Ticks are the most widespread carriers of zoonotic pathogens and are second only to mosquitoes globally as vectors of human diseases (de la Fuente et al. 2008). Ticks harbor various pathogenic and nonpathogenic microorganisms including viruses, bacteria, protozoa, helminths, and fungi (Estrada-Pena 2015). The socioeconomic impact of ticks and tick-borne diseases has increased in recent years because of current trends in climate and unpredictable long-term seasonal changes (Michelet et al. 2016; Papa et al. 2017). In the tropics and the Caribbean, ticks are one of the most important vectors of disease organisms of veterinary and medical importance (Gondard et al. 2017).

Rhipicephalus sanguineus Latreille, known as the brown dog tick, has a worldwide distribution (Dantas-Torres 2010). This tick is known to be the parasite of dogs, but despite its relative host specificity, it has parasitized other livestock mammals and humans occasionally (Filipe Dantas-Torres 2008). This tick inflicts direct injury to its host and plays a major role in the transmission of infections and zoonotic agents such as Coxiella burnetii (Derrick) Philip responsible for Q fever, Ehrlichia canis (Donatien and Lestoquard) responsible for ehrlichiosis in dogs, Rickettsia conorii Brumpy responsible for boutonneuse fever in dogs, and Rickettsia rickettsia (Wolbach) responsible for spotted fever in humans (Demma et al. 2005; Parola et al. 2005; Walker et al. 2000). Additionally, both Candidatus Neoehrlichia mikurensis (Gofton et al.) an emerging pathogen of humans, and Rickettsia conorii subsp. israelensis, the causative agent of Mediterranean spotted fever, were detected in R. sanguineus ticks in Nigeria (Kamani et al. 2013).

Blood-sucking arthropod vectors habor symbiotic microorganisms that play vital roles in determining the transmission of pathogens (Ahantarig et al. 2013). For instance, the Rickettsia-like symbionts in ticks are functionally vital for the population dynamics, physiology, and transmission of pathogenic rickettsiae (Childs and Paddock 2002). Ticks can be host to a mixture of symbiotic bacteria (Morimoto et al. 2006; Perotti et al. 2006), and many of these endosymbiotic intracellular bacteria seem to be nonpathogenic to mammals (Burgdorfer et al. 1973; Rowley et al. 2004). Most of these endosymbionts are found mainly in the ovaries or Malpighian tubules of ticks (Noda et al. 1997). Symbionts in ticks can be classified into two groups: obligate symbionts that play a vital role for the survival of the host and are mostly transmitted vertically, and facultative (secondary) symbionts that are dispensable (Noda et al. 1997; Perlman et al. 2006). Francisella-, Coxiella-, and Rickettsia-like organisms are among the vertically transmitted bacterial symbionts in ticks (Noda et al. 1997; Sun et al. 2000) and may be responsible for mild symptoms of certain clinical conditions (Apperson et al. 2008). They can also influence the colonies of other tick-borne microbes and affect their mode of transmission (de la Fuente et al. 2003; Macaluso et al. 2002). However, the exact function of tick endosymbionts in the transmission of the pathogens that cause severe infections remain poorly understood (Ahantarig et al. 2013).

A previous study in Nigeria molecularly screened three species of ticks (R. sanguineus, R. turanicus Neumann, and Haemaphysalis longicornis Neumann) for human and animal vector-borne pathogens by polymerase chain reaction (PCR) and sequencing (Kamani et al. 2013). The DNA of Rickettsia conorii ssp. israelensis and Candidatus Neoehrlichia mikurensis were detected in R. sanguineus (Kamani et al. 2013). However, to the best of our knowledge, this is the first broad overview of the bacterial microbiome in R. sanguineus in Nigeria. Thus, this study aims to characterize the bacterial microbiome in R. sanguineus from two states in Nigeria using 16S rRNA high-throughput sequencing (IonS5TM XL sequencing platform).

