Expression of the Tpanxb1 Gene from Taenia pisiformis and Its Potential Diagnostic Value by Dot-ELISA Author(s): Deying Yang, Lin Chen, Xuhang Wu, Xuan Zhou, Mei Li, Zuqin Chen, Xiang Nong, Xiaobin Gu, Xuerong Peng, and Guangyou Yang Source: Journal of Parasitology, 100(2):246-250. 2014. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/13-304.1 URL: http://www.bioone.org/doi/full/10.1645/13-304.1
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J. Parasitol., 100(2), 2014, pp. 246–250 Ó American Society of Parasitologists 2014
Expression of the Tpanxb1 Gene from Taenia pisiformis and Its Potential Diagnostic Value by Dot-ELISA Deying Yang*, Lin Chen*, Xuhang Wu, Xuan Zhou, Mei Li, Zuqin Chen, Xiang Nong, Xiaobin Gu, Xuerong Peng†, and Guangyou Yang, Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, Sichuan Province, China; *These authors contributed equally to this work; †College of Life and Basic Science, Sichuan Agricultural University, Ya’an 625014, China. Correspondence should be sent to: [email protected]
development (Hofmann et al., 2010) include annexin B1, B2, and B3 isolated from Taenia solium cysticercus (Yan et al., 2004; Gao et al., 2007; Lu et al., 2010). In our present study, a new annexin homolog, Tpanxb1, was screened from the transcriptome of adult T. pisiformis, its immunoinformatic data were analyzed, and its potential diagnostic value was evaluated by dot-ELISA. Taenia pisiformis eggs were artificially hatched as described by Rajasekariah et al. (1980). Total RNA was extracted from the harvested oncospheres using Trizol reagent (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions, and transcribed into cDNA using a BD SMARTTM RACE cDNA amplification kit (Invitrogen) following the manufacturer’s instructions. The Tpanxb1 unigene (360 bp) was screened from the transcriptome of adult T. pisiformis using genespecific primers based on the partial N-terminal and C-terminal sequences: 3 0 -RACE F1 5 0 -GCAATAAAGAAGGAGACGACGAGAG-3 0 and F2 5 0 -TGAGGGCTGATACCGATTTGGGAAG-3 0 , and 5 0 -RACE R1 5 0 AGCCAATGAGTTTCAAGCAGAGAGT-3 0 and R2 5 0 -ATGGCAAAGTGGAGCAGTTCGG-3 0 . PCR was performed in a 25 ll final volume, including 12.5 ll of PCR mixture (Invitrogen), 0.4 lM of each primer (forward and reverse), 1 ll of cDNA template, and 9.5 ll of ddH2O. The amplification conditions consisted of an initial denaturing step at 94 C for 5 min, followed by 35 cycles of amplification at 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min, and a final extension step at 72 C for 30 min. The PCR products were cloned into a pMD19-T vector (TaKaRa, Dalian, China) and sequenced using an ABI PRISMTM 377XL DNA Sequencer (Applied Biosystems Inc., Foster City, California) with universal forward and reverse primers. Sequence splicing of the 3 0 -RACE and 5 0 -RACE fragment was performed by DNAStar software. A BLASTx alignment was analyzed between Tpanxb1 and the non-redundant (NR) protein NCBI database. The condensed phylogenetic tree was reconstructed using the neighbor-joining (NJ) method in MEGA 4.0 (Tamura et al., 2007) based on the BLASTx result. Parameters for tree construction included the p-distance index and 1,000 bootstrap resampling. The expression cDNA of Tpanxb1 was amplified by F3 5 0 CGCGGATCCATGGCCTACTGTCGCTCCCTG-3 0 and R3 5 0 CCGCTCGAGCTATGCGGAGCCAATGAGTTT-3 0 . The PCR conditions were the same as described above. The Tpanxb1-pET32a prokaryotic expression vector was constructed by inserting Tpanxb1 cDNA into the BamH1 and Xhol sites of the expression pET32a vector, and expressed in Escherichia coli BL21 (DE3) induced with 0.8 M isopropyl-b-D-thiogalactoside (IPTG). The recombinant Tpanxb1 (rTpanxb1) proteins were purified by Ni-IDA SefinoseTM Resin (Bio-Rad, Hercules, California), and the concentration of the purified protein was determined by biophotometer (Eppendorf, Hamburg, Germany). The rTpanxb1 protein was separated on a 12% SDS-PAGE and electrophoretically transferred to a nitrocellulose filter membrane (Sigma, St. Louis, Missouri). The remaining Western blot procedure has been described by Bian et al. (2011). Here, we used a dilution of rabbit antiserum (1:100) (obtained 50 days post-experimental infection with T. pisiformis oncosphere provided by the Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, China) and negative serum as control, and horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (1:5,000 dilution, Sigma).
