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ARTICLE IN PRESSG ModelTBDIS-412; No. of Pages 5

Ticks and Tick-borne Diseases xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

j ourna l h o me page: w ww.elsev ier .com/ locate / t tbd is

riginal article

haracterization of two strains of Anaplasma marginale isolated fromattle in Rio de Janeiro, Brazil, after propagation in tick cell culture

runa A. Baêtaa, Carla C.D.U. Ribeiroa, Rafaella C. Teixeiraa, Alejandro Cabezas-Cruzb,c,ygia M.F. Passosd, Erich Zweygarthe,f, Adivaldo H. Fonsecaa,∗

Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro, BrazilCenter for Infection and Immunity of Lille (CIIL), INSERM U1019 – CNRS UMR 8204, Université Lille Nord de France, Institut Pasteur de Lille, Lille, FranceSaBio, Instituto de Investigación de Recursos Cinegéticos, IREC-CSIC-UCLM-JCCM, Ciudad Real 13005, SpainDepartamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, BrazilInstitute for Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, GermanyDepartment of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X4, Onderstepoort 0110, South Africa

r t i c l e i n f o

rticle history:eceived 30 July 2014eceived in revised form 30 October 2014ccepted 3 November 2014vailable online xxx

eywords:naplasma marginale

a b s t r a c t

IDE8 tick cell cultures have been used for the isolation and propagation of several isolates of Anaplasmamarginale. The genetic heterogeneity of A. marginale strains in cattle is diverse in endemic regions world-wide and the analyses of msp1˛ (major surface protein 1 alpha) gene sequences have allowed theidentification of different strains. This study reports the isolation and propagation of two new isolates ofA. marginale in IDE8 cells from blood of two cattle and their morphological and molecular characteriza-tion using light microscopy and the msp1˛ gene, respectively. Small colonies were observed in cytospinsmears of each of the isolates 60 days after culture initiation. Based on msp1˛ sequence variation, the

ick cell culturesolation

sp1� gene

two isolates were found to be separate strains and were named AmRio1 and AmRio2. Analysis of msp1˛microsatellite in both strains resulted in a single genotype, genotype E. The amino acid sequence of oneMSP1� tandem repeat from the strain AmRio1 resulted in a new sequence (named 162) with one aminoacid change. The results of these phylogenetic analyses demonstrated that A. marginale strains from Braziland Argentina formed two large clusters of which one was less divergent that the other.

ntroduction

Anaplasma marginale is an intraerythrocytic rickettsial agenthat causes bovine anaplasmosis in cattle worldwide. Bovinenaplasmosis is endemic in Brazil and the disease is characterizedy anemia, weight loss, fever, abortion and even deaths in acuteases (Vidotto et al., 1998). A. marginale is biologically transmit-ed by ticks, mechanically by infective blood on fomites or on the

outhparts of biting insects, and transplacentally from dams toheir calves (Aubry and Geale, 2011).

The genetic heterogeneity of A. marginale strains is high in cat-

Please cite this article in press as: Baêta, B.A., et al., Characterization

de Janeiro, Brazil, after propagation in tick cell culture. Ticks Tick-born

le from endemic regions worldwide (de la Fuente et al., 2005,007; Pohl et al., 2013; Cabezas-Cruz et al., 2013). Three out ofix major surface proteins (MSPs), MSP5, MSP4 and MSP1� have

∗ Corresponding author. Tel.: +55 2126821103.E-mail addresses: babaeta@hotmail.com (B.A. Baêta), carlauzedo@yahoo.com.br

C.C.D.U. Ribeiro), rafaella.medvet@hotmail.com (R.C. Teixeira),lejandrocabezascruz@yahoo.es (A. Cabezas-Cruz), lygiapassos@yahoo.comL.M.F. Passos), adivaldofonseca@yahoo.com (A.H. Fonseca).

ttp://dx.doi.org/10.1016/j.ttbdis.2014.11.003877-959X/© 2014 Published by Elsevier GmbH.