Materials and Methods

Study area. This study was performed in 2 of 36 states randomly selected from the agro-ecologic zones of the Federal Republic of Nigeria. Tick sampling was conducted on dogs in the two states: Plateau state (between latitude N 9°55′ and N 9°57′and longitude E 8°52′ and E 8°55′) (Okorukwu et al. 2016) and Benue state (between latitude N 6°25′ and N 8°8′ and longitude E 7°47′ and E 10°0′) (Agada and Nirupama 2015), which are located in the Guinea savannah vegetative belt of Nigeria. Although the climate in Benue is typically tropical with a temperature range of 21° to 34°C, Plateau state enjoys a near temperate climate known as the Tropical savannah climate with an average monthly temperature range of 21°C to 25°C and the nighttime temperature sometimes dropping as low as 16°C. The mean annual precipitation in Plateau and Benue is approximately 1,400 and 1,173 mm, respectively. The vegetation cover in both states is mainly savannah composed of plains of grasses interrupted by trees (Ogungbenro and Morakinyo 2014).

Ticks collection. Ticks were collected from dogs that were brought by their owners to the veterinary clinics in Plateau and Benue states in Nigeria. The dogs were examined for ectoparasites after obtaining oral permission from the owners. Ticks were collected from the body of dogs with tweezers and transferred into 70% ethanol in labeled Eppendorf tubes. The ticks used were semiengorged adult female ticks with body weight ranges of 160 to 170 mg. Ticks were identified in the laboratory using taxonomic descriptions and morphologic keys (Apanaskevich et al. 2007; Walker et al. 2014) and then surface sterilized with 30% hydrogen peroxide followed by 70% ethanol before being stored at –80°C.

Genomic DNA extraction and PCR amplification. Genomic DNA was extracted from each group of adult ticks (six from Plateau and six from Benue) by the sodium dodecyl sulfate method, and genomic DNA purity and concentration were detected by agarose gel electrophoresis. The appropriate amount of sample DNA was transferred into a centrifuge tube and was diluted to 1 ng/µl with sterile water. Using diluted genomic DNA as a template, PCR was used to ensure amplification based on the selection of sequencing regions using specific primers with barcode using Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Ipswish, MA) and high-fidelity enzymes for efficiency and accuracy. The primer corresponding area is 16S V4 region primers (515F and 806R) for the identification of bacterial diversity.

Mixing and purification of PCR products. Agarose (2%) gel electrophoresis was used to detect the PCR product. The samples were mixed thoroughly according to the concentration of the PCR product. Then, the PCR product was purified by electrophoresis with a 1×Tris-acetate-EDTA (TAE) concentration of 2% agarose gel band using a GeneJET Glue Recovery Kit (Thermo Fisher Scientific, Waltham, MA).

Library construction and sequencing. The DNA library was constructed using Thermofisher's Ion Plus Fragment Library Kit (Thermo Fisher Scientific). The library was subjected to Qubit quantification and library testing and then sequenced using Thermofischer's Ion S5TMXL (Thermo Fisher Scientific). The sequences from ticks in Plateau State and Benue State have been deposited in NCBI under accessions numbers SAMN13506797 and SAMN13506798, respectively.

Data analysis. Low-quality partial cut on the reads was performed using Cutapt (V1.9.1, http://cutadapt.readthedocs.io/en/stable/) (Jiao et al. 2016). The resulting data files were sorted according to sample specific multiplex identifiers (MID) tags. MID tag barcodes and primers were trimmed, and then low-quality and short sequence reads (<150 bp) were removed (https://github.com/torognes/vsearch/) (Qin et al. 2012). To detect and finally remove the chimeric sequences, alignment with the species annotation database was carried out, and the final valid data (clean reads) were obtained (Martin 2011).