ABSTRACT: Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in rabbits that results in economic losses. To date, there has been limited information available on the early detection of infection by this parasite. This study describes a dot-ELISA method based on an autologous antigen annexin B1 (Tpanxb1). Its potential for serodiagnosis of rabbit cysticercosis was also evaluated. Western blot analysis revealed that the recombinant Tpanxb1 (rTpanxb1) protein could be specifically recognized by rabbit anti-sera. In serum trials, the antibodies could be detected by dot-ELISA using rTpanxb1 at 14 days post-infection. The positive response was present for up to 49 days post-infection. Based on the necropsy results of 169 rabbit samples, the relative sensitivity and specificity of the dot-ELISA were 94.55% and 92.86%, respectively. This study provides a foundation for studying the immunological function of annexin and its application to control Taenia cestodes.
Cysticercosis, caused by the larvae of Taenia pisiformis, is an important parasitic disease of rabbits with a worldwide distribution (Saeed et al., 2006; Mart ´ınez-Moreno et al., 2007; Lahmar et al., 2008; Bagrade et al., 2009). Rabbit-producing countries include Italy, France, Spain, Russia, China, and India (Wang, 2002). China is the world’s largest producer of rabbit meat (Chen et al., 2010), and T. pisiformis has become one of the most common rabbit-infecting parasites, severely affecting rabbit breeding (Zhang et al., 2007). It has a complex 2-host life cycle, including canines as definitive hosts and rabbits as intermediate hosts. Cysticerci usually parasitize the gastric omentum majus and the mesentery of rabbits, while the adults reside in the intestines of dogs and foxes (Bagrade et al., 2009). Rabbits infected with T. pisiformis have a weakened immunological resistance and are susceptible to infections with other diseases. They appear emaciated and may die from severe secondary infections (Zhou et al., 2008). The clinical manifestations are not very pronounced in the early infection period, which makes the disease difficult to diagnose. Until now, no effective diagnostic method has been reported. Some studies have shown that the crude oncosphere or mature metacestode antigens can be used to detect anti-T. pisiformis antibodies based on indirect enzymelinked immunosorbent assay (ELISA) or indirect fluorescent antibody test (IFAT) (Craig, 1984; Wang et al., 2009). However, due to the limited availability of crude parasite antigens, the use of native antigen is difficult in practice. The transcriptome data of adult T. pisiformis have recently been reported, which now provide a source for screening potential antigen genes to diagnose and prevent this disease (Yang et al., 2012). The annexins are a multi-gene family of calcium-dependent phospholipid-binding proteins that are widely distributed in eukaryotes (Moss and Morgan, 2004). Annexins usually contain a conserved protein core characterized by high alpha-helix content and a variable N-terminal domain (Lu et al., 2010). Previous studies have revealed a high degree of biochemical and functional diversity in different annexins (Donato and Marie, 1999; Gerke and Moss, 2002; Yan et al., 2004). Parasite annexins that have been suggested as new potential molecules for drug and vaccine DOI: 10.1645/13-304.1 246
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FIGURE 1. SDS-PAGE analysis of expression and Western blot of purified rTpanxb1. Lane M, molecular weight markers (kDa); lane 1, crude lysate of BL21 transformed with pET32a after 3 hr incubation; lane 2, crude lysate of BL21 transformed with Tpanxb1-pET32a before incubation; lane 3, sediment lysate of recombinant Tpanxb1; lane 4, supernatant lysate of recombinant Tpanxb1; lane 5, purified rTpanxb1; lane 6, identification of rabbit cysticercosis from anti-sera by Western blot; lane 7, identification of rabbit cysticercosis from negative sera by Western blot.
