PARASITOLOGICAL AND MOLECULAR STUDIES ON TICKS AND BABESIASPECIES INFECTION OF DOGS IN THE FEDERAL CAPITAL TERRITORY, NIGERIA

ABSTRACT

The study aimed at determining the prevalence of Babesia species infection in dogs in the Federal Capital Territory, Abuja, Nigeria andidentification of ticks of the sampled animals. It further evaluated the parasitological and molecular characteristics of the ticks and Babesia species. A total of 480 asymptomatic dogs were sampled for tick and blood, using standard method. Ticks collected were identified to generic level using morphological features in a dissecting microscope. Thick and thin blood smears were prepared, Geimsa stained and examined microscopically, for intra-erythrocytic merozoites of Babesia spp. Haematological parameters of sampled dogs were evaluated using automated haematology analyser. Genomic DNA was extracted from 30 Rhipicephalus spp. of ticks and 15 B. canispositive whole blood, followed by partial amplification of 18S rRNA gene using genus specific primers. The PCR products were purified and sequenced. The chromatograms were trimmed in ApE software, and aligned to generate a consensus sequence for each isolate. The nucleotide sequence BLAST analysis was carried out in NCBI database. Correct sequences of 9R. sanguineus and 3B. c. vogeliwere obtained and deposited in the GenBank using Bankit tool.Multiple sequence alignments was carried out using ClustralW in MEGA7.0 software.Phylogenetic trees were constructed for Rhipicephalus and Babesia species using neighbour-joining and maximum likelihood methods respectively. The results showed an overall prevalence of 10.8% and 3.1% B. canis infection in dogs for thick and thin smears techniques respectively. The prevalence of Babesia infection in FCT based on sampled location was highest (12.5%) in Gwagwalada, Abuja Municipal and Kwali Area Councils and lowest (6.3%) in Abaji Area Council. The prevalence of B.canis infectionbased on sex showed that males had a higher (13.7%) than the females with 8.3% infection rate. The prevalence based on breed showed that exotic breeds had the highest (12.9%) while the cross breed had the lowest of 9.4% B. canis infection rate The prevalence based on purpose showed that hunting dogs had the highest (11.3%) while the pet dogs recorded no Babesiaspp. infections. The prevalence based on season showed that the rainy season had higher (14.6%) than the dry season with 7.1% of B. canis infection. The prevalence of Babesia infection based on tick infestations showed that tick infested dogs had higher (17.1%) than dogs without ticks, with 3.5% infection rate. The percentage of tick attachment based on predilection sites showed that the ear region had the highest (45%), while the scrotal and the mammary region had the lowest with 4.0%. There was significant (p < 0.05) association between rate of Babesia infection and age group, purpose, season and tick infestation. No significant association was observed between Babesiainfection and sex, breed, and sampled locations. The haematological parameters showed higher values for packed cell volume, haemoglobin concentration, red blood cell count and platelets in Babesia negative dogs.However,red blood cell count, lymphocytes and monocytes values were lower when compared with Babesia positive dogs. Out of the 2,043 ticks collected from sampled dogs, 99.9% were Rhipicephalus spp. and 0.1% were Amblyomma spp. The PCR amplified at 173bp and 612bp gene segments on gel electrophorogram for Rhipicephalus spp. andB. canisrespectively. The BLAST analysis showed 96-100% and 100% similarity with R. sanguineus and B. c. vogelirespectively in the GenBank. The phylogenetic trees showed that sequences of R. sanguineusisolates from the study area(accession numbers: KY 799078 – KY799086) clustered togather while the sequences of B. c. vogeli isolates (accession numbers: MF000388, MF000389, MF000390), separated into two different clades. The study concludes that R. sanguineus was the common ticks infesting dogs and B. canis vogeli was the subspecies of B. canis infectingthe sampled dogs in the study area.