Operational taxonomic unit clustering and species annotation. For clustering, all clean reads of the samples, UPARSE software (Uparse v7.0.1001, http://www.drive5.com/uparse/) (Rognes et al. 2016) was used with a default of 97% identity. By this, operational taxonomic units (OTUs) are formed, and the representative sequence of the OTUs was selected using the highest frequency according to its algorithm principle. Specimen annotation of the OTUs sequences and species annotation analysis (set threshold of 0.8–1) was carried out using the Mothur method and SSU rRNA gene database (Edgar 2013) of SILVA132 (http://www.arb-silva.de/) (Haas et al. 2011), and, thus, the taxonomic information and composition of each sample at each level were classified to the kingdom, phylum, class, order, family, genus, and species. To obtain the phylogenetic relationships for all OTU sequences, fast multisequence alignments were performed using MUSCLE (version 3.8.31, http://www.drive5.com/muscle/) (Wang et al. 2007).

Sample complexity analysis (alpha diversity). The QIIME software, version 1.9.1 (Caporaso et al. 2010) was used for the calculation of the observed OTUs, Chao1 (for the index of the total number of the species), Shannon (index for microbiome diversity), Simpson (microbiome diversity index), abundance-based coverage estimators, Good's coverage (for the sequencing depth), and phylogenietic diversity (PD) whole tree index (for the display of PD). R software (version 2.15.3) was used to create the dilution curve, rank abundance curve, species accumulation curve, and alpha diversity analysis index.

Multisample comparative analysis (beta diversity). The Unifrac distance was calculated using Qiime software, version 1.9.1, and the unweighted pair group method with arithmetic mean sample clustering tree was constructed. Principal component analysis (PCA), principal coordinates analysis (PCoA), and nonmetric multidimensional scaling (NMDS) maps were drawn using R software (version 2.15.3). R software's ade4 package and the ggplot2 software package were used for PCA analysis. W software's weighted gene coexpression network analysis, stats, and ggplots software packages were used for PCoA analysis, whereas, the R software's vegan software package was used for NMDS analysis. Differences between the beta diversity index groups were analyzed with R software, and parameters were tested with a nonparametric test.

Ethical considerations. All institutional and national guidelines for the care and use of animals were followed. This study was approved by the Animal Ethics Committee of Hebei Normal University, complying with the Animal Protection Law of the People's Republic of China (Protocol IACUC-157031).

Results

After sequencing, 171,042 raw reads were generated from adult R. sanguineus samples from the Plateau and Benue states of Nigeria. Clean reads (160,149) were clustered into 1,230 distinct OTUs. Among them, 857 OTUs were obtained from the Plateau samples and 373 OTUs were obtained from Benue samples (Table 1). The number of observed species found to be common between Plateau R. sanguineus and Benue R. sanguineus is 293. The number of species found to be distinct to Benue R. sanguineus is 39, whereas 555 species were found to be distinct to Plateau R. sanguineus (Fig. 1). The results revealed higher microbial diversity in Plateau state samples of R. sanguineus. Three hundred genera of bacteria belonging to 22 phyla were detected after pooling and sequencing, although 25 groups of those belong to unidentified genera.

Table 1 Sequences and alpha diversity from 16S rRNA sequencing of Rhipicephalus sanguineus.

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Multiple indices were adopted to ascertain that the current sequencing revealed the diversity of the real microbial communities. Shannon index revealed the diversity of the microbiome, the Chao1 estimator showed the index of the total number of species, observed species revealed the total number of the species, and the Good's coverage represented the sequencing depth (Table 1). The PD was displayed in the phylogenetic tree. Thus, these results indicate that all OTUs characterized from our samples adequately represented the microbial communities.

Microbial community of R. sanguineus. The taxonomic abundance of all OTUs was summarized at different taxonomic levels (phylum, class, order, family, genus, and species). At the phylum level, 22 phyla were present. Actinobacteria (47.39%) was the most abundant phylum in the Benue samples, followed by Proteobacteria (43.87%) and Firmicutes (8.21%), whereas Fusobacteria (38.14%) was the most abundant phylum in Plateau samples, followed by Bacteroidetes (17.57%) and Firmicutes (17.54%). Proteobacteria were in 17% of the samples from Plateau state. The relative abundance of the top 30 genera of microbes in adult R. sanguineus are shown in Table 2, whereas the top 30 characterized microbes at the genus level are shown in Fig. 2. In general, Cetobacterium (35.86%) was the most abundant genus, followed by an unidentified genus under Corynebacteriaceae (29.94%) and Stenotrophomonas (19.52%) (Fig. 2).