Dot-ELISA was carried out with the soluble rTpanxb1 protein. The dilution of antigens and positive sera sourced from T. pisiformis–infected rabbits were 50 ng, 100 ng, 200 ng, and 1:50, 1:100, and 1:200, respectively. The test was standardized according to the technique described by Pina ˜ et al. (2011). ELISA dots were detected by HRP-DAB (Invitrogen), and the reaction was stopped with distilled water. Two independent observers checked all the color reactions to judge the color intensity of the reactions. In addition to the background color, tan-yellow reactions were considered as positive results. To determine the earliest diagnostic time of rabbit cysticercosis, experimental sera against T. pisiformis were detected by dot-ELISA as described above. Blood samples were collected from 7 (60-day-old) healthy female New Zealand white rabbits (obtained from Laboratory Animal Centre of Sichuan Agricultural University, Ya’an, China). Each of 7 rabbits was orally infected with 5,000 mature viable T. pisiformis eggs. Sera samples were collected at 7 days intervals by venipuncture and stored at 20 C. Rabbits were humanely sacrificed at 49 days post-infection with ketamine (50 mg/kg) and sodium pentobarbital (100 mg/kg) (Sigma) (Betancourt et al., 2012). All animals were handled in strict accordance with animal protection law of the People’s Republic of China (a draft of an animal protection law in China released on 18 September 2009). All experimental protocols were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Veterinary College, Sichuan Agricultural University, Ya’an, China. Serum samples (n ¼ 169) from rabbits were collected from the local slaughterhouse and tested by dot-ELISA. The rabbit necropsy included the examination of T. pisiformis cysticerci in the abdominal cavity. The percentage sensitivity was calculated as dot-ELISA positive 3 100/true positive, and the percentage specificity was calculated as dot-ELISA negative 3 100/true negative (Varghese et al., 2012).
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Rabbit sera (n ¼ 22; provided by the Department of Parasitology, Sichuan Agricultural University) was used to test cross-reactions to the following infections: Sarcoptes scabiei (7 cases), Psoroptid communis (8 cases), Eimeria spp. (4 cases), and Passalurus ambiguus (3 cases). The protocol for the dot-ELISA tests was the same as described above. The entire cDNA region of Tpanxb1 (TpcC1 is renamed as Tpanxb1 in this paper, GenBank ID: AFJ24969) was 1,044 bp long and consisted of 347 amino-acid residues. The primary structure analysis of Tpanxb1 showed a 92% amino-acid sequence similarity with T. solium annexin B1 (Tsanxb1), and 38–46% with other species of annexins, such as anxa8 (38% with Homo sapiens), anxa7, anxa13, and anxb9. The NJ tree showed that Tpanxb1 and Tsanxb1 were in the same cluster with 100% bootstrap value (data not shown). The rTpanxb1 protein was successfully expressed in E. coli strain BL21 (DE3). The molecular weight of the soluble recombinant protein was determined to be 58 kDa. The rTpanxb1 protein was recognized by rabbit T. pisiformis cysticercosis anti-sera with a very intensely stained band (Fig. 1). Based on the continuous dilution of rTpanxb1 antigen and anti-sera dot-ELISA tests, 100 ng of recombinant antigen and 1:100 of rabbit sera in each strip were determined to be the best dilution combination. The antibody of experimental sera can be detected at 14 days post-infection. The positive response was maintained for 49 days post-infection (Fig. 2), when the rabbits were humanely sacrificed. Furthermore, the detectable results from the sera of the 7 experimental rabbits were similar. Of the 169 slaughterhouse serum samples, 33.14% (56/169) were positive by dotELISA test, and 31.95% (54/169) were positive by necropsy. The relative sensitivity and specificity of dot-ELISA using rTpanxb1 for diagnosis were 94.55% and 92.86%, respectively. None of the 22 sera samples from rabbits infected with S. scabiei, Ps. communis, Eimeria spp., and P. ambiguus was found positive in the dot-ELISA. Annexins are speculated to participate in a broad range of important biological processes, such as interaction with cytoskeletal proteins, inhibition of phospholipase A2, anti-coagulation, and membrane trafficking and fusion (Gao et al., 2007). Tpanxb1 and Tsanxb1 are homologous according to the high degree of sequence similarity (92%) and gathering in 1 cluster of the NJ tree. Annexin B1 from T. solium may belong to the secreted group of annexins (Gao et al., 2007). It has been proposed that Taenia annexin B1 mimics a common host pathway to reduce the inflammatory response to