701, amplifying a 629–632-bp fragment of SFG
*Rickettsia* [2,6].

For each PCR reaction, one positive control (*Rickettsia montanensis* DNA) and 2 negative controls (sterile water containing DNA extracted from uninfected ticks maintained in laboratory colonies) were included, with the exception of the *Rickettsia africana*-specific qPCR reaction in which *Rickettsia africana* DNA was used as positive control.

The resulting PCR products were purified and directly
sequenced using a BigDye Terminator Cycle Sequencing
Kit (Perkin Elmer Applied Biosystems, USA) and an ABI
PRISM automated sequencer (Applied Biosystems, USA),
as previously described [6].

Sequence editing and phylogenetic analyses

Sequences were analyzed using Chromas Pro (version
1.49 beta Technelysium Pty Ltd, Tewantin, Australia)
and compared with sequences available in the GenBank
database using NCBI BLAST. Multiple sequence align-
ments were noted at the nucleotide and amino acid
levels using ClustalW implemented in BioEdit software.
The sequences matched with sequences from other *gltA*
and *ompA* rickettsiae retrieved from the GenBank data-
base. *Rickettsia conorii* and *Rickettsia rickettsii* sequences
have been chosen as root sequences. Accession numbers
of the GenBank sequences used for the genetic analyses
are indicated in the phylogenetic trees. The selection of
the DNA substitution model that best fit the data was
performed with MEGA 5.2 and was considered for
phylogenetic analyses. We selected different models of
nucleotide substitution using the corrected Akaike in-
formation criterion. Bayesian phylogenetic inference (BI)
was carried out using Mr Bayes 3.1.2 [19] with two inde-
pendent runs of four incrementally heated, Metropolis
Coupled Markov chain Monte Carlo (MC) inquiries be-
ginning with a random tree. MC queries were run for
1,000,000 generations with trees and associated model parameters sampled every 200 generations. The initial 1000 trees in each run were discarded as burn-in samples, and the harmonic mean of the likelihood was calculated by combining each of the two independent runs.

## Statistical analysis

A statistical analysis was performed to compare the prevalence of *Rickettsia africanae* between the islands using the Mantel-Haenszel test implemented in Epi-info version 3.5 followed by the Yates correction option. Differences were considered statistically significant for P values <0.05.

## Results

### PCR detection of rickettsia

### Ticks collected on autochthonous cattle

Rickettsial DNA was detected in 14% (67/477) of ticks from the Union of the Comoros. Table 1 presents the distribution of samples positive for *Rickettsia* by tick species and location. The mean Ct ± SD value of *gltA* amplification by qPCR of positive tick samples was 28.34 ± 3.11. All positive samples were also positive for an *Rickettsia africanae*-specific qPCR with a mean Ct ± SD value of 28.51 ± 2.03. On Grande Comore Island, 90% (60/67) of *Amblyomma variegatum* ticks tested positive for *Rickettsia africanae*, with Ct values averaging 28.67 ± 1.95, while 1.9% (2/105) of *Rhipicephalus* spp. ticks were positive, with a Ct averaging 28.88 ± 1.8 (Figure 1). The prevalence of *Rickettsia africanae* on Anjouan Island was 8.12% (13/160), and a significant difference in prevalence was observed between Anjouan and Grande Comore 32.48% (51/157) (Mantel-Haenszel test, P value < 0.001). Among the positive samples from Anjouan, 61.5% (8/13) were obtained from *Amblyomma variegatum* ticks, while the remainders were from *Rhipicephalus microplus* (Table 1). In Mohéli, one of the five regions we visited had cattle carrying infected ticks (Figure 1). The prevalence of *Rickettsia africanae* on this island was 1.9% (3/160), which was also significantly different from that found on Grande Comore (Mantel-Haenszel test, P-value <0.001) and Anjouan (Mantel-Haenszel test, P-value = 0.016). All positive ticks from Mohéli were *Amblyomma variegatum* (Table 1), with positive PCRs displaying an average Ct of 25.24 ± 1.97.

