Introduction

Babesia canis (sensu lato) is a tick-borne hemoprotozoan parasite that induces anemia, fever, jaundice, hemoglobinuria, and sometimes fatal symptoms in dogs (Boozer and Macintire 2003). The pathogen belongs to the Piroplasmidae and there are three described subspecies, B. canis canis, B. canis vogeli and B. canis rossi (Uilenberg et al. 1989). They are each transmitted by a different tick species, Dermacentor reticulatus, Rhipicephalus sanguineus (s.l.) and Haemaphysalis elliptica (previously lumped with Haemaphysalis leachi), respectively (Uilenberg 2006; Apanaskevich et al. 2007). There are also differences in the geographical distribution, antigenicity and pathogenicity to dogs of each subspecies (Schetters et al. 1997). Babesia canis (sensu stricto) is found in the Palaearctic region coinciding with the distribution of its vector tick D. reticulatus, B. vogeli has a global distribution similar to that of its vector R. sanguineus (s.l.) throughout tropical, subtropical and Mediterranean areas, and B. rossi is thought to be restricted to sub-Saharan Africa (Irwin 2009; Solano-Gallego and Baneth 2011; Jongejan et al. 2018). Genotyping has confirmed the existence of three separate species (Zahler et al. 1998; Carret et al. 1999), B. canis, B. vogeli and B. rossi (Allsopp and Allsopp 2006; Solano-Gallego and Baneth 2011). B. rossi is known to be the most pathogenic of the three species and causes the most severe disease manifestations in canine hosts (Jacobson 2006; Penzhorn 2011).

Canine babesiosis has a high prevalence in Nigeria with molecular studies confirming the presences of all the three species (Sasaki et al. 2007; Kamani et al. 2010, 2013, Adamu et al. 2014). These studies reported higher prevalence of B. rossi (2–38%) than B. vogeli (0.3–1%). Curiously, several studies on the ectoparasites of dogs in Nigeria have reported R. sanguineus, the vector of B. vogeli, as the predominant tick species infesting dogs and not H. elliptica, the vector of B. rossi (Kamani et al. 2013; Adamu et al. 2014). In fact, some studies did not find Haemaphysalis spp. ticks on the dogs examined but found them positive for Babesia spp. (Okoli et al. 2006; Adamu et al. 2012; Opara et al. 2017). These findings raise the question as to whether R. sanguineus is involved in the natural transmission of B. rossi in dogs as well. Recently H. hystricis was incriminated as a novel vector for B. gibsoni in Taiwan by first amplifying the DNA of the pathogen in the tick followed by the demonstration of its transovarian passage to the tick progenies (Jongejan et al. 2018). Therefore, the aim of this study was to detect and characterize the DNA of B. rossi in R. sanguineus ticks removed from dogs as a preliminary step in the evaluation of its possible involvement in the epidemiology of this infection in Nigeria.

Materials and methods

A total of 66 ticks were collected from 31 dogs manifesting clinical signs of tick-borne diseases that were referred to veterinary hospitals in Jos, the capital city of Plateau State of Nigeria, from January to June 2011. The living ticks were immersed in 70% ethanol and kept at − 20 °C until processing. Morphological identification was done using a binocular compound microscope and standard taxonomic keys for adult ticks (Walker et al. 2003). After identification, the DNA was isolated from individual ticks by using the QIAamp DNA Miniprep kit (Qiagen) according to the manufacturer’s instructions.

The oligonucleotide primers used for amplification and sequencing of the 18S ribosomal RNA gene (rDNA) of canine babesial species were designed using primer design software (Primer Select, DNASTAR, Madison, WI, USA) and related sequence information available in the GenBank database: B. rossi (accession numbers L19079 and DQ111760), B. vogeli (HQ148663 and AY072925), B. canis (AY072926), and Babesia gibsoni (JQ910685). The primers B18S-F1 (5′-CGGTGAAACTGCGAATGGCT-3′) and B18S-R1 (5′-TAACCAGCGCTAGTTAGCAGG-3′), B18S-F2 (5′-ATTACCCAATCCCGACACGGG-3′) and B18S-R2 (5′-TGTCTGGACCTGGTGAGTTTC-3′) were used respectively, in a nested PCR for the first and second amplifications targeting the 693 bp of the 18S ribosomal DNA (18S rDNA) gene of Babesia spp.

