International Journal for Parasitology 42 (2012) 187–195

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International Journal for Parasitology

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Functional genomics studies of Rhipicephalus (Boophilus) annulatus ticksin response to infection with the cattle protozoan parasite, Babesia bigemina q

Sandra Antunes a, Ruth C. Galindo b, Consuelo Almazán c, Natasha Rudenko d, Marina Golovchenko d,Libor Grubhoffer d, Varda Shkap e, Virgílio do Rosário a, José de la Fuente b,f, Ana Domingos a,g,⇑a Instituto de Higiene e Medicina Tropical, Rua da Junqueira 100, 1349-008 Lisboa, Portugalb Instituto de Investigación en Recursos Cinegéticos IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, Spainc Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Tamaulipas, Km. 5 carretera Victoria-Mante, CP 87000 Ciudad Victoria, Tamaulipas, Mexicod Institute of Parasitology of the Academy of Sciences of the Czech Republic, Ceske Budejovice 37005, Czech Republice Kimron Veterinary Institute, P.O. Box 12, Bet Dagan, 50250, Israelf Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USAg Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Rua da Junqueira 100, 1349-008 Lisboa, Portugal

a r t i c l e i n f o

Article history:Received 13 October 2011Received in revised form 5 December 2011Accepted 6 December 2011Available online 8 January 2012

Keywords:TickGenomicsBabesiaRhipicephalusBoophilusRNA interferenceVaccine

0020-7519/$36.00 � 2012 Australian Society for Paradoi:10.1016/j.ijpara.2011.12.003

q Note: Expressed sequences tags presented withinGenBank under the Accession Nos. JK489362–JK4894⇑ Corresponding author at: Instituto de Higiene e

Junqueira 100, 1349-008 Lisboa, Portugal. Tel.: +351 221 05.

E-mail address: adomingos@ihmt.unl.pt (A. Domin

a b s t r a c t

Ticks are obligate haematophagous ectoparasites of wild and domestic animals as well as humans, con-sidered to be second worldwide to mosquitoes as vectors of human diseases, but the most important vec-tors of disease-causing pathogens in domestic and wild animals. Babesia spp. are tick-borne pathogensthat cause a disease called babesiosis in a wide range of animals and in humans. In particular, Babesiabovis and Babesia bigemina are transmitted by cattle ticks, Rhipicephalus (Boophilus) annulatus and Rhipi-cephalus microplus, which are considered the most important cattle ectoparasites with major economicimpacts on cattle production. The objectives of this study were to identify R. annulatus genes differen-tially expressed in response to infection with B. bigemina. Functional analyses were conducted on selectedgenes by RNA interference in both R. annulatus and R. microplus ticks. Eight hundred randomly selectedsuppression-subtractive hybridisation library clones were sequenced and analysed. Molecular functionGene Ontology assignments showed that the obtained tick sequences encoded for proteins with differentcellular functions. Differentially expressed genes with putative functions in tick–pathogen interactionswere selected for validation of SSH results by real-time reverse transcription-PCR. Genes encoding forTROSPA, calreticulin, ricinusin and serum amyloid A were over-expressed in B. bigemina-infected tickswhile Kunitz-type protease inhibitor 5 mRNA levels were down-regulated in infected ticks. Functionalanalysis of differentially expressed genes by double stranded RNA-mediated RNAi showed that underthe conditions of the present study knockdown of TROSPA and serum amyloid A significantly reducedB. bigemina infection levels in R. annulatus while in R. microplus, knockdown of TROSPA, serum amyloidA and calreticulin also reduced pathogen infection levels when compared with controls. Several studieshave characterised the tick–pathogen interface at the molecular level. However, to our knowledge this isthe first report of functional genomics studies in R. annulatus infected with B. bigemina. The resultsreported here increase our understanding of the role of tick genes in Babesia infection/multiplication.

� 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Ticks are obligate haematophagous ectoparasites of wild anddomestic animals and humans classified in the subclass Acari,order Parasitiformes, suborder Ixodida. These arthropods are

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Medicina Tropical, Rua da13652600; fax: +351 21 363

gos).

distributed worldwide from Arctic to tropical regions and are con-sidered as relevant as mosquitoes as vectors of human diseases,but the most important vectors of disease-causing pathogens indomestic and wild animals (Kompen, 2005; de la Fuente et al.,2008a,b).

Tick-borne pathogens of the genus Babesia are Apicomplexanparasites responsible for babesiosis, a disease that affects a widerange of animals and occasionally humans. The major economicimpact of babesiosis is on the cattle industry, caused by infectionwith Babesia bovis and Babesia bigemina. Rhipicephalus (Boophilus)spp. ticks are their principal vectors and are considered one ofthe most important cattle ectoparasites due to their direct impact

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188 S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195

and pathogen transmission that affects leather, meat and milk pro-duction (Bock et al., 2004).

Control of tick infestations has been primarily based on the useof acaricides. However, application of acaricides has had limitedefficacy in reducing tick infestations and is often accompanied byserious drawbacks, including the selection of acaricide-resistantticks, environmental contamination and contamination of milkand meat products with acaricide residues (Graf et al., 2004).

Alternatives to conventional acaricide treatments have beendeveloped in recent years as represented by tick vaccines. TheBm86 tick vaccine antigen, which was used in the first and onlycommercially available cattle tick vaccines, reduced tick numbers,weight and reproductive performance of female ticks which re-sulted in reduction of cattle tick populations over time (de laFuente and Kocan, 2006, 2007a; Willadsen, 2006).

Reduction of tick infestations has been the goal of tick vaccinedevelopment. However, reduction of tick-borne pathogen trans-mission represents an equally important goal. Recent studies dem-onstrated that tick vaccines reduce tick pathogen infection whenusing antigens found to be related in pathogen infection/multipli-cation, illustrating the complexity of tick–pathogen co-evolution(de la Fuente et al., 2011). Developing vaccines by targeting bothpathogen transmission and tick infestations will likely be a feasibleand productive strategy (de la Fuente et al., 2006, 2011; Labudaet al., 2006).

