genetic diversity and molecular phylogeny of anaplasma marginale isolates from minas gerais, brazil

Veterinary Parasitology 121 (2004) 307–316

Genetic diversity and molecular phylogeny ofAnaplasma marginaleisolates from

Minas Gerais, Brazil�

José de la Fuentea,∗, Lygia M.F. Passosb,Ronald A. Van Den Busschec, Múcio F.B. Ribeirod,

E.J. Facury-Filhoe, Katherine M. Kocanaa Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University,

250 McElroy Hall, Stillwater, OK 74078, USAb Departamento de Medicina Veterinária Preventiva, Escola de Veterinária,

Universidade Federal de Minas Gerais (UFMG), CP 567,Belo Horizonte 31170-220, Minas Gerais, Brazil

c Department of Zoology and Collection of Vertebrates, Oklahoma State University, Stillwater, OK 74078, USAd Departamento de Parasitologia-ICB, Universidade Federal de Minas Gerais (UFMG), CP 567, Belo

Horizonte 31170-220, Minas Gerais, Brazile Departamento de Cl´ınica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas

Gerais (UFMG), CP 567, Belo Horizonte 31170-220, Minas Gerais, Brazil

Received 1 August 2003; received in revised form 17 February 2004; accepted 17 February 2004

Abstract

Anaplasma marginale(Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is en-demic in tropical and subtropical regions of the world, and many isolates ofA. marginalemay occurin a given geographic area. Phylogenetic relationships have been reported forA. marginaleisolatesfrom the US using gene and protein sequences of MSP1a andmsp4. These studies demonstratedthatmsp4sequences, but not MSP1a DNA or protein sequences, provide phylogeographic infor-mation and also that MSP1a sequences are highly heterogeneous amongA. marginalepopulations.However, little information is available on the genetic diversity ofA. marginaleisolates from otherregions of the world. The present study was undertaken to examine genetic variation among 10isolates ofA. marginaleobtained from infected cattle in the State of Minas Gerais, Brazil, whereA.marginaleis endemic. Neighbor-joining analysis ofmsp4sequences of Brazilian and New Worldisolates ofA. marginalefrom Argentina, Mexico and the US provided bootstrap support for a Latin

� The GenBank accession numbers formsp1α andmsp4sequences of Brazilian isolates ofA. marginaleareAY283198–AY283200 and AY283189–AY283197, respectively.

∗ Corresponding author. Tel.:+1-405-744-0372; fax:+1-405-744-5275.E-mail address:[email protected] (J. de la Fuente).

0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2004.02.021

308 J. de la Fuente et al. / Veterinary Parasitology 121 (2004) 307–316

American clade. The sequences of the MSP1a repeats of four Brazilian isolates ofA. marginalewere compared to sequences of Latin American and US isolates. The MSP1a repeated sequencesof Latin American isolates ofA. marginalehad nine repeat forms,α–�, which have not beenreported previously in North American isolates ofA. marginale. Furthermore, the repeated formsτ, σ andµ were only present in the Brazilian isolates. The results demonstrated that the geneticheterogeneity observed among isolates ofA. marginaleis common in endemic areas, independentof the predominant tick vector and is consistent with previous studies in whichmsp4providedphylogeographic information aboutA. marginaleisolates, while MSP1a was found not to be auseful marker for phylogeographic characterization ofA. marginaleisolates.© 2004 Elsevier B.V. All rights reserved.

Keywords: Anaplasma marginale; Rickettsia; Major surface protein; Evolution; Tick;Boophilus

1. Introduction

Anaplasma marginaleis a rickettsial pathogen that causes the disease anaplasmosis incattle (reviewed byKocan et al., 2003). Feeding ticks effect biological transmission of thisobligate intraerythrocytic organism, while mechanical transmission occurs when infectedblood is transferred to susceptible cattle by biting flies or blood contaminated fomites. Manygeographic isolates ofA. marginalehave been identified which differ in biology, morphol-ogy, protein sequence and antigenic characteristics (reviewed byKocan et al., 2003) andhave been characterized by the major surface protein (MSP) 1a which varies in sequence andmolecular weight due to different numbers of tandem 28–29 amino acid repeats (reviewedby de la Fuente et al., 2001a).