The amplification, sequencing and BLAST analyses of *ompA* and *gltA* genes from positive DNA samples extracted from *Amblyomma variegatum*, *Rhipicephalus microplus* and *Rhipicephalus appendiculatus* ticks confirmed the presence of *Rickettsia africanae* in 14% of our samples. The sequence analysis of *ompA* genes obtained from *Rhipicephalus* spp. samples revealed 99.0 to 99.6% nucleotide identity with *Rickettsia africanae* detected from *Rhipicephalus evertsi evertsi* in Senegal (GenBank accession numbers JN043509), while *R. africanae* sequenced from *Amblyomma variegatum* showed 98 to 99.7% average identity with *R. africanae* detected in Ethiopia from the same tick species (GenBank accession numbers CP001612). The sequencing of *gltA* genes obtained from *Amblyomma variegatum* and *Rhipicephalus* spp. showed 99.1 to 99.42% average identity with the published sequences of *Rhipicephalus africanae* that were amplified from *Amblyomma variegatum* collected in the West Indies (GenBank accession number HM050288).

### Ticks collected from imported cattle

Of the ticks collected from cattle imported from Tanzania, 77.14% (27/35) were positive for *Rickettsia* spp., with a Ct average of 27.75 ± 4.13. All of the samples detected positive for *Rickettsia* spp. were also positive for *Rickettsia africanae* by qPCR (Figure 1), with Ct averages of 26.7 ± 4.67.

The sequencing of *ompA* genes obtained from *Amblyomma variegatum* collected from imported cattle showed an average of 98% alignment with published sequences of *Rickettsia africanae* from *Amblyomma variegatum* collected in Antigua (GenBank accession number EU622980).

The sequence analysis of the *gltA* gene obtained from *Amblyomma variegatum* ticks revealed 98.0 to 99.0% nucleotide identity with the *gltA* gene from *Rickettsia africanae* detected in *Amblyomma variegatum* from Ethiopia (GenBank accession numbers CP001612) and in *Rhipicephalus evertsi evertsi* from Senegal (GenBank accession numbers HM050288), respectively.

## Phylogenetic analysis

The best DNA substitution model fitting the data was determined to be HKY for both the *gltA* and *ompA* sequences. The Bayesian Inference tree based on *gltA* sequences

<table><caption>Table 1 Prevalence of *Rickettsia africanae* by species and regions</caption><thead><tr><th>Species<br>Regions</th><th>Amblyomma<br>variegatum</th><th>Rhipicephalus<br>microplus</th><th>Rhipicephalus<br>appendiculatus</th><th>Number of ticks<br>tested by region</th></tr></thead><tbody><tr><td>Grande Comore</td><td>94.23% (49/52)</td><td>8% (1/13)</td><td>1% (1/92)</td><td>157</td></tr><tr><td>Anjouan</td><td>89% (8/9)</td><td>3.3% (5/151)</td><td>-</td><td>160</td></tr><tr><td>Mohéli</td><td>7% (3/28)</td><td>0% (0/132)</td><td>-</td><td>160</td></tr><tr><td>Ticks collected from imported cattle</td><td>77.14% (27/35)</td><td>-</td><td>-</td><td>35</td></tr><tr><td>Total from the Union of the Comoros</td><td>65.17% (58/89)</td><td>2.7% (8/296)</td><td>1% (1/95)</td><td>477</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>Figure 1 Prevalence of infected ticks by island and regions.</figcaption></figure>

(Figure 2A) showed a distinct phylogenetic Comorian clade (posterior probability > 0.968) in which one well-supported group of sequences (posterior probability > 0.999) included sequences from *Rickettsia africanae* infecting ticks sampled in Grande Comore and Anjouan but not Mohéli, and a *Rickettsia africanae* sequence obtained from a tick imported from Tanzania. The Bayesian Inference tree based on *ompA* sequences (Figure 2B) showed a similar tree topology, which consolidate our analysis.

Consensus sequences of *Rickettsia africanae* infecting
ticks sampled on Grande Comore, Mohéli, Anjouan and
Tanzania were generated and aligned to determine nu-
cleotide mutations specific to a geographic origin. No
specific mutation was observed for the *ompA* genes (data
not shown); interestingly, the *gltA* sequences originating
from Mohéli have 5 specific non-synonymous mutations
(Table 2). The same analysis was conducted to construct
consensus sequences of *Rickettsia africanae* isolated from

