INTRODUCTION
Babesiosis is a tick-borne haemoparasitic disease affecting a wide range of domestic and wild animals and is caused by the genus Babesia, which comprises many species of parasites that infect a large range of erythrocytes and various vertebrate hosts (Vannier and Krause, Reference Vannier and Krause2009). Many Babesia species are highly pathogenic for animals. Ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants in tropical, subtropical and temperate regions. The economic losses in sheep and goat production due to babesiosis are significant in tropical and subtropical areas, where it has increasingly attracted more attention (Luo and Yin, Reference Luo and Yin1997; Ahmed et al. Reference Ahmed, Yin, Schnittger and Jongejan2002). Three Babesia species are known to infect small ruminants: Babesia ovis, Babesia motasi, and Babesia crassa (Uilenberg, Reference Uilenberg2006).
In China, several Babesia isolates have been described to infect small ruminants, most of which belong to B. motasi-like species, which has the similar morphology characteristics and form a sister clade with B. motasi species based on phylogenetic trees of the 18S rRNA and internal transcribed spacer (ITS) gene sequences (Liu et al. Reference Liu, Yin, Guan, Schnittger, Liu, Ma, Dang, Liu, Ren, Bai, Ahmed and Luo2007; Niu et al. Reference Niu, Luo, Guan, Liu, Ma, Liu, Gao, Ren, Li, Qiu and Yin2009). The other isolate, Babesia sp. Xinjiang, was isolated from sheep experimentally infected with Rhipicephalus sanguineus and Hyalomma anatolicum anatolicum collected from pastures in China (Guan et al. Reference Guan, Yin, Luo, Lu, Zhang, Ma, Yuan, Lu, Wang and Muhe2001). The demonstrated vector of this Babesia species is H. a. anatolicum. The pathogenic and morphological characteristics of this species are totally different from the other Babesia species of small ruminants previously reported worldwide, including China. It is a large parasite with various morphological forms in infected erythrocytes: single and paired piriform (the most common shapes), ring form, three-leafed shaped, rod shaped, budding forms and oval forms (Guan et al. Reference Guan, Ma, Moreau, Liu, Lu, Bai, Luo, Jorgensen, Chauvin and Yin2009).
Although the Babesia parasites are specifically parasitic on a large range of obligate host red blood cells, the pathological changes caused by Babesia species are similar to those observed in Plasmodium falciparum, and the mechanism of host cell entry is thought to be conserved (Besteiro et al. Reference Besteiro, Dubremetz and Lebrun2011). Parasites in the genus Babesia are transmitted when sporozoites are released into the vertebrate host with the saliva during tick feeding, and then the apicomplexan parasites could display a machinery to invade into host cells. Clinical symptoms of the disease might appear when the merozoites invade and replicate within host erythrocytes, and reach high parasitaemia (Yokoyama et al. Reference Yokoyama, Okamura and Igarashi2006). The vaccine development targeted on the molecules of the extracellular merozoites during invasion process might be a significant strategy against babesiosis.
The Babesia rhoptry-associated protein-1 (RAP-1) protein was first described in Babesia bigemina (designated p58) (McElwain et al. Reference McElwain, Perryman, Davis and McGuire1987) and was then characterized in all examined Babesia species: B. bovis (Suarez et al. Reference Suarez, Palmer, Jasmer, Hines, Perryman and McElwain1991), B. divergens (Rodriguez et al. Reference Rodriguez, Alhassan, Ord, Cursino-Santos, Singh, Gray and Lobo2014), Babesia canis, B. ovis (Dalrymple et al. Reference Dalrymple, Casu, Peters, Dimmock, Gale, Boese and Wright1993), Babesia orientalis (Yu et al. Reference Yu, He, Zhang, Cheng, Hu, Miao, Huang, Fan, Khan, Zhou, Hu and Zhao2014), Babesia gibsoni (Zhou et al. Reference Zhou, Jia, Nishikawa, Fujisaki and Xuan2007), B. motasi-like (Niu et al. Reference Niu, Bonsergent, Guan, Yin and Malandrin2013, Reference Niu, Valentin, Bonsergent and Malandrin2014) and Babesia sp. Xinjiang (Niu et al. Reference Niu, Marchand, Yang, Bonsergent, Guan, Yin and Malandrin2015). Seven different rap-1 genes (five rap-1aα and two rap-1aβ) in Babesia sp. Xinjiang have been recently described, and at least three rap-1aα and two rap-1aβ were transcribed in vitro (Niu et al. Reference Niu, Marchand, Yang, Bonsergent, Guan, Yin and Malandrin2015).