Molecular interactions at the tick–pathogen interface ensuresurvival and development of both the pathogen and tick vector.While recent studies have demonstrated that tick gene expressionis modified in response to pathogen infection (Macaluso et al.,2003; Mulenga et al., 2003; Nene et al., 2004; Rudenko et al.,2005; de la Fuente et al., 2007b,c; Villar et al., 2010; Zivkovicet al., 2010a; Mercado-Curiel et al., 2011), information on the func-tion of differentially expressed genes is limited (de la Fuente et al.,2007b,c, 2008; Kocan et al., 2009; Villar et al., 2010; Zivkovic et al.,2010a,b; Mercado-Curiel et al., 2011). RNA interference (RNAi) hasbeen shown to be a useful tool for functional characterisation ofgenes involved in tick–host–pathogen interactions and selectionof candidate tick protective antigens (de la Fuente et al., 2007b).

The objectives of this study were the identification of Rhipiceph-alus (Boophilus) annulatus genes differentially expressed inresponse to infection with B. bigemina by using suppression-sub-tractive hybridisation (SSH), which allows the identification of rareand novel differentially expressed genes (Diatchenko et al., 1996,1999). The results of the SSH studies were validated by real-timereverse transcription (RT)-PCR. Functional analyses were con-ducted by RNAi on selected genes in both R. annulatus and Rhipi-cephalus microplus ticks to determine the putative role of thesegenes in B. bigemina–tick interactions.

This study is a fundamental contribution towards the under-standing of the pathogen–tick interface and will likely contributeto the development of new generation pathogen transmission-blocking vaccines designed to prevent transmission and reduceexposure of vertebrate hosts to tick-borne Babesia parasites.

2. Material and methods

2.1. Ticks

The non-infected and B. bigemina-infected R. annulatus ticksused for construction of the SSH library were provided by theKimron Veterinary Institute, Israel. The Anaplasma- and Babesia-free R. annulatus (Mercedes strain, Texas, USA) and R. microplus(Media Joya strain, CENAPA, Mexico) ticks used for the RNAi exper-iments were obtained from a laboratory colony maintained at theUniversity of Tamaulipas, Mexico. Originally, these tick strains

were collected from infested cattle. Ticks were maintained oncattle at the tick rearing facilities at the Kimron VeterinaryInstitute or the University of Tamaulipas. Animal ethics approvalwas given for this work. Larvae were kept off-host in an incubatorat 20–25 �C with 95% relative humidity and 12 h light:12 h darkphotoperiod. Cattle were cared for in Israel and Mexico inaccordance with standards specified in the Guide for Care andUse of Laboratory Animals.

2.2. Non-infected and B. bigemina-infected ticks for SSH libraryconstruction

Two 3–4 months old male Friesian calves, free of babesiosis,were used to obtain B. bigemina-infected R. annulatus female ticks.Prior to tick infestation, calves were tested for antibodies to Babesiaspp. infection using an immunoflourescence assay (Shkap et al.,2005) and kept under strict tick-free conditions. One calf was inoc-ulated i.v. with cryopreserved 2 � 108 B. bigemina (Moledet strain).To obtain infected ticks, R. annulatus ticks were fed on the infectedcalf. Engorged adult female ticks were collected from both the in-fected and non-infected calf after feeding and maintained at 28 �Cand 80% humidity.

2.3. cDNA library construction and SSH

Ticks were twice rinsed individually in distiled water, once in75% (v/v) ethanol and once more in water. Each tick was dissectedand whole internal organs were placed in a 2 ml tube with 1 ml ofTri Reagent (Sigma–Aldrich, St. Louis, MO, USA). Total RNA andDNA were isolated according to the manufacturer’s protocol. APCR was performed to detect the presence of B. bigemina in thesampled ticks using primers Bbi400F: 50-AGCTTGCTTTCA-CAACTCGCC-30 and Bbi400R: 50-TTGGTGCTTTGACCGACGACAT-30

which amplify a 400 bp fragment within the conserved region ofthe five rap-1a paralogous genes (Suarez et al., 2003).

Total RNA was isolated from nine R. annulatus engorged femalesinfected with B. bigemina and nine engorged non-infected ticks.RNA quality was checked by gel electrophoreses to confirm theintegrity of RNA preparations. Two pools corresponding to the in-fected and non-infected tick populations were made. Poly A+ RNAwas isolated using the FastTrack� 2.0 mRNA Isolation Kit (Invitro-gen life Technologies, Carlsbad, CA, USA). The cDNA was synthes-ised and the SSH library was constructed using the PCR-Select™cDNA subtraction Kit (Clontech-Takara, Mountain View, CA, USA).Briefly, double stranded cDNA from both groups (infected andnon-infected ticks) was digested with RsaI. Part of the digestedcDNA was ligated with Adapter 1 and part with the Adapter 2R;the rest of the cDNA was saved to be used as a driver in hybridisa-tion. The forward subtracted library was made by hybridisingadapter ligated cDNA from B. bigemina infected ticks as the testerin the presence of non-infected tick cDNA as the driver. This reac-tion was designed to produce clones that are upregulated in in-fected ticks. Differentially expressed cDNAs were PCR amplifiedwith Advantage PCR polymerase mix, cloned using TOPO TA Clon-ing� Kit for sequencing (Invitrogen life Technologies, Carlsbad, CA,USA), transformed into One Shot� TOP10 cells (Invitrogen lifeTechnologies, Carlsbad, CA, USA) and plated on Lennox broth(LB)/agar with ampicillin (100 lg/ml), X-gal (5-bromo-4-chloro-indolyl-beta D-galactoside) (40 lg/ml) and IPTG (isopropyl-beta-thio galactopyranoside) (0.2 mM). Transformed cells were grown,the number of recombinant colonies was estimated and the pres-ence of the inserts was confirmed by PCR using the universal T3and T7 primers. The colonies having the confirmed inserts werethen inoculated into LB/ampicillin and grown overnight for plas-mid DNA purification.