<figure><img src="image_4.png" /><figcaption>**Figure 2** Phylogenetic tree inferred from the comparison of *Rickettsia africanae* strains from specimens tested and selected GenBank *Rickettsia africanae* sequences. Posterior probabilities are expressed in percentages and indicated at branch nodes. The sequences generated in this study are highlighted in bold. **A:** Phylogenetic tree based on the *gltA* citrate synthase-encoding gene. **B:** Phylogenetic tree based on the *ompA* outer membrane protein A gene. Abbreviation: *Rh* = *Rhipicephalus*; *Hy* = *Hyalomma*; *D* = *Dermacentor*.</figcaption></figure>
<table><caption>Table 2 Non-synonymous mutations in *Rickettsia africanae* *gltA* Mohéli sequences</caption><thead><tr><th>Nucleotide position (U59733)</th><th>560</th><th>566</th><th>635</th><th>692</th><th>1067</th></tr></thead><tbody><tr><td>Codon sequence</td><td>GAT → AAT</td><td>CCG → TCG</td><td>CCA → GCA</td><td>AAT → TAT</td><td>AGA → GGA</td></tr><tr><td>Amino-acid mutation in Mohéli *gltA* sequence</td><td>D → N</td><td>P → S</td><td>P → A</td><td>N → Y</td><td>R → G</td></tr></tbody></table>

distinct tick species, but no specific mutation was observed in either the *ompA* or *gltA* gene (data not shown).

## Discussion

*A. variegatum* is the main vector of *Rickettsia africanae*, a spotted fever group (SFG) *Rickettsia* bacterium in sub-Saharan Africa [9,20,21], though it is also considered a competent vector for other human and animal pathogens [22], including the highly virulent Crimean-Congo hemorrhagic fever virus [23]. In the present study, we show for the first time the presence of *Rickettsia africanae* in cattle ticks collected from the Union of the Comoros. The confirmation of the presence of this *Rickettsia* spp. provides background for further epidemiologic and clinical investigations of tick-borne diseases in the Union of the Comoros. Indeed, other than *Theileria parva*, a parasitic protozoan that is the causative agent of the East Coast fever in cattle and that was previously detected in *Rhipicephalus appendiculatus* [24], no other tick-borne pathogens have been detected in this country.

We provide evidence for *Rickettsia africanae* infection in *Amblyomma variegatum* ticks from all three islands of the Union of the Comoros. Although the *Amblyomma variegatum* tick infection rate varied among the islands as observed in Grande Comore and Anjouan, further study needs to confirm this result. The presence of *Rickettsia africanae*-infected ticks in Grande Comore and Anjouan is congruent with the geographic distribution of *Amblyomma variegatum* [8]. The high prevalence of *Rickettsia africanae* (90%) in *Amblyomma variegatum* collected in all study sites of Grande Comore showed endemicity of this bacterium and that this tick species is the reservoir of *R. africanae* in the Archipelago. Ticks of the genus *Amblyomma* are considered to be the main vectors for *Rickettsia africanae* although this bacterium has recently been found infecting other genera, including *Rhipicephalus* [21,22,25]. In this study, we also found *Rhipicephalus* spp. ticks carrying *Rickettsia africanae* DNA although the presence rate was substantially lower (2%) than the rate measured in *Amblyomma variegatum* (65.17%). However, our data do not provide direct evidence for the vector competence of the *Rhipicephalus* (*Boophilus* microplus genus for *Rickettsia africanae* because *Rhipicephalus*-positive ticks were always collected on animals infested with *A. variegatum* ticks that tested positive for *R. africanae*. Thus, we can hypothesize that *Rhipicephalus* ticks that were positive for *Rickettsia africanae* acquired the pathogen during a blood meal, but this does not prove that these species of ticks are competent vectors

for *Rickettsia africanae*. Further experimental evaluations of vector competence clearly need to be carried out in order to establish the vector competence of *Rhipicephalus* ticks.

Ticks sampled on cattle imported from Tanzania showed a prevalence of 77.14% for *Rickettsia africanae*, which is coherent with serological studies previously carried out in Tanzania that also showed high seroprevalence levels [26]. *Rickettsia africanae* polymorphism did not show any island or tick host species structuration. Comorian haplotypes were closely related with African haplotypes, thus strengthening the hypothesis of an African origin for *Rickettsia africanae* in the Comoros archipelago. Phylogenetic trees could not determine the origin of *Rickettsia africanae* infection in the ticks imported from Tanzanian cattle. However, the polymorphism of *ompA* and *gltA* is notionarily weak and sequencing of additional markers may help in resolving this issue. Citrate synthase is a component of nearly all living cells and is one enzyme of the citric acid cycle, a key metabolic pathway that plays a key role in energy production [27]. The *OmpA* gene plays a role in a protective immune response and is considered as a good candidate for phylogenetic analysis for most of the SFG *Rickettsiae* [2]. These gene sequences showed no clear differences between east African and Comorian bacterial haplotypes, thus supporting an African/Comorian *Rickettsia africanae* metapopulation, although the use of additional markers, or the full sequencing of bacterial isolates would obviously provide more robust information.

