Introduction Genetic Characterization of Coxiella Burnetii in Amblyomma Varigatum Ticks from North-central Nigeria: Public Health Importance

coxiellosis have been associated with about 40 different tick species [6]. Even though a few of these Ticks are important vectors of various pathogenic tick species have been shown to be able to harbor and agents that cause disease in humans and animals; some maintain C. burnetii, they are not the primary vectors of of these agents, such as Coxiella burnetii, are considered this pathogen for transmission to animal or humans as emerging vector-borne pathogens as well as agents since their role in the natural cycle of C. burnetii of bioterrorism [1]. C. burnetii is an obligate intracellular, remains to be defined till date [4]. However, they are gram-negative, gamma proteobacteria that infect and thought to acquire the pathogen during a blood meal on cause a worldwide zoonoses, Q fever, in humans and infected animals and may transmit the bacterium to animals. Ticks act as reservoirs and responsible for the other mammals during the next blood meal or by transmission of the pathogen to animals through their aerogenic spread of dried tick faecal excretions [3,8]. bite or fecal contamination [2, 3], and the major source Amongst the tick genera that serve as reservoir for of dissemination of the pathogen in the environment as this pathogen, the genus Amblyomma seem to have a a result of the high concentration of the pathogen in tick high prevalence and capacity to maintain the Coxiella feces, saliva, and coxal fluid [4]. The infected domestic and Coxiella-like symbionts as observed in studies animals and pets through their milk, urine, feces, involving A. americanum and A. cajennense [9,10,11]. placental, and birth fluids are the main source of human The importance of this genus in the maintenance of infection [5,6]. tick-borne infectious pathogens and as vector of Although Inhalation of aerosolized C. burnetii several zoonotic agents requires that species in the organisms is the most important route of infection in genus be properly studied. humans, ingestion of raw milk or fresh dairy products In Nigeria, Amblyomma variegatum ticks are the can also cause infection [7]. Tick transmitted livestock largest, most abundant, and destructive on domestic animals, and pets, and are associated with the transmission of diseases such as anaplasmosis, babesiosis, heartwater, and rickettsiosis, which are of Abstract Aim: The purpose of this pilot study was to genetically identify and characterize Coxiella burnetii from Amblyomma varigatum ticks collected on cattle in North central Nigeria. Materials and Methods: A …


Introduction
coxiellosis have been associated with about 40 different tick species [6].Even though a few of these Ticks are important vectors of various pathogenic tick species have been shown to be able to harbor and agents that cause disease in humans and animals; some maintain C. burnetii, they are not the primary vectors of of these agents, such as Coxiella burnetii, are considered this pathogen for transmission to animal or humans as emerging vector-borne pathogens as well as agents since their role in the natural cycle of C. burnetii of bioterrorism [1].C. burnetii is an obligate intracellular, remains to be defined till date [4].However, they are gram-negative, gamma proteobacteria that infect and thought to acquire the pathogen during a blood meal on cause a worldwide zoonoses, Q fever, in humans and infected animals and may transmit the bacterium to animals.Ticks act as reservoirs and responsible for the other mammals during the next blood meal or by transmission of the pathogen to animals through their aerogenic spread of dried tick faecal excretions [3,8].bite or fecal contamination [2,3], and the major source Amongst the tick genera that serve as reservoir for of dissemination of the pathogen in the environment as this pathogen, the genus Amblyomma seem to have a a result of the high concentration of the pathogen in tick high prevalence and capacity to maintain the Coxiella feces, saliva, and coxal fluid [4].The infected domestic and Coxiella-like symbionts as observed in studies animals and pets through their milk, urine, feces, involving A. americanum and A. cajennense [9,10,11].placental, and birth fluids are the main source of human The importance of this genus in the maintenance of infection [5,6].
tick-borne infectious pathogens and as vector of Although Inhalation of aerosolized C. burnetii several zoonotic agents requires that species in the organisms is the most important route of infection in genus be properly studied.humans, ingestion of raw milk or fresh dairy products In Nigeria, Amblyomma variegatum ticks are the can also cause infection [7].Tick transmitted livestock largest, most abundant, and destructive on domestic animals, and pets, and are associated with the transmission of diseases such as anaplasmosis, babesiosis, heartwater, and rickettsiosis, which are of great importance to the livestock [12,13].The notoriety and have a tropical climate and a moderate annual of this tick in terms of damage to the skin of animals rainfall mean of about 1311.75cm[19].The area is during disease transmission and as pest has earned it home to some of the normadic Fulani pastoralists who the name "koti" (´dangerous tick´) amongst the Fulani are known to traverse from their origin in the semi-arid herdsmen of Nigeria, and thus is accorded much (Northern) parts of Nigeria to the sub-humid zones in attention during acaricidal treatment and the routine the South in search of trade and pasture for their hand de-ticking exercise [14].
animals.Some of these normads are beginning to settle Studies have shown that the collection of ticks and live in sedentary households for easy access to from hosts or vegetation and analysis by genus-specific regular markets in peri-urban areas where they trade and/or species specific PCR is an efficient method for their products [20].It is from animals in several of such the assessment of tick-borne pathogens since data from sedentary pastoralist communities that the tick samples such analysis are useful in the risk assessment of were collected for the study.emerging tick-borne diseases in a geographic area Sample collection: A total of 40 A. variegatum tick [15,16].
samples were collected from the body of cattle on the The presence of C. burnetti in other tick species basis of accessibility and convenience as a result of the have been reported [3,4].However, the importance of ethno-religious conflicts inherent in the areas sampled.