S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195 189

2.4. Sequence analysis and database search

Plasmid DNAs were purified using the Illustra plasmid PrepMini Spin Kit (GE HealthCare, Buckinghamshire, UK) and 800clones from the SSH library were randomly selected and sequencedat the Department of Genome Sciences, University of Washington,USA. The cDNA Annotation System software (dCAS; Bioinformaticsand Scientific IT Program (BSIP), Office of Technology InformationSystems (OTIS), National Institute of Allergy and Infectious Dis-eases (NIAID), Bethesda, MD, USA) (http://exon.niaid.nih.gov)(Guo et al., 2009) was used for automated sequence clean up,assembly, blasting against the non-redundant (nr) sequence data-base and databases of tick-specific sequences (http://www.ncbi.nlm.nih.gov and http://www.vectorbase.org/index.php) and GeneOntology (GO) molecular function assignments. Protein ontologywas also analysed using the protein reference database (http://www.proteinlounge.com). Nucleotide sequences were alignedusing the programme AlignX (Vector NTI Suite V 5.5, InforMax,North Bethesda, MD, USA) and protein sequences were alignedusing the CLUSTAL 2.1 multiple sequence alignment tool (EMBL-EBI; http://www.ebi.ac.uk/Tools/). The phylogram was constructedwith protein sequences using the Neighbor-Joining method (Saitouand Nei, 1987). The evolutionary distances were computed usingthe Poisson correction method (Zuckerkandl and Pauling, 1965)and expressed in the units of the number of amino acid substitu-tions per site (EMBL-EBI; http://www.ebi.ac.uk/Tools/). Sequenceswere deposited in GenBank with Accession Nos. JK489362–JK489457.

Table 1Sequences of primers used for real-time reverse transcription-PCR.

Gene Upstream/down

Hebrain-like AGAACTCTCTGCTCTTGATGAGA

Mucin-like ACCGTCGCCTACGGACGTAGAAT

Calreticulin TGAGAGTCTTGCGTCATCCTCCT

Kunitz-type proteinase inhibitor SHPI-1 chain CCAAGAGTTGCACACATTTCAGG

Protease inhibitor carrapatin ACACTACCCTAATCGGAAAGTAA

Microplusin-like TCACTTCCAGGAACTCTGAGCTCA

GP80 precursor CCAACTCGCTCAGTTGGACACCA

Microplusin preprotein AGAAGTGCACAGCAACAACTGA

Kunitz-type protease inhibitor 5 (KTPI) AGCAGCACGTGTGACCAAGACT

Chaperonin, similar CGCACAAGAATCGGGAGGACAT

Von Willebrand factor GACATGAAAGGAGTGGTTACCA

TROSPA GTTGGACACCAGCCCAAGCGCA

Serum amyloid A GGCAGGTACTTTTGGGTGGTAA

Similar to 50–30 exoribonuclease 1 TGGGCTTCTTTTTCATCCACGTGT

Ricinusin GAAACTGCAAGCTCTTCAGGGTG

Aspartic protease TCCAGCAATGGCAAGGCATGCA

Subolesin GATGCACTGGTCACAGTCCGAG

b-actina GACATCAAGGACGTTGCCGATG

16S rRNAb GACAAGAAGACATCCAACATCGA

a Described previously by Zivkovic et al. (2010a).b Described previously by Zivkovic et al. (2010b).

2.5. Real-time RT-PCR

RNA from nine infected B. bigemina ticks and nine non-infectedticks were used for real-time RT-PCR analysis. Primers were de-signed based on the sequences determined for selected candidatedifferentially expressed genes (Table 1) using Primer3 v. 0.4.0(Whitehead Institute for Biomedical Research, Cambridge, MA,USA). The PCR was performed using the iScript SYBR Green RT-PCR Kit (BioRad, Hercules, CA, USA) in a BioRad IQ5 thermo-cyclerfollowing the manufacturer’s recommendations. The mRNA levelswere normalised separately against mRNA levels of two house-keeping genes, tick b-actin or 16S rRNA using the ddCT(2�CTtarget gene�CThousekeeping gene ) method (Livak and Schmittgen, 2001;Schefe et al., 2006) as in similar previous studies (Zivkovic et al.,2010a,b). In all cases, the mean of the duplicate values was usedand data from infected and non-infected ticks were comparedusing the Student’s t-test (P = 0.05).

2.6. RNA interference in ticks

Gene-specific double-stranded (ds)RNA was synthesised basedon identified R. annulatus sequences and used to knockdown theexpression of selected genes in R. annulatus and R. microplus ticksinjected with dsRNA. Specific primers containing T7 promoter se-quences (50-TAATACGACTCACTATAGGGTACT-30) at the 50-endwere synthesised (Table 2) and the MEGAscript RNAi Kit (Ambion,Austin, TX, USA) was used to synthesise dsRNA according to themanufacturer’s instructions. The resulting dsRNA was purified,

stream primer sequence 50–30 PCR annealing conditions

GAGGCTTG 51 �C/30 sTGCGTGAGG

GATATGAC 51 �C/30 sTCGGGTTCATGGGGAAGG 51 �C/30 sTCTTGCTCCAGAGGTTC 51 �C/30 s

TGGTGCAAGCGCTGGA 51 �C/30 s

CCCTTGCAGGGTCCATT 51 �C/30 sAGGGCAAGAGACCTTC 51 �C/30 s

GCCAGTTCTGCCCCTAGA 51 �C/30 sACTGCCTCATCCTTCTTT 51 �C/30 sTGCGAACAGGCTGACAGT 51 �C/30 sTGAGTTTGTAGTGCGTGA 51 �C/30 s

GGGCACTCAGCCAGTTCT 51 �C/30 sTAAATAAGACCACTGCAT 51 �C/30 sAAGCACTCCGCAGACTT 51 �C/30 sGCTCTCATCTGTCACCA 51 �C/30 sGGCAGTAG

TGAATGAAA 51 �C/30 sAGTCAAGAAGACGAGAGA 55 �C/30 sTGGCAGATGAAGCT(TC)TGC 55 �C/30 sGTGAT(GC)CCTA 42 �C/30 sGGT

Table 2Sequences of primers used for double-stranded RNA synthesis.

Gene Upstream/downstream primer sequence 50–30a PCR annealing conditions Fragment size (bp)

TROSPA TGGCGGTGGATATGGAGG 51 �C/30 s 496CGTTGAGCTCGCCCTTTC

Aspartic protease CAAGGCATGCAAGTCAAG 51 �C/30 s 458GTAGGCACACGGCATTCC

Serum amyloid A GCGATTCGCCCTTGAGCG 51 �C/30 s 324GTCTACGATTCGCCCTTAG

Kunitz-type protease inhibitor 5 (KTPI) CGCTGCAGTGCTTCAATCAGCA 55 �C/30 s 248TTCGCCCTTAGCGTGGTCGC

Ricinusin ATGAAGCCCACGAAGCCCCG 55 �C/30 s 323CATGGTGGGCCGCTTCAGGG

Calreticulin TTCGCCCTTAGCGTGGTCGC 55 �C/30 s 526GGTCGCGGCCGAGGTACAAA

Subolesin GACTGGGACCCCTTGCACAGT 55 �C/30 s 370CGAGTTTGGTAGATAGCACA

a All primers contained T7 promoter sequences (50-TAATACGACTCACTATAGGGTACT-30) at the 50 end.

