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Development and evaluation of a chemiluminescence immunoassay for detecting tropical theileriosis
Acta Tropica 202 (2020) 105245
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Development and evaluation of a chemiluminescence immunoassay for detecting tropical theileriosis
T
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Li Zhia, Liu Junlonga, , Ma Quanyinga, Xu Jianlina, Wang Jinminga, Liu Aihonga, Li Youquana, ⁎ Guan Guiquana, Luo Jianxuna, Yin Honga,b, a
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu, 730046, P. R. China b Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, P. R. China
A R T I C LE I N FO
A B S T R A C T
Keywords: Theileria annulata SVSP CLIA tropical theileriosis
Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection.
1. Introduction Theileria annulata is a tick-borne apicomplexan parasite causing tropical theileriosis, which predominantly infects cattle in tropical and subtropical areas around the world (Uilenberg et al., 1981; Bishop et al., 2004; Sivakumar et al., 2014). Although numerous Theileria species of tick-transmitted parasites have been identified in wild and domestic ruminants, only Theileria species (T. parva, T. annulata and T. lestoquardi) that have the ability to transform host cells, which are responsible for severe clinical diseases in animals (Mans et al., 2015; Nene and Morrison, 2016; Brown, 1997). In the worldwide, more than 0.25 billion cattle were infected with T. annulata, which led to economic losses of more than 0.8 billion US$ (Brown, 1997; Radositis et al., 1997;Sudan et al., 2014, 2015). To date, T. annulata is the only Theileria species to cause acute lymphoproliferative disease in China where T. parva is not present. The incidence and prevalence of tropical theileriosis ranges from 1.59% to 90% in endemic regions in China
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based on previous studies (Luo and Lu, 1997; Liu et al., 2015; Aihemaiti et al., 2017). Therefore, the disease causes substantial economic burden in the sustainable development of the livestock industry. In the late 1970s, the CLIA was developed and widely applied in numerous fields of life sciences (Halmann et al., 1977; Mirasoli and Michelini, 2014). Due to its high sensitivity and signal-to-noise ratio, low background fluorescence signal and wide linear range, the CLIA has been extensively used for diagnosing of various diseases, including hepatitis B virus (HBV) infection (Yang et al., 2016), human immunodeficiency virus (HIV) infection (Alonso et al., 2014; Cui et al., 2015), and foot and mouth disease virus (FMDV) infection (Liu et al., 2018). However, to date, there are no related reports on use of the CLIA method to detect T. annulata infection. The subtelomeric variable secreted proteins (SVSPs) family is the largest gene family in both T. parva and T. annulata, which may be associated with immune evasion, host transformation and invasion (Schmuckli et al., 2009; Weir et al., 2010; Tretina et al., 2015). The
Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (H. Yin).
https://doi.org/10.1016/j.actatropica.2019.105245 Received 25 August 2019; Received in revised form 24 October 2019; Accepted 24 October 2019 Available online 30 October 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
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SVSPs are mainly expressed in the T. annulata and T. parva schizont stage, and the majority of the SVSPs of T. annulata are secreted into the host cell cytoplasm (Pain et al., 2005; Shiels et al., 2006; Schmuckli et al., 2009). However, can the genes of this family be used a diagnostic antigen for tropical theileriosis? In the present study, a recombinant SVSP protein was used to develop a CLIA assay to detect tropical theileriosis, and the specificity and sensitivity of the CLIA were also evaluated. The diagnostic performance of the assay was determined by comparing it with ELISA. In addition, small-scale serological screening of serum samples from four endemic regions was conducted using the CLIA to further identify the reliability.
After washing the strips three times with TBST buffer for 10 min each time, the reference serum samples (1:100 dilution) were then added to the designed strips, and incubated overnight at 4 °C. Following washing with TBST buffer three times, the strips were probed with rabbit antibovine IgG secondary antibody (1:10000 dilution) (HRP-IgG; SigmaAldrich, Missouri, USA) at room temperature for 1 h. The strips were washed four times with TBST for 10 min each time, and the signal bands for the strips were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Massachusetts, USA).
2. Materials and methods
A checkerboard titration was performed in 96-well CLIA white plates (Thermo Fisher Scientific, Massachusetts, USA) to determine the optimum dilutions of the coating protein, serum, secondary antibody. The reaction time for serum and secondary antibody were selected at 5 min, 10 min, 15 min and 20 min for 37 °C, respectively. In addition, each screening assay contained standard positive and negative serum and blank controls. Briefly, the plate was coated with rSVSP450 protein (22.3 ng/well, 11.15 ng/well, 5.58 ng/well, and 2.79 ng/well) diluted in carbonated buffer (pH 9.6) and incubated at 4°C overnight or 37 °C for 2 h. After washing the plate with PBST, the wells were blocked with 5% gelatin (Sigma-Aldrich, Missouri, USA) and 5% horse serum (Gibco, New York, USA) in PBS buffer, and incubated at 37 °C for 2 h. After washing the plate with PBST three times, 100 μL of positive or negative standard sera diluted using the serum dilution buffer at the ratio of 1:10, 1:20, 1:40 and 1:80, respectively, were added to the wells and incubated at 37 °C for 10 min. After washing the plate three times with PBST, 100 μL of serial diluted HRP-conjugated rabbit anti-bovine IgG (1:10,000, 1:20,000, 1:40,000 and 1:80,000) was added to each well and incubated at 37 °C for 10 min. Finally, following five washes, 100 μL per/well of substrate solution A (Luminol; Thermo Fisher Scientific, Massachusetts, USA) and solution B (Luminol enhancer; Thermo Fisher Scientific, Massachusetts, USA) were added and incubated at room temperature for 5 min. The chemiluminescence (CL) values of the plate were measured with a VarioskanTM LUX Multimode Microplate Reader (Thermo Fisher Scientific, Massachusetts, USA).