Table 2 Relative abundance of the top 30 genera of microbes in adult Rhipicephalus sanguineus.

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Table 2 Continued.

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The relative abundance of the characterized microbial species was summarized to further explore the diversity of microbes associated with R. sanguineus. The results indicated that Corynebacterium xerosis (Lehmann and Neumann) was the most abundant species, accounting for 28.27% of the total bacteria identified, followed by Pseudomonas geniculate (Wright) Chester (13.01%), and Acinetobacter baumannii (Bouvet and Grimont) (12.52%) (Fig. 3). The top 30 characterized microbes were further hierarchically clustered at the genus level as shown in the heatmap, and the results indicated that R. sanguineus from Plateau state samples harbored more diversity of microorganisms (Fig. 4). An evolutionary tree of characterized microbes of top 100 genera (Fig. 5) showed that Cetobacterium, which belongs to the phylum Fusobacteria, was the most abundant genus.

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Discussion

Rhipicephalus sanguineus is the most prevalent species parasitizing dogs in Nigeria and accounted for 95.2% of the ticks collected from dogs from all the states of Nigeria (Adamu et al. 2014; Kamani et al. 2019). This is most likely because of its affinity for dogs (Walker et al. 2000) and/or its high survival rate in a wide range of environmental conditions (Dantas-Torres et al. 2013; Gray et al. 2013; Hekimoğlu et al. 2016; Papadopoulos et al. 1996; Zemtsova et al. 2016). High prevalence of endosymbionts in R. sanguineus feeding on dogs in two Nigerian states were recorded in the present study. Similarly, Vila et al. (2019) reported a 92% prevalence of endosymbionts in different ticks species feeding on dogs including R. sanguineus found in Spain.

In this study, 300 genera (25 groups of those belong to unidentified genera) of bacteria belonging to 22 phyla were detected, indicating that R. sanguineus harbors a diversity of microorganisms. It is noteworthy that the 16S rRNA sequencing approach comes with some flaws that have to do with poor species differentiation in some bacteria families, which implies that diversity may be under-represented (Jovel et al. 2016). Additionally, 16S rRNA sequencing is weaker in detecting archaea and microbial eukarya (Walters et al. 2011), but they are not the focus of this study. The multiple indices used to analyze the sequencing showed that the microbial communities of R. sanguineus were well explored. The difference in the number of OTUs of the ticks obtained in the two regions was substantial, with Plateau state samples having more OTUs. A possible explanation could be that Plateau state enjoys a near temperate climate known as the tropical savannah climate with an average monthly temperature range of between 21°C and 25°C and the nighttime temperature dropping to as low as 16°C, which could be more favorable for ticks, quite unlike Benue state with a typical temperature range of between 21°C and 34°C (Ogungbenro and Morakinyo 2014). This was corroborated by the study that investigated the effect of temperature on the microbial diversity of Ixodus scapularis (Say) ticks that showed that an increase in temperature resulted in decreased microbial diversity (Thapa et al. 2019).

Ticks are the most important arthropods after mosquitoes that transmit pathogens to humans and both domestic and wild animals worldwide (de la Fuente et al. 2008). However, they also harbor many nonpathogenic microorganisms. Although much exploration has been conducted on the pathogenic microbiomes in ticks, knowledge on nonpathogenic agents in ticks is usually underrated. The impact of the nonpathogenic microorganisms on their tick hosts can be disadvantageous, neutral, or favorable and may be functionally vital to the nutritional adaptation, reproduction, immunity, and overall biological system of the tick (Bonnet et al. 2017).