Ticks are amongst the most important arthropod ecto-parasites of animals, being adapted to different climates and host species (Dantas-Torres et al., 2012). Hard ticks (order Ixodida, family Ixodidae) represent the most diverse group, occurring in tropical, temperate and even arctic regions (Estrada-Peña et al., 2012). Their medical and veterinary importance is mostly due to their great capacity to transmit infectious agents to vertebrate animals, which may cause a diverse range of conditions, commonly referred to as Tick-Borne Diseases (TBDs). As haematophagous obligate parasites, ticks are major vectors for a wide range of pathogens (e.g., viruses, bacteria and protozoa) of companion animals, livestock and humans (de la Fuente et al., 2008; Dantas-Torres et al., 2012). Tick-borne pathogens include species of Babesia, Anaplasma, Rickettsia, Ehrlichia, Theileria and Coxiella (Uilenberg, 1995).Babesia species are tick-borne Apicomplexan protozoan parasites of erythrocytes that infect a wide range of vertebrate hosts (Irwin, 2009), resulting to a disease condition known as babesiosis, with a worldwide distribution (Parnell et al., 2008; Otranto et al., 2009). The Babesia parasites replicate in the red blood cells and initiate a mechanism of antibody-mediated cytotoxic destruction of circulating erythrocytes (Zygner et al., 2007).

The disease is characterised by fever, splenomegaly, inappetence, weakness, lethargy, generalised lymphadenopathy, anaemia, haemoglobinuria and collapse associated with intra-and extravascular haemolysis, hypoxic injury, systemic inflammation, thrombocytopenia, and pigmenturia (Irwin, 2009). Canine Babesia infection (formarly canine piroplasmosis) is one of the most important tick-borne diseases of domestic and wild canidae (Abd Rani, 2011). It is characterized by a wide range of clinical manifestations; from subclinical disease to life-threatening conditions, with severity of clinical signs, depending on the species involved and host immune response against the infection (Otranto et al., 2010; De Tommasi et al., 2013). The diagnosis of canine Babesia infections and identification of each species has traditionally been based on host specificity and the morphology of the intra-erythrocytic forms in stained blood smear. The Babesiacanis and Babesiagibsoni are the major species infecting dogs, causing anaemia. Babesia canis is the large piroplasm (4-5µm), usually occurs as a single pear-shaped piroplasm or in pairs or in multiple merozoites divided by binary fission within the erythrocyte (Soulsby, 1982).

Recent advances in molecular biology techniques like Polymerase Chain Reaction (PCR) have made it possible to detect and identify piroplasms with greater sensitivity and specificity than the traditional methods (Birkenheuer et al., 2003 and Jeff eries et al., 2003). Molecular biology studieshave revealed that, on the basis of diff erences in the geographical distribution, vector specificity, and antigenic properties (Hauschild et al., 1995), B.canis is subdivided into three subspecies: namely, B. canis canis transmitted by Dermacentorreticulatus (in Europe), B.canis vogeli transmitted by Rhipicephalussanguineus (in tropical and sub-tropical regions), and B.canis rossi transmitted by Haemaphysaliselliptica (in South Africa)(Birkenheuer et al., 1999). There are also differences in their pathogenicity, as B.canis rossi causes a frequently fatal infection in domestic dogs, even after treatment; B. canis vogeli gives a moderate and often clinically inapparent infection, and B. canis canis infections result in a more variable pathogenicity intermediate between B. canis rossi and B. canis vogeli (Uilenberg et al., 2006). Babesia gibsoni is the small Babesia that commonly appears as individual ring forms or pyriform bodies ranging between 1.0 and 2.5µm in size (Conrad et al., 1991). The B. gibsoni was found to be associated with infection of dogs in Asia, North America, northern and eastern Africa, and Europe (Conrad et al., 1991). Dogs are most common domestic animals seen in almost every human settlement performing valuable roles in the society. In Nigeria, dogs are kept as pets, guards, for hunting, herding, breeding, as well as a source of animal protein among some ethnic groups (Aiyedun and Olugasa, 2012; Hambolu et al., 2014). Canine Babesia infection was first encountered in Nigeria, in 1962, and documented in the Veterinary Department annual report, as a disease associated frequently with higher severity among imported dogs than the indigenous breeds Leeflang and Ilemobade (1977). Several publications have shown different prevalence reports of the infection in different parts of the country based on microscopy (Ajayi et al., 2009; Ogo et al., 2011; Adamu et al., 2012; Okubanjo et al., 2013; Konto et al., 2014). Recent studies using molecular techniqueshave shown the presence of all the three Babesia canis subspecies:Babesia canis,Babesia vogeli and Babesia rossi inNigeria (Sasaki et al., 2007; Kamani et al., 2010; Takeet et al., 2017) with evidence of co-infections with Ehrlichia canis, Hepatozoon canis, Anaplasma platys, Theileria sp., Candidatus Neoehrlichia mikurensis and Rickettsia conorii israelensis(Kamani et al. 2013; Adamu et al. 2014). In the Federal Capital Territory, only few reports ofBabesiacanisinfections exist (Obeta et al., 2009; Jegede et al., 2014). Molecular techniques are being utilized in the diagnosis and in differentiating different infections caused by various Babesia subspecies (Oyamada et al., 2005).

