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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

pidemiology and evolution of the genetic variability of Anaplasmaarginale in South Africa

welani M. Mutshembelea,b, Alejandro Cabezas-Cruzc,d,∗, Moses S. Mtshali a,b,riel M.M. Thekisoeb, Ruth C. Galindoc, José de la Fuentec,e

Research and Scientific Services Department, National Zoological Gardens of South Africa, P.O. Box 754, Pretoria 0001, South AfricaDepartment of Zoology and Entomology, University of the Free State, QwaQwa Campus, Private Bag x13, Phuthaditjhaba 9866, South AfricaSaBio, Instituto de Investigación en Recursos Cinegéticos IRES-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, SpainCenter for Infection and Immunity of Lille (CIIL), INSERM U1019, CNRS UMR 8204, Université Lille Nord de France, Institut Pasteur de Lille, Lille, FranceDepartment of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA

r t i c l e i n f o

rticle history:eceived 5 March 2014eceived in revised form 2 April 2014ccepted 17 April 2014vailable online xxx

eywords:. marginaleSP1a

revalenceelective pressuresSP1a vaccines

a b s t r a c t

Bovine anaplasmosis caused by infection of cattle with Anaplasma marginale has been considered tobe endemic in South Africa, an assumption based primarily on the distribution of the tick vectors of A.marginale and serological studies on the prevalence of anaplasmosis in Limpopo, Free State, and NorthWest. However, molecular evidence of the distribution of anaplasmosis has only been reported in theFree State province. In order to establish effective control measures for anaplasmosis, epidemiologicalsurveys are needed to define the prevalence and distribution of A. marginale in South Africa. In addition,a proposed control strategy for anaplasmosis is the development of an A. marginale major surface protein1a (MSP1a)-based vaccine. Nevertheless, regional variations of this gene would need to be characterizedprior to vaccine development for South Africa. The objectives of the present study were therefore toconduct a national survey of the prevalence of A. marginale in South Africa, followed by an evaluation of thediversity and evolution of msp1a in South African strains of A. marginale. To accomplish these objectives,species-specific PCR was used to test 250 blood samples from cattle collected from all South Africanprovinces (including 26 districts and municipalities), except the Free State province where similar studieswere reported previously. The prevalence of A. marginale ranged from 65% to 100%, except in NorthernCape province where A. marginale was not detected. A correlation was found between the prevalence andgenetic diversity of A. marginale MSP1a. Additionally, the genetic diversity of the A. marginale MSP1a wasfound to evolve under negative and positive selection, and 23 new tandem repeats in South Africa wereshown to have evolved from the extant tandem repeat 4. Despite the MSP1a genetic variability, some

types of tandem repeats were found to be conserved among the A. marginale strains, and low-variablepeptides in MSP1a tandem repeats were subsequently identified. The results of this research confirmedthat anaplasmosis is endemic in South Africa. The results of the molecular characterization of the MSP1acan then be used as the basis for development of new and novel vaccines for anaplasmosis control inSouth Africa.

ntroduction

Bovine anaplasmosis is a non-contagious tick-borne disease

Please cite this article in press as: Mutshembele, A.M., et al., Epidemioloin South Africa. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016

aused by infection of cattle with Anaplasma marginale, an obligatentraerythrocytic bacterium classified in the family Anaplasmat-ceae, order Rickettsiales (Dumler et al., 2001). This pathogen is

∗ Corresponding author at: Center for Infection and Immunity of Lille (CIIL),NSERM U1019, CNRS UMR 8204, Université Lille Nord de France, Institut Pasteure Lille, Lille, France. Tel.: +33 631 235 191.

E-mail address: cabezasalejandrocruz@gmail.com (A. Cabezas-Cruz).

ttp://dx.doi.org/10.1016/j.ttbdis.2014.04.011877-959X/© 2014 Elsevier GmbH. All rights reserved.

© 2014 Elsevier GmbH. All rights reserved.

transmitted biologically by ticks, mechanically by biting insects andblood-contaminated fomites and from cow to calf via transplacen-tal transmission (Aubry and Geale, 2011). Five tick species havebeen shown experimentally to transmit A. marginale in South Africa,including Rhipicephalus microplus, R. decoloratus, R. evertsi evertsi, R.simus, and Hyalomma marginatum rufipes (as reviewed by de Waal,2000). Acute disease in cattle is characterized by weight loss, fever,abortion, low milk production, and in some cases death. The ani-

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

mals that recover from the disease become persistently infectedand serve as reservoir of infection for mechanical transmission andbiological transmission by ticks (Kocan et al., 2003). Anaplasmosisis widespread in South Africa and, as estimated by de Waal (2000),

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99)% of the total cattle population is at risk of acquiring A. marginalenfection.

