First Molecular Identification of Rickettsia aeschlimannii and Rickettsia africae in Ticks from Ghana

ABSTRACT. The threats from vector-borne pathogens transmitted by ticks place people (including deployed troops) at increased risk for infection, frequently contributing to undifferentiated febrile illness syndromes. Wild and domesticated animals are critical to the transmission cycle of many tick-borne diseases. Livestock can be infected by ticks, and serve as hosts to tick-borne diseases such as rickettsiosis. Thus, it is necessary to identify the tick species and determine their potential to transmit pathogens. A total of 1,493 adult ticks from three genera—Amblyomma, Hyalomma, and Rhipicephalus—were identified using available morphological keys and were pooled (n = 541) by sex and species. Rickettsia species were detected in 308 of 541 (56.9%) pools by genus-specific quantitative polymerase chain reaction assay (Rick17b). Furthermore, sequencing of the outer membrane protein A and B genes (ompA and ompB) of random samples of Rickettsia-positive samples led to the identification of Rickettsia aeschlimannii and Rickettsia africae with most R. africae DNA (80.2%) detected in pools of Amblyomma variegatum. We report the first molecular detection and identification of the rickettsial pathogens R. africae and R. aeschlimannii in ticks from Ghana. Our findings suggest there is a need to use control measures to prevent infections from occurring among human populations in endemic areas in Ghana. This study underscores the importance of determining which vector-borne pathogens are in circulation in Ghana. Further clinical and prevalence studies are needed to understand more comprehensively the clinical impact of these rickettsial pathogens contributing to human disease and morbidity in Ghana.


INTRODUCTION
Ticks are currently considered second in importance to mosquitoes as human infectious disease vectors in the world. 1 They are the vectors of the spotted fever group rickettsiae, [2][3][4] which have been identified as significant agents in human tick-borne infections worldwide.In particular, African tick-bite fever (ATBF) has raised concerns beyond the continent because it is considered one of the leading causes of fever among travelers returning from sub-Saharan Africa. 5,6frican tick-bite fever is caused by the pathogen Rickettsia africae and is transmitted by Amblyomma variegatum and Amblyomma hebraeum, the predominant and aggressive tick species in Africa. 7The majority of ATBF cases are reported in South Africa (. 80%), with R. africae infection transmitted by A. variegatum approximately 70% of the time and A. hebraeum approximately 30%. 8However, R. africae is reported to be widely distributed across the continent, as it has either been isolated or detected by polymerase chain reaction (PCR) in Kenya, Chad, Burundi, Mali, Senegal, Niger, Sudan, and in most South African countries. 8,9Despite the increasing information available on ticks in other parts of the world, and extensive work in some parts of West Africa such as Senegal 9 and Nigeria, 10,11 there are limited published data on tick species, their presence, prevalence, distribution, and the tick-borne pathogens they transmit in Ghana.
In Ghana, many household pets and livestock are commonly infested with ticks, making it a possible risk zone for ATBF. 12However, to date, R. africae has not been identified in ticks from Ghana; Rickettsia felis has been the only Rickettsia species found in Ghana and was obtained from blood samples of febrile children in the Ashanti region of Ghana. 13ecent studies of ticks collected in different areas of Ghana have shown that A. variegatum ticks are predominant, 14,15 suggesting that R. africae may be present but has yet to be detected.Data from this study will be beneficial in guiding force health protection for both the U.S. and Ghanaian Armed Forces, as well as in enhancing global health security countermeasures.This study sought to characterize A. variegatum and other tick species, and to determine their potential role in the transmission of pathogenic Rickettsia species in Ghana.