In the present study, the gene encoding full-length and C-terminal truncated RAP-1aα2 in Babesia sp. Xinjiang was cloned and expressed in Escherichia coli and evaluated for its potential use as a diagnostic and vaccine candidate. The presence of antibodies directed against native and recombinant RAP-1a during the course of infection was detected using Western blotting and enzyme-linked immunosorbent assay (ELISA). The identification and localization of native RAP-1aα2 in Babesia sp. Xinjiang erythrocytes were detected by Western blotting and indirect fluorescent antibody test (IFAT) and its serodiagnostic performance as an antigen was evaluated by ELISA.
MATERIALS AND METHODS
Parasites
Biologically cloned Babesia sp. Xinjiang lines were derived from in vitro culture by limiting dilution, as described previously (Malandrin et al. Reference Malandrin, Jouglin, Moreau and Chauvin2009), and cryopreserved at the vector and vector-borne disease (VVBD) laboratory of Lanzhou Veterinary Research Institute (LVRI) (CAAS Lanzhou, China).
Sera
Sera collected from two sheep (Nos. 3201 and 026), which were infected by Babesia sp. Xinjiang were used to evaluate antibody kinetics. Sera collected from sheep Nos. 3216, 08026, 08040 and 08020, which were infected with Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian), Babesia sp. Tianzhu and Babesia sp. Hebei, respectively, as well as the sera collected from sheep experimentally infected with Theileria luwenshuni, Theileria uilenbergi and Anaplasma ovis, or bovine B. bovis and B. bigemina, were used as controls to evaluate cross-reactivity.
The sera samples (n = 110) were selected from ten sheep, 3–12 weeks post-infection (wpi), experimentally infected with Babesia sp. Xinjiang and used as positive samples to evaluate the sensitivity of the ELISA and a mixture of these sera was used as the positive control serum (Guan et al. Reference Guan, Ma, Liu, Ren, Wang, Yang, Li, Liu, Du, Li, Liu, Zhu, Yin and Luo2012b ).
Sera from 197 lambs, purchased from the Babesia-free region in Jingtai County (Gansu Province of China) from 2005 to 2010 were collected. The genomic DNA samples corresponded to these serum samples were tested by different methods and has never found an animal positive. A mixture of these sera was used as the negative control serum to evaluate the specificity of the ELISA (Guan et al. Reference Guan, Ma, Liu, Ren, Wang, Yang, Li, Liu, Du, Li, Liu, Zhu, Yin and Luo2012b ).
A total of 3690 serum samples were randomly collected from clinically healthy sheep from 59 different locations in 23 Chinese provinces between 2010 and 2015. All of the blood samples were collected in tubes and transported to the laboratory on ice, where the serum was separated and stored at −20 °C until further use.
Recombinant protein expression and purification of the XJrRAP-1aα2
Genomic Babesia sp. Xinjiang DNA encoding full-length and the C-terminal truncated version of XJrRAP-1aα2 protein were amplified using the modified primers based on the RAP-1aα2 gene sequence (GenBank accession number: KF811194), rRAP-1aα-full-F: 5′-CGCATATGGGTTCGTCACTATCAGAATGT-3′, or rRAP-1aα-CT-F: 5′-CGCATATGATCGCCATTCCAACAAAAGA-3′ and rRAP-1aα-R: 5′-GCCAAGCTTTTCTTGAGATACCTCATCCT-3′ to include NdeI and HindIII (New England BioLabs, USA) enzyme restriction sites (underlined) on the 5′ end and sub-cloned into the pET-30a vector. The resulting plasmids (pET-30a – XJRAP-1aα2 and pET-30a – XJRAP-1aα2 CT) were confirmed by enzyme restriction digestion and sequencing, and subsequently transformed into the E. coli BL21 (DE3 strain) according to the manufacturer's instructions for protein expression.