## Conclusion

The PCR assays and sequence analyses provide new information on the epidemiology of ticks infected with *Rickettsia africanae* in the Union of the Comoros. The detection of *Rickettsia africanae* in ticks collected from live cattle imported from Tanzania represents a risk to local farms. Our results strengthen the need for an evaluation of the burden of ATBF in the human populations in contact with cattle. Together with the previous investigation reporting the introduction of a tick species of veterinary importance new to the country, the present study clearly shows that because of the insularity, ticks and parasites introduction into the country represents a risk of biological invasion. Therefore, increased vigilance is required to limit this risk [28].

## Competing interests

The authors declare that they have no competing interests.
## Authors' contributions

AY performed the experiments and drafted the manuscript with the input from all co-authors, CS provided constant intellectual support in the course of the experiments, TK participated in the experiments. ST performed the phylogenetic analysis, EL provided the biological material. PT conceived the study. PP conceived the study and gave approval of the final version. All authors read and approved the final version of the manuscript.

## Acknowledgements

We wish to thank Frederic Stachurski for his contribution in the achievement of the prevalence distribution map of *Rickettsia africana* in the Union of the Comoros.

## Author details

¹Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, Inserm 1095, WHO Collaborative Center for Rickettsioses and Other Arthropod-borne Bacterial Diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385 Marseille cedex 5, France. ²Centre de Recherche et de Veille sur les Maladies Emergentes dans l'Océan Indien (CRVOI)-Plateforme de Recherche CYROI- 2, rue Maxime Rivière, 97490 Ste Clotilde, La Reunion, France. ³Université de La Réunion, Ste Clotilde, La Réunion, France.

Received: 4 March 2014 Accepted: 9 September 2014

Published: 22 September 2014

## References

1. Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D: Update on tick-borne rickettsioses around the world: a geographic approach. *Clin Microbiol Rev* 2013, 26:657-702.

2. Fournier PE, Roux V, Raoult D: Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. *Int J Syst Bacteriol* 1998, 48(Pt 3):839-849.

3. Parola P, Vestris G, Martinez D, Brochier B, Roux V, Raoult D: Tick-borne rickettiosis in Guadeloupe, the French West Indies: isolation of *Rickettsia africana* from *Amblyomma variegatum* ticks and serosurvey in humans, cattle, and goats. *Am J Trop Med Hyg* 1999, 60:888-893.

4. Parola P, Barre N: *Rickettsia africana*, the agent of African tick-bite fever: an emerging pathogen in the West Indies and reunion island (Indian ocean). *Bull Soc Pathol Exot* 2004, 97:193-198.

5. Parola P: Rickettsioses in sub-Saharan Africa. *Ann N Y Acad Sci* 2006, 1078:42-47.

6. Mura A, Socolovschi C, Ginesta J, Lafrance B, Magnan S, Rolain JM, Davoust B, Raoult D, Parola P: Molecular detection of spotted fever group rickettsiae in ticks from Ethiopia and Chad. *Trans R Soc Trop Med Hyg* 2008, 102:945-949.

7. Ndip LM, Fokam EB, Bouyer DH, Ndip RN, Titanji VP, Walker DH, McBride JW: Detection of *Rickettsia africana* in patients and ticks along the coastal region of Cameroon. *Am J Trop Med Hyg* 2004, 71:363-366.

8. Eldin C, Mediannikov O, Davoust B, Cabre O, Barre N, Raoult D, Parola P: Emergence of *Rickettsia africana*, Oceania. *Emerg Infect Dis* 2011, 17:100-102.

9. Kelly PJ: *Rickettsia africana* in the West Indies. *Emerg Infect Dis* 2006, 12:224-226.

10. Jensenius M, Fournier PE, Kelly P, Myrvang B, Raoult D: African tick bite fever. *Lancet Infect Dis* 2003, 3:557-564.

11. Barre N, Garris G, Camus E: Propagation of the tick *Amblyomma variegatum* in the Caribbean. *Rev Sci Tech* 1995, 14:841-855.

12. Fournier PE, El Karkouri K, Leroy Q, Robert C, Giumelli B, Renesto P, Socolovschi C, Parola P, Audic S, Raoult D: Analysis of the *Rickettsia africana* genome reveals that virulence acquisition in rickettsia species may be explained by genome reduction. *BMC Genomics* 2009, 10:166.

