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Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence
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Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence Bruna T. Silvestre a , Júlia A.G. Silveira a , Rodrigo M. Meneses b , Elias J. Facury-Filho b , Antônio U. Carvalho b , Múcio F.B. Ribeiro a,∗ a b
Department of Parasitology, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Department of Clinical and Veterinary Surgery, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 17 June 2015 Received in revised form 7 August 2015 Accepted 2 September 2015 Available online xxx Keywords: Anaplasma marginale UFMG3 Vertical transmission Genetic characterization Low virulence
a b s t r a c t Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia species Anaplasma marginale and results in great economic losses in tropical and subtropical regions. Vertical transmission is an important phenomenon that contributes to the persistence of different strains of the agent within the same herd. The identification of new strains and genetic characterization studies are essential to understanding their epidemiology and virulence and for vaccine development. The aim of this study was to perform molecular and phylogenetic characterizations of a new vertically transmitted strain from A. marginale and to evaluate its virulence by experimental inoculation of rickettsia-free calves. Thirty newborn Holstein calves were subjected to molecular tests for the detection of A. marginale, Babesia bovis and Babesia bigemina. Calves positive for A. marginale (n = 3) were splenectomized and monitored for the clinical manifestations of anaplasmosis. Blood samples from one of the calves that presented rickettsemia of 42.8% and spontaneous recovery of clinical parameters were used for molecular and phylogenetic characterization (msp1a gene), and inoculum production was used for the evaluation of virulence. This strain was identified as UFMG3. Three tandem repeat forms (13 and MGI19) were identified from the analysis of the msp1a gene, in which the form MGI19 appeared twice. Analysis of these repeats revealed the presence of the sequences QASTSS and SSASGQQQESS and of aspartic acid (D) at position 20 of both repeats. Phylogenetic analysis showed a close relationship among the UFMG3, MGI19 and UFMG2 strains. For virulence evaluation, six Holstein calves were inoculated intravenously with 2 × 107 A. marginale UFMG3-infected erythrocytes. The calves showed maximum rickettsemia of 5.1%, a moderate decrease in packed cell volume and spontaneous recovery of clinical parameters without the need for treatment. The results of experimental inoculation suggest that the strain A. marginale UFMG3 has low virulence and potential application for use as a live vaccine against A. marginale. © 2015 Elsevier GmbH. All rights reserved.
Introduction Bovine anaplasmosis is a major constraint to cattle production in several countries, generating significant economic losses in livestock worldwide (Kocan et al., 2010; Aubry and Geale, 2011). The disease is caused by the rickettsia Anaplasma marginale (Theiler, 1910) and is characterized by anemia, jaundice, fever, dehydration, weight loss, abortion and death (Kocan et al., 2003; Marana et al., 2009).
∗ Corresponding author. E-mail address: [email protected] (M.F.B. Ribeiro).
In South America, A. marginale is biologically transmitted by the tick Rhipicephalus microplus and mechanically by biting flies and contaminated fomites (Guglielmone, 1995; Kocan et al., 2003). The vertical transmission of A. marginale has been reported causing persistent infections in calves without clinical signs of disease, suggesting the low virulence of these isolates (Swift and Paumer, 1976; Passos and Lima, 1984; Salabarria and Pino, 1988; Ribeiro et al., 1995; Benesi et al., 1999; Gonc¸alves et al., 2005; Grau et al., 2013). However, clinical anaplasmosis in newborn calves has been reported, suggesting differences in the virulence of these isolates (Bird, 1973; Paine and Miller, 1977; Norton et al., 1983; Passos and Lima, 1984; Benesi et al., 1999; Gonc¸alves et al., 2005; Pypers et al., 2011; Pohl et al., 2013).
http://dx.doi.org/10.1016/j.ttbdis.2015.09.001 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001
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The identification of different geographic strains of A. marginale has been performed with regard to virulence, genetic characteristics and transmissibility by ticks. The genetic diversity of these strains has been identified by differences in molecular weight and in the constitution of the MSP1a protein, based on the varying number of tandem repeats located in the N-terminal region of the protein (de la Fuente et al., 2005, 2007). The number and the constitution of tandem repeats varies among geographic strains of A. marginale, and this region has been recognized as a stable genetic marker (de la Fuente et al., 2007). Understanding the genetic diversity and characteristics of A. marginale strains is of fundamental importance for the study of epidemiology, virulence, and the development of effective vaccines (Lis et al., 2015), which require a standardized inoculum containing samples of low virulence (Ribeiro et al., 1997; Coelho, 2007; Bastos et al., 2010). However, there are few samples of low-virulence isolates, and none from vertical transmission. Considering that the characterization and use of live samples of low-virulence isolates is a promising strategy for preventing and reducing clinical manifestations, the aim of this study was to perform molecular and phylogenetic characterizations of a new vertically transmitted strain from A. marginale and to evaluate its virulence by experimental inoculation of rickettsia-free calves.
Fig. 1. Phylogenetic tree based of msp1a gene of Anaplasma marginale Brazilian isolates. The tree was constructed using the Neighbor Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method. The GenBank accession numbers of the respective sequences used for the phylogenetic analysis are shown. UFMG3* is the sequence from the present study.
