INTRODUCTION
Babesiosis is one of the most common tick-borne haemoparasitic diseases in wild and domesticated animals in tropical, subtropical and temperate regions worldwide. Babesiosis is caused by infection and subsequent intraerythrocytic multiplication of apicomplexan parasites of the genus Babesia. In China, Babesia cf. motasi is a widespread pathogen of small ruminants that is transmitted via the tick vectors Haemaphysalis qinghaiensis and H. longicornis (Guan et al. Reference Guan, Yin, Luo, Lu, Zhang, Gao and Lu2002, Reference Guan, Moreau, Liu, Hao, Ma, Luo, Chauvin and Yin2010a ; Wang et al. Reference Wang, Ma, Liu, Ren, Li, Liu, Li, Yin, Luo and Guan2013).
Vaccination is currently the best control strategy for preventing babesiosis, and the different live vaccines developed are able to efficiently control the disease. The first live B. bovis vaccine was produced in Australia using virulent B. bovis strains attenuated by multiple rapid passages and exhibited apparently reduced virulence (Callow et al. Reference Callow, Mellors and McGregor1979; Bock et al. Reference Bock, de Vos, Kingston, Shiels and Dalgliesh1992; Pipano, Reference Pipano1995). An Australian chilled tick fever vaccine and three attenuated live vaccines using B. bovis strains have been produced, offering protection against B. bovis and B. bigemina. However, occasional reversion of live attenuated parasites to the virulent state has been observed by Australian researchers (Gohil et al. Reference Gohil, Herrmann, Günther and Cooke2013).
The clinical symptoms of babesiosis appear when Babesia merozoites invade and replicate within host erythrocytes and reach a high parasitaemia level (Yokoyama et al. Reference Yokoyama, Okamura and Igarashi2006). Initially, the merozoites attach to the host RBC surface in any orientation. After re-orientation, the parasites form tight junctions between the RBC surface and the apical region, and, subsequently, the invasion is initiated, followed by the internalization of the parasite within infected RBCs. RBC invasion is mediated by proteins located both on the merozoite surface coat and in organelles. Proteins involved in attachment include variable merozoite surface antigens, and proteins involved in invasion, such as associated membrane antigen 1 (AMA-1), thrombospondin-related adhesive protein, rhoptry-associated protein-1 (RAP-1) and spherical body proteins, are secreted by micronemes, rhoptries and dense granules (Lobo et al. Reference Lobo, Rodríguez and Cursino-Santos2012). These proteins involved in RBC invasion could be targets for developing recombinant or subunit vaccines to protect against babesiosis (Gohil et al. Reference Gohil, Herrmann, Günther and Cooke2013).
After synthesis, the AMA-1 protein is stored in microneme organelles and is later transported to the parasite surface immediately prior to or during host-cell invasion (Healer et al. Reference Healer, Crawford, Ralph, McFadden and Cowman2002). AMA-1 has been characterized in several Babesia species, including B. bovis (Gaffar et al. Reference Gaffar, Yatsuda, Franssen and de Vries2004), B. bigemina (Torina et al. Reference Torina, Agnone, Sireci, Mosqueda, Blanda, Albanese, La Farina, Cerrone, Cusumano and Caracappa2010), B. divergens (Montero et al. Reference Montero, Rodríguez, Oksov and Lobo2009; Tonkin et al. Reference Tonkin, Crawford, Lebrun and Boulanger2013), B. orientalis (He et al. Reference He, Fan, Hu, Miao, Huang, Zhou, Hu and Zhao2015) and B. gibsoni (Zhou et al. Reference Zhou, Yang, Zhang, Nishikawa, Fujisaki and Xuan2006). AMA-1, which is essential for host cell invasion, is a structurally conserved type I integral membrane protein with the following three characteristic structures: (i) an N-terminal, cysteine-rich ectodomain, (ii) a single transmembrane domain and (iii) a C-terminal cytoplasmic tail (Gaffar et al. Reference Gaffar, Yatsuda, Franssen and de Vries2004).