2.3. Optimization of the CLIA protocol
2.1. Serum samples 2.1.1. T. annulata-negative serum samples Serum samples (n = 302) obtained from domestic cattle in Yuzhong and Wuwei regions of Gansu province in China during 2018 were detected using two indirect ELISA methods based on sporozoite and macroschizont gene 2 (Spm2) (GeneBank accession no: Y15795) protein and major piroplasma surface protein (MPSP) (GeneBank accession no: GQ180193.1) of T. sinensis, respectively (Tian et al., 2018; Zhao et al., 2017). Of these serum samples, 158 samples negative for T. annulata were selected and used to evaluate the cut-off value and specificity. 2.1.2. T. annulata-positive serum samples A total of 196 serum samples were collected from domestic cattle in Gannan and Wuwei regions in China between 2017 and 2018. Of these serum samples, T. annulata-positive samples (n = 152) were determined using indirect ELISA (Tian et al., 2018). And an additional 90 T. annulata-positive serum samples were obtained from cattle, which were experimentally challenged with T. annulata (Kashi strain) (Ma et al., 2019) in our laboratory. Therefore, the obtained 242 T. annulata-positive serum samples were used to estimate the cut-off value and sensitivity of the CLIA. 2.1.3. Standard positive and negative serum samples T. annulata-positive serum samples obtained in our laboratory (Zhao et al., 2017; Tian et al., 2018) were tested using indirect ELISA (Tian et al., 2018) and IFA (Rajendran and Ray., 2014, the high titer positive serum samples were served as the standard positive samples. In addition, the T. annulata-negative serum samples, which were stored in our laboratory (Zhao et al., 2017; Tian et al., 2018) were tested using both indirect ELISAs (Zhao et al., 2017; Tian et al., 2018) and IFA (Rajendran and Ray., 2014), and marked as the standard negative samples. Both positive and negative serum samples were used to develop the CLIA method for detecting antibodies against T. annulata.
With the optimum coating antigen concentration, serum dilution, secondary antibody dilution, reaction time and other conditions were identified, T. annulata-negative sera (n = 158) and T. annulata -positive sera (n = 242) were detected with the CLIA method. The obtained data were used to calculate the percent positive (PP) using the formula based on the study (Liu et al., 2018). The cut-off value, specificity and sensitivity of the CLIA were evaluated by performing receiver-operating characteristic (ROC) interactive dot diagram analysis using MedCalc statistical software.
2.2. Preparation of the recombinant protein (SVSP450)
2.5. Validation of the CLIA
The histidine-tagged recombinant protein (SVSP450) was prepared by DETABIO Inc. (Nanjing, China) according to the SVSP950450 gene (SVSP450) sequence (GenBank accession no: XM950450). The obtained recombinant protein was analyzed by SDS–PAGE and western blotting with the anti-histidine tag monoclonal antibody (Sigma-Aldrich, Missouri, USA). The concentration of the protein was detected by the Bradford Protein assay, which was 0.446 mg/mL. The reactivity and cross-reactivity of the recombinant protein was tested with the T. annulata-positive sera and positive sera for T. sergenti, T. sinensis, Babesia bovis, and B. bigemina stored in our laboratory using western blotting. A total of 2 μg rSVSP450 protein was separated by 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Massachusetts, USA). The membrane was then cut into strips and blocked with 5% BSA (Amresco, Washington, USA) in TBST for 2 h at room temperature.
To evaluate the diagnostic performance of the developed CLIA assay, 180 field serum samples obtained from Tianzhu county in Gansu province were tested using both CLIA and rSpm2 ELISA to determine the concordance rate. In addition, four field collected serum samples from cattle in Xinjiang province, other four serum samples obtained from Hubei province were selected and detected in triplicate to evaluate the reproducibility of the CLIA under optimal parameters. The coefficients of variation (CV) of inter-and intra -assay for the CLIA were also measured.
2.4. Identification of the cut-off value, specificity and sensitivity of the assay
2.6. Serological epidemiology of field serum samples A total of 565 field-collected serum samples from Shanxi, Xinjiang, Hubei and Zhangye region of Gansu provinces were detected using the 2
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according to the checkerboard titration results. The blocking buffer used was PBS containing 5% gelatin and 5% horse serum, and the incubation time for the serum samples and secondary antibody was 10 min at 37 °C, respectively. The detection time required for the CLIA was only 25 min, excluding the coating time (2 h) and blocking time (2 h). Based on the PP values of T. annulata–negative sera (n = 158) and T. annulata–positive sera (n = 242), the specificity and sensitivity were determined. The cut-off value of the assay was evaluated by performing a ROC curve analysis (Fig. 3A and 3B). The area under the curve was 0.997 (standard error, SE = 0.00150), the 95% confidence interval was 0.986–1.000, and the Youden index value was 0.9621, using the ROC analysis in our study, which demonstrated that the CLIA showed good discriminatory power. The MedCalc statistics software revealed that the optimal values for Sn and Sp were 98.8% (95% CI: 96.4–99.7) and 97.5% (95% CI: 93.6–99.3), respectively when the cut-off value of PP for the CLIA was 26.1%. Field serum samples (n = 180) obtained from the Tianzhu county of Gansu province in 2017 were used to evaluate the diagnostic performances of the CLIA and the indirect ELISA. The results are shown in Table 1. The CLIA showed that the seroprevalence of T. annulata in cattle was 38.8% and was 30.6% for the rSpm2 ELISA. The concordance rate for the CLIA and ELISA was 72.8%. As shown in Tables 2 and 3, the intra-assay coefficient of variation (CV) of eight field serum samples was between 1.117% and 7.627%, while inter-assay coefficient of variation (CV) of these samples ranged from 1.423% to 10.528%.
