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Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants Zhijie Liu a , Youquan Li a , Dia Eldin A. Salih b , Jianxun Luo a , Jabbar S. Ahmed c , Ulrike Seitzer c , Hong Yin a,∗ a State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Grazing Animal Diseases MOA, Key Laboratory of Veterinary Parasitology of Gansu Province, LVRI, CAAS, Lanzhou, PR China b Veterinary Research Institute, Khartoum, Sudan c Forschungszentrum Borstel, Parkallee 22, 23845 Borstel, Germany
a r t i c l e
i n f o
Article history: Received 13 July 2013 Received in revised form 8 March 2014 Accepted 5 May 2014
Keywords: rTuIP ELISA Theileria uilenbergi Theileria luwenshuni Validation
a b s t r a c t An enzyme-linked immunosorbent assay (ELISA) based on a recombinant Theileria uilenbergi immunodominant protein (rTuIP) was validated for detection of antibodies in 188 positive and 198 negative reference serum samples, respectively. The cut-off value was determined at 32.7% with 95% and 90% accuracy levels by two-graphic receiver-operating characteristic (TG-ROC). The equal diagnostic sensitivity (Se) and specificity (Sp) were calculated to be 98.4%. Further validation of the repeatability with positive and negative reference samples indicated the reliable performance of the assay. Monitoring the antibody dynamics of sheep experimentally infected with Theileria luwenshuni showed the efficient detection of antibody response against the pathogen at the early infection stage and up until two months post infection. Application of this assay for detection of antibody in field sera from previous unknown Theileria endemic regions in Suizhou and Guiyang showed 17.8% and 11.6% seroprevalence, respectively, and presence of the pathogen was confirmed by identification of the 18S rRNA gene in the corresponding blood of the seropositive animals. These data support that the rTuIP ELISA could be a useful tool to study the epidemiology of theileriosis caused by T. uilenbergi and/or T. luwenshuni. © 2014 Published by Elsevier B.V.
1. Introduction Theileria uilenbergi and Theileria luwenshuni are very closely related protozoan parasites, which cause theileriosis of small ruminants in the northwest of China (Yin et al., 2007). Both cause severe disease which is often lethal and responsible for a severe restriction for the development of the small ruminant livestock industry in its
∗ Corresponding author. Tel.: +86 931 8342515; fax: +86 931 8342515. E-mail address: [email protected] (H. Yin).
endemic regions, especially regarding the use of exotic animals (Luo and Yin, 1997; Yin et al., 2007). In recent years, the development of nuclear acid based diagnostic tools such as polymerase chain reaction (PCR) (Yin et al., 2008), reverse line blot (RLB) (Schnittger et al., 2004) and loopmediated isothermal amplification (LAMP) (Liu et al., 2008) has allowed not only to efficiently detect these pathogens, but also to differentiate between them. Likewise, several enzyme-linked immunosorbent assays (ELISA) have been established. The crude antigen (merozoite lysate) based ELISA showed high sensitivity for detection of antibodies against both T. uilenbergi and T. luwenshuni (Gao et al.,
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Please cite this article in press as: Liu, Z., et al., Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.010
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2002), but it also showed a cross-reaction with antibodies against Babesia ovis infection and has the inherent problem in obtaining large antigen amounts based on time intensive and expensive sophisticated procedures involving infection of experimental animals and purification of the merozoites. Contrarily, recombinant protein based ELISAs have the advantage of the preparation of large batches amenable to standardization and increased specificity. Three recombinant protein based ELISAs have been established for detection of small ruminant theileriosis in China, the antigens used were heat shock protein 70 from merozoite of Theileria sp. China (TcHSP70) (Miranda et al., 2006), T. uilenbergi immunodominant protein (TuIP) (Liu et al., 2010) and Clone 9 from a merozoite cDNA library of T. uilenbergi (Abdo et al., 2010). Among these tests, the recombinant TuIP (rTuIP) based ELISA showed a high sensitivity and was able to detect the early and persistent infection with T. uilenbergi. It was also the most extensively characterized regarding absence of cross reaction with related pathogens, found only to show cross-reactivity with antibody against the closely related pathogen T. luwenshuni (Liu et al., 2010). When six known T. luwenshuni positive serum samples were tested, four gave positive results while two remained negative (Liu et al., 2010). No serological test is on the horizon regarding the unambiguous and specific detection of T. uilenbergi or T. luwenshuni. As the pathogens are very closely related, a serological assay detecting both is nonetheless helpful in assessing prevalence and carrying out epidemiological surveys of small ruminant theileriosis. The aim of the present study was to further analyse the rTuIP ELISA for detection of the antibody response of T. luwenshuni infection in experimentally infected animals and to validate the test for field study by determining the cut-off value using two graphic receiver operating characteristic (TG-ROC), and finally to apply this method on field samples from a region previously unknown to be endemic for small ruminant theileriosis caused by T. uilenbergi or T. luwenshuni.
