Genetic diversity and molecular phylogeny of Anaplasma marginale isolates from Minas Gerais, Brazil

Veterinary Parasitology 121 (2004) 307–316

Genetic diversity and molecular phylogeny of Anaplasma marginale isolates from Minas Gerais, Brazil夽 José de la Fuente a,∗ , Lygia M.F. Passos b , Ronald A. Van Den Bussche c , Múcio F.B. Ribeiro d , E.J. Facury-Filho e , Katherine M. Kocan a a

c

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, 250 McElroy Hall, Stillwater, OK 74078, USA b Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), CP 567, Belo Horizonte 31170-220, Minas Gerais, Brazil Department of Zoology and Collection of Vertebrates, Oklahoma State University, Stillwater, OK 74078, USA d Departamento de Parasitologia-ICB, Universidade Federal de Minas Gerais (UFMG), CP 567, Belo Horizonte 31170-220, Minas Gerais, Brazil e 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 endemic in tropical and subtropical regions of the world, and many isolates of A. marginale may occur in a given geographic area. Phylogenetic relationships have been reported for A. marginale isolates from the US using gene and protein sequences of MSP1a and msp4. These studies demonstrated that msp4 sequences, but not MSP1a DNA or protein sequences, provide phylogeographic information and also that MSP1a sequences are highly heterogeneous among A. marginale populations. However, little information is available on the genetic diversity of A. marginale isolates from other regions of the world. The present study was undertaken to examine genetic variation among 10 isolates of A. marginale obtained from infected cattle in the State of Minas Gerais, Brazil, where A. marginale is endemic. Neighbor-joining analysis of msp4 sequences of Brazilian and New World isolates of A. marginale from Argentina, Mexico and the US provided bootstrap support for a Latin 夽 The GenBank accession numbers for msp1α and msp4 sequences of Brazilian isolates of A. marginale are AY283198–AY283200 and AY283189–AY283197, respectively. ∗ Corresponding author. Tel.: +1-405-744-0372; fax: +1-405-744-5275. E-mail address: jose [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

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American clade. The sequences of the MSP1a repeats of four Brazilian isolates of A. marginale were compared to sequences of Latin American and US isolates. The MSP1a repeated sequences of Latin American isolates of A. marginale had nine repeat forms, α–, which have not been reported previously in North American isolates of A. marginale. Furthermore, the repeated forms τ, σ and µ were only present in the Brazilian isolates. The results demonstrated that the genetic heterogeneity observed among isolates of A. marginale is common in endemic areas, independent of the predominant tick vector and is consistent with previous studies in which msp4 provided phylogeographic information about A. marginale isolates, while MSP1a was found not to be a useful marker for phylogeographic characterization of A. marginale isolates. © 2004 Elsevier B.V. All rights reserved. Keywords: Anaplasma marginale; Rickettsia; Major surface protein; Evolution; Tick; Boophilus

1. Introduction Anaplasma marginale is a rickettsial pathogen that causes the disease anaplasmosis in cattle (reviewed by Kocan et al., 2003). Feeding ticks effect biological transmission of this obligate intraerythrocytic organism, while mechanical transmission occurs when infected blood is transferred to susceptible cattle by biting flies or blood contaminated fomites. Many geographic isolates of A. marginale have been identified which differ in biology, morphology, protein sequence and antigenic characteristics (reviewed by Kocan et al., 2003) and have been characterized by the major surface protein (MSP) 1a which varies in sequence and molecular weight due to different numbers of tandem 28–29 amino acid repeats (reviewed by de la Fuente et al., 2001a). Recent research with A. marginale has focused on MSPs that are involved in interactions with 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 phylogeographic patterns of A. marginale (de la Fuente et al., 2001a,d, 2002, 2003b,c; Kano et al., 2002; Lew et al., 2002). These MSPs are involved in host–pathogen interactions and may evolve more rapidly than other nuclear genes because of selective pressures exerted by host immune systems. Of the six A. marginale MSPs that have been identified and characterized, only three (MSP1a, MSP4 and MSP5) are encoded by single genes. Because these MSPs do not appear to undergo antigenic variation in cattle or ticks (Bowie et al., 2002), they were posted to be more stable genes for phylogenetic studies. MSP1a, encoded by msp1α, has been reported to be an adhesin for bovine erythrocytes and tick cells and to be involved in adhesion, infection and transmission of A. marginale by Dermacentor spp. (McGarey et al., 1994; McGarey and Allred, 1994; de la Fuente et al., 2001b,c, 2003a). Molecular characterization of A. marginale isolates from the US demonstrated that MSP1a gene and protein sequences are highly variable in isolates within and between different geographic locations and evidenced that msp1α is under positive selection pressure and is not a marker for geographic isolates (Palmer et al., 2001; de la Fuente et al., 2003b). Although the specific function of MSP4 is currently not known, our previous analysis of the msp4 gene from A. marginale isolates demonstrated sufficient sequence variation to support its use in phylogeographic studies (de la Fuente et al., 2001d, 2002, 2003b,c).

