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An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats Yu Wang a,1 , Huaming Nie a,1 , Xiaobin Gu a , Tao Wang a , Xing Huang a , Lin Chen a , Weimin Lai a , Xuerong Peng b , Guangyou Yang a,∗ a b
Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China Department of Chemistry, College of Science, Sichuan Agricultural University, Ya’an 625014, China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 11 June 2015 Accepted 13 June 2015 Keywords: Tapeworm Taenia multiceps TmHSP 70 Clone Expression Immunodiagnosis Indirect ELISA
a b s t r a c t Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 g/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Taenia multiceps is a parasitic cestode, the larval stage of which is the causative agent of coenurus cerebralis, a zoonosis that affects both ruminant animals and people worldwide (Ghazaei, 2007). The impact of the disease on animal health is considerable and causes severe socio-economic consequences for ruminant farming (Nie et al., 2013). Often, the clinical symptoms of infection include: circling, ataxia, drowsiness, head pressing, blindness and coma (Wu et al., 2012). Effective diagnosis is central in implementing control programs. Unlike imaging methods including radiology and ultrasonography, immunodiagnosis can be used for primary diagnosis and for follow-up examinations of infected animals after pharmacological treatment (Komnenou et al., 2000; Tirgari et al., 1987). For example, ELISA, a flexible immunodiagnostic method, can accommodate
∗ Corresponding author. Fax: +86 835 2885302. E-mail addresses: [email protected], [email protected] (G. Yang). 1 These authors contributed equally to this work.
large number of samples and is becoming the predominant serologic assay for the field surveillance.To date, an ELISA has been developed by applying a purified recombinant protein coating that improved the specificity of diagnosis (An et al., 2011). Notably, the target antigens that have good immunogenic properties and potential of serologic diagnosis are still lacking. Prior to numerous studies showed that the heat shock proteins (HSPs) within different parasites, including Trypanosoma, Trichomonas and Haemonchus, are ideal (Leeuwen, 1995; Bozner, 1997; Toaldo et al., 2001). For example, HSP60, Eg2HSP70 and HSP70, with their encoded proteins deemed immunodominant antigens, have been described in Echinococcus granulosus. (Martinez et al., 1999). Given the lack of information on the characteristics of HSPs of T. Multiceps and the importance of this parasite in ruminant farming, it is crucial to develop a rapid, sensitive and specific serological method to improve diagnosis of coenurus cerebralis. In this study, therefore, we aimed to develop a novel, rapid tool to diagnose T. multiceps infections using a recombinant protein. Specifically, we developed an indirect ELISA based on purified TmHSP70 heat shock protein and tested its potential of serological diagnosis in the field.
http://dx.doi.org/10.1016/j.vetpar.2015.06.020 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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2. Materials and methods
were visualized using Diaminobenzidine (DAB; TIANGEN, Beijing, China).
2.1. Parasite and serum preparation 2.5. Development of the TmHSP ELISA A total of 30 blood samples were collected from 30 moribund goats presenting with neurological symptoms of coenurosis from a goat farm in Sichuan province, China. Goats which were identified by surgical puncture to be affected by coenurosis were sacrificed by farmers. All the organs of the dead goats were inspected for cysts. T. multiceps protoscoleces were aspirated from cysts and then stored at 4 ◦ C in sterile normal saline prior to RNA extraction. Cysticercus tenuicollis sera (6 samples) were isolated from naturally infected goats, while negative sera (30 samples) collected from healthy goats with no cysts by autopsy. All the goats were from goat farms in Sichuan Province. 2.2. Cloning of TmHSP from cDNA Total RNA was extracted using Trizol reagent (Huashun, Shanghai, China) according to the manufacturer’s instructions. The obtained RNA was reverse-transcribed (Fermentas, Shenzhen, China) and processed according to the manufacturer’s instructions. The cDNA sequence of TmHSP used for designing primers was derived from unigene 18109 of the assembled T. multiceps transcriptome dataset (accession number JR934847 in Transcriptome Shotgun Assembly Sequence Database at NCBI). The open reading frame (ORF) of the nucleotide sequence was analyzed by Open Reading Frame Finder (http://www. ncbi.nlm.nih.gov/projects/gorf/). The amino acid sequences predicted by oligo (dT)18 primer (MBI Fermentas, Germany) were found with previously reported sequences in GenBank by the BLAST network server of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The forward primer, TmHSP-F 5 -CCGGAATTCATGGGTCTCAAAGAAACAG3 , contained a BamHI site, while the reverse primer, TmHSP-R 5 -CCGCTCGAGTCAGAATAGAGCCATTAACTC-3 , contained a XhoI site. The PCR products were digested with BamHI and XohI (TaKaRa, Dalian, China) and inserted into similarly digested pET32a(+) (Novagen, Madison, USA) to generate recombinant expression plasmid.The secondary structure of TmHSP was analyzed by use of DNAstar (Lasergene, USA). 