Nigerian Veterinary Journal 38(3). 2017 Ogo et al

NIGERIAN VETERINARY JOURNAL ISSN 0331-3026

Nig. Vet. J., September 2017 Vol 38 (3): 260-267. ORIGINAL ARTICLE

Morphological and Molecular Characterization of variegatum (: ) from Nigeria

Ogo, N. I.1; Okubanjo, O. O. 2; Inuwa, H. M. 3 and Agbede, R. I. S.4

1National Veterinary Research Institute, Vom, Plateau State. 2Department of Veterinary Parasitology and Entomology, Ahmadu Bello University, Zaria, Nigeria. 3Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria. 4Department of Veterinary Parasitology and Entomology, University of Abuja, FCT, Nigeria. *Corresponding author: Email: [email protected]; Tel No:+234 8034521514

SUMMARY The association of most -borne pathogens with specific tick species has made it imperative that proper identification and characterization of such tick vectors is necessary for the purpose of developing effective tick and tick-borne control strategies. This study was undertaken to identify and characterize Amblyomma species ticks collected from cattle in Plateau State, North-Central, Nigeria. They were morphologically identified using diagnostic characters. Further confirmation and characterization was done genetically using a 460bp-long partial fragment of the 16S rRNA gene amplified by polymerase chain reaction (PCR). The amplified fragment was cloned and sequenced for the phylogenetic dendogram. All the examined ticks were identified as A. variegatum which was confirmed by 16S rRNA gene sequences analysis, and phylogenetic inferences showed a 99% similarity and grouping to A. variegatum of African origin. However, the A. variegatum sequences from Nigeria were clustered into 2 groups, but formed a distinct clade from the A. variegatum sequence from Ethiopia. This study was able to conclusively identify and characterize A. variegatum ticks from the study areas by utilizing morphology and molecular genotyping based on sequencing of the 16S rRNA gene.

Key words: Amblyomma variegatum, Morphology, 16S rRNA, PCR, Sequencing.

INTRODUCTION The genus Amblyomma has 130 valid 2008). Most of the Amblyomma spp ticks so species most of which are known to transmit far studied are known to accidentally a wide variety of bacterial, fungal, parasitize humans and potentially act as protozoan, rickettsial, and viral agents in vectors of zoonoses (Marrelli et al., 2007), man, companion and domestic . and consequently are of significant medical Guglielmone et al., 2010). Besides their and veterinary importance. Of the above manifested abilities as vectors, they also number of Amblyomma species in the world, inflict notable dermatitis and predispose six species (A. gemma, A. lepidum, A. their hosts to other -borne maculatum, A. nuttalli, A. splendidum and infestations such as myiasis. (Molia et al., A. variegatum) have been reported in