© 2014 Published by Elsevier GmbH.

been extensively used for the molecular characterization of A.marginale (Aubry and Geale, 2011). The msp5 gene is highly con-served among Anaplasma spp. and therefore can only be used toestablish that organisms are in the genus Anaplasma and cannotbe used for identification of the species. Furthermore, MSP5-basedserologic tests result in cross reactions between Anaplasma species(as reviewed Kocan et al., 2012). However, previous analysis ofthe msp4 gene from A. marginale isolates demonstrated sufficientsequence variation to support its use in phylogeographic stud-ies (de la Fuente et al., 2003b). Analyses of msp1 (major surfaceprotein 1 alpha) gene sequences have allowed the identificationof A. marginale strains worldwide (Cabezas-Cruz et al., 2013) anddespite the msp1 genetic diversity, this gene is considered as astable genetic marker conserved during acute and persistent rick-ettsemia in cattle and also during multiplication in ticks (Palmeret al., 2001; Bowie et al., 2002; de la Fuente et al., 2003c). Further-more, MSP1� is an adhesin for bovine erythrocytes and tick cells.

of two strains of Anaplasma marginale isolated from cattle in Rioe Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.11.003

Binding residues are localized in the tandem repeated region of thisprotein, thus, tandem repeats provide information regarding ticktransmissibility phenotypes of A. marginale strains (de la Fuenteet al., 2003a).

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Tick cell lines have provided an in vitro system for the studyf tick–pathogen interactions and have reduced the use of cattle. marginale research (Bell-Sakyi et al., 2007). One of the mostidespread applications of tick cell lines is its use for isolation

nd propagation of several economically important tick-borneathogens. The first continuous in vitro culture system for theathogen A. marginale was established in the Ixodes scapularis cell

ine IDE8 from infected bovine erythrocytes (Munderloh et al.,996). Subsequently, this system has been used for the isolationnd propagation of several isolates of A. marginale (Bastos et al.,009; Blouin et al., 2002).

The present study reports the isolation and propagation of twotrains of A. marginale in IDE8 cells and their characterizationhrough light microscopy and sequence analysis of the msp1 gene.hese two strains are the first A. marginale isolates from cattle inio de Janeiro to be characterized after being propagated in tick cellulture.

aterials and methods

solation of A. marginale

Blood samples were collected from two naturally infected cattlewithout clinical signs) with an approximate 10% parasitemia thatere from the farm of Universidade Federal Rural do Rio de Janeiro,io de Janeiro, Brazil. The blood were collected in EDTA and placedt 4 ◦C and then transported to Germany for propagations in tickell culture. Therefore a delay of 7 days occurred before the sam-les were processed. The samples were processed as described bylouin et al. (2000). Briefly, the red blood cells were washed threeimes by centrifugation (700 × g for 10 min) and resuspension inhysiological phosphate-buffered saline solution (PBS). After eachentrifugation, the buffy coat were removed. The red blood cell pel-et were resuspended (1:1) in Leibovitz L-15 medium using DMSO10%) as cryoprotectant and then cryopreserved at −80 ◦C.

After 48 h, the samples were quickly thawed, suspended in L15edium and centrifuged at 9000 × g for 10 min. The pellet was sus-

ended in 5 mL of Leibovitz L15B medium containing 5% inactivatedetal bovine serum, NaHCO3 (2.5 g/L) and HEPES (Munderloh et al.,996). The pH was adjusted to 7.4. A sample of each animal was

ndividually inoculated into 25 cm2 flasks containing monolayersf IDE8 cells and incubated at 34 ◦C. The first medium change waserformed after 24 h with the removal of the all medium, subse-uent changes were made twice a week with the renewal of 60% ofhe culture supernatant.