190 S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195

quantified by spectrometry and checked on a 0.5X TBE (Tris base,Boric acid and Ethylenediaminetetraacetic acid), 1.2% (w/v) aga-rose gel.

Freshly moulted R. annulatus and R. microplus adult female tickswere injected with 0.4 ll of dsRNA (1 � 1011–1 � 1012 molecules/ll) in the lower right quadrant of the ventral surface of the tickexoskeleton (de la Fuente et al., 2006). Thirty female ticks pergroup were injected using a Hamilton syringe with a 2.54 cm, 33gauge needle. Control ticks were injected with R. microplus subole-sin dsRNA (positive control) or buffer (10 mM Tris–HCl, pH 7,1 mM EDTA) alone (negative control). Ticks were allowed to feedin eight separated patches (six test groups and two controls) on acalf that was experimentally infected with 2 � 108 B. bigemina(field strain from Chiapas, Mexico). Cattle infection was demon-strated by visual examination of blood smears and PCR. Unat-tached ticks were removed 2 days after infestation. All attachedticks were removed after 7 days of feeding and held in a humiditychamber for 4 days to allow ticks to digest the blood meal. Tickswere dissected and whole internal organs were stored in RNAlater(Ambion) for total RNA and DNA extraction as described previouslyin Section 2.3. Gene knockdown was analysed by real-time RT-PCRusing sequence-specific primers (Table 1) by comparing mRNA lev-els between dsRNA-injected and control ticks. The B. bigeminainfection levels were determined by quantitative PCR (qPCR) ofthe 18S rDNA gene (GenBank AY603402) using primers 50-AATAA-CAATACAGGGCTTTCGTCT-30 and 50-AACGCGAGGCTGAAATACAACT-30 and normalising against tick 16S rDNA gene using the ddCTmethod (Livak and Schmittgen, 2001; Schefe et al., 2006). Tickmortality was evaluated as the ratio of dead ticks to the total num-ber of ticks allowed to feed on the calf. The mRNA levels, B. bigem-ina infection in ticks and female tick weight after feeding werecompared between dsRNA and saline-injected control ticks by aStudent’s t-test (P = 0.05). To analyse tick mortality, the Chi-squaretest (P = 0.05) was used with the null hypothesis that tick mortalitywas not dependent on gene knockdown.

3. Results

3.1. Identification of candidate differentially expressed genes in R.annulatus ticks in response to B. bigemina infection

SSH was used to identify R. annulatus tick genes differentiallyexpressed in response to B. bigemina infection. Eight hundred ran-domly selected SSH library clones were sequenced and analysed.After eliminating clones with poor quality sequences, 752 se-quences (average length ± S.D., 562 ± 297 bp) were assembled into96 unigenes (87 contigs and nine singlets) representing unique ex-

pressed sequence tags (ESTs). On average, the number of sequencesper unigene was 8.3, which suggested a low diversity in our data-set. Assembled ESTs resulted in 41 (43%) ESTs with unknown func-tion or without any identity to sequence databases. Significantidentity to genes with functional annotation was confirmed forone (1%) Babesia T2Bo hypothetical protein EST, five (5%) ESTs re-lated to the vertebrate host and 49 (51%) ESTs with similarity totick sequence databases. Of the 49 ESTs with similarity to tick se-quences, seven (14%) corresponded to protease inhibitors. Molecu-lar function GO assignments, together with protein ontology andavailable tick sequence databases, showed that the obtained ticksequences encoded for proteins with different molecular functionssuch as cell structure, defence, transport, signal transduction andregulation, synthesis, energy metabolism and enzymatic processes(Fig. 1).

3.2. Differential gene expression in B. bigemina-infected R. annulatusticks

Sixteen candidate differentially expressed genes with putativefunctions in tick–pathogen interactions were selected for valida-tion of SSH results by real-time RT-PCR using RNA from non-in-fected and B. bigemina-infected ticks (Table 3). Of the sixteengenes analysed, five were confirmed as differentially expressed inB. bigemina-infected ticks. Genes encoding for homologues of TRO-SPA, calreticulin, ricinusin and serum amyloid A proteins wereover-expressed in infected ticks while Kunitz-type protease inhib-itor 5 (KTPI) mRNA levels were down-regulated in infected ticks(Table 3).

3.3. Sequence analysis of tick genes differentially expressed in responseto B. bigemina infection

Additional sequence analysis was conducted on R. annulatusESTs confirmed as differentially expressed in response to B. bigem-ina infection. Rhipicephalus annulatus EST68 (GenBank AccessionNo. JK489429) sequence analysis showed that TROSPA is a highlyconserved gene in ticks, with 78% (128/165 amino acids) homologybetween R. annulatus, Ixodes ricinus, Ixodes scapularis and Ixodespersulcatus protein sequences (Fig. 2). For calreticulin, R. annulatusEST21 (JK489382) showed a 99% (226/229 nucleotides) identity toR. microplus calreticulin precursor (AF420211) 30-end coding regionwith 97% (73/75 amino acids) homology to protein COOH-terminalregion. The R. annulatus EST84 (JK489445) showed the highesthomology (97%; 97/100 amino acids) to I. ricinus ricinusin(ABB79785) and to a lesser extent to I. scapularis microplusin pre-protein-like (AAY66716) and R. microplus microplusin (AAO48942)

Fig. 1. Functional grouping of tick genes differentially expressed in Babesia bigemina-infected Rhipicephalus (Boophilus) annulatus based on Gene Ontology molecular functionassignments.

Table 3Differential gene expression in Babesia bigemina-infected Rhipicephalus (Boophilus)annulatus ticks.

Gene Infected/non-infected ratio(mean ± S.D.)