In the present study, the complete sequence of AMA-1 of Babesia sp. BQ1 (Lintan) was characterized and compared with homologous sequences of other apicomplexan parasites. In addition, recombinant expression of a conserved central region of BLTAMA-1, including domains I and II, were expressed in vitro and the immunoreactivity of these proteins were tested.
MATERIALS AND METHODS
Parasites
Babesia sp. (BQ1) Lintan was initially isolated from a sheep infested with adult H. qinghaiensis ticks from Lintan, Gansu Province in China (Guan et al. Reference Guan, Yin, Luo, Lu, Zhang, Gao and Lu2002).
gDNA and total RNA extraction
Genomic DNA was extracted from 300 µL of Babesia sp. BQ1 (Lintan) infected blood samples (parasitaemia about 5%) using a QIAamp DNA Blood Mini Kit (Gentra, USA), according to the manufacturer's instructions. The DNA samples were stored at −20 °C until further use.
Total RNA was extracted by lysing Babesia sp. BQ1 (Lintan)-infected RBCs using a standard TRIzol reagent protocol (Life Technologies), followed by chloroform extraction, precipitation with isopropyl alcohol and ethanol and DNase I treatment (Amplification Grade; Life Technologies, Invitrogen, Carlsbad, CA, USA).
Analysis of the complete mRNA transcript (cDNA clone) encoding AMA-1
The amino acid sequences of AMA-1 from B. bovis, B. bigemina, B. divergens, B. orientalis and B. gibsoni (GenBank accession numbers: ACM44018, ADP02976, ACC96234, AHW45797 and ACY07255, respectively) were aligned. A set of degenerate primers (BLTF: 5′-ACM AAR TAY AGG TAY CCH and BLTR: GWA RTA VGT DCC ACA RCT-3′) corresponding to amino acid sequences TKYRYP and SCGTYY was used to amplify partial Babesia sp. (BQ1) Lintan ama-1 sequences.
Several forward and reverse gene-specific primers were designed based on the partial sequences obtained and synthesized to amplify the full-length cDNA using 5′ and 3′ RACE. The primers (BLTama-1fullF: 5′-ATG CAG TGC ATA GTG AGA AAG T-3′ and BLTama-1fullR: 5′-TCA ATT GAT CTT GGT TAG GTG G-3′) were designed from obtained partial 5′ and 3′ putative sequences to amplify the entire open reading frame (ORF) of the Babesia sp. BQ1 (Lintan) ama-1 gene. The gene fragment encoding the BLTAMA-1 extracellular region of BLTAMA-1-DI/DII corresponding to amino acids G116 to E428 was PCR amplified using specific primers (BLTrAMA1-F: 5′-GCC TCT AGA GGT GGT AAG CAT TAC CGC ATG-3′ and BLTrAMA1-R: 5′-GCC GGA TCC TTC CAG CGG GGA TCC AAG A-3′) modified to include Xba I and BamH I sites (underlined).
Amplification of an 807 bp fragment of the ama-1 gene was achieved using degenerate primers followed by cloning into the pGEM-T easy vector (Promega, USA) and sequencing. Orthologous sequences from B. ovata (GenBank accession number KT312795), B. bigemina (GenBank accession number HM54372), B. bovis (GenBank accession number FJ588027), B. orientalis (GenBank accession number KJ196379), B. divergens (GenBank accession number EU486539) and B. gibsoni (GenBank accession number FJ800574) were identified by BLAST analysis.
Rapid amplification of 5′- and 3′-RACE-Ready cDNA from total parasite RNA was obtained using SMARTer® RACE 5′/3′ Kit (Clontech Laboratories, USA), according to the manufacturer's instructions. The PCR products were purified and cloned into the pGEM-T easy vector (Promega, USA), followed by sequencing. The ORF was determined using ORF Finder (www.ncbi.nlm.nih.gov/gorf). The full-length gene was further amplified and identified with a pair of specific primers, using cDNA and gDNA as templates.
