Accepted Manuscript Title: Genetic diversity and molecular phylogeny of Anaplasma marginale studied longitudinally under natural transmission conditions in Rio de Janeiro, Brazil Author: Jenevaldo Barbosa Silva Luiz Ricardo Gonc¸alves Alessandro de Mello Varani Marcos Rog´erio Andr´e Rosangela Zacarias Machado PII: DOI: Reference:
S1877-959X(15)00070-9 http://dx.doi.org/doi:10.1016/j.ttbdis.2015.04.002 TTBDIS 470
To appear in: Received date: Revised date: Accepted date:
30-6-2014 26-3-2015 8-4-2015
Please cite this article as: Silva, J.B., Gonc¸alves, L.R., Varani, A.M., Andr´e, M.R., Machado, R.Z.,Genetic diversity and molecular phylogeny of Anaplasma marginale studied longitudinally under natural transmission conditions in Rio de Janeiro, Brazil, Ticks and Tick-borne Diseases (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Genetic diversity and molecular phylogeny of Anaplasma marginale studied
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longitudinally under natural transmission conditions in Rio de Janeiro, Brazil
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Jenevaldo Barbosa Silva1, Luiz Ricardo Gonçalves1, Alessandro de Mello Varani2,
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Marcos Rogério André1, Rosangela Zacarias Machado1*
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Jaboticabal SP, Brazil.
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Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias,
Departamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias,
Jaboticabal SP, Brazil.
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*Corresponding author:
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Rosangela Zacarias Machado ([email protected])
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Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade
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de Ciências Agrárias e Veterinárias FCAV-UNESP, Via de Acesso Prof. Paulo Donato
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Castellane s/n, 14884-900, Jaboticabal, SP, Brasil. Phone: +55 16 3209-2663.
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ABSTRACT- Anaplasma marginale is the most prevalent tick-borne pathogen in cattle
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in tropical and subtropical regions of the world. Major Surface Protein 1a (MSP1a) has
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been found to be a stable genetic marker for identifying A. marginale isolates within
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geographical regions. It is conserved in cattle during infection and tick-borne
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transmission of the pathogen. The aim of the present longitudinal study was to
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determine occurrences of genetic diversity associated with high prevalence of A.
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marginale under natural transmission conditions. Twenty calves were evaluated every
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three months during the first year of life. Rickettsemia levels due to A. marginale,
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measured as the number of msp1α copies/ml in the blood of positive calves, ranged
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from 2.06 x 103 to 4.36 1012. The numbers of MSP1a tandem repeats among MSP1a
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tandem repeats were 3 and 6. The predominant msp1α microsatellite was E, and another
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MSP1a tandem repeat was found that presented genotype G. Nineteen different MSP1a
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tandem repeats of A. marginale were found circulating in animals. The MSP1a tandem
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repeats 4-63-27 (27.5%), 78-242-25-31 (n = 21.6%) and τ-102-15 (n = 17.6%) were the
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ones most commonly observed. Twenty-two MSP1a tandem repeats resulted in new
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sequences with amino acid changes, as shown in this study. Thirty sequences were
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found for the first time in Brazil. Glycine, glutamate, serine and alanine amino acids
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were found at position 20. During the study, 80% (16/20) of the animals were infected
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by more than one genotype. Three animals were born infected, with MSP1a tandem
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repeats 4-63-27, 78-242-25-31 and τ-102-15, thus indicating occurrence of transplacental
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transmission. In the phylogenetic analysis, nineteen different MSP1a tandem repeats of
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A. marginale were found in the cattle, which suggested that many MSP1a tandem
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repeats and high variation in MSP1a were occurring.
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KEYWORDS: Brazil, Cattle, Genotype, MSP1a, Tandem repeats
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INTRODUCTION
Anaplasma marginale (Rickettsiales: Anaplasmataceae) is the most prevalent
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tick-borne pathogen in the world, distributed on six continents, and is responsible for
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high morbidity and mortality among cattle in temperate, subtropical and tropical regions
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(Kocan et al., 2010). Bacteria of the genus Anaplasma are obligate intracellular
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pathogens that may be transmitted biologically by ticks, mechanically by blood sucking
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flies or infected blood in fomites, and also transplacentally (Aubry and Geale, 2011).
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Brazil can be classified as an area of endemic stability for occurrences of bovine
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anaplasmosis, from north to south and from east to west, with prevalence ranging from
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16.3% in the semi-arid zone of the state of Sergipe (Oliveira et al., 1992) to close to
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100% in the states of Minas Gerais, Bahia, Paraíba and Paraná (Ribeiro and Reis, 1981;
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Araújo et al., 1995; Madruga et al., 1994; Vidotto et al., 1995). In the state of Rio de
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Janeiro, a serological survey showed that 98.21% of the cattle were positive for A.
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marginale (Souza et al., 2000).
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Major Surface Protein 1 alpha (MSP1a) is a heterodimer composed of MSP1a
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(100 kDa) and MSP1b (105 kDa). These proteins are structurally unrelated and bonded
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non-covalently and they are exposed on the surface of A. marginale (Barbet and Allred,
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1991). MSP1a is formed by a single polymorphic gene composed by one region that is
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conserved and another region that is variable and contains a mutable number of units in
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tandem repeats (Allred et al., 1990). MSP1b is coded by a mutagenic family composed
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of two genes (msp1b1 and msp1b2) and three genes (msp1b1pg, msp1b2pg and
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msp1b3pg) that seem to recombine such that the genetic and antigenic diversity is
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increased (Barbet and Allred, 1991).
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Phylogenetic studies have identified several strains of A. marginale worldwide
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that differ in their morphology, amino acid sequence, antigenic characteristics and
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ability to be transmitted by ticks (Chaves et al., 2012; Cabezas-Cruz et al., 2013). The
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genetic diversity of MSP1a tandem repeats of A. marginale has previously been
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characterized based on the repeated amino acid sequence of MSP1a (Allred et al., 1990;
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de la Fuente et al., 2007). In addition, this sequence contains T and B cell epitopes that
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are important in the protective immune response (Brown et al., 2001; Brown et al.,
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2002).
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Research involving MSP1a tandem repeats is a promising route for acquiring
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comprehensive knowledge of the extensive worldwide diversity of A. marginale
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(Cabezas-Cruz et al., 2013). A single nomenclature has been created based on the data
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available in GenBank (de la Fuente et al., 2007). Studies in non-endemic regions have
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shown that this repeat sequence exhibits little variation (Palmer et al., 2004). However,
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in areas where anaplasmosis occurs endemically, MSP1a tandem repeats of A.
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marginale may exhibit high variability in their tandem repeats, and some animals can be
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infected by more than one MSP1a tandem repeat (de la Fuente et al., 2001; Palmer et
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al., 2001).
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Although significant variations in MSP1a tandem repeats have been observed in
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cattle in Brazil and Argentina (Vidotto et al., 2006; Ruybal et al., 2009; Pohl et al.
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2013), no study has yet been conducted involving cattle herds in an area of South
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America where multiple MSP1a tandem repeats of A. marginale circulate
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simultaneously. Therefore, the aim of the present longitudinal study was to determine
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occurrences of genetic diversity associated with high prevalence of A. marginale under
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natural transmission conditions.
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MATERIALS AND METHODS
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Study site and cattle population. A longitudinal study was conducted in 2012 and 2013
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at the Seropédica Experimental Station, Empresa de Pesquisa Agropecuária do Estado do
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Rio de Janeiro (Pesagro-Rio; Agricultural and Livestock Research Corporation of the State
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of Rio de Janeiro). The experimental area is located in the metropolitan microregion of the
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city of Rio de Janeiro (latitude 22º45’ S, longitude 43º41’ W, and altitude 33 m). This
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area’s average annual temperature is approximately 22.7 ºC, and it receives an average of
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1291.7 mm of precipitation per year. This region is characterized by two well-defined
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seasonal periods. The dry period (March to September) has lower temperatures and
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rainfall, which leads to a reduction in the population of vectors; the rainy period (October
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to February) has higher temperatures and rainfall, resulting in an increased number of
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vectors.
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In the herd used in this study, the serological prevalence of A. marginale was 70%
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and no clinical cases of this disease had been observed over the preceding three years
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(Silva and Fonseca, 2014). Pesagro-Rio had a herd of 410 animals, composed of 60 calves,
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70 heifers and 280 cows, with the potential to produce between 1,000 and 4,000 kg of milk
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per lactation. The herd was divided into groups according to the animals’ ages and
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physiological state. Each group of animals was kept in a different area of the experimental
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station. The calves aged 0 to 2 months were kept in a shed, in individual pens, and had
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access to an area of 0.5 ha of Brachiaria humidicula from the age of 15 days onwards. At
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this stage, they received 4 kg of milk per day. From 3 to 6 months of age, the calves
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received 4 kg of milk per day and were kept during the day in an area of 1.5 ha of
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Brachiaria decumbens and brought in at night, to individual pens. Between the ages of 7
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and 12 months, the calves were transferred to an area of 3 ha of Brachiaria decumbens and
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Panicum maximum, where they were kept during the day, and were brought into a
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collective pen at night.
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Twenty heifers (Bos taurus taurus x Bos taurus indicus) were evaluated every three
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months from birth until they reached 12 months of age, from May 2012 to May 2013. The
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first blood sampling was performed after the calves had ingested the colostrum, i.e. not
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more than one hour after their birth. Thus, during the study period, it was possible to
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perform five observations on each animal (at birth and at the ages of 3, 6, 9 and 12
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months), producing a total of 100 samples.
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Whole blood samples were collected from the caudal or jugular veins of individual
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calves. To prepare the serum samples, the blood samples collected were incubated at room
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temperature for 1 h and then centrifuged at 1000 × g for 15 minutes. Giemsa-stained blood
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smears were made for further microscopic examination. Blood and serum samples were
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stored at -20 ºC. DNA was extracted from 200 µl of each of the 100 whole blood samples
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using a QIAamp DNA blood mini-kit (Qiagen, Madison, WI, USA), in accordance with
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the manufacturer’s instructions.
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Antigen production. An A. marginale isolate from a calf in Jaboticabal in the state of
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São Paulo, Brazil (Andrade et al., 2004) was used to infect a calf for crude ELISA and
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IFAT antigen production. For this purpose, a 3-month-old splenectomized calf was
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inoculated with 200 ml of A. marginale-infected blood (1.0 x 107 infected
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erythrocytes/ml).
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erythrocytes/ml) was observed 7 days after the experimental infection. After the blood
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had been collected and processed for crude ELISA/IFAT antigen production (Machado
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et al., 1997), the experimentally infected animal was treated with oxytetracycline
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administered intramuscularly (20 mg/kg).
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Enzyme-Linked Immunosorbent Assay (ELISA) and Indirect Fluorescent
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Antibody Test (IFAT). These were performed as previously described by Machado et
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al. (1997) and Andrade et al. (2004).
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Semi-nested msp1α PCR. A semi-nested PCR (nPCR) was used to amplify the msp1α
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sequence (Lew et al., 2002). The reactions were performed using the primers 1733F (5'-
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TGT GCTTATGGCAGACATTTCC-3'), 3134R (5'-TCACGGTCAAAACCTTTGCTT
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ACC-3') and 2957R (5'-AAACCTTGTAGCCCCAACTTATCC-3').
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Quantitative PCR for detection and quantitation of A. marginale. Quantitative real-
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time PCR was used (qPCR), as described by Carelli et al. (2007) for the gene msp1β of
rickettsemia
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A. marginale, with the aim of estimating the parasitemia by means of absolute
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quantification (number of copies/µL). Serial dilutions were performed with the aim of
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constructing standards with different concentrations of plasmid DNA containing the
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target sequence (2.0 x 107 copies/µL to 2.0 x 100 copies/µL). The number of plasmid
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copies was determined in accordance with the formula (X g/µL DNA/[plasmid size (bp)
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x 660]) x 6.022 x 1023 x plasmid copies/µL. The amplification reactions were performed
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using a final total reaction volume of 10 µL, containing a mixture of 1.0 µL of sample
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DNA, 0.2 µL of probe, 0.9 µL of each primers, 5.0 µL of PCR buffer (IQ Multiplex
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Power Mix®, BioRad) and 2.0 µL of ultra-pure sterile water (Nuclease-Free Water ®,
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Promega).