Several bacteria genera that are obligate symbionts can coexist with many facultative symbionts that include maternally inherited symbionts in ticks (Duron et al. 2017). Similar to the present study, the facultative symbiont diversity in ticks include many nonpathogenic microbes such Coxiella, Pseudomonas, Wolbachia, Midichloria, Enterobacteriaceae, or Rhizobiales groups (Abraham et al. 2017; Andreotti et al. 2011; Carpi et al. 2011; Narasimhan et al. 2014; Qiu et al. 2014; Williams-Newkirk et al. 2014).

Coxiella spp. (Proteobacteria) is a common endosymbiont found in various tick species, which we found to be true for R. sanguineus in the present study. Vila et al. (2019) found Coxiella spp. endosymbionts in R. sanguineus, Ixodes hexagonus (Leach), and I. ricinus (Linnaeus). In I. ricinus, and Rhipicephalus spp. tick populations, six bacteria genera coexist (Duron et al. 2017). It has been reported that Coxiella spp. have definite tropism for the ovaries and Malpighian tubules of the abovementioned tick species (Bonnet et al. 2017; Duron et al. 2015; Guizzo et al. 2017). The rationale behind its infection of the ovary is for the purpose of vertical transmission, whereas its possible role in nutrition, osmoregulation, or excretion is responsible for their occupation of the distal part of Malpighian tubules (Klyachko et al. 2007; Lalzar et al. 2014; Machado-Ferreira et al. 2011). Interestingly, in the Rhipicephalus genus, previous studies have revealed evolutionary stable associations that exist between some Coxiella spp. and their tick host, which have been present for ages, and such relationships are co-cladogenetically solid such that a consistent host–symbiont phylogenies have been formed (Duron et al. 2017). Coxiella spp. are ubiquitous in some genera of ticks to the extent of validating the hypothesis of its obligate endosymbiontic status (Duron et al. 2015, 2017; Guizzo et al. 2017; Papa et al. 2017). Although Coxiella spp. associations could probably exhibit conditional mutualism in these species, there is no sufficient experimental evidence to confirm these roles (Vila et al. 2019).

Sometimes, the detection of a maternally transmitted bacterium can be as a result of cross-contamination as for several I. ricinus studies. The presence of infected endoparasitoid wasp, Ixodiphagus hookeri (Howard), can cause Wolbachia and Arsenophonus infections in tick tissues necessitating the application of caution during the assessment of tick symbionts (Plantard et al. 2012; Bohacsova et al. 2016). Nonpathogenic microorganisms can obstruct the reproduction and transmission of a tick-borne pathogen (TBP) and, by implication, become a determinant factor for the abundance and diversity of TBPs in tick populations, in addition to their transmission to vertebrate hosts (Bonnet et al. 2017), and this can be of paramount import to veterinary and public health.

The microorganisms from R. sanguineus characterized in the present study have minimal veterinary and public health importance. A possible explanation could be that most dogs being brought to veterinary clinics in Nigeria are high-priced exotic dogs that often receive conventional ectoparasiticides in the form of tick collars, powders, and pour-on or injectable preparations such as ivermectin, permethrin, and cypermethrin, among others (Kamani et al. 2019). This is not the case in South Africa where clinical complications and deaths of dogs are associated with Babesia rossi (Nuttall) (Penzhorn 2011) infections probably because of the presence of the wild African canid species Lycaon pictus (Temminck) and Canis mesomelas (Schreber) (black-backed jackals), responsible for the maintenance of the life cycle of B. rossi. In Nigeria, the wild canid populations are no longer easily found in the wildlife population (Fanshawe et al. 1991), and thus, the virulence of B. rossi has probably been lost in Nigeria over time by natural selection, indicating the loss of the BrEMA 1 gene and its related virulent genotypes (Adamu et al. 2014).

This study provides baseline information on the bacterial diversity of R. sanguineus in Nigeria, and it reveals the predominance of nonpathogenic microorganisms in R. sanguineus in Nigeria. Knowledge of the role of many nonpathogenic microorganisms inhabiting ticks remains limited. This warrants further research to explore the functional and ecological implications of these microorganisms, which will provide basic knowledge for integrated control of ticks and tick-borne diseases.