cysticercosis (Hofmann et al., 2010). These predictions suggest that the biological function of Tpanxb1 may be similar to Tsanxb1. A recent study described a new dot immunogold filtration assay based on recombinant cC1 (annexin B1) that was used to detect antibodies to cysticercus cellulosae in patient sera with 95% sensitivity and 100% specificity (Yang and Lan, 2008). Thus, the Tpanxb1 gene was selected to develop a diagnostic test for rabbit T. pisiformis cysticercosis. Dot-ELISA is a sensitive method that has been used in many research and clinical laboratories for the detection of some helminthic and bacterial infections (Jiang et al., 2004; Ferrer et al., 2007; Sharma et al., 2007; Prasad et al., 2008; Swarna and Parija, 2008; Varghese et al., 2012). Compared with other methods, dot-ELISA is an economic and effective detection technique that can utilize either recombinant or parasite-specific antigens, and it does not need mass reagents, long incubation times, and extra equipment. Multiple laboratory diagnostic tools have been used to detect T. solium taeniasis and cysticercosis, such as antigen-ELISA, tongue inspection, the formalin-ether technique, and enzyme-linked immunoelectrotransfer blot (Rodriguez-Hidalgo et al., 2006). The further use of complementary diagnostic methods for a better understanding of the epidemiology of T. solium has been suggested. Previous research has shown that a positive antibody test would indicate infection, active or past, or a transient antibody response (Garcia et al., 2001; Fujimoto et al., 2005). Thus, the dot-ELISA of rTpanxb1 protein as an antibody detection method was constructed for diagnosing rabbit T. pisiformis cysticercosis. The early infective stage (at 7 wk post-infection) involves adherence of the oncosphere to and migration across the intestinal wall, followed by transport of the oncosphere to the liver parenchyma via the circulatory
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FIGURE 2. Detection of infected rabbit experimental sera with rTpanxb1 by dot-ELISA. (A) negative control sera; (B) positive anti-sera; (C) sera at 0 day post-infection; (D) sera at 7 days post-infection; (E) sera at 14 days post-infection; (F) sera at 21 days post-infection; (G) sera at 28 days postinfection; (H) sera at 35 days post-infection; (I) sera at 42 days post-infection; (J) sera at 49 days post-infection.
system. Through this process, the oncosphere finally migrates to the abdominal cavity of rabbits (Miguel-Angel et al., 2011). Few studies have evaluated the effectiveness of experimental infections with different numbers of T. pisiformis eggs, with heterogeneous results reported, such as infection with 2,000 and 3,300 eggs (Gemmell, 1965; Craig, 1984; ToralBastida et al., 2011; Betancourt et al., 2012). Circulating antibodies were detected after 2 wk and 3 wk post-infection by Craig (1984) and Wang et al. (2009), respectively. In this study, we found that the antibodies of experimental sera could be detected by 14 days post-infection with 5,000 eggs, which was consistent with a previous report describing the timing of the emergence of circulating antibodies in rabbits infected with T. pisiformis (Craig, 1984). This result might be relevant to the infection
time of rabbit cysticercosis, despite the comparatively huge dose of 5,000 T. pisiformis eggs. In our study, high degrees of sensitivity (94.55%) and specificity (92.86%) were shown for the 169 serum samples, and there was no crossreaction between the rTpanxb1 protein and anti-sera against 4 other parasite species. These findings suggest that the dot-ELISA using the rTpanxb1 protein would be a good potential candidate for the diagnosis of rabbit T. pisiformis cysticercosis under field conditions. There are currently 3 tapeworm species known to infect rabbits, including Taenia pisiformis, T. serialis (Lucas, 2010), and Cittotaenia pectinata (Allan et al., 1999; Boag et al., 2001). Of these, T. pisiformis is the most common cestode species. In the future, it would be interesting to
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further explore the possible cross-reactivity of T. pisiformis Tpanxb1 with the other 2 tapeworm’s antigens. In conclusion, the present study demonstrated that rTpanxb1 is a potentially valuable diagnostic antigen for dot-ELISA. Our study provides a foundation for the biological function of annexin and application for control of the Taenia cestode. We are grateful to Dr. Sanjie Cao (College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, China) for providing instruments. This study was supported by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (grant IRT0848).