For testing repeatability and stability, 10 positive sera of sheep experimentally infected with T. uilenbergi from previous reports were used (Seitzer et al., 2008; Liu et al., 2010). They were serum 58 and 70 days post infection from five sheep experimentally infected either by inoculating blood infected with T. uilenbergi Lintan isolate (animals 1236, 1219) or infected by attachment of ticks collected in an endemic region (animals 1229, 1250, 1240). The corresponding negative serum samples were collected before experimental infection using the criteria mentioned above. These samples were divided into aliquots for evaluation of the repeatability and stability of the rTuIP ELISA. The animal experiments were performed in Lanzhou Veterinary Research Institute according to the approved institutional animal care and use committee guidelines. Each of sheep Nos. 14, 30, 94 and 135 was infected by inoculation of 5 ml blood infected with T. luwenshuni Wenyuan isolate, which was originally isolated from an ill sheep in Wenyuan County in Gansu Province and then identified as T. luwenshuni by PCR (Yin et al., 2008). Each of sheep Nos. 15, 39, 90 and 133 was infected by attaching 50 adult Haemaphysalis qinghaiensis ticks which were molted from clean nymphs engorged on a T. luwenshuni Wenyuan isolate infected sheep. The successful infection of these animals was confirmed by microscopic detection of the merozoites in the Giemsa-stained thin blood smears and by PCR detection of T. luwenshuni genomic DNA in the blood using a previously described assay (Yin et al., 2008). The field sera and corresponding blood samples from Guizhou (n = 95, goats) and Hubei (n = 45, goats) were collected in 2010. DNA was extracted from the fieldblood samples using genomic DNA extraction kit (Qiagen, Germany) following the manufacturer’s instructions. The field samples from Tianzhu (n = 39, sheep) and Madang (n = 67, sheep) in Gansu Province were collected in 2005 in areas known as endemic regions of T. uilenbergi and T. luwenshuni. 2.2. ELISA protocol
2. Materials and methods 2.1. Source of samples The reference positive samples were 188 field ovine sera from a main Theileria endemic region, Lintan County in Gansu Province, and these samples were tested positive by the merozoite antigen ELISA (Guo et al., 2007). The reference negative samples were 198 ovine sera from experimental sheep from 2007 to 2012. These animals were purchased from commercial farms in a Theileria nonendemic region, Jingtai County in Gansu Province. They were born in winter (November–January), which guarantees they were free from ticks. The animals were free of piroplasms as confirmed by microscopic examination of blood smears and PCR amplification of genomic DNA isolated from blood samples for detection of Theileria (Yin et al., 2008), Babesia (Liu et al., 2007) and Anaplasma (de la Fuente et al., 2007) before use. Both positive and negative reference samples were screened for normality using the d-Kolmogorov–Smirnov test.
The ELISA procedure was described previously by Liu et al. (2010). Briefly, 100 l of sera diluted 1:400 in diluent buffer (1% BSA (bovine serum albumin) in PBS (phosphate buffered saline: 137 mM NaCl, 10 mM Na2 HPO4 , 2.7 mM KCl, pH 7.4)) were applied in duplicate for testing field samples and in triplicate for evaluation of repeatability in the coated plates. Control positive (C+) and control negative (C−) sera were applied in four replicates and four wells were always used as a conjugate control (CC), receiving only the diluent buffer. For the second antibody, 100 l of a peroxidase conjugate of monoclonal anti-goat/sheep IgG clone GT-34 (A-9452, Sigma) diluted 1:15,000 in the diluent buffer was added to all the wells and incubated. After washing, 100 l of freshly prepared substrate solution [H2 O2 /tetramethylbenzi-dine (TMB, T0440-1L, Sigma, USA) in citric acid buffer (pH 4.0)] was added for color development and 100 l 1 N H2 SO4 was added for terminating the reaction. Finally, the absorbance at 450 nm was measured using an ELISA reader (microplate reader Model 680, Bio-Rad, USA).
Please cite this article in press as: Liu, Z., et al., Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.010
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Fig. 1. TG-ROC analysis of the rTuIP ELISA results using 188 positive and 198 negative reference serum samples. The intermediate range (IR) is determined by the cut-off values at 95% sensitivity (Se) and 95% specificity (Sp). The accuracy level is shown as a horizontal line.
The obtained optical density (OD) values were expressed as percent positivity (PP) of the internal positive control as follows: [(average OD value of testing samples − average OD value of blank controls)/(average OD value of the positive controls − average OD value of blank controls)] × 100 (Liu et al., 2010). For repeatability and stability testing, three batches of ELISA plates (10 in each batch) were coated on three different days and incubated at 4 ◦ C overnight. The plates were then washed and dried in a fume hood. The dried plates were sealed and stored at 4 ◦ C until stability testing every three months. In each test, duplicate plates were used for testing the 10 positive and 10 negative serum samples. The obtained OD values were converted to percent positivity and the overall mean and standard deviation were calculated. Comparison of the means in all tests was conducted using one-way ANOVA.
amplification was performed in an automatic DNA thermocycler (PE Company, USA) with a final volume of 30 l containing 3 l of 10 × PCR buffer (10 mM TRIS–HCl, 50 mM KCl, 1.5 mM MgCl2, 0.01%gelatin), 200 M of each deoxynucleoside triphosphate (dNTP), 10 M of each primer, 100 ng of genomic DNA and 1.5 U of Taq polymerase. The cycling conditions of the PCR were 3 min at 94 ◦ C for denaturation followed by 35 cycles at 94 ◦ C for 30 s, 59 ◦ C for 1 min and 72 ◦ C for 1 min. The final extension step was 7 min at 72 ◦ C. The PCR products were visualized by UV transillumination in a 1.0% agarose gel containing 0.5 g/ml ethidium bromide. The positive PCR fragments were cloned into pGEM-T vector (Promega, Shanghai, China) and subjected to sequencing in Sangon Biotech Company (Shanghai, China) for analysis of sequences.
2.3. Cut-off determination
3.1. Cut-off determination using TG-ROC
The 188 positive and 198 negative reference samples were tested using the above described ELISA protocol. The OD value was converted to positive percentage and these data were used for the cut-off determination using TG-ROC analysis of the computational methods for diagnostic tests (CMDT) software (Greiner et al., 1995; Salih et al., 2005). In this method, the specificity and sensitivity were plotted against the cut-off and the point of intersection of the two curves was taken as the value of the cut-off where both specificity and sensitivity are equal. Two alternative cutoff values, which were the lower and the upper limits of the intermediate range (IR) were defined at an accuracy level of 90 and 95%, and the valid range proportion (VRP) was calculated. The defined cut-off was expressed using percent positivity.
The results of the d-Kolmogorov–Smirnov test indicated that the OD values of the positive and negative reference samples were not normally distributed; therefore, the cut-off, specificity and sensitivity of the rTuIP ELISA were calculated using the non-parametric approach in the TG-ROC analysis. The cut-off was found to be 32.7% using percent positivity (PP) values and both the specificity and sensitivity were 98.4% (Fig. 1). The valid range proportion (VRP) was 1.052 and the intermediate range (IR) was 0 at 95% confidence interval (CI), while at 90% CI they were 1 and 0, respectively. The overlap between positive and negative reference samples is shown in Fig. 2.