The bacterial cultures containing His6-fusion proteins of rXJRAP-1aα2 and rXJRAP-1aα2/CT were harvested after induction with 1 mm isopropy-β-D-thiogalactoside (IPTG) for15 °C for 16 h and lysed by ultrasonication in binding buffer (50 mm Tris, 6 m guanidine–HCl, pH 8·0) containing 1 mm phenylmethylsulfonyl fluoride (PMSF) and purified from the E. coli. The target protein was then eluted using urea followed by buffer containing increasing concentrations of imidazole (20, 50 and 250 mm). The recombinant proteins were then assessed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–10% PAGE). Recombinant protein expression was then analysed by Western blotting.
Preparation of anti-XJrRAP-1aα2 CT-specific rabbit immune serum
Purified XJrRAP-1aα2 CT was used to raise antibodies in New Zealand Rabbits (2 kg each). The sera were collected from each rabbit before the first immunization as a negative control. The rabbits were subcutaneously injected with 400 µg of purified rXJRAP-1aα2/CT protein emulsified in Freund's complete adjuvant (FCA; Sigma). Booster injections with the same amount of protein in Freund's incomplete adjuvant (FIA; Sigma) were administered on days 15, 20 and 28. The sera from the immunized rabbits were then collected 15 days after the last injection and purified and stored at −20 °C until further use.
Immunoblot analysis
The recombinant proteins (XJrRAP-1aα2 and XJrRAP-1aα2 CT) were separated on SDS–18% PAGE and then transferred to nitrocellulose (NC) membranes. The membrane was cut into 0·25 cm strips and incubated in blocking solution [5% skimmed milk powder in Tris-buffered saline (pH 7·6) with 0·1% Tween-20 (TBST)], for overnight at 4 °C on a shaker. After three washes, the NC strips were incubated for 1 h with each tested serum diluted at 1:100 in TBST. After three washes with TBST, the strips were incubated for 2 h 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). The reactions were developed using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) liquid substrate system (B1911-100ML, Sigma).
IFAT and confocal laser microscopic observation
The native Babesia sp. Xinjiang RAP-1 was identified in merozoites by IFAT using a procedure previously described by Moitra et al. (Reference Moitra, Zheng, Anantharaman, Banerjee, Takeda, Kozakai, Lepore, Krause, Aravind and Kumar2015). Babesia sp. Xinjiang-infected red blood cells (iRBC) were washed using serum-free medium, and the iRBC membrane was stained using PKH26 (MINI26, Sigma-Aldrich) according to the manufacturer's protocol. The iRBCs were fixed in a buffer containing 4% paraformaldehyde and 10 mm piperazine-N, N-bis (2-ethanesulfonic acid) in PBS (PIPES) at pH 6·4 on coverslips for 30 min at room temperature. The coverslips were washed in PBS and then blocked for 1 h in 3% bovine serum albumin (BSA in PBS containing 0·25% Triton X-100). The coverslips were stained at 4 °C overnight with either pre-immune serum or purified anti-rXJRAP-1aα2/CT sera (1:100) diluted in blocking buffer without Triton X-100. The coverslips were washed three times in PBS and stained for 1 h with FITC-conjugated anti-rabbit IgG secondary antibody (Sigma; F0382; 1:80). The coverslips were then stained for 10 min in 1 µg mL−1 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma: D8417) and examined for reactivity using confocal microscopy.