Recent research withA. marginalehas focused on MSPs that are involved in interactionswith vertebrate and invertebrate host cells (McGarey et al., 1994; McGarey and Allred,1994; de la Fuente et al., 2001b,c, 2003a) and have been used to elucidate phylogeographicpatterns ofA. marginale(de la Fuente et al., 2001a,d, 2002, 2003b,c; Kano et al., 2002; Lewet al., 2002). These MSPs are involved in host–pathogen interactions and may evolve morerapidly than other nuclear genes because of selective pressures exerted by host immunesystems. Of the sixA. marginaleMSPs that have been identified and characterized, onlythree (MSP1a, MSP4 and MSP5) are encoded by single genes. Because these MSPs donot appear to undergo antigenic variation in cattle or ticks (Bowie et al., 2002), they wereposted to be more stable genes for phylogenetic studies. MSP1a, encoded bymsp1α, hasbeen reported to be an adhesin for bovine erythrocytes and tick cells and to be involvedin adhesion, infection and transmission ofA. marginaleby Dermacentorspp. (McGareyet al., 1994; McGarey and Allred, 1994; de la Fuente et al., 2001b,c, 2003a). Molecularcharacterization ofA. marginaleisolates from the US demonstrated that MSP1a gene andprotein sequences are highly variable in isolates within and between different geographiclocations and evidenced thatmsp1α is under positive selection pressure and is not a markerfor geographic isolates (Palmer et al., 2001; de la Fuente et al., 2003b). Although thespecific function of MSP4 is currently not known, our previous analysis of themsp4genefrom A. marginaleisolates demonstrated sufficient sequence variation to support its use inphylogeographic studies (de la Fuente et al., 2001d, 2002, 2003b,c).

J. de la Fuente et al. / Veterinary Parasitology 121 (2004) 307–316 309

A. marginaleis endemic in Brazil, and in other regions of Latin America, where the maintick vector isBoophilus microplus(Guglielmone, 1995; Vidotto et al., 1998; Braz et al.,2000). Two morphologically distinctA. marginaleisolates have been reported in Brazil,based on the presence of an inclusion appendage (Patarroyo et al., 1987, 1994; Ribeiroet al., 1997). Recently, antigenic diversity among Brazilian isolates ofA. marginalewasdemonstrated using monoclonal antibodies (Kano et al., 2002; Gonçalves Ruiz et al., 2002a;Oliveira et al., 2003). Genetic diversity among Brazilian isolates ofA. marginalehas beenstudied byFerreira et al. (2001)using repetitive DNA elements in the rickettsial genome.Tick transmission and electron microscopy studies demonstrated that the tailless isolatemultiplied in epithelial cells ofB. microplus(Ribeiro and Lima, 1996), while a tailed isolatewas not infective for ticks (Gonçalves Ruiz et al., 2002b). These studies documented theoccurrence of antigenic, genetic, morphological and biological differences among Brazilianisolates ofA. marginalebut did not explore the genetic diversity of the MSP sequences.

In this study, we examine the genetic variation among isolates ofA. marginaleobtainedfrom infected cattle in the State of Minas Gerais, Brazil, using MSP4 and MSP1a nucleotideand protein sequences.

2. Materials and methods

2.1. A. marginale isolates

Ten Brazilian isolates ofA. marginalewere analyzed in this study. The isolate Brazil (dela Fuente et al., 2002, Table 1) corresponds to the Brazilian tailed isolate ofA. marginale(isolate A.m.-MG inGonçalves Ruiz et al., 2002a,b) and was originally obtained from anacutely infected cow from Pará de Minas, Minas Gerais (Ribeiro et al., 1997). The isolateBrazil was inoculated into a splenectomized calf and blood collected at peak rickettsemia

Table 1Amplified sequences ofA. marginaleisolates from Minas Gerais, Brazil, and their origin

Isolate Origin (location and date) Genes (no. of repeatsfor MSP1a)

GenBank accessionnumber

Brazil Para de Minas, Minas Gerais,1991 (Ribeiro et al., 1997).Characterized byGonçalvesRuiz et al. (2002a,b)andde laFuente et al. (2002)