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Nigeria (Nuttal et al., 1926; Mohammed, pathogens at different taxonomic levels 1974; Talabi et al., 2011; Tonjura et al., (Chao et al., 2009; Chitimia et al., 2010). 2012; Eyo et al., 2014). However, none of Molecular characterisation and Phylogenetic these studies described the morphological studies on ticks is imperative as such study features of the different species mentioned, has not been hitherto carried out in the study nor were they subjected to any molecular area. This preliminary study, thus focused method for confirmation. Also, there are no on the use of morphological and molecular reference specimen of these species (except tools to identify and characterize A. for A. variegatum) in the archives for variegatum ticks collected from cattle in the comparison and differential diagnosis. Thus, Guinea Savanna vegetation zone of Plateau casting doubts on the authenticity of the state, Nigeria. We also inferred the mentioned species. phylogenetic position of sequences of A. The importance of different stages and variegatum species from our study areas species of ticks in disease transmission and with respect to other Amblyomma species as reservoir of infectious pathogens in nature from other parts of the world based on the requires proper identification of ticks which analysis of mitochondrial 16S rRNA gene is also necessary for field research on tick sequences of the ticks. ecology, and for effective control measures, and veterinary intervention. Traditionally, MATERIALS AND METHODS tick identification is achieved using several Tick Collection and Preservation developmental stages (larvae, nymphs and Partially fed Amblyomma sp. ticks (n=200) adults) based on the external anatomy and were collected from 50 cattle owned by morphological features of the ticks. This Fulani pastoralists in three Local method of identification could be laborious, Government Areas (LGA) of Plateau State, difficult and sometimes impossible Nigeria. These local government areas especially when using phenotypic include: (Barkin Ladi; 9°32´00´´N– characteristics which sometimes overlap 8°54´00´´E, Mangu; 9°18´00´´N– between individual species or when dealing 9°11´34´´E, and Jos-South; 9°47´00´´N– with the immature stages or females of some 8°51´00´´) which have high concentration of tick species (Nava et al., 2009). These pastoralists in the state (Majekodunmi et al., difficulties have led to the use of other 2014). The vegetation of our study areas techniques either solely or in combination consists of short trees, grasses and plateau with the phenotypic characteristics in the type mosaic vegetation, and lies within the identification and understanding of the Northern Guinea Savannah zone. The area relationship of ticks (Mangold et al., 1998). records an average daily temperature range Advances in molecular biology techniques of 23 °C–27 °C and has a tropical climate have made the identification of tick species with a moderate annual rainfall mean of using any of the tick stages or eggs, easy about 1311.75 cm (Odumodu, 1983). (Zahler et al., 1995). Such techniques Ticks were carefully removed by grasping include DNA sequencing of molecular with a blunt curved forceps as close to the markers such as internal-transcribed-spacers skin over the scutum and pulled outward (ITS 1&2), 16S, 12S, and 18S rDNA genes with a steady even pressure, ensuring that (Norris et al., 1999; Labruna et al., 2007; the mouth parts were completely removed Marrelli et al., 2007). Previous studies have with the whole tick. Damaged ticks were demonstrated that these genes provide useful removed and safely disposed while the intact markers for the genetic identification and ticks were cleaned by first sterilizing them characterization studies, and the in sodium hypochlorite and subsequently phylogenetic relationships of ticks and their rinsing twice in distilled water then