For the confirmation of the morphological identification of ticks, primers TQ16S-1F (5′-CTGCTCAATGATTTTTTAAATTGCTGTGG-3′) and TQ16S-2R (5′-ACGCTGTTATCCCTAGAG-3′) were used for the amplification of the 320-bp of the tick mitochondrial 16S rDNA (Black and Piesman 1994; Nava et al. 2012).

Polymerase chain reaction (PCR) was performed in a total volume of 25 μl containing 2 μl of extracted DNA, 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.625 U Hotstartaq DNA Polymerase (Qiagen), and 12.5 pmol of each primer in the reaction buffer provided by the manufacturer (Qiagen). The reaction conditions were one cycle of 5 min at 95 °C, 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 90 s at 72 °C each, followed by an extension step for 5 min at 72 °C.

The resulting PCR products were electrophoresed on a 1.2% agarose gel stained with ethidium bromide to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 kb Plus DNA Ladder, Promega). In each case, the single amplified product of the expected size was column purified using the QIAquick PCR Purification Kit (Qiagen) and directly sequenced by using an ABI PRISM 3730 capillary sequencer (Applied Biosystems) and the Dye Terminator Cycler Sequencing Kit (Applied Biosystems). The representative nucleotide sequence for the partial region of the 18S rDNA of B. rossi was deposited in GenBank under accession number JN982342. The partial sequences of mitochondrial 16S rRNA gene from ticks were deposited in GenBank under accession numbers from JN982352 to JN982359.

The BLAST program (http://www.ncbi.nlm.nih.gov/BLAST) was used for the comparison and the analysis of sequence data obtained in this study with those previously deposited in GenBank.

Results

A total of 66 ticks were collected and all of them were morphologically determined as R. sanguineus adults. All tick samples were subjected to DNA extraction followed by a nested PCR amplification with the 18S rDNA-gene. Among 66 samples examined, 8 (12.1%) were positive for Babesia spp. Sequencing results for those amplicons showed that all 18S rDNA sequences of 693 bp were identical to each other and bore 99.3–99.9% identities with the corresponding sequences of B. rossi from South Africa (GenBank Accession No. L19079), eastern Sudan (DQ111760), and Nigeria (AB303071-75), 95.4% identity with that of B. canis (AY072926), and 94.8% identity with that of B. vogeli (AY072925).

To verify the previous morphological identification of the ticks from which DNAs were derived at molecular level, those B. rossi-positive DNA samples were further analyzed with a PCR using species-specific primers based on the tick mitochondrial 16S rDNA. BLAST analyses of the resulting sequences of PCR amplicons (320 bp) from all samples confirmed their affiliation of R. sanguineus with 98.2–99.1% identities with R. sanguineus from USA (AF081829) (Black and Roehrdanz 1998; Nava et al. 2012). In contrast, these sequences bore only 83.1–83.6% similarities with that of H. elliptica (HM068958).

Discussion

Babesia parasites are naturally transmitted only by ticks, and in the case of canine babesiosis there is very strong vector specificity (Uilenberg 2006). As a consequence, the prevalence of babesiosis is dependent on the presence of the tick vector in the environment. For a long time it was widely accepted that H. elliptica (previously regarded as synonymous with H. leachi), the southern African yellow dog tick, is the only tick species that can transmit B. rossi to dogs (Schetters et al. 2009; Schoeman 2009; Penzhorn 2011; Matijatko et al. 2012). In surveys conducted in Nigeria, however, the prevalence of 2–38% was observed for B. rossi compared to 0.3–1.0% B. vogeli but the predominant tick species recovered from the infected dogs was R. sanguineus, and Haemaphysalis spp. ticks (H. elliptica or H. leachi) was rarely detected (Sasaki et al. 2007; Kamani et al. 2013; Adamu et al. 2014). Similarly, several other studies attest to the cosmopolitan occurrence of R. sanguineus and the rarity of Haemaphysalis spp. on dogs in Nigeria (Okoli et al. 2006; Adamu et al. 2012; Aquino et al. 2016; Opara et al. 2017). The disparity therefore between the high prevalence of B. rossi infection of dogs and the rarity of its acclaimed Haemaphysalis spp. vector in Nigeria may be a pointer to the possible involvement of other vector(s) in the epidemiology of this disease. Therefore, the detection of B. rossi DNA in R. sanguineus in this study coupled with increasing reports incriminating it in the transmission of more pathogens prompt us to probe the possible role of this catholic tick in the epidemiology of B. rossi in Nigeria.