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Sequence of the A. marginale msp1α microsatellite. A microsatellite is located at the
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5'-untranslated region (UTR) of the msp1α gene between the putative Shine-Dalgarno
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(GTAGG) sequence and the translation initiation codon (ATG) (de la Fuente et al.,
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2001). Its structure is GTAGG (G/ATTT)m (GT)n T ATG (Estrada-Peña et al., 2009).
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An analysis of the repeat sequences was performed in accordance with the nomenclature
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proposed by de la Fuente et al. (2007). The SD-ATG distance was calculated using the
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formula (4 × m) + (2 × n) + 1.
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Phylogenetic analysis. The phylogenetic analysis was performed using msp1α
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nucleotide sequences that were aligned with MAFFT (v7) and configured for highest
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accuracy (Katoh and Standley, 2013). After alignment, regions with gaps were removed
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from the alignment. Phylogenetic trees were reconstructed using the maximum
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likelihood (ML) and neighbor-joining (NJ) methods as implemented in PhyML (v3.0
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aLRT) (Anisimova and Gascuel, 2006; Guindon and Gascuel, 2003) and PHYLIP
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(v3.66) (Felsenstein, 1989), respectively. The reliability of the internal branching of the
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ML and NJ trees was assessed using the bootstrapping method (1000 bootstrap
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replicates). The graphical representation and editing of the phylogenetic trees were done
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using TreeDyn (v 198.3) (Chevenet et al., 2006).
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RESULTS
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A. marginale prevalence and bacteremia in calves. The animals were diagnosed
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positive for A. marginale during the first year of life by means of blood smears, ELISA,
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IFAT and qPCR (Table 1). The prevalence of A. marginale in blood smear samples
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ranged from 10% (newborn animals) to 80% (animals at 90 days of age). The
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serological variation in ELISA/IFAT was 35% (newborn animals) to 70% (360-day-old
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animals), while qPCR ranged from 15% (among newborns) to 100% (animals at 90
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days of age). Quantifying the number of MSP1a A. marginale copies per ml of blood
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allowed us to determine which animals may have been acutely and chronically infected.
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The rickettsemia levels in the positive animals ranged from 2.06 x 103 to 4.36 x 1012
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DNA copies per ml of blood (Table 1). Among the tests used, the following
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concordances were observed: qPCR/ELISA 57% (50/88), qPCR/IFAT 55% (48/87),
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qPCR/blood smear 65% (54/83), ELISA/blood smear 70% (45/64), ELISA/IFAT 95%
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(52/55), IFAT/blood smear 71% (44/62) and qPCR/ELISA/IFAT/blood smear 41%
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(36/88).
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Analysis of A. marginale msp1a sequences in calves. The msp1α gene was amplified
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using nPCR and sequenced in 51 samples from calves. The sequence analysis on the
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MSP1a microsatellites of A. marginale indicated that the genotypes E and G were
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present (Table 2). Three animals were born infected with MSP1a tandem repeats 4-63-
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27 [1.50 x 104], 78-242-25-31 [2.06 x 103] and τ-102-15 [1.12 x 105], thus indicating
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occurrence of transplacental transmission. Major and minor rickettsemia was observed
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in animals infected by MSP1a tandem repeats 78-242-25-31 [4.36 x 1012 and 2.06 x
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103]. The rickettsemia levels among the animals showed a correlation with age and were
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highest at three months of age. However, we did not observe any relationship between
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rickettsemia and the A. marginale genotype infecting the animals. Twenty-two new
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sequences were described in this study, which were labeled as 165 to 186 (Table 4). The
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analysis on the repeated MSP1a sequences also produced 30 sequences that were
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described for the first time in Brazil. Ten percent (2/20) of the animals (Rio16 and
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Rio19) were positive in only a single sample. Fifteen percent (3/20) of the animals
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(Rio1, Rio11 and Rio14) remained infected with only one MSP1a tandem repeat of A.
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marginale in multiple samples. However, 75% (15/20) of the positive animals tested
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positive in two to four samples and more than one infecting MSP1a tandem repeat of A.
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marginale was found during the study. One A. marginale-positive animal (Rio9)
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presented four different MSP1a tandem repeats in four samples.
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Occurrence of the most common A. marginale MSP1a tandem repeats. The number
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of repeat MSP1a sequences of A. marginale ranged from three to six. The most
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commonly observed sequences were those with three (41%) and five (37%) repeats. The
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most prevalent MSP1a tandem repeats were 4-63-27 (27,5%), 78-242-25-31 (21,6%)
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and τ-102-15 (17,6%) (Table 3). The most common repeat sequences were 24 (n = 27),
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4 (n = 24), 10 (n = 22), 63 (n = 21) and 27 (n = 21). The new repeat sequences are listed
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in Table 4. Only one animal (Rio15b) had an entire sequence of unprecedented repeats
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(178-179-180-181-182). Although most of the samples exhibited three repeats, 53%
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(9/17) of those that included new sequences in their structure were five-repeat
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sequences.
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The MSP1a tandem repeats of A. marginale identified in this study exhibited
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variation in the MSP1a sequence of 23-29 amino acids in the tandem repeats located in
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the N-terminal region of the protein. The new sequence 167 showed deletion of the
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amino acids QQQESS located between positions 9 and 14 (Table 4). Alanine, serine,
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aspartate and glycine amino acids were found at position 20, and glycine was the one
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most often found. Glutamine was predominant at position 21, but histidine and
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glutamate were also observed. Two newly described sequences had glycine and valine
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amino acids at positions 31 and 32. No relationship between age and the number of
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MSP1a tandem repeats of A. marginale was observed (Figure 1). However, high
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variation in amino acids was found in the MSP1a tandem repeats of A. marginale with 4
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and 6 tandem repeats present in animals aged nine to 12 months (Figure 1). Analysis on
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the MSP1a tandem repeat sequences at positions 4, 9, 11, 15, 25, 30 and 31 found only
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one amino acid at each position with no variability (Figure 2). Positions 1, 7, 8, 20 and
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21 exhibited high variability, with five to six different amino acids present at these
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positions (Figure 2).
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Phylogenetic analysis. The phylogenetic analysis identified high divergence between
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the samples studied. All of the analyses (NJ, PA and ML) yielded similar topologies and
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the same relationships for all of the major clades that were identified in this study and
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represented in the NJ tree (Fig. 3). Based on the tandem repeats of Msp1a of the 51
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samples evaluated, 19 different MSP1a tandem repeats of A. marginale were identified
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and used to construct the phylogenetic tree. Based on the 2D structure of MSP1a, there
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were two different clusters. Twelve different MSP1a tandem repeats of A. marginale
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were pooled in the τ-related cluster (57-92% bootstrap support), and seven different
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MSP1a tandem repeats were pooled in the α-related cluster (72-98% bootstrap support).
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During the study, different MSP1a tandem repeats of A. marginale (e.g. Rio9, Rio10
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and Rio15) that infected the same animal were located in distinct clusters. The average
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diversity among the A. marginale samples was 0.9%. The MSP1a tandem repeats of A.
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marginale identified in this study differed from other MSP1a tandem repeats that have
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already been identified worldwide.
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Anaplasma marginale is endemic in Brazil, and outbreaks of anaplasmosis cause
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economic losses to the cattle industry in this country (Vidotto et al., 2008; Kocan et al.,
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2010). However, only four studies on the genetic diversity of this pathogen have
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previously been conducted in Brazil (Ferreira et al., 2001; de la Fuente et al. 2004;
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Vidotto et al., 2006; Pohl et al. 2013).
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Endemic stability, as defined by Mahoney and Ross (1972), such that
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seroprevalence can be relied on as an indicator of exposure to ticks and to the diseases
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transmitted by them, requires knowledge of the infestation rate, serial seroprevalence
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and disease frequency among cattle caused by the various genotypes (Jonsson et al.,
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2012). In our study, it was clearly seen that the sensitivity of the serological tests
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(sensitivity 98%), taking into consideration the exposure rate, ranged from 40% to 65%
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for A. marginale, i.e. below the level of 75% that was defined by Mahoney and Ross
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(1972). However, all calves were A. marginale qPCR-positive. Thus, enzootic stability
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exists based on the absence of clinical signs of bovine anaplasmosis. Therefore, the
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association between A. marginale prevalence and occurrences of clinical disease and/or
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mortality is more complex than what was previously established for the Babesia bovis-
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Rhipicephalus microplus system in Australia by Mahoney and Ross, in 1972.
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Although studies have proven that transplacental transmission of A. marginale
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occurs in Brazil (Grau et al., 2013), this was the first study have proven that
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transplacental transmission of the strains 4-63-27, 78-242-25-31 and τ-102-15 occurs. In
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this study, although 19 different MSP1a tandem repeats of A. marginale were
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circulating in the cattle tested, only three were transmitted through the placenta. Studies
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have suggested that transplacental transmission can occur when cows have acute
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anaplasmosis during pregnancy (Zaugg and Kuttler, 1984) or may be due to constant
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inoculations in endemic areas (Potgieter and Vanrensburg, 1987). In the present study,
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infection in newborn calves caused by these three MSP1a tandem repeats of A.
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marginale (-63-27, 78-242-25-31 and τ-102-15) was detected by PCR. MSP1a tandem
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repeat τ-102-15 was previously reported to be circulating in Brazil (Vidotto et al., 2006).
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Hence, our confirmation that this MSP1a tandem repeat is dominant (detected in 17.6%
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of the animals) and can be transmitted through the placenta suggests that this type of
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transmission may have greater importance in these regions than has been supposed until
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now.
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The prevalence of genotype E over G observed in this study may indicate that
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genotype E is better adapted and thus may be more efficient for infecting reservoir
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hosts. Both genotype G and genotype E have SD-ATG distances of 23 nucleotides.
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These microsatellite genotypes have been correlated with high levels of MSP1α protein
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expression (Estrada-Peña et al., 2009), thus suggesting that these MSP1a tandem repeats
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identified in calves present high infectivity potential. Estrada-Peña et al. (2009)
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evaluated the distribution of nine different genotypes in four distinct ecosystems around
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the world and noted that in South America, especially in Brazil and Argentina, genotype
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E has been found to be the most common type. However, genotypes B, C, D and G were
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previously detected in Argentina and Brazil (de la Fuente et al., 2004; Vidotto et al.,
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2006; Ruybal et al., 2009; Pohl et al., 2013). Genotype G is the most frequently found
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genotype around the world and has been seen to be the most prevalent type in the
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ecoregions of South Africa and parts of the USA and Mexico (Estrada-Peña et al.,
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2009).
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When we compared the repeat sequences found in this study with already-known
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sequences (Cabezas-Cruz et al., 2013), we were able to describe 22 new repeat
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sequences and also to report occurrences of 30 replicates for the first time in Brazil.
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Repeat sequences of MSP1a vary geographically among MSP1a tandem repeats of A.
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marginale and are functionally important in infection with and biological transmission
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of this pathogen (McGarey and Allred, 1994; de la Fuente et al., 2007). Furthermore,
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analysis on repeat sequences provides evolutionary information about A. marginale
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lineages and is used to characterize the genetic diversity of the pathogen (de la Fuente et
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al., 2001, 2007; Palmer et al., 2001, 2004). Thus, maintaining a database characterizing
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the structure of this gene region is crucial for new studies aiming to correlate these
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repeat sequences with the antigenic characteristics of this pathogen.