ELISA
The optimum coating concentration of XJrRAP-1aα2 CT (100 µL at 2 µg mL−1) in coating buffer (0·1 m carbonate/bicarbonate, pH 9·6) was distributed and adsorbed in 96-well flat-bottom microplates at 4 °C overnight. The plates were then blocked with 200 µL of 2% gelatin in PBST at 37 °C for 1 h. After washing, sera diluted with 1:100 were distributed in duplicate, and the plates were incubated for 1 h at 37 °C. After three washes, 100 µL of monoclonal anti-goat/sheep IgG-peroxidase (Sigma; A9452; dilution: 1:1000) was added to each well and incubated for 1 h at 37 °C. The reaction was detected with 100 µL of 1-Step™ Ultra TMB-ELISA (34028–250 mL; Thermo Scientific) for 15–25 min at room temperature and then halted by adding 100 µL of 2 m H2SO4. The plates were read at 450 nm with an automated ELISA plate reader (Bio-RAD Model 680 microplate reader, USA).
Statistical analysis
The 95% confidence intervals (95% CIs) for the overall prevalence values of Babesia sp. Xinjiang infection were calculated using IBM SPSS Statistics version 19.0.
RESULTS
Expression of recombinant XJRAP-1aα2 protein
The genes encoding XJrRAP-1aα2 (aa 22–447) and XJrRAP-1aα2 CT (aa 313–447) were successfully amplified by PCR. The pET-30a-XJRAP-1aα2 and pET-30a-XJRAP-1aα2 CT recombinant plasmids were identified by enzyme restriction analysis and subsequently confirmed by sequencing using specific primers; the insert sequences have also been verified (data not shown). The E. coli lysates containing recombinant protein were analysed by SDS–10% PAGE and Western blotting using anti-His6 serum (THETM His Tag Antibody, mAb, Mouse, GenScript, A00186). The results show that XJrRAP-1aα2 and XJrRAP-1aα2 CT were expressed as apparent approximately 52-kDa and 19-kDa proteins, respectively (Fig. 1).
Fig. 1. SDS–18% PAGE (panels A and B) and Western blot (panels C and D) analysis (ECL) of the expressed and purified rXJRAP-1aα2 and rXJRAP-1aα2 CT proteins with His6-tag expressed in E. coli. M: molecular weight marker. Lanes 1 and 2: SDS–18% PAGE analysis for rXJRAP-1aα2 and rXJRAP-1aα2 CT proteins; lanes 3 and 4: Western blot analysis for rXJRAP-1aα2 and rXJRAP-1aα2 CT proteins using monoclonal anti-His6 Tag antibody in mouse.
Evaluation of the specificity of XJrRAP-1aα2 and XJrRAP-1aα2 CT via Western blotting
The reactivity of each positive serum sample from infected sheep with XJrRAP-1aα2 and XJrRAP-1aα2CT was analysed by Western blotting. Strong reactions with the expected size of XJrRAP-1aα2 CT (approximately 19 kDa) were observed with sera from Babesia sp. Xinjiang infected sheep (No. 3201). In contrast, the sera positive for other Babesia, Theileria and Anaplasma species giving a negative reaction with the RAP-1aα2 CT gave a positive reaction under identical conditions when tested with their corresponding antigens (data not shown) (Fig. 2). However, the cross-reactivity of positive serum from B. motasi-like and T. uilenbergi was observed with full length of XJrRAP-1aα2 (data not shown).
Fig. 2. Western blot analysis of the recombinant RAP-1aα2 CT in Babesia sp. Xinjiang. M: molecular weight marker. Lanes 1–10: sera positive for Babesia sp. Xinjiang, Babesia sp. BQ1 (Lintan), Babesia sp. Tianzhu, Babesia sp. BQ1 (Ningxian), Babesia sp. Hebei, B. bigemina, B. bovis, T. luwenshun, T. uilenbergi and A. ovis; lane 11: pool of negative sera; lane 12: positive control (anti-XJrRAP-1aα2 CT rabbit immune serum). The sera positive for other pathogens (lanes 2–10) giving a negative reaction with the RAP-1aα2 CT gave a positive reaction under identical conditions when tested with their corresponding antigens (data not shown).