2.4. PCR and sequencing The PCR with primers 989/rDNA-AS and rDNA-S/990 (Allsopp et al., 1993; Yin et al., 2004) was applied to verify for the presence of T. uilenbergi or T. luwenshuni in the corresponding DNA samples of ELISA-positive field samples from Guizhou and Hubei provinces. PCR
3. Results
3.2. Repeatability and stability of the rTuIP ELISA Fig. 3 illustrates the PP values of the same negative and positive sera obtained from twelve different tests. The PP values of the negative sera remained negative and the positive sera remained positive in the tests which were repeated over a period of nine months on three different batches of antigen-coated plates. Comparison of the means was conducted with one-way ANOVA. The variation for the positive samples among months 1, 3, 6 was not significant
Please cite this article in press as: Liu, Z., et al., Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.010
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deposited in the Genbank under accession Nos. HQ844674, JQ923446, JQ923447. The BLAST alignment in the NCBI (Altschul et al., 1990) with these sequences showed 99% identity to the sequences of T. luwenshuni (Genbank accession Nos. AY262115, AY262118). 4. Discussion
Fig. 2. Scatter plot of percentage positivity (PP) rTuIP ELISA test results illustrating the overlap between positive and negative values according to the cut-off value used (32.7%). A total of 188 positive and 198 negative reference sera were tested.
Fig. 3. Repeatability and stability of three batches of rTuIP ELISA (1, 2, 3) tested every three months using 10 known positive and 10 known negative sera. Percent positivity distribution is indicated by the mean ± standard deviation.
(P > 0.05); and significant difference for negative samples in the same month was not observed but between months was observed (P < 0.01). 3.3. Use of rTuIP ELISA for testing antibody response to infection with T. luwenshuni All the experimental animals showed the presence of specific antibodies against rTuIP protein within 3 weeks after inoculation with T. luwenshuni-infected blood or infestation with T. luwenshuni-infected ticks. The titer of the antibodies declined at around 8 weeks post infection (Fig. 4). 3.4. Application of rTuIP in field sample investigation The sample locations are as shown in Fig. 5, the corresponding test results are shown in Table 1. Eight out of 45 samples (17.8%) tested positive in Suizhou in Central China, 11 out of 95 (11.6%) were positive in Guiyang in Southwest China. In the known endemic region, 38 out of 39 samples were positive in Tianzhu with a positivity of 97.4% and 65 out of 67 were positive in Madang with a positivity of 97.0%. Corresponding DNA samples for the positive samples from Suizhou and Guiyang were tested by the PCR (Yin et al., 2004). One PCR product from Suizhou and two PCR products from Guiyang samples were sequenced, and the obtained 18S rRNA sequences were
It is well recognized that sensitivity (Se) and specificity (Sp) of ELISA are inversely related depending on the choice of the cut-off value (Greiner and Gardner, 2000). When increasing values of a measurement are associated with disease, higher cut-off values are generally associated with lower Se and a higher Sp (and vice versa, i.e. lower cut-off values are associated with higher Se and lower SP) (Greiner and Gardner, 2000). In our previous study, the cut-off of the rTuIP ELISA was derived using the Gaussian (normal) distribution method, which is defined as the mean plus two standard deviations (2SD) of the negative reference sample. The rationale of the 2SD procedure is to establish a cut-off value providing a Sp of 97.5% (Greiner et al., 2000). The procedure is clearly not adequate if the test values follow a skewed or multimodal distribution, and the other disadvantage is that it does not consider the Se (Greiner et al., 2000). The TG-ROC, an approach modified from the conventional ROC was applied in this study. In this method, the cut-off is defined based on the frequency distributions (Jacobson, 1996) and the test Se and Sp can be read directly from scatterplots of the data. The rTuIP ELISA is intended for investigation of the seroprevalence of small ruminant theileriosis caused by T. uilenbergi or T. luwenshuni in regions where prevalence of the disease is unknown. Thus we assume that the prevalence in the target population is about 50% and that the costs of false-positive and false-negative test results are equivalent. With this criterion, the cut-off was set at 32.7% using the PP values and both Se and Sp were 98.4%. Giving equal weights to Se and Sp fully exploits the information provided by the diagnostic test (Greiner et al., 2000). The determined cut-off was considered to be suitable for field investigation according to the OIE recommendations (Norval, 1992). The TuIP was derived from a merozoite cDNA library of T. uilenbergi. When the rTuIP ELISA was previously used for detecting antibodies from T. luwenshuni infected animals, four of six known infected animals were tested positive (Liu et al., 2010). It was presumed that TuIP might share similar epitopes in T. luwenshuni due to its close phylogenetic relationship to T. uilenbergi (Schnittger et al., 2003; Yin et al., 2004). When a batch of TuIP specific PCR primers was used to identify the TuIP gene from both T. uilenbergi and T. luwenshuni, positive results were only observed in T. uilenbergi (data not shown), thus the identification of an orthologous gene in T. luwenshuni remains to be reached. This point will hopefully be clarified with the accomplishing of a genome project of T. luwenshuni. Testing of further sera from animals experimentally infected with T. luwenshuni in this study indicated that specific anti-rTuIP antibodies can be recorded at the third week after infection. In confirmation of the previous study (Liu et al., 2010), however, a low antibody titer in some of the experimental animals indicates a low analytical Se of the
Please cite this article in press as: Liu, Z., et al., Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.010
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Fig. 4. Specific anti-rTuIP serologic response over time in sheep experimentally infected with Theileria luwenshuni Wenyuan isolate as assessed in the ELISA. Sheep Nos. 14, 30, 94, 135 were infected by blood inoculation with T. luwenshuni Wenyuan isolate. Sheep Nos. 15, 39, 90, 133 were infected by attaching 50 adult Haemaphysalis qinghaiensis ticks which were moulted from clean nymphs engorged on a T. luwenshuni Wenyuan isolate infected sheep.
