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Characterization of Anaplasma ovis strains using the major surface protein 1a repeat sequences

Anaplasma ovis is one of the tick-transmitted pathogens of small ruminants. It causes ovine anaplasmosis and widely distributed in the world. In contrast to extensive worldwide genetic diversity of A. marginale and A. phagocytophilum, there are few reports on the classification of A. ovis strains. This study was conducted to investigate the occurrence and characterize A. ovis strains from goats and sheep from 12 provinces in China. The occurrence of A. ovis DNA was tested in 552 goats and sheep, by PCR based on the msp4 gene. Positive samples were used for the amplification of the msp1a gene of A. ovis. The Msp1a amino acid repeats were further identified and used for the characterization of A. ovis strains. The results showed that 79 (14.3%) goats and sheep were positive for A. ovis. The infection rates of A. ovis among different study sites ranged from 0 to 100%, and were significantly higher in sheep (26.6%, 45/169) than in goats (8.9%, 34/383) (χ 2 = 21.403, df = 1, P < 0.001). The msp4 gene sequences of these isolates were 99.8–100% identical to each other, and they represented two sequence types. Forty-four partial msp1a gene sequences containing the repeat sequences were obtained from A. ovis-positive samples. After translation to amino acid sequences, 24 Msp1a repeats with 33 to 47 amino acids, which corresponded to 19 genotypes of A. ovis, were recognized in goats and sheep in China. Anaplasma ovis is widely distributed in the investigated geographical regions. The msp4 gene of A. ovis had high sequence identity and was unable to be used to discriminate different strains. The Msp1a could be used as a genetic marker for characterizing A. ovis, and 19 genotypes of A. ovis were recognized in domestic small ruminants in China. The present study revealed, for the first time, the genetic diversity of A. ovis based on the analyses of Msp1a amino acid repeats.


Background
Anaplasma are obligate intracellular Gram-negative rickettsial bacteria of medical and veterinary interest in both tropical and subtropical regions [1]. The disease caused by Anaplasma spp. has been recognized over a century, and is still an important issue worldwide [2,3]. Since disclosure of zoonotic potential of A. phagocytophilum in 1994, there has been great interest in these bacteria [1,4]. Until recently, six species have been recognized in the genus Anaplasma: Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Anaplasma centrale (A. marginale centrale), Anaplasma platys and Anaplasma ovis [5]. Anaplasma carpa has recently been described and considered as an emerging zoonotic pathogen in China [6]. The members in the genus Anaplasma differ in their cellular tropism, vectors, host range and pathogenicity [5].
Ovine anaplasmosis is caused by A. ovis, which is an obligate intra-erythrocytic pathogen of small ruminants [5,7]. The causative agent was first described in sheep in 1912, and is widely distributed in Asia, Africa, Europe and the USA [7,8]. This organism infects sheep, goats and some wild ruminants [9,10]. Recently, an A. ovis variant was detected in a patient in Cyprus, indicated the zoonotic potential of this agent [11]. The life-cycle of A. ovis involves vertebrates and ticks, and animals can develop persistent infections and serve as reservoir hosts [12].
Currently, the identification and characterization of A. ovis mainly relies on the analysis of 16S rRNA and msp4 genes; however, these genes are highly conserved among heterologous strains [3,13]. In previous reports, the major surface protein 1a (Msp1a), encoded by the msp1a gene, has been recognized as a stable molecular marker for classifying strains of A. marginale [14]. It has been revealed that A. marginale Msp1a could have evolved on the strength of immune selection pressure and differs among strains due to variable sequences and numbers of tandem amino acid repeats located in the N-terminal region of the protein [15]. The repeated region of A. marginale Msp1a contains the adhesion domain for tick cells and erythrocytes, which is essential for the invasion and transmission of the organism [15]. Previous reports have reported that immunization of cattle with Msp1a induces partial protection when challenged with A. marginale [15,16]. Recently, Msp1a has also been identified in A. centrale, although attempts on other Anaplasma species have been performed [17]. In this study, we investigated the occurrence of A. ovis in small domestic animals in China, and identified the msp1a gene from A. ovis-positive samples. The A. ovis isolates identified herein were subsequently characterized based on the Msp1a amino acid repeats.

Sample collection and DNA preparation
Blood samples were obtained from March to September between 2011 and 2015 in 24 counties from 12 provinces of China (Table 1). Five hundred and fifty-two asymptomatic small ruminants (sheep, n = 169; goats, n = 383) were randomly selected in two to three sampling sites from each county included in this study. Blood samples were collected from the jugular vein of individual animals and collected in a sterile 10 ml vacutainer EDTA tubes and stored at 4°C. DNA was prepared from 300 μl of blood by using the Gentra Puregene Blood Kit (Qiagen, Beijing, China) following the manufacturer's instructions.