Currently, antimicrobial drugs are not available for the elim-nation of persistent infections in cattle. Although the Worldrganization for Animal Health proposed the use of enrofloxacin,

midocarb, and oxytetracycline for the elimination of persistent. marginale infections in cattle, these antimicrobial drugs wereot found to eliminate the persistent A. marginale infectionsCoetzee et al., 2005, 2006). Vaccines have been used as an alter-ative method for control of anaplasmosis. A live vaccine using A.arginale ssp. centrale (A. centrale), a subspecies of relatively lowathogenicity, is available in South Africa, Australia, Israel, and Latinmerica, but this vaccine has proven to be only partially effective,nd reports of vaccination failure are not uncommon (Kocan et al.,003). While anaplasmosis still constitutes a problem for cattle pro-uction in South Africa, sales of vaccines for bovine anaplasmosis

n South Africa have reduced from 800,000 to 200,000 doses over2 years (1976–1998) (de Waal, 2000).

MSP1a has been shown to have potential use as a vaccine antigenecause this protein contains both neutralization-sensitive (Palmert al., 1987; Allred et al., 1990) and immunodominant epitopesGarcia-Garcia et al., 2004). Recently, the use of MSP1a for vaccineevelopment for A. marginale has regained new attention. The Nerminus tandem repeated region of this protein was used in immu-ization trials in cattle against A. marginale (Torina et al., 2014)nd laboratory animal models (Santos et al., 2013; Silvestre et al.,014) and demonstrated promising results. MSP1a is one of 6 MSPshat have been described in A. marginale. This protein is encodedy a single-copy gene, msp1a, which is conserved during the mul-iplication of the parasite in cattle and ticks (Kocan et al., 2003)nd has been useful for epidemiological studies of A. marginale in

Please cite this article in press as: Mutshembele, A.M., et al., Epidemioloin South Africa. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016

arious regions of the world (Ruybal et al., 2009; Almazán et al.,008). MSP1a contains tandem repeats at the N terminus of therotein, which present functional residues that serve as adhesinsor bovine erythrocytes and tick cells, a prerequisite for infection of

ig. 1. Map of South African areas included in the study. Map of South Africa showing thauteng, KwaZulu-Natal, Eastern Cape, Western Cape, and Northern Cape). Endemic, epidaal, 2000). The main tick species involved in the transmission of A. marginale in the sam

data collected from de Waal, 2000).

PRESSborne Diseases xxx (2014) xxx–xxx

host cells (McGarey et al., 1994; de la Fuente et al., 2003a). Whilethe tandem repeats of MSP1a are highly variable, repeats are com-monly represented among worldwide strains (Cabezas-Cruz et al.,2013) and have been shown to evolve under positive selection (dela Fuente et al., 2003b). However, the specific codon positions thatevolve under positive or negative selection have not been reported.For development of MSP1a-based vaccines, some characteristics ofMSP1a should be taken into consideration, including (i) the extantgenetic variability of MSP1a, (ii) the evolution of MSP1a geneticdiversity, and (iii) the conservative nature of some tandem repeatsamong different isolates.

In this study, molecular evidence is provided regarding theprevalence of A. marginale in 8 of the 9 South African provinces.Additionally, evolution of the genetic diversity of A. marginalemsp1a in South Africa was studied, demonstrating that differentcodon positions of this gene evolved under positive or nega-tive selection, likely due to immune selection and transmissionfitness. Finally, these studies demonstrated the low variabilityof some tandem repeats commonly found among A. marginalestrains. Collectively, results of this research will contribute towarddevelopment of new and novel vaccines for control of bovineanaplasmosis in South Africa and other regions of the world.

Materials and methods

Study site and samples collection

Blood samples were collected for these studies from May 2011to July 2013 in 26 districts and municipalities from 8 South Africanprovinces (Fig. 1). Coordinates of the collection sites are provided

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

in Table 1, as well as the farm production systems. Cattle for thesestudies were randomly selected, and blood samples were collectedonly from adult animals after the owner’s consent. While infor-mation regarding age or sex of the animals was not recorded,

e provinces that were included in the study (Limpopo, Mpumalanga, North West,emic, and A. marginale-free areas are coloured differentially (data collected from depled areas are shown: Rhipicephalus microplus, R. decoloratus, and R. evertsi evertsi

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Table 1Sampling sites, farming system, and coordinates in the South African provinces.