Table 1 Application of rTuIP ELISA for the detection of antibody to Theileria uilenbergi and/or T. luwenshuni in field sera (caprine and ovine) from previous unknown and known Theileria endemic regions. Sampling site (province/location)
Sample size
ELISA positive (%)
Confirmation
Hubei* Guizhou* Gansu
45 (Caprine) 95 (Caprine) 39 (Ovine) 67 (Ovine)
8 (17.8) 11 (11.6) 38 (97.4) 65 (97.0)
PCR, sequencing PCR, sequencing PCR, microscopic Examination of blood films
*
Suizhou Guiyang Tianzhu Madang
Previously unknown endemic regions.
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Fig. 5. Map of the main endemic region of Theileria uilenbergi and T. luwenshuni and the indication of the sampling sites for this study.
rTuIP ELISA in detection of T. luwenshuni infection, and false negative results can be anticipated. Moreover, the antibody titer dropped close or down to the cut-off value at the 9th week post infection, indicating that this ELISA is only efficient for detection of anti-T. luwenshuni antibody within two months post infection. This period is shorter than that described for the rTuIP ELISA for the detection of T. uilenbergi-specific antibody, which is possible for at least three months after infection (Liu et al., 2010), or the Tams-ELISA that can detect T. annulata antibody one year post infection (Gubbels et al., 2000). Thus, when applying the rTUIP ELISA for the study of the presence of small ruminant theileriosis in the field, it has to be kept in mind that the prevalence may be underestimated. The manner of testing the repeatability and stability of the rTuIP was to give insight into whether antigencoated plates can be preserved and applied in field studies for detection of samples from different sites at different time points. Although all positive reference samples and all negative reference samples were tested as such for the different time points, significantly increasing values from the negative reference samples was observed. Application of the preserved plates for field sample investigation might thus bear the risk of giving false positive results if stored for long periods of time. However, preserved plates with the advantages of using homogenous coating antigen and easy-to-share between laboratories is still promising for application in field sample investigations, where storage conditions need to be investigated for optimization of shelf life. Although theileriosis caused by T. luwenshuni and T. uilenbergi has to date only been reported in the northwest of China (Luo and Yin, 1997), the identification of the 18S rRNA sequences from field samples from different hosts have indicated that similar parasites might be present in Spain (Nagore et al., 2004), Japan (Ikawa et al., 2011), South Korea (Han et al., 2009) as well as in Central and South China (Ge et al., 2012; He et al., 2012; Li et al., 2012). A validated rTuIP ELISA as presented in this study represents a possible
alternative tool for serological investigation for the presence of these parasites worldwide if they are confirmed to be T. uilenbergi or T. luwenshuni. Application of the rTuIP ELISA for testing the sera from sheep in known endemic regions, Madang, Tianzhu in Gansu Province, showed about 97% positivity, which is consistent with previous investigation results (Guo et al., 2007; Liu et al., 2010). Application of the assay in Suizhou of Hubei Province and Guiyang of Guizhou Province showed 17.8% and 11.6% seroprevalence of Theileria infection in the investigated sites. Successful identification of the Theileria 18S rRNA gene from the corresponding blood DNA of the Theileria serum positive samples confirmed the accuracy of the rTuIP ELISA. Haemaphysalis longicornis, the transmitting tick vector of ovine Theileria, has also been found in Suizhou in Hubei Province, indicating that Suizhou is a potential new endemic region of small ruminant theileriosis; the transmission vector in Guizhou Province remains to be elucidated in future studies. In conclusion, the validated rTuIP ELISA can be regarded as a useful tool for studying the epidemiology of theileriosis caused by T. uilenbergi and T. luwenshuni. It needs to be emphasized that the ELISA has been validated using the negative samples from animals under controlled conditions which may not represent the actual field situation. It will thus be of great interest to further validate this method using samples from larger numbers of animals kept under field conditions.
Acknowledgments The research was financially supported by PIROVAC (KBBE-3-245145) and EPIZONE (FOOD-CT-2006-016236) of the European Commission, Brussels, Belgium. This study was also facilitated by NSFC(No. 30800820; No. 30972182, No. 31072130, No. 31001061), the 973 Program (2010CB530206), the Key Project of Gansu Province (1002NKDA035), the NBCITS MOA, (CARS-38), Specific Fund for Sino-Europe Cooperation, MOST China, State Key Laboratory of Veterinary Etiological Biology Project (SKLVEB2008ZZKT019).