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J. Parasitol., 100(2), 2014, pp. 246–250 Ó American Society of Parasitologists 2014
Expression of the Tpanxb1 Gene from Taenia pisiformis and Its Potential Diagnostic Value by Dot-ELISA Deying Yang*, Lin Chen*, Xuhang Wu, Xuan Zhou, Mei Li, Zuqin Chen, Xiang Nong, Xiaobin Gu, Xuerong Peng†, and Guangyou Yang, Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, Sichuan Province, China; *These authors contributed equally to this work; †College of Life and Basic Science, Sichuan Agricultural University, Ya’an 625014, China. Correspondence should be sent to: [email protected]
development (Hofmann et al., 2010) include annexin B1, B2, and B3 isolated from Taenia solium cysticercus (Yan et al., 2004; Gao et al., 2007; Lu et al., 2010). In our present study, a new annexin homolog, Tpanxb1, was screened from the transcriptome of adult T. pisiformis, its immunoinformatic data were analyzed, and its potential diagnostic value was evaluated by dot-ELISA. Taenia pisiformis eggs were artificially hatched as described by Rajasekariah et al. (1980). Total RNA was extracted from the harvested oncospheres using Trizol reagent (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions, and transcribed into cDNA using a BD SMARTTM RACE cDNA amplification kit (Invitrogen) following the manufacturer’s instructions. The Tpanxb1 unigene (360 bp) was screened from the transcriptome of adult T. pisiformis using genespecific primers based on the partial N-terminal and C-terminal sequences: 3 0 -RACE F1 5 0 -GCAATAAAGAAGGAGACGACGAGAG-3 0 and F2 5 0 -TGAGGGCTGATACCGATTTGGGAAG-3 0 , and 5 0 -RACE R1 5 0 AGCCAATGAGTTTCAAGCAGAGAGT-3 0 and R2 5 0 -ATGGCAAAGTGGAGCAGTTCGG-3 0 . PCR was performed in a 25 ll final volume, including 12.5 ll of PCR mixture (Invitrogen), 0.4 lM of each primer (forward and reverse), 1 ll of cDNA template, and 9.5 ll of ddH2O. The amplification conditions consisted of an initial denaturing step at 94 C for 5 min, followed by 35 cycles of amplification at 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min, and a final extension step at 72 C for 30 min. The PCR products were cloned into a pMD19-T vector (TaKaRa, Dalian, China) and sequenced using an ABI PRISMTM 377XL DNA Sequencer (Applied Biosystems Inc., Foster City, California) with universal forward and reverse primers. Sequence splicing of the 3 0 -RACE and 5 0 -RACE fragment was performed by DNAStar software. A BLASTx alignment was analyzed between Tpanxb1 and the non-redundant (NR) protein NCBI database. The condensed phylogenetic tree was reconstructed using the neighbor-joining (NJ) method in MEGA 4.0 (Tamura et al., 2007) based on the BLASTx result. Parameters for tree construction included the p-distance index and 1,000 bootstrap resampling. The expression cDNA of Tpanxb1 was amplified by F3 5 0 CGCGGATCCATGGCCTACTGTCGCTCCCTG-3 0 and R3 5 0 CCGCTCGAGCTATGCGGAGCCAATGAGTTT-3 0 . The PCR conditions were the same as described above. The Tpanxb1-pET32a prokaryotic expression vector was constructed by inserting Tpanxb1 cDNA into the BamH1 and Xhol sites of the expression pET32a vector, and expressed in Escherichia coli BL21 (DE3) induced with 0.8 M isopropyl-b-D-thiogalactoside (IPTG). The recombinant Tpanxb1 (rTpanxb1) proteins were purified by Ni-IDA SefinoseTM Resin (Bio-Rad, Hercules, California), and the concentration of the purified protein was determined by biophotometer (Eppendorf, Hamburg, Germany). The rTpanxb1 protein was separated on a 12% SDS-PAGE and electrophoretically transferred to a nitrocellulose filter membrane (Sigma, St. Louis, Missouri). The remaining Western blot procedure has been described by Bian et al. (2011). Here, we used a dilution of rabbit antiserum (1:100) (obtained 50 days post-experimental infection with T. pisiformis oncosphere provided by the Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, China) and negative serum as control, and horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (1:5,000 dilution, Sigma).