MSP1a (5) MSP4 AF428092 AF428082

Brazil 3 Herd B, Minas Gerais, 2003 MSP4 AY283189Brazil 4 Herd C, Minas Gerais, 2003 MSP4 AY283190Brazil 5 Herd D, Minas Gerais, 2003 MSP1a (3) MSP4 AY283198 AY283191Brazil 6 Herd D, Minas Gerais, 2003 MSP4 AY283192Brazil 7 Herd E, Minas Gerais, 1992 MSP4 AY283193Brazil 8 Herd F, Minas Gerais, 1998 MSP4 AY283194Brazil 9 Herd G, Minas Gerais, 1997 MSP1a (4) MSP4 AY283199 AY283195Brazil 10 Herd H, Minas Gerais, 2003 MSP4 AY283196Brazil 12 Herd J, Minas Gerais, 2003 MSP1a (4) MSP4 AY283200 AY283197


3 weeks after inoculation was prepared as stabilate and stored in liquid nitrogen for furtherexperiments. Nine new isolates ofA. marginale(Brazil 3–10 and Brazil 12;Table 1) wereobtained from infected crossbred (Holstein x Gir) cattle in Minas Gerais, Brazil, and usedfor this study from naturally or experimentally infected cattle.A. marginaleisolates frominfected cattle and bison that were reported previously were used for sequence analysis andphylogenetic comparison (de la Fuente et al., 2001b, 2002, 2003b,c), except for an isolatefrom Puerto Rico for whichmsp1α (AY191826) andmsp4(AY191827) sequences wereobtained from the GenBank.A. marginaleisolates included 24 isolates from US cattle,named according to the US state in which they were isolated, except for those isolatesfrom Oklahoma which were named after the city from which they were obtained (de laFuente et al., 2003b) and two isolates from North American bison from the US and Canada,respectively (de la Fuente et al., 2003c). Also included were seven isolates from LatinAmerica (Mexico, Yucatan, Morelos and Veracruz from Mexico; Salta and Virasoro fromArgentina, and an isolate from Puerto Rico).

2.2. Polymerase chain reaction (PCR), sequence alignment and phylogenetic analysis

A. marginaleDNA was extracted from infected blood and themsp1α andmsp4geneswere amplified by PCR and sequenced as previously reported (de la Fuente et al., 2001d,2003b). The msp4coding region was completely sequenced. Formsp1α gene, only thefragment containing the tandem repeats in the variable region of the gene was sequencedin all the isolates (de la Fuente et al., 2001d, 2002, 2003b).

The A. marginale msp1α variable region and themsp4coding region were used forsequence alignment. Phylogenetic analysis was conducted withmsp4sequences. Multi-ple sequence alignment was performed using the program AlignX (Vector NTI Suite V5.5, InforMax, North Bethesda, MD, USA) with an engine based on the Clustal W al-gorithm (Thompson et al., 1994). Nucleotides were coded as unordered, discrete charac-ters with five possible character-states: A, C, G, T or N and gaps were coded as missingdata. Phylogenetic analysis was conducted using a distance-based (Kimura 2 parameter)neighbor-joining method followed by branch-swapping as implemented in PAUP∗4.0b4a(Swofford, 2000). Stability or accuracy of inferred topology(ies) were assessed via bootstrapanalysis (Felsenstein, 1985) of 500 iterations.

3. Results

Seven differentmsp4sequences were obtained from 10 isolates ofA. marginalefromMinas Gerais, Brazil. Silent single nucleotide substitutions differentiatedmsp4sequences,except for aT × G mutation at position 656 (position 1 corresponds to adenine at transla-tion initiation codon) of the Brazil isolate, which resulted in aV × G amino acid changeat position 219.