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Liu, Z., Wang, Z., Yin, H., Luo, J., Zhang, B., Kullmann, B., Abdo, J., Salih, D., Ahmed, J., Seitzer, U., 2010. Identification of Theileria uilenbergi immunodominant protein for development of an indirect ELISA for diagnosis of ovine theileriosis. Int. J. Parasitol. 40, 591–598. Luo, J., Yin, H., 1997. Theileriosis of sheep and goats in China. Trop. Anim. Health Prod. 29, 8S–10S. Miranda, J., Bakheit, M.A., Liu, Z., Yin, H., Mu, Y., Guo, S., Beyer, D., Oliva, A., Ahmed, J.S., Seitzer, U., 2006. Development of a recombinant indirect ELISA for the diagnosis of Theileria sp. (China) infection in small ruminants. Parasitol. Res. 98, 561–567. Nagore, D., Garcia-Sanmartin, J., Garcia-Perez, A.L., Juste, R.A., Hurtado, A., 2004. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int. J. Parasitol. 34, 1059–1067. Norval, R.A., 1992. In: Perry, B.D., Young, A.S. (Eds.), The Epidemiology of Theileriosis in Africa. Academic Press, New York, NY, p. 481. Salih, D.E., Ahmed, J.S., Bakheit, M.A., Ali, E.B., El Hussein, A.M., Hassan, S.M., Shariff, O.E., Fadl, M., Jongejan, F., 2005. Validation of the indirect TaSP enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle. Parasitol. Res. 97, 302–308. Schnittger, L., Yin, H., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S., 2003. Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol. Res. 91, 398–406. Schnittger, L., Yin, H., Qi, B., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S., 2004. Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. Parasitol. Res. 92, 189–196. Seitzer, U., Liu, Z., Yin, H., Beyer, D., Kullmann, B., Miranda, J., Ahmed, J., 2008. Immune response of Theileria sp.-infected sheep to recombinant Theileria proteins. Ann. N.Y. Acad. Sci. 1149, 186–190. Yin, H., Liu, Z., Guan, G., Liu, A., Ma, M., Ren, Q., Luo, J., 2008. Detection and differentiation of Theileria luwenshuni and T. uilenbergi infection in small ruminants by PCR. Transbound. Emerg. Dis. 55, 233–237. Yin, H., Luo, J., Schnittger, L., Lu, B., Beyer, D., Ma, M., Guan, G., Bai, Q., Lu, C., Ahmed, J., 2004. Phylogenetic analysis of Theileria species transmitted by Haemaphysalis qinghaiensis. Parasitol. Res. 92, 36–42. Yin, H., Schnittger, L., Luo, J., Seitzer, U., Ahmed, J.S., 2007. Ovine theileriosis in China: a new look at an old story. Parasitol. Res. 101 (Suppl 2), S191L S195.
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Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants Zhijie Liu a , Youquan Li a , Dia Eldin A. Salih b , Jianxun Luo a , Jabbar S. Ahmed c , Ulrike Seitzer c , Hong Yin a,∗ a State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Grazing Animal Diseases MOA, Key Laboratory of Veterinary Parasitology of Gansu Province, LVRI, CAAS, Lanzhou, PR China b Veterinary Research Institute, Khartoum, Sudan c Forschungszentrum Borstel, Parkallee 22, 23845 Borstel, Germany
a r t i c l e
i n f o
Article history: Received 13 July 2013 Received in revised form 8 March 2014 Accepted 5 May 2014
Keywords: rTuIP ELISA Theileria uilenbergi Theileria luwenshuni Validation
a b s t r a c t An enzyme-linked immunosorbent assay (ELISA) based on a recombinant Theileria uilenbergi immunodominant protein (rTuIP) was validated for detection of antibodies in 188 positive and 198 negative reference serum samples, respectively. The cut-off value was determined at 32.7% with 95% and 90% accuracy levels by two-graphic receiver-operating characteristic (TG-ROC). The equal diagnostic sensitivity (Se) and specificity (Sp) were calculated to be 98.4%. Further validation of the repeatability with positive and negative reference samples indicated the reliable performance of the assay. Monitoring the antibody dynamics of sheep experimentally infected with Theileria luwenshuni showed the efficient detection of antibody response against the pathogen at the early infection stage and up until two months post infection. Application of this assay for detection of antibody in field sera from previous unknown Theileria endemic regions in Suizhou and Guiyang showed 17.8% and 11.6% seroprevalence, respectively, and presence of the pathogen was confirmed by identification of the 18S rRNA gene in the corresponding blood of the seropositive animals. These data support that the rTuIP ELISA could be a useful tool to study the epidemiology of theileriosis caused by T. uilenbergi and/or T. luwenshuni. © 2014 Published by Elsevier B.V.
1. Introduction Theileria uilenbergi and Theileria luwenshuni are very closely related protozoan parasites, which cause theileriosis of small ruminants in the northwest of China (Yin et al., 2007). Both cause severe disease which is often lethal and responsible for a severe restriction for the development of the small ruminant livestock industry in its
∗ Corresponding author. Tel.: +86 931 8342515; fax: +86 931 8342515. E-mail address: [email protected] (H. Yin).
endemic regions, especially regarding the use of exotic animals (Luo and Yin, 1997; Yin et al., 2007). In recent years, the development of nuclear acid based diagnostic tools such as polymerase chain reaction (PCR) (Yin et al., 2008), reverse line blot (RLB) (Schnittger et al., 2004) and loopmediated isothermal amplification (LAMP) (Liu et al., 2008) has allowed not only to efficiently detect these pathogens, but also to differentiate between them. Likewise, several enzyme-linked immunosorbent assays (ELISA) have been established. The crude antigen (merozoite lysate) based ELISA showed high sensitivity for detection of antibodies against both T. uilenbergi and T. luwenshuni (Gao et al.,
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Please cite this article in press as: Liu, Z., et al., Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.010
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2002), but it also showed a cross-reaction with antibodies against Babesia ovis infection and has the inherent problem in obtaining large antigen amounts based on time intensive and expensive sophisticated procedures involving infection of experimental animals and purification of the merozoites. Contrarily, recombinant protein based ELISAs have the advantage of the preparation of large batches amenable to standardization and increased specificity. Three recombinant protein based ELISAs have been established for detection of small ruminant theileriosis in China, the antigens used were heat shock protein 70 from merozoite of Theileria sp. China (TcHSP70) (Miranda et al., 2006), T. uilenbergi immunodominant protein (TuIP) (Liu et al., 2010) and Clone 9 from a merozoite cDNA library of T. uilenbergi (Abdo et al., 2010). Among these tests, the recombinant TuIP (rTuIP) based ELISA showed a high sensitivity and was able to detect the early and persistent infection with T. uilenbergi. It was also the most extensively characterized regarding absence of cross reaction with related pathogens, found only to show cross-reactivity with antibody against the closely related pathogen T. luwenshuni (Liu et al., 2010). When six known T. luwenshuni positive serum samples were tested, four gave positive results while two remained negative (Liu et al., 2010). No serological test is on the horizon regarding the unambiguous and specific detection of T. uilenbergi or T. luwenshuni. As the pathogens are very closely related, a serological assay detecting both is nonetheless helpful in assessing prevalence and carrying out epidemiological surveys of small ruminant theileriosis. The aim of the present study was to further analyse the rTuIP ELISA for detection of the antibody response of T. luwenshuni infection in experimentally infected animals and to validate the test for field study by determining the cut-off value using two graphic receiver operating characteristic (TG-ROC), and finally to apply this method on field samples from a region previously unknown to be endemic for small ruminant theileriosis caused by T. uilenbergi or T. luwenshuni.