2.3. Protein expression and purification Recombinant expression plasmid pET32a(+) was transformed into E. coli BL21 (DE3) competent cells. For the expression of the fusion protein, transformed cells were grown at 37 ◦ C in liquid LB medium containing 50 g/mL ampicillin. When the OD600 reached approximately 0.6, cultures were induced with 1 mM isopropyl thiogalactoside (IPTG) at 37 ◦ C for 3 h. The expressed recombinant proteins were purified using Ni2+ affinity chromatography (BioRad, Hercules, CA, USA) according the manufacturer’s instructions. 2.4. Western blot analyses The recombinant protein was separated by SDS-PAGE (12%) and transferred to a nitrocellulose filter (0.2 m, Bio-Rad). Non-specific binding sites were blocked with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline (TBS, pH 7.4) overnight at 4 ◦ C. The serum of goats infected with T. multiceps was used at a dilution of 1:200 in TBS containing 0.1% (v/v) Tween (TBST) and 5% (w/v) BSA. The membrane was incubated with serum for 2 h at room temperature. Following a wash with TBST, the rabbit anti-goat IgG-HRP conjugate (Boster Bio-project Co, Wuhan, China) was used as the secondary antibody. After further washing, immunoreactive bands
The 96-well ELISA plates were coated with 100 L of two-fold diluted rTmHSP (ranging from 5.40 g /well to 0.09 g/well) in 0.1 M carbonate buffer (pH 9.6) for 12 h at 4 ◦ C. All wells were washed by 300 L of PBS containing 0.05% Tween-20 (PBST) and then each well was blocked with 5% skimmed milk (skimmed milk in PBS) for 1 h at 37 ◦ C. The sera of infected and uninfected goats were diluted in PBS to dilutions of 1:20, 1:40, 1:80, 1:160, 1:320 or 1:640, then added to each well and incubated for 1 h at 37 ◦ C. After three washes with PBST, the rabbit anti-goat IgG-HRP conjugate (100 L; 1:3000) was added to the wells and the plates were incubated for a further 1 h at 37 ◦ C (Boster Bio-project Co). Color reactions were developed by the addition of 100 L of the substrate TMB (TIANGEN). This process was stopped with 100 L of 2 M H2 SO4 . The optical density (OD) of each well was measured at 450 nm (OD450). We then determined the dilutions of rTmHSP antigen and goat serum that gave rise to the maximum difference at OD450 values between positive and negative sera (P/N), and the cutoff value. Negative and blank controls were included on each plate. The cutoff value was calculated as the mean +3 standard deviations from the OD450 value of the negative sera. 2.6. Specificity and sensitivity of the TmHSP ELISA To determine the specificity of the TmHSP ELISA, we evaluated potential cross-reactivity with six C. tenuicollis sera (co-infections with these two tapeworms are common in goats). Three T. multiceps-positive served as positive controls. To evaluate sensitivity, the 96-well plates were coated with optimal concentrations of purified rTmHSP as described above. Three positive serum samples with twofold serial dilutions from 1:20 to 1:20480 were then added to each well and incubated with the antigen. The sensitivity of the TmHSP ELISA was defined as the highest dilution of the positive serum that produced an OD450 value higher than the cutoff value. 2.7. Repeatability and reproducibility of the TmHSP ELISA To evaluate the repeatability and reproducibility of the TmHSP ELISA, six positive serum samples were used. Every sample was tested simultaneously to assess the intra-assay variability (repeatability), and then tested consecutively to assess the inter-assay variability (reproducibility). Repeatability and reproducibility tests were conducted three times and their corresponding mean OD450, standard deviation (SD), and coefficient of variation (CV) were calculated. 2.8. Clinical testing of the TmHSP ELISA We assessed the clinical serodiagnostic potential of our TmHSP ELISA on 60 field-held goats, 30 of which were infected with T. multiceps and 30 of which were uninfected. The positive diagnostic rate was calculated by referring to the cutoff value. Each goat was tested twice. 3. Results 3.1. Characteristics of TmHSP70 and antibody reactivity The entire TmHSP cDNA sequence comprised 408 bp (GenBank accession number GU205474) and showed an initiation and a stop codon. Analysis of the nucleotide sequence revealed 90% nucleotide
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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Fig. 2. The specificity of indirect ELISA. T. multiceps represent positive sera of T. multiceps, C. tenuicollis represent positive sera of C. tenuicollis. Fig. 1. SDS-PAGE and Western blotting of rTmHSP. Lanes shown are as follows: M = protein markers; 1 and 2 = purified recombinant protein, Coomassie blue staining; 3 and 4 = Western blot of expressed rTmHSP probed with serum of Taeniainfected goats; 5 = Western blot of expressed rTmHSP probed with normal goat serum.