MATERIALS AND METHODS
Tick sampling, identification, and processing.Adult ticks were collected from cattle during a tick survey carried out between August 2017 and March 2018 from seven sites in the southern and northern sectors of Ghana (Figure 1).Ticks were identified using morphological keys 16 and pooled (five individuals or less) according to species, sex, and collection site for bacterial nucleic acid detection.
Nucleic acid was extracted using a QIAamp Viral RNA Mini Kit 17 (QIAGEN, Valencia, CA) following the manufacturer's instruction without adding carrier RNA to maintain DNA content.The tick DNA preparations were screened by a genusspecific quantitative real-time polymerase chain reaction (qRT-PCR) assay (Rick17b), with primers targeting the gene encoding the 17-kDa antigen of Rickettsia DNA as described previously. 18Rickettsia-positive pools were tested by a species-specific qRT-PCR assay (RafriG) for R. africae as described previously. 19The negative control was nucleasefree water whereas the positive control was Rickettsia DNA from a field isolate.Random samples that were positive by genus-specific qRT-PCR were selected from different collection sites in the southern and northern sectors for further characterization using primers targeting the outer membrane protein A gene (ompA) 20 and outer membrane protein B gene (ompB). 21The amplification products were purified using the QIAquick PCR Purification kit (QIAGEN) and were sequenced using the Applied Biosystem 3730XL (Applied Biosystems, Foster City, CA).
Sequences obtained from our study were used to query the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) to retrieve reference sequences for comparison.Sequences were aligned using ClustalW implemented in MEGA X. Tree model inference and phylogeny were conducted simultaneously in IQ-TREE (version 1.6.1),executing 1,000 bootstrap replicates.Tree visualization was done in FigTree (version 1.4.4).
The infection rates in the tick pools were calculated using PoolScreen 2.0.(version 2.0.1). 22
In addition, 12 PCR products were sequenced successfully and compared with those available in the GenBank database using BLAST analyses.The sequences of ompA-and ompBpositive amplicons gave the same identification results.The BLAST search showed that one of the sequences was 99% identical to an R. africae isolate from Benin, and 11 sequences were 100% identical to Rickettsia aeschlimannii isolates from China and Spain (Figures 2 and 3).
The sequences from bacterial DNA preparations in our study were compared with other sequences in the NCBI database, and corresponding hit sequences were used to generate the phylogenetic trees shown in Figures 2 and 3   ).The sequences selected were $ 98% similar to the ompA gene sequence of R. aeschlimannii Ghana 1 and 2 after the BLAST analysis.Rickettsia aeschlimannii (GHA1 and GHA2) detected in ticks in our study were not identical to each other.A single nucleotide change was observed in the ompA DNA sequence when sequence alignments were performed, and this could be responsible for the divergence observed between the two Ghana ompA sequences.