For testing repeatability and stability, 10 positive sera of sheep experimentally infected with T. uilenbergi from previous reports were used (Seitzer et al., 2008; Liu et al., 2010). They were serum 58 and 70 days post infection from five sheep experimentally infected either by inoculating blood infected with T. uilenbergi Lintan isolate (animals 1236, 1219) or infected by attachment of ticks collected in an endemic region (animals 1229, 1250, 1240). The corresponding negative serum samples were collected before experimental infection using the criteria mentioned above. These samples were divided into aliquots for evaluation of the repeatability and stability of the rTuIP ELISA. The animal experiments were performed in Lanzhou Veterinary Research Institute according to the approved institutional animal care and use committee guidelines. Each of sheep Nos. 14, 30, 94 and 135 was infected by inoculation of 5 ml blood infected with T. luwenshuni Wenyuan isolate, which was originally isolated from an ill sheep in Wenyuan County in Gansu Province and then identified as T. luwenshuni by PCR (Yin et al., 2008). Each of sheep Nos. 15, 39, 90 and 133 was infected by attaching 50 adult Haemaphysalis qinghaiensis ticks which were molted from clean nymphs engorged on a T. luwenshuni Wenyuan isolate infected sheep. The successful infection of these animals was confirmed by microscopic detection of the merozoites in the Giemsa-stained thin blood smears and by PCR detection of T. luwenshuni genomic DNA in the blood using a previously described assay (Yin et al., 2008). The field sera and corresponding blood samples from Guizhou (n = 95, goats) and Hubei (n = 45, goats) were collected in 2010. DNA was extracted from the fieldblood samples using genomic DNA extraction kit (Qiagen, Germany) following the manufacturer’s instructions. The field samples from Tianzhu (n = 39, sheep) and Madang (n = 67, sheep) in Gansu Province were collected in 2005 in areas known as endemic regions of T. uilenbergi and T. luwenshuni. 2.2. ELISA protocol
2. Materials and methods 2.1. Source of samples The reference positive samples were 188 field ovine sera from a main Theileria endemic region, Lintan County in Gansu Province, and these samples were tested positive by the merozoite antigen ELISA (Guo et al., 2007). The reference negative samples were 198 ovine sera from experimental sheep from 2007 to 2012. These animals were purchased from commercial farms in a Theileria nonendemic region, Jingtai County in Gansu Province. They were born in winter (November–January), which guarantees they were free from ticks. The animals were free of piroplasms as confirmed by microscopic examination of blood smears and PCR amplification of genomic DNA isolated from blood samples for detection of Theileria (Yin et al., 2008), Babesia (Liu et al., 2007) and Anaplasma (de la Fuente et al., 2007) before use. Both positive and negative reference samples were screened for normality using the d-Kolmogorov–Smirnov test.
The ELISA procedure was described previously by Liu et al. (2010). Briefly, 100 l of sera diluted 1:400 in diluent buffer (1% BSA (bovine serum albumin) in PBS (phosphate buffered saline: 137 mM NaCl, 10 mM Na2 HPO4 , 2.7 mM KCl, pH 7.4)) were applied in duplicate for testing field samples and in triplicate for evaluation of repeatability in the coated plates. Control positive (C+) and control negative (C−) sera were applied in four replicates and four wells were always used as a conjugate control (CC), receiving only the diluent buffer. For the second antibody, 100 l of a peroxidase conjugate of monoclonal anti-goat/sheep IgG clone GT-34 (A-9452, Sigma) diluted 1:15,000 in the diluent buffer was added to all the wells and incubated. After washing, 100 l of freshly prepared substrate solution [H2 O2 /tetramethylbenzi-dine (TMB, T0440-1L, Sigma, USA) in citric acid buffer (pH 4.0)] was added for color development and 100 l 1 N H2 SO4 was added for terminating the reaction. Finally, the absorbance at 450 nm was measured using an ELISA reader (microplate reader Model 680, Bio-Rad, USA).
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Fig. 1. TG-ROC analysis of the rTuIP ELISA results using 188 positive and 198 negative reference serum samples. The intermediate range (IR) is determined by the cut-off values at 95% sensitivity (Se) and 95% specificity (Sp). The accuracy level is shown as a horizontal line.
The obtained optical density (OD) values were expressed as percent positivity (PP) of the internal positive control as follows: [(average OD value of testing samples − average OD value of blank controls)/(average OD value of the positive controls − average OD value of blank controls)] × 100 (Liu et al., 2010). For repeatability and stability testing, three batches of ELISA plates (10 in each batch) were coated on three different days and incubated at 4 ◦ C overnight. The plates were then washed and dried in a fume hood. The dried plates were sealed and stored at 4 ◦ C until stability testing every three months. In each test, duplicate plates were used for testing the 10 positive and 10 negative serum samples. The obtained OD values were converted to percent positivity and the overall mean and standard deviation were calculated. Comparison of the means in all tests was conducted using one-way ANOVA.
amplification was performed in an automatic DNA thermocycler (PE Company, USA) with a final volume of 30 l containing 3 l of 10 × PCR buffer (10 mM TRIS–HCl, 50 mM KCl, 1.5 mM MgCl2, 0.01%gelatin), 200 M of each deoxynucleoside triphosphate (dNTP), 10 M of each primer, 100 ng of genomic DNA and 1.5 U of Taq polymerase. The cycling conditions of the PCR were 3 min at 94 ◦ C for denaturation followed by 35 cycles at 94 ◦ C for 30 s, 59 ◦ C for 1 min and 72 ◦ C for 1 min. The final extension step was 7 min at 72 ◦ C. The PCR products were visualized by UV transillumination in a 1.0% agarose gel containing 0.5 g/ml ethidium bromide. The positive PCR fragments were cloned into pGEM-T vector (Promega, Shanghai, China) and subjected to sequencing in Sangon Biotech Company (Shanghai, China) for analysis of sequences.