Fig. 1.. Identification and analysis of purified rSVSP450 protein. (A) SDS-PAGE analysis of the recombinant protein. Lane M, PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, USA); Lane 1, the purified rSVSP450 protein (2 μg). (B) Western blotting analysis of the rSVSP450 protein (2 μg) with antihistidine (Anti-His) tag monoclonal antibody. Lane M, PageRuler Prestained Protein Ladder; lane 1, anti-histidine tag monoclonal antibody from mouse. Anti-His tag antibody and HRP-conjugated secondary antibody were both diluted in TBST containing 5% BSA at 1:5000.
CLIA in small scale. The CLIA protocol was conducted as described above. 3. Results
3.3. Small scale seroprevalence of tropical theileriosis based on the CLIA
3.1. Identification of the recombinant protein
In the present study, a total of 565 field collected serum samples from four provinces were detected for tropical theileriosis using the CLIA method. In Table 4, the results showed that 53.3% to 67.3% of the sera samples were positive for T. annulata infection.
The SDS-PAGE and western blotting results demonstrated that the recombinant SVSP450 protein had high purity and strong reaction with the anti-histidine tag antibody, which indicated the exact expression of the protein in E. coli (Fig. 1A and 1B). Specific test results showed that the recombinant protein had good immunoreactivity with T. annulatapositive sera, while no cross-reactivity with T. sergenti, T. sinensis, B. bovis, and B. bigemina-positive sera was observed (Fig. 2).
4. Discussion Although the indirect immunofluorescence assay (IFA) is the gold standard recommended by the OIE (2000) for the detection of T. annulata antibodies in cattle, this method is sophisticated, labor intensive and time-consuming compared with alternative methods. In previous studies, numerous serological assays, including indirect ELISAs and competitive ELISA targeting various antigen genes (Salih et al., 2005; Renneker et al., 2009; Abdo et al., 2010; Rajendran and Ray, 2014; Tian et al., 2018) for sero-monitoring Theileria infection, have been extensively established. Despite the development of an indirect ELISA
3.2. Development and evaluation of the CLIA Following optimization of the reaction conditions for the CLIA, the coating antigen concentration was determined to be 27.9 ng/mL that was much less than in other assays, while the optimal dilutions of the serum and secondary antibody were 1:10 and 1:80000, respectively,
Fig. 2.. Immunoreactivity analysis of the rSVSP450 protein with sera against bovine piroplasms. Lane M, PageRuler Prestained Protein Ladder; lane 1, positive sera against T. annulata; lane 2, positive sera against T. sergenti; lane 3, positive sera against T. sinensis; lane 4, positive sera against B. bigemina; lane 5, positive sera against B. bovis. Four different positive serum samples were diluted in TBST containing 5% BSA at 1:100, and secondary antibody was at the ratio of 1:10,000 in TBST containing 5% BSA. 3
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Fig. 3.. Validation of the CLIA by the ROC analysis. (A) Interactive dot diagram analysis. 0 = T. annulata-negative sera and 1 = T. annulata- positive sera; (B) Every point on ROC graph indicated the sensitivity and specificity at a particular criterion Table 1. Comparison of the CLIA and rSpm2 ELISA Serum samples information
Total samples
Cattle Concordance rate
CLIA Positive 70/180
180
Negative 110/180
a b c d
1a 7.9 11.6 49.3 111.8 70.4 29.8 99.2 45.6
2 6.8 12.4 50.5 110.6 70.7 31.7 99.8 47.2
3 7.2 11.1 55.2 115.5 71.9 33.4 98.4 46.0
Xb 7.3 11.7 51.6 112.6 71 31.6 99.1 46.2
SDc 0.556 0.655 3.118 2.554 0.793 1.801 0.702 0.832
CV%d 7.627 5.604 6.035 2.267 1.117 5.693 7.085 1.799
Percent positive value. Average percent positive value. Standard deviation. Coefficient of variation.
Table 3. Inter-batch reproducibility test of the CLIA Serum samples 1 2 3 4 5 6 7 8 a b c d
1a 9.1 15.2 58.1 107.5 62.4 42.1 86.5 32.7
2 9.9 13.9 56.4 104.6 65.2 44.6 83.4 28.4
3 11.2 14.3 48.7 106.8 63.9 45.9 82.6 30.9
Xb 10.1 14.5 54.4 106.3 63.8 44.2 84.1 30.7
SDc 1.059 0.665 5.008 1.513 1.401 1.931 2.059 2.159
Negative 125(91)/180
accession EU032540‐EU032577), and allelic variants were identified within individual parasite isolates and in isolates from different geographical regions (Schnittger et al., 2002; Ali et al., 2008). In addition, although the ELISA based on rSpm2 has good specificity and sensitivity, the assay was still needed to further validate its reliability (Tian et al., 2018). Therefore, a species-specific diagnostic antigen is urgently needed to investigate T. annulata infection in regions where the T. parva is not present. In our study, the results of western blotting demonstrated that the rSVSP450 protein has good immunoreactivity and no cross-reactivity with the other four bovine piroplasms positive serum samples. In addition, there is no related report or study for T. parva infection in China, and T. parva infection mainly occurs in East Africa. Therefore, rSVSP450 can be used as a specific sero-diagnostic candidate antigen to develop a serological assay for detecting T. annulata infection in areas where T. parva infection is not present. Compared with the rSpm2 ELISA, the CLIA based on rSVSP450 protein showed a significantly reduced assay time for investigating tropical theileriosis. We also evaluated the diagnostic performance of the CLIA using 180 field serum samples, which had a concordance rate of 72.8% with the ELISA. Whereas, it is still needed to further assess the correlation between CLIA and rSpm2 ELISA, even with IFA using more serum samples in the future research. On the basis of previous studies (Jacobson., 1996; Salih et al., 2005), the identified negative serum samples (n = 1000) and identified positive serum samples (n = 300) were used to compensate many factors, which may affect the specificity and sensitivity of diagnostic assays. In our study, only T. annulata positive serum samples (n = 242) and T. annulata negative serum samples (n = 158) were used to evaluate the cut-off value, specificity and sensitivity of the CLIA. Therefore, we need more positive and negative serum samples to calculate the above parameters of the developed CLIA in the future study. The detection results of serum samples from selected areas showed that the positive rate for T. annulata infection was ranged from 53.3% to 67.3%. Compared with a previous survey, which include different diagnostic assays, the prevalence of T. annulata was between 30% and
Table 2. Intra-batch reproducibility test of the CLIA Serum samples 1 2 3 4 5 6 7 8
rSpm2 ELISA Positive 55(40)/180 72.8% (131/180)
CV%d 10.528 4.602 9.207 1.423 2.195 4.369 2.447 7.041
Percent positive value. Average percent positive value. Standard deviation. Coefficient of variation.