Ricinusin 1.8 ± 0.4a

Kunitz-type protease inhibitor 5 (KTPI) 0.5 ± 0.1a

Aspartic protease 0.9 ± 0.2Von Willebrand factor 1.2 ± 0.3TROSPA 2.3 ± 0.5a

Chaperonin 1.1 ± 0.2Serum amyloid A 2.1 ± 0.4a

Similar to 50–30 exoribonuclease 1 1.2 ± 0.2Microplusin preprotein 1.5 ± 0.4Hebrain like 1.3 ± 0.4Mucin 0.8 ± 0.2Calreticulin 3.5 ± 0.7a

Kunitz-type protease inhibitor SHIP-1chain

0.9 ± 0.1

Carrapatin inhibitor 0.9 ± 0.3Microplusin-like 0.6 ± 0.1GP80 precursor 0.8 ± 0.2

The mRNA levels of selected differentially expressed genes were determined byreal-time reverse transcription-PCR. The mRNA levels were normalised against tickb-actin transcripts using the ddCT method (2�CTtarget�CTb-actin ). The infected/non-infected ratios represent the expression fold-change in the infected ticks calculatedby dividing the mean of normalised mRNA levels in infected ticks (n = 9) by themean of the normalised mRNA level in uninfected control ticks (n = 9). In all cases,the mean of the duplicate values was used and data from infected (n = 9) and non-infected ticks (n = 9) were compared using the Student’s t-test.

a P < 0.05.

S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195 191

(Fig. 3). The R. annulatus EST81 (JK489442) showed 46–49% homol-ogy to the previously reported Ornithodorus parkeri (EF633889)and I. scapularis (XM_002407273, XM_002416454) serum amyloidA protein-like sequences. Four different R. annulatus ESTs (EST24,EST25, EST28, EST29, EST42) showed homology to KTPIs, with amaximum 62% (32/52 amino acids) homology to I. scapularis serineproteinase inhibitor (XM_002434100).

3.4. Functional analysis of tick genes differentially expressed inresponse to B. bigemina infection

The five genes confirmed to be differentially expressed in in-fected ticks (TROSPA, calreticulin, ricinusin, serum amyloid A andKTPI), together with one gene for which mRNA levels were similarbetween infected and non-infected ticks (aspartic protease) andsubolesin, previously shown to be involved in tick innate immunity(Zivkovic et al., 2010b; de la Fuente et al., 2011), were selected forfunctional studies using dsRNA-mediated RNAi in both R. annulatusand R. microplus. The effect of gene knockdown on B. bigeminainfection levels and tick weight and mortality was evaluated(Tables 4–6).

Under the conditions undertaken in this study gene knockdownafter dsRNA-mediated RNAi was demonstrated for all genes in R.annulatus, while in R. microplus the silencing of subolesin andaspartic protease genes was not demonstrated (Tables 4 and 5).Knockdown of TROSPA and serum amyloid A significantly reducedB. bigemina infection levels by 83% and 66%, respectively, in R.annulatus compared with control ticks (Table 4). In R. microplus,knockdown of TROSPA and serum amyloid A also reduced patho-gen infection levels by 70% and 86%, respectively, while calreticulinknockdown resulted in 73% lower infection levels compared withcontrols (Table 5). Subolesin knockdown did not affect B. bigeminainfection levels in R. annulatus ticks (Table 4).

The possible knockdown effect of selected genes on tick weightand mortality was determined and statistically analysed. KTPIknockdown reduced female tick weight in both R. annulatus andR. microplus (Table 6). For other genes such as TROSPA, ricinusinand calreticulin, gene knockdown resulted in lower tick weightcompared with controls in one of the tick species only (Table 6).The effect of subolesin knockdown was characterised in R. annula-tus only and resulted in the reduction of tick weight comparedwith controls (Table 6). Tick mortality was not affected indsRNA-injected ticks compared with controls. For all genes andin both tick species, R. annulatus and R. microplus, the null hypoth-esis was accepted (P > 0.05), suggesting that none of the studiedgenes had a role in tick survival.

4. Discussion

Several studies have characterised the tick–pathogen interfaceat the molecular level (Macaluso et al., 2003; Mulenga et al.,2003; Nene et al., 2004; Rudenko et al., 2005; de la Fuente et al.,2007b, c; Villar et al., 2010; Zivkovic et al., 2010a; Mercado-Curielet al., 2011). However, to our knowledge this is the first report ofdifferential expression of genes in an R. annulatus tick populationinfected with B. bigemina. In this work we characterised R. annula-tus genes differentially expressed in response to B. bigemina infec-tion using SSH and real-time RT-PCR. Genes confirmed asdifferentially expressed in infected ticks were functionally charac-terised using a RNAi approach to analyse their role during patho-gen infection in the tick vector.

The SSH analysis used in this study to identify genes differen-tially expressed in R. annulatus in response to B. bigemina infectiondid not result in a large variety of ESTs. These results are similar tothose obtained with Anaplasma marginale and probably reflecttick–pathogen co-evolution (de la Fuente et al., 2007c; Villaret al., 2010; Zivkovic et al., 2010a; Mercado-Curiel et al., 2011).As expected, a large percentage of the identified ESTs did not showidentity to known sequences with functional annotation. However,confirmed differentially expressed ESTs with predicted function

Rhipicephalus annulatus VDMEAMEAAMAA - - AMVAT- DTVASSAASAMATEATVAMDTASLSLPLQLSP 49Ixodes ricinus MAAMEAMAVDMEAMAAAMAA - - AMVAT- DTVASSAASAMATEATVAMDTASLSLPLQLSP 57Ixodes persulcatus MAAMEAMAAAMAAMEAMAAT- DTVASSAASATATEATVAMDTASLSLPLQLSP 52 Ixodes scapularis MVAMEAMAAMEVMVAAMAATADTVASSAASATATEATVAMDTASLSLPLQLSP 53

**** * ** ** ********** *********************Rhipicephalus annulatus RSLPQSSLSATAATVATDTVVSSADTEVTDTEDSAATVSATATLSMLPQLSPRSLPQSSL 109Ixodes ricinus RSLPQSSLSATAATVATDTVVSSADTEVTDTEDSAATVSATASLSMLPQLSPRSLPQSSL 117Ixodes persulcatus RSLHQSSLSATAAMVAMDTVVSSADTEVTDTEDSAAMVSVTASLSMLPRSSPGSLPQSSL 112Ixodes scapularis RSLPQSSLSATAATVATDTVVSSADTEVSDTEDSAATVSATASLSMLPQSSPRSLPQSSL 113

*** ********* ** *********** ******* ** ** ***** ** *******

Rhipicephalus annulatus SATATEASVTADMADTATDTKQFISKGNQHFFAASYLCAWADQSAAGS 157Ixodes ricinus SATATEASVTADMADTATDTKQFISKGNQHFFAASYLCAWADQSAAGS 165Ixodes persulcatus SATATEASAATDMADTATDTRQFISKGNQHFFAASYLCAWADQSAAGS 160Ixodes scapularis SATAATVDSVTDMADTAMDTKQFISKGNEHFFAASYLCAWADQSAAGS 161

**** ****** ** ******* *******************Fig. 2. Alignment of TROSPA multiple amino acid sequences. Sequences aligned included Rhipicephalus (Boophilus) annulatus EST68 (JK489429), Ixodes ricinus (ABU43150),Ixodes scapularis (AAO43095) and Ixodes persulcatus (BAK09229). Asterisks denote amino acids conserved in all sequences analysed.