2.3. Cut-off determination
3.1. Cut-off determination using TG-ROC
The 188 positive and 198 negative reference samples were tested using the above described ELISA protocol. The OD value was converted to positive percentage and these data were used for the cut-off determination using TG-ROC analysis of the computational methods for diagnostic tests (CMDT) software (Greiner et al., 1995; Salih et al., 2005). In this method, the specificity and sensitivity were plotted against the cut-off and the point of intersection of the two curves was taken as the value of the cut-off where both specificity and sensitivity are equal. Two alternative cutoff values, which were the lower and the upper limits of the intermediate range (IR) were defined at an accuracy level of 90 and 95%, and the valid range proportion (VRP) was calculated. The defined cut-off was expressed using percent positivity.
The results of the d-Kolmogorov–Smirnov test indicated that the OD values of the positive and negative reference samples were not normally distributed; therefore, the cut-off, specificity and sensitivity of the rTuIP ELISA were calculated using the non-parametric approach in the TG-ROC analysis. The cut-off was found to be 32.7% using percent positivity (PP) values and both the specificity and sensitivity were 98.4% (Fig. 1). The valid range proportion (VRP) was 1.052 and the intermediate range (IR) was 0 at 95% confidence interval (CI), while at 90% CI they were 1 and 0, respectively. The overlap between positive and negative reference samples is shown in Fig. 2.
2.4. PCR and sequencing The PCR with primers 989/rDNA-AS and rDNA-S/990 (Allsopp et al., 1993; Yin et al., 2004) was applied to verify for the presence of T. uilenbergi or T. luwenshuni in the corresponding DNA samples of ELISA-positive field samples from Guizhou and Hubei provinces. PCR
3. Results
3.2. Repeatability and stability of the rTuIP ELISA Fig. 3 illustrates the PP values of the same negative and positive sera obtained from twelve different tests. The PP values of the negative sera remained negative and the positive sera remained positive in the tests which were repeated over a period of nine months on three different batches of antigen-coated plates. Comparison of the means was conducted with one-way ANOVA. The variation for the positive samples among months 1, 3, 6 was not significant
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deposited in the Genbank under accession Nos. HQ844674, JQ923446, JQ923447. The BLAST alignment in the NCBI (Altschul et al., 1990) with these sequences showed 99% identity to the sequences of T. luwenshuni (Genbank accession Nos. AY262115, AY262118). 4. Discussion
Fig. 2. Scatter plot of percentage positivity (PP) rTuIP ELISA test results illustrating the overlap between positive and negative values according to the cut-off value used (32.7%). A total of 188 positive and 198 negative reference sera were tested.
Fig. 3. Repeatability and stability of three batches of rTuIP ELISA (1, 2, 3) tested every three months using 10 known positive and 10 known negative sera. Percent positivity distribution is indicated by the mean ± standard deviation.
(P > 0.05); and significant difference for negative samples in the same month was not observed but between months was observed (P < 0.01). 3.3. Use of rTuIP ELISA for testing antibody response to infection with T. luwenshuni All the experimental animals showed the presence of specific antibodies against rTuIP protein within 3 weeks after inoculation with T. luwenshuni-infected blood or infestation with T. luwenshuni-infected ticks. The titer of the antibodies declined at around 8 weeks post infection (Fig. 4). 3.4. Application of rTuIP in field sample investigation The sample locations are as shown in Fig. 5, the corresponding test results are shown in Table 1. Eight out of 45 samples (17.8%) tested positive in Suizhou in Central China, 11 out of 95 (11.6%) were positive in Guiyang in Southwest China. In the known endemic region, 38 out of 39 samples were positive in Tianzhu with a positivity of 97.4% and 65 out of 67 were positive in Madang with a positivity of 97.0%. Corresponding DNA samples for the positive samples from Suizhou and Guiyang were tested by the PCR (Yin et al., 2004). One PCR product from Suizhou and two PCR products from Guiyang samples were sequenced, and the obtained 18S rRNA sequences were
It is well recognized that sensitivity (Se) and specificity (Sp) of ELISA are inversely related depending on the choice of the cut-off value (Greiner and Gardner, 2000). When increasing values of a measurement are associated with disease, higher cut-off values are generally associated with lower Se and a higher Sp (and vice versa, i.e. lower cut-off values are associated with higher Se and lower SP) (Greiner and Gardner, 2000). In our previous study, the cut-off of the rTuIP ELISA was derived using the Gaussian (normal) distribution method, which is defined as the mean plus two standard deviations (2SD) of the negative reference sample. The rationale of the 2SD procedure is to establish a cut-off value providing a Sp of 97.5% (Greiner et al., 2000). The procedure is clearly not adequate if the test values follow a skewed or multimodal distribution, and the other disadvantage is that it does not consider the Se (Greiner et al., 2000). The TG-ROC, an approach modified from the conventional ROC was applied in this study. In this method, the cut-off is defined based on the frequency distributions (Jacobson, 1996) and the test Se and Sp can be read directly from scatterplots of the data. The rTuIP ELISA is intended for investigation of the seroprevalence of small ruminant theileriosis caused by T. uilenbergi or T. luwenshuni in regions where prevalence of the disease is unknown. Thus we assume that the prevalence in the target population is about 50% and that the costs of false-positive and false-negative test results are equivalent. With this criterion, the cut-off was set at 32.7% using the PP values and both Se and Sp were 98.4%. Giving equal weights to Se and Sp fully exploits the information provided by the diagnostic test (Greiner et al., 2000). The determined cut-off was considered to be suitable for field investigation according to the OIE recommendations (Norval, 1992). The TuIP was derived from a merozoite cDNA library of T. uilenbergi. When the rTuIP ELISA was previously used for detecting antibodies from T. luwenshuni infected animals, four of six known infected animals were tested positive (Liu et al., 2010). It was presumed that TuIP might share similar epitopes in T. luwenshuni due to its close phylogenetic relationship to T. uilenbergi (Schnittger et al., 2003; Yin et al., 2004). When a batch of TuIP specific PCR primers was used to identify the TuIP gene from both T. uilenbergi and T. luwenshuni, positive results were only observed in T. uilenbergi (data not shown), thus the identification of an orthologous gene in T. luwenshuni remains to be reached. This point will hopefully be clarified with the accomplishing of a genome project of T. luwenshuni. Testing of further sera from animals experimentally infected with T. luwenshuni in this study indicated that specific anti-rTuIP antibodies can be recorded at the third week after infection. In confirmation of the previous study (Liu et al., 2010), however, a low antibody titer in some of the experimental animals indicates a low analytical Se of the
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Fig. 4. Specific anti-rTuIP serologic response over time in sheep experimentally infected with Theileria luwenshuni Wenyuan isolate as assessed in the ELISA. Sheep Nos. 14, 30, 94, 135 were infected by blood inoculation with T. luwenshuni Wenyuan isolate. Sheep Nos. 15, 39, 90, 133 were infected by attaching 50 adult Haemaphysalis qinghaiensis ticks which were moulted from clean nymphs engorged on a T. luwenshuni Wenyuan isolate infected sheep.