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Nigerian Veterinary Journal 38(3). 2017 Ogo et al preserved in 70% ethanol into which tested alongside and if there was a reaction 5% glycerine was added. the entire mixture were discarded. Morphological identification to species level The amplicons from the above PCR where was done with the aid of a Zeiss electrophoresed on 1.5% agarose gel, stereomicroscope at X60 magnification and stained with SYBR Safe DNA gel stain Olympus microscope at ×100 magnification. (Invitrogen, Eugene, OR, USA) and size of Diagnostic characters of the different parts the amplified fragments were checked by of the ticks (hypostome, dorsum, legs and comparing the fragments to a standard spurs, including the shapes and positions of molecular weight marker (1 kb MassRuler®, the anal groove, festoons, genital aperture Fermentas). The amplified DNAs were and spiracles) were carefully observed and purified using Purelink™ PCR purification used in drawing conclusions according to kit (Invitrogen™, USA), then cloned into the guidelines for tick identification as pGEM-T vector (Promega, Madison) and provided by Okello-Onen et al., (1999) and transformed into JM109 high-efficiency Walker et al., (2003). Ten (10) competent Escherichia coli cells. Plasmid representative tick samples (5 females and 5 DNA was purified with the QIAprep® Spin males) from the study areas were used for Miniprep kit (Qiagen, Valencia, CA) whilst molecular studies. a Pvu II enzyme kit (Fermentas®, Vilnius) was used to digest the plasmid DNA for size DNA Extraction from Ticks evaluation on 1% agarose prior to Total genomic DNA (50 ng/µL – 80 ng/µL) sequencing. At least two clones from each was isolated from the whole ticks. Briefly, purified plasmid were submitted for ticks from the areas under study were sequence confirmation in an automatic individually dissected using separate sterile sequencer (3730 DNA analyzer, Applied scalpel blades to remove the internal organs. Biosystems®, Carlsbad, CA). DNA was extracted from all the organs using TriReagent (Sigma–Aldrich, Madrid, Sequence and Phylogenetic Studies Spain) according to the manufacturer’s BLAST search for previously reported instructions. Isolated DNA were run on 1% sequences that are identical to the sequences agarose gel to evaluate their integrity. DNA in this study was done using the NCBI concentration was determined with a database Nanodrop ND-1000 spectrophotometer (http://www.ncbi.nlm.nih.gov/BLAST). (Wilmington, DE, USA), and then stored at Multiple alignments were carried out using −20 ◦C until used for further analysis. Clustal W algorithm (Thompson et al., 1994). The output of the tree was PCR Amplification, Cloning and constructed with the MEGA 4.0 program Sequencing of the 16S rRNA Gene (Tamura et al., 2007). For pairwise Primer sets and conditions for Tick 16S comparisons, the following 16S rRNA rRNA gene (forward 16S+1 5ʹ- CTG CTC sequences of Amblyomma species deposited AAT GAT TTT TTA AAT T-3ʹ and reverse in the GenBank were used; A. hebraeum 16S−1 5ʹ-CCG GTC TGA ACT CAG ATC (L34316), A. americanum (L34313), A. AAG TA-3ʹ) designed by Black & Piesman, variegatum (L34315) and A. cajennense (1994) that target the 460bp fragment of 16S (L34317), while Ixodes scapularis (L34293) rRNA genes for ticks were used. DNA from was used as out group. The distance matrix A. americanum was used as positive control; was calculated by use of Kimura-2 whereas negative controls containing the parameters, whilst 1000 bootstrap replicates reaction mix without DNA were always were used to estimate the reliabilities of the nodes on the neighbour-joining tree. A

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Nigerian Veterinary Journal 38(3). 2017 Ogo et al bootstrap value of 70% was considered boostrap support value of 99% comprised significant evidence for phylogenetic three sequences (JF949794, JF949795 and grouping (Hills and Bulls, 1993). JF949798) while the second cluster with 71% boostrap value had one sequence RESULTS (JF949799). Also, the high boostrap value All the 200 (100%) sampled ticks were supports the close relationship between the morphologically identified as A. variegatum A. variegatum ticks and A. hebraeum based on the very colourful scutum which (L34316), both of which are of African covered the entire dorsum of the male and origin. the anterior one third of the female. The punctations on the scutum were small to medium in size; the basis capituli were DISCUSSION rectangular and had posterior margins Tick species belonging to the genus slightly concave shaped. The ticks examined Amblyomma are known to be reservoirs and had long palps with 3 segments; the vectors of several disease pathogens that can hypostome were long with dentitions. The cause considerable economic losses in festoons of the ticks were prominent and livestock in Nigeria, as in most tropical and dark coloured, where as the marginale subtropical regions of the world (Ogo et al., grooves were continous. The legs had 2012; Vesco et al., 2013). The notoriety of brownish colours with white bands at the these ticks in terms of damage to the skin of extremities, the coxae I had two unequal animals and disease transmission has earned spurs, while coxae IV had a single stout and them the name “koti” (´dangerous tick´) long spur (the spur on coxae IV of female amongst the Fulani herdsmen of Nigeria, ticks were short and not as prominent as in and are accorded much attention during the male) The spiracles were large, acaricidal treatment and the routine hand de- triangular, with rounded edges and located ticking exercise (Bayer & Maina 1984). behind coxae IV (Plate 1). The identification of ticks in Nigeria is PCR analysis of the isolated DNA indicates usually achieved using morphological amplification of 460bp. Blast results for our characteristics. However, when sampled cloned sample sequences across the ticks become damaged, or closely related DDBJ/EMBL/GenBank databases shows species are available this method becomes that all the submitted samples had 99% inadequate and may require a more decisive similarity to mitochondrial A. variegatum molecular based method. Our inability to 16S rRNA, Genbank accession numbers access previously reported species of L34312 and L34315. All the seven Amblyomma in Nigeria limited this study as sequences of A. variegatum obtained in this it did not afford us the opportunity to draw study were deposited in the GenBank under inferences. However, the presence of several accession numbers: JF949794 - JF949801. diagnostic characteristic features on the To access the phylogenetic relationships of samples studied phenotypically identified A. variegatum ticks from Nigeria, a them as A. variegatum. Confirmation of the neighbour-joining tree was constructed species using PCR analysis of the 16S rRNA using 16S rRNA nucleotide sequence gene marker produced 460bp fragments alignments based on Kimura two-parameter which were consistent with the range of model (Figure 1). All the A. variegatum tick fragment lengths of previously reported sequences were clustered into one group studies (Guglielmone et al., 2013) on ticks with 100% boostrap value. However, the in the same genus using the 16S rRNA gene. Nigerian A. variegatum ticks were separated This target gene is highly conserved, with into two clusters. The first cluster with a large numbers of registered sequences in the