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The dominant strain was 4-63-27, and this was possibly associated with low
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occurrence of blood-sucking dipterans and high infestations by R. (B.) microplus ticks
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in this study area. Some tandem repeats, such as 27 and 13, have been found to be
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present in samples of the pathogen circulating in Latin America and South Africa, the
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common areas of Rhipicephalus (Boophilus) spp. ticks (de la Fuente et al., 2007). These
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results are consistent with the geographic distribution of the tick Rhipicephalus (B.)
314
microplus (Scoles et al., 2005), which is most likely the main vector of this pathogen in
315
tropical regions. However, other species of ticks and mechanical transmission may also
316
play important roles in the growth of this pathogen (Kocan et al., 2004).
te
Ac ce p
317
d
308
The most commonly observed sequences in the first replicate of MSP1a were 4
318
and τ. These results support previous studies showing that in South America, circulating
319
MSP1a tandem repeats of A. marginale usually present sequences 4, 8, 16, 56, 60, 64,
320
67, γ, π and τ in the first replicate (Estrada-Peña et al., 2009). In samples described on
321
this continent, the most frequent sequences in the first replicate are 16, α, τ (Vidotto et
13 Page 13 of 34
322
al., 2006), 72 and α (Pohl et al., 2013) in Brazil; and, α, τ and B (Ruybal et al., 2009) in
323
Argentina. The results shown in the present study corroborate those found by Cabezas-Cruz
325
et al. (2013), in which only serine was found at position 4 and glycine at position 31 in
326
tandem repeats of MSP1a. The positions in which the highest variation of amino acids
327
was observed were 1, 20 and 28. However, we observed that valine, arginine, alanine,
328
aspartate and asparagine amino acids were present at position 22, whereas the analysis
329
conducted by Cabezas-Cruz et al. (2013) found no variations in this position, with only
330
alanine being present.
us
cr
ip t
324
In our study, sample Rio5b exhibited a deletion of the QQQESS sequence
332
consisting of 23 amino acids in the first tandem repeats. This result may indicate that
333
MSP1a tandem repeats of A. marginale are present in these cattle, transmitted by blood-
334
sucking dipterans, since MSP1a contains B and T cell epitopes that are sensitive to
335
neutralization (Allred et al., 1990). Thus, studies have shown that MSP1a tandem
336
repeats of A. marginale that lack the amino acids included between positions 4 and 14
337
(SSAGGQQQESS) are unable to infect the cells of ticks (Blouin et al., 2003) and
338
cannot be transmitted by Dermacentor variabilis ticks (Kocan et al., 2003).
M
d
te
Ac ce p
339
an
331
To determine which selective pressures could be triggering MSP1a
340
diversification in A. marginale from cattle, the ratio ω was calculated, showing that the
341
codon at position 10 from tandem repeat 4 was evolving under negative selection.
342
Interestingly, this amino acid position is present in an immunodominant B-cell epitope
343
previously described for A. marginale MSP1a (Garcia-Garcia et al., 2004). These results
344
suggest that this tandem repeat, which is present in the most common MSP1a tandem
345
repeat of A. marginale found in cattle, may be under selective pressure from the host
346
immune system.
14 Page 14 of 34
Among geographic isolates, the MSP1a repeat sequences of A. marginale
348
located between the first and last repeat sequences are highly conserved (de la Fuente et
349
al., 2001). However, our results showed significant variation in amino acid sequences
350
for all replicates. Even the samples with four or more replicates exhibited variation in
351
their repeat sequences. These results were also found by Palmer et al. (2001) in cattle in
352
eastern Oregon, USA. These authors demonstrated that although only one genotype was
353
circulating, the A. marginale population was genetically heterogeneous.
cr
ip t
347
In our study, the most likely explanation for the genetic heterogeneity of A.
355
marginale is that the high diversity is a result of distinct biological and mechanical
356
transmission processes, each introducing different genotypes of A. marginale into cattle.
357
The causes of this occurrence are still unknown, but it reflects a population with high
358
genetic diversity in endemic areas (de la Fuente et al., 2001). This same assumption was
359
made previously by Palmer et al. (2001) in endemic areas of the USA, where it was
360
shown that bovine reservoirs harbor genetically heterogeneous A. marginale, thus
361
suggesting that different genotypes are maintained by transmission within the reservoir
362
cattle. In the region where our study was conducted, R. microplus ticks complete
363
between three and five cycles per year (Kasai et al., 2000) and can be infected by more
364
than one MSP1a tandem repeat of A. marginale over time and, during the feeding
365
process, transmit them to new hosts.
an
M
d
te
Ac ce p
366
us
354
Our results show that most animals were infected only by the MSP1a tandem
367
repeat 4-63-27, which we assume is dominant. Some animals were infected by other
368
minority MSP1a tandem repeats, and other animals were superinfected by multiple
369
MSP1a tandem repeats of A. marginale. Genetic diversity does not result in evolution
370
unless a variant with increased aptitude arises under conditions that promote imbalance
371
and favor fast random evolution (de la Fuente et al., 2001). Hence, wild-type sequences,
15 Page 15 of 34
or the master sequence in this situation, would remain unchanged (de la Fuente et al.,
373
1999). Our results show that more than one genotype was established in animals, thus
374
indicating that there was no occurrence of selection for specific variants, or a "genetic
375
divide" process, as alleged by de la Fuente et al. (2001). These findings corroborate the
376
results of Ueti et al. (2012) and Palmer and Brayton (2013), which showed that different
377
genotypes coexist in the same ecosystem and can parasitize the same animal at the same
378
time, thereby characterizing an occurrence of superinfection, which is common in
379
natural infections in tropical countries. The results from this study are supported by the
380
theory of Palmer and Brayton (2013), who showed that several MSP1a tandem repeats
381
can circulate in the same cattle, such that most animals are parasitized by a dominant
382
MSP1a tandem repeat and some animals are parasitized by more than one MSP1a
383
tandem repeat.
M
an
us
cr
ip t
372
The new tandem repeats observed in this study have broad similarity to existing
385
sequences and are located in the same cluster. This finding suggests that the new
386
tandem repeats may have originated recently from existing tandem repeats, and this
387
provides evidence for genetic diversification of A. marginale in cattle. In summary, we
388
suggest that calves kept under natural conditions can be infected by more than one
389
MSP1a tandem repeat of A. marginale throughout their lives. Furthermore, even in the
390
same cattle, circulation of various MSP1a tandem repeats of A. marginale can occur,
391
which are kept under constant variation of MSP1a tandem repeats.
te
Ac ce p
392
d
384
393
CONFLICT OF INTEREST STATEMENT
394
None of the authors of this work has a financial or personal relationship with other
395
people or organizations that would inappropriately influence or bias the content of this
396
paper.
16 Page 16 of 34
ACKNOWLEDGEMENTS
398
We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for
399
their financial support (Process #2012/21371-4) and the Coordination Office for
400
Improvement of Higher-Education Staff (CAPES) for the J. B. Silva fellowship. We
401
thank the Germania Farm for access to the study animals.
ip t
397
cr
402
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Fluminense. Pesq Vet Bras, 20, 97-101.
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2360.
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Vidotto, M.C., Vidotto, O., Andrade, G.M., Palmer, G., Mcelwain, T., Knowles, D.P.
557
1998. Seroprevalence of Anaplasma marginale in cattle in Paraná State, Brazil, by
558
MSP-5 competitive ELISA. Ann N Y Acad Sci. 849, 424-426.
559
Vidotto, M. C., Kano, F.S., Gregori, F., Vidotto, O. 2006. Phylogenetic analysis of
560
Anaplasma marginale strains from Parana State, Brazil, using the msp1-alfa and
561
msp4 genes. Journal of Veterinary Medicine. B, Infect Dis Vet Public Health, 53,
562
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23 Page 23 of 34
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Zaugg, J.L., Kuttler, K.L. 1984. Bovine anaplasmosis: in utero transmission and the
564
immunologic significance of ingested colostral antibodies. Am J Vet Res, 45, 440-
565
443.
567
ip t
566
FIGURE CAPTIONS
cr
568
TABLE 1. Frequency (%) of animals positive for A. marginale. Anaplasma
570
marginale was detected through direct examination (blood smears), serological tests
571
(ELISA/IFAT) and molecular analysis (qPCR) among naturally infected calves in the
572
state of Rio de Janeiro, Brazil.
an
us
569
M
573
TABLE 2. Organization of MSP1a tandem repeats and A. marginale strains
575
isolated from cattle in the state of Rio de Janeiro, Brazil. A. marginale strain
576
identification was based on msp1α and included microsatellite genotype (tandem repeat
577
structure). Superscripts represent the number of times that the tandem repeats were
578
repeated.
te
Ac ce p
579
d
574
580
TABLE 3. Occurrence of the most common A. marginale strains. The most frequent
581
A. marginale strains, observed in 51 samples. All animals were naturally infected in the
582
state of Rio de Janeiro, Brazil.
583 584
TABLE 4. Sequences of newly described A. marginale MSP1a tandem repeats. The
585
one-letter code is used to name the different amino acids of the tandem repeats.
586
Conserved amino acid positions and deletions/insertions (-) are shown. The new MSP1a
587
tandem repeats 165-186 were named in accordance with the system proposed by de la
24 Page 24 of 34
588
Fuente et al. (2007) and updated by Cabezas-Cruz et al. (2013). Tandem repeat
589
sequence A is used as a model for comparison.
590
FIGURE 1. MSP1a tandem repeats. Analysis on MSP1a microsatellite sequences in
592
different A. marginale strains detected at different ages. All MSP1a tandem repeats
593
ranged from 1 to 6 and the ages of the animals ranged from newborn to 12 months
594
(three-month intervals between observations).
us
595
cr
ip t
591
FIGURE 2. Amino acid variability and frequency in MSP1a tandem repeats. The
597
amino acids were calculated per amino acid position in the MSP1a tandem repeats using
598
the following formula: Variability = number of different amino acids at a given
599
position/frequency of the most common amino acid at that position. The one-letter
600
amino acid code was used to name the amino acids, and the most frequent amino acid at
601
each position was colored in gray.
M
d
te
602
an
596
FIGURE 3. Distribution and phylogenetic analysis on A. marginale strains
604
identified in cattle. In the left panel, the locations of Rio de Janeiro (Brazil), Azaria,
605
Israel, Mississippi, Idaro, St. Meries, California, Okeechobee, Oklahoma, Florida,
606
Virginia and Porto Rico are shown. The strains isolated from cattle are identified as in
607
the legend of the figure. The right panel shows the consensus tree from the ML and NJ
608
phylogenetic analyses. The numbers above and below the internal branches represent
609
bootstrap values (1000 replicates) for ML and NJ, respectively. Only bootstrap values
610
higher than 50 are shown. The MSP1a GenBank accession numbers of the respective
611
sequences used in the phylogenetic tree are shown. The strains identified in this study in
Ac ce p
603
25 Page 25 of 34
612
cattle are shown as in Table 2. The four phylogenetic clusters shown contained different
613
patterns of MSP1a tandem repeat 2D structures.
Ac ce p
te
d
M
an
us
cr
ip t
614
26 Page 26 of 34
614
Table 1
615 Frequency of positivity (%) Age (days) IFAT
qPCR
Rickettsemia*
1
10
40
35
15
1.04 x 101
90
80
50
50
100
180
70
60
55
100
270
60
55
55
360
50
70
65
ip t
ELISA
4,36 x 1012
cr
7,91 x 104
100
1,54 x 105
100
5,23 x 105
us
616
Blood smear
* DNA copies per ml of blood
Ac ce p
te
d
M
an
617
27 Page 27 of 34
TABLE 2.