(mean = 4.98%). All the CVs were 0.446 was 1:10240 (mean = 0.462). 3.4. Repeatability and reproducibility of the TmHSP ELISA The coefficients of variation (CVs) of the repeatability in intra-assay ranged from 2.21–6.23% (mean = 4.14%), while the CVs of the reproducibility in inter-assay ranged from 2.72–6.45%
Sixty field-held goats were subjected to verify the reliability of the TmHSP ELISA as a clinical serodiagnostic tool. The ELISA successfully confirmed infection in all 30 of the infected goats, the remaining were diagnosed as negative, consistent with the clinical examination. This indicated that our novel ELISA was an efficient and reliable tool to diagnose T. multiceps infections in goats. 4. Discussion Proteins elicited by heat shock (HSPs) were first identified in the chromosomal in the salivary gland tissue of Drosophila busckii in 1962 (Snoeckx et al., 2001). Parasites undergo heat-stress after infecting animals, as they adapt to drastic environmental changes during their life cycles. Several studies on HSP expression in parasites have demonstrated that HSPs may play a fundamental role in the virulence of parasites, as evidenced by the physiological function of HSPs as chaperones. Recently, HSPs have been used as antigens in the genetic engineering of vaccines, and in the diagnosis and identification of parasitic worms (Huang and Jiang, 2006). HSPs with a molecular weight of 70 kDa (HSP70) have been identified as dominant antigens that are recognized by the immune system in a large spectrum of parasites. Colebrook and Lightowlers (1997) demonstrated, however, that specific antibodies reactive with E. granulosus HSP70 (EgHSP70) were found in 60% of hydatid patients but in only 20% of healthy control donors (Colebrook and Lightowlers, 1997). Thus, in host-pathogen interactions, the stress proteins of pathogens can assume a role of immunological importance. The accurate diagnosis of coenurosis in goats is essential for its effective control(Christodoulopoulos, 2007). Previous studies using ultrasonographic examination have successfully detected T. multiceps in sheep (An et al., 2011). In addition, some parasitologists have proposed that oncosphere secretory and protoscolex molecules could diagnose C. cerebralis at early stages (Yao and Zhou, 2001). However, these assays use natural worm extracts as antigens cannot be produced as commercial products. Other studies have also contributed to progress in the serodiagnosis of T. multiceps infection in sheep (Paltrinieri et al., 2010; Demeler et al., 2012). In this study, the novel ELISA based on the purified rTmHSP exhibited high sensitivity with antisera against T. multiceps detected at dilutions as low as 1:10240. Moreover, as demonstrated
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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by cross-reactivity testing, this ELISA exhibited high specificity for T. multiceps sera. These results indicate that rTmHSP70 can detect T. multiceps infections and could, therefore, be used to develop an ELISA-based serological test for the diagnosis of coenurosis in goats and other ruminants. Competing interests The authors declare that they have no competing interests. Authors’ contributions Yu Wang and Huaming Nie participated in the design of study, wrote the manuscript and performed the statistical analysis; Xiaobin Gu, Weimin Lai and Xuerong Peng contributed to animal care and experiments; Tao Wang, Xing Huang and Lin Chen contributed with the design and analyses of serodiagnostic trials; Guangyou Yang conceived of the study, participated in its design and coordination; All authors read and approved the final manuscript. Acknowledgments We are grateful to Dr Sanjie Cao (College of Veterinary Medicine, Sichuan Agricultural University, China) for providing instruments and constructive suggestions. This project was supported by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (Grant no. IRT0848). References An, X., Yang, G., Wang, Y., Mu, J., Yang, A., Gu, X., Yang, Y., Wei, L., Wen, J., Wang, S., 2011. Prokaryotic expression of Tm7 gene of Taenia multiceps and establishment of indirect ELISA using the expressed protein. Acta Vet. Zootechn. Sin. 42, 1302–1308.