ABSTRACT: Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in rabbits that results in economic losses. To date, there has been limited information available on the early detection of infection by this parasite. This study describes a dot-ELISA method based on an autologous antigen annexin B1 (Tpanxb1). Its potential for serodiagnosis of rabbit cysticercosis was also evaluated. Western blot analysis revealed that the recombinant Tpanxb1 (rTpanxb1) protein could be specifically recognized by rabbit anti-sera. In serum trials, the antibodies could be detected by dot-ELISA using rTpanxb1 at 14 days post-infection. The positive response was present for up to 49 days post-infection. Based on the necropsy results of 169 rabbit samples, the relative sensitivity and specificity of the dot-ELISA were 94.55% and 92.86%, respectively. This study provides a foundation for studying the immunological function of annexin and its application to control Taenia cestodes.
Cysticercosis, caused by the larvae of Taenia pisiformis, is an important parasitic disease of rabbits with a worldwide distribution (Saeed et al., 2006; Mart ´ınez-Moreno et al., 2007; Lahmar et al., 2008; Bagrade et al., 2009). Rabbit-producing countries include Italy, France, Spain, Russia, China, and India (Wang, 2002). China is the world’s largest producer of rabbit meat (Chen et al., 2010), and T. pisiformis has become one of the most common rabbit-infecting parasites, severely affecting rabbit breeding (Zhang et al., 2007). It has a complex 2-host life cycle, including canines as definitive hosts and rabbits as intermediate hosts. Cysticerci usually parasitize the gastric omentum majus and the mesentery of rabbits, while the adults reside in the intestines of dogs and foxes (Bagrade et al., 2009). Rabbits infected with T. pisiformis have a weakened immunological resistance and are susceptible to infections with other diseases. They appear emaciated and may die from severe secondary infections (Zhou et al., 2008). The clinical manifestations are not very pronounced in the early infection period, which makes the disease difficult to diagnose. Until now, no effective diagnostic method has been reported. Some studies have shown that the crude oncosphere or mature metacestode antigens can be used to detect anti-T. pisiformis antibodies based on indirect enzymelinked immunosorbent assay (ELISA) or indirect fluorescent antibody test (IFAT) (Craig, 1984; Wang et al., 2009). However, due to the limited availability of crude parasite antigens, the use of native antigen is difficult in practice. The transcriptome data of adult T. pisiformis have recently been reported, which now provide a source for screening potential antigen genes to diagnose and prevent this disease (Yang et al., 2012). The annexins are a multi-gene family of calcium-dependent phospholipid-binding proteins that are widely distributed in eukaryotes (Moss and Morgan, 2004). Annexins usually contain a conserved protein core characterized by high alpha-helix content and a variable N-terminal domain (Lu et al., 2010). Previous studies have revealed a high degree of biochemical and functional diversity in different annexins (Donato and Marie, 1999; Gerke and Moss, 2002; Yan et al., 2004). Parasite annexins that have been suggested as new potential molecules for drug and vaccine DOI: 10.1645/13-304.1 246
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FIGURE 1. SDS-PAGE analysis of expression and Western blot of purified rTpanxb1. Lane M, molecular weight markers (kDa); lane 1, crude lysate of BL21 transformed with pET32a after 3 hr incubation; lane 2, crude lysate of BL21 transformed with Tpanxb1-pET32a before incubation; lane 3, sediment lysate of recombinant Tpanxb1; lane 4, supernatant lysate of recombinant Tpanxb1; lane 5, purified rTpanxb1; lane 6, identification of rabbit cysticercosis from anti-sera by Western blot; lane 7, identification of rabbit cysticercosis from negative sera by Western blot.