Ixodes ricinus MKCSVCLLVLCSLALFVSAEEADGAHEAHEAPVAPTPTQSPYCHLDDAHLTALTECVGRG 60Rhipicephalus annulatus XXXXXXXXXXXXXXXXXXAEEADGAHEAHEAPVAPTPTQSPYCHLDDAHLTALTECVGRG 60Ixodes scapularis MKCSVCILVLCSLALFVSAEEA - - - - - - HEAPEAPTPTQSPYCHLDDEHLTALTTCVGHG 54Rhipicephalus microplus MKAIFVSALLVVALVA - - - - - - - - - - - STSAHHQELCTKGDDALVTELECIRLR 43

* * * * * *

Ixodes ricinus MTEALRTKLQAVTTSLSCENMVCTLRKLCEQEPLSTVS - -VFNDEEKDEFRALGAECRSP 118Rhipicephalus annulatus MTEALRTKLQAVTTSLSCENMVCTLRKLCEQEPLSTVS - -VFNEEEKDEFRTLGAGCRSP 118Ixodes scapularis MTEALRTKLQAVTTSLSCENTVCTLRKLCEQEPLSTVS - - VFNDEEKHEFRTLAAGCHSP 112Rhipicephalus microplus ISPETNAAFDNAVQQLNCLNRACAYRKMCATNNLEQAMSVYFTNEQIKEIHDAATACDP 102

* * * * ** * * * * * * Ixodes ricinus ATAHPEEAHPEAAHHDA 135Rhipicephalus annulatus ATAHPEEAHPEAAHHDA 135Ixodes scapularis TTAHPEGAHHEA - - - - - 124Rhipicephalus microplus - EAHHEHDH - - - - - - - - 110

** * *

A

B Rhipicephalus microplus

Rhipicephalus annulatus

Ixodes scapularis

Ixodes ricinus

0.2

Ixodes ricinus MKCSVCLLVLCSLALFVSAEEADGAHEAHEAPVAPTPTQSPYCHLDDAHLTALTECVGRG 60Rhipicephalus annulatus XXXXXXXXXXXXXXXXXXAEEADGAHEAHEAPVAPTPTQSPYCHLDDAHLTALTECVGRG 60Ixodes scapularis MKCSVCILVLCSLALFVSAEEA - - - - - - HEAPEAPTPTQSPYCHLDDEHLTALTTCVGHG 54Rhipicephalus microplus MKAIFVSALLVVALVA - - - - - - - - - - - STSAHHQELCTKGDDALVTELECIRLR 43

* * * * * *

Ixodes ricinus MTEALRTKLQAVTTSLSCENMVCTLRKLCEQEPLSTVS - -VFNDEEKDEFRALGAECRSP 118Rhipicephalus annulatus MTEALRTKLQAVTTSLSCENMVCTLRKLCEQEPLSTVS - -VFNEEEKDEFRTLGAGCRSP 118Ixodes scapularis MTEALRTKLQAVTTSLSCENTVCTLRKLCEQEPLSTVS - - VFNDEEKHEFRTLAAGCHSP 112Rhipicephalus microplus ISPETNAAFDNAVQQLNCLNRACAYRKMCATNNLEQAMSVYFTNEQIKEIHDAATACDP 102

* * * * ** * * * * * * Ixodes ricinus ATAHPEEAHPEAAHHDA 135Rhipicephalus annulatus ATAHPEEAHPEAAHHDA 135Ixodes scapularis TTAHPEGAHHEA - - - - - 124Rhipicephalus microplus - EAHHEHDH - - - - - - - - 110

** * *

Rhipicephalus microplus

Rhipicephalus annulatus

Ixodes scapularis

Ixodes ricinus

Rhipicephalus microplus

Rhipicephalus annulatus

Ixodes scapularis

Ixodes ricinus

Fig. 3. Analysis of ricinusin orthologue sequences. (A) Amino acid sequence alignment of Rhipicephalus (Boophilus) annulatus EST84 (JK489445), Ixodes ricinus ricinusin(ABB79785), Ixodes scapularis microplusin preprotein-like (AAY66716) and Rhipicephalus microplus microplusin (AAO48942). Asterisks denote amino acids conserved amongall sequences analysed. (B) Unrooted phylogram inferred using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of theevolutionary distances used to infer the phylogenetic tree.

192 S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195

suggested that these genes are involved in pathogen infection/mul-tiplication and tick response to infection.

TROSPA was first described in I. scapularis as a receptor for Bor-relia burgdorferi, showing an enormous potential as a vaccine anti-gen to control bacterial infection in ticks (Pal et al., 2004; Hoviuset al., 2007). Anti-TROSPA antibodies or gene knockdown reducedB. burgdorferi adherence to the I. scapularis gut in vivo, preventing

efficient colonisation of the vector and subsequently reducingpathogen transmission to the mammalian host (Pal et al., 2004).In I. scapularis, TROSPA mRNA levels increased following spiro-chaete infection and decreased in response to tick engorgement,events that are temporally linked to B. burgdorferi infection andtransmission by the tick vector (Pal et al., 2004). Our resultsshowed that the R. annulatus gene with high sequence identity to

Table 4Babesia bigemina infection levels after gene knockdown by RNA interference in Rhipicephalus (Boophilus) annulatus ticks.

Gene n Gene silencing (% mean ± S.D.) B. bigemina infection levels (mean ± S.D.) Test/control (mean ± S.D.)