South Africanprovinces

Farming system Sampling sites Map coordinates

Limpopo Communal Capricorn Dist., Aganang Municipality 23◦40′ S, 29◦5′ EMpumalanga Communal Ehlanzeni South Dist. 25◦27′00′′ S, 30◦58′59′′ EGauteng Commercial West Rand Dist., Merafong Municipality, Khutsong South, Carltonville 26◦20′1′′ S, 27◦19′39′′ E

26◦22′S, 27◦24′ ENorth West Communal Moretele Dist., Maubane 25◦16′ S, 28◦15′ EKwaZulu-Natal Communal Pietermaritzburg Chota, Umhlati Dist., Albert Falls, Shallow drift,

Umgungundlovu Dist, Richmond Municipality, Ndaleni Dip Tank29◦29′17.16′′ S, 30◦26′27.78′′ E29◦28′33.5′′ S, 30◦26′11.3′′ E29◦52′55.3′′ S, 30◦40′56.2′′ E

Eastern Cape Mix Amathole Dist., Nkokobe Municipality, Middledrift, Nxuba Municipality 26◦38′39′′ E, 32◦46′52′′ S26◦51′25′′ E, 32◦41′12′′ S27◦11′58′′ E, 32◦52′37′′ S26◦26′40′′ E, 32◦44′25′′ S26◦18′18′′ E, 32◦42′04′′ S

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Mix Stellenbosch Dist., Boland

Northern Cape Communal John Taolo Gaetsewe Dist., Ga

accination histories were not available for these animals. Bloodamples were collected by tail venipuncture using an 18-gaugeeedle and sterile 10 ml vacutainer EDTA tubes and stored at 4 ◦C.

NA extraction

Genomic DNA was extracted from cattle blood samples using ZRenomic DNATM Tissue Miniprep (Zymo Research, CA, USA). DNAas resuspended in DNA elution buffer and stored at −20 ◦C. The

oncentration of DNA was determined using the NanoDrop® ND-000 (NanoDrop Technologies Inc., Wilmington, USA).

. marginale species-specific PCR

A specific set of primers was used to amplify msp1aene – 1733F-(5′-TGTGCTTATGGCAGACATTTCC-3′), 2957R-(5′-AACCTTGTAGCCCCAACTTATCC-3′) (Lew et al., 2002). Primersere synthesized and supplied by Inqaba Biotech [Inqaba Biotech-ical Industries (Pty) Ltd., Pretoria, Gauteng, South Africa]. PCReactions were prepared using Master Mix (Thermo ScientificreamTaq Green PCR), 0.1–1.0 �M of Forward and Reverse primersnd 1 �g of DNA template in 25 �l final volume. The amplifica-ion cycles consisted of 40 cycles of 1 min at 94 ◦C, 1 min at 65 ◦C,nd 2 min at 72 ◦C. Amplified products were separated in 1% TBE89 mM Tris–Borate, 2 mM EDTA, pH 8) agarose gel using 1 kb lad-er as a DNA size marker (1 kb DNA ladder, Life Sciences, FermentasmbH, Germany). DNA was visualized by gel staining in 0.5 �g/mlelRed (Fermentas GmbH, Germany) under UV illumination andhotographed.

NA sequencing

PCR products containing only single amplification productsf msp1a were sequenced at Inqaba Biotech facilities [Inqabaiotechnical Industries (Pty) Ltd., Pretoria, Gauteng, South Africa].he termination reactions were performed using BigDye VER3.1ABI, Life Technologies, CA, USA) according to manufacturer’snstructions. The labelled fragments were purified using Zymoesearch sequencing clean-up kit (Zymo Research, CA, USA) and

Please cite this article in press as: Mutshembele, A.M., et al., Epidemioloin South Africa. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016

ubsequently analyzed on a 3500×L Genetic Analyzer (ABI, Lifeechnologies, CA, USA). The sequences obtained in this study wereubmitted to GenBank and provided with accession numbers forsp1a (KC470153-KC470196) gene.

33 44 28.6 S, 18 59 53 E

yana Municipality, Kuruman, Zero Farm 27◦18′53′′ S, 23◦42′15′′ E

Sequence analysis

To identify the msp1a gene sequences obtained in our study,the database Nucleotide collection (nr/nt) using Megablast (opti-mize for highly similar sequences) from the BLAST server wasused (Zhang et al., 2000). Protein homology and identity analysiswere performed using the multiple-alignment program ClustalW(Thompson et al., 1994). The MSP1a tandem repeats found in thisstudy were reported previously (Cabezas-Cruz et al., 2013).