method targeting the major piroplasm surface protein for detecting antibodies against bovine Theileria (T. annulata, T. sergenti, T. sinensis) infection in China (Zhao et al., 2017), this method cannot differentiate between different Theileria species in the case of coinfection. The T. annulatesurface protein (TaSP) gene was polymorphic (GenBank 4
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Table 4. Small-scale seroprevalence of T. annulata in China using CLIA Province Gansu Xinjiang Shaanxi Hubei
Region Zhangye Bole Yanglin Suizhou
Herds Cattle Cattle Cattle Cattle
Date 5/7/2017 18/4/2018 16/4/2018 25/4/2018
Total samples 263 183 60 59
Positive samples 177 118 32 36
Prevalence (%) 67.3 64.5 53.3 66.1
References
50% (Guo et al., 2013; Cao et al., 2011; Aihemaiti et al., 2017); thus, the seroprevalence of T. annulata in these areas has increased in recent years. Most notably, the seroprevalence of T. annulata infection in the serum samples (183) collected from the Bole region of Xinjiang province was 64.5% with the CLIA method, which is remarkably higher than that in the previous survey. Climate change, and more suitable breeding habits for ticks may be the reasons for the marked increase in the prevalence of tropical theileriosis in Xinjiang province. Our serological survey also revealed the high seroprevalence of T. annulata, which varied from 53.3% to 67.3% in the other three provinces, Gansu, Shaanxi and Hubei, which are located in western and central China. Based on previous studies, these three provinces are not areas of vectors prevalence for T. annulata. Therefore, the high positive rate for T. annulata in these areas might be due to increasing inter-provincial cattle transportation. On the other hand, the progressive distribution of T. annulata in various regions and latitudes has correlated with the unique geographical features of China. In addition, the extreme climate conditions due to rainfall, dry season, high humidity, environmental pollution, and potential interactions between vectors and cattle, have provided the ideal breeding conditions for the vectors, which may also be responsible for the spread of tropical theileriosis. Therefore, continuous serological surveillance is necessary to accurately evaluate the threat of T. annulata infection for susceptible cattle herds in China where T. parva infection is absence in the future. In conclusion, a CLIA method based on the rSVSP450 protein was developed in the present study, which showed high sensitivity, specificty, reproductivity and reliability for detecting antibodies against T. annulata. Compared with alternative ELISA methods, the CLIA method required a much shorter assay time, which is more suitable for epidemiological investigations of tropical theileriosis. The CLIA method can also be combined with other diagnostic assays, including etiological and serological techniques, and even new vaccines to facilitate the prevention and control of increasing serious tropical theileriosis.
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Ethical approval All of animal experiments in the study were approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. In the present study, all experimental animals used were dealt with according to the Animal Ethics Procedures and Guidelines of the People's Republic of China (SYXK2010-0001). Declaration of competing interest All authors declare that they have no competing conflicts of interest. Acknowledgement This study was supported by the grants of National Key Research and Development Program of China (2017YFD050403), 973 Program (2015CB150300), ASTIP(CAAS-ASTIP-2016-LVRI), and NBCIS(CARS37), Central Public-interest Scientific Institution Basal Research Fund (1610312016009), and Jiangsu Co-innovation Center programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses. 5
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s11250-015-0808-5. Sudan, V., Sharma, R.L., Yadav, R., Borah, M.K., 2014. Turning sickness in a riverine buffalo naturally infected with Theileria annulata and its successful therapeutic management. Comp. Clin. Pathol 23 (1), 39–42. https://doi.org/10.1007/s00580012-1566-7. Tian, Z.C., Du, X.Y., Du, J.Z., Gao, S.D., Yu, R.M., Muhammad, A.H., Liu, G.Y., Luo, J.X., Yin, H., 2018. Development of an indirect ELISA based on the recombinant spm2 protein for detection of tropical theileriosis. Acta Tropica 182, 232–236. https://doi. org/10.1016/j.actatropica.2018.03.003. Tretina, K., Gotia, H.T., Mann, D.J., Silva, J.C., 2015. Theileria-transformed bovine leukocytes have cancer hallmarks. Trends Parasitol 31 (7), 306–314. https://doi.org/10. 1016/j.pt.2015.04.001. Uilenberg, G., 1981. Theilerial species of domestic livestock. Advances in the control of Theileriosis. Springer, the Netherlands. Weir, W., Tülin, K., Baird, M., Tait, A., Shiels, B.R., 2010. Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata. BMC Genomics 11 (1), 42. https://doi.org/10.1186/1471-2164-11-42. Yang, L., Song, L.W., Fang, L.L., Wu, Y., Ge, S.X., Li, H., Yuan, Q., Zhang, J., Xi, N.S., 2016. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection. J. Virol. Methods 228, 55–59. https://doi.org/ 10.1016/j.jviromet.2015.11.013. Zhao, S.Y., Liu, J.L., Zhao, H.X., Li, Y.Q., Xie, J.R., Liu, A.H., Luo, J.X., Yin, H., 2017. Evaluating an indirect rmpsp enzyme-linked immunosorbent assay for the detection of bovine Theileria infection in China. Parasitol. Res 116 (2), 667–676. https://doi. org/10.1007/s00436-016-5332-7. Radositis, O.M., 1997. Veterinary medicine. a text book of the diseases of cattle, sheep, pigs, goats and horses[M]. Saunders, London.