KJ398348 KJ398349 KJ398350 KJ398351 KJ398352 KJ398353 KJ398354 KJ398355 KJ398356 KJ398357 KJ398358 KJ398359 KJ398360 KJ398361 KJ398362 KJ398363 KJ398364 KJ398365 KJ398366 KJ398367 KJ398368 KJ398369 KJ398370 KJ398371 KJ398372 KJ398373 KJ398374 KJ398375 KJ398376 KJ398377 KJ398378 KJ398379 KJ398380 KJ398381 KJ398382 KJ398383 KJ398384 KJ398385 KJ398386 KJ398387 KJ398388 KJ398389 KJ398390 KJ398391 KJ398392 KJ398393 KJ398394 KJ398396
an M
d
te
Infection Acute Acute Chronic Acute Acute Chronic Acute Acute Chronic Acute Acute Chronic Chronic Acute Acute Acute Acute Acute Acute Acute Acute Chronic Acute Acute Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic
ip t
Rio1a / E - (78, 242, 25, 31) Rio1b / E - (78, 242, 25, 31) Rio1c / E - (78, 242, 25, 31) Rio2a / E - (4, 63, 27) Rio2b / E - (4, 63, 27) Rio2c / E - (τ, 102, 15) Rio3a / E - (τ, 102, 15) Rio3b / E - (4, 63, 27) Rio3c / E - (τ, 102, 15) Rio4a / E - (4, 63, 27) Rio4b / E - (4, 63, 27) Rio4c / E - (4, 63, 27) Rio4d / E - (165, 102, 166) Rio5a / E - (78, 242, 25, 31) Rio5b / E - (167, 168, β2, 169, 170) Rio6a / E - (78, 242, 171, 31) Rio6b / E - (4, 63, 27) Rio7a / E - (4, 63, 27) Rio7b / E - (78, 242, 25, 31) Rio8a / E - (78, 242, 25, 31) Rio8b / E - (78, 242, 25, 31) Rio8c / E - (4, 63, 4) Rio9a / E - (4, 63, 3) Rio9b / E - (78, 24, 172, 24, 173) Rio9c / E - (α, 174, β) Rio9d / E - (175, 63, 27) Rio10a / E - (τ, 102, 176) Rio10b / E - (174, 176, β2, Ʈ) Rio11a / E - (78, 242, 25, 31) Rio11b / E - (78, 242, 25, 31) Rio12a / E - (4, 63, 27) Rio12b / E - (τ, 102, 15) Rio13a / E - (4, 63, 27) Rio13a / E - (4, 63, 27) Rio13c / E - (τ, 102, 15) Rio14a / E - (4, 63, 27) Rio14b / E - (4, 63, 27) Rio15a / E - (4, 63, 177) Rio15b / E - (178, 179, 180, 181, 182) Rio16a / G - (163, 1643, 61) Rio17a / E - (4, 63, 27) Rio17b / E - (78, 242, 25, 31) Rio17c / E - (176, 174, β2, Ʈ) Rio18a / E - (78, 242, 25, 31) Rio18b / E - (τ, 102, 15) Rio18c / E - (182, 24, 183, 184, 185) Rio19a / E - (τ, 102, 15) Rio20a / E - (τ, 102, 15)
Rickettsemia (msp1α copies/ml) 4.36 x 1012 1.40 x 1012 2.06 x 103 2.22 x 1012 6.76 x 1011 1.02 x 104 8.13 x 1011 8.20 x 1011 2.17 x 104 5.06 x 1010 2.33 x 1011 1.37 x 104 4.19 x 104 5.89 x 1011 4.06 x 1010 8.68 x 1010 5.36 x 1011 3.76 x 1011 7.76 x 1010 6.76 x 1011 2.95 x 1011 2.07 x 104 7.68 x 1011 5.21 x 1011 2.00 x 105 5.12 x 104 1.32 x 105 1.76 x 104 4.13 x 104 5.08 x 104 1.33 x 105 3.20 x 104 1.50 x 104 2.73 x 104 5.17 x 104 1.69 x 105 3.04 x 105 9.29 x 104 3.27 x 104 4.64 x 104 3.50 x 104 8.76 x 105 1.74 x 105 4.81 x 105 5.25 x 104 5.46 x 106 1.12 x 105 6.53 x 105
cr
GenBank accession number
us
Strain identification and structure of msp1a tandem repeats
Ac ce p
617
28 Page 28 of 34
618
Rio20b / E - (174, 176, β2, 186) Rio20c / E - (τ, 102, 15)
KJ398397 KJ398398
3.94 x 105 2.48 x 103
Chronic Chronic
Strain identification is based on msp1α and includes microsatellite genotype – (tandem repeats structure).
620
Superscripts represent the number of times that a tandem repeats are repeated. Infection of animals was
621
calculated by Eriks et al. (1993). The new MSP1a tandem repeat 165-186 was named in accordance with
622
the system proposed by de la Fuente et al. (2007) and updated by Cabezas-Cruz et al. (2013).
cr
ip t
619
Ac ce p
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d
M
an
us
623
29 Page 29 of 34
TABLE 3
Strain
Structure of MSP1a tandem repeats
Nº of strains
Frequency
1ª commom
4-63-27
14
27,5 %
2ª commom
78-242-25-31
11
21,6 %
3ª commom
τ-102-15
9
17,6 %
ip t
623
The most common tandem repeats found among all the A. marginale strains are underlined and there were
625
found more than 27 (24), 24, (4), 22 (10), 21 (63) and 21 (27).
cr
624
626
Ac ce p
te
d
M
an
us
627
31 Page 30 of 34
TABLE 4.
629
M
an
ip t cr
us
DDSSSASGQQQESSVSSQSE-ASTSSQLG-TDSSSASGQQQESSVLSPSGHVRTSSQLG-ADSSSGRGQQQESGVLSQSGQASTSSQLG-ADSSSASG------VLSQSGEATTSAQLR-TDSSSAGDQPQGSGVSSQSGQASTSAQLR-TDSSSATDQQQESGVSSQSGQASTSA---VG TDSSSASAQQQESSVSSHTD-RSTSSQ--VG ADSSSASGQQQESSVLSQSSQASTSSQLR-ADSSSAGNQQQESSVLSQSGQASTSSQSG-ADSSSAGNQQQESSVSSQSD-ASTSSDYG-TDSSSAGDQQQGSGVSSQSGQASTSSQLR-TDSSSASGQQQESSVLSQSGHASTSSQLG-ADSSSASGQQQESGVLSQSAQASTSSQLG-ADSSSASGQQQESSVLSQSDHASTSSQLG-DDSSSADDQQQESSVSSQSG-DSTSSQLG-TDSSSAGDQQQESSVSSQSG-DSTSSQLG-TDSSSAQHQQQESNVSSQTG-NSTSSQLG-NDTSSAGHQQQESNVSSQSG-DSTSSQLG-TDSSTAGDQQQESSVSSQSG-ASTSSQLG-VDSSSAGDQQQESSVSSQSG-DSTSSQLG-TDSSSAGDQQQESSVSSQSG-DSTSSQLG-TDNSSASGQQQENSVLSQSSQASTSSQLG-DDSSSAGNQQQESSVLPQSGQASTSSQLG--
Number of Amino Acid 28 29 29 23 29 28 29 29 28 28 29 29 29 29 28 28 28 28 28 28 28 29 29
Ac ce p
628
Tandem repeats
te
New sequences A 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
d
627
32 Page 31 of 34
3 Months
6 Months
9 Months
12 Months
4-63-27
an
Tandem repeats
Newborn
3
4-63-27
4
τ-102-15
not detected
5
2
78-24 -25-31
α-174-β
175-63-27
M
d
not detected
2
τ-10 -15 2
τ-10 -176
2
165-10 -176 178-179-180-181-182
2
78-24 -171-31
2
176-174-β -Γ
182-24-183-184-185 2
174-176-β -186
not detected
2
167-168-β -169-170
not detected
ep te
not detected
2
78-24 -174-31
2
τ-10 -15
Ac c
6
4-63-27
4-63-164
2
78-24 -25-31
us
cr
ip t
Figure
Page 32 of 34
Figure
Figure 2
Average of amino acids variability 0,61 Proportion of variable/conserved 8,66 51 samples and 66 2
1
ip t
Shannon variability
3
0
V
Q
K
C
Y
F
I
R
0,00
A
T
0,60
0,35
D
L
S
N
us
P
0,03 0,90
0,00
0,99 1,00
0,01
0,00
0,00
1,00 0,01
0,99
0,00 0,13
M
1,00 0,01
1,00
0,88
0,97 0,00
ed
0,12
0,00
0,00
0,00 0,65 0,21
0,33 0,65
0,01
0,98
0,02
0,86
0,01
0,91
0,13
0,23 0,87 0,00
0,01
0,99
0,00
0,20
0,01 0,95
0,03
0,00
0,14
0,66 0,02
0,01
0,00
0,01
0,98
0,99
0,01
0,00
1,00
0,01 0,99
0,00
0,10
0,09
0,77 0,13
E
0,01
1,00
0,00
H
1,00
an
0,00
G
0,01
0,01
0,98 0,00
0,00
0,85 0,05
0,00
0,14
0,00 0,95
1,00
1,00
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A S T S S Q L G V G
Frequency of amino acid position W
ce pt
AAP*
cr
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 D D S S S A S G Q Q Q E S S V S S Q S E -
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure
Page 34 of 34
S1877-959X(15)00070-9 http://dx.doi.org/doi:10.1016/j.ttbdis.2015.04.002 TTBDIS 470
To appear in: Received date: Revised date: Accepted date:
30-6-2014 26-3-2015 8-4-2015
Please cite this article as: Silva, J.B., Gonc¸alves, L.R., Varani, A.M., Andr´e, M.R., Machado, R.Z.,Genetic diversity and molecular phylogeny of Anaplasma marginale studied longitudinally under natural transmission conditions in Rio de Janeiro, Brazil, Ticks and Tick-borne Diseases (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Genetic diversity and molecular phylogeny of Anaplasma marginale studied
2
longitudinally under natural transmission conditions in Rio de Janeiro, Brazil
3
Jenevaldo Barbosa Silva1, Luiz Ricardo Gonçalves1, Alessandro de Mello Varani2,
5
Marcos Rogério André1, Rosangela Zacarias Machado1*
7
1
8
Jaboticabal SP, Brazil.
9
2
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Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias,
Departamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias,
Jaboticabal SP, Brazil.
an
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6
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4
M
11
*Corresponding author:
13
Rosangela Zacarias Machado ([email protected])
14
Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade
15
de Ciências Agrárias e Veterinárias FCAV-UNESP, Via de Acesso Prof. Paulo Donato
16
Castellane s/n, 14884-900, Jaboticabal, SP, Brasil. Phone: +55 16 3209-2663.
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ABSTRACT- Anaplasma marginale is the most prevalent tick-borne pathogen in cattle
19
in tropical and subtropical regions of the world. Major Surface Protein 1a (MSP1a) has
20
been found to be a stable genetic marker for identifying A. marginale isolates within
21
geographical regions. It is conserved in cattle during infection and tick-borne
22
transmission of the pathogen. The aim of the present longitudinal study was to
23
determine occurrences of genetic diversity associated with high prevalence of A.
24
marginale under natural transmission conditions. Twenty calves were evaluated every
25
three months during the first year of life. Rickettsemia levels due to A. marginale,
1 Page 1 of 34
measured as the number of msp1α copies/ml in the blood of positive calves, ranged
27
from 2.06 x 103 to 4.36 1012. The numbers of MSP1a tandem repeats among MSP1a
28
tandem repeats were 3 and 6. The predominant msp1α microsatellite was E, and another
29
MSP1a tandem repeat was found that presented genotype G. Nineteen different MSP1a
30
tandem repeats of A. marginale were found circulating in animals. The MSP1a tandem
31
repeats 4-63-27 (27.5%), 78-242-25-31 (n = 21.6%) and τ-102-15 (n = 17.6%) were the
32
ones most commonly observed. Twenty-two MSP1a tandem repeats resulted in new
33
sequences with amino acid changes, as shown in this study. Thirty sequences were
34
found for the first time in Brazil. Glycine, glutamate, serine and alanine amino acids
35
were found at position 20. During the study, 80% (16/20) of the animals were infected
36
by more than one genotype. Three animals were born infected, with MSP1a tandem
37
repeats 4-63-27, 78-242-25-31 and τ-102-15, thus indicating occurrence of transplacental
38
transmission. In the phylogenetic analysis, nineteen different MSP1a tandem repeats of
39
A. marginale were found in the cattle, which suggested that many MSP1a tandem
40
repeats and high variation in MSP1a were occurring.