Bozner, P., 1997. Immunological detection and subcellular localization of Hsp70 and Hsp60 homologs in Trichomonas vaginalis. J. Parasitol. 83, 224–229. Christodoulopoulos, G., 2007. Two rare clinical manifestations of coenurosis in sheep. Vet. Parasitol. 143, 368–370. Colebrook, A.L., Lightowlers, M.W., 1997. Serological reactivity to heat shock protein 70 in patients with hydatid disease. Parasite Immunol. 19, 41–46. Demeler, J., Schein, E., von Samson-Himmelstjerna, G., 2012. Advances in laboratory diagnosis of parasitic infections of sheep. Vet. Parasitol. 189, 52–64. Ghazaei, C., 2007. Evaluation therapeutic effects of antihelminthic agents albendazole, fenbendazole and praziquantel against coenurosis in sheep. Small Ruminant Res. 71, 48–51. Huang, J., Jiang, C., 2006. Progress on study of stress protein of parasites. Int. Med. Parasite Dis. 33, 317–321. Komnenou, A., Dessiris, A., Giadinis, N., 2000. Surgical treatment of coenurosis (gid) in sheep. Vet. Rec. 147, 242–244. Leeuwen, M.A.W., 1995. Heat-shock and stress response of the parasitic nematode Haemonchus contortus. Parasitol. Res. 81, 706–709. Martinez, J., Perez-Serrano, J., Bernadina, W.F., 1999. Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 Levels Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 Levels. Exp. Parasitol. 93, 171–180. Nie, H., Xie, Y., Fu Yan, Yang, Y., Gu, X., Wang, S., Peng, X., Lai, W., Peng, X., Yang, G., 2013. Cloning and characterization of the fatty acid-binding protein gene from the protoscolex of Taenia multiceps. Parasitol. Res. 112, 1833–1839. Paltrinieri, S., Varcasia, A., Cazzaniga, S., Giordano, A., Pipia, A.P., Marrosu, R., A, 2010. Brain creatine kinase isoenzyme (CK-BB) as a possible biomarker for the diagnosis in vivo of ovine coenurosis in a naturally infected flock. Small Ruminant Res. 94, 180–184. Snoeckx, L.H.E.H., Cornelussen, R.N., Van Nieuwenhoven, F.A., Reneman, R.S., Van der Vusse, G.J., 2001. Heat shock proteins and cardiovascular pathophysiology. Physiol. Rev. 81, 1461–1497. Tirgari, M., Howard, B., A, 1987. Clinical and radiographical diagnosis of coenurosis cerebralis in sheep and its surgical treatment. Vet. Rec. 120, 173–178. Toaldo, C., Steindel, M., Sousa, M., Tavares, C., 2001. Molecular karyotype and chromosomal localization of genes encoding -tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli. Mem. Inst. Oswaldo Cruz 96, 113–121. Wu, X., Fu, Y., Yang, D., Zhang, R., Zheng, W., Nie, H., Xie, Y., Yan, N., Hao, G., Gu, X., Wang, S., Peng, X., Yang, G., 2012. Detailed transcriptome description of the neglected cestode Taenia multiceps. PLoS One 79, 45830. Yao, X., Zhou, W., 2001. Study on the application of dot-ELISA for diagnosing coenurosis in sheep. Jilin Anim. Sci. Vet. Med. 12, 3–5.