A. variegatum ticks in the Nigerian livestock industry
Each tick was carefully removed by grasping with a in terms of damage to hides and skin, and their blunt curved forceps as close to the skin surface as penchant to readily feed on man (especially the possible and pulled upward with a steady even pastoralists) and increase their risk of exposure to pressure, ensuring that the mouth parts were diseases of public health significance [17,18] completely removed with the whole tick.Damaged influenced our study.
ticks were removed and safely disposed while the In this paper, we present the result of a survey in intact ticks were cleaned by first sterilizing them in which the purpose was to use molecular methods and bleach and subsequently rinsing twice in distilled water phylogenetics to identify and characterize the zoonotic and then preserved in 70% ethanol.pathogen C. burnetii in A. variegatum ticks collected from pastoralist herds in North central Nigeria.The DNA samples within the Northern Guinea savanna region; with plain from A. variegatum ticks were subjected to a nested lands and hills measuring up to 3000ft above sea level PCR using oligonucleotide primers synthesized at some points, daily temperature range of 23°C-27°C, commercially by Sigma-Aldrich Biotechnology, L.P., least two clones from each purified plasmid were Germany.Primary and secondary PCR amplifications submitted for sequence confirmation in an automatic were carried out with primer designates; Q5 (5´-GCG sequencer (3730 DNA analyzer, Applied Biosystem®).GGT GAT GGT ACC ACA ACA-3´) and Q3 (5´-GGC DNA sequence analysis: Comparison of our sequences AAT CAC CAA TAA GGG CCG-3´) ; Q6 (5´-TT GCT with sequences previously deposited in the gene bank GGA ATG AAC CCC A-3´) and Q4 (5´-TC AAG CTC was done using the basic local alignment search tool CGC ACT CAT G-3´) respectively.The GenBank accession numbers of the C. burnetii WI, USA), 1µl of 10mM dNTPs, 1 µl (10 pmol) each of heat shock protein antigenic polypeptide (htpB) gene the primers (forward and reverse), 3 µl of 25mM sequences used to construct the neigbour-joining MgSO4, 1 µl of DNA sample (50 ng/µl), 1 µl of Tfl tree [23] includes; JN871859, JN871850, JN871863 Polymerase (Promega, Madison, WI, USA) and 32 µl and EF547935, while Ehrlichia spp., (JF949769) was nuclease-free water.The reaction mixture for the used as an outgroup.The distance matrix was secondary nested PCR was the same as the first PCR calculated by use of Kimura-2 parameters, whilst 1000 except that 0.5 µl of the PCR product (amplicons) from bootstrap replicates were used to estimate the the first reaction was used as the DNA template, reliabilities of the branches on the tree.The output of together with the Q6 and Q4 primers.Conditions for the tree was constructed with the MEGA 4.1 program the reactions were an initial denaturation of 94°C for 3 [24].minutes, 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and extension