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Development and evaluation of a chemiluminescence immunoassay for detecting tropical theileriosis
T
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Li Zhia, Liu Junlonga, , Ma Quanyinga, Xu Jianlina, Wang Jinminga, Liu Aihonga, Li Youquana, ⁎ Guan Guiquana, Luo Jianxuna, Yin Honga,b, a
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu, 730046, P. R. China b Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, P. R. China
A R T I C LE I N FO
A B S T R A C T
Keywords: Theileria annulata SVSP CLIA tropical theileriosis
Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection.
1. Introduction Theileria annulata is a tick-borne apicomplexan parasite causing tropical theileriosis, which predominantly infects cattle in tropical and subtropical areas around the world (Uilenberg et al., 1981; Bishop et al., 2004; Sivakumar et al., 2014). Although numerous Theileria species of tick-transmitted parasites have been identified in wild and domestic ruminants, only Theileria species (T. parva, T. annulata and T. lestoquardi) that have the ability to transform host cells, which are responsible for severe clinical diseases in animals (Mans et al., 2015; Nene and Morrison, 2016; Brown, 1997). In the worldwide, more than 0.25 billion cattle were infected with T. annulata, which led to economic losses of more than 0.8 billion US$ (Brown, 1997; Radositis et al., 1997;Sudan et al., 2014, 2015). To date, T. annulata is the only Theileria species to cause acute lymphoproliferative disease in China where T. parva is not present. The incidence and prevalence of tropical theileriosis ranges from 1.59% to 90% in endemic regions in China
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based on previous studies (Luo and Lu, 1997; Liu et al., 2015; Aihemaiti et al., 2017). Therefore, the disease causes substantial economic burden in the sustainable development of the livestock industry. In the late 1970s, the CLIA was developed and widely applied in numerous fields of life sciences (Halmann et al., 1977; Mirasoli and Michelini, 2014). Due to its high sensitivity and signal-to-noise ratio, low background fluorescence signal and wide linear range, the CLIA has been extensively used for diagnosing of various diseases, including hepatitis B virus (HBV) infection (Yang et al., 2016), human immunodeficiency virus (HIV) infection (Alonso et al., 2014; Cui et al., 2015), and foot and mouth disease virus (FMDV) infection (Liu et al., 2018). However, to date, there are no related reports on use of the CLIA method to detect T. annulata infection. The subtelomeric variable secreted proteins (SVSPs) family is the largest gene family in both T. parva and T. annulata, which may be associated with immune evasion, host transformation and invasion (Schmuckli et al., 2009; Weir et al., 2010; Tretina et al., 2015). The
Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (H. Yin).
https://doi.org/10.1016/j.actatropica.2019.105245 Received 25 August 2019; Received in revised form 24 October 2019; Accepted 24 October 2019 Available online 30 October 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
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SVSPs are mainly expressed in the T. annulata and T. parva schizont stage, and the majority of the SVSPs of T. annulata are secreted into the host cell cytoplasm (Pain et al., 2005; Shiels et al., 2006; Schmuckli et al., 2009). However, can the genes of this family be used a diagnostic antigen for tropical theileriosis? In the present study, a recombinant SVSP protein was used to develop a CLIA assay to detect tropical theileriosis, and the specificity and sensitivity of the CLIA were also evaluated. The diagnostic performance of the assay was determined by comparing it with ELISA. In addition, small-scale serological screening of serum samples from four endemic regions was conducted using the CLIA to further identify the reliability.
After washing the strips three times with TBST buffer for 10 min each time, the reference serum samples (1:100 dilution) were then added to the designed strips, and incubated overnight at 4 °C. Following washing with TBST buffer three times, the strips were probed with rabbit antibovine IgG secondary antibody (1:10000 dilution) (HRP-IgG; SigmaAldrich, Missouri, USA) at room temperature for 1 h. The strips were washed four times with TBST for 10 min each time, and the signal bands for the strips were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Massachusetts, USA).