Materials and methods Obtaining an A. marginale congenital sample Thirty newborn male Holstein calves from a farm in InhaúmaMG (19◦ 29 27 South, 44◦ 23 24 West) were subjected to molecular tests of nested PCR (nPCR) for the detection of A. marginale (gene msp4), Babesia bovis and Babesia bigemina, according to Silveira et al. (2013). A. marginale DNA was detected in three calves, which were kept in a tie stall barn and were sprayed weekly with cypermethrin (Flytick® – Vallée, Montes Claros, MG, Brazil) to keep them free of ticks and biting flies. Throughout the experimental period, all animals were fed with powdered milk, commercial feed and water ad libitum. All procedures were approved by the Ethics Committee on Animal Use – CEUA/UFMG (Protocol number 41/2006). The animals were monitored daily through clinical examination and measurement of rectal temperature and weekly through blood smears and determination of the packed cell volume (PCV). Up to 70 days of age, the animals showed negative results for A. marginale in the clinical evaluations performed, though they remained positive by nPCR. The three calves were splenectomized and monitored again. One of the calves presented maximum rickettsemia of 42.8%, with spontaneous recovery of clinical parameters (PCV ≥ 24%). Blood samples from this calf were then used for molecular and phylogenetic characterization and for inoculum production for virulence evaluation. Inoculum production At peak rickettsemia, blood samples were collected from the jugular vein into EDTA tubes and were cryopreserved in liquid nitrogen using dimethyl sulfoxide (DMSO). The vertically transmitted A. marginale strain was identified as UFMG3. Molecular analysis and characterization of the UFMG3 strain Blood samples with EDTA were collected from the jugular vein on each calf’s day of birth and after their splenectomies for DNA extraction (kit Wizard Genomic DNA Purification, Promega). Detection of A. marginale DNA was performed via nPCR using primers complementary to the region of the msp4 gene (Silveira et al., 2013).
To assess the genetic diversity of isolated UFMG3 by sequences of tandem repeats, primers for the msp1a gene of A. marginale were used according to Bastos et al. (2009). The analyses of tandem repeats were performed according to the nomenclature proposed by Cabezas-Cruz et al. (2013). Positives products from the PCR reactions were purified using a QIAquick PCR Purification Kit (Qiagen Biotecnolo-gia Brasil, São Paulo, Brazil) according to the recommendations of the manufacturer. The purified amplicons were sequencing using an Applied Biosystems model ABI3130 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) and the Applied Biosystems BigDye® Direct Cycle Sequencing Kit (v. 3.1), with the POP-7TM polymer as the separating matrix and the primers employed in the PCR reaction. Sequences were aligned, edited, and analyzed using MEGA 6.0 (Tamura et al., 2013). The identity of each sequence was confirmed by comparison with sequences available on GenBank using BLAST software (Altschul et al., 1990). Phylogenetic and molecular evolutionary analyses were conducted using MEGA 6.0 and the program “Electropherogram quality analysis” (http://asparagin. cenargen.embrapa.br/phph/). The phylogenetic tree was constructed by analyzing msp1a gene fragments from Brazilian A. marginale strains deposited in GenBank (Fig. 1). Nucleotide sequences were aligned with MUSCLE from de MEGA 6.0 package (Tamura et al., 2013). Each alignment was analyzed using the Neighbor Joining (NJ) method and the evolutionary distances were computed using the Maximum Composite Likelihood method in MEGA 6.0 software. Internal branch confidence was assessed by the bootstrapping method using 1000 bootstrap replicates.
Evaluation of the virulence Experimental design Six male Holstein calves, 90–100 days old and A. marginaleand B. bovis/B. bigemina-free, were inoculated intravenously with 2 × 107 cryopreserved A. marginale UFMG3-infected erythrocytes. As a control group, two calves were inoculated intravenously with saline to ensure the absence of transmission in the barn during the experimental phase.
Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001
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Table 1 MSP1a tandem repeat sequences in the A. marginale UFMG3 strain. Anaplasma marginale genotype
Encoded sequence DDSSSASGQQQESSVSSQSE-ASTSSQLG–
Repeat form A*
UFMG 3
T**************L***DQ********** AE*************L***DQ********** AE*************L***DQ**********
13* MGI19** MGI19**
A. marginale strain
Country of origin
GenBank accession No.
Structure of MSP1a tandem repeats
UFMG 3
Brazil
KF005076.1
13
* **
MGI19
No. of repeats MGI19
3
Sequences reported by Cabezas-Cruz et al. (2013). Sequences reported by Silveira et al. (2012).
Clinical and hematological parameters Throughout the experimental period, all animals (n = 8) were monitored daily by clinical examination through measurements of rectal temperature, heart and respiratory rate, ruminal movements and clinical score. Blood smears were performed for the determination of rickettsemia and were stained with stain type Romanowsky (IICA, 1987). The PCV was measured using the microhematocrit from blood samples collected in EDTA tubes from the jugular vein. The PCV and blood smears were initially performed every 48 h to detect rickettsemia. At 20 days post inoculation (dpi), PCV and rickettsemia were performed daily until the end of the experiment, which occurred 61 dpi, when the median values of PCV reached 24%, considered lower limit of normality (Radostits et al., 2007). The incubation period (IP) was defined as the interval between inoculation and the beginning of rickettsemia. The patent period (PP) was measured from the day when A. marginale was first detected until the day of less PCV, and the convalescent period (CP) the interval time between the days of less PCV until recovery (24%). The times of the evaluations were defined from the first day of observation of erythrocytes infected with A. marginale in the blood smears, designated day 0 (zero). The previous and subsequent data were referenced from this day. Statistical analysis The Lilliefors test was used to determine the normality of the data. The variables did not follow a normal distribution and were subjected to the Friedman test to observe the existence of differences between the times (Sampaio, 2010). Statistical analysis was performed using the software InfoStat® version 2008 and median values obtained among the times were plotted in scatter plots. Results Detection of calves with congenital transmission of A. marginale (UFMG3) Three of thirty newborn calves (10%) subjected to nPCR for gene msp4 of A. marginale were diagnosed positive. After splenectomy, a calf exhibited rickettsemia due to A. marginale, peaking at 42.8%. The calf recovered clinical parameters spontaneously, without the need for treatment. Molecular analysis and characterization of the UFMG3 strain The molecular identity of the UFMG3 strain was performed by sequencing of the msp4 and msp1a gene fragments of A. marginale, obtained via nPCR. BLASTN analysis of the amplicon sequence from the UFMG3 msp4 gene showed high identity (98%) with others A. marginale strains deposited in GenBank (AY665999.1, AY665999.1). After msp4 gene result, this strain was tested by PCR for the msp1a