41
KEYWORDS: Brazil, Cattle, Genotype, MSP1a, Tandem repeats
43 44
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42
ip t
26
INTRODUCTION
Anaplasma marginale (Rickettsiales: Anaplasmataceae) is the most prevalent
45
tick-borne pathogen in the world, distributed on six continents, and is responsible for
46
high morbidity and mortality among cattle in temperate, subtropical and tropical regions
47
(Kocan et al., 2010). Bacteria of the genus Anaplasma are obligate intracellular
48
pathogens that may be transmitted biologically by ticks, mechanically by blood sucking
49
flies or infected blood in fomites, and also transplacentally (Aubry and Geale, 2011).
2 Page 2 of 34
Brazil can be classified as an area of endemic stability for occurrences of bovine
51
anaplasmosis, from north to south and from east to west, with prevalence ranging from
52
16.3% in the semi-arid zone of the state of Sergipe (Oliveira et al., 1992) to close to
53
100% in the states of Minas Gerais, Bahia, Paraíba and Paraná (Ribeiro and Reis, 1981;
54
Araújo et al., 1995; Madruga et al., 1994; Vidotto et al., 1995). In the state of Rio de
55
Janeiro, a serological survey showed that 98.21% of the cattle were positive for A.
56
marginale (Souza et al., 2000).
cr
ip t
50
Major Surface Protein 1 alpha (MSP1a) is a heterodimer composed of MSP1a
58
(100 kDa) and MSP1b (105 kDa). These proteins are structurally unrelated and bonded
59
non-covalently and they are exposed on the surface of A. marginale (Barbet and Allred,
60
1991). MSP1a is formed by a single polymorphic gene composed by one region that is
61
conserved and another region that is variable and contains a mutable number of units in
62
tandem repeats (Allred et al., 1990). MSP1b is coded by a mutagenic family composed
63
of two genes (msp1b1 and msp1b2) and three genes (msp1b1pg, msp1b2pg and
64
msp1b3pg) that seem to recombine such that the genetic and antigenic diversity is
65
increased (Barbet and Allred, 1991).
an
M
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66
us
57
Phylogenetic studies have identified several strains of A. marginale worldwide
67
that differ in their morphology, amino acid sequence, antigenic characteristics and
68
ability to be transmitted by ticks (Chaves et al., 2012; Cabezas-Cruz et al., 2013). The
69
genetic diversity of MSP1a tandem repeats of A. marginale has previously been
70
characterized based on the repeated amino acid sequence of MSP1a (Allred et al., 1990;
71
de la Fuente et al., 2007). In addition, this sequence contains T and B cell epitopes that
72
are important in the protective immune response (Brown et al., 2001; Brown et al.,
73
2002).
3 Page 3 of 34
Research involving MSP1a tandem repeats is a promising route for acquiring
75
comprehensive knowledge of the extensive worldwide diversity of A. marginale
76
(Cabezas-Cruz et al., 2013). A single nomenclature has been created based on the data
77
available in GenBank (de la Fuente et al., 2007). Studies in non-endemic regions have
78
shown that this repeat sequence exhibits little variation (Palmer et al., 2004). However,
79
in areas where anaplasmosis occurs endemically, MSP1a tandem repeats of A.
80
marginale may exhibit high variability in their tandem repeats, and some animals can be
81
infected by more than one MSP1a tandem repeat (de la Fuente et al., 2001; Palmer et
82
al., 2001).
us
cr
ip t
74
Although significant variations in MSP1a tandem repeats have been observed in
84
cattle in Brazil and Argentina (Vidotto et al., 2006; Ruybal et al., 2009; Pohl et al.
85
2013), no study has yet been conducted involving cattle herds in an area of South
86
America where multiple MSP1a tandem repeats of A. marginale circulate
87
simultaneously. Therefore, the aim of the present longitudinal study was to determine
88
occurrences of genetic diversity associated with high prevalence of A. marginale under
89
natural transmission conditions.
M
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te
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90
an
83
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MATERIALS AND METHODS
92
Study site and cattle population. A longitudinal study was conducted in 2012 and 2013
93
at the Seropédica Experimental Station, Empresa de Pesquisa Agropecuária do Estado do
94
Rio de Janeiro (Pesagro-Rio; Agricultural and Livestock Research Corporation of the State
95
of Rio de Janeiro). The experimental area is located in the metropolitan microregion of the
96
city of Rio de Janeiro (latitude 22º45’ S, longitude 43º41’ W, and altitude 33 m). This
97
area’s average annual temperature is approximately 22.7 ºC, and it receives an average of
98
1291.7 mm of precipitation per year. This region is characterized by two well-defined
4 Page 4 of 34
seasonal periods. The dry period (March to September) has lower temperatures and
100
rainfall, which leads to a reduction in the population of vectors; the rainy period (October
101
to February) has higher temperatures and rainfall, resulting in an increased number of
102
vectors.
ip t
99
In the herd used in this study, the serological prevalence of A. marginale was 70%
104
and no clinical cases of this disease had been observed over the preceding three years
105
(Silva and Fonseca, 2014). Pesagro-Rio had a herd of 410 animals, composed of 60 calves,
106
70 heifers and 280 cows, with the potential to produce between 1,000 and 4,000 kg of milk
107
per lactation. The herd was divided into groups according to the animals’ ages and
108
physiological state. Each group of animals was kept in a different area of the experimental
109
station. The calves aged 0 to 2 months were kept in a shed, in individual pens, and had
110
access to an area of 0.5 ha of Brachiaria humidicula from the age of 15 days onwards. At
111
this stage, they received 4 kg of milk per day. From 3 to 6 months of age, the calves
112
received 4 kg of milk per day and were kept during the day in an area of 1.5 ha of
113
Brachiaria decumbens and brought in at night, to individual pens. Between the ages of 7
114
and 12 months, the calves were transferred to an area of 3 ha of Brachiaria decumbens and
115
Panicum maximum, where they were kept during the day, and were brought into a
116
collective pen at night.
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103
Twenty heifers (Bos taurus taurus x Bos taurus indicus) were evaluated every three
118
months from birth until they reached 12 months of age, from May 2012 to May 2013. The
119
first blood sampling was performed after the calves had ingested the colostrum, i.e. not
120
more than one hour after their birth. Thus, during the study period, it was possible to
121
perform five observations on each animal (at birth and at the ages of 3, 6, 9 and 12
122
months), producing a total of 100 samples.
5 Page 5 of 34
Whole blood samples were collected from the caudal or jugular veins of individual
124
calves. To prepare the serum samples, the blood samples collected were incubated at room
125
temperature for 1 h and then centrifuged at 1000 × g for 15 minutes. Giemsa-stained blood
126
smears were made for further microscopic examination. Blood and serum samples were
127
stored at -20 ºC. DNA was extracted from 200 µl of each of the 100 whole blood samples
128
using a QIAamp DNA blood mini-kit (Qiagen, Madison, WI, USA), in accordance with
129
the manufacturer’s instructions.
130
Antigen production. An A. marginale isolate from a calf in Jaboticabal in the state of
131
São Paulo, Brazil (Andrade et al., 2004) was used to infect a calf for crude ELISA and
132
IFAT antigen production. For this purpose, a 3-month-old splenectomized calf was
133
inoculated with 200 ml of A. marginale-infected blood (1.0 x 107 infected
134
erythrocytes/ml).
135
erythrocytes/ml) was observed 7 days after the experimental infection. After the blood
136
had been collected and processed for crude ELISA/IFAT antigen production (Machado
137
et al., 1997), the experimentally infected animal was treated with oxytetracycline
138
administered intramuscularly (20 mg/kg).
139
Enzyme-Linked Immunosorbent Assay (ELISA) and Indirect Fluorescent
140
Antibody Test (IFAT). These were performed as previously described by Machado et
141
al. (1997) and Andrade et al. (2004).
142
Semi-nested msp1α PCR. A semi-nested PCR (nPCR) was used to amplify the msp1α
143
sequence (Lew et al., 2002). The reactions were performed using the primers 1733F (5'-
144
TGT GCTTATGGCAGACATTTCC-3'), 3134R (5'-TCACGGTCAAAACCTTTGCTT
145
ACC-3') and 2957R (5'-AAACCTTGTAGCCCCAACTTATCC-3').
146
Quantitative PCR for detection and quantitation of A. marginale. Quantitative real-
147
time PCR was used (qPCR), as described by Carelli et al. (2007) for the gene msp1β of
rickettsemia
peak
(1.0
x
107
A.
marginale-infected
Ac ce p
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The
M
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6 Page 6 of 34
A. marginale, with the aim of estimating the parasitemia by means of absolute
149
quantification (number of copies/µL). Serial dilutions were performed with the aim of
150
constructing standards with different concentrations of plasmid DNA containing the
151
target sequence (2.0 x 107 copies/µL to 2.0 x 100 copies/µL). The number of plasmid
152
copies was determined in accordance with the formula (X g/µL DNA/[plasmid size (bp)
153
x 660]) x 6.022 x 1023 x plasmid copies/µL. The amplification reactions were performed
154
using a final total reaction volume of 10 µL, containing a mixture of 1.0 µL of sample
155
DNA, 0.2 µL of probe, 0.9 µL of each primers, 5.0 µL of PCR buffer (IQ Multiplex
156
Power Mix®, BioRad) and 2.0 µL of ultra-pure sterile water (Nuclease-Free Water ®,
157
Promega).
158
Sequence of the A. marginale msp1α microsatellite. A microsatellite is located at the
159
5'-untranslated region (UTR) of the msp1α gene between the putative Shine-Dalgarno
160
(GTAGG) sequence and the translation initiation codon (ATG) (de la Fuente et al.,
161
2001). Its structure is GTAGG (G/ATTT)m (GT)n T ATG (Estrada-Peña et al., 2009).
162
An analysis of the repeat sequences was performed in accordance with the nomenclature
163
proposed by de la Fuente et al. (2007). The SD-ATG distance was calculated using the
164
formula (4 × m) + (2 × n) + 1.
165
Phylogenetic analysis. The phylogenetic analysis was performed using msp1α
166
nucleotide sequences that were aligned with MAFFT (v7) and configured for highest
167
accuracy (Katoh and Standley, 2013). After alignment, regions with gaps were removed
168
from the alignment. Phylogenetic trees were reconstructed using the maximum
169
likelihood (ML) and neighbor-joining (NJ) methods as implemented in PhyML (v3.0
170
aLRT) (Anisimova and Gascuel, 2006; Guindon and Gascuel, 2003) and PHYLIP
171
(v3.66) (Felsenstein, 1989), respectively. The reliability of the internal branching of the
172
ML and NJ trees was assessed using the bootstrapping method (1000 bootstrap
Ac ce p
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M
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148
7 Page 7 of 34
173
replicates). The graphical representation and editing of the phylogenetic trees were done
174
using TreeDyn (v 198.3) (Chevenet et al., 2006).
175
RESULTS
177
A. marginale prevalence and bacteremia in calves. The animals were diagnosed
178
positive for A. marginale during the first year of life by means of blood smears, ELISA,
179
IFAT and qPCR (Table 1). The prevalence of A. marginale in blood smear samples
180
ranged from 10% (newborn animals) to 80% (animals at 90 days of age). The
181
serological variation in ELISA/IFAT was 35% (newborn animals) to 70% (360-day-old
182
animals), while qPCR ranged from 15% (among newborns) to 100% (animals at 90
183
days of age). Quantifying the number of MSP1a A. marginale copies per ml of blood
184
allowed us to determine which animals may have been acutely and chronically infected.