2. Materials and methods
A checkerboard titration was performed in 96-well CLIA white plates (Thermo Fisher Scientific, Massachusetts, USA) to determine the optimum dilutions of the coating protein, serum, secondary antibody. The reaction time for serum and secondary antibody were selected at 5 min, 10 min, 15 min and 20 min for 37 °C, respectively. In addition, each screening assay contained standard positive and negative serum and blank controls. Briefly, the plate was coated with rSVSP450 protein (22.3 ng/well, 11.15 ng/well, 5.58 ng/well, and 2.79 ng/well) diluted in carbonated buffer (pH 9.6) and incubated at 4°C overnight or 37 °C for 2 h. After washing the plate with PBST, the wells were blocked with 5% gelatin (Sigma-Aldrich, Missouri, USA) and 5% horse serum (Gibco, New York, USA) in PBS buffer, and incubated at 37 °C for 2 h. After washing the plate with PBST three times, 100 μL of positive or negative standard sera diluted using the serum dilution buffer at the ratio of 1:10, 1:20, 1:40 and 1:80, respectively, were added to the wells and incubated at 37 °C for 10 min. After washing the plate three times with PBST, 100 μL of serial diluted HRP-conjugated rabbit anti-bovine IgG (1:10,000, 1:20,000, 1:40,000 and 1:80,000) was added to each well and incubated at 37 °C for 10 min. Finally, following five washes, 100 μL per/well of substrate solution A (Luminol; Thermo Fisher Scientific, Massachusetts, USA) and solution B (Luminol enhancer; Thermo Fisher Scientific, Massachusetts, USA) were added and incubated at room temperature for 5 min. The chemiluminescence (CL) values of the plate were measured with a VarioskanTM LUX Multimode Microplate Reader (Thermo Fisher Scientific, Massachusetts, USA).
2.3. Optimization of the CLIA protocol
2.1. Serum samples 2.1.1. T. annulata-negative serum samples Serum samples (n = 302) obtained from domestic cattle in Yuzhong and Wuwei regions of Gansu province in China during 2018 were detected using two indirect ELISA methods based on sporozoite and macroschizont gene 2 (Spm2) (GeneBank accession no: Y15795) protein and major piroplasma surface protein (MPSP) (GeneBank accession no: GQ180193.1) of T. sinensis, respectively (Tian et al., 2018; Zhao et al., 2017). Of these serum samples, 158 samples negative for T. annulata were selected and used to evaluate the cut-off value and specificity. 2.1.2. T. annulata-positive serum samples A total of 196 serum samples were collected from domestic cattle in Gannan and Wuwei regions in China between 2017 and 2018. Of these serum samples, T. annulata-positive samples (n = 152) were determined using indirect ELISA (Tian et al., 2018). And an additional 90 T. annulata-positive serum samples were obtained from cattle, which were experimentally challenged with T. annulata (Kashi strain) (Ma et al., 2019) in our laboratory. Therefore, the obtained 242 T. annulata-positive serum samples were used to estimate the cut-off value and sensitivity of the CLIA. 2.1.3. Standard positive and negative serum samples T. annulata-positive serum samples obtained in our laboratory (Zhao et al., 2017; Tian et al., 2018) were tested using indirect ELISA (Tian et al., 2018) and IFA (Rajendran and Ray., 2014, the high titer positive serum samples were served as the standard positive samples. In addition, the T. annulata-negative serum samples, which were stored in our laboratory (Zhao et al., 2017; Tian et al., 2018) were tested using both indirect ELISAs (Zhao et al., 2017; Tian et al., 2018) and IFA (Rajendran and Ray., 2014), and marked as the standard negative samples. Both positive and negative serum samples were used to develop the CLIA method for detecting antibodies against T. annulata.
With the optimum coating antigen concentration, serum dilution, secondary antibody dilution, reaction time and other conditions were identified, T. annulata-negative sera (n = 158) and T. annulata -positive sera (n = 242) were detected with the CLIA method. The obtained data were used to calculate the percent positive (PP) using the formula based on the study (Liu et al., 2018). The cut-off value, specificity and sensitivity of the CLIA were evaluated by performing receiver-operating characteristic (ROC) interactive dot diagram analysis using MedCalc statistical software.
2.2. Preparation of the recombinant protein (SVSP450)
2.5. Validation of the CLIA
The histidine-tagged recombinant protein (SVSP450) was prepared by DETABIO Inc. (Nanjing, China) according to the SVSP950450 gene (SVSP450) sequence (GenBank accession no: XM950450). The obtained recombinant protein was analyzed by SDS–PAGE and western blotting with the anti-histidine tag monoclonal antibody (Sigma-Aldrich, Missouri, USA). The concentration of the protein was detected by the Bradford Protein assay, which was 0.446 mg/mL. The reactivity and cross-reactivity of the recombinant protein was tested with the T. annulata-positive sera and positive sera for T. sergenti, T. sinensis, Babesia bovis, and B. bigemina stored in our laboratory using western blotting. A total of 2 μg rSVSP450 protein was separated by 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Massachusetts, USA). The membrane was then cut into strips and blocked with 5% BSA (Amresco, Washington, USA) in TBST for 2 h at room temperature.
To evaluate the diagnostic performance of the developed CLIA assay, 180 field serum samples obtained from Tianzhu county in Gansu province were tested using both CLIA and rSpm2 ELISA to determine the concordance rate. In addition, four field collected serum samples from cattle in Xinjiang province, other four serum samples obtained from Hubei province were selected and detected in triplicate to evaluate the reproducibility of the CLIA under optimal parameters. The coefficients of variation (CV) of inter-and intra -assay for the CLIA were also measured.