Bioinformatic analysis
The sequences obtained in the present study were identified using BLASTn and PSI-BLAST [non-redundant (NR) protein database] programs. A multiple sequence alignment was performed using Clustal W 2·0·12: Multiple alignment. The per cent identity value among Chinese isolates was calculated after Clustal W alignment using DNAStar (Version 4.01, Madison, WI, USA) The phylogenetic analysis was conducted with MEGA 7·0·18 software (Kumar et al. Reference Kumar, Stecher and Tamura2016). The presence of potential transmembrane helices and signal peptides in the BLTAMA1 protein were predicted using the TMHMM Server v. 2·0 and the SignalIP 4·1 server (http://www.expasy.org/resources).
Recombinant expression, purification of BLTrAMA-1-DI/DII and production of anti- BLTrAMA-1-DI/DII polyclonal antibodies
The extracellular region of BLT-AMA1 containing DI/II, was amplified and cloned into the pUC57 vector. Plasmid with the verified sequence was selected and digested using Xba I and BamH I restriction enzymes and cloned into the pET-30a expression vector (Genscript) according to the manufacturer's instructions. The recombinant plasmid pET-30a-AMA1-DI/DII, containing 939 bp fragment, was confirmed by sequencing.
Recombinant protein pET-30a-AMA-1-DI/DII was expressed in BL21 (DE3) cells in the presence of kanamycin (50 µg mL−1) by the addition of 1 mm isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h or 15 °C overnight. The bacterial culture was evaluated by SDS–PAGE and Western-blot analyses. One litre of bacterial culture was further induced with 0·5 mm IPTG using stored strain at 15 °C overnight. The bacterial cultures were harvested by centrifugation (8000 rpm, 10 min) and lysed by ultrasonication in binding buffer (50 mm Tris–HCl, 150 mm NaCl, 8 m urea; pH 8·0) containing PMSF (phenylmethylsulfonyl fluoride). The recombinant protein was purified as inclusion bodies; the pellet was re-suspended in solubilization buffer [20 mm Tris–HCl (pH 8·0), 50 mm NaCl and 1 mm DTT (dithiothreitol), 8 m urea] containing increasing concentrations of imidazole (20, 50 and 500 mm). The samples were centrifuged and collected. Recombinant protein expression was subsequently analysed using SDS–PAGE and Western-blot analyses. The purity of the recombinant protein was more than 85% according to SDS–PAGE gel scanning analysis.
Three New Zealand rabbits (2·3 kg each) were subcutaneously injected with 500 µg purified BLTrAMA-1/DI/DII protein with Freund's complete adjuvant (FCA, Saint Louis, Missouri, USA, Sigma). Booster injections containing the same amount of protein in Freund's incomplete adjuvant (FIA, Saint Louis, Missouri, USA, Sigma) were administered on days 15, 20 and 28. Sera were collected from the immunized rabbits at 15 days after the last injection, purified (Protein A-affinity Purified, Genscript, China) and stored at −20 °C until further use. For the negative control, sera were collected from each rabbit prior to the first injections.