at 72°C for 1 minute, and a final extension of 72°C for 5
Of the 40 partially fed A. variegatum ticks screened minutes.Conditions for the second PCR amplification for C. burnetii using a specific nested PCR, we were are as described above with the exception of the able to detect the pathogen DNA in 10 ticks samples.annealing temperature which was set at 52°C.This positive results from these C. burnetii-specific Amplification reactions were carried out in a PCR assays provide molecular evidence for C. burnetii thermocycler MyCycler 170-9711 (BioRad, Hercules, infection in the ticks, and with an infection rate of 25% CA, USA).Amplicons from the reactions were for the areas under investigation.The ticks collected visualized after electrophoresis on a 1% agarose gel from the various areas had different levels of infection, stained with SYBR Safe DNA gel stain (Invitrogen, with ticks from Barkin ladi and Lafia areas having the Eugene, OR, USA) and compared to a DNA molecular highest infection rate of 40% each.marker.
BLAST search for the sequenced amplicons Cloning and sequencing of the htpB gene: Like in most using the DDBJ/EMBL/ GenBank databases showed molecular biology studies where large quantities of similarity of 99%-100% to the partial sequence of C. DNA molecule can be isolated in pure form for detailed burnetii heat shock protein B gene (htpB) isolated from molecular analysis, we ensured that the DNA isolated goat milk (GenBank accession numbers: EU888861from A. variegatum ticks used in this study were EU888863) and Lions (EF547935).The sequences actually that of C. burnetii by cloning it into the derived from our study are deposited in the GenBank pGMET vector and purifying before sequencing.
under accession numbers JQ346185 -JQ346188.Amplified products from the PCR reactions were Phylogenetic analysis using neighbor-joining indicates purified using a Purelink PCR purification kit (Invitrogen, the grouping of C. burnetii sequencesfrom our study USA) according to the manufacturer's instructions, areas with that collected from Oyo state, South-western then ligated into pGEM®-T Vector System I Nigeria and Spain (Figure -2).(Promega, Madison, USA) and transformed into

JM109 high efficiency competent Escherichia coli
Previous studies on the presence of C. burnetii cells according to the manufacturer's protocol.The in Nigeria were centered on the serological analysis transformed cells were subsequently plated on X-gal/ of human sera [25]; fresh milk samples from cows; and IPTG plates.The small, colourless positive transformants suckling calves under different management systems were selected and cultured in LB-Ampicillin medium [26,5].In these studies, the prevalence rate of infection at 37 °C overnight.Purification of the plasmid DNA for the human sera was 3%, while prevalence for both was done with the QIAprep® Spin Miniprep kit the milk and animal samples ranged from 24.3% -(Qiagen, USA) while Proteus vulgaris II enzyme (Pvu 59.8%.II) kit (Fermentas®) was used to digest the Plasmid However, Reye et al., [22] recently used PCR to DNA for evaluation of their size on 1% agarose prior to establish the prevalence of 27% for C. burnetii sequencing.For every PCR sample that was cloned, at infection in ticks collected from the vegetation and infected tick collection and possibly include samples livestock in Oyo state, South West, Nigeria.In this from the pastoralists for analysis.study, we sampled regions of the country that have a outbreak in this population.
Neighbour-joining tree of 1000 replicates with Kimura 2 parameters from htpB gene sequences for 3 characterized C. burnetii isolates (highlighted in red) from North Central Nigeria.The tree illustrates the phylogenetic placement of C. burnetii detected in A. variegatum ticks from 2 states in North central Nigeria and those in ticks from South west Nigeria and Spain.