Dot-ELISA was carried out with the soluble rTpanxb1 protein. The dilution of antigens and positive sera sourced from T. pisiformis–infected rabbits were 50 ng, 100 ng, 200 ng, and 1:50, 1:100, and 1:200, respectively. The test was standardized according to the technique described by Pina ˜ et al. (2011). ELISA dots were detected by HRP-DAB (Invitrogen), and the reaction was stopped with distilled water. Two independent observers checked all the color reactions to judge the color intensity of the reactions. In addition to the background color, tan-yellow reactions were considered as positive results. To determine the earliest diagnostic time of rabbit cysticercosis, experimental sera against T. pisiformis were detected by dot-ELISA as described above. Blood samples were collected from 7 (60-day-old) healthy female New Zealand white rabbits (obtained from Laboratory Animal Centre of Sichuan Agricultural University, Ya’an, China). Each of 7 rabbits was orally infected with 5,000 mature viable T. pisiformis eggs. Sera samples were collected at 7 days intervals by venipuncture and stored at 20 C. Rabbits were humanely sacrificed at 49 days post-infection with ketamine (50 mg/kg) and sodium pentobarbital (100 mg/kg) (Sigma) (Betancourt et al., 2012). All animals were handled in strict accordance with animal protection law of the People’s Republic of China (a draft of an animal protection law in China released on 18 September 2009). All experimental protocols were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Veterinary College, Sichuan Agricultural University, Ya’an, China. Serum samples (n ¼ 169) from rabbits were collected from the local slaughterhouse and tested by dot-ELISA. The rabbit necropsy included the examination of T. pisiformis cysticerci in the abdominal cavity. The percentage sensitivity was calculated as dot-ELISA positive 3 100/true positive, and the percentage specificity was calculated as dot-ELISA negative 3 100/true negative (Varghese et al., 2012).
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Rabbit sera (n ¼ 22; provided by the Department of Parasitology, Sichuan Agricultural University) was used to test cross-reactions to the following infections: Sarcoptes scabiei (7 cases), Psoroptid communis (8 cases), Eimeria spp. (4 cases), and Passalurus ambiguus (3 cases). The protocol for the dot-ELISA tests was the same as described above. The entire cDNA region of Tpanxb1 (TpcC1 is renamed as Tpanxb1 in this paper, GenBank ID: AFJ24969) was 1,044 bp long and consisted of 347 amino-acid residues. The primary structure analysis of Tpanxb1 showed a 92% amino-acid sequence similarity with T. solium annexin B1 (Tsanxb1), and 38–46% with other species of annexins, such as anxa8 (38% with Homo sapiens), anxa7, anxa13, and anxb9. The NJ tree showed that Tpanxb1 and Tsanxb1 were in the same cluster with 100% bootstrap value (data not shown). The rTpanxb1 protein was successfully expressed in E. coli strain BL21 (DE3). The molecular weight of the soluble recombinant protein was determined to be 58 kDa. The rTpanxb1 protein was recognized by rabbit T. pisiformis cysticercosis anti-sera with a very intensely stained band (Fig. 1). Based on the continuous dilution of rTpanxb1 antigen and anti-sera dot-ELISA tests, 100 ng of recombinant antigen and 1:100 of rabbit sera in each strip were determined to be the best dilution combination. The antibody of experimental sera can be detected at 14 days post-infection. The positive response was maintained for 49 days post-infection (Fig. 2), when the rabbits were humanely sacrificed. Furthermore, the detectable results from the sera of the 7 experimental rabbits were similar. Of the 169 slaughterhouse serum samples, 33.14% (56/169) were positive by dotELISA test, and 31.95% (54/169) were positive by necropsy. The relative sensitivity and specificity of dot-ELISA using rTpanxb1 for diagnosis were 94.55% and 92.86%, respectively. None of the 22 sera samples from rabbits infected with S. scabiei, Ps. communis, Eimeria spp., and P. ambiguus was found positive in the dot-ELISA. Annexins are speculated to participate in a broad range of important biological processes, such as interaction with cytoskeletal proteins, inhibition of phospholipase A2, anti-coagulation, and membrane trafficking and fusion (Gao et al., 2007). Tpanxb1 and Tsanxb1 are homologous according to the high degree of sequence similarity (92%) and gathering in 1 cluster of the NJ tree. Annexin B1 from T. solium may belong to the secreted group of annexins (Gao et al., 2007). It has been proposed that Taenia annexin B1 mimics a common host pathway to reduce the inflammatory response to cysticercosis (Hofmann et