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats Yu Wang a,1 , Huaming Nie a,1 , Xiaobin Gu a , Tao Wang a , Xing Huang a , Lin Chen a , Weimin Lai a , Xuerong Peng b , Guangyou Yang a,∗ a b
Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China Department of Chemistry, College of Science, Sichuan Agricultural University, Ya’an 625014, China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 11 June 2015 Accepted 13 June 2015 Keywords: Tapeworm Taenia multiceps TmHSP 70 Clone Expression Immunodiagnosis Indirect ELISA
a b s t r a c t Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 g/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Taenia multiceps is a parasitic cestode, the larval stage of which is the causative agent of coenurus cerebralis, a zoonosis that affects both ruminant animals and people worldwide (Ghazaei, 2007). The impact of the disease on animal health is considerable and causes severe socio-economic consequences for ruminant farming (Nie et al., 2013). Often, the clinical symptoms of infection include: circling, ataxia, drowsiness, head pressing, blindness and coma (Wu et al., 2012). Effective diagnosis is central in implementing control programs. Unlike imaging methods including radiology and ultrasonography, immunodiagnosis can be used for primary diagnosis and for follow-up examinations of infected animals after pharmacological treatment (Komnenou et al., 2000; Tirgari et al., 1987). For example, ELISA, a flexible immunodiagnostic method, can accommodate
∗ Corresponding author. Fax: +86 835 2885302. E-mail addresses: [email protected], [email protected] (G. Yang). 1 These authors contributed equally to this work.
large number of samples and is becoming the predominant serologic assay for the field surveillance.To date, an ELISA has been developed by applying a purified recombinant protein coating that improved the specificity of diagnosis (An et al., 2011). Notably, the target antigens that have good immunogenic properties and potential of serologic diagnosis are still lacking. Prior to numerous studies showed that the heat shock proteins (HSPs) within different parasites, including Trypanosoma, Trichomonas and Haemonchus, are ideal (Leeuwen, 1995; Bozner, 1997; Toaldo et al., 2001). For example, HSP60, Eg2HSP70 and HSP70, with their encoded proteins deemed immunodominant antigens, have been described in Echinococcus granulosus. (Martinez et al., 1999). Given the lack of information on the characteristics of HSPs of T. Multiceps and the importance of this parasite in ruminant farming, it is crucial to develop a rapid, sensitive and specific serological method to improve diagnosis of coenurus cerebralis. In this study, therefore, we aimed to develop a novel, rapid tool to diagnose T. multiceps infections using a recombinant protein. Specifically, we developed an indirect ELISA based on purified TmHSP70 heat shock protein and tested its potential of serological diagnosis in the field.
http://dx.doi.org/10.1016/j.vetpar.2015.06.020 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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were visualized using Diaminobenzidine (DAB; TIANGEN, Beijing, China).
2.1. Parasite and serum preparation 2.5. Development of the TmHSP ELISA A total of 30 blood samples were collected from 30 moribund goats presenting with neurological symptoms of coenurosis from a goat farm in Sichuan province, China. Goats which were identified by surgical puncture to be affected by coenurosis were sacrificed by farmers. All the organs of the dead goats were inspected for cysts. T. multiceps protoscoleces were aspirated from cysts and then stored at 4 ◦ C in sterile normal saline prior to RNA extraction. Cysticercus tenuicollis sera (6 samples) were isolated from naturally infected goats, while negative sera (30 samples) collected from healthy goats with no cysts by autopsy. All the goats were from goat farms in Sichuan Province. 2.2. Cloning of TmHSP from cDNA Total RNA was extracted using Trizol reagent (Huashun, Shanghai, China) according to the manufacturer’s instructions. The obtained RNA was reverse-transcribed (Fermentas, Shenzhen, China) and processed according to the manufacturer’s instructions. The cDNA sequence of TmHSP used for designing primers was derived from unigene 18109 of the assembled T. multiceps transcriptome dataset (accession number JR934847 in Transcriptome Shotgun Assembly Sequence Database at NCBI). The open reading frame (ORF) of the nucleotide sequence was analyzed by Open Reading Frame Finder (http://www. ncbi.nlm.nih.gov/projects/gorf/). The amino acid sequences predicted by oligo (dT)18 primer (MBI Fermentas, Germany) were found with previously reported sequences in GenBank by the BLAST network server of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The forward primer, TmHSP-F 5 -CCGGAATTCATGGGTCTCAAAGAAACAG3 , contained a BamHI site, while the reverse primer, TmHSP-R 5 -CCGCTCGAGTCAGAATAGAGCCATTAACTC-3 , contained a XhoI site. The PCR products were digested with BamHI and XohI (TaKaRa, Dalian, China) and inserted into similarly digested pET32a(+) (Novagen, Madison, USA) to generate recombinant expression plasmid.The secondary structure of TmHSP was analyzed by use of DNAstar (Lasergene, USA). 