Cultures were monitored daily by direct observation undernverted microscope and weekly by microscopic examination ofiemsa-stained cytospin smears (Watson, 1966).

haracterization of the A. marginale strains

After the detection of the first small bacterial colonies in IDE8ytospin smears of each flask, aliquots of the infected culturesere collected for molecular analysis. DNA from blood samples

nd cultures was extracted using QIAamp® DNA Mini Kit (QIA-EN, Biotecnologia Brasil, São Paulo, Brazil) and quantified with apectrophotometer (NanoDrop® ND-1000). Initially, real-time PCRas performed targeting the msp1 gene, following the protocol

s described by Carelli et al. (2007), with modifications accord-ng to Pohl et al. (2013). In order to determine the genotype ofhe A. marginale strains, a PCR targeting the msp1 gene was

Please cite this article in press as: Baêta, B.A., et al., Characterization

de Janeiro, Brazil, after propagation in tick cell culture. Ticks Tick-born

erformed according to Lew et al. (2002). The reactions were per-ormed using the 1733F (5′-TGTGCTTATGGCAGACATTTCC-3′) and957R primers (5′-AAACCTTGTAGCCCCAACTTATCC-3′). The ampli-cation cycle had denaturation at 94 ◦C for 5 min, followed by 40

PRESSe Diseases xxx (2014) xxx–xxx

cycles at 94 ◦C for 30 s, 60 ◦C for 1 min and 72 ◦C for 1.5 min and afinal extension at 72 ◦C for 7 min. PCR products were separated in2% agarose gels and stained with ethidium bromide, 100 bp markerwas used for molecular size determination (Ladder Mix1/2 load-ing dye, Fermentas Life Science, Sinapse Biotecnologia, São Paulo,Brazil).

Purification of the PCR products was performed using a GFX PCRDNA kit (GE Healthcare Biosciences, São Paulo, Brazil) followingthe manufacturer’s recommendations. After purification, the DNAsamples were sequenced in the capillary type platform ABI 3730DNA Analyser Equipment (Applied Biosystems, Life technologiesdo Brasil, São Paulo, Brazil) and sequences were analyzed withthe Analysis 5.3.1 program. The results were evaluated withChroma©Lites (www.technelysium.com.au/chromas lite.html)and the similarities of the sequences were searched by BLASTnanalysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in GenBank.

The isolated strains were identified based on the sequencesof the msp1 tandem repeats (Cabezas-Cruz et al., 2013; dela Fuente et al., 2007) and the 5′-UTR microsatellite (Estrada-Pena et al., 2009). Briefly, the 5′-UTR microsatellite is locatedbetween the putative Shine–Dalgarno (SD) sequence (GTAGG)and the translation initiation codon (ATG) with the structure:GTAGG (G/A TTT) m (GT) n T ATG (microsatellite sequence isshown in bold letters), the SD–ATG distances were calculatedin nucleotides as (4 × m) + (2 × n) + 1. Nucleotide sequences weretranslated to amino acid (aa) sequences using ExPASy translationtool (http://web.expasy.org/translate).

The phylogenetic analysis

The phylogenetic analyses were conducted with MSP1a aminoacid sequences aligned with MAFFT (v7) configured for the high-est accuracy (Katoh and Standley, 2013). After alignment, regionswith gaps were removed from the alignment. Phylogenetic treeswere reconstructed using maximum likelihood (ML), neighbor join-ing (NJ) and Bayesian inference (MB) methods as implemented inPhyML (v3.0 aLRT) (Gascuel and Steel, 2006; Guindon and Gascuel,2003), PHYLIP (v3.66) (Felsenstein, 1989) and MrBayes (v3.1.2),respectively. The reliability for the internal branches of ML wasassessed using the bootstrapping method (1000 bootstrap repli-cates) and the approximate likelihood ratio test (aLRT–SH-Like)(Gascuel and Steel, 2006). Reliability for the NJ tree was assessedusing bootstrapping method (1000 bootstrap replicates). 10 000generations of Markov Chain Monte Carlo (MCMC) chains were runfor MrBayes. Graphical representation and editing of the phyloge-netic trees were performed with TreeDyn (v198.3) (Chevenet et al.,2006).