Immunoblotting analysis
Two micrograms per lane of BLTrAMA-1-DI/DII or 5 µg per lane of native soluble antigens from Babesia sp. BQ1 (Lintan) merozoites (BQMA, Guan et al. Reference Guan, Chauvin, Rogniaux, Luo, Yin and Moreau2010b ) were separated by SDS–PAGE (12%) gel and subsequently transferred to nitrocellulose membranes (BioRad) at 24 V and 50 W for 35 min. The membranes were then cut into 3-mm strips and blocked in 5% (w/v) skimmed milk in Tris-buffered saline (pH 7·6) with 0·1% Tween-20 (TBST) overnight at 4 °C on a shaker. The strips were probed with serum (diluted at 1:100 in TBST), obtained as described by Guan et al. (Reference Guan, Chauvin, Rogniaux, Luo, Yin and Moreau2010b ), from sheep (No. 3216, 3 weeks post-infection) experimentally infected with Babesia sp. BQ1 (Lintan) (Guan et al. Reference Guan, Chauvin, Rogniaux, Luo, Yin and Moreau2010b ), with serum from a rabbit immunized with the BLTrAMA-1 protein, or serum from a Babesia-free sheep or rabbit prior to immunization (negative control) for 1 h. After washing three times with TBST, the strips were incubated with secondary antibodies (monoclonal anti-goat/sheep IgG-alkaline phosphatase conjugate, Sigma, A8062, dilution: 1:5000 or polyclonal anti-rabbit IgG–alkaline phosphatase conjugate, Sigma, A9919, dilution: 1:5000) for 2 h. Positive blots were developed using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid substrate system (B1911-100ML, Sigma).
RESULTS
Cloning and sequencing of the ama-1 gene of Babesia sp. BQ1 (Lintan)
The full-length ama-1 gene from Babesia sp. BQ1 (Lintan) cDNA was amplified using 5′ and 3′ RACE. The sequence obtained is 2066 bp and contains a predicted ORF of 1785 bp, a 208 bp 5′ UTR and a 72 bp 3′ UTR following a 30 bp poly (A) tail. The full-length ama-1 gene was also amplified from Babesia sp. BQ1 (Lintan) gDNA. Comparison of the sequence with the cDNA indicated no introns in the ama-1 gene. The sequence has been deposited in GenBank under accession number KX570629.
AMA-1 amino acid sequences from different parasite species were compared, as shown in Table 1. The results indicated that the AMA-1 protein of Babesia sp. BQ1 (Lintan) has significant identity with AMA-1 of other Babesia parasites, i.e. a maximum per cent identity of 84·4% with B. ovata and 77·9% identity with B. bigemina, 54·1% with B. divergens, 53·5% with B. orientalis, 52·1% with B. bovis and 50·3% with B. gibsoni. In addition, Babesia sp. BQ1 (Lintan) AMA-1 shows 21·3–42·1% identity with AMA-1 proteins of Theileria species and other apicomplexan parasites.
Table 1. Per cent identity of AMA-1 amino acid sequences between Babesia sp. BQ1 (Lintan) and other 16 apicomplexan parasites deduced after CLUSTAL W alignment
Characterization and sequence comparison of the AMA-1 protein
Sequence analysis using the SignalP 4·1 program predicts a 23-aa signal sequence (MQCIVRKLSLLAMPVVIAGMLSAE) in the BLTAMA-1 protein. The program TMHMM version 1.0 predicts an extracellular region (ectodomain) from M1 to K511, a single hydrophobic C-terminal transmembrane helix from A512 to N531, and a small cytoplasmic tail from R532 to N594 (Fig. 1).
Fig. 1. Multiple alignment of AMA-1 amino acid sequences from Babesia sp. BQ1 (Lintan) (BLT), B. divergence (Bdi, GenBank accession number: ACC96234), B. orientalis (Bor, GenBank accession number: AHW45797), B. bigemina (Bbi, GenBank accession number: ADP02976), B. bovis (Bbo, GenBank accession number: ACM44018), B. gibsoni (Bgi, GenBank accession number: ACY07255), B. microti (Bmi, GenBank accession number: AGJ72906) Plasmodium vivax (Pvi, GenBank accession number: AAC16731), P. falciparum (Pfa, GenBank accession number: XP001348015), P. berghei (Pbe, GenBank accession number: AAC47192), P. knowlesi (Pkn, GenBank accession number: XP002259339), Eimeria maxima (Ema, GenBank accession number: CBL80633), Toxoplasma gondii (Tgo, GenBank accession number:AAB65410) and Neospora caninum (Nca, GenBank accession number: XP_003886059). The signal peptide cleavage site is indicated by a black arrow. Domains I (red), II (blue) and III (green) by the formation of disulphide bridges between conserved cysteine residues (indicated by solid circular) were marked. The transmembrane helices are marked by orange dotted line and a cytoplasmic tail is indicated by purple dotted line. The DI/DII region for recombinant protein expression was marked by pink arrows.