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A. marginale is endemic in Brazil, and in other regions of Latin America, where the main tick vector is Boophilus microplus (Guglielmone, 1995; Vidotto et al., 1998; Braz et al., 2000). Two morphologically distinct A. marginale isolates have been reported in Brazil, based on the presence of an inclusion appendage (Patarroyo et al., 1987, 1994; Ribeiro et al., 1997). Recently, antigenic diversity among Brazilian isolates of A. marginale was demonstrated using monoclonal antibodies (Kano et al., 2002; Gonçalves Ruiz et al., 2002a; Oliveira et al., 2003). Genetic diversity among Brazilian isolates of A. marginale has been studied by Ferreira et al. (2001) using repetitive DNA elements in the rickettsial genome. Tick transmission and electron microscopy studies demonstrated that the tailless isolate multiplied in epithelial cells of B. microplus (Ribeiro and Lima, 1996), while a tailed isolate was not infective for ticks (Gonçalves Ruiz et al., 2002b). These studies documented the occurrence of antigenic, genetic, morphological and biological differences among Brazilian isolates of A. marginale but did not explore the genetic diversity of the MSP sequences. In this study, we examine the genetic variation among isolates of A. marginale obtained from infected cattle in the State of Minas Gerais, Brazil, using MSP4 and MSP1a nucleotide and protein sequences.

2. Materials and methods 2.1. A. marginale isolates Ten Brazilian isolates of A. marginale were analyzed in this study. The isolate Brazil (de la Fuente et al., 2002, Table 1) corresponds to the Brazilian tailed isolate of A. marginale (isolate A.m.-MG in Gonçalves Ruiz et al., 2002a,b) and was originally obtained from an acutely infected cow from Pará de Minas, Minas Gerais (Ribeiro et al., 1997). The isolate Brazil was inoculated into a splenectomized calf and blood collected at peak rickettsemia Table 1 Amplified sequences of A. marginale isolates from Minas Gerais, Brazil, and their origin Isolate

Origin (location and date)

Genes (no. of repeats for MSP1a)

GenBank accession number

Brazil

Par´a de Minas, Minas Gerais, 1991 (Ribeiro et al., 1997). Characterized by Gonçalves Ruiz et al. (2002a,b) and de la Fuente et al. (2002) Herd B, Minas Gerais, 2003 Herd C, Minas Gerais, 2003 Herd D, Minas Gerais, 2003 Herd D, Minas Gerais, 2003 Herd E, Minas Gerais, 1992 Herd F, Minas Gerais, 1998 Herd G, Minas Gerais, 1997 Herd H, Minas Gerais, 2003 Herd J, Minas Gerais, 2003

MSP1a (5) MSP4

AF428092 AF428082

MSP4 MSP4 MSP1a (3) MSP4 MSP4 MSP4 MSP4 MSP1a (4) MSP4 MSP4 MSP1a (4) MSP4

AY283189 AY283190 AY283198 AY283191 AY283192 AY283193 AY283194 AY283199 AY283195 AY283196 AY283200 AY283197

Brazil 3 Brazil 4 Brazil 5 Brazil 6 Brazil 7 Brazil 8 Brazil 9 Brazil 10 Brazil 12