Identification of native XJRAP-1 proteins
The IFAT results show that native rhoptry proteins could be interacted with serum from a rabbit immunized by XJrRAP-1aα2 CT with Babesia sp. Xinjiang merozoites. Non-specific fluorescence signalling was detected using serum from pre-immunized rabbits (Fig. 3).
Fig. 3. Immunofluorescence microscopy analysis. Babesia sp. Xinjiang-iRBC were stained with the rabbit anti-XJrRAP-1aα2 CT antibody (A–D) and pre-immune serum (E–H). Nuclei were counterstained with DAPI (blue, A and E). Anti-XJrRAP-1aα2 CT antibody (green) reacted with native RAP-1 on merozoites (B and F). Overlaid image of fluorescent green reactivity and blue DAPI staining of nuclei (C and G). Merged images of fluorescent green reactivity, RBCs staining with PKH26 (red) and blue DAPI staining are shown in the right (D and H). Arrowheads showed the representative merozoites. Bar, 5 µ m (A–D), 10 µ m (E–H).
Cross-reactivity with other haemoparasites via ELISA
The sera collected from Babesia sp. Xinjiang and other haemoparasites-infected animals were used to evaluate cross-reactivity with the rRAP-1aα2 CT in the ELISA. The tests were repeated in triplicate, and the mean AbRs [AbR % = (Sample mean OD − Negative control mean OD)/(Positive control mean OD − Negative control mean OD) × 100] and s.d. were calculated with Excel 2007. The results indicate that no cross-reactivity was observed with sera against other Babesia, Theileria and A. ovis parasites (Fig. 4).
Fig. 4. Cross-reactivity of XJrRAP-1aα2 CT of Babesia sp. Xinjiang with the sera positive for Babesia, Theileria and Anaplasma infective to sheep or cattle in ELISA. XJ: Babesia sp. Xinjiang; LT: Babesia sp. BQ1 (Lintan); TZ: Babesia sp. Tianzhu; NX: Babesia sp. BQ1 (Ningxian); HB: Babesia sp. Hebei; T.u: T. uilenbergi; T.l: T. luwenshuni; A.o: A. ovis; B.bi: B. bigemina and B. bo: B. bovis; PC: positive control (anti-XJrRAP-1aα2 CT rabbit immune serum). AbR % = (Sample mean OD − Negative control mean OD)/(Positive control mean OD − Negative control mean OD) × 100).
Kinetics of anti-Babesia sp. Xinjiang antibodies in experimentally infected sheep via ELISA
The kinetics of antibody production against RAP-1a have been studied via ELISA using XJrRAP-1aα2 CT and sera from two infected intact sheep (Nos. 3201 and 026) collected before (0 weeks post-infection, WPI0) and after infection (from WPI1 to WPI12). A significant increase in antibodies produced against RAP-1a was observed after infection and with similar kinetics between each individual sheep. In general, antibodies were produced at 1 week post-inoculation and continued to increase through 3 weeks (WPI3), after which slight decreases were observed through 12 weeks (WPI4–12) (Fig. 5).
Fig. 5. Kinetics of anti-Babesia sp. Xinjiang antibodies in two sheep (Nos. 3201 and 026) infected experimentally. AbR % = (Sample mean OD − Negative control mean OD)/(Positive control mean OD − Negative control mean OD) × 100)
Evaluation of the specificity, sensitivity and positive threshold value of the ELISA in detecting XJrRAP-1aα2 CT
MedCalc statistical software was used to evaluate the sensitivity and specificity, as well as the positive threshold value of the ELISA, by testing 110 sera of Babesia sp. Xinjiang positive and 197 sera of Babesia sp. Xinjiang negative. The specific antibody mean rate (AbR %) was calculated for each serum sample. The results indicate that the positive threshold value was 29·10%, corresponding to 94·3% sensitivity (95% CI = 80·1–97·8) and 95·1% specificity (95% CI = 82·3–99·6). The numbers of false-positive and false-negative sera were 3 and 14, respectively (Fig. 6).