Clustal W alignment of multiple AMA-1 sequences, including Babesia sp. BQ1 (Lintan) and other Babesia species (B. bigemina, B. bovis, B. divergens, B. orientalis, B. gibsoni and B. microti), some plasmodia (Plasmodium species: P. falciparum, P. vivax, P. berghei and P. knowlesi), and some coccidia (Toxoplasma gondii, Eimeria maxima and Neospora caninum) revealed the characteristic features of AMA-1: three conserved extracellular domains (DI–DIII), containing 14 conserved cysteines, including six in DI, four in DII and four in DIII (Fig. 1).
Phylogenetic analysis
A phylogenetic tree, based on babesial AMA-1 sequences and homologues from other related apicomplexans deposited in GenBank, including 10 species of piroplasmida, four species of Plasmodia and three species of Conoidasida, was constructed using the Neighbour-Joining method (Fig. 2). The results showed that these apicomplexan parasites could be divided into two major clades, with high bootstrap values. One branch corresponds to two vertebrate blood-infecting clades (Piroplasmida and Plasmodia), and the other corresponds to Conoidasida species. Within Piroplasmida, Babesia sp. BQ1 (Lintan), with B. bigemina and B. ovata infecting for ruminants, form one clade; B. bovis and B. orientalis, which infect Bovidae animals, form another clade, with a highly significant bootstrap value. In contrast, B. divergens and B. gibsoni, which infect cattle and dogs, respectively, form another clade, with a lower bootstrap value that is over 50%. Babesia microti was found outside other Babesia species in a single cluster of Piroplasmida, with a high bootstrap value. All Piroplasmida sequences clustered, with a highly significant bootstrap value, into a clade different from Plasmodia and Conoidasida sequences, containing all analysed Babesia and Theileria species. These results indicate the validity of tree construction based on the sequence of AMA-1.
Fig. 2. Phylogenetic tree of the AMA-1 amino acid sequences of Babesia sp. BQ1 (Lintan) and other apicomplexan parasites. The accession numbers were showed after parasite species name and the species of Plasmodia and Conoidasida AMA-1 amino acid sequences were used as outgroups. The BLTAMA-1 sequence obtained in this study was indicated with bold triangle. The analysis involved 18 amino acid sequences. The tree was inferred using the Neighbour-Joining method of MEGA7·0·18; bootstrap values are shown at each branch point. Numbers above the branch demonstrate bootstrap support from 1000 replications. All sites of the alignment containing insertions–deletions, missing data were eliminated from the analysis (option ‘complete deletion’). The optimal tree with the sum of branch length = 3·64528684. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. There were a total of 274 positions in the final dataset.
Cloning, expression and purification of the DI/DII region of BLTrAMA-1
The gene encoding the extracellular region of BLTAMA-1 was successfully cloned into the pET-30a expression vector and expressed in Eschrichia coli BL21 (DE3). The recombinant plasmid pET-30a-BLTAMA-1-DI/DII was identified by restriction analysis and subsequently confirmed by sequencing using specific primers. The expressed recombinant proteins were analysed by SDS–PAGE (Fig. 3A) and Western-blot (Fig. 3B) using anti-His6 serum (THETM His Tag Antibody, mAb, Mouse, GenScript, A00186). The predicted mass of the recombinant protein (His-BLTAMA-1-DI/DII) is ~35 kDa (Fig. 3).