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3 weeks after inoculation was prepared as stabilate and stored in liquid nitrogen for further experiments. Nine new isolates of A. marginale (Brazil 3–10 and Brazil 12; Table 1) were obtained from infected crossbred (Holstein x Gir) cattle in Minas Gerais, Brazil, and used for this study from naturally or experimentally infected cattle. A. marginale isolates from infected cattle and bison that were reported previously were used for sequence analysis and phylogenetic comparison (de la Fuente et al., 2001b, 2002, 2003b,c), except for an isolate from Puerto Rico for which msp1α (AY191826) and msp4 (AY191827) sequences were obtained from the GenBank. A. marginale isolates included 24 isolates from US cattle, named according to the US state in which they were isolated, except for those isolates from Oklahoma which were named after the city from which they were obtained (de la Fuente 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 Latin America (Mexico, Yucatan, Morelos and Veracruz from Mexico; Salta and Virasoro from Argentina, and an isolate from Puerto Rico). 2.2. Polymerase chain reaction (PCR), sequence alignment and phylogenetic analysis A. marginale DNA was extracted from infected blood and the msp1α and msp4 genes were amplified by PCR and sequenced as previously reported (de la Fuente et al., 2001d, 2003b). The msp4 coding region was completely sequenced. For msp1α gene, only the fragment containing the tandem repeats in the variable region of the gene was sequenced in all the isolates (de la Fuente et al., 2001d, 2002, 2003b). The A. marginale msp1α variable region and the msp4 coding region were used for sequence alignment. Phylogenetic analysis was conducted with msp4 sequences. Multiple sequence alignment was performed using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD, USA) with an engine based on the Clustal W algorithm (Thompson et al., 1994). Nucleotides were coded as unordered, discrete characters with five possible character-states: A, C, G, T or N and gaps were coded as missing data. 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 bootstrap analysis (Felsenstein, 1985) of 500 iterations.

3. Results Seven different msp4 sequences were obtained from 10 isolates of A. marginale from Minas Gerais, Brazil. Silent single nucleotide substitutions differentiated msp4 sequences, except for a T × G mutation at position 656 (position 1 corresponds to adenine at translation initiation codon) of the Brazil isolate, which resulted in a V × G amino acid change at position 219. Phylogenetic relationships between Brazilian and New World isolates from Argentina, Mexico and the US were inferred using msp4 sequences. Alignment of msp4 DNA sequences resulted in 854 bp of which 170 bp were variable. Bootstrap analysis revealed support (78%)

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Fig. 1. Unrooted neighbor-joining tree of New World A. marginale isolates using msp4 coding sequences, constructed with PAUP∗ using Kimura 2 parameter corrected distance with numbers along branches reflecting the percentage of 500 bootstrap iterations for each clade.

for a clade containing isolates of A. marginale from Latin America, including the isolates from 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 other MSP1a sequences reported for New World A. marginale isolates (Fig. 2). The sequences of Brazilian isolates of A. marginale demonstrated the presence of 3–5 tandem repeats in the amino terminal portion of the protein as described for other A. marginale isolates (Fig. 2B). The MSP1a repeat sequences of Latin American isolates of A. marginale had nine repeat

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forms, ␣– (Fig. 2A), which were not present in the sequenced North American isolates of A. marginale (Fig. 2). Furthermore, the repeat forms τ, σ and µ were present only in the Brazilian isolates (Fig. 2B). 4. Discussion As we have demonstrated in previous studies (de la Fuente et al., 2001d, 2002, 2003b,c), msp4 sequences provided phylogeographic patterns for A. marginale isolates on a broad (A)

Repeat form A B C D E F G H I J K L M N O P Q R S T U V W α ß Γ π Σ σ µ τ Φ

Encoded sequence DDSSSASGQQQESSVSSQSE-ASTSSQLGA*****G************DQ********* A*****G************GQ********* A****************************G A***************************** T******************GQ********* *******************GQ******S** T******************GQ******S** *******************GQ********* A***L*G************DQ********* A*G***G************DQ********* AG****D************DQ********* A******************GQ********* T******************DQ********* ----**G************DQ********* T*************G****GQ**H*A*S** A******************DQ********* A*****G***H********DQ*******W* A*G***G************DQ********* AG****G************DQ********* *******************DQ********* A*****G***-********DQ********* T******************GQ******SR* A*******------*L***GQ********* T*****GD***G*G*****GQ********* T******************D-********* A*****G************GQ******F** A*****G*********************** A*****G******I*****DH********* A*******L**********GQ********* T**************L*P*GQ********* T*****************************