Ancestor reconstruction

The reconstruction of the ancestral amino acid sequence wasperformed using a Neighbor Joining Tree under the Dayhoff modelof substitutions which was estimated to be the best model fit-ting the actual data. Three reconstruction methods were used:joint (Pupko et al., 2000), marginal (Yang et al., 1995) and sam-ple (Nielsen, 2002) which are implemented in the Datamonkeywebserver.

Results

Isolation and propagation of A. marginale from Rio de Janeiro

of two strains of Anaplasma marginale isolated from cattle in Rioe Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.11.003

Sixty days after culture initiation, small bacterial colonies wereobserved in cytospin smears prepared from a sample collectedfrom each flask (Fig. 1A). On day 81, these colonies were vis-ible in 70% of the cells, with several colonies in the same cell

ARTICLE IN PRESSG ModelTTBDIS-412; No. of Pages 5

B.A. Baêta et al. / Ticks and Tick-borne Diseases xxx (2014) xxx–xxx 3

Fig. 1. Examination of Giemsa-stained cytospin smears of IDE8 cells infected with A. marginale from Rio de Janeiro, Brazil. (A) Formation of small colonies in the cells; (B)s lony r

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Fig. 1B). The formation of large colony was followed by the dis-uption of the vacuole membrane releasing the organisms intohe culture medium (Fig. 1C). The first subculture was performed1 days after culture initiation. Subsequently, subcultures wereerformed each time the culture reached an infection rate ofpproximately 70%. The isolated strains were named AmRio1 andmRio2.

olecular characterization of the A. marginale strains

All flasks were positive for A. marginale in the real-time PCR andonventional PCR. From the msp1 PCR an amplicon of approxi-ately 1.1 kb was isolated corresponding to the size of the targetedsp1 gene fragment.

The msp1 microsatellite analysis resulted in one genotype fromoth strains, genotype E. The microsatellite sequences producedD–ATG distances of nucleotides (Table 1).

In this study, five different msp1 tandem repeats were iden-ified among the two isolates from Rio de Janeiro. The aminocid sequence of one MSP1� tandem repeat, from strain AmRio1,esulted in a new sequence (named 162) with one amino acidhange as shown in Table 2. Both isolates had three different msp1˛andem repeats and the number of repeats was five. The structures

Please cite this article in press as: Baêta, B.A., et al., Characterization

de Janeiro, Brazil, after propagation in tick cell culture. Ticks Tick-born

f MSP1a tandem repeats of isolates were different, AmRio 1 (162--17-F-F) and AmRio2 (�-�-�-�-F). Moreover, the msp1 sequenceas found to be the same after six passages in vitro.

able 1he msp1 microsatellite analysis of A. marginale from Rio de Janeiro strains.

Isolates Genotype m* n** SD–ATG distance (nucleotide)

AmRio1 E 2 7 23AmRio2 E 2 7 23

he microsatellite was located between the Shine–Dalgarno (SD; sequence inrackets) and the translation initiation codon (ATG) with the structure: GTAGGG/ATTT) m* (GT) n** T ATG. The SD–ATG distance was calculated in nucleotidess (4 × m) + (2 × n) + 1.

eleasing corpuscles. Arrows indicate the colonies and rupture of a colony.