gene, to molecular characterization through tandem analysis of the translated gene product (Cabezas-Cruz et al., 2013). After this analysis, the molecular sequence of UFMG3 was deposited in GenBank with the accession number KF005076.1. Three MSP1a repeat forms (13 and MGI19) were identified, in which the form MGI19 appeared twice (Table 1). Analysis of these repeats allowed the identification of the sequences QASTSS and SSASGQQQESS and of aspartic acid (D) at position 20 in the three tandem repeats. Phylogenetic analysis revealed the formation of a cluster with a high percentage of internal branches among UFMG3, MGI19 and UFMG2 isolates and another between the UFMG1 and Parana2 strains. Other sequences analyzed showed lower branch values (Fig. 1). Evaluation of virulence from strain A. marginale UFMG3 Incubation period, patency period and convalescence period All of the six calves inoculated with the strain A. marginale UFMG3 presented patent rickettsemia, with an average of 20 days IP, nine days PP and 31 days CP. No calves required treatment, and all spontaneously recovered. Calves of control group remained negative for A. marginale during the experiment. Rickettsemia and PCV The kinetics of rickettsemia and PCV values in the calves inoculated with the strain A. marginale UFMG3 are shown in Fig. 2. After IP, the animals showed ascendant rickettsemia until the 6th day, when they reached a peak of 3.78%. From the 8th day, the values showed gradual reduction. At the 13th dpi, another rise in rickettsemia levels was observed, reaching a peak of 5.1% at the 19th dpi. Subsequently, rickettsemia levels showed a steady reduction until the end of the experiment, when they reached 0.18%. The PCV levels showed a gradual decrease after the start of rickettsemia, reaching the lowest value at the 7th dpi (13.5%). No statistical difference was observed among days analyzed. Clinical parameters Increased temperature was observed during peak rickettsemia, reaching 40.2 ◦ C and 40.1 ◦ C at the 7th and 18th dpi, respectively. The heart rate increased significantly (P < 0.05) at the 7th and 8th dpi, and although the respiratory rate did not show a significant difference, a higher value (50 strokes per minute – spm) was observed on the 7th day after the start of rickettsemia. In the same period, ruminal movements showed a significant reduction (P < 0.05) (9 mov/5 min). The clinical score of calves inoculated with the UFMG3 strain was not significantly different, being considered normal throughout the experimental period. Discussion The understanding of A. marginale epidemiology, including the molecular characterization and virulence studies of different
Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001
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geographic strains, provides important knowledge for the development and implementation of appropriate measures to prevent and control bovine anaplasmosis (Pohl et al., 2013). The use of live samples of A. marginale in the vaccination protocol requires lowvirulence standardized samples (Ribeiro et al., 1997; Coelho, 2007; Bastos et al., 2010), making studies of genetic characterization and virulence essential tools for greater understanding and application of these samples. In this work, we performed molecular and phylogenetic characterization and evaluated the virulence of a new strain from A. marginale (UFMG3), obtained from a calf with vertical transmission. The farm from which the newborn calves were obtained is a dairy herd with recognized enzootic stability of A. marginale and with no occurrence of reported cases of anaplasmosis in adult animals (data not shown). In this condition, 10% of calves born from chronically infected cows were positive for A. marginale at birth by nPCR (msp4). These calves were followed for 70 days, and none showed clinical signs of anaplasmosis. Blood smears were negative, even though their nPCR results remained positive. Similar findings were also described by Potgieter and Van Rensburg, (1987) and Grau et al. (2013), identifying incidence rates of 15.6% and 10.5%, respectively, for the vertical transmission of A. marginale, with no clinical signs of disease. According to Pohl et al. (2013), vertical transmission is an important phenomenon that contributes to the persistence of various strains of A. marginale within the same herd. After splenectomy, only one calf showed high rickettsemia and spontaneous recovery of clinical parameters without the need for treatment. As this isolate induced moderate symptoms in the splenectomized calf and the packed cell volume values spontaneously reached normal, we suggest that this UFMG3 strain presents low virulence. Upon genomic sequencing of the UFMG3 strain for the msp4 gene of A. marginale, we observed a high similarity to other A. marginale strains deposited in GenBank (AY665999.1, AY665999.1). Presence of three repeat forms in tandem (13 and MGI19) were detected in the analysis of fragment of the msp1a gene. These forms differ from tandem repeats found in newborn calves from Rio de Janeiro, Brazil, by Silva et al. (2015), suggesting that several repeat forms of A. marginale can be transmitted vertically. The MSP1a repeat forms analysis contributed to a better understanding of the genetic diversity of A. marginale within specific regions and provided information on the evolution and host-pathogenvector interactions of A. marginale (de la Fuente et al., 2007, 2010). The MGI19 form, identified twice, was originally obtained from a wild brocket deer (Mazama gouazoubira) from Minas Gerais, the home state of the UFMG3 strain (Silveira et al., 2012). Samples from deer are considered of low virulence and as possible candidates for immunogens for cattle (Kuttler, 1984). The identification of this
repetition in the UFMG3 strain may be an additional indicator of low virulence. The amino acid sequences QASTSS and SSASGQQQESS, present in the tandem repeats of the MSP1a protein, represent an epitope recognized by neutralizing monoclonal antibodies and an immunodominant B cell epitope, respectively. Neutralizing B cell epitopes are important for the development of natural or induced protective immune responses in cattle (reviewed by Cabezas-Cruz and de la Fuente, 2015). These sequences were identified in the UFMG3 strain and may represent important markers of its future use as an immunogen. Furthermore, the peptide sequence of MSP1a from the UFMG3 strain presented three tandem repeats with aspartic acid (D) at position 20. According to de la Fuente et al. (2003), this fact demonstrates the high binding capacity of this isolate to bind to tick cell extracts (TCEs). These data suggest the capacity of the UFMG3 strain to propagate in IDE8 tick cell culture and its likely infection and transmission by ticks (Cabezas-Cruz et al., 2013). Comparatively, the Brazilian UFMG1 and UFMG2 strains of A. marginale also feature the amino acid D at position 20 in tandem sequences. The UFMG1 strain, obtained from a naturally infected calf (Ribeiro et al., 1997) and considered of low virulence, has the amino acid D at position 20 in three out of four tandem repeats. The UFMG2 strain, which induces high morbidity and mortality of susceptible calves (Bastos et al., 2010), also encodes D in all repeats. The UFMG1, UFMG2 (Bastos et al., 2009; Lasmar et al., 2012) and UFMG3 strains (unpublished data) are all cultivated in IDE8 tick cells culture, demonstrating the relevance of the amino acid at position 20 to the binding capacity to TCE. The phylogenetic tree produced using the NJ method (Fig. 1) shows that the cluster formed by UFMG3, MGI19 and UFMG2 reinforces the phylogenetic relation between UFMG3 and MGI19, as demonstrated in the analysis of tandem repeats. The UFMG2 strain also encodes the repeat form 13 (Bastos et al., 2010), which further reinforces the relation of strains of this cluster. However, the UFMG2 strain is considered a sample of high virulence (Bastos et al., 2010), unlike the virulence demonstrated here for the UFMG3 strain. To assess the capacity of the UFMG3 strain to induce clinical manifestations of bovine anaplasmosis, six calves were inoculated with the UFMG3 strain and monitored for clinical parameters. The results of rickettsemia, packed cell volume, IP, PP and CP obtained in this study demonstrate that the UFMG3 strain, vertically transmitted, has comparable virulence to other A. marginale strains of low virulence (Benavides et al., 2000; Rodríguez et al., 2000; Coelho, 2007; Bastos et al., 2010), as it was able to induce rickettsemia peaks, followed by spontaneous recovery of clinical parameters without the need for treatment.
