Acta Tropica 148 (2015) 8–12
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Development of a recombinant OppA-based indirect hemagglutination test for the detection of antibodies against Haemophilus parasuis Shengli Chen, Yuefeng Chu, Ping Zhao, Ying He, Yingna Jian, Yongsheng Liu, Zhongxin Lu ∗ State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou 730046, Gansu, People’s Republic of China
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Article history: Received 20 January 2015 Received in revised form 28 March 2015 Accepted 11 April 2015 Available online 21 April 2015 Keywords: Haemophilus parasuis OppA Indirect hemagglutination test Antibody detection
a b s t r a c t An indirect hemagglutination (IHA) test that could detect antibodies against Haemophilus parasuis was developed. The full-length cDNA sequence of the oligopeptide permease ABC transporter membrane protein (OppA) gene was cloned, and inserted into the prokaryotic expression vector pET-30a(+) to construct recombinant plasmid pET-30a-OppA. The recombinant OppA protein was expressed partly in soluble form in Escherichia coli BL21 (DE3) and then purified by Ni2+ column. Furthermore, the recombinant OppA protein was used as an antigen to develop an IHA assay for detecting antibodies against H. parasuis. Results showed that this IHA test could detect species-specific antibodies against H. parasuis. Compared with currently available ELISA, the IHA test had a sensitivity of 85.0% and a specificity of 95.0%. The overall agreement between these two methods was 90.0%. The developed IHA test was used to evaluate the seroprevalence of H. parasuis in Hubei Province, China. The H. parasuis seroprevalence rate ranged from 5.5% to 26.2% in 325 tested clinical serum samples that were collected from three different pig farms in Hubei Province, China. The IHA test developed in this study will greatly contribute to the epidemiological surveys and immunization surveillance of H. parasuis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Haemophilus parasuis is the causative agent of Glässer’s disease, which is characterized by fibrinous polyserositis, arthritis, and meningitis. This disease has caused great economic loss in the pig industry worldwide, including China (Cai et al., 2005). This bacterium has great serovar diversity; 15 serovars of H. parasuis have been defined, and some strains cannot be typed by serotyping (Kielstein and Rapp-Gabrielson, 1992). Numerous field isolates have been identified in China (Cai et al., 2005; Chu et al., 2011; Zhang et al., 2012), and serovars 4, 5, and 12 were considered the most prevalent H. parasuis strains (Cai et al., 2005; Zhang et al., 2012). Given the serovar diversity and limited cross-serovar protection of this microorganism, diagnosing and controlling H. parasuis infection are difficult. The traditional diagnosis of H. parasuis infections is based on clinical signs, pathological findings, and bacteriologic culture (Oliveira and Pijoan, 2004). Several etiologic diagnosis methods, such as
∗ Corresponding author. Tel.: +86 931 8342676; fax: +86 931 8340977. E-mail address: [email protected] (Z. Lu). http://dx.doi.org/10.1016/j.actatropica.2015.04.009 0001-706X/© 2015 Elsevier B.V. All rights reserved.
immunohistochemistry (Segales et al., 1997), oligonucleotidespecific capture plate hybridization (OSCPH) assay (Calsamiglia et al., 1999), polymerase chain reaction (PCR) (Oliveira et al., 2001), in-situ hybridization (Jung and Chae, 2004), improved speciesspecific PCR (Angen et al., 2007), real-time PCR (Turni et al., 2010), loop-mediated isothermal amplification (Chen et al., 2010; Wang et al., 2010), and plate agglutination (Guo et al., 2010), were developed for rapid diagnosis of H. parasuis. Several serological methods, such as complement fixation (Takahashi et al., 2001), ELISA (Miniats et al., 1991; Solano-Aguilar et al., 1999), and indirect hemagglutination (IHA), were developed to detect antibodies against H. parasuis, but the results were often inconsistent and inaccurate (Miniats et al., 1991). The early developed IHA, which employed the supernatant of sonicated whole cells or boiled cells as coating antigens for sheep red blood cells (SRBCs), was unreliable for the assessment of protective immunity against H. parasuis (Miniats et al., 1991). IHA based on H. parasuis antigen (serovars 4, 5, and 12) has been applied to investigate the most prevalent serovars of H. parasuis infection in China (Zhang et al., 2014); however, it is unable to detect antibodies against all serovars of H. parasuis. The oligopeptide permease ABC transporter membrane protein (OppA), which belongs to the ATP-binding cassette (ABC) transporter family, has previously been identified as a target for the
S. Chen et al. / Acta Tropica 148 (2015) 8–12