Phylogenetic analysis

In order to determine the phylogenetic relationship between theA. marginale strains isolated in this study and other strains fromnearby regions, we performed ML, NJ and MrBayes phylogeneticanalyses (Fig. 2). The strains AmRio1 and AmRio2 presented a dif-ferent tandem repeat composition, however, they fall in the samephylogenetic cluster (Cluster �) with strains previously reported inArgentina and Brazil (Fig. 2). A. marginale strains from this phylo-genetic cluster present high abundance of tandem repeats � and �which were found only in AmRio2. Although both strains, AmRio1and AmRio2, were isolated in Rio de Janeiro, strain AmRio2 seemsto be closer to strain (C-F-N) from Minas Gerais (de la Fuenteet al., 2004) (genetic distance between AmRio2 and strain (C-F-N)is 0.043) than to AmRio1 (genetic distance between AmRio1 andAmRio2 is 0.065). Interestingly, the evolution of the two clusters(Cluster � and Cluster �) seems to be different. Compared to theancestor (blue circle in Cluster �), two of the strains in Cluster �increase the number of tandem repeats (from 5 in the ancestor to7 in strain �-11-10-10-11-10-15 and 6 in strain 23-24-25-26-27-27) while in Cluster �, to which AmRio1 and AmRio2 belong, allthe strain have lost tandem repeats since the separation from theancestor (red circle in Cluster �) (Fig. 2).

Discussion

Two Brazilian A. marginale strains were propagated in IDE8 tickcell culture. First colonies were detected 60 days after culture ini-tiation. The time until first detection, however, was considerablylonger than reported in other studies (Bastos et al., 2009; Blouinet al., 2000; Munderloh et al., 1996) where periods between 8 and34 days were observed. The reasons for this could have been man-ifold. In the present experiments the percentage of infected ery-

of two strains of Anaplasma marginale isolated from cattle in Rioe Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.11.003

throcytes of the starting inoculum was only about 10% in contrastto a range of 30–64% of those reported by the above-mentionedauthors. Furthermore, the relatively long transport from Brazil toGermany might have had a negative influence on the survival of

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Table 2New sequence of MSP1� tandem repeat (named 162) found in A. marginale isolates from Rio de Janeiro (AmRio1 strain) with one amino acid change.

Repeat Encoded sequence

17 T D S S S A S G Q Q Q E S G V S S Q S G Q A S T S S Q L G162 A . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The one letter amino acid code was used to depict the differences found in MSP1a repeats. Points indicate identical amino acids. The repeat form 17 was taken as a model tocompare the new repeat.

Fig. 2. Phylogenetic analyses were conducted using ML, NJ and MrBayes. The topologies obtained with the three methods were very similar, therefore only one is show(NJ). The sequences AmRio1 and AmRio2 from the A. marginale strains isolated in this study are highlighted (asterisk). MSP1� amino acid sequences from different strainsof A. marginale reported in Brazil (Br) (de la Fuente et al., 2004; Pohl et al., 2013) and Argentina (Ar) (Ruybal et al., 2009) were used to perform the phylogenetic analyses.The two clusters were named Cluster � and Cluster �. The position and tandem repeat composition of the ancestral MSP1a sequences is shown for the two clusters( th new( nal brh eat co

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circles and squares). The ancestor of Cluster � present two tandem repeats that wiTDSSSAGNQQQESSVLPQSGQASTSSQLG). The numbers above and below the interigher that 50 are shown. The MSP1� GenBank accession numbers and tandem rep

he organisms and thus slowing down culture development. Oth-rwise, the development of the colonies in the tick cells was similaro that already described (Bastos et al., 2009; Blouin et al., 2000).

The sequence of the msp1 gene of A. marginale from both cat-le remained the same after passage in IDE8 tick cell line. Previoustudies demonstrated that the sequence of MSP1� also remainedhe same throughout successive passages in IDE8 cells or transmis-ion by ticks (Bastos et al., 2009; Bowie et al., 2002).

Several geographic isolates of A. marginale have been preparedorldwide, presenting genetic diversity among them. The geo-

raphic identity of these isolates has been based on the msp1 geneAllred et al., 1990; de la Fuente et al., 2001, 2002). Among differenttrains, MSP1� is involved in the adhesion and transmission of A.arginale by ticks and varies among geographic strains in the num-

er and sequence of amino-terminal tandem repeats influencing inhe infectivity of isolate (de la Fuente et al., 2005, 2007).