Fig. 3. Expression and purification of recombinant BLT-AMA-1-DI/DII protein. A: SDS–PAGE (Coomassie stained) analysis of recombinant protein produced in E. coli. M: molecular weight marker (Genscript, M00516). Lane 1, recombinant protein before induction; lane 2: supernatant of cell lysate of recombinant protein induced by 1 mm IPTG for 16 h at 15 °C; lane 3: cell lysate pellet of recombinant protein induced by 1 mm IPTG for 16 h at 15 °C after ultrasonication; lane 4: supernatant of cell lysate of recombinant protein induced by 1 mm IPTG for 4 h at 37 °C; lane 5: cell lysate pellet of recombinant protein induced by 1 mm IPTG for 4 h at 37 °C after ultrasonication; lane 6: purified recombinant BLT-AMA-1-DI/DII. B: Western-blot analysis. M: molecular weight marker. Lanes 1: Purified recombinant BLT-rAMA-1-DI/DII protein was detected by using monoclonal anti-His6 Tag antibody in mouse.
Antigenic characterization of BLTrAMA-1 and native BLTAMA-1 by immunoblot analysis
Sera from sheep (No. 3216) infected with Babesia sp. BQ1 (Lintan) and the sera from rabbits immunized with BLTrAMA-1-DI/DII were used to identify BLTrAMA-1 (~35 kDa) by Western-blot analysis, whereas the sera obtained from sheep or rabbits prior to infection did not react with BLTrAMA-1 (Fig. 4A). No reaction of the pET-30a vector control with positive control sera from sheep or rabbits was observed (Fig. 4B).
Fig. 4. Western-blot analysis for recombinant pET-30a-AMA1-DI/DII (A), control strain harboring pET-30a (B) and native BQMA (C) recognized by sheep serum against Babesia sp. BQ1 (Lintna) or rabbit serum anti-BLTrAMA-1-DI/DII. M: molecular weight marker; Lane 1: Positive serum from infected sheep (No. 3216) for Babesia sp. BQ1 (Lintan); Lane 2: Positive serum from immunized rabbits anti-BLTrAMA-1-DI/DII; Lane 3: Serum from pre-immunized sheep (No. 3216); Lane 4: Serum from before immunized rabbit. Native BLTAMA-1 in lane 1 was indicated by arrow.
To identify native AMA-1 in Babesia sp. BQ1 (Lintan) merozoites, antibodies against recombinant BLTAMA-1 obtained from rabbits and sera obtained from sheep (No. 3216) infected with Babesia sp. BQ1 (Lintan) were examined for reactivity with native BQMA. By Western-blot, the native AMA-1 protein was detected at a size of approximately 65 kDa in the lysate of Babesia sp. BQ1 (Lintan)-infected sheep RBC s (Fig. 4C) but not in the lysate from uninfected sheep RBCs (data not shown). These results indicate that AMA-1 could induce antibody production in sheep infected by Babesia sp. BQ1 (Lintan).
DISCUSSION
In the present study, we report the cloning, recombinant expression, genetic and biological characterization of the ama-1 gene from Babesia sp. BQ1 (Lintan). The results confirmed that AMA-1 in the sheep parasite Babesia sp. BQ1 (Lintan) resembled AMA-1 orthologs of other apicomplexan parasites sharing a similar domain organization. AMA-1 possesses an N-terminal signal sequence followed by an ectodomain region, a single transmembrane region, and a short cytoplasmic domain.