1.2 Statement of Research Problem

Current global changes, such as climate change, deforestation, changes in land use, urbanization, increased trade and travel are affecting animal host populations worldwide (Dantas-Torres, 2015), favouring the establishment of ticks and their associated pathogens into previously free areas. Due to the ability of ticks to survive under different climatic conditions and ecological niches, they have a cosmopolitan distribution and their control is still a major challenge for veterinarians and pet owners (Cafarchia et al., 2015). Dantas-Torreset al. (2012), reported that among ixodid ticks, Rhipicephalus sanguineus sensu lato (s.l.) is a vector of Ehrlichia canis, Babesia vogeli and Hepatozoon canis in dogs and Rickettsia conorii and Rickettsia rickettsii in humans. The Rhipicephalussanguineus, (brown dog tick), is a good example of ‗parasite globalization‘ as is considered the most cosmopolitan species among the families, transmitting a wide range of pathogens to dogs and other animals including humans (Fourie et al., 2013). It can transmit a variety of pathogens to dogs, including Babesia canis vogeli, Ehrlichia canis, Anaplasma platys and Hepatozoon canis and also capable of transmitting pathogens to humans such as Rickettsia conorii, the agent of Mediterranean spotted fever (Chomel, 2011). Reports have shown that Babesiainfections in dogs occur worldwide (Solano-Gallego and Baneth 2011; Schnittger et al., 2012). There are over a hundred species of Babesia infecting vertebrate hosts and twelve have been documented to infect dogs (Irwin, 2010). There is a speculation that all vertebrates, including human, can be infected with Babesia, largely depending on their suitability as hosts for tick vectors (Schnittger et al., 2012). Some TBDs of companion animals are zoonotic (Cito et al., 2015) and may place human owners at risk of infection (Rijks et al., 2015). In Nigeria, studies have shown that Rhipicephalus, Boophilus,Hyalomma and Amblyomma species can infest dogs (Konto et al., 2014). However, the Rhipicephalus sanguineus is the most common ticks infesting dogs while Babesia is the predominant haemoparasite of dogs in the country (Amuta et al., 2010; Opara et al., 2017). The tick fauna was reported to peaks around the rainy and is attributed to the high humidity of 80% and optimum temperature of 27⁰C required for the hatching of eggs (Natala et al., 2009). Adamu et al. (2014), recorded 72% prevalence of haemoparasites infections in dogs from Jos, Plateau State. Amuta et al. (2010), reported 10.2% prevalence of canine babesiosis in Makurdi, Nigeria. Sasaki et al. (2007), reported the presence of Babesia canis rossi and B canis vogeli in 2.0% and 0.3% respectively using nested PCR and sequence analysis. Kamani et al. (2010), reported for the first time in an untraveled Nigerian dog the presence of Babesia canis canis on the African continent, as a co-infection with Babesia canis rossi, which is a rare occurrence. In areas where canine tick-borne diseases are endemic, dogs can be simultaneously infected with more than one tick-borne pathogen (Cardoso et al., 2010). Infection with Babesia canisis common in Nigerian dogs due to high prevalence of the tick vector, Rhipencephalus sanguineus (Abdullahi et al., 1990).Studies have shown an increasing prevalence of tick-borne pathogens in local dogs, with a recent report of Candidatus Neoerlichiamikurensis as an emerging disease agent of humans, in Rhipicephalus sanguineus and Haemaphysalis leachi ticks (Kamani et al., 2013). Rickettsia conorii israelensis, the causative agent of Mediterranean spotted fever was also detected in Rhipicephalus sanguineus ticks using molecular techniques (Kamani et al., 2013). An earlier study of 400 randomly sampled dogs, from different parts of Nigeria, showed 2.3% positive for B. canis rossi, while a single dog was positive for B. canis vogeli (Sasaki et al., 2007). Of the 500 blood samples from dogs presented to Veterinary Clinics in Ibadan, Oyo State; 26.0% were infected with B. caniswhile 20.2% were infected with B. gibsoni under microscopic examination (Oduye and Dipeolu, 1976). Babesia canis (sensulato) infection had earlier been reported in dogs from Zaria, Kaduna State (Useh et al., 2003). A low prevalence of 2.8% B. canis infection was recorded in a thin blood-smear survey of slaughtered dogs in Maiduguri, Borno State (Adamu et al., 2012). A prevalence of 4.8% to 14.6% B. canis rossi infection was recorded in dogs presented to veterinary hospitals in four states, comprising; Rivers, Plateau, Kaduna and Nasarrawa (Kamani et al., 2010). Other reports include B. canis vogeli infectionin a dog in Kaduna;B. canis (sensu stricto) and B. canis rossi co-infection in a dog domiciled in Vom, Plateau State (Kamani et al., 2010). These documented reports being the first confirmation of the occurrence of B. canis canis in a region where the tick-vector, Dermacentorreticulates, had not been reported, have stimulated a renewed interest in the epidemiology of canine babesiosis in Nigeria (Kamani et al., 2010).