2.4. Identification of the cut-off value, specificity and sensitivity of the assay
2.6. Serological epidemiology of field serum samples A total of 565 field-collected serum samples from Shanxi, Xinjiang, Hubei and Zhangye region of Gansu provinces were detected using the 2
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according to the checkerboard titration results. The blocking buffer used was PBS containing 5% gelatin and 5% horse serum, and the incubation time for the serum samples and secondary antibody was 10 min at 37 °C, respectively. The detection time required for the CLIA was only 25 min, excluding the coating time (2 h) and blocking time (2 h). Based on the PP values of T. annulata–negative sera (n = 158) and T. annulata–positive sera (n = 242), the specificity and sensitivity were determined. The cut-off value of the assay was evaluated by performing a ROC curve analysis (Fig. 3A and 3B). The area under the curve was 0.997 (standard error, SE = 0.00150), the 95% confidence interval was 0.986–1.000, and the Youden index value was 0.9621, using the ROC analysis in our study, which demonstrated that the CLIA showed good discriminatory power. The MedCalc statistics software revealed that the optimal values for Sn and Sp were 98.8% (95% CI: 96.4–99.7) and 97.5% (95% CI: 93.6–99.3), respectively when the cut-off value of PP for the CLIA was 26.1%. Field serum samples (n = 180) obtained from the Tianzhu county of Gansu province in 2017 were used to evaluate the diagnostic performances of the CLIA and the indirect ELISA. The results are shown in Table 1. The CLIA showed that the seroprevalence of T. annulata in cattle was 38.8% and was 30.6% for the rSpm2 ELISA. The concordance rate for the CLIA and ELISA was 72.8%. As shown in Tables 2 and 3, the intra-assay coefficient of variation (CV) of eight field serum samples was between 1.117% and 7.627%, while inter-assay coefficient of variation (CV) of these samples ranged from 1.423% to 10.528%.
Fig. 1.. Identification and analysis of purified rSVSP450 protein. (A) SDS-PAGE analysis of the recombinant protein. Lane M, PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, USA); Lane 1, the purified rSVSP450 protein (2 μg). (B) Western blotting analysis of the rSVSP450 protein (2 μg) with antihistidine (Anti-His) tag monoclonal antibody. Lane M, PageRuler Prestained Protein Ladder; lane 1, anti-histidine tag monoclonal antibody from mouse. Anti-His tag antibody and HRP-conjugated secondary antibody were both diluted in TBST containing 5% BSA at 1:5000.
CLIA in small scale. The CLIA protocol was conducted as described above. 3. Results
3.3. Small scale seroprevalence of tropical theileriosis based on the CLIA
3.1. Identification of the recombinant protein
In the present study, a total of 565 field collected serum samples from four provinces were detected for tropical theileriosis using the CLIA method. In Table 4, the results showed that 53.3% to 67.3% of the sera samples were positive for T. annulata infection.
The SDS-PAGE and western blotting results demonstrated that the recombinant SVSP450 protein had high purity and strong reaction with the anti-histidine tag antibody, which indicated the exact expression of the protein in E. coli (Fig. 1A and 1B). Specific test results showed that the recombinant protein had good immunoreactivity with T. annulatapositive sera, while no cross-reactivity with T. sergenti, T. sinensis, B. bovis, and B. bigemina-positive sera was observed (Fig. 2).
4. Discussion Although the indirect immunofluorescence assay (IFA) is the gold standard recommended by the OIE (2000) for the detection of T. annulata antibodies in cattle, this method is sophisticated, labor intensive and time-consuming compared with alternative methods. In previous studies, numerous serological assays, including indirect ELISAs and competitive ELISA targeting various antigen genes (Salih et al., 2005; Renneker et al., 2009; Abdo et al., 2010; Rajendran and Ray, 2014; Tian et al., 2018) for sero-monitoring Theileria infection, have been extensively established. Despite the development of an indirect ELISA
3.2. Development and evaluation of the CLIA Following optimization of the reaction conditions for the CLIA, the coating antigen concentration was determined to be 27.9 ng/mL that was much less than in other assays, while the optimal dilutions of the serum and secondary antibody were 1:10 and 1:80000, respectively,
Fig. 2.. Immunoreactivity analysis of the rSVSP450 protein with sera against bovine piroplasms. Lane M, PageRuler Prestained Protein Ladder; lane 1, positive sera against T. annulata; lane 2, positive sera against T. sergenti; lane 3, positive sera against T. sinensis; lane 4, positive sera against B. bigemina; lane 5, positive sera against B. bovis. Four different positive serum samples were diluted in TBST containing 5% BSA at 1:100, and secondary antibody was at the ratio of 1:10,000 in TBST containing 5% BSA. 3
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Fig. 3.. Validation of the CLIA by the ROC analysis. (A) Interactive dot diagram analysis. 0 = T. annulata-negative sera and 1 = T. annulata- positive sera; (B) Every point on ROC graph indicated the sensitivity and specificity at a particular criterion Table 1. Comparison of the CLIA and rSpm2 ELISA Serum samples information
Total samples
Cattle Concordance rate
CLIA Positive 70/180
180
Negative 110/180
a b c d
1a 7.9 11.6 49.3 111.8 70.4 29.8 99.2 45.6
2 6.8 12.4 50.5 110.6 70.7 31.7 99.8 47.2
3 7.2 11.1 55.2 115.5 71.9 33.4 98.4 46.0
Xb 7.3 11.7 51.6 112.6 71 31.6 99.1 46.2
SDc 0.556 0.655 3.118 2.554 0.793 1.801 0.702 0.832
CV%d 7.627 5.604 6.035 2.267 1.117 5.693 7.085 1.799
Percent positive value. Average percent positive value. Standard deviation. Coefficient of variation.