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Plate 1: Adult male (A) and female (D) A. variegatum showing medium sized punctations (1) on their coloured scutum; B shows banded legs (2), two unequal spurs on coxae 1(3), and a single long spur on coxae IV (4) of a male tick; E, short spur on coxae IV (5) of a female tick and triangular shaped spiracles (6); C, F shows the long hypostome and genital aperture (7) and (8) respectively

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GenBank, and is suitable for species identification and validation. In this study, we phylogenetically established the status of A. variegatum ticks from Plateau State, Nigeria, based on the partial sequence of 16S rRNA gene. Results of the phylogenetic tree showed a clustering of A. variegatum tick sequences into two groups with 99% and 71% boostrap values. Based on the above values, the possibility of having a diverse population of A. variegatum ticks in our study area cannot be ruled out. However, a more detailed phylogenetic study of this tick species needs to be undertaken to confirm any Figure 1: Phylogenetic relationships of 16S rRNA gene intraspecies variation. sequences of A. variegatum ticks from Nigeria and 5 This study provides the first Amblyomma species based on neighbour-joining reconstruction genetic characterization of using the Kimura two-parameter distance. Bootstrap values are the mitochondrial 16S rRNA shown above the nodes. The scale bar indicates the nucleotide gene of A. variegatum ticks substitution per site. A. variegatum sequences detected in this collected from Nigeria, and study (JF949795, JF949794, JF949798 and JF949799) are supports the use of both highlighted in red. Ixodes scapularis (GenBank number morphological and molecular L34293) was used as outgroup species methods in conjunction with bioinformatics tools for the BLACK, W.C., and PIESMAN, J. (1994). identification of ticks in Phylogeny of hard ticks and soft- Nigeria. ticks taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences. ACKNOWLEDGEMENT Proceedings of the National Authors are grateful to the management of Academy Science. 91: 10034-10038. National Veterinary Research Institute, CHAO, L., WU, W. and SHIH, C. (2009). Vom, Plateau State, Nigeria for their Molecular analysis of Ixodes permission to conduct this study. granulatus, a possible vector tick for Borrelia burgdorferi sensu lato in REFERENCES Taiwan. Experimental & Applied BAYER, W., and MAINA, J.A. (1984). Acarology. 48: 329–344. Seasonal pattern of tick load in CHITIMIA, L., LIN, R.Q., COSOROABA, Bunaji cattle in the subhumid zone of I., WU, X.Y., SONG, H.Q., YUAN, Nigeria. Veterinary Parasitology. 15: Z.G., and ZHU, X.Q. (2010). 301-307. Genetic characterization of ticks from Southwestern Romania by

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