al., 2010). These predictions suggest that the biological function of Tpanxb1 may be similar to Tsanxb1. A recent study described a new dot immunogold filtration assay based on recombinant cC1 (annexin B1) that was used to detect antibodies to cysticercus cellulosae in patient sera with 95% sensitivity and 100% specificity (Yang and Lan, 2008). Thus, the Tpanxb1 gene was selected to develop a diagnostic test for rabbit T. pisiformis cysticercosis. Dot-ELISA is a sensitive method that has been used in many research and clinical laboratories for the detection of some helminthic and bacterial infections (Jiang et al., 2004; Ferrer et al., 2007; Sharma et al., 2007; Prasad et al., 2008; Swarna and Parija, 2008; Varghese et al., 2012). Compared with other methods, dot-ELISA is an economic and effective detection technique that can utilize either recombinant or parasite-specific antigens, and it does not need mass reagents, long incubation times, and extra equipment. Multiple laboratory diagnostic tools have been used to detect T. solium taeniasis and cysticercosis, such as antigen-ELISA, tongue inspection, the formalin-ether technique, and enzyme-linked immunoelectrotransfer blot (Rodriguez-Hidalgo et al., 2006). The further use of complementary diagnostic methods for a better understanding of the epidemiology of T. solium has been suggested. Previous research has shown that a positive antibody test would indicate infection, active or past, or a transient antibody response (Garcia et al., 2001; Fujimoto et al., 2005). Thus, the dot-ELISA of rTpanxb1 protein as an antibody detection method was constructed for diagnosing rabbit T. pisiformis cysticercosis. The early infective stage (at 7 wk post-infection) involves adherence of the oncosphere to and migration across the intestinal wall, followed by transport of the oncosphere to the liver parenchyma via the circulatory
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FIGURE 2. Detection of infected rabbit experimental sera with rTpanxb1 by dot-ELISA. (A) negative control sera; (B) positive anti-sera; (C) sera at 0 day post-infection; (D) sera at 7 days post-infection; (E) sera at 14 days post-infection; (F) sera at 21 days post-infection; (G) sera at 28 days postinfection; (H) sera at 35 days post-infection; (I) sera at 42 days post-infection; (J) sera at 49 days post-infection.
system. Through this process, the oncosphere finally migrates to the abdominal cavity of rabbits (Miguel-Angel et al., 2011). Few studies have evaluated the effectiveness of experimental infections with different numbers of T. pisiformis eggs, with heterogeneous results reported, such as infection with 2,000 and 3,300 eggs (Gemmell, 1965; Craig, 1984; ToralBastida et al., 2011; Betancourt et al., 2012). Circulating antibodies were detected after 2 wk and 3 wk post-infection by Craig (1984) and Wang et al. (2009), respectively. In this study, we found that the antibodies of experimental sera could be detected by 14 days post-infection with 5,000 eggs, which was consistent with a previous report describing the timing of the emergence of circulating antibodies in rabbits infected with T. pisiformis (Craig, 1984). This result might be relevant to the infection
time of rabbit cysticercosis, despite the comparatively huge dose of 5,000 T. pisiformis eggs. In our study, high degrees of sensitivity (94.55%) and specificity (92.86%) were shown for the 169 serum samples, and there was no crossreaction between the rTpanxb1 protein and anti-sera against 4 other parasite species. These findings suggest that the dot-ELISA using the rTpanxb1 protein would be a good potential candidate for the diagnosis of rabbit T. pisiformis cysticercosis under field conditions. There are currently 3 tapeworm species known to infect rabbits, including Taenia pisiformis, T. serialis (Lucas, 2010), and Cittotaenia pectinata (Allan et al., 1999; Boag et al., 2001). Of these, T. pisiformis is the most common cestode species. In the future, it would be interesting to
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further explore the possible cross-reactivity of T. pisiformis Tpanxb1 with the other 2 tapeworm’s antigens. In conclusion, the present study demonstrated that rTpanxb1 is a potentially valuable diagnostic antigen for dot-ELISA. Our study provides a foundation for the biological function of annexin and application for control of the Taenia cestode. We are grateful to Dr. Sanjie Cao (College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, China) for providing instruments. This study was supported by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (grant IRT0848).
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