Codon-based phylogenetic analysis of tandem repeats

Codon-based alignment was performed using the codon suiteserver (Schneider et al., 2005, 2007). Detection of selection pressureon individual codons was calculated using 2 methods, single like-lihood ancestor counting (SLAC) and fixed effects likelihood (FEL)(Pond and Frost, 2005), used in the Datamonkey webserver (Delportet al., 2010; Pond and Frost, 2005). Positive and negative selectionswere assigned to codon where ω = dN (non-synonymous substi-tutions)/dS (synonymous substitutions) ratio was higher or lowerthan 1, respectively. The reconstruction of the ancestral amino acidsequence was performed using a neighbour joining tree rootedin tandem repeat 83 under the Dayhoff model of substitutionswhich was estimated to be the best model fitting the actual data.Three reconstruction methods were used: Joint (Pupko et al., 2000),marginal (Yang et al., 1995), and sample (Nielsen, 2002), which arealso used in the Datamonkey webserver.

Amino acid variability, composition and genetic diversity index(GDI)

The amino acid variability was calculated in the variabilityserver (Garcia-Boronat et al., 2008) using the Shannon entropy (H)formula (Shannon, 1948) as follows:

H = −M∑

i=1

Pilog2 Pi

where Pi is the fraction of residues with a certain type of aminoacid i, and M is the number of types of amino acid for a position.The proportion of variable over conserved positions was calculatedas the number of positions with more than 0 of Shannon variabil-ity divided by the number of positions with 0 Shannon variability.

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

Amino acids were also classified regarding biochemistry propertiesto know negative charged, positively charged, uncharged-polar,and non-polar. For comparison purposes, Shannon variability wasadditionally calculated in 28 and 43 MSP1a sequences available

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Table 2Observed prevalences of Anaplasma marginale in different provinces of South Africa.

South Africanprovinces

No. of bloodsamples collectedper province

Prevalence of A.marginale,msp1a-positivePCR (%)

No. of msp1asequenced

Limpopo 20 13 (65) 8Mpumalanga 21 21 (100) 2Gauteng 20 20 (100) 5North West 33 24 (72) 7KwaZulu-Natal 55 50 (90) 9Eastern Cape 40 40 (100) 3Western Cape 16 14 (87.5) 11Northern Cape 45 0 (0) 0

icftsd

R

M

StmeeasoeWmewbwsspiaw2wrtpacttlbApgoci

Fig. 2. Newly reported sequences of MSP1a tandem repeats. The one-letter aminoacid code was used to depict MSP1a repeat sequences. Dots indicate identical aminoacids, and gaps indicate deletion/insertions. The ID of each repeat form was assigned

Free Statea 215 129 (51.3%) 29

a Data collected from Mtshali et al. (2007).

n GenBank from Venezuela and the USA, respectively. To furtherharacterize the genetic diversity of MSP1a, a GDI was calculatedor each A. marginale strain as follows: number of different MSP1aandem repeats divided by the total number of tandem repeats pertrain. 1 and 0 were considered maximum and minimum geneticiversity, respectively.

esults and discussion

olecular evidence of A. marginale prevalence in South Africa

Bovine anaplasmosis has been considered to be endemic inouth Africa, an assumption based primary on the distribution ofhe tick vectors (Ndou et al., 2010) and the seroprevalence of A.arginale which was determined only in the Free State (Dreyer

t al., 1998), Limpopo (Rikhotso et al., 2005), and North West (Ndout al., 2010) provinces. Molecular evidence of endemic bovinenaplasmosis has not been reported in most of South Africa. Thistudy was therefore designed to investigate the molecular evidencef A. marginale infection in cattle from Mpumalanga, Gauteng, East-rn Cape, Limpopo, North West, KwaZulu-Natal, Eastern Cape, andestern Cape provinces (Table 2). Molecular diagnosis of anaplas-osis was determined previously in Free State province (Mtshali

t al., 2007). In the present study, species-specific msp1a primersere used for PCR assays on 250 DNA samples obtained from cattle

lood. The results of these PCR studies confirmed that A. marginale isidespread in South Africa, but with a variable prevalence in all the

ampled provinces, except for Northern Cape in which no positiveamples were detected (Table 2). The absence of A. marginale-ositive PCR in Northern Cape is not surprising because this area

s considered to be free of tick vectors (Mtshali and Mtshali, 2013),nd the prevalence of Babesia spp., another tick-borne pathogen,as also found to be very low in this province (Mtshali and Mtshali,