185
The rickettsemia levels in the positive animals ranged from 2.06 x 103 to 4.36 x 1012
186
DNA copies per ml of blood (Table 1). Among the tests used, the following
187
concordances were observed: qPCR/ELISA 57% (50/88), qPCR/IFAT 55% (48/87),
188
qPCR/blood smear 65% (54/83), ELISA/blood smear 70% (45/64), ELISA/IFAT 95%
189
(52/55), IFAT/blood smear 71% (44/62) and qPCR/ELISA/IFAT/blood smear 41%
190
(36/88).
191
Analysis of A. marginale msp1a sequences in calves. The msp1α gene was amplified
192
using nPCR and sequenced in 51 samples from calves. The sequence analysis on the
193
MSP1a microsatellites of A. marginale indicated that the genotypes E and G were
194
present (Table 2). Three animals were born infected with MSP1a tandem repeats 4-63-
195
27 [1.50 x 104], 78-242-25-31 [2.06 x 103] and τ-102-15 [1.12 x 105], thus indicating
196
occurrence of transplacental transmission. Major and minor rickettsemia was observed
197
in animals infected by MSP1a tandem repeats 78-242-25-31 [4.36 x 1012 and 2.06 x
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103]. The rickettsemia levels among the animals showed a correlation with age and were
199
highest at three months of age. However, we did not observe any relationship between
200
rickettsemia and the A. marginale genotype infecting the animals. Twenty-two new
201
sequences were described in this study, which were labeled as 165 to 186 (Table 4). The
202
analysis on the repeated MSP1a sequences also produced 30 sequences that were
203
described for the first time in Brazil. Ten percent (2/20) of the animals (Rio16 and
204
Rio19) were positive in only a single sample. Fifteen percent (3/20) of the animals
205
(Rio1, Rio11 and Rio14) remained infected with only one MSP1a tandem repeat of A.
206
marginale in multiple samples. However, 75% (15/20) of the positive animals tested
207
positive in two to four samples and more than one infecting MSP1a tandem repeat of A.
208
marginale was found during the study. One A. marginale-positive animal (Rio9)
209
presented four different MSP1a tandem repeats in four samples.
210
Occurrence of the most common A. marginale MSP1a tandem repeats. The number
211
of repeat MSP1a sequences of A. marginale ranged from three to six. The most
212
commonly observed sequences were those with three (41%) and five (37%) repeats. The
213
most prevalent MSP1a tandem repeats were 4-63-27 (27,5%), 78-242-25-31 (21,6%)
214
and τ-102-15 (17,6%) (Table 3). The most common repeat sequences were 24 (n = 27),
215
4 (n = 24), 10 (n = 22), 63 (n = 21) and 27 (n = 21). The new repeat sequences are listed
216
in Table 4. Only one animal (Rio15b) had an entire sequence of unprecedented repeats
217
(178-179-180-181-182). Although most of the samples exhibited three repeats, 53%
218
(9/17) of those that included new sequences in their structure were five-repeat
219
sequences.
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The MSP1a tandem repeats of A. marginale identified in this study exhibited
221
variation in the MSP1a sequence of 23-29 amino acids in the tandem repeats located in
222
the N-terminal region of the protein. The new sequence 167 showed deletion of the
9 Page 9 of 34
amino acids QQQESS located between positions 9 and 14 (Table 4). Alanine, serine,
224
aspartate and glycine amino acids were found at position 20, and glycine was the one
225
most often found. Glutamine was predominant at position 21, but histidine and
226
glutamate were also observed. Two newly described sequences had glycine and valine
227
amino acids at positions 31 and 32. No relationship between age and the number of
228
MSP1a tandem repeats of A. marginale was observed (Figure 1). However, high
229
variation in amino acids was found in the MSP1a tandem repeats of A. marginale with 4
230
and 6 tandem repeats present in animals aged nine to 12 months (Figure 1). Analysis on
231
the MSP1a tandem repeat sequences at positions 4, 9, 11, 15, 25, 30 and 31 found only
232
one amino acid at each position with no variability (Figure 2). Positions 1, 7, 8, 20 and
233
21 exhibited high variability, with five to six different amino acids present at these
234
positions (Figure 2).
235
Phylogenetic analysis. The phylogenetic analysis identified high divergence between
236
the samples studied. All of the analyses (NJ, PA and ML) yielded similar topologies and
237
the same relationships for all of the major clades that were identified in this study and
238
represented in the NJ tree (Fig. 3). Based on the tandem repeats of Msp1a of the 51
239
samples evaluated, 19 different MSP1a tandem repeats of A. marginale were identified
240
and used to construct the phylogenetic tree. Based on the 2D structure of MSP1a, there
241
were two different clusters. Twelve different MSP1a tandem repeats of A. marginale
242
were pooled in the τ-related cluster (57-92% bootstrap support), and seven different
243
MSP1a tandem repeats were pooled in the α-related cluster (72-98% bootstrap support).
244
During the study, different MSP1a tandem repeats of A. marginale (e.g. Rio9, Rio10
245
and Rio15) that infected the same animal were located in distinct clusters. The average
246
diversity among the A. marginale samples was 0.9%. The MSP1a tandem repeats of A.
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247
marginale identified in this study differed from other MSP1a tandem repeats that have
248
already been identified worldwide.
249
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Anaplasma marginale is endemic in Brazil, and outbreaks of anaplasmosis cause
252
economic losses to the cattle industry in this country (Vidotto et al., 2008; Kocan et al.,
253
2010). However, only four studies on the genetic diversity of this pathogen have
254
previously been conducted in Brazil (Ferreira et al., 2001; de la Fuente et al. 2004;
255
Vidotto et al., 2006; Pohl et al. 2013).
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Endemic stability, as defined by Mahoney and Ross (1972), such that
257
seroprevalence can be relied on as an indicator of exposure to ticks and to the diseases
258
transmitted by them, requires knowledge of the infestation rate, serial seroprevalence
259
and disease frequency among cattle caused by the various genotypes (Jonsson et al.,
260
2012). In our study, it was clearly seen that the sensitivity of the serological tests
261
(sensitivity 98%), taking into consideration the exposure rate, ranged from 40% to 65%
262
for A. marginale, i.e. below the level of 75% that was defined by Mahoney and Ross
263
(1972). However, all calves were A. marginale qPCR-positive. Thus, enzootic stability
264
exists based on the absence of clinical signs of bovine anaplasmosis. Therefore, the
265
association between A. marginale prevalence and occurrences of clinical disease and/or
266
mortality is more complex than what was previously established for the Babesia bovis-
267
Rhipicephalus microplus system in Australia by Mahoney and Ross, in 1972.
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268
Although studies have proven that transplacental transmission of A. marginale
269
occurs in Brazil (Grau et al., 2013), this was the first study have proven that
270
transplacental transmission of the strains 4-63-27, 78-242-25-31 and τ-102-15 occurs. In
271
this study, although 19 different MSP1a tandem repeats of A. marginale were
11 Page 11 of 34
circulating in the cattle tested, only three were transmitted through the placenta. Studies
273
have suggested that transplacental transmission can occur when cows have acute
274
anaplasmosis during pregnancy (Zaugg and Kuttler, 1984) or may be due to constant
275
inoculations in endemic areas (Potgieter and Vanrensburg, 1987). In the present study,
276
infection in newborn calves caused by these three MSP1a tandem repeats of A.
277
marginale (-63-27, 78-242-25-31 and τ-102-15) was detected by PCR. MSP1a tandem
278
repeat τ-102-15 was previously reported to be circulating in Brazil (Vidotto et al., 2006).
279
Hence, our confirmation that this MSP1a tandem repeat is dominant (detected in 17.6%
280
of the animals) and can be transmitted through the placenta suggests that this type of
281
transmission may have greater importance in these regions than has been supposed until
282
now.
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The prevalence of genotype E over G observed in this study may indicate that
284
genotype E is better adapted and thus may be more efficient for infecting reservoir
285
hosts. Both genotype G and genotype E have SD-ATG distances of 23 nucleotides.
286
These microsatellite genotypes have been correlated with high levels of MSP1α protein
287
expression (Estrada-Peña et al., 2009), thus suggesting that these MSP1a tandem repeats
288
identified in calves present high infectivity potential. Estrada-Peña et al. (2009)
289
evaluated the distribution of nine different genotypes in four distinct ecosystems around
290
the world and noted that in South America, especially in Brazil and Argentina, genotype
291
E has been found to be the most common type. However, genotypes B, C, D and G were
292
previously detected in Argentina and Brazil (de la Fuente et al., 2004; Vidotto et al.,
293
2006; Ruybal et al., 2009; Pohl et al., 2013). Genotype G is the most frequently found
294
genotype around the world and has been seen to be the most prevalent type in the
295
ecoregions of South Africa and parts of the USA and Mexico (Estrada-Peña et al.,
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2009).
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When we compared the repeat sequences found in this study with already-known
298
sequences (Cabezas-Cruz et al., 2013), we were able to describe 22 new repeat
299
sequences and also to report occurrences of 30 replicates for the first time in Brazil.
300
Repeat sequences of MSP1a vary geographically among MSP1a tandem repeats of A.
301
marginale and are functionally important in infection with and biological transmission
302
of this pathogen (McGarey and Allred, 1994; de la Fuente et al., 2007). Furthermore,
303
analysis on repeat sequences provides evolutionary information about A. marginale
304
lineages and is used to characterize the genetic diversity of the pathogen (de la Fuente et
305
al., 2001, 2007; Palmer et al., 2001, 2004). Thus, maintaining a database characterizing
306
the structure of this gene region is crucial for new studies aiming to correlate these
307
repeat sequences with the antigenic characteristics of this pathogen.
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The dominant strain was 4-63-27, and this was possibly associated with low
309
occurrence of blood-sucking dipterans and high infestations by R. (B.) microplus ticks
310
in this study area. Some tandem repeats, such as 27 and 13, have been found to be
311
present in samples of the pathogen circulating in Latin America and South Africa, the
312
common areas of Rhipicephalus (Boophilus) spp. ticks (de la Fuente et al., 2007). These
313
results are consistent with the geographic distribution of the tick Rhipicephalus (B.)
314
microplus (Scoles et al., 2005), which is most likely the main vector of this pathogen in
315
tropical regions. However, other species of ticks and mechanical transmission may also
316
play important roles in the growth of this pathogen (Kocan et al., 2004).
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The most commonly observed sequences in the first replicate of MSP1a were 4
318
and τ. These results support previous studies showing that in South America, circulating
319
MSP1a tandem repeats of A. marginale usually present sequences 4, 8, 16, 56, 60, 64,
320
67, γ, π and τ in the first replicate (Estrada-Peña et al., 2009). In samples described on
321
this continent, the most frequent sequences in the first replicate are 16, α, τ (Vidotto et
13 Page 13 of 34
322
al., 2006), 72 and α (Pohl et al., 2013) in Brazil; and, α, τ and B (Ruybal et al., 2009) in
323
Argentina. The results shown in the present study corroborate those found by Cabezas-Cruz
325
et al. (2013), in which only serine was found at position 4 and glycine at position 31 in
326
tandem repeats of MSP1a. The positions in which the highest variation of amino acids
327
was observed were 1, 20 and 28. However, we observed that valine, arginine, alanine,
328
aspartate and asparagine amino acids were present at position 22, whereas the analysis
329
conducted by Cabezas-Cruz et al. (2013) found no variations in this position, with only
330
alanine being present.