Phylogenetic relationships between Brazilian and New World isolates from Argentina,Mexico and the US were inferred usingmsp4sequences. Alignment ofmsp4DNA sequencesresulted in 854 bp of which 170 bp were variable. Bootstrap analysis revealed support (78%)


Wetumka1997

Californi

a

Idaho

St.Maries

Illinois

TexasMissouriOregon

Glencoe1

Glencoe2

Gle

ncoe

3

New

Castle

Okm

ulgeeStigler

Stillwater

Bison

Buffalo

BR

AZIL9

BRAZIL8&12

BRAZIL3

Mexico

Morelos

Salta

VeracruzVirasoro Yucatan Puerto R

ico

Brazil BR

AZI

L10

BR

AZI

L4,5

,&6

BR

AZ

IL7 OKCity

Flor

ida

Mis

siss

ippi

Virg

inia

Pawh

uska

1960

Cushing2

Cushing

Okeechobee

Stillwater2

71

54

78

51

63 60

Fig. 1. Unrooted neighbor-joining tree of New WorldA. marginaleisolates usingmsp4coding sequences, con-structed with PAUP∗ using Kimura 2 parameter corrected distance with numbers along branches reflecting thepercentage of 500 bootstrap iterations for each clade.

for a clade containing isolates ofA. marginalefrom Latin America, including the isolatesfrom Brazil (Fig. 1).

The sequence of the MSP1a repeats region was obtained for isolates Brazil 5, 9 and 12,and compared to the sequence of the isolate Brazil, also from Minas Gerais, and to all otherMSP1a sequences reported for New WorldA. marginaleisolates (Fig. 2). The sequences ofBrazilian isolates ofA. marginaledemonstrated the presence of 3–5 tandem repeats in theamino terminal portion of the protein as described for otherA. marginaleisolates (Fig. 2B).The MSP1a repeat sequences of Latin American isolates ofA. marginalehad nine repeat


forms,�–� (Fig. 2A), which were not present in the sequenced North American isolatesof A. marginale(Fig. 2). Furthermore, the repeat formsτ, σ andµ were present only in theBrazilian isolates (Fig. 2B).

4. Discussion

As we have demonstrated in previous studies (de la Fuente et al., 2001d, 2002, 2003b,c),msp4sequences provided phylogeographic patterns forA. marginaleisolates on a broad

(A)

Repeat form Encoded sequence A DDSSSASGQQQESSVSSQSE-ASTSSQLG- B A*****G************DQ********* C A*****G************GQ********* D A****************************G E A***************************** F T******************GQ********* G *******************GQ******S** H T******************GQ******S** I *******************GQ********* J A***L*G************DQ********* K A*G***G************DQ********* L AG****D************DQ********* M A******************GQ********* N T******************DQ********* O ----**G************DQ********* P T*************G****GQ**H*A*S** Q A******************DQ********* R A*****G***H********DQ*******W* S A*G***G************DQ********* T AG****G************DQ********* U *******************DQ********* V A*****G***-********DQ********* W T******************GQ******SR*

A*******------*L***GQ********* α

ΓπΣσ

τΦ

ß T*****GD***G*G*****GQ********* T******************D-********* A*****G************GQ******F** A*****G*********************** A*****G******I*****DH*********

µ A*******L**********GQ********* T**************L*P*GQ********* T*****************************

Fig. 2. Sequence of MSP1a tandem repeats in New World isolates ofA. marginale. (A) The one letter aminoacid code was used to depict the different sequences found in MSP1a repeats. Repeat forms A–J were designatedafter Palmer et al. (2001). Asterisks indicate identical amino acids. Gaps indicate deletions/insertions. (B) Thestructure of the MSP1a repeats region was represented for New World isolates ofA. marginaleusing the repeatforms described in (A).


(B)