013). The provinces with the highest prevalences of A. marginaleere Mpumalanga, Gauteng, and Eastern Cape with an infection

ate of 100%. In the other provinces, A. marginale infections in cat-le ranged from 65% to 90% (Table 2 and Fig. 3). Differences in therevalence of A. marginale were not observed between commercialnd communal farming systems. The prevalence of A. marginale inattle from South Africa can be considered high when comparedo the prevalence of A. marginale in cattle from other regions ofhe world, for example, in Brazil, a recent study found 70% preva-ence of A. marginale in a cattle herd (Pohl et al., 2013). A studyy de la Fuente et al. (2005) showed a range from 25% to 100% of. marginale prevalence in cattle herds from Italy. Analysis of therevalence and genetic diversity of A. marginale MSP1a in different

Please cite this article in press as: Mutshembele, A.M., et al., Epidemioloin South Africa. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016

eographic regions constitutes an important step toward devel-pment of effective MSP1a-based vaccines because the antigenicomposition of the vaccine should contain MSP1a variants presentn different regions of the world.

previously in Cabezas-Cruz et al. (2013). Tandem repeat A was used as a model foramino acid comparison.

A. marginale prevalence and MSP1a genetic diversity

The sequence of A. marginale MSP1a was analyzed in 44 strains(Table 3). We found 52 different types of tandem repeats amongSouth African strains (including those reported by Mtshali et al.(2007), from Free State province), 23 of which were describedfor the first time (Fig. 2). Using a GDI, the genetic diversity perA. marginale strain was described (Table 3), and the average ofgenetic diversity was calculated per province. A polynomial cor-relation (R2 = 0.76) occurred between the GDI and the prevalenceof anaplasmosis per province (Fig. 3). Interestingly, provinces with100% of A. marginale prevalence were not the provinces with thehighest or lowest GDI, rather these provinces were between arange of genetic diversity (0.82–0.87). Two driving forces, immuneselection and transmission fitness, have been suggested to impactgenetic diversification of A. marginale (Palmer and Brayton, 2013).Immune pressure in cattle may induce greater genetic diversityin A. marginale populations in order to insure persistent infec-tion. At the same time, the development of antigenic variationwas suggested to have a transmission cost (Palmer and Brayton,2013). Anaplasma marginale strains with lower transmission fit-ness due to high genetic variability would be at risk of eliminationfrom the population (Palmer and Brayton, 2013). Furthermore,high incidence of ticks correlated with increased MSP1a geneticvariability in Argentina (Ruybal et al., 2009). These apparently con-tradictory aspects may be reconciled if increased tick transmissioncontributes to greater circulation of A. marginale strains in the cattlepopulation, favouring the interaction between A. marginale strainsand potentially resistant hosts. In this study, the observed range

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

of GDI, in which 100% prevalence (an indicator of transmissionefficiency) exist, may reflect a positive balance between geneticdiversity and biological transmission by ticks taking into accountthat, as shown in Fig. 1, all the studied areas except for Northern

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ape is infested by ticks. Notably, most of the new tandem repeatshown in Fig. 2 were sequenced from KwaZulu-Natal provincehich has 90% prevalence of A. marginale. When KwaZulu-Natalas excluded from the correlation analysis between A. marginalerevalence and GDI (Fig. 3), the polynomial regression increasedrom R2 = 0.76 to R2 = 0.98. A possible explanation for this result maye that the tandem repeats diversification found among KwaZulu-atal MSP1a provided a degree of transmission fitness of the

trains present in that area. Another explanation could be thatattle immunity is increasing in the area, inducing A. marginaleiversification for antigenic variation. One interesting question thatemains unanswered is how genetic diversity of A. marginale msp1amerges in a specific region.

volution of MSP1a genetic diversity

In order to analyze the evolution of MSP1a genetic diversity

Please cite this article in press as: Mutshembele, A.M., et al., Epidemioloin South Africa. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016

bserved in our samples, the tandem repeats (Table 3) were classi-ed as “frequent” (present more than 22 times) or “rare” (present

ess than 10 times) based on the frequency of their appearancemong South African A. marginale strains, including those from Free

able 3naplasma marginale strains and MSP1a tandem repeats organization.