PCR reactions
Specific DNA of A. ovis was detected by PCR based on msp4 gene with primer set MSP45 (5′-GGG AGC TCC TAT GAA TTA CAG AGA ATT GTT TAC-3′) and MSP43 (5′-CCG GAT CCT TAG CTG AAC AGA ATC  TTG C-3′) as described previously, which generated a product of 869 bp [13]. The DNA of A. ovis strain Haibei (GenBank accession no. GQ483471) and sterile water were used as the positive and negative control, respectively. Amplification products were analyzed by 1.0% agarose gel electrophoresis. The msp1a gene was further amplified from A. ovis-positive samples. Primers AoM-sp1aF (5′-CGT TTC CAT GTG CTA CAA TGC CG-3′) and AoMsp1aR (5′-GCT GTT CGC TAT CGC AGT CTG TG-3′) were designed based on the A. ovis strain Haibei genome sequence (GenBank accession no. CP007596, unreleased) to target repeat sequences within the msp1a gene. The PCR reaction system is consistent with the amplification of msp4 gene. Thermal cycling conditions include 94°C for 4 min, 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension step at 72°C for 5 min.

Sequence and statistical analysis
The amplified fragments of msp4 and msp1a genes were purified and cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). At least two recombinants were sequenced from each amplification (Genscript, Nanjing, China). The msp4 gene sequences have been deposited in GenBank (accession numbers KY807127 and KY807128) and were analyzed by the BLASTn search and the Clus-talW software (DNAStar, Madison, WI, USA). The msp1a gene sequences were edited and translated to amino acids by using CLC Genomics Workbench 7.5.1 (Qiagen, Aarhus, Denmark). The amino acid repeat sequences were identified and named Aon. These repeats were aligned using the ClustalV method in the MegAlign software. Statistical analysis was performed with a Chi-square test in Predictive for Analytics Software Statistics 18 (PASW, SPSS Inc., Chicago, IL, USA), and a difference was considered statistically significant at P < 0.05.
Molecular characterization of Anaplasma has relied mainly on analyses of various gene loci [3]. The target genes used to determine the genetic diversity of A. ovis include the 16S rRNA and msp4 genes, and several genotypes and genetic variants have been identified in previous reports [22,25,[28][29][30][31][32]. However, these molecular markers were found to be highly conserved and not informative enough to delineate A. ovis isolates [3,13,22]. In this study, we also found that the msp4 gene of A. ovis isolates identified from goats and sheep shared high sequence similarity (99.8-100%), and were unable to reveal the genetic characterization of these isolates.
The major surface proteins of the members in the genus Anaplasma have been well characterized, especially in A. marginale and A. phagocytophilum [33,34]. The Msp1a has been extensively used as a molecular marker for characterizing A. marginale strains on the basis of the variable Nterminal region, containing the repeated peptides [15]. To date, over 200 A. marginale Msp1a tandem repeats have been identified, and a great number of strains from different countries have been classified into a variety of genotypes [3,15,17]. In this study, we examined A. ovis-positive samples for Msp1a genotype, and 24 Msp1a repeats with 33-47 amino acids, which corresponded to 19 A. ovis genotypes identified in goats and sheep in China. The structure of Msp1a tandem repeat and the amino acid sequences vary among strains, which has also been shown for A. marginale.
It has been reported that the Msp1a of A. marginale interact with vertebrate host and tick cells and have evolved on the strength of immune pressure [15]. This study revealed high genetic diversity of A. ovis isolates in small domestic ruminants in China, suggesting that msp1a gene of A. ovis may also have evolved more obviously than other genes. The A. ovis strains identified in this study had two to three Msp1a repeats, some of which were shared between different strains. However, no significant association was observed between specific tandem repeats and host or geographical regions in this study, since some repeats were identified in both goats and sheep and distributed extensively (repeat Ao6, Ao8 and Ao10 identified in goats and sheep from several provinces). Moreover, same genotypes of A. ovis were found in several provinces (Ao1/Ao6, Ao2/Ao6, etc.); this may be attributed to the animal movement between those provinces.
To date, characterizing A. marginale strains based on MSP1a repeat sequences has been well studied. The present study, for the first time, revealed the genetic diversity of A. ovis using Msp1a repeats in goats and sheep in China. Due to the wide distribution of A. ovis, more studies should be conducted in vertebrate and invertebrate hosts from different countries, which will ultimately Fig. 1 The Msp1a amino acid repeat sequences of A. ovis geographical strains identified from goats and sheep. The ID of each repeat type was named as Ao1-24, which were aligned using the ClustalV method in the MegAlign software. The one letter code was used to reveal the different amino acid sequences of Msp1a repeats. The variable amino acids are highlighted on a black background and gaps indicate deletions/insertions provide more evolutionary and phylogenetic information about A. ovis strains.

Conclusions
Anaplasma ovis was molecularly detected in goats and sheep from 12 provinces in China, with an overall infection rate of 14.3%. The msp4 gene of A. ovis had high sequence identity and was unable to be used to discriminate different strains. The Msp1a could be used as a genetic marker for characterizing A. ovis, and 24 Msp1a repeats with 33-47 amino acids, which corresponded to 19 genotypes of A. ovis, were identified in goats and sheep in China. The present study provided the first evidence of genetic diversity of A. ovis based on the analyses of Msp1a repeats.

Additional file
Additional file 1: Table S1. Partial msp1a amino-acid sequences containing repeat sequences of A. ovis strains analyzed in this study. (DOCX 31 kb)

Abbreviations
Msp: Major surface protein; UV: ultraviolet