Table 3. Inter-batch reproducibility test of the CLIA Serum samples 1 2 3 4 5 6 7 8 a b c d
1a 9.1 15.2 58.1 107.5 62.4 42.1 86.5 32.7
2 9.9 13.9 56.4 104.6 65.2 44.6 83.4 28.4
3 11.2 14.3 48.7 106.8 63.9 45.9 82.6 30.9
Xb 10.1 14.5 54.4 106.3 63.8 44.2 84.1 30.7
SDc 1.059 0.665 5.008 1.513 1.401 1.931 2.059 2.159
Negative 125(91)/180
accession EU032540‐EU032577), and allelic variants were identified within individual parasite isolates and in isolates from different geographical regions (Schnittger et al., 2002; Ali et al., 2008). In addition, although the ELISA based on rSpm2 has good specificity and sensitivity, the assay was still needed to further validate its reliability (Tian et al., 2018). Therefore, a species-specific diagnostic antigen is urgently needed to investigate T. annulata infection in regions where the T. parva is not present. In our study, the results of western blotting demonstrated that the rSVSP450 protein has good immunoreactivity and no cross-reactivity with the other four bovine piroplasms positive serum samples. In addition, there is no related report or study for T. parva infection in China, and T. parva infection mainly occurs in East Africa. Therefore, rSVSP450 can be used as a specific sero-diagnostic candidate antigen to develop a serological assay for detecting T. annulata infection in areas where T. parva infection is not present. Compared with the rSpm2 ELISA, the CLIA based on rSVSP450 protein showed a significantly reduced assay time for investigating tropical theileriosis. We also evaluated the diagnostic performance of the CLIA using 180 field serum samples, which had a concordance rate of 72.8% with the ELISA. Whereas, it is still needed to further assess the correlation between CLIA and rSpm2 ELISA, even with IFA using more serum samples in the future research. On the basis of previous studies (Jacobson., 1996; Salih et al., 2005), the identified negative serum samples (n = 1000) and identified positive serum samples (n = 300) were used to compensate many factors, which may affect the specificity and sensitivity of diagnostic assays. In our study, only T. annulata positive serum samples (n = 242) and T. annulata negative serum samples (n = 158) were used to evaluate the cut-off value, specificity and sensitivity of the CLIA. Therefore, we need more positive and negative serum samples to calculate the above parameters of the developed CLIA in the future study. The detection results of serum samples from selected areas showed that the positive rate for T. annulata infection was ranged from 53.3% to 67.3%. Compared with a previous survey, which include different diagnostic assays, the prevalence of T. annulata was between 30% and
Table 2. Intra-batch reproducibility test of the CLIA Serum samples 1 2 3 4 5 6 7 8
rSpm2 ELISA Positive 55(40)/180 72.8% (131/180)
CV%d 10.528 4.602 9.207 1.423 2.195 4.369 2.447 7.041
Percent positive value. Average percent positive value. Standard deviation. Coefficient of variation.
method targeting the major piroplasm surface protein for detecting antibodies against bovine Theileria (T. annulata, T. sergenti, T. sinensis) infection in China (Zhao et al., 2017), this method cannot differentiate between different Theileria species in the case of coinfection. The T. annulatesurface protein (TaSP) gene was polymorphic (GenBank 4
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Table 4. Small-scale seroprevalence of T. annulata in China using CLIA Province Gansu Xinjiang Shaanxi Hubei
Region Zhangye Bole Yanglin Suizhou
Herds Cattle Cattle Cattle Cattle
Date 5/7/2017 18/4/2018 16/4/2018 25/4/2018
Total samples 263 183 60 59
Positive samples 177 118 32 36
Prevalence (%) 67.3 64.5 53.3 66.1
References
50% (Guo et al., 2013; Cao et al., 2011; Aihemaiti et al., 2017); thus, the seroprevalence of T. annulata in these areas has increased in recent years. Most notably, the seroprevalence of T. annulata infection in the serum samples (183) collected from the Bole region of Xinjiang province was 64.5% with the CLIA method, which is remarkably higher than that in the previous survey. Climate change, and more suitable breeding habits for ticks may be the reasons for the marked increase in the prevalence of tropical theileriosis in Xinjiang province. Our serological survey also revealed the high seroprevalence of T. annulata, which varied from 53.3% to 67.3% in the other three provinces, Gansu, Shaanxi and Hubei, which are located in western and central China. Based on previous studies, these three provinces are not areas of vectors prevalence for T. annulata. Therefore, the high positive rate for T. annulata in these areas might be due to increasing inter-provincial cattle transportation. On the other hand, the progressive distribution of T. annulata in various regions and latitudes has correlated with the unique geographical features of China. In addition, the extreme climate conditions due to rainfall, dry season, high humidity, environmental pollution, and potential interactions between vectors and cattle, have provided the ideal breeding conditions for the vectors, which may also be responsible for the spread of tropical theileriosis. Therefore, continuous serological surveillance is necessary to accurately evaluate the threat of T. annulata infection for susceptible cattle herds in China where T. parva infection is absence in the future. In conclusion, a CLIA method based on the rSVSP450 protein was developed in the present study, which showed high sensitivity, specificty, reproductivity and reliability for detecting antibodies against T. annulata. Compared with alternative ELISA methods, the CLIA method required a much shorter assay time, which is more suitable for epidemiological investigations of tropical theileriosis. The CLIA method can also be combined with other diagnostic assays, including etiological and serological techniques, and even new vaccines to facilitate the prevention and control of increasing serious tropical theileriosis.
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Ethical approval All of animal experiments in the study were approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. In the present study, all experimental animals used were dealt with according to the Animal Ethics Procedures and Guidelines of the People's Republic of China (SYXK2010-0001). Declaration of competing interest All authors declare that they have no competing conflicts of interest. Acknowledgement This study was supported by the grants of National Key Research and Development Program of China (2017YFD050403), 973 Program (2015CB150300), ASTIP(CAAS-ASTIP-2016-LVRI), and NBCIS(CARS37), Central Public-interest Scientific Institution Basal Research Fund (1610312016009), and Jiangsu Co-innovation Center programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses. 5
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