A. marginalestrain

Origin Structure of MSP1a tandem repeats

LP-7 Limpopo 34 159

LP-10 Limpopo 27 13 3

LP-30 Limpopo 27 13 3

LP-34 Limpopo 34 13 3

LP-37 Limpopo 27 13 13

LP-46 Limpopo 3 38

LP-50 Limpopo 34 13 13

MP-C2 Mpumalanga 34 13 158

MP-C5 Mpumalanga 15 15 100

NW-C2 North West 27 13 4

NW-C4 North West 27 13 4

NW-C5 North West 82 13 79

NW-C1-160312 North West 34 13 3

NW-C4-160312 North West 34 36 38

GP-C1 Gauteng 82 13 4

GP-C2 Gauteng 34 27 3

GP-C5 Gauteng 3 4 4

GP-C1112105 Gauteng 34 37

GP-C4117105 Gauteng 3 36 38

GP-C7117105 Gauteng 34 13 13

GP-C1817105 Gauteng 34 13 37

KZN-D KwaZulu-Natal 42 43 25

KZN-F KwaZulu-Natal 42 43 25

KZN-K KwaZulu-Natal 27 13 4

KZN-Y KwaZulu-Natal 143 144 145

KZN-MM KwaZulu-Natal 42 43 25

KZN-14 KwaZulu-Natal 142 43 25

KZN-19 KwaZulu-Natal 141 140 140

KZN-49 KwaZulu-Natal 147 148 149

KZN-51 KwaZulu-Natal 147

EC-22 Eastern Cape 27 13 4

EC-23 Eastern Cape 151 152 4

EC-24 Eastern Cape 27 13 4

WC-4 Western Cape 40 Q Q

WC-6 Western Cape 3 4 4

WC-7 Western Cape M M M

WC-8 Western Cape 34 4 37

WC-10 Western Cape 154

WC-11 Western Cape 40 Q Q

WC-12 Western Cape 27 13 37

WC-13 Western Cape M Q M

WC-14 Western Cape 155 36 38

WC-15 Western Cape 160 13 37

WC-16 Western Cape 34 13 4

ore common tandem repeats are highlighted (bold) and mentioned in order of abundana Genetic diversity index of MSP1a (GDI) was calculated as follows: number of differen

enetic diversity. The average of GDI (AVE-GDI) and standard deviation (STDEV) for each

PRESSorne Diseases xxx (2014) xxx–xxx 5

State province reported by Mtshali et al. (2007). The unique tan-dem repeats found in this study were classified as rare, repeated,most of them, one time in only one strain (Fig. 2). In contrast, tan-dem repeats 3, 4, 13, 34, Q, and 37 had a high frequency (Table 3)and were also reported in A. marginale strains from Israel (3, 4),South America (4, 13), and Europe (Q). Repeat sequences 34 and37 were abundant only in South Africa with rare exceptions (asreviewed by Cabezas-Cruz et al., 2013). Considering this, we wantedto test whether the tandem repeats newly described in this study(Fig. 2) originated from extant A. marginale MSP1a tandem repeatsor had evolved from a tandem repeat that was lost after tandemrepeat differentiation. To test this hypothesis, we reconstructedthe ancestral state (see ‘Materials and methods’) of the new tan-dem repeats presented in Fig. 2. Surprisingly, the ancestral stateof all the new tandem repeats was tandem repeat 4 (Fig. 4) sug-gesting that all of the newly described tandem repeats from SouthAfrican A. marginale strains evolved from this tandem repeat. In

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

addition, this evidence suggests that these repeated sequences mayconstitute a group of recently evolved tandem repeats specific toSouth Africa and, in fact, these repeats have not been reportedelsewhere (Cabezas-Cruz et al., 2013). The mechanism for the

GDIa AVE-GDI/STDEV

1 0.917/0.14436 1

138 137 0.75

10.667

37 1 0.875/0.17783 0.754 37 0.8 0.960/0.08937 14 37 136 38 13 14 37 0.8 0.826/0.17338 13 3 38 0.7144 37 0.6

110.6671

161 31 1 0.919/0.12831 31 0.84 37 0.8146 131 131 1

0.667150 1

14 37 0.8 0.867/0.1154 153 0.8

1m 0.75 0.741/0.28637 0.75M 0.25

11

Q Q 37 0.4291

Q M 0.41

4 161 113 13 4 37 0.571

ce, from higher to lower: 13, 4, 37 and 3, 34 have the same frequency, respectively.t MSP1a tandem repeats/total number of tandem repeats per strain. 1 is maximumregion is shown.

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Fig. 3. Correlation between msp1a genetic diversity and A. marginale prevalence inSouth Africa. The prevalences of anaplasmosis in different South African provinceswere plotted against the average of genetic diversity index (GDI). GDI was calcu-lated for each strain as the number of different tandem repeats divided by the totalnumber of tandem repeats. A polynomial correlation was found between these 2parameters with R2 = 0.76. Provinces with 100% prevalence (Gauteng, Eastern Cape,and Mpumalanga) are in the range of 0.82–0.87 GDI while regions having less than100% prevalence are out of this range.

Fig. 4. Reconstruction of ancestral amino acid sequence and amino acid positions undeTandem repeat 4, sequence marked with *) of the new tandem repeats found in South Afrand sample (see ‘Materials and methods’ for details). Positions that evolve under negativis indicated (arrow). The residues of the immunodominant B-cell epitope (Garcia-Garcia eAllred et al., 1990) (second box) are also shown.