Fig. 2. Rickettsemia and PCV values from calves inoculated with the Anaplasma marginale strain UFMG3. The data plotted in the graph represent the median values obtained in each day analyzed. No statistical difference was observed among the days analyzed (p > 0.05).
Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001
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Conclusions The occurrence of vertical transmission in a herd with enzootic stability is an important factor in the epidemiology of bovine anaplasmosis. The UFMG3 strain, obtained from a newborn calf, demonstrates the presence of high genetic diversity of this rickettsia in endemic areas for the disease, such as Brazil. The results of experimental inoculation in calves presented here suggest that the A. marginale UFMG3 strain has low virulence, characterized by low levels of rickettsemia, a moderate decrease in packed cell volume, and spontaneous recovery of clinical parameters without the need for treatment. Therefore, the UFMG3 strain has potential for use as a live vaccine against A. marginale. Further studies are needed to verify the induced protection in cattle vaccinated with the UFMG3 strain and subjected to natural challenge under field conditions. Acknowledgments This study was funded by CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior) and FAPEMIG (Fundac¸ão de Amparo a Pesquisa de Minas Gerais). The authors wish to thank Dr. Élida Mara Leite Rabelo for assistance with laboratory work. We thank American Journal Experts reviewers for improving of English writing and for all contributions that allowed us to execute the present study. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Aubry, P., Geale, D.W., 2011. A review of bovine anaplasmosis. Transbound. Emerg. Dis. 58, 1–30. Bastos, C.V., Passos, L.F.M., Facury Filho, E.J., Rabelo, E.M., de la Fuente, J., Ribeiro, M.F.B., 2010. Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves. Vet. J. 186, 374–378. Bastos, C.V., Passos, L.F.M., Vasconcelos, M.M.C., Ribeiro, M.F.B., 2009. In vitro establishment and propagation of a Brazilian strain of Anaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells. Braz. J. Microb. 9, 399–403. Benavides, E., Vizcaino, O., Britto, C.M., 2000. Attenuated trivalent vaccine against babesiosis and anaplasmosis in Colombia. Ann. N.Y. Acad. Sci. 916, 613–616. Benesi, F.J., Howard, D.L., Sá, C.S.C., Junior, E.H.B., 1999. Relato de um caso de transmissão transplacentária de anaplasmose bovina. Observac¸ões clínico-laboratoriais. R. Bras. Ci. Vet. 6, 175–176. Bird, J.E., 1973. Neonatal anaplasmosis in a calf. J. S. Afr. Vet. Assoc. 44, 69–70. Cabezas-Cruz, A., de la Fuente, J., 2015. Anaplasma marginale major surface protein 1a: a marker of strain diversity with implications for control of bovine anaplasmosis. Ticks Tick-borne Dis. 6 (3), 205–210. Cabezas-Cruz, A., Passos, L.M.F., Lis, K., Kenneil, R., Valdés, J.J., Ferrolho, J., Tonk, M., ˜ Pohl, A.E., Grubhoffer, L., Zweygarth, E., Shkap, V., Ribeiro, M.F.B., Estrada-Pena, A., Kocan, K.M., de la Fuente, J., 2013. Functional and immunological relevance of Anaplasma marginale major surface protein 1a sequence and structural analysis. PLoS ONE 8 (6), e65243. Coelho, L.C.T., (Dissertation) 2007. Anaplasmose bovina: parâmetros clínicos e de patologia clínica em bezerros infectados experimentalmente. Federal University of Minas Gerais, pp. 65. de la Fuente, J., Garcia-Garcia, J.C., Blouin, E.F., Kocan, K.M., 2003. Characterization of the functional domain of major surface protein 1a involved in adhesion of the rickettsia Anaplasma marginale to host cells. Vet. Microbiol. 91, 265–283. de la Fuente, J., Kocan, K.M., Blouin, E.F., Zivkovic, Z., Naranjo, V., Almazán, C., Esteves, E., Jongejan, F., Daffre, S., Mangold, A.J., 2010. Functional genomics and evolution of tick-Anaplasma interactions and vaccine development. Vet. Parasitol. 167, 175–186. de la Fuente, J., Ruybal, P., Mtshali, M.S., Naranjo, V., Shuqing, L., Mangold, A.J., Rodríguez, S.D., Jiménez, R., Vicente, J., Moretta, R., Torina, A., Almazán, C., Mbati, P.M., de Echaide, S.T., Farber, M., Rosario-Cruz, R., Gortazar, C., Kocan, K.M., 2007. Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences. Vet. Microbiol. 119, 382–390. de la Fuente, J., Torina, A., Naranjo, V., Caracappa, S., Vicente, J., Mangold, A.J., Vicari, D., Alongi, A., Scimeca, S., Kocan, K.M., 2005. Genetic diversity of
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Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence Bruna T. Silvestre a , Júlia A.G. Silveira a , Rodrigo M. Meneses b , Elias J. Facury-Filho b , Antônio U. Carvalho b , Múcio F.B. Ribeiro a,∗ a b
Department of Parasitology, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Department of Clinical and Veterinary Surgery, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 17 June 2015 Received in revised form 7 August 2015 Accepted 2 September 2015 Available online xxx Keywords: Anaplasma marginale UFMG3 Vertical transmission Genetic characterization Low virulence
a b s t r a c t Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia species Anaplasma marginale and results in great economic losses in tropical and subtropical regions. Vertical transmission is an important phenomenon that contributes to the persistence of different strains of the agent within the same herd. The identification of new strains and genetic characterization studies are essential to understanding their epidemiology and virulence and for vaccine development. The aim of this study was to perform molecular and phylogenetic characterizations of a new vertically transmitted strain from A. marginale and to evaluate its virulence by experimental inoculation of rickettsia-free calves. Thirty newborn Holstein calves were subjected to molecular tests for the detection of A. marginale, Babesia bovis and Babesia bigemina. Calves positive for A. marginale (n = 3) were splenectomized and monitored for the clinical manifestations of anaplasmosis. Blood samples from one of the calves that presented rickettsemia of 42.8% and spontaneous recovery of clinical parameters were used for molecular and phylogenetic characterization (msp1a gene), and inoculum production was used for the evaluation of virulence. This strain was identified as UFMG3. Three tandem repeat forms (13 and MGI19) were identified from the analysis of the msp1a gene, in which the form MGI19 appeared twice. Analysis of these repeats revealed the presence of the sequences QASTSS and SSASGQQQESS and of aspartic acid (D) at position 20 of both repeats. Phylogenetic analysis showed a close relationship among the UFMG3, MGI19 and UFMG2 strains. For virulence evaluation, six Holstein calves were inoculated intravenously with 2 × 107 A. marginale UFMG3-infected erythrocytes. The calves showed maximum rickettsemia of 5.1%, a moderate decrease in packed cell volume and spontaneous recovery of clinical parameters without the need for treatment. The results of experimental inoculation suggest that the strain A. marginale UFMG3 has low virulence and potential application for use as a live vaccine against A. marginale. © 2015 Elsevier GmbH. All rights reserved.