13. Yssouf A, Lagadec E, Bakari A, Foray C, Stachurski F, Cardinale E, Plantard O, Tortosa P: Colonization of Grande Comore Island by a lineage of *Rhipicephalus appendiculatus* ticks. *Parasit Vectors* 2011, 4:38.

14. Walker AR, Bouattour A, Camicas J, Estrada-Pena A, Horak IG, Pegram RG, Preston PM: *Ticks of Domestic Animals in Africa: A Guide to Identification of Species*, Bioscience Reports. Edinburgh, United Kingdom: 2003.

15. Socolovschi C, Pages F, Raoult D: *Rickettsia felis* in *Aedes albopictus* mosquitoes, Libreville, Gabon. *Emerg Infect Dis* 2012, 18:1687-1689.

16. Bechah Y, Socolovschi C, Raoult D: Identification of rickettsial infections by using cutaneous swab specimens and PCR. *Emerg Infect Dis* 2011, 17:83-86.

17. Socolovschi C, Gomez J, Marie JL, Davoust B, Guigal PM, Raoult D, Parola P: *Ehrlichia canis* in *Rhipicephalus sanguineus* ticks in the ivory coast. *Ticks Tick Borne Dis* 2012, 3:411-413.

18. Kernif T, Djerbouh A, Mediannikov O, Ayach B, Rolain JM, Raoult D, Parola P, Bitam I: *Rickettsia africana* in *Hyalomma dromedarii* ticks from sub-Saharan Algeria. *Ticks Tick Borne Dis* 2012, 3:377-379.

19. Ronquist F, Huelsenbeck JP: *MrBayes 3: Bayesian phylogenetic inference under mixed models*. *Bioinformatics* 2003, 19:1572-1574.

20. Lynen G, Zeman P, Bakuname C, Di Giulio G, Mtui P, Sanka P, Jongejan F: Cattle ticks of the genera *Rhipicephalus* and *amblyomma* of economic importance in Tanzania: distribution assessed with GIS based on an extensive field survey. *Exp Appl Acarol* 2007, 43:303-319.

21. Macaluso KR, Davis J, Alam U, Korman A, Rutherford JS, Rosenberg R, Azad AF: Spotted fever group rickettsiae in ticks from the Masai Mara region of Kenya. *Am J Trop Med Hyg* 2003, 68:551-553.

22. Mediannikov O, Diatta G, Zolia Y, Balde MC, Kohar H, Trape JF, Raoult D: Tick-borne rickettsiae in Guinea and Liberia. *Ticks Tick Borne Dis* 2012, 3:43-48.

23. Camicas JL, Cornet JP, Gonzalez JP, Wilson ML, Adam F, Zeller HG: *Crimean-Congo hemorrhagic fever* in Senegal. Latest data on the ecology of the CCHF virus. *Bull Soc Pathol Exot* 1994, 87:11-16.

24. De Deken R, Martin V, Saido A, Madder M, Brandt J, Geysen D: An outbreak of east coast fever on the Comoros: a consequence of the import of immunised cattle from Tanzania? *Vet Parasitol* 2007, 143:245-253.

25. Portillo A, Perez-Martinez L, Santibanez S, Blanco JR, Ibarra V, Oteo JA: Detection of *Rickettsia africana* in *Rhipicephalus (Boophilus) decoloratus* ticks from the Republic of Botswana, south Africa. *Am J Trop Med Hyg* 2007, 77:376-377.

26. Prabhu M, Nicholson WL, Roche AJ, Kersh GJ, Fitzpatrick KA, Oliver LD, Massung RF, Morrissey AB, Bartlett JA, Onyango JJ, Maro VP, Kinabo GD, Saganda W, Crump JA: Q fever, spotted fever group, and typhus group rickettsioses among hospitalized febrile patients in northern Tanzania. *Clin Infect Dis* 2011, 53:e8-e15.

27. Roux V, Rydkina E, Eremeeva M, Raoult D: Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. *Int J Syst Bacteriol* 1997, 47:252-261.

28. Tortosa P, Pascalis H, Guernier V, Cardinale E, Le Corre M, Goodman SM, Dellagi K: Deciphering arboviral emergence within insular ecosystems. *Infect Genet Evol* 2012, 12:1333-1339.

doi:10.1186/1756-3305-7-444

Cite this article as: Yssouf et al.: First molecular detection of *Rickettsia africana* in ticks from the Union of the Comoros. *Parasites & Vectors* 2014 7:444.

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