The results of our phylogenetic analysis showed that the popu-ation of A. marginale in Brazil and Argentina form two big clusters.hese two clusters have one interesting difference: Cluster � is moreariable than Cluster �. This conclusion is based on the followingrguments:

Cluster � has 8 strains with 16 different tandem repeats, whileCluster � has 14 strains with only 19 different tandem repeats.

Please cite this article in press as: Baêta, B.A., et al., Characterization

de Janeiro, Brazil, after propagation in tick cell culture. Ticks Tick-born

Thus, the proportion of different tandem repeats is higher in Clus-ter �.

Out of 8 strains in Cluster �, only 2 fall in the same branch ofthe tree, while in Cluster �, 10 out of 14 fall in the same branch.

amino acid sequences, ASRI (ADSSSASGQQQESSVLSPSGQASTSSQSGVG) and ASRIIanches represent statistical support values as indicated in the figure. Only valuesmposition of the respective sequences used in the phylogenetic tree are shown.

Thus, it seems that lineages in Cluster � differ faster from parentallineages than in Cluster �.

- Only 2 tandem repeats (� and 13) from the ancestor of Cluster � arekept in the strains of this cluster, the other three tandem repeats(ASR-I, ASR-II and 154) were lost during the genetic diversifica-tion of this group; in contrast, all the tandem repeats in Cluster�, except one (tandem repeat 3), are kept in the strains of thiscluster. Thus, tandem repeats replacement is faster in Cluster �that in Cluster �.

- Another difference is that after the diversification from the ances-tor, some strains in Cluster � gained tandem repeats, while allstrains in Cluster � have lost tandem repeats. Thus, increase in thenumber of tandem repeats is associated to genetic diversificationof MSP1a in these A. marginale strains.

These results suggest that lineages belonging to these two clus-ters may be under different selection pressures. An increase inthe genetic diversity of MSP1a was observed in areas of Argentinawere R. microplus was present (Ruybal et al., 2009). Another studyby Estrada-Pena et al. (2009) showed a lowest percentage of con-served amino acids in MSP1a from ecoregions with R. microplus. Thetandem repeats of MSP1a have been implicated in pathogen-tickinteraction and the amino acid position 20, implicated in the adhe-sion to tick cell extracts (de la Fuente et al., 2003a), was recently

of two strains of Anaplasma marginale isolated from cattle in Rioe Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.11.003

found to be under positive selection (Mutshembele et al., 2014).In addition, this molecule contains a neutralization-sensitive epi-tope (Allred et al., 1990) and an immuno-dominant B-cell epitope(Garcia-Garcia et al., 2004) that were found to be under purifying

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nd positive selection (Mutshembele et al., 2014). Thus, two majorvolutionary forces may be shaping a diversity of MSP1a: hostmmune system and pathogen-tick interactions. New combinationsf tandem repeats may give adaptive advantage to A. marginale inegions with high host immunity and transmission by ticks.

Finally, assuming that all strains in Cluster � have a MSP1� com-on ancestor with tandem repeat structure (3-�-�-�-�-�-�-N),e could consider AmRio1 as a highly divergent strain compared

o AmRio2 that still conserve 3 tandem repeats type � from thencestor. However, we do not have enough evidence to certainlyay whether this diversification is related or not to transmissiony R. microplus. Further transmission studies are needed to deter-ine whether these newly reported strains are transmissible by R.icroplus and, in case of transmissible phenotype, whether AmRio1ave some transmission fitness differences with AmRio2 related tohese differences in MSP1a sequences.

onclusions

We successfully isolated two strains of A. marginale using IDE8ick cell cultures. The strains were genetically divergent and may besed in future for the development of anti-A. marginale vaccines andiagnostics tests. In addition, these strains may be useful to test theypothesis regarding the tick-transmissibility genotype and MSP1aariability.

cknowledgements

To the National Council for Scientific and Technological Devel-pment (CNPq), and to the Coordination Office for Improvement ofigher-Education Staff (CAPES), for their financial support.

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