The BLTAMA-1 structure of the ectodomain region is divided into three domains (DI, DII and DIII) (Fig. 1). In a previous study, domains I and II, containing 10 conserved cysteine residues (six in D1 and four in DII) in all of apicomplexan AMA-1 proteins, were proposed to have an important function in adhesion to proteins or glycoprotein receptors (Pizarro et al. Reference Pizarro, Vulliez-Le Normand, Chesne-Seck, Collins, Withers-Martinez, Hackett, Blackman, Faber, Remarque, Kocken, Thomas and Bentley2005). Domain III was relatively variable and located adjacent to the parasite surface, suggesting that this region might be under fewer selective constraints or that this variability is associated with immune evasion (Moitra et al. Reference Moitra, Zheng, Anantharaman, Banerjee, Takeda, Kozakai, Lepore, Krause, Aravind and Kumar2015). In addition, within highly variable domain III of the extracellular region, four conserved cysteines are observed in most Babesia spp., including Babesia sp. BQ1 (Lintan). Exception are that of B. microti, which contains only two cysteines and Plasmodium AMA-1, which contains six cysteines (Hodder et al. Reference Hodder, Crewther, Matthew, Reid, Moritz, Simpson and Anders1996). In general, the analysis of the protein structure of Babesia sp. BQ1 (Lintan) AMA-1 is consistent with the common features of apicomplexan parasites AMA-1, as described above.
The constructed phylogenetic tree based on all apicomplexan parasite AMA-1 amino acid sequences indicates that Babesia sp. BQ1 (Lintan) AMA-1 is closely related to that of B. ovata and B. bigemina. The per cent identity between Babesia sp. BQ1 (Lintan) and B. ovata (84·4%) and B. bigemina (77·9%) also reflects the evolutionary relationships of these parasites. Interestingly, these findings are consistent with previous phylogenetic analyses based on 18S rRNA, whereby B. bigemina and B. motasi (Lack et al. Reference Lack, Reichard and Van Den Bussche2012) as well as B. ovata, B. crassa and B. major were grouped together (Schnittger et al. Reference Schnittger, Rodriguez, Florin-Christensen and Morrison2012), and on RAP-1, whereby B. bigemina and Babesia cf. motasi [Babesia sp. BQ1 (Lintan)] were grouped in the same clade (Niu et al. Reference Niu, Bonsergent, Guan, Yin and Malandrin2013). In general, the AMA-1 proteins of Piroplasmida are more closely related to those of Plasmodia than to those of Conoidasida. In addition, the phylogenetic analysis indicated that B. microti AMA-1 forms a separate branch from all other piroplasm species. This finding is consistent with phylogenetic analyses based on AMA-1 (Moitra et al. Reference Moitra, Zheng, Anantharaman, Banerjee, Takeda, Kozakai, Lepore, Krause, Aravind and Kumar2015) and the eta (η) subunit of the chaperonin-containing t-complex polypeptide l (CCT η) gene (Nakajima et al. Reference Nakajima, Tsuji, Oda, Zamoto-Niikura, Wei, Kawabuchi-Kurata, Nishida and Ishihara2009), suggesting that B. microti is distinct from all other Babesia species and represents a sister group to Theileria and Babesia.
In the present study, a recombinant protein based on the BLTAMA-1 conserved region containing domains I and II, was constructed and expressed. Sera produced from rabbits immunized with the BLTrAMA-1-DI/DII protein specifically recognized parasite-produced native AMA-1 protein, with the expected size of BLTAMA-1; this result indicates that Babesia sp. BQ1 (Lintan) merozoites expressed the AMA-1 protein. Sera (3 weeks post-infection) from Babesia sp. BQ1 (Lintan) experimentally infected sheep strongly reacted with BLTrAMA-1-DI/DII, suggesting that the antibody responses of AMA-1 are elicited early during sheep Babesia sp. BQ1 (Lintan) infection. However, kinetic studies of humoral responses against B. divergens AMA-1 indicated that antibody production against extracellular DI and DII of BdAMA-1 is weak and late, between more than 3 and 5 months post-infection during experimental infection of sheep by B. divergens (Moreau et al. Reference Moreau, Bonsergent, Al Dybiat, Gonzalez, Lobo, Montero and Malandrin2015). Rabbit sera against the BLTrAMA-1-DI/DII protein did not react with uninfected sheep RBCs and the serum obtained prior to infection of sheep did not react with BLTrAMA-1 in Western-blot analyses. These finding suggest that the AMA-1 protein is present in Babesia sp. BQ1 (Lintan) and that BLTrAMA-1 is a potential diagnostic antigen for detecting Babesia sp. BQ1 (Lintan) and/or other isolates of Babesia cf. motasi in China, as AMA-1 is considered highly conserved among different isolates of the same species (Torina et al. Reference Torina, Agnone, Sireci, Mosqueda, Blanda, Albanese, La Farina, Cerrone, Cusumano and Caracappa2010; Moreau et al. Reference Moreau, Bonsergent, Al Dybiat, Gonzalez, Lobo, Montero and Malandrin2015).