A. marginale isolate Structure of MSP1a tandem repeats No. of repeats

Florida A B B B B B B B 8

Idaho D D D D D E 6

Virginia A B 2

Washington B B B C 4

Wetumka, OK K C H 3

Cushing, OK L C B C 4

Cushing 2, OK S N N F H 5

Glencoe 1, OK S F N F H 5

Glencoe 2, OK B M F H 4

Glencoe 3, OK T B C 3

Stillwater, OK S F F F H 5

Stillwater 2, OK L B C C 4

Stillwater 68, OK S B M F H 5

Oklahoma City, OK U 1

Okmulgee, OK S B V C 4

Stigler, OK T B B C 4

Pawhuska, OK I H 2

New Castle, OK L B C B 4

St. Maries, ID J B B 3

California B B C 3

Okeechobee, FL L B C B C 5

Mississippi D D D D E 5

Missouri B B B B 4

Illinois M N B M H 5

Texas O B M P 4

South Dakota A F H 3

Oregon A F H 3

Canadian bison D Q Q R 4

U.S. bison (buffalo) K B M F W 5

Yucatán T C B B C B 7

Mexico ß ß 4

Morelos ß ß 4

Veracruz ß ß 4

Brazil 9 ß M 4

Brazil 12 ß ß N 4

Brazil 5 C F N 3

Brazil B B Q µ 5

Virasoro B Q B C 5

Salta B B M 3

Puerto Rico E 6

Fig. 2. (Continued).


geographic scale, in which the Latin AmericanA. marginaleisolates clustered into a separateclade.

A. marginaleis endemic in Brazil, and like in most Latin American countries, the mainvector for its biological transmission isB. microplus(Guglielmone, 1995). In North Amer-ica, the main tick vectors areDermacentorspp. (reviewed byKocan et al., 2003). In aprevious study,de la Fuente et al. (2001d)presented evidence of the co-evolution ofD.variabilis–A. marginalein the United States using MSP1a and MSP4 sequences. The evo-lutionary history of vector-pathogen interactions could be reflected in the sequences ofMSP4 and MSP1a, which has been implicated in vector-pathogen interactions (McGareyet al., 1994; McGarey and Allred, 1994; de la Fuente et al., 2001b,c, 2003a). Therefore, theinteraction betweenA. marginaleandB. micropluscould influence the grouping of LatinAmerican isolates ofA. marginaleinto a clade separated from North American isolates,which are mainly transmitted byDermacentorspp. Similarly, vector-pathogen interactionscould influence the presence of particular MSP1a repeat sequences in Latin American iso-lates ofA. marginale. However, in geographic regions of Latin America whereB. microplushas been eradicated, anaplasmosis remains a problem (Guglielmone, 1995), suggestingthat mechanical transmission and/or other tick species are involved in transmission ofA.marginale. In tropical regions, the climatic conditions favor the life cycle of hematophagousDiptera, which are known to be efficient mechanical vectors ofA. marginale. Furthermore,characterization of a Brazilian isolate ofA. marginale(isolate Brazil in this study) nottransmissible byB. microplus(Gonçalves Ruiz et al., 2002b) suggests a role for mechanicaltransmission in the epidemiology ofA. marginalein Brazil.

As demonstrated in previous studies (de la Fuente et al., 2002, 2003b), MSP1a did notprovide phylogeographic information. Furthermore, within the State of Minas Gerais, thedifferent sequences for MSP1a repeats ofA. marginaleisolates provided evidence of geneticheterogeneity at this locus and reinforced our previous conclusion that MSP1a sequencesare not a marker for geographic isolates ofA. marginale. This heterogeneity probably re-flects cattle movement and maintenance of different genotypes by independent transmissionevents (Palmer et al., 2001; de la Fuente et al., 2003b). Similar results have been documentedfor Mexican isolates ofA. marginale(Rodrıguez et al., 2000; de la Fuente et al., 2002).

The results of this study demonstrated genetic heterogeneity among Brazilian isolatesof A. marginaleby use of MSP1a and MSP4 sequences, and suggest that this finding iscommon in endemic areas, independent of the predominant tick vector. The results furtherconfirm that MSP1a is not a marker for the characterization of geographic isolates ofA.marginale, while use ofmsp4provides useful phylogeographic information.

Acknowledgements

This research was supported by the Project No. 1669 of the Oklahoma Agricultural Ex-periment Station, the Endowed Chair for Food Animal Research (K.M. Kocan, College ofVeterinary Medicine, Oklahoma State University) and NIH Centers for Biomedical ResearchExcellence through a subcontract to J. de la Fuente from the Oklahoma Medical ResearchFoundation, and the Oklahoma Center for the Advancement of Science and Technology,Applied Research Grant, AR00(1)-001. Joy Yoshioka (Department of Veterinary Pathobi-


ology, Oklahoma State University) is acknowledged for editing the manuscript. Sue AnnHudiburg and Janet J. Rogers (Core Sequencing Facility, Department of Biochemistry andMolecular Biology, Noble Research Center, Oklahoma State University) are acknowledgedfor oligonucleotide synthesis and DNA sequencing, respectively.