Introduction Bovine anaplasmosis is a major constraint to cattle production in several countries, generating significant economic losses in livestock worldwide (Kocan et al., 2010; Aubry and Geale, 2011). The disease is caused by the rickettsia Anaplasma marginale (Theiler, 1910) and is characterized by anemia, jaundice, fever, dehydration, weight loss, abortion and death (Kocan et al., 2003; Marana et al., 2009).
∗ Corresponding author. E-mail address: [email protected] (M.F.B. Ribeiro).
In South America, A. marginale is biologically transmitted by the tick Rhipicephalus microplus and mechanically by biting flies and contaminated fomites (Guglielmone, 1995; Kocan et al., 2003). The vertical transmission of A. marginale has been reported causing persistent infections in calves without clinical signs of disease, suggesting the low virulence of these isolates (Swift and Paumer, 1976; Passos and Lima, 1984; Salabarria and Pino, 1988; Ribeiro et al., 1995; Benesi et al., 1999; Gonc¸alves et al., 2005; Grau et al., 2013). However, clinical anaplasmosis in newborn calves has been reported, suggesting differences in the virulence of these isolates (Bird, 1973; Paine and Miller, 1977; Norton et al., 1983; Passos and Lima, 1984; Benesi et al., 1999; Gonc¸alves et al., 2005; Pypers et al., 2011; Pohl et al., 2013).
http://dx.doi.org/10.1016/j.ttbdis.2015.09.001 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001
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The identification of different geographic strains of A. marginale has been performed with regard to virulence, genetic characteristics and transmissibility by ticks. The genetic diversity of these strains has been identified by differences in molecular weight and in the constitution of the MSP1a protein, based on the varying number of tandem repeats located in the N-terminal region of the protein (de la Fuente et al., 2005, 2007). The number and the constitution of tandem repeats varies among geographic strains of A. marginale, and this region has been recognized as a stable genetic marker (de la Fuente et al., 2007). Understanding the genetic diversity and characteristics of A. marginale strains is of fundamental importance for the study of epidemiology, virulence, and the development of effective vaccines (Lis et al., 2015), which require a standardized inoculum containing samples of low virulence (Ribeiro et al., 1997; Coelho, 2007; Bastos et al., 2010). However, there are few samples of low-virulence isolates, and none from vertical transmission. Considering that the characterization and use of live samples of low-virulence isolates is a promising strategy for preventing and reducing clinical manifestations, the aim of this study was to perform molecular and phylogenetic characterizations of a new vertically transmitted strain from A. marginale and to evaluate its virulence by experimental inoculation of rickettsia-free calves.
Fig. 1. Phylogenetic tree based of msp1a gene of Anaplasma marginale Brazilian isolates. The tree was constructed using the Neighbor Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method. The GenBank accession numbers of the respective sequences used for the phylogenetic analysis are shown. UFMG3* is the sequence from the present study.
Materials and methods Obtaining an A. marginale congenital sample Thirty newborn male Holstein calves from a farm in InhaúmaMG (19◦ 29 27 South, 44◦ 23 24 West) were subjected to molecular tests of nested PCR (nPCR) for the detection of A. marginale (gene msp4), Babesia bovis and Babesia bigemina, according to Silveira et al. (2013). A. marginale DNA was detected in three calves, which were kept in a tie stall barn and were sprayed weekly with cypermethrin (Flytick® – Vallée, Montes Claros, MG, Brazil) to keep them free of ticks and biting flies. Throughout the experimental period, all animals were fed with powdered milk, commercial feed and water ad libitum. All procedures were approved by the Ethics Committee on Animal Use – CEUA/UFMG (Protocol number 41/2006). The animals were monitored daily through clinical examination and measurement of rectal temperature and weekly through blood smears and determination of the packed cell volume (PCV). Up to 70 days of age, the animals showed negative results for A. marginale in the clinical evaluations performed, though they remained positive by nPCR. The three calves were splenectomized and monitored again. One of the calves presented maximum rickettsemia of 42.8%, with spontaneous recovery of clinical parameters (PCV ≥ 24%). Blood samples from this calf were then used for molecular and phylogenetic characterization and for inoculum production for virulence evaluation. Inoculum production At peak rickettsemia, blood samples were collected from the jugular vein into EDTA tubes and were cryopreserved in liquid nitrogen using dimethyl sulfoxide (DMSO). The vertically transmitted A. marginale strain was identified as UFMG3. Molecular analysis and characterization of the UFMG3 strain Blood samples with EDTA were collected from the jugular vein on each calf’s day of birth and after their splenectomies for DNA extraction (kit Wizard Genomic DNA Purification, Promega). Detection of A. marginale DNA was performed via nPCR using primers complementary to the region of the msp4 gene (Silveira et al., 2013).