Several studies of Plasmodium AMA-1 as a potential vaccine candidate have reported promising vaccine trials conducted in animal-model systems (Collins et al. Reference Collins, Pye, Crewter, Vandeberg, Galland, Sulzer, Kemp, Edwards, Coppel, Sullivan, Morris and Anders1994); in particular, recombinant Plasmodium AMA-1 containing only the ectodomain provided complete protection against challenge (Lal et al. Reference Lal, Hughes, Oliveira, Nelson, Bloland, Oloo, Hawley, Hightower, Nahlen and Udhayakumar1996). In B. bovis, the use of specific antibodies directed against certain synthetic peptides (Gaffar et al. Reference Gaffar, Yatsuda, Franssen and de Vries2004) and conserved DI and DII regions of the AMA-1 protein inhibited parasite invasion, potentially serving as a vaccine candidate against B. bovis infection (Salama et al. Reference Salama, Terkawi, Kawai, Aboulaila, Nayel, Mousa, Zaghawa, Yokoyama and Igarashi2013). In B. divergens, antibodies against DI and DII of AMA-1 have a potent inhibitory effect on merozoite invasion, decreasing invasion by ~50% (Montero et al. Reference Montero, Rodríguez, Oksov and Lobo2009). Because the structure of AMA-1 is conserved among apicomplexans, the data obtained in this study provide a basis for further investigation of the function of the AMA-1 protein in the Babesia cf. motasi group and suggest this protein's potential as a vaccine for controlling ovine babesiosis in China and even worldwide.
Further work is necessary to obtain the sequences of other sheep Babesia species (B. motasi, B. ovis and Babesia sp. Xinjiang) and to investigate polymorphism of AMA-1 sequences between isolates of the same species or different sheep-infection Babesia species. The kinetics of antibody production against Babesia sp. BQ1 (Lintan) during sheep infection and cross-reaction with other haemoparasites should to be further studied.
In conclusion, this study is the first to report the ama-1 gene sequence in the sheep parasite Babesia sp. BQ1 (Lintan) (Babesia cf. motasi group) and show a sequence comparison of its orthologues from different apicomplexans. We cloned and expressed the BLTAMA-1-DI/DII region and detected the presence of antibodies directed against native and recombinant forms of AMA-1. Future studies should examine sequence polymorphisms of ama-1 in different isolates of the Babesia cf. motasi group for the purpose of potential vaccine development, the process by which BLTAMA-1 mediates RBC invasion, the potential of BLTAMA-1 for disease diagnosis and development of a common vaccine against different isolates of Babesia cf. motasi.
ACKNOWLEDGEMENTS
The authors would like to express their appreciation for the good suggestions of editor and two anonymous reviewers, which were very useful for improved this manuscript.
FINANCIAL SUPPORT
This study was supported financially by the NSFC (Grant numbers 31502054 to Q. N.; 31372432 to J. L.); ASTIP (to H.Y.); FRIP (Grant number 2015ZL009), CAAS (to Q. N.); the 973 Program (Grant number 2015CB150300 to J. L.); and the Jiangsu Co-innovation Center Programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, State Key Laboratory of Veterinary Etiological Biology Project (to H. Y.).