Fig. 6. Evaluation of specificity, sensitivity and positive threshold value of XJrRAP-1aα2 CT with ELISA using antibody mean rates (AbR %) of positive and negative sera. Spec: specificity, Sens: sensitivity. 0 = negative sera; 1 = positive sera.
Identification of XJrRAP-1aα2 CT protein as a potential antigen for the serological epidemiology of Babesia sp. Xinjiang infection by ELISA
A total of 3690 field serum samples from 59 different regions of 23 Chinese provinces were detected by the ELISA to evaluate the prevalence of infection with Babesia sp. Xinjiang. The results of the ELISA for positive sample screenings are summarized in Table 1. Sera samples infected with Babesia sp. Xinjiang were examined in 57 prefectures of 23 surveyed provinces. The average positive rate was 30·43% (1123/3690). The sero-prevalence ranged from 0 to 73·58%. The significantly high positive rates exceeded 50% in eight of 59 surveyed prefectures, mainly from North China (Table 1).
Table 1. Prevalence of Babesia sp. Xinjiang in field samples collected from 23 provinces by detected antibodies produced from rRAP-1aα2 CT with ELISA
DISCUSSION
According to molecular taxonomy and phylogenetic reconstruction based on the 18S rRNA gene, Babesia sp. Xinjiang was closely related to Babesia recently described from wild ruminants in South Africa and with Babesia pecorum isolated from red deer in Spain (Oosthuizen et al. Reference Oosthuizen, Allsopp, Troskie, Collins and Penzhorn2009; Jouglin et al. Reference Jouglin, Fernández-de-Mera, de la Cotte, Ruiz-Fons, Gortázar, Moreau, Bastian, de la Fuente and Malandrin2014). Presently, a Babesia sp. Xinjiang-like parasite (which shared 99·5% identity with the original strain of Babesia sp. Xinjiang) was isolated using an in vitro culture system from one of 19 sheep blood samples collected from Dunhuang city, Western Gansu Province, China (Guan et al. Reference Guan, Ma, Liu, Du, Ren, Li, Wang, Liu, Yin and Luo2012a ). Moreover, a recent report of sero-epidemiological investigation by ELISA for the infections of small ruminants by Babesia sp. Xinjiang has indicated that this Babesia species was widespread in 22 provinces of China (Guan et al. Reference Guan, Ma, Liu, Ren, Wang, Yang, Li, Liu, Du, Li, Liu, Zhu, Yin and Luo2012b ). These findings indicated that ovine babesiosis caused by Babesia sp. Xinjiang presented a tendency to expand in China.