To assess the genetic diversity of isolated UFMG3 by sequences of tandem repeats, primers for the msp1a gene of A. marginale were used according to Bastos et al. (2009). The analyses of tandem repeats were performed according to the nomenclature proposed by Cabezas-Cruz et al. (2013). Positives products from the PCR reactions were purified using a QIAquick PCR Purification Kit (Qiagen Biotecnolo-gia Brasil, São Paulo, Brazil) according to the recommendations of the manufacturer. The purified amplicons were sequencing using an Applied Biosystems model ABI3130 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) and the Applied Biosystems BigDye® Direct Cycle Sequencing Kit (v. 3.1), with the POP-7TM polymer as the separating matrix and the primers employed in the PCR reaction. Sequences were aligned, edited, and analyzed using MEGA 6.0 (Tamura et al., 2013). The identity of each sequence was confirmed by comparison with sequences available on GenBank using BLAST software (Altschul et al., 1990). Phylogenetic and molecular evolutionary analyses were conducted using MEGA 6.0 and the program “Electropherogram quality analysis” (http://asparagin. cenargen.embrapa.br/phph/). The phylogenetic tree was constructed by analyzing msp1a gene fragments from Brazilian A. marginale strains deposited in GenBank (Fig. 1). Nucleotide sequences were aligned with MUSCLE from de MEGA 6.0 package (Tamura et al., 2013). Each alignment was analyzed using the Neighbor Joining (NJ) method and the evolutionary distances were computed using the Maximum Composite Likelihood method in MEGA 6.0 software. Internal branch confidence was assessed by the bootstrapping method using 1000 bootstrap replicates.
Evaluation of the virulence Experimental design Six male Holstein calves, 90–100 days old and A. marginaleand B. bovis/B. bigemina-free, were inoculated intravenously with 2 × 107 cryopreserved A. marginale UFMG3-infected erythrocytes. As a control group, two calves were inoculated intravenously with saline to ensure the absence of transmission in the barn during the experimental phase.
Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001
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Table 1 MSP1a tandem repeat sequences in the A. marginale UFMG3 strain. Anaplasma marginale genotype
Encoded sequence DDSSSASGQQQESSVSSQSE-ASTSSQLG–
Repeat form A*
UFMG 3
T**************L***DQ********** AE*************L***DQ********** AE*************L***DQ**********
13* MGI19** MGI19**
A. marginale strain
Country of origin
GenBank accession No.
Structure of MSP1a tandem repeats
UFMG 3
Brazil
KF005076.1
13
* **
MGI19
No. of repeats MGI19
3
Sequences reported by Cabezas-Cruz et al. (2013). Sequences reported by Silveira et al. (2012).
Clinical and hematological parameters Throughout the experimental period, all animals (n = 8) were monitored daily by clinical examination through measurements of rectal temperature, heart and respiratory rate, ruminal movements and clinical score. Blood smears were performed for the determination of rickettsemia and were stained with stain type Romanowsky (IICA, 1987). The PCV was measured using the microhematocrit from blood samples collected in EDTA tubes from the jugular vein. The PCV and blood smears were initially performed every 48 h to detect rickettsemia. At 20 days post inoculation (dpi), PCV and rickettsemia were performed daily until the end of the experiment, which occurred 61 dpi, when the median values of PCV reached 24%, considered lower limit of normality (Radostits et al., 2007). The incubation period (IP) was defined as the interval between inoculation and the beginning of rickettsemia. The patent period (PP) was measured from the day when A. marginale was first detected until the day of less PCV, and the convalescent period (CP) the interval time between the days of less PCV until recovery (24%). The times of the evaluations were defined from the first day of observation of erythrocytes infected with A. marginale in the blood smears, designated day 0 (zero). The previous and subsequent data were referenced from this day. Statistical analysis The Lilliefors test was used to determine the normality of the data. The variables did not follow a normal distribution and were subjected to the Friedman test to observe the existence of differences between the times (Sampaio, 2010). Statistical analysis was performed using the software InfoStat® version 2008 and median values obtained among the times were plotted in scatter plots. Results Detection of calves with congenital transmission of A. marginale (UFMG3) Three of thirty newborn calves (10%) subjected to nPCR for gene msp4 of A. marginale were diagnosed positive. After splenectomy, a calf exhibited rickettsemia due to A. marginale, peaking at 42.8%. The calf recovered clinical parameters spontaneously, without the need for treatment. Molecular analysis and characterization of the UFMG3 strain The molecular identity of the UFMG3 strain was performed by sequencing of the msp4 and msp1a gene fragments of A. marginale, obtained via nPCR. BLASTN analysis of the amplicon sequence from the UFMG3 msp4 gene showed high identity (98%) with others A. marginale strains deposited in GenBank (AY665999.1, AY665999.1). After msp4 gene result, this strain was tested by PCR for the msp1a
gene, to molecular characterization through tandem analysis of the translated gene product (Cabezas-Cruz et al., 2013). After this analysis, the molecular sequence of UFMG3 was deposited in GenBank with the accession number KF005076.1. Three MSP1a repeat forms (13 and MGI19) were identified, in which the form MGI19 appeared twice (Table 1). Analysis of these repeats allowed the identification of the sequences QASTSS and SSASGQQQESS and of aspartic acid (D) at position 20 in the three tandem repeats. Phylogenetic analysis revealed the formation of a cluster with a high percentage of internal branches among UFMG3, MGI19 and UFMG2 isolates and another between the UFMG1 and Parana2 strains. Other sequences analyzed showed lower branch values (Fig. 1). Evaluation of virulence from strain A. marginale UFMG3 Incubation period, patency period and convalescence period All of the six calves inoculated with the strain A. marginale UFMG3 presented patent rickettsemia, with an average of 20 days IP, nine days PP and 31 days CP. No calves required treatment, and all spontaneously recovered. Calves of control group remained negative for A. marginale during the experiment. Rickettsemia and PCV The kinetics of rickettsemia and PCV values in the calves inoculated with the strain A. marginale UFMG3 are shown in Fig. 2. After IP, the animals showed ascendant rickettsemia until the 6th day, when they reached a peak of 3.78%. From the 8th day, the values showed gradual reduction. At the 13th dpi, another rise in rickettsemia levels was observed, reaching a peak of 5.1% at the 19th dpi. Subsequently, rickettsemia levels showed a steady reduction until the end of the experiment, when they reached 0.18%. The PCV levels showed a gradual decrease after the start of rickettsemia, reaching the lowest value at the 7th dpi (13.5%). No statistical difference was observed among days analyzed. Clinical parameters Increased temperature was observed during peak rickettsemia, reaching 40.2 ◦ C and 40.1 ◦ C at the 7th and 18th dpi, respectively. The heart rate increased significantly (P < 0.05) at the 7th and 8th dpi, and although the respiratory rate did not show a significant difference, a higher value (50 strokes per minute – spm) was observed on the 7th day after the start of rickettsemia. In the same period, ruminal movements showed a significant reduction (P < 0.05) (9 mov/5 min). The clinical score of calves inoculated with the UFMG3 strain was not significantly different, being considered normal throughout the experimental period. Discussion The understanding of A. marginale epidemiology, including the molecular characterization and virulence studies of different