Table 1 Application of rTuIP ELISA for the detection of antibody to Theileria uilenbergi and/or T. luwenshuni in field sera (caprine and ovine) from previous unknown and known Theileria endemic regions. Sampling site (province/location)
Sample size
ELISA positive (%)
Confirmation
Hubei* Guizhou* Gansu
45 (Caprine) 95 (Caprine) 39 (Ovine) 67 (Ovine)
8 (17.8) 11 (11.6) 38 (97.4) 65 (97.0)
PCR, sequencing PCR, sequencing PCR, microscopic Examination of blood films
*
Suizhou Guiyang Tianzhu Madang
Previously unknown endemic regions.
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Fig. 5. Map of the main endemic region of Theileria uilenbergi and T. luwenshuni and the indication of the sampling sites for this study.
rTuIP ELISA in detection of T. luwenshuni infection, and false negative results can be anticipated. Moreover, the antibody titer dropped close or down to the cut-off value at the 9th week post infection, indicating that this ELISA is only efficient for detection of anti-T. luwenshuni antibody within two months post infection. This period is shorter than that described for the rTuIP ELISA for the detection of T. uilenbergi-specific antibody, which is possible for at least three months after infection (Liu et al., 2010), or the Tams-ELISA that can detect T. annulata antibody one year post infection (Gubbels et al., 2000). Thus, when applying the rTUIP ELISA for the study of the presence of small ruminant theileriosis in the field, it has to be kept in mind that the prevalence may be underestimated. The manner of testing the repeatability and stability of the rTuIP was to give insight into whether antigencoated plates can be preserved and applied in field studies for detection of samples from different sites at different time points. Although all positive reference samples and all negative reference samples were tested as such for the different time points, significantly increasing values from the negative reference samples was observed. Application of the preserved plates for field sample investigation might thus bear the risk of giving false positive results if stored for long periods of time. However, preserved plates with the advantages of using homogenous coating antigen and easy-to-share between laboratories is still promising for application in field sample investigations, where storage conditions need to be investigated for optimization of shelf life. Although theileriosis caused by T. luwenshuni and T. uilenbergi has to date only been reported in the northwest of China (Luo and Yin, 1997), the identification of the 18S rRNA sequences from field samples from different hosts have indicated that similar parasites might be present in Spain (Nagore et al., 2004), Japan (Ikawa et al., 2011), South Korea (Han et al., 2009) as well as in Central and South China (Ge et al., 2012; He et al., 2012; Li et al., 2012). A validated rTuIP ELISA as presented in this study represents a possible
alternative tool for serological investigation for the presence of these parasites worldwide if they are confirmed to be T. uilenbergi or T. luwenshuni. Application of the rTuIP ELISA for testing the sera from sheep in known endemic regions, Madang, Tianzhu in Gansu Province, showed about 97% positivity, which is consistent with previous investigation results (Guo et al., 2007; Liu et al., 2010). Application of the assay in Suizhou of Hubei Province and Guiyang of Guizhou Province showed 17.8% and 11.6% seroprevalence of Theileria infection in the investigated sites. Successful identification of the Theileria 18S rRNA gene from the corresponding blood DNA of the Theileria serum positive samples confirmed the accuracy of the rTuIP ELISA. Haemaphysalis longicornis, the transmitting tick vector of ovine Theileria, has also been found in Suizhou in Hubei Province, indicating that Suizhou is a potential new endemic region of small ruminant theileriosis; the transmission vector in Guizhou Province remains to be elucidated in future studies. In conclusion, the validated rTuIP ELISA can be regarded as a useful tool for studying the epidemiology of theileriosis caused by T. uilenbergi and T. luwenshuni. It needs to be emphasized that the ELISA has been validated using the negative samples from animals under controlled conditions which may not represent the actual field situation. It will thus be of great interest to further validate this method using samples from larger numbers of animals kept under field conditions.
Acknowledgments The research was financially supported by PIROVAC (KBBE-3-245145) and EPIZONE (FOOD-CT-2006-016236) of the European Commission, Brussels, Belgium. This study was also facilitated by NSFC(No. 30800820; No. 30972182, No. 31072130, No. 31001061), the 973 Program (2010CB530206), the Key Project of Gansu Province (1002NKDA035), the NBCITS MOA, (CARS-38), Specific Fund for Sino-Europe Cooperation, MOST China, State Key Laboratory of Veterinary Etiological Biology Project (SKLVEB2008ZZKT019).
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