The N-terminal regions of RAP-1, as well as their sequences, are more conserved at the species level among geographically distant isolates than the C-terminal region and several C-terminal sequences encode conserved or degenerate repetitive motifs in many Babesia species (Dalrymple et al. Reference Dalrymple, Casu, Peters, Dimmock, Gale, Boese and Wright1993; Hötzel et al. Reference Hötzel, Suarez, McElwain and Palmer1997; Terkawi et al. Reference Terkawi, Amornthep, Ooka, Aboge, Jia, Goo, Nelson, Yamagishi, Nishikawa, Igarashi, Kawazu, Fujisakik and Xuan2009; Bhoora et al. Reference Bhoora, Quan, Zweygarth, Guthrie, Prinsloo and Collins2010). In Babesia sp. Xinjiang RAP-1, the sequence is identical among copies over the first 300 and the last 30 amino acids, with few differences in the sequences of the repeats of the C-terminus of the protein. The prediction of B-cell epitopes in all RAP-1a using software demonstrated the presence of B-cell epitopes primarily in the repeat region of the polymorphic C-terminus (Niu et al. Reference Niu, Marchand, Yang, Bonsergent, Guan, Yin and Malandrin2015). In general, repetitive regions of a protein are thought to mediate important functions in parasite survival and immune evasion (Mendes et al. Reference Mendes, Lobo, Rodrígues, Rodrígues-Luiz, daRocha, Fujiwara, Teixeira and Bartholomeu2013). The variable C-terminal region among Babesia species could induce a high humoral response; therefore, it could be considered as a useful antigen for specific diagnostic and epidemiological investigation purposes. The C-terminal truncated region of the RAP-1a protein has been widely used as a specific diagnostic antigen to differentiate Babesia infection (Boonchit et al. Reference Boonchit, Xuan, Yokoyama, Goff, Wagner and Igarashi2002, Reference Boonchit, Alhassan, Chan, Xuan, Yokoyama, Ooshiro, Goff, Waghela, Wagner and Igarashi2006; Zhou et al. Reference Zhou, Jia, Nishikawa, Fujisaki and Xuan2007).
In the present study, the XJrRAP-1aα2 proteins were subjected to Western blot analysis, and cross-reactivity was observed between full-length XJrRAP-1aα2 and serum samples infected with B. motasi-like and T. uilenbergi parasites. Previous studies in bovine Babesia species also indicated that the full-length recombinant RAP-1a from B. bovis could cross-react with B. bigemina due to the conserved features in the N-terminus of RAP-1 (Boonchit et al. Reference Boonchit, Xuan, Yokoyama, Goff, Wagner and Igarashi2002). By contrast, XJrRAP-1aα2 CT could be specifically recognized by the serum from Babesia sp. Xinjiang infection only by Western blot analysis. Recombinant RAP-1a constructed on the C-terminal region in B. bovis and B. bigemina has been used as a diagnostic antigen to develop an ELISA method and could improve the sensitivity and specificity of antibody detection (Terkawi et al. Reference Terkawi, Huyen, Shinuo, Inpankaew, Maklon, Aboulaila, Ueno, Goo, Yokoyama, Jittapalapong, Xuan and Igarashi2011).
Serological diagnostic testing based on the immunoreactive proteins generated from parasite genes is an essential tool used for the control of babesiosis (Ooka et al. Reference Ooka, Terkawi, Cao, Aboge, Goo, Luo, Li, Nishikawa, Igarashi and Xuan2012; Sevinc et al. Reference Sevinc, Cao, Zhou, Sevinc, Ceylan and Xuan2015a ). Recombinant antigens (merozoite antigen, RAP-1 and spherical body proteins (SBPs)) used in serological assays have been widely and effectively used for the diagnosis of bovine, equine and canine babesiosis (Boonchit et al. Reference Boonchit, Xuan, Yokoyama, Goff, Wagner and Igarashi2002; Huang et al. Reference Huang, Xuan, Yokoyama, Xu, Suzuki, Sugimoto, Nagasawa, Fujisaki and Igarashi2003; Zhou et al. Reference Zhou, Jia, Nishikawa, Fujisaki and Xuan2007; Terkawi et al. Reference Terkawi, Huyen, Shinuo, Inpankaew, Maklon, Aboulaila, Ueno, Goo, Yokoyama, Jittapalapong, Xuan and Igarashi2011; Mandal et al. Reference Mandal, Banerjee, Kumar, Garg, Ram and Raina2016). In the ovine Babesia parasite, a crude B. bovis antigen and a synthetic B. bovis-derived