1.3 Justification for the Research

Despite huge resources committed towards tick control projects, especially for companion animals and livestock, the incidence of TBDs has been reported to be on the increase globally (Dantas-Torres et al., 2012).Reports have shown that due to the ability of ticks to survive under different climatic conditions and ecological niches, they have a cosmopolitan distribution and their control is still a major challenge for veterinarians and pet owners (Cafarchia et al., 2015). The efforts towards the production of effective anti-tick vaccines have not been successful and dogs appear to develop no immunity against ticks (Nuttall et al., 2006). While our understanding of Babesia is improving, diagnosis and treatment of Babesia infections remain challenging. The morphological similarity between species and subspecies of Babesia has led to much confusion over accurate diagnosis using microscopy (Jefferies et al., 2007).The use of molecular tools; such as PCR and sequence analyses has increased the understanding of the distribution of TBDs and the role of ticks in the infection (Dantas-Torres et al., 2012). The techniques also offers an effective and rapid means of differentiating between species (Jefferies et al., 2007; Gulanber et al., 2006).Cross antigenicity seen in the B. canis subspecies is thought to be responsible for vaccine failures in the field, thus the possibility of developing potent vaccines against canine babesiosis will be dependent on the proper identification of the subspecies (Schetters et al., 1995). The pathogenicity of Babesia is believed to vary in different regions due to host factors and/or differences in the species present. Therefore, accurate identification of tick vectors and theBabesia species infections is an important factor in the monitoring, management and control of tick-borne diseases (Abdel Aziz et al., 2014). Combination of PCR and nucleotide sequence analysis may increase information about subspecies or strains of the parasite. Knowledge obtained from cross-species or cross-genus comparisons may be helpful in improving existing prophylaxis, treatment, and management of the disease and may provide a better understanding of their underlying biology and evolution (Lau, 2009). Being companion animals, the diseases of dogs are of great concern, both directly because of financial burden for owners and the negative impact on animal health, and indirectly because of transmission of pathogens to humans and animals (Fourie et al., 2013). It has also been reported that Nigeria does not consider canine babesiosis as a reportable disease and as such does not enforce screening of dogs imported into the country, which may possibly introduce foreign tick vectors and their associated parasites/diseases (Ogo et al., 2011). There is a paucity of information on the molecular characterisation of ticks and Babesia spp of dogs in the study area. This research was conducted with the aim of determing the prevalence distributions of Babesia species infection of dogs in the Federal Capital Territory, identify the ticks found on them using morphological features, evaluate the haematological indices of sampled dogs, and further characterize the ticks and Babesia sp. in the blood of infected dogs using Polymerase Chain Reaction and Sequence analysis.

1.4 Research Questions

1. What is the prevalence of Babesia infection in dogs in FCT?
2. What are the tick species infesting sampled dogs in the study area?
3. What are the haematological indices of dogs sampled for ticks and Babesia in the FCT?
4. What is the molecular characteristics ticks and Babesia species from sampled dogs in FCT?

5. What is the relationship of the sequences of ticks and Babesia from dogs in the FCT, with reference sequences in the GenBank?

1.5 Aim of the Study

To conduct parasitological and molecular study on ticks and Babesia speciesin dogs in Federal Capital Territory, Abuja, Nigeria.

1.6 The Specific Objectives of the Study

1. Determine the prevalence of Babesia in dogs in the FCT
2. Identify tick species in sampled dogs in the FCT using morphological features.
3. Determine the haematological parameters of sampled dogs in the study area.
4. To evaluate the molecular characteristics of ticks and Babesia of the sampled dogs in FCT using Molecular techniques.
5. To determine the relationship of the sequences of ticks and Babesiafrom the study area with reference sequences in the GenBank.