2.3. Protein expression and purification Recombinant expression plasmid pET32a(+) was transformed into E. coli BL21 (DE3) competent cells. For the expression of the fusion protein, transformed cells were grown at 37 ◦ C in liquid LB medium containing 50 g/mL ampicillin. When the OD600 reached approximately 0.6, cultures were induced with 1 mM isopropyl thiogalactoside (IPTG) at 37 ◦ C for 3 h. The expressed recombinant proteins were purified using Ni2+ affinity chromatography (BioRad, Hercules, CA, USA) according the manufacturer’s instructions. 2.4. Western blot analyses The recombinant protein was separated by SDS-PAGE (12%) and transferred to a nitrocellulose filter (0.2 m, Bio-Rad). Non-specific binding sites were blocked with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline (TBS, pH 7.4) overnight at 4 ◦ C. The serum of goats infected with T. multiceps was used at a dilution of 1:200 in TBS containing 0.1% (v/v) Tween (TBST) and 5% (w/v) BSA. The membrane was incubated with serum for 2 h at room temperature. Following a wash with TBST, the rabbit anti-goat IgG-HRP conjugate (Boster Bio-project Co, Wuhan, China) was used as the secondary antibody. After further washing, immunoreactive bands
The 96-well ELISA plates were coated with 100 L of two-fold diluted rTmHSP (ranging from 5.40 g /well to 0.09 g/well) in 0.1 M carbonate buffer (pH 9.6) for 12 h at 4 ◦ C. All wells were washed by 300 L of PBS containing 0.05% Tween-20 (PBST) and then each well was blocked with 5% skimmed milk (skimmed milk in PBS) for 1 h at 37 ◦ C. The sera of infected and uninfected goats were diluted in PBS to dilutions of 1:20, 1:40, 1:80, 1:160, 1:320 or 1:640, then added to each well and incubated for 1 h at 37 ◦ C. After three washes with PBST, the rabbit anti-goat IgG-HRP conjugate (100 L; 1:3000) was added to the wells and the plates were incubated for a further 1 h at 37 ◦ C (Boster Bio-project Co). Color reactions were developed by the addition of 100 L of the substrate TMB (TIANGEN). This process was stopped with 100 L of 2 M H2 SO4 . The optical density (OD) of each well was measured at 450 nm (OD450). We then determined the dilutions of rTmHSP antigen and goat serum that gave rise to the maximum difference at OD450 values between positive and negative sera (P/N), and the cutoff value. Negative and blank controls were included on each plate. The cutoff value was calculated as the mean +3 standard deviations from the OD450 value of the negative sera. 2.6. Specificity and sensitivity of the TmHSP ELISA To determine the specificity of the TmHSP ELISA, we evaluated potential cross-reactivity with six C. tenuicollis sera (co-infections with these two tapeworms are common in goats). Three T. multiceps-positive served as positive controls. To evaluate sensitivity, the 96-well plates were coated with optimal concentrations of purified rTmHSP as described above. Three positive serum samples with twofold serial dilutions from 1:20 to 1:20480 were then added to each well and incubated with the antigen. The sensitivity of the TmHSP ELISA was defined as the highest dilution of the positive serum that produced an OD450 value higher than the cutoff value. 2.7. Repeatability and reproducibility of the TmHSP ELISA To evaluate the repeatability and reproducibility of the TmHSP ELISA, six positive serum samples were used. Every sample was tested simultaneously to assess the intra-assay variability (repeatability), and then tested consecutively to assess the inter-assay variability (reproducibility). Repeatability and reproducibility tests were conducted three times and their corresponding mean OD450, standard deviation (SD), and coefficient of variation (CV) were calculated. 2.8. Clinical testing of the TmHSP ELISA We assessed the clinical serodiagnostic potential of our TmHSP ELISA on 60 field-held goats, 30 of which were infected with T. multiceps and 30 of which were uninfected. The positive diagnostic rate was calculated by referring to the cutoff value. Each goat was tested twice. 3. Results 3.1. Characteristics of TmHSP70 and antibody reactivity The entire TmHSP cDNA sequence comprised 408 bp (GenBank accession number GU205474) and showed an initiation and a stop codon. Analysis of the nucleotide sequence revealed 90% nucleotide
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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Fig. 2. The specificity of indirect ELISA. T. multiceps represent positive sera of T. multiceps, C. tenuicollis represent positive sera of C. tenuicollis. Fig. 1. SDS-PAGE and Western blotting of rTmHSP. Lanes shown are as follows: M = protein markers; 1 and 2 = purified recombinant protein, Coomassie blue staining; 3 and 4 = Western blot of expressed rTmHSP probed with serum of Taeniainfected goats; 5 = Western blot of expressed rTmHSP probed with normal goat serum.