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geographic strains, provides important knowledge for the development and implementation of appropriate measures to prevent and control bovine anaplasmosis (Pohl et al., 2013). The use of live samples of A. marginale in the vaccination protocol requires lowvirulence standardized samples (Ribeiro et al., 1997; Coelho, 2007; Bastos et al., 2010), making studies of genetic characterization and virulence essential tools for greater understanding and application of these samples. In this work, we performed molecular and phylogenetic characterization and evaluated the virulence of a new strain from A. marginale (UFMG3), obtained from a calf with vertical transmission. The farm from which the newborn calves were obtained is a dairy herd with recognized enzootic stability of A. marginale and with no occurrence of reported cases of anaplasmosis in adult animals (data not shown). In this condition, 10% of calves born from chronically infected cows were positive for A. marginale at birth by nPCR (msp4). These calves were followed for 70 days, and none showed clinical signs of anaplasmosis. Blood smears were negative, even though their nPCR results remained positive. Similar findings were also described by Potgieter and Van Rensburg, (1987) and Grau et al. (2013), identifying incidence rates of 15.6% and 10.5%, respectively, for the vertical transmission of A. marginale, with no clinical signs of disease. According to Pohl et al. (2013), vertical transmission is an important phenomenon that contributes to the persistence of various strains of A. marginale within the same herd. After splenectomy, only one calf showed high rickettsemia and spontaneous recovery of clinical parameters without the need for treatment. As this isolate induced moderate symptoms in the splenectomized calf and the packed cell volume values spontaneously reached normal, we suggest that this UFMG3 strain presents low virulence. Upon genomic sequencing of the UFMG3 strain for the msp4 gene of A. marginale, we observed a high similarity to other A. marginale strains deposited in GenBank (AY665999.1, AY665999.1). Presence of three repeat forms in tandem (13 and MGI19) were detected in the analysis of fragment of the msp1a gene. These forms differ from tandem repeats found in newborn calves from Rio de Janeiro, Brazil, by Silva et al. (2015), suggesting that several repeat forms of A. marginale can be transmitted vertically. The MSP1a repeat forms analysis contributed to a better understanding of the genetic diversity of A. marginale within specific regions and provided information on the evolution and host-pathogenvector interactions of A. marginale (de la Fuente et al., 2007, 2010). The MGI19 form, identified twice, was originally obtained from a wild brocket deer (Mazama gouazoubira) from Minas Gerais, the home state of the UFMG3 strain (Silveira et al., 2012). Samples from deer are considered of low virulence and as possible candidates for immunogens for cattle (Kuttler, 1984). The identification of this
repetition in the UFMG3 strain may be an additional indicator of low virulence. The amino acid sequences QASTSS and SSASGQQQESS, present in the tandem repeats of the MSP1a protein, represent an epitope recognized by neutralizing monoclonal antibodies and an immunodominant B cell epitope, respectively. Neutralizing B cell epitopes are important for the development of natural or induced protective immune responses in cattle (reviewed by Cabezas-Cruz and de la Fuente, 2015). These sequences were identified in the UFMG3 strain and may represent important markers of its future use as an immunogen. Furthermore, the peptide sequence of MSP1a from the UFMG3 strain presented three tandem repeats with aspartic acid (D) at position 20. According to de la Fuente et al. (2003), this fact demonstrates the high binding capacity of this isolate to bind to tick cell extracts (TCEs). These data suggest the capacity of the UFMG3 strain to propagate in IDE8 tick cell culture and its likely infection and transmission by ticks (Cabezas-Cruz et al., 2013). Comparatively, the Brazilian UFMG1 and UFMG2 strains of A. marginale also feature the amino acid D at position 20 in tandem sequences. The UFMG1 strain, obtained from a naturally infected calf (Ribeiro et al., 1997) and considered of low virulence, has the amino acid D at position 20 in three out of four tandem repeats. The UFMG2 strain, which induces high morbidity and mortality of susceptible calves (Bastos et al., 2010), also encodes D in all repeats. The UFMG1, UFMG2 (Bastos et al., 2009; Lasmar et al., 2012) and UFMG3 strains (unpublished data) are all cultivated in IDE8 tick cells culture, demonstrating the relevance of the amino acid at position 20 to the binding capacity to TCE. The phylogenetic tree produced using the NJ method (Fig. 1) shows that the cluster formed by UFMG3, MGI19 and UFMG2 reinforces the phylogenetic relation between UFMG3 and MGI19, as demonstrated in the analysis of tandem repeats. The UFMG2 strain also encodes the repeat form 13 (Bastos et al., 2010), which further reinforces the relation of strains of this cluster. However, the UFMG2 strain is considered a sample of high virulence (Bastos et al., 2010), unlike the virulence demonstrated here for the UFMG3 strain. To assess the capacity of the UFMG3 strain to induce clinical manifestations of bovine anaplasmosis, six calves were inoculated with the UFMG3 strain and monitored for clinical parameters. The results of rickettsemia, packed cell volume, IP, PP and CP obtained in this study demonstrate that the UFMG3 strain, vertically transmitted, has comparable virulence to other A. marginale strains of low virulence (Benavides et al., 2000; Rodríguez et al., 2000; Coelho, 2007; Bastos et al., 2010), as it was able to induce rickettsemia peaks, followed by spontaneous recovery of clinical parameters without the need for treatment.