The tick R. sanguineus accounts for the 10% of 867 known tick species implicated to transmit different pathogens (Jongejan and Uilenberg 2004). They are known vectors of pathogens such as B. vogeli, B. gibsoni, Ehrlichia canis, Anaplasma platys, Hepatozoon canis, Mycoplasma haemocanis, and Rickettsia conorii in domestic and wild animals in spite of their relatively low anthropophily (Palmas et al. 2001; Dantas-Torres 2008). In the present study, B. rossi DNA was detected in 12% of R. sanguineus ticks collected from dogs presenting clinical signs compatible with tick-borne disease. Ordinarily, the detection of pathogen DNA in an engorged tick removed from a host should not be used as a proof of vector competence. It could be that the tick ingests the pathogen from its host. But this may not always be the case as shown in an earlier study where 258 ticks remove from dogs in Nigeria were screened for various pathogens. None of the ticks examined was positive for Babesia spp. DNA even though their hosts were infected with Babesia spp. and DNA of other pathogens, Ehrlichia, Hepatozoon, Anaplasma and Rickettsia spp. were detected in them (Kamani et al. 2013). Therefore, the finding in the present study deserves some consideration under the prevailing scenario where the disease is prevalent but its acclaimed vector is rare. Although the absence of evidence is not evidence of absence, the fact remains that ticks of the H. leachi-group are rarely found on or are absent from dogs in Nigeria. Thus the detection of B. rossi DNA in R. sanguineus (s.l.) in the present study should be considered as a strong circumstantial evidence suggestive of the possible role of this tick in the epidemiology of B. rossi in Nigeria. More so, Babesia spp. could be transmitted transstadially or transovarially (Uilenberg 2006; Chauvin et al. 2009).

Considering that R. sanguineus is the most widespread arthropod tick throughout the tropics and subtropics because of its specialized feeding on domestic dogs (Walker et al. 2003), we thought that the finding present herein deserve further investigations to elucidate the epidemiological implications on B. rossi infections in dogs globally. Currently B. rossi is mainly confined to sub-Saharan Africa (Solano-Gallego and Baneth 2011) with few sporadic cases occurring in areas outside the African continent (Fritz 2010; Allison et al. 2011; Chomel 2011). Therefore, the circumstantial evidence from this report highlights a potential risk for transmission of B. rossi to dogs living in other geographical areas in the world via the spread of the infested R. sanguineus ticks. Further epidemiological surveillance and controls are necessary to prevent such a possibility. If possible, B. rossi should be included in the routine diagnosis of dogs with compatible clinical signs and hematological abnormalities in those countries, especially in regions with suitable environmental conditions supporting the presence of R. sanguineus ticks.

In conclusion, this study confirms the presence of B. rossi DNA in R. sanguineus ticks in Nigeria where B. rossi infection is prevalent, but the acclaimed vector tick, H. elliptica is rarely reported. We therefore ask, is this tick involved in the transmission of B. rossi to dogs in Nigeria? Based on prevailing evidences, the scientific community is inclined to answer negatively. However, there is the need to provide an evidence based data before accepting or dismissing the idea. In the interim, while experimental transmission studies might not be feasible, the use of molecular methods to determine the transovarial passage of B. rossi in R. sanguineus will be the next step in answering this question.