antigen (11C5) are described to detect B. ovis antibodies by the development of an ELISA (Duzgun et al. Reference Duzgun, Wright, Waltisbuhl, Gale, Goodger, Dargie, Alabay and Cerci1991), and few novel recombinant proteins have been identified as candidate diagnostic antigens: B. ovis secreted antigen 1 and 2 (BoSA1 and BoSA2) for B. ovis detection (Sevinc et al. Reference Sevinc, Cao, Zhou, Sevinc, Ceylan and Xuan2015a , Reference Sevinc, Cao, Xuan, Sevinc and Ceylan b ) and BQP35 and heat shock protein 90 (HSP90) for Babesia sp. BQ1 (Lintan) (B. motasi-like species) detection (Guan et al. Reference Guan, Moreau, Liu, Ma, Rogniaux, Liu, Niu, Li, Ren, Luo, Chauvin and Yin2012c , Reference Guan, Liu, Liu, Li, Niu, Gao, Chauvin, Yin and Moreau2015). Western blotting and an ELISA based on rBQP35 showed cross-reactivity between Lintan and Tianzhu isolates of B. motasi-like (Guan et al. Reference Guan, Moreau, Liu, Ma, Rogniaux, Liu, Niu, Li, Ren, Luo, Chauvin and Yin2012c ), whereas rBQHSP90 could not differentiate among any ovine Babesia species (Guan et al. Reference Guan, Liu, Liu, Li, Niu, Gao, Chauvin, Yin and Moreau2015). Until now, for Babesia sp. Xinjiang, only one report described that an ELISA was recently developed using soluble merozoite antigens (BXJMA) from the in vitro culture of Babesia sp. in Xinjiang, eight common proteins were recognized by antibodies, one of them was recognized by anti-B. bovis, -B. bigemina and -Babesia U sp. Kashi antibodies (Guan et al. Reference Guan, Ma, Liu, Ren, Wang, Yang, Li, Liu, Du, Li, Liu, Zhu, Yin and Luo2012b ). No report was focused on the production of the recombinant immunological antigens of Babesia sp. Xinjiang and their use as antigens in any serological test to detect ovine babesiosis, caused by this parasite. The result in this study suggest that rRAP-1aα2 CT could be replaced native crude parasite antigens, which require large parasite reproduction from the sacrifice of experimentally infected animals or the harvesting of cultures in vitro (Böse et al. Reference Böse, Jorgensen, Dalgliesh, Friedhoff and de Vos1995).
According to the results of Western blotting and ELISA, the serum of Babesia sp. Xinjiang experimentally infected sheep interacted with rRAP-1aα2 CT, indicating that the antibody responses of RAP-1 may be elicited in a Babesia sp. Xinjiang infection. High levels of antibodies against RAP-1 could be attained for 2·5 months. Moreover, IFAT analysis showed that immunofluorescence signals were observed using rabbit antiserum raised against XJrRAP-1aα2 CT in Babesia sp. Xinjiang, which is indicated the expression of native rhoptry proteins in Babesia sp. Xinjiang merozoites. These data indicated that the RAP-1 proteins exist in Babesia sp. Xinjiang and suggested that XJrRAP-1aα2 CT could be a potential specific antigen for the detection of Babesia sp. Xinjiang antibodies.
Until now, there is no standard Babesia sp. Xinjiang ELISA assay based on specifically recombinant protein reported. In the present study, based on the calculated specificity, sensitivity and positive threshold value, the results of a sero-investigation indicated that Babesia sp. Xinjiang infections were widespread in China. The results similar to a recent report, and a positive rate as high as 68·13% was found from the Shandong Province (Guan et al. Reference Guan, Ma, Liu, Ren, Wang, Yang, Li, Liu, Du, Li, Liu, Zhu, Yin and Luo2012b ). The positive samples in our study were from the Shaanxi and Jilin Provinces, with 29·73 and 16·24% rates, respectively; however, no positive samples were found in these two provinces in a previous study (Guan et al. Reference Guan, Ma, Liu, Ren, Wang, Yang, Li, Liu, Du, Li, Liu, Zhu, Yin and Luo2012b ). The distribution of Babesia sp. Xinjiang infection in small ruminants shows a tendency to expand.
ACKNOWLEDGEMENTS
The authors would like to thank the editor and two anonymous reviewers for provided constructive comments, 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 Programme (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.).