Fig. 2. Rickettsemia and PCV values from calves inoculated with the Anaplasma marginale strain UFMG3. The data plotted in the graph represent the median values obtained in each day analyzed. No statistical difference was observed among the days analyzed (p > 0.05).
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Conclusions The occurrence of vertical transmission in a herd with enzootic stability is an important factor in the epidemiology of bovine anaplasmosis. The UFMG3 strain, obtained from a newborn calf, demonstrates the presence of high genetic diversity of this rickettsia in endemic areas for the disease, such as Brazil. The results of experimental inoculation in calves presented here suggest that the A. marginale UFMG3 strain has low virulence, characterized by low levels of rickettsemia, a moderate decrease in packed cell volume, and spontaneous recovery of clinical parameters without the need for treatment. Therefore, the UFMG3 strain has potential for use as a live vaccine against A. marginale. Further studies are needed to verify the induced protection in cattle vaccinated with the UFMG3 strain and subjected to natural challenge under field conditions. Acknowledgments This study was funded by CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior) and FAPEMIG (Fundac¸ão de Amparo a Pesquisa de Minas Gerais). The authors wish to thank Dr. Élida Mara Leite Rabelo for assistance with laboratory work. We thank American Journal Experts reviewers for improving of English writing and for all contributions that allowed us to execute the present study. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Aubry, P., Geale, D.W., 2011. A review of bovine anaplasmosis. Transbound. Emerg. Dis. 58, 1–30. Bastos, C.V., Passos, L.F.M., Facury Filho, E.J., Rabelo, E.M., de la Fuente, J., Ribeiro, M.F.B., 2010. Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves. Vet. J. 186, 374–378. Bastos, C.V., Passos, L.F.M., Vasconcelos, M.M.C., Ribeiro, M.F.B., 2009. In vitro establishment and propagation of a Brazilian strain of Anaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells. Braz. J. Microb. 9, 399–403. Benavides, E., Vizcaino, O., Britto, C.M., 2000. Attenuated trivalent vaccine against babesiosis and anaplasmosis in Colombia. Ann. N.Y. Acad. Sci. 916, 613–616. Benesi, F.J., Howard, D.L., Sá, C.S.C., Junior, E.H.B., 1999. Relato de um caso de transmissão transplacentária de anaplasmose bovina. Observac¸ões clínico-laboratoriais. R. Bras. Ci. Vet. 6, 175–176. Bird, J.E., 1973. Neonatal anaplasmosis in a calf. J. S. Afr. Vet. Assoc. 44, 69–70. Cabezas-Cruz, A., de la Fuente, J., 2015. Anaplasma marginale major surface protein 1a: a marker of strain diversity with implications for control of bovine anaplasmosis. Ticks Tick-borne Dis. 6 (3), 205–210. Cabezas-Cruz, A., Passos, L.M.F., Lis, K., Kenneil, R., Valdés, J.J., Ferrolho, J., Tonk, M., ˜ Pohl, A.E., Grubhoffer, L., Zweygarth, E., Shkap, V., Ribeiro, M.F.B., Estrada-Pena, A., Kocan, K.M., de la Fuente, J., 2013. Functional and immunological relevance of Anaplasma marginale major surface protein 1a sequence and structural analysis. PLoS ONE 8 (6), e65243. Coelho, L.C.T., (Dissertation) 2007. Anaplasmose bovina: parâmetros clínicos e de patologia clínica em bezerros infectados experimentalmente. Federal University of Minas Gerais, pp. 65. de la Fuente, J., Garcia-Garcia, J.C., Blouin, E.F., Kocan, K.M., 2003. Characterization of the functional domain of major surface protein 1a involved in adhesion of the rickettsia Anaplasma marginale to host cells. Vet. Microbiol. 91, 265–283. de la Fuente, J., Kocan, K.M., Blouin, E.F., Zivkovic, Z., Naranjo, V., Almazán, C., Esteves, E., Jongejan, F., Daffre, S., Mangold, A.J., 2010. Functional genomics and evolution of tick-Anaplasma interactions and vaccine development. Vet. Parasitol. 167, 175–186. de la Fuente, J., Ruybal, P., Mtshali, M.S., Naranjo, V., Shuqing, L., Mangold, A.J., Rodríguez, S.D., Jiménez, R., Vicente, J., Moretta, R., Torina, A., Almazán, C., Mbati, P.M., de Echaide, S.T., Farber, M., Rosario-Cruz, R., Gortazar, C., Kocan, K.M., 2007. Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences. Vet. Microbiol. 119, 382–390. de la Fuente, J., Torina, A., Naranjo, V., Caracappa, S., Vicente, J., Mangold, A.J., Vicari, D., Alongi, A., Scimeca, S., Kocan, K.M., 2005. Genetic diversity of
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Please cite this article in press as: Silvestre, B.T., et al., Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.09.001