DISCUSSION
Similar to previous studies in Ghana, A. variegatum was the predominant tick species. 23,24Amblyomma variegatum, an important vector in transmitting various rickettsial and viral pathogens, is infected with Crimean-Congo hemorrhagic fever 14 and Dugbe viruses 15 in Ghana.It was previously thought that A. variegatum was the only reservoir for Rickettsia spp. in sub-Saharan Africa. 3However, more recent studies have identified increased diversity in both Rickettsia spp.and the associated tick species harboring them. 25,26Our study demonstrates the same, with the identification of R. aeschlimannii and R. africae DNA in multiple tick species.
The prevalence of Rickettsia spp. in ticks collected from cattle in our study was greater than that reported from cattle in Zambia (18.6%) 27 and Nigeria (12.5%), 11 and from different animal species, including cattle from northern Senegal (5.8%). 28The high rate of infection observed in our study could be a result of the number of susceptible livestock present at the various sampling sites.The more livestock infected with Rickettsia spp., the greater the rate at which ticks will be infected during blood feeding and will potentially  transmit to animal handlers.Further species identification of the Rickettsia-positive pools demonstrated a high prevalence of R. africae infection in the A. variegatum and Rhipicephalus species.Transovarial and trans-stadial transmission of R. africae has been demonstrated in A. variegatum. 29Thus, in the presence of cattle reservoirs, A. variegatum poses a significant risk to the human population.In Africa, although R. africae infection is common, it is rare to find reports of ATBF in indigenous people. 7This could be a result of chronic and recurrent exposure conferring some level of immunity.It could also be that cases are mild and unreported, or that effective diagnostic methods for Rickettsia are not available.However, ATFB is one of the most frequently reported causes of febrile illness among travelers returning from Africa. 9The most common clinical symptoms include fever, rash, headache, chills, lymphangitis, and fatigue. 30However, the nonspecific presentation can present as an undifferentiated febrile illness in travelers and individuals living in areas where the pathogen is endemic.
Sequencing analysis revealed that R. aeschlimannii, a pathogenic agent in the spotted fever group, 31 was present in 11 of 12 samples.This pathogen was first identified and characterized after it was isolated from Hyalomma marginatum in Morocco. 32Since then, R. aeschlimannii has been identified frequently in other Hyalomma tick species in various West African countries, including Côte d'Ivoire, Nigeria, Senegal, Mali, and Niger, 25,33,34 and is reported infrequently in Amblyomma and Rhipicephalus ticks. 3As in other areas of West Africa, our study identified R. aeschlimannii in H. rufipes ticks in Ghana.In addition, it provides evidence of R. aeschlimannii in A. variegatum and Rhipicephalus spp.Because the ticks assessed were collected from livestock, the presence of R. aeschlimannii may have been a result of the presence of the agent within the blood meal.This requires further blood meal analysis to determine hosts and/or reservoirs of the pathogen.
The pathogenicity of R. aeschlimannii in humans is not well understood, but based on limited reports of human infection, it appears to mimic Mediterranean spotted fever, 35 with symptoms ranging from fever and sore throat to myalgias, maculopapular rashes, and acute hepatitis. 36Although no human infections with R. aeschlimannii have been reported in Ghana, this does not discount the circulation of this pathogen in the population, resulting from its documented presence in at least three tick species and the lack of routine testing in febrile patients.
This first reported molecular detection of R. aeschlimannii and R. africae in ticks collected in Ghana highlights the potential risk of infection and illness among animal handlers, within the community at large, and among travelers and deployed military personnel.Additional surveillance studies need to be performed to access the prevalence and distribution of ticks, the transmission of tick-borne pathogens, and their public health importance.
A limitation of the pooling method used in our study is that the identified Rickettsia species cannot be associated with individual tick species.Furthermore, pooling could have caused a reduction in the concentration of Rickettsia DNA, leading to false negatives.Engorged ticks that could not be identified to the species level but were subjected to pathogen screening could have introduced bias into the analysis.

CONCLUSION
This study reports the dominance of Amblyomma variegatum ticks in sampled sites that harbored Rickettsia species of clinical significance to the U.S. and Ghanaian military, as well as to tropical medicine.Interestingly, for the first time in Ghana, our report identifies the presence of R. aeschlimannii and R. africae.However, confirming the vector and existence of these pathogens in Ghana is only the first step in determining the clinical impact of human disease in Ghana.Further studies assessing etiologies of undifferentiated fever are needed to determine the prevalence of the contribution of these rickettsial agents to human disease transmission in Ghana.

FIGURE 2 .
FIGURE 2. Phylogenetic analysis of the Rickettsia africae sequence from Ghana (red) and others from different geographic origins.The tree was constructed from a partial ompA gene segment (567 bp).Tree model inference and phylogeny were conducted simultaneously in IQ-TREE (version 1.6.1),executing 1,000 bootstrap replicates.The reference sequences included in the analyses are shown by their GenBank accession number, country of origin, and/or isolation date and host.Critical nodes are labeled with bootstrap values.The tree was visualized in FigTree (version 1.4.4),https://github.com/rambaut/figtree/releases.

FIGURE 3 .
FIGURE 3. Phylogenetic analysis of two Ghana Rickettsia aeschlimannii sequences (red) and others from different geographic origins.The tree was constructed from a partial ompA gene segment (529 bp).Tree model inference and phylogeny were conducted simultaneously in IQ-TREE (version 1.6.1),executing 1,000 bootstrap replicates.The reference sequences included in the analyses are shown by their GenBank accession number, country of origin, and/or isolation date and host.Critical nodes are labeled with bootstrap values.The tree was visualized in FigTree (version 1.4.4).

TABLE 1
Infection rates of Rickettsia spp.and Rickettsia africae among pooled tick samples