(mean = 4.98%). All the CVs were 0.446 was 1:10240 (mean = 0.462). 3.4. Repeatability and reproducibility of the TmHSP ELISA The coefficients of variation (CVs) of the repeatability in intra-assay ranged from 2.21–6.23% (mean = 4.14%), while the CVs of the reproducibility in inter-assay ranged from 2.72–6.45%
Sixty field-held goats were subjected to verify the reliability of the TmHSP ELISA as a clinical serodiagnostic tool. The ELISA successfully confirmed infection in all 30 of the infected goats, the remaining were diagnosed as negative, consistent with the clinical examination. This indicated that our novel ELISA was an efficient and reliable tool to diagnose T. multiceps infections in goats. 4. Discussion Proteins elicited by heat shock (HSPs) were first identified in the chromosomal in the salivary gland tissue of Drosophila busckii in 1962 (Snoeckx et al., 2001). Parasites undergo heat-stress after infecting animals, as they adapt to drastic environmental changes during their life cycles. Several studies on HSP expression in parasites have demonstrated that HSPs may play a fundamental role in the virulence of parasites, as evidenced by the physiological function of HSPs as chaperones. Recently, HSPs have been used as antigens in the genetic engineering of vaccines, and in the diagnosis and identification of parasitic worms (Huang and Jiang, 2006). HSPs with a molecular weight of 70 kDa (HSP70) have been identified as dominant antigens that are recognized by the immune system in a large spectrum of parasites. Colebrook and Lightowlers (1997) demonstrated, however, that specific antibodies reactive with E. granulosus HSP70 (EgHSP70) were found in 60% of hydatid patients but in only 20% of healthy control donors (Colebrook and Lightowlers, 1997). Thus, in host-pathogen interactions, the stress proteins of pathogens can assume a role of immunological importance. The accurate diagnosis of coenurosis in goats is essential for its effective control(Christodoulopoulos, 2007). Previous studies using ultrasonographic examination have successfully detected T. multiceps in sheep (An et al., 2011). In addition, some parasitologists have proposed that oncosphere secretory and protoscolex molecules could diagnose C. cerebralis at early stages (Yao and Zhou, 2001). However, these assays use natural worm extracts as antigens cannot be produced as commercial products. Other studies have also contributed to progress in the serodiagnosis of T. multiceps infection in sheep (Paltrinieri et al., 2010; Demeler et al., 2012). In this study, the novel ELISA based on the purified rTmHSP exhibited high sensitivity with antisera against T. multiceps detected at dilutions as low as 1:10240. Moreover, as demonstrated
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
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by cross-reactivity testing, this ELISA exhibited high specificity for T. multiceps sera. These results indicate that rTmHSP70 can detect T. multiceps infections and could, therefore, be used to develop an ELISA-based serological test for the diagnosis of coenurosis in goats and other ruminants. Competing interests The authors declare that they have no competing interests. Authors’ contributions Yu Wang and Huaming Nie participated in the design of study, wrote the manuscript and performed the statistical analysis; Xiaobin Gu, Weimin Lai and Xuerong Peng contributed to animal care and experiments; Tao Wang, Xing Huang and Lin Chen contributed with the design and analyses of serodiagnostic trials; Guangyou Yang conceived of the study, participated in its design and coordination; All authors read and approved the final manuscript. Acknowledgments We are grateful to Dr Sanjie Cao (College of Veterinary Medicine, Sichuan Agricultural University, China) for providing instruments and constructive suggestions. This project was supported by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (Grant no. IRT0848). References An, X., Yang, G., Wang, Y., Mu, J., Yang, A., Gu, X., Yang, Y., Wei, L., Wen, J., Wang, S., 2011. Prokaryotic expression of Tm7 gene of Taenia multiceps and establishment of indirect ELISA using the expressed protein. Acta Vet. Zootechn. Sin. 42, 1302–1308.