development of vaccines against pathogenic bacteria (Garmory and Titball, 2004), such as Yersinia pestis (Tanabe et al., 2006). This study aimed to explore the possibility of using the OppA protein as the antigen to sensitize SRBCs, and to develop an IHA test for evaluating H. parasuis seroprevalence in China. 2. Materials and methods 2.1. Strains and serum samples The 15 reference strains of H. parasuis and a serovar 1 Australian field isolate, HS145, were kindly provided by Dr. Pat Blackall, Qld DPI, Animal Research Institute, Australia. These strains and three isolates of H. parasuis from China, Hps30, Hps31 and Hps0818, were used for hyperimmune antiserum production by rabbits as previously described (Rafiee and Blackall, 2000). Antisera against Actinobacillus pleuropneumoniae (serovars 1, 3, and 7), Pasteurella multocida (serovars B1, B2, D2, and D3), and Bordetella bronchiseptica, as well as the rabbit anti-OppA polyclonal serum, were prepared and stored in our laboratory. Antiserum against Mycoplasma hyopneumoniae was provided by Jiangsu Academy of Agricultural Sciences, Nanjing, China. Antiserum for Streptococcus suis serotype 2 was provided by Wuhan Keqian Animal Biological Products Co., Ltd. (Wuhan, China). Escherichia coli BL21 (DE3)lysate polyclonal antibody was a product of Proteintech Group Inc. (Chicago, IL, USA). A total of 325 clinical pig serum samples were collected from three different pig farms located in Suizhou, Yangxin, and Wuxue of Hubei, China. All animal studies were approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute. 2.2. Construction of recombinant plasmid containing the OppA gene of H. parasuis Based on the nucleotide sequence of H. parasuis strain SH0165 (GenBank accession no: NC 011852.1), the primers OppA-F (5 GGCGAATTCATGCAAACAACCTTTACC-3 , underlined is EcoRI site) and OppA-R (5 -CCCTCGAGTTACTGCTTAATGATATA-3 , underlined is XhoI site) were designed to amplify the 1638 bp full-length OppA gene. DNA of the Nagasaki strain (serovar 5 reference strain) was extracted from a culture in tryptic soy broth using a TIANamp bacterial DNA kit (TianGen Corporation, Beijing, China) and further used as the PCR template. The PCR conditions were as follows: 94 ◦ C for 5 min, followed by 35 cycles (94 ◦ C for 50 s, 51 ◦ C for 45 s, and 72 ◦ C for 2 min), and a final 10 min elongation step at 72 ◦ C. The PCR product was then inserted into the TA cloning vector (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. Plasmid DNA-containing OppA of H. parasuis was purified with a TIANprep Mini Plasmid Kit (TianGen Corporation, Beijing, China) and sequenced. Furthermore, the TA plasmid-containing OppA was digested with EcoR1 and XhoI, and inserted into similarly digested pET-30a(+) to construct the recombinant plasmid pET-30a-OppA.
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protein expression conditions were optimized at different temperatures (37 ◦ C, 25 ◦ C, 20 ◦ C, and 16 ◦ C) and various concentrations of isopropyl -d-thiogalactopyranoside (0.01–2 mmol/l). The expression level was tested by SDS-PAGE and stained with Coomassie brilliant R250. Expressed protein was further purified with a Ni2+ column in His-Bind Kits (Gen Script, Nanjing, China). Protein concentration was estimated using a Nano assay. An aliquot of the purified protein was stored at −70 ◦ C for later use. 2.4. SDS-PAGE and Western blot analysis The purity and identity of protein were analyzed by 12% SDSPAGE and then stained with Coomassie brilliant R250. The antigenic protein was determined by Western blot. In brief, purified protein was separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking at room temperature for 2 h with 10% skimmed milk (Becton, Dickinson and Company, New Jersey, USA) in TBST, the membrane was incubated with the 1:200 diluted rabbit anti-H. parasuis polyclonal serum at room temperature for 2 h. The membrane was then washed and incubated with 1:5000 diluted HRP-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China), which was used to detect the bound antibodies. Colorimetric reaction was observed using the diaminobenzidine enhanced liquid substrate system (TianGen Corporation, Beijing, China). 2.5. IHA test For the IHA test, the purified OppA protein was used as the antigen. Rabbit anti-OppA polyclonal serum, healthy rabbit serum, and PBS (0.15 mol/l, pH 7.2) were employed as positive control serum, negative control serum, and dilution control, respectively; an antigen control was also included. The optimal antigen concentration (1.25, 2.5, 5, 10, 20, 40, 60, 80, and 100 g/ml), temperature (56 ◦ C and 37 ◦ C), and time of sensitized SRBCs (15, 30, 45, and 60 min), as well as sensitization buffer pH (pH 6.4 and pH 7.2), were evaluated using the titration test. The sensitized SRBCs were washed three times with PBS (0.15 mol/l, pH 7.2) with 1% healthy rabbit serum, and centrifuged at 3000 r/min for 10 min. The cell pellet was then resuspended with PBS (0.15 mol/l, pH 7.2) with 1% healthy rabbit serum at 10% concentration, and diluted to a final 1% for the IHA test. The specificity, sensitivity, reproducibility, and stability of the established IHA assay were determined. 2.6. Comparison of IHA and commercial ELISA The developed IHA and commercial ELISA kit (BioChek) were compared. The sensitivity, specificity and the agreement ratio were determined by testing 160 pig serum samples. These samples contained 80 positive samples and 80 negative samples for anti-H. parasuis antibodies, which were detected by ELISA. 2.7. Application of IHA IHA was used to evaluate the seroprevalence of H. parasuis in three different pig farms from three cities in Hubei Province, China.
2.3. Expression and purification of recombinant OppA protein 3. Results Recombinant plasmid pET-30a-OppA was transformed into E. coli BL21 (DE3) competent cells to produce the recombinant strain E. coli BL21 (DE3). For protein expression, recombinant E. coli BL21 (DE3) cells were grown on LB agar containing 50 g/ml kanamycin. A 4 ml overnight LB culture (containing 50 g/ml kanamycin) originating from a single colony from the agar was inoculated into 400 