References

Bowie, M.V., de la Fuente, J., Kocan, K.M., Blouin, E.F., Barbet, A.F., 2002. Conservation of major surface protein1 genes of the ehrlichial pathogenAnaplasma marginaleduring cyclic transmission between ticks and cattle.Gene 282, 95–102.

Braz Jr, C.J., Ribeiro, M.F., Lima, J.D., Passos, L.M., 2000. Development of an ELISA system for detectionof anti-Anaplasma marginaleantibodies in cattle in Brazil. J. Vet. Med. B Infect. Dis. Vet. Public Health 47,241–248.

de la Fuente, J., Garcia-Garcia, J.C., Blouin, E.F., Rodrıguez, S.D., Garcıa, M.A., Kocan, K.M., 2001a. Evolutionand function of tandem repeats in the major surface protein 1a of the ehrlichial pathogenAnaplasma marginale.Anim. Health Res. Rev. 2, 163–173.

de la Fuente, J., Garcia-Garcia, J.C., Blouin, E.F., Kocan, K.M., 2001b. Differential adhesion of major surfaceproteins 1a and 1b of the ehrlichial cattle pathogenAnaplasma marginaleto bovine erythrocytes and tick cells.Int. J. Parasitol. 31, 145–153.

de la Fuente, J., Garcia-Garcia, J.C., Blouin, E.F., Kocan, K.M., 2001c. Major surface protein 1a effects tickinfection and transmission of the ehrlichial pathogenAnaplasma marginale. Int. J. Parasitol. 31, 1705–1714.

de la Fuente, J., Van Den Bussche, R.A., Kocan, K.M., 2001d. Molecular phylogeny and biogeography of NorthAmerican isolates ofAnaplasma marginale(Rickettsiaceae: Ehrlichieae). Vet. Parasitol. 97, 65–76.

de la Fuente, J., Van Den Bussche, R.A., Garcia-Garcia, J.C., Rodrıguez, S.D., Garcıa, M.A., Guglielmone, A.A.,Mangold, A.J., Passos, L.M., Ribeiro, M.F., Blouin, E.F., Kocan, K.M., 2002. Phylogeography of New Worldisolates ofAnaplasma marginaleRickettsiaceae: Ehrlichieae based on major surface protein sequences. Vet.Microbiol. 88, 275–285.

de la Fuente, J., Garcia-Garcia, J.C., Blouin, E.F., Kocan, K.M., 2003a. Characterization of the functional domainof major surface protein 1a involved in adhesion of the rickettsiaAnaplasma marginaleto host cells. Vet.Microbiol. 91, 265–283.

de la Fuente, J., Van Den Bussche, R.A., Prado, T., Kocan, K.M., 2003b.Anaplasma marginalemajor surfaceprotein 1( genotypes evolved under positive selection pressure but are not markers for geographic isolates. J.Clin. Microbiol. 41, 1609–1616.

de la Fuente, J., Golsteyn Thomas, E.J., Van Den Bussche, R.A., Hamilton, R.G., Tanaka, E.E., Druhan, S.E.,Kocan, K.M., 2003c. Characterization ofAnaplasma marginaleisolated from North American bison. Appl.Environ. Microbiol. 69, 5001–5005.

Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.Ferreira, A.M., Suzart, S., Vidotto, O., Knowles, D.P., Vidotto, M.C., 2001. Use of repetitive DNA elements to

define genetic relationships amongAnaplasma marginaleisolates. FEMS Microbiol. Lett. 197, 139–143.Gonçalves Ruiz, P.M., Passos, L.M.F., Martins, M.S., Patarroyo, J.H., Ribeiro, M.F.B., 2002a. Antigenic

characterization of morphologically distinctAnaplasma marginaleisolates using a panel of monoclonalantibodies. Vet. Parasitol. 107, 169–177.