Bozner, P., 1997. Immunological detection and subcellular localization of Hsp70 and Hsp60 homologs in Trichomonas vaginalis. J. Parasitol. 83, 224–229. Christodoulopoulos, G., 2007. Two rare clinical manifestations of coenurosis in sheep. Vet. Parasitol. 143, 368–370. Colebrook, A.L., Lightowlers, M.W., 1997. Serological reactivity to heat shock protein 70 in patients with hydatid disease. Parasite Immunol. 19, 41–46. Demeler, J., Schein, E., von Samson-Himmelstjerna, G., 2012. Advances in laboratory diagnosis of parasitic infections of sheep. Vet. Parasitol. 189, 52–64. Ghazaei, C., 2007. Evaluation therapeutic effects of antihelminthic agents albendazole, fenbendazole and praziquantel against coenurosis in sheep. Small Ruminant Res. 71, 48–51. Huang, J., Jiang, C., 2006. Progress on study of stress protein of parasites. Int. Med. Parasite Dis. 33, 317–321. Komnenou, A., Dessiris, A., Giadinis, N., 2000. Surgical treatment of coenurosis (gid) in sheep. Vet. Rec. 147, 242–244. Leeuwen, M.A.W., 1995. Heat-shock and stress response of the parasitic nematode Haemonchus contortus. Parasitol. Res. 81, 706–709. Martinez, J., Perez-Serrano, J., Bernadina, W.F., 1999. Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 Levels Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 Levels. Exp. Parasitol. 93, 171–180. Nie, H., Xie, Y., Fu Yan, Yang, Y., Gu, X., Wang, S., Peng, X., Lai, W., Peng, X., Yang, G., 2013. Cloning and characterization of the fatty acid-binding protein gene from the protoscolex of Taenia multiceps. Parasitol. Res. 112, 1833–1839. Paltrinieri, S., Varcasia, A., Cazzaniga, S., Giordano, A., Pipia, A.P., Marrosu, R., A, 2010. Brain creatine kinase isoenzyme (CK-BB) as a possible biomarker for the diagnosis in vivo of ovine coenurosis in a naturally infected flock. Small Ruminant Res. 94, 180–184. Snoeckx, L.H.E.H., Cornelussen, R.N., Van Nieuwenhoven, F.A., Reneman, R.S., Van der Vusse, G.J., 2001. Heat shock proteins and cardiovascular pathophysiology. Physiol. Rev. 81, 1461–1497. Tirgari, M., Howard, B., A, 1987. Clinical and radiographical diagnosis of coenurosis cerebralis in sheep and its surgical treatment. Vet. Rec. 120, 173–178. Toaldo, C., Steindel, M., Sousa, M., Tavares, C., 2001. Molecular karyotype and chromosomal localization of genes encoding -tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli. Mem. Inst. Oswaldo Cruz 96, 113–121. Wu, X., Fu, Y., Yang, D., Zhang, R., Zheng, W., Nie, H., Xie, Y., Yan, N., Hao, G., Gu, X., Wang, S., Peng, X., Yang, G., 2012. Detailed transcriptome description of the neglected cestode Taenia multiceps. PLoS One 79, 45830. Yao, X., Zhou, W., 2001. Study on the application of dot-ELISA for diagnosing coenurosis in sheep. Jilin Anim. Sci. Vet. Med. 12, 3–5.
Please cite this article in press as: Wang, Y., et al., An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.020