TROSPA 7 60 ± 30 6.55E-06 ± 1.13E-07 0.17 ± 0.03a

Aspartic protease 20 88 ± 13 3.75E-05 ± 9.33E-06 0.97 ± 0.06Serum amyloid A 16 59 ± 30 1.33E-05 ± 4.10E-06 0.34 ± 0.04a

Kunitz-type protease inhibitor 5 (KTPI) 8 100 ± 0 1.84E-05 ± 1.91E-06 0.49 ± 0.14Ricinusin 17 65 ± 25 2.64E-05 ± 5.94E-06 0.68 ± 0.03Calreticulin 8 68 ± 33 2.46E-05 ± 5.37E-06 0.66 ± 0.26Subolesin 9 61 ± 31 2.86E-05 ± 2.14E-06 0.74 ± 0.31Control 19 – 3.86E-05 ± 7.07E-06 –

Thirty female ticks per group were injected with double stranded (ds)RNA or saline control. Ticks were allowed to feed in eight separated patches on a calf experimentallyinfected with B. bigemina. All attached ticks (n = 7–20) were removed after 7 days of feeding and held in a humidity chamber for 4 days to allow ticks to digest the blood meal.Gene knockdown was analysed by real-time reverse transcription (RT)-PCR by comparing mRNA levels between dsRNA-injected and control ticks. The B. bigemina infectionlevels were determined by quantitative PCR of the 18S rRNA gene and normalised against tick 16S rRNA using the ddCT method (2�CTtarget�CTb-actin ). The mRNA levels and B.bigemina infection in ticks were compared between dsRNA and saline-injected control ticks by a Student’s t-test.

a P < 0.05.

Table 5Babesia bigemina infection levels after gene knockdown by RNA interference in Rhipicephalus (Boophilus) microplus ticks.

Gene n Gene silencing (% mean ± S.D.) B. bigemina infection levels (mean ± S.D.) Test/control (mean ± S.D.)

TROSPA 9 97 ± 6 2.77E-05 ± 1.07E-05 0.30 ± 0.07a

Aspartic protease 5 0 ± 0 ND NDSerum amyloid A 10 48 ± 31 1.31E-05 ± 2.40E-06 0.14 ± 0.00a

Kunitz-type protease inhibitor 5 (KTPI) 14 93 ± 17 9.60E-04 ± 6.36E-04 11.35 ± 8.97Ricinusin 12 94 ± 8 1.81E-04 ± 5.87E-05 2.07 ± 1.01Calreticulin 14 93 ± 6 2.47E-05 ± 4.67E-06 0.28 ± 0.10a

Subolesin 9 0 ± 0 ND NDControl 17 – 9.09E-05 ± 1.58E-05 –

Thirty female ticks per group were injected with double stranded (ds)RNA or saline control. Ticks were allowed to feed in eight separated patches on a calf experimentallyinfected with B. bigemina. All attached ticks (n = 5–17) were removed after 7 days of feeding and held in a humidity chamber for 4 days to allow ticks to digest the blood meal.Gene knockdown was analysed by real-time reverse trancription (RT)-PCR by comparing mRNA levels between dsRNA-injected and control ticks. The B. bigemina infectionlevels were determined by quantitative PCR of the 18S rRNA gene and normalised against tick 16S rRNA using the ddCT method ((2�CTtarget�CT16s ). The mRNA levels and B.bigemina infection in ticks were compared between dsRNA and saline-injected control ticks by a Student’s t-test. ND, not determined because gene knockdown was notdemonstrated.

a P < 0.05.

Table 6Female tick weight after gene knockdown by RNA interference in Rhipicephalus(Boophilus) annulatus and Rhiphicephalus microplus ticks.

Gene Tick weight (mean ± S.D.) (mg)

R. annulatus R. microplus

TROSPA 95 ± 90 30 ± 16a

Aspartic protease 72 ± 53 NDSerum amyloid A 148 ± 118 115 ± 122Kunitz-type protease inhibitor 5 (KTPI) 37 ± 11a 22 ± 11a

Ricinusin 49 ± 42a 110 ± 110Calreticulin 37 ± 26a 146 ± 136Subolesin 21 ± 14a NDControl 89 ± 84 62 ± 48

Thirty female ticks per group were injected with double stranded RNA or salinecontrol. Ticks were allowed to feed in eight separated patches on a calf experi-mentally infected with Babesia bigemina. All attached ticks were removed after7 days of feeding, weighed and held in a humidity chamber for 4 days to allow ticksto digest the blood meal. Tick mortality was evaluated as the ratio of dead ticks tothe total number of placed ticks on the calf. Female tick weight after feeding wascompared between dsRNA and saline-injected control ticks by a Student’s t-test.ND, not determined because gene knockdown was not demonstrated.

a P < 0.05.

S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195 193

Ixodes spp. TROSPA was over-expressed in B. bigemina-infectedticks and played a similar role in both R. annulatus and R. microplusby leading to a significant lower infection after gene knockdown.These results suggested the possibility that B. bigemina uses a TRO-SPA orthologue receptor for infection of Rhipicephalus tick cells andencouraged research for the characterisation of this molecule inBabesia–tick interactions and development of transmission block-ing vaccines.

Calreticulin, a major endoplasmic reticulum calcium-bindingprotein, was over-expressed in B. bigemina-infected R. annulatus.This result is corroborated by a previous study where this proteinwas shown to be upregulated in ovaries of infected R. microplusticks (Rachinsky et al., 2007). Gene knockdown under the condi-tions undertaken here reduced pathogen infection in R. microplusbut not in R. annulatus ticks. The possible use of this protein inthe development of protective immunity against parasites wassuggested previously (Ferreira et al., 2002). Bovines immunisedwith a R. microplus recombinant calreticulin protein failed to pro-duce a hyperimmune serum against this molecule, showing lowimmunogenicity of this protein which could possibly be surpassedby the use of adjuvants and conjugation with highly immunogenicproteins (Ferreira et al., 2002). Calreticulin was found to be se-creted in Amblyomma spp., Dermacentor spp. and R. microplus sali-va, suggesting a role for this protein during tick blood feeding(Jaworski et al., 1995; Ferreira et al., 2002), a result supported hereafter gene knockdown in R. annulatus which resulted in reducedtick weight. These results suggested that calreticulin and calciummetabolism have a role during tick feeding and may be requiredfor Babesia infection in some tick species.