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In our study, sample Rio5b exhibited a deletion of the QQQESS sequence
332
consisting of 23 amino acids in the first tandem repeats. This result may indicate that
333
MSP1a tandem repeats of A. marginale are present in these cattle, transmitted by blood-
334
sucking dipterans, since MSP1a contains B and T cell epitopes that are sensitive to
335
neutralization (Allred et al., 1990). Thus, studies have shown that MSP1a tandem
336
repeats of A. marginale that lack the amino acids included between positions 4 and 14
337
(SSAGGQQQESS) are unable to infect the cells of ticks (Blouin et al., 2003) and
338
cannot be transmitted by Dermacentor variabilis ticks (Kocan et al., 2003).
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To determine which selective pressures could be triggering MSP1a
340
diversification in A. marginale from cattle, the ratio ω was calculated, showing that the
341
codon at position 10 from tandem repeat 4 was evolving under negative selection.
342
Interestingly, this amino acid position is present in an immunodominant B-cell epitope
343
previously described for A. marginale MSP1a (Garcia-Garcia et al., 2004). These results
344
suggest that this tandem repeat, which is present in the most common MSP1a tandem
345
repeat of A. marginale found in cattle, may be under selective pressure from the host
346
immune system.
14 Page 14 of 34
Among geographic isolates, the MSP1a repeat sequences of A. marginale
348
located between the first and last repeat sequences are highly conserved (de la Fuente et
349
al., 2001). However, our results showed significant variation in amino acid sequences
350
for all replicates. Even the samples with four or more replicates exhibited variation in
351
their repeat sequences. These results were also found by Palmer et al. (2001) in cattle in
352
eastern Oregon, USA. These authors demonstrated that although only one genotype was
353
circulating, the A. marginale population was genetically heterogeneous.
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In our study, the most likely explanation for the genetic heterogeneity of A.
355
marginale is that the high diversity is a result of distinct biological and mechanical
356
transmission processes, each introducing different genotypes of A. marginale into cattle.
357
The causes of this occurrence are still unknown, but it reflects a population with high
358
genetic diversity in endemic areas (de la Fuente et al., 2001). This same assumption was
359
made previously by Palmer et al. (2001) in endemic areas of the USA, where it was
360
shown that bovine reservoirs harbor genetically heterogeneous A. marginale, thus
361
suggesting that different genotypes are maintained by transmission within the reservoir
362
cattle. In the region where our study was conducted, R. microplus ticks complete
363
between three and five cycles per year (Kasai et al., 2000) and can be infected by more
364
than one MSP1a tandem repeat of A. marginale over time and, during the feeding
365
process, transmit them to new hosts.
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Our results show that most animals were infected only by the MSP1a tandem
367
repeat 4-63-27, which we assume is dominant. Some animals were infected by other
368
minority MSP1a tandem repeats, and other animals were superinfected by multiple
369
MSP1a tandem repeats of A. marginale. Genetic diversity does not result in evolution
370
unless a variant with increased aptitude arises under conditions that promote imbalance
371
and favor fast random evolution (de la Fuente et al., 2001). Hence, wild-type sequences,
15 Page 15 of 34
or the master sequence in this situation, would remain unchanged (de la Fuente et al.,
373
1999). Our results show that more than one genotype was established in animals, thus
374
indicating that there was no occurrence of selection for specific variants, or a "genetic
375
divide" process, as alleged by de la Fuente et al. (2001). These findings corroborate the
376
results of Ueti et al. (2012) and Palmer and Brayton (2013), which showed that different
377
genotypes coexist in the same ecosystem and can parasitize the same animal at the same
378
time, thereby characterizing an occurrence of superinfection, which is common in
379
natural infections in tropical countries. The results from this study are supported by the
380
theory of Palmer and Brayton (2013), who showed that several MSP1a tandem repeats
381
can circulate in the same cattle, such that most animals are parasitized by a dominant
382
MSP1a tandem repeat and some animals are parasitized by more than one MSP1a
383
tandem repeat.
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The new tandem repeats observed in this study have broad similarity to existing
385
sequences and are located in the same cluster. This finding suggests that the new
386
tandem repeats may have originated recently from existing tandem repeats, and this
387
provides evidence for genetic diversification of A. marginale in cattle. In summary, we
388
suggest that calves kept under natural conditions can be infected by more than one
389
MSP1a tandem repeat of A. marginale throughout their lives. Furthermore, even in the
390
same cattle, circulation of various MSP1a tandem repeats of A. marginale can occur,
391
which are kept under constant variation of MSP1a tandem repeats.
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CONFLICT OF INTEREST STATEMENT
394
None of the authors of this work has a financial or personal relationship with other
395
people or organizations that would inappropriately influence or bias the content of this
396
paper.
16 Page 16 of 34
ACKNOWLEDGEMENTS
398
We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for
399
their financial support (Process #2012/21371-4) and the Coordination Office for
400
Improvement of Higher-Education Staff (CAPES) for the J. B. Silva fellowship. We
401
thank the Germania Farm for access to the study animals.
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Madruga, C.R., Honer, M.R., Andreotti, R., Araújo, F.R., Santarém, V. Prevalência de
504
Anaplasma marginale em três regiões do estado da Paraíba. In: CONGRESSO
505
INTERNACIONAL
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PORTUGUESA, 6, 1994, Salvador, BA. Anais... Bahia : Escola de Medicina
507
Veterinária da Universidade Federal da Bahia, 1994. p.350-352.
509
MEDICINA
VETERINÁRIA
EM
LÍNGUA
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Mahoney, D.F. and Ross, D.R. 1972. Epizootiological factors in the control of bovine
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M
503
babesiosis. Aust. Vet. J. 48, 292–298
510
McGarey, D.J., Allred, D.R., 1994. Characterization of hemagglutinating components
511
on the Anaplasma marginale initial body surface and identification of possible
512
adhesins. Infect Immun.62, 4587–4593.
513
Oliveira AA, de Pedreira PAS, Almeida MFR 1992. Doenças de bezerro. II
514
Epidemiologia da anaplasmose no estado de Sergipe. Arq Bras Med Vet Zootec 44,
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377-386.
21 Page 21 of 34
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Palmer, G.H., Rurangirwa, F.R., McElwain, T.F. 2001. Strain composition of the
517
Ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian
518
reservoir for tick transmission. J Clin Microbiol, 39, 631–635. Palmer, G.H., Knowles Jr., D.P., Rodriguez, J.L., Gnad, D.P., Hollis, L.C., Marston, T.,
520
Brayton, K.A., 2004. Stochastic transmission of multiple genotypically distinct
521
Anaplasma marginale strains in a herd with high prevalence of Anaplasma
522
infection. J. Clin.Microbiol. 42, 5381–5384.
524
cr
Palmer, G. H. and Brayton, K. A. 2013. Antigenic variation and transmission fitness as
us
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ip t
519
drivers of bacterial strain structure. Cell Microbiol, 14, 1-7.
Pohl, A.E., Cabezas-Cruz, A., Ribeiro, M.F.B., Silveira, J.A.G., Silaghi, C., Pfister, K.,
526
Passos, L.M.F., 2013. Detection of genetic diversity of Anaplasma marginale
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isolates in Minas Gerais. Brazil. Rev. Bras. Parasitol. Vet. 22, 129-135.
M
an
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Potgieter, F.T., Vanrensburg, L. 1987. The persistence of colostral Anaplasma
529
marginale antibodies and incidence of in utero transmission of Anaplasma
530
infections in calves under laboratory conditions. Onderstepoort J Vet Res, 54, 557-
531
560.
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te
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Ribeiro, M.F.B., Reis, R. 1981. Prevalência da anaplasmose em quatro regiões do estado de Minas Gerais. Arq Esc Vet UFMG, 33, 57-62.
534
Ruybal P., Moretta, R., Perez, A., Petrigh, R., Zimmer, P., Alcaraz, E., Echaide, I.,
535
Torioni de Echaide, S., Kocan, K.M., de la Fuente, J., Farber, M. 2009. Genetic
536
diversity of Anaplasma marginale in Argentina. Vet Parasitol, 162, 176–180.
537
Scoles, G.A., McElwain, T.F., Rurangirwa, F.R., Knowles, D.P., Lysyk, T.J., 2006. A
538
Canadian bison isolate of Anaplasma marginale (Rickettsiales, Anaplasmataceae) is
539
not transmissible by Dermacentor andersoni (Acari, Ixodidae), whereas ticks from
22 Page 22 of 34
540
two Canadian D. andersoni populations are competent vectors of a US strain. J. Med.
541
Entomol. 43, 971–975.
543
Silva, J.B. and Fonseca, A.H. 2014. Risk factors for anaplasmosis in dairy cows during the peripartum. Trop Anim Health Prod, 46, 461-465.
ip t
542
Souza, J.C.P., Soares, C.O., Massard, C.L., Scofield, A., Fonseca, A.H. 2000.
545
Soroprevalência de Anaplasma marginale em bovinos na mesorregião Norte
546
Fluminense. Pesq Vet Bras, 20, 97-101.
cr
544
Ueti, M.W., Tan, Y., Broschat, S.L., Castañeda Ortiz, E.J., Camacho-Nuez, M.,
548
Mosqueda, J.J., Scoles, G.A., Grimes, M., Brayton, K.A., Palmer, G.H. 2012.
549
Superinfection under conditions of natural with a high prevalence of pathogen strain
550
Expansion of variant diversity associated transmission. Infect Immun, 80, 2354–
551
2360.
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an
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547
Vidotto, O. Yamamura, M.H., Andrade, G.M., Barbosa, C.S., Freire, R.L., Vidotto,
553
M.C. 1995. Ocorrência de Babesia bigemina, B. bovis, e Anaplasma marginale em
554
rebanhos de bovinos leiteiros da região de Londrina, PR. Rev Bras Parasitol Vet, 4,
555
184.
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te
d
552
556
Vidotto, M.C., Vidotto, O., Andrade, G.M., Palmer, G., Mcelwain, T., Knowles, D.P.
557
1998. Seroprevalence of Anaplasma marginale in cattle in Paraná State, Brazil, by
558
MSP-5 competitive ELISA. Ann N Y Acad Sci. 849, 424-426.
559
Vidotto, M. C., Kano, F.S., Gregori, F., Vidotto, O. 2006. Phylogenetic analysis of
560
Anaplasma marginale strains from Parana State, Brazil, using the msp1-alfa and
561
msp4 genes. Journal of Veterinary Medicine. B, Infect Dis Vet Public Health, 53,
562
404-411.
23 Page 23 of 34
563
Zaugg, J.L., Kuttler, K.L. 1984. Bovine anaplasmosis: in utero transmission and the
564
immunologic significance of ingested colostral antibodies. Am J Vet Res, 45, 440-
565
443.
567
ip t
566
FIGURE CAPTIONS
cr
568
TABLE 1. Frequency (%) of animals positive for A. marginale. Anaplasma
570
marginale was detected through direct examination (blood smears), serological tests
571
(ELISA/IFAT) and molecular analysis (qPCR) among naturally infected calves in the
572
state of Rio de Janeiro, Brazil.
an
us
569
M
573
TABLE 2. Organization of MSP1a tandem repeats and A. marginale strains
575
isolated from cattle in the state of Rio de Janeiro, Brazil. A. marginale strain
576
identification was based on msp1α and included microsatellite genotype (tandem repeat
577
structure). Superscripts represent the number of times that the tandem repeats were
578
repeated.
te
Ac ce p
579
d
574
580
TABLE 3. Occurrence of the most common A. marginale strains. The most frequent
581
A. marginale strains, observed in 51 samples. All animals were naturally infected in the
582
state of Rio de Janeiro, Brazil.
583 584
TABLE 4. Sequences of newly described A. marginale MSP1a tandem repeats. The
585
one-letter code is used to name the different amino acids of the tandem repeats.