Table 4Sites that evolved under positive and negative selection in the new tandem repeats from

Codons Method SLAC

ω (dN/dS) p value

1 5.49 0.043

6 1.58 0.311

16 1.39 0.371

20 2.86 0.172

28 2.22 0.197

8 −1.66 0.373

10 −6.81 0.037

25 −3.57 0.134

PRESSborne Diseases xxx (2014) xxx–xxx

generation of genetic diversity of A. marginale could be gene dupli-cation followed by either mutation or mismatch repair as proposedby Palmer and Brayton (2013). The variable number of tandemrepeats found among MSP1a sequences suggests that tandemrepeat duplication due to homologous recombination may occurin order to give a “substrate” for “testing” competitive advantageof new tandem repeats variants. As demonstrated by this research,new tandem repeats shown in Fig. 2 evolved from an initial tan-dem repeat 4 which served as a template for genetic variation.To test whether tandem repeat 4 evolved to the new forms underselective pressures, the ratio ω (see ‘Materials and methods’) wascalculated for each codon position of the new tandem repeats fromSouth Africa. We found that the diversification of tandem repeat 4in South African strains occurred under both positive and negativeselective pressures (Table 4 and Fig. 4). Surprisingly, the positionsevolving under negative selection (Fig. 4, negative signs), 8 and 10,were reported before to be present in an immunodominant B-cellepitope present in MSP1a (Fig. 4, first boxed area, Garcia-Garciaet al., 2004), and position 25, also evolving under negative selec-

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

tion, was found previously in a neutralization-sensitive epitope(Fig. 4, second boxed area; Palmer et al., 1987; Allred et al., 1990).In contrast, one of the positions found to be evolving under positive

r positive and negative selection. The reconstruction of the ancestral state (TR 4:ica (Fig. 2) was performed using 3 reconstruction methods, namely: joint, marginal,e (−) and positive (+) selection are shown (see Table 4). Amino acid at position 20t al., 2004) (first box) and the neutralization-sensitive epitope (Palmer et al., 1987;

South Africa.

Method FEL Type of selection

ω (dN/dS) p value

Infinite 0.012 PositiveInfinite 0.093 PositiveInfinite 0.196 PositiveInfinite 0.133 PositiveInfinite 0.079 Positive−5.14 0.236 Negative−13.89 0.016 Negative−6.56 0.110 Negative

IN PRESSG ModelT

Tick-borne Diseases xxx (2014) xxx–xxx 7

sblcdgoTtcrGaBmd((d7msrsnt

smnivtstmufa

A

aooifaTAUaSitbwmiltasmc

Fig. 5. MSP1a amino acid variability, composition, and low-variable peptides. Thefigure shows the amino acid variability and composition among the tandem repeatsin 3 different countries: Venezuela (A), USA (B), and South Africa (C). Venezuelashows a high proportion of variable/conserved sites and a high average of amino acidvariability while South Africa and the USA show middle and lower values, respec-tively. Different colours in columns depict different biochemical properties in theamino acid composition: negative (green), positive (red), uncharged-polar (beige),and non-polar (blue); proportion of deleted positions is shown in yellow. Consen-sus sequences of low-variable peptides are shown (*) for the USA and South Africa.The region of the immunodominant B-cell epitope from A. marginale (Garcia-Garciaet al., 2004) is boxed in the low-variable peptide from South Africa. (For interpre-tation of the references to colour in this figure legend, the reader is referred to the

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election was the amino acid position 20 (Fig. 4, arrow), which haseen implicated in the binding of MSP1a to tick cells extract (de

a Fuente et al., 2003a). These findings support the hypothesis dis-ussed above that immune escape and tick transmission are bothriving forces of A. marginale MSP1a genetic diversification and sug-est that tick transmission and immune escape would be triggersf the observed diversification of tandem repeat 4 in South Africa.his trend of purifying selection/negative selection (or deletion ofhe unfit) for sites involved in immune recognition by the host wasonfirmed, in wider frame, by the following data: (i) Most of theesidues of the MSP1a immunodominant B-cell epitope (Garcia-arcia et al., 2004) were deleted in the tandem repeats forms �nd 108. The tandem repeat � is widespread in strains from Mexico,razil, Venezuela, Argentina, and Taiwan and is also present in theost common A. marginale strains of the world which have the tan-

em repeat composition �, �, �, �, � (Cabezas-Cruz et al., 2013).ii) The first glutamine (Q) of the neutralization-sensitive epitopePalmer et al., 1987; Allred et al., 1990) is deleted in several tan-em repeats (A, D, E, �, �, �, 5–9, 14, 31, 36, 52, 57, 60–66, 69–72,6, 84–86, 95–99, 105, 107, 116–119, 129–131, 136–139) from A.arginale strains reported previously (Cabezas-Cruz et al., 2013). As

een in Fig. 4, Q was deleted in some of South African new tandemepeats (146, 147, 148, 149, and 150). Collectively, this evidenceuggests that purifying selection is most likely one of the mecha-isms that A. marginale had evolved to escape immune recognitionoward MSP1a.