Ricinusin, longicin and microplusin are tick antimicrobial pep-tides. These proteins are included in the group of defensins, awell-conserved defence mechanism that is among the most impor-tant components of tick innate immunity (Tsuji et al., 2007; Silvaet al., 2009). Longicin has been shown to be a defensin againstBabesia gibsoni infection in Haemaphysalis longicornis (Tsuji et al.,2007). Ricinusin was induced in R. annulatus in response to B.bigemina infection. Although ricinusin mRNA levels were signifi-cantly higher in infected than in non-infected ticks, under the

194 S. Antunes et al. / International Journal for Parasitology 42 (2012) 187–195

conditions of this study, gene knockdown did not affect pathogeninfection, thus suggesting that this molecule is not essential to con-trol B. bigemina infection in Rhipicephalus spp. ticks.

The expression of serum amyloid A increased in R. annulatusticks infected with B. bigemina and gene knockdown resulted inlower infection levels in both R. annulatus and R. microplus withoutaffecting tick weight after feeding. These results suggested thatserum amyloid A may be part of tick response to the stress pro-duced by Babesia infection but at the same time it was necessaryfor pathogen infection/multiplication in Rhipicephalus spp. ticks.Serum amyloid A is involved in host response to tissue injuryand inflammation, which can increase their concentration over1,000 fold and have various physiological effects including anti-platelet activity (Urieli-Shoval et al., 2000). A serum amyloid Aputative protein was recently identified in the sialome of the softtick Ornithodoros parkeri (Francischetti et al., 2008) and in the I.scapularis genome (XM_002416454). However, a role for these pro-teins in tick pathogen infection and multiplication has not beenpreviously described.

The Kunitz-type protease inhibitors were the most representedESTs in our dataset. KTPIs are proteins of approximately 20 kDawith one or two disulphide bonds and a single reactive site (Majorand Constabel, 2008). However, some proteins belonging to theKunitz family do not act as protease inhibitors or may havelectin-like carbohydrate-binding or invertase inhibitor activity(McCoy and Kortt, 1997; Macedo et al., 2004). Thus, determininga precise function of Kunitz-type inhibitors (KTI)-like proteins can-not be based only on primary sequence similarities, but requiresin vitro assays for confirmation. Some KTIs are involved in tick de-fence mechanisms against pathogen infection, presumably viainhibition of microbial proteinases (Sasaki and Tanaka, 2008). InDermacentor variabilis, tick cell invasion by Rickettsia montanensisis limited by a KTPI (Ceraul et al., 2011). Although KTPIs werefound to be upregulated in other cases (Rachinsky et al., 2007) inthe present study when induced in B. bigemina-infected ticks, KTPImRNA levels were lower in infected R. annulatus than in non-infected ticks, probably suggesting a mechanism by which thepathogen manipulates gene expression to increase infection/multi-plication. However, KTPI knockdown did not affect B. bigeminainfection in ticks but the effect of other KTPIs on B. bigemina infec-tion was not studied. Interestingly, KTPI knockdown in R. annulatusand R. microplus reduced tick weight after feeding compared withcontrols, suggesting a role for this protein during tick feeding.

Subolesin is a candidate tick protective antigen initially discov-ered in I. scapularis and conserved in many tick species. Subolesinplays an important role in the immune response to pathogen infec-tion through the control of genes involved in innate immunity(Almazán et al., 2003; Goto et al., 2008; Galindo et al., 2009;Zivkovic et al., 2010b; de la Fuente et al., 2011). In previous exper-iments, we showed that subolesin knockdown reduced B. bigeminainfection in R. microplus (Merino et al., 2011a). However, hereinsubolesin knockdown did not affect B. bigemina infection in R.annulatus. The discrepancy between these results could be due tothe fact that here adult ticks were injected with dsRNA beforeinfestation while in the previous experiment dsRNA was injectedinto replete females to infest cattle with resulting larvae (Merinoet al., 2011b). As in previous experiments, subolesin knockdownreduced R. annulatus female tick weight after feeding (Almazánet al., 2010). The effect of gene knockdown on pathogen infectioncould suggest genes necessary for pathogen infection/multiplica-tion in the tick, or at least in some cases, genes affecting tickweight after RNAi may reduce the amount of blood ingested byticks and thus the number of pathogens ingested during feeding.

RNAi was used in this study to analyse the effect of knockdownof selected genes on B. bigemina infection in ticks. Gene knockdownwas carried out using R. annulatus sequences in both R. annulatus

and R. microplus ticks due to the high similarity between their genesequences. In R. annulatus but not in R. microplus, gene knockdownwas successful for all of the genes tested. Although dsRNA-mediated RNAi has been shown to function in R. annulatus usingR. microplus sequences, the resulting phenotype was not similarbetween both tick species (Almazán et al., 2010). These results sug-gested that for some genes, sequence identity might not be suffi-ciently high for efficient gene knockdown in R. microplus using R.annulatus-derived dsRNAs. The RNAi off-target effects (OTEs)(Scacheri et al., 2004) cannot be ruled out in our gene knockdownexperiments, as reported previously in R. microplus (Lew-Taboret al., 2011). The absence of full tick genomic data and the lackof a confirmed tick RNAi pathway can underestimate the OTEs incurrent tick RNAi experiments (Lew-Tabor et al., 2011). Despitethis, the use of long dsRNAs as gene knockdown treatments in tickshas been accepted as a routine method for validation/support oftick gene function (de la Fuente et al., 2007b; Smith et al., 2009;Merino et al., 2011b).

The present study identified new genes involved in the tickinfection/multiplication of B. bigemina, improving our understand-ing of the molecular mechanisms involved in tick–pathogen inter-actions. The results reported here increased our understanding ofthe role of tick genes in Babesia infection/multiplication, which isfundamental to development of novel tick control measures. Someof the R. annulatus genes discovered in this study such as serumamyloid A, calreticulin and TROSPA could contribute to the devel-opment of novel vaccines designed to reduce tick infestations andprevent or minimise pathogen infection in ticks and transmissionto vertebrate hosts.

Acknowledgements

This work was supported by the Fundação para a Ciência eTecnologia (FCT, Portugal) project PTDC/CVT/112050/2009 and bythe Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC to JF). Sandra Antunes would like to acknowledgethe FCT for her PhD Grant SFRH/BD/48251/2008.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ijpara.2011.12.003.

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