586
Conserved amino acid positions and deletions/insertions (-) are shown. The new MSP1a
587
tandem repeats 165-186 were named in accordance with the system proposed by de la
24 Page 24 of 34
588
Fuente et al. (2007) and updated by Cabezas-Cruz et al. (2013). Tandem repeat
589
sequence A is used as a model for comparison.
590
FIGURE 1. MSP1a tandem repeats. Analysis on MSP1a microsatellite sequences in
592
different A. marginale strains detected at different ages. All MSP1a tandem repeats
593
ranged from 1 to 6 and the ages of the animals ranged from newborn to 12 months
594
(three-month intervals between observations).
us
595
cr
ip t
591
FIGURE 2. Amino acid variability and frequency in MSP1a tandem repeats. The
597
amino acids were calculated per amino acid position in the MSP1a tandem repeats using
598
the following formula: Variability = number of different amino acids at a given
599
position/frequency of the most common amino acid at that position. The one-letter
600
amino acid code was used to name the amino acids, and the most frequent amino acid at
601
each position was colored in gray.
M
d
te
602
an
596
FIGURE 3. Distribution and phylogenetic analysis on A. marginale strains
604
identified in cattle. In the left panel, the locations of Rio de Janeiro (Brazil), Azaria,
605
Israel, Mississippi, Idaro, St. Meries, California, Okeechobee, Oklahoma, Florida,
606
Virginia and Porto Rico are shown. The strains isolated from cattle are identified as in
607
the legend of the figure. The right panel shows the consensus tree from the ML and NJ
608
phylogenetic analyses. The numbers above and below the internal branches represent
609
bootstrap values (1000 replicates) for ML and NJ, respectively. Only bootstrap values
610
higher than 50 are shown. The MSP1a GenBank accession numbers of the respective
611
sequences used in the phylogenetic tree are shown. The strains identified in this study in
Ac ce p
603
25 Page 25 of 34
612
cattle are shown as in Table 2. The four phylogenetic clusters shown contained different
613
patterns of MSP1a tandem repeat 2D structures.
Ac ce p
te
d
M
an
us
cr
ip t
614
26 Page 26 of 34
614
Table 1
615 Frequency of positivity (%) Age (days) IFAT
qPCR
Rickettsemia*
1
10
40
35
15
1.04 x 101
90
80
50
50
100
180
70
60
55
100
270
60
55
55
360
50
70
65
ip t
ELISA
4,36 x 1012
cr
7,91 x 104
100
1,54 x 105
100
5,23 x 105
us
616
Blood smear
* DNA copies per ml of blood
Ac ce p
te
d
M
an
617
27 Page 27 of 34
TABLE 2.
KJ398348 KJ398349 KJ398350 KJ398351 KJ398352 KJ398353 KJ398354 KJ398355 KJ398356 KJ398357 KJ398358 KJ398359 KJ398360 KJ398361 KJ398362 KJ398363 KJ398364 KJ398365 KJ398366 KJ398367 KJ398368 KJ398369 KJ398370 KJ398371 KJ398372 KJ398373 KJ398374 KJ398375 KJ398376 KJ398377 KJ398378 KJ398379 KJ398380 KJ398381 KJ398382 KJ398383 KJ398384 KJ398385 KJ398386 KJ398387 KJ398388 KJ398389 KJ398390 KJ398391 KJ398392 KJ398393 KJ398394 KJ398396
an M
d
te
Infection Acute Acute Chronic Acute Acute Chronic Acute Acute Chronic Acute Acute Chronic Chronic Acute Acute Acute Acute Acute Acute Acute Acute Chronic Acute Acute Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic
ip t
Rio1a / E - (78, 242, 25, 31) Rio1b / E - (78, 242, 25, 31) Rio1c / E - (78, 242, 25, 31) Rio2a / E - (4, 63, 27) Rio2b / E - (4, 63, 27) Rio2c / E - (τ, 102, 15) Rio3a / E - (τ, 102, 15) Rio3b / E - (4, 63, 27) Rio3c / E - (τ, 102, 15) Rio4a / E - (4, 63, 27) Rio4b / E - (4, 63, 27) Rio4c / E - (4, 63, 27) Rio4d / E - (165, 102, 166) Rio5a / E - (78, 242, 25, 31) Rio5b / E - (167, 168, β2, 169, 170) Rio6a / E - (78, 242, 171, 31) Rio6b / E - (4, 63, 27) Rio7a / E - (4, 63, 27) Rio7b / E - (78, 242, 25, 31) Rio8a / E - (78, 242, 25, 31) Rio8b / E - (78, 242, 25, 31) Rio8c / E - (4, 63, 4) Rio9a / E - (4, 63, 3) Rio9b / E - (78, 24, 172, 24, 173) Rio9c / E - (α, 174, β) Rio9d / E - (175, 63, 27) Rio10a / E - (τ, 102, 176) Rio10b / E - (174, 176, β2, Ʈ) Rio11a / E - (78, 242, 25, 31) Rio11b / E - (78, 242, 25, 31) Rio12a / E - (4, 63, 27) Rio12b / E - (τ, 102, 15) Rio13a / E - (4, 63, 27) Rio13a / E - (4, 63, 27) Rio13c / E - (τ, 102, 15) Rio14a / E - (4, 63, 27) Rio14b / E - (4, 63, 27) Rio15a / E - (4, 63, 177) Rio15b / E - (178, 179, 180, 181, 182) Rio16a / G - (163, 1643, 61) Rio17a / E - (4, 63, 27) Rio17b / E - (78, 242, 25, 31) Rio17c / E - (176, 174, β2, Ʈ) Rio18a / E - (78, 242, 25, 31) Rio18b / E - (τ, 102, 15) Rio18c / E - (182, 24, 183, 184, 185) Rio19a / E - (τ, 102, 15) Rio20a / E - (τ, 102, 15)
Rickettsemia (msp1α copies/ml) 4.36 x 1012 1.40 x 1012 2.06 x 103 2.22 x 1012 6.76 x 1011 1.02 x 104 8.13 x 1011 8.20 x 1011 2.17 x 104 5.06 x 1010 2.33 x 1011 1.37 x 104 4.19 x 104 5.89 x 1011 4.06 x 1010 8.68 x 1010 5.36 x 1011 3.76 x 1011 7.76 x 1010 6.76 x 1011 2.95 x 1011 2.07 x 104 7.68 x 1011 5.21 x 1011 2.00 x 105 5.12 x 104 1.32 x 105 1.76 x 104 4.13 x 104 5.08 x 104 1.33 x 105 3.20 x 104 1.50 x 104 2.73 x 104 5.17 x 104 1.69 x 105 3.04 x 105 9.29 x 104 3.27 x 104 4.64 x 104 3.50 x 104 8.76 x 105 1.74 x 105 4.81 x 105 5.25 x 104 5.46 x 106 1.12 x 105 6.53 x 105
cr
GenBank accession number
us
Strain identification and structure of msp1a tandem repeats
Ac ce p
617
28 Page 28 of 34
618
Rio20b / E - (174, 176, β2, 186) Rio20c / E - (τ, 102, 15)
KJ398397 KJ398398
3.94 x 105 2.48 x 103
Chronic Chronic
Strain identification is based on msp1α and includes microsatellite genotype – (tandem repeats structure).
620
Superscripts represent the number of times that a tandem repeats are repeated. Infection of animals was
621
calculated by Eriks et al. (1993). The new MSP1a tandem repeat 165-186 was named in accordance with
622
the system proposed by de la Fuente et al. (2007) and updated by Cabezas-Cruz et al. (2013).
cr
ip t
619
Ac ce p
te
d
M
an
us
623
29 Page 29 of 34
TABLE 3
Strain
Structure of MSP1a tandem repeats
Nº of strains
Frequency
1ª commom
4-63-27
14
27,5 %
2ª commom
78-242-25-31
11
21,6 %
3ª commom
τ-102-15
9
17,6 %
ip t
623
The most common tandem repeats found among all the A. marginale strains are underlined and there were
625
found more than 27 (24), 24, (4), 22 (10), 21 (63) and 21 (27).
cr
624
626
Ac ce p
te
d
M
an
us
627
31 Page 30 of 34
TABLE 4.
629
M
an
ip t cr
us
DDSSSASGQQQESSVSSQSE-ASTSSQLG-TDSSSASGQQQESSVLSPSGHVRTSSQLG-ADSSSGRGQQQESGVLSQSGQASTSSQLG-ADSSSASG------VLSQSGEATTSAQLR-TDSSSAGDQPQGSGVSSQSGQASTSAQLR-TDSSSATDQQQESGVSSQSGQASTSA---VG TDSSSASAQQQESSVSSHTD-RSTSSQ--VG ADSSSASGQQQESSVLSQSSQASTSSQLR-ADSSSAGNQQQESSVLSQSGQASTSSQSG-ADSSSAGNQQQESSVSSQSD-ASTSSDYG-TDSSSAGDQQQGSGVSSQSGQASTSSQLR-TDSSSASGQQQESSVLSQSGHASTSSQLG-ADSSSASGQQQESGVLSQSAQASTSSQLG-ADSSSASGQQQESSVLSQSDHASTSSQLG-DDSSSADDQQQESSVSSQSG-DSTSSQLG-TDSSSAGDQQQESSVSSQSG-DSTSSQLG-TDSSSAQHQQQESNVSSQTG-NSTSSQLG-NDTSSAGHQQQESNVSSQSG-DSTSSQLG-TDSSTAGDQQQESSVSSQSG-ASTSSQLG-VDSSSAGDQQQESSVSSQSG-DSTSSQLG-TDSSSAGDQQQESSVSSQSG-DSTSSQLG-TDNSSASGQQQENSVLSQSSQASTSSQLG-DDSSSAGNQQQESSVLPQSGQASTSSQLG--
Number of Amino Acid 28 29 29 23 29 28 29 29 28 28 29 29 29 29 28 28 28 28 28 28 28 29 29
Ac ce p
628
Tandem repeats
te
New sequences A 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
d
627
32 Page 31 of 34
3 Months
6 Months
9 Months
12 Months
4-63-27
an
Tandem repeats
Newborn
3
4-63-27
4
τ-102-15
not detected
5
2
78-24 -25-31
α-174-β
175-63-27
M
d
not detected
2
τ-10 -15 2
τ-10 -176
2
165-10 -176 178-179-180-181-182
2
78-24 -171-31
2
176-174-β -Γ
182-24-183-184-185 2
174-176-β -186
not detected
2
167-168-β -169-170
not detected
ep te
not detected
2
78-24 -174-31
2
τ-10 -15
Ac c
6
4-63-27
4-63-164
2
78-24 -25-31
us
cr
ip t
Figure
Page 32 of 34
Figure
Figure 2
Average of amino acids variability 0,61 Proportion of variable/conserved 8,66 51 samples and 66 2
1
ip t
Shannon variability
3
0
V
Q
K
C
Y
F
I
R
0,00
A
T
0,60
0,35
D
L
S
N
us
P
0,03 0,90
0,00
0,99 1,00
0,01
0,00
0,00
1,00 0,01
0,99
0,00 0,13
M
1,00 0,01
1,00
0,88
0,97 0,00
ed
0,12
0,00
0,00
0,00 0,65 0,21
0,33 0,65
0,01
0,98
0,02
0,86
0,01
0,91
0,13
0,23 0,87 0,00
0,01
0,99
0,00
0,20
0,01 0,95
0,03
0,00
0,14
0,66 0,02
0,01
0,00
0,01
0,98
0,99
0,01
0,00
1,00
0,01 0,99
0,00
0,10
0,09
0,77 0,13
E
0,01
1,00
0,00
H
1,00
an
0,00
G
0,01
0,01
0,98 0,00
0,00
0,85 0,05
0,00
0,14
0,00 0,95
1,00
1,00
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A S T S S Q L G V G
Frequency of amino acid position W
ce pt
AAP*
cr
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 D D S S S A S G Q Q Q E S S V S S Q S E -
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure
Page 34 of 34