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

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(B)

A. marginale isolate Florida Idaho Virginia Washington Wetumka, OK Cushing, OK Cushing 2, OK Glencoe 1, OK Glencoe 2, OK Glencoe 3, OK Stillwater, OK Stillwater 2, OK Stillwater 68, OK Oklahoma City, OK Okmulgee, OK Stigler, OK Pawhuska, OK New Castle, OK St. Maries, ID California Okeechobee, FL Mississippi Missouri Illinois Texas South Dakota Oregon Canadian bison U.S. bison (buffalo) Yucatán Mexico Morelos Veracruz Brazil 9 Brazil 12 Brazil 5 Brazil Virasoro Salta Puerto Rico

Structure of MSP1a tandem repeats A D A B K L S S B T S L S U S T I L J B L D B M O A A D K T

C B B E

B D B B C C N F M B F B B B B H B B B B D B N B F F Q B C ß ß ß ß ß F B B B

B D

B D

B H B N N F C F C M

C C F F H F C F

V B

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Fig. 2. (Continued ).

B

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No. of repeats B

8 6 2 4 3 4 5 5 4 3 5 4 5 1 4 4 2 4 3 3 5 5 4 5 4 3 3 4 5 7 4 4 4 4 4 3 5 5 3 6

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geographic scale, in which the Latin American A. marginale isolates clustered into a separate clade. A. marginale is endemic in Brazil, and like in most Latin American countries, the main vector for its biological transmission is B. microplus (Guglielmone, 1995). In North America, the main tick vectors are Dermacentor spp. (reviewed by Kocan et al., 2003). In a previous study, de la Fuente et al. (2001d) presented evidence of the co-evolution of D. variabilis–A. marginale in the United States using MSP1a and MSP4 sequences. The evolutionary history of vector-pathogen interactions could be reflected in the sequences of MSP4 and MSP1a, which has been implicated in vector-pathogen interactions (McGarey et al., 1994; McGarey and Allred, 1994; de la Fuente et al., 2001b,c, 2003a). Therefore, the interaction between A. marginale and B. microplus could influence the grouping of Latin American isolates of A. marginale into a clade separated from North American isolates, which are mainly transmitted by Dermacentor spp. Similarly, vector-pathogen interactions could influence the presence of particular MSP1a repeat sequences in Latin American isolates of A. marginale. However, in geographic regions of Latin America where B. microplus has been eradicated, anaplasmosis remains a problem (Guglielmone, 1995), suggesting that mechanical transmission and/or other tick species are involved in transmission of A. marginale. In tropical regions, the climatic conditions favor the life cycle of hematophagous Diptera, which are known to be efficient mechanical vectors of A. marginale. Furthermore, characterization of a Brazilian isolate of A. marginale (isolate Brazil in this study) not transmissible by B. microplus (Gonçalves Ruiz et al., 2002b) suggests a role for mechanical transmission in the epidemiology of A. marginale in Brazil. As demonstrated in previous studies (de la Fuente et al., 2002, 2003b), MSP1a did not provide phylogeographic information. Furthermore, within the State of Minas Gerais, the different sequences for MSP1a repeats of A. marginale isolates provided evidence of genetic heterogeneity at this locus and reinforced our previous conclusion that MSP1a sequences are not a marker for geographic isolates of A. marginale. This heterogeneity probably reflects cattle movement and maintenance of different genotypes by independent transmission events (Palmer et al., 2001; de la Fuente et al., 2003b). Similar results have been documented for Mexican isolates of A. marginale (Rodr´ıguez et al., 2000; de la Fuente et al., 2002). The results of this study demonstrated genetic heterogeneity among Brazilian isolates of A. marginale by use of MSP1a and MSP4 sequences, and suggest that this finding is common in endemic areas, independent of the predominant tick vector. The results further confirm that MSP1a is not a marker for the characterization of geographic isolates of A. marginale, while use of msp4 provides useful phylogeographic information.

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

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

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