Gonçalves Ruiz, P.M., Passos, L.M.F., Ribeiro, M.F.B., 2002. Lack of transmission of a BrazilianAnaplasmamarginale isolate with appendage byBoophilus microplus. In: Proceedings of the Fourth InternationalConference on Ticks and Tick-borne Pathogens, University of Alberta, Banff, Canada, p. 41.

Guglielmone, A.A., 1995. Epidemiology of babesiosis and anaplasmosis in South and Central America. Vet.Parasitol. 57, 109–119.

Kano, F.S., Vidotto, O., Pacheco, R.C., Vidotto, M.C., 2002. Antigenic characterization ofAnaplasma marginaleisolates from different regions of Brazil. Vet. Microbiol. 87, 131–138.

Kocan, K.M., de la Fuente, J., Guglielmone, A.A., Meléndez, R.D., 2003. Antigens and alternatives for control ofAnaplasma marginaleinfection in cattle. Clin. Microbiol. Rev. 16, 698–712.


Lew, A.E., Bock, R.E., Minchin, C.M., Masaka, S., 2002. Amsp1α polymerase chain reaction assay for specificdetection and differentiation ofAnaplasma marginaleisolates. Vet. Microbiol. 86, 325–335.

McGarey, D.J., Allred, D.R., 1994. Characterization of hemagglutinating components on theAnaplasma marginaleinitial body surface and identification of possible adhesins. Infect. Immunol. 62, 4587–4593.

McGarey, D.J., Barbet, A.F., Palmer, G.H., McGuire, T.C., Allred, D.R., 1994. Putative adhesins ofAnaplasmamarginale: major surface polypeptides 1a and 1b. Infect. Immunol. 62, 4594–4601.

Oliveira, J.B., Madruga, C.R., Schenk, M.A.M., Kessler, R.H., Miguita, M., Araújo, F.R., 2003. Antigeniccharacterization of Brazilian isolates ofAnaplasma marginale. Mem. Inst. Oswaldo Cruz 98, 395–400.

Palmer, G.H., Rurangirwa, F.R., McElwain, T.F., 2001. Strain composition of the ehrlichiaAnaplasma marginalewithin persistently infected cattle, a mammalian reservoir for tick transmission. J. Clin. Microbiol. 39, 631–635.

Patarroyo, J.H.S., Viloria, M.I.V., Ribeiro, M.F.B., Santos, J.L., Faria, J.E., 1987.Anaplasma marginale:ultraestrutura de uma amostra de origem brasileira. Arq. Bras. Med. Vet. Zoot. 39, 839–847.

Patarroyo, J.H., Henckel, D.J., Prates, A.A., Mafra, C.L., 1994. Antigenic profile of a pure isolate ofAnaplasmamarginaleof Brazilian origin, using a western blot technique. Vet. Parasitol. 52, 129–137.

Ribeiro, M.F.B., Lima, J.D., 1996. Morphology and development ofAnaplasma marginalein midgut of engorgedfemale ticks ofBoophilus microplu. Vet. Parasitol. 61, 31–39.

Ribeiro, M.F., Passos, L.M., Guimaraes, A.M., 1997. Ultrastructure ofAnaplasma marginalewith an inclusionappendage, isolated in Minas Gerais State, Brazil. Vet. Parasitol. 70, 271–277.

Rodrıguez, S.D., Garcıa Ortız, M.A., Hernández Salgado, G., Santos Cerda, N.A., Aboytes Torres, R., CantoAlarcón, G.J., 2000.Anaplasma marginaleinactivated vaccine: dose titration against a homologous challenge.Comp. Immunol. Microbiol. Infect. Dis. 23, 239–252.

Swofford, D.L., 2000. PAUP∗ phylogenetic analysis using parsimony (∗and other methods), Version 4.02b. SinauerAssociates Inc. Publishers, Sunderland, MA.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.Nucl. Acid Res. 22, 4673–4680.

Vidotto, M.C., Vidotto, O., Andrade, G.M., Palmer, G., McElwain, T., Knowles, D.P., 1998. Seroprevalence ofAnaplasma marginalein cattle in Parana state, Brazil, by MSP-5 competitive ELISA. Ann. NY Acad. Sci. 849,424–426.

genetic diversity and molecular phylogeny of anaplasma marginale isolates from minas gerais, brazil

Documents