Tandem repeat 4 from MSP1a was also found in A. marginaletrains isolated from an anaplasmosis outbreak in Mexico where R.icroplus was implicated as tick vector (Almazán et al., 2008), andew tandem repeats were reported also in this study. It should be

nteresting to test whether these newly described Mexican MSP1aariants evolved from tandem repeat 4. We consider these findingso be relevant to the development of MSP1a-based vaccines anduggest that MSP1a-based vaccination should be combined withick control strategies in order to minimize genetic diversity of A.arginale MSP1a. A recent study was reported that combined these of a tick-protective antigen with MSP1a in the same vaccineormulation that was directed toward control both tick infestationsnd anaplasmosis (Torina et al., 2014).

mino acid variability and low-variable MSP1a peptides

Genetic variability could also be analyzed by calculating themino acid variability in each position of the tandem repeat. Inrder to compare the MSP1a genetic variability in South Africa withther regions of the world, we calculated the amino acid variabil-ty for 29 amino acid positions from all the MSP1a tandem repeatsound in South Africa, in the USA, and in Venezuela which werevailable in GenBank and collected by Cabezas-Cruz et al. (2013).he amino acid variability of MSP1a tandem repeats from Southfrica can be seen in Fig. 5 in comparison with those from theSA (low) and Venezuela (high). In agreement with this, the aver-ge of amino acid variability was 0.30, 0.42, and 0.72 for USA,outh Africa, and Venezuela, respectively (Fig. 5A–C). In compar-son, the proportion of variable over-conserved positions in MSP1aandem repeats was higher in Venezuela (29) compared to those ofoth the USA (0.85) and South Africa (2.2). Epitopes from MSP1aere found to induce a protective immune response against A.arginale (Santos et al., 2013). Considering this, using the variabil-

ty server (Garcia-Boronat et al., 2008), we explored whether someow-variable peptides could be found in MSP1a from South Africa,he USA, and Venezuela. We observed that the amino acid vari-

Please cite this article in press as: Mutshembele, A.M., et al., Epidemioloin South Africa. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016

bility of MSP1a tandem repeats from South Africa and the USAupported the existence of low-variable peptides (Fig. 5), whichay be useful in the development of peptide-based MSP1a vac-

ines, while the high amino variability of MSP1a tandem repeats

web version of this article.)

from Venezuela does not support this type of low-variable peptide.Interestingly, the low variability peptide in South Africa overlapswith the position of the immunodominant B-cell epitope reportedpreviously for MSP1a (Garcia-Garcia et al., 2004).

Conclusions

In this molecular study, bovine anaplasmosis was confirmed tobe widespread in South Africa. The genetic diversity of A. marginaleMSP1a was found to occur through the evolution of extant tandemrepeats which, under positive and negative selection, diversify tonew tandem repeat variants that are likely constricted by 2 forces:the host immune system and tick transmission. Furthermore, com-mon MSP1a tandem repeats were present among A. marginalestrains in South Africa and other regions of the world which in

gy and evolution of the genetic variability of Anaplasma marginale/j.ttbdis.2014.04.011

combination with the use of low-variable MSP1a peptides will con-tribute to the development of MSP1a-based vaccines for the controlof anaplasmosis.

ING ModelT

8 Tick-

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D

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ARTICLETBDIS-334; No. of Pages 8

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onflict of interest

The authors declare no conflict of interest.

cknowledgements

This research was funded by National Research Foundation. Theuthors are grateful to the Directors of Department of Agriculturend Veterinary Institute from Mpumalanga, North West, Kwa-Zuluatal, Eastern Cape, and Western Cape (Prof Nomfundo Mnisi, Drlilo, Dr Songelwayo Chisi, Dr L Mrwebi, Dr Ronald Sinclair) with

he assistance from the Veterinary Technicians (Mr Toypat Mdluli,s Keneilwe Constance Kaotane, Mr Jan Masethe Maime, Mr Anil

uresh, Mr Erwin Lucas) and the farmers who facilitated sampleollection. Dr Chris Marufu for sample donations from Eastern Cape.s Sinesipho Ntanta (DST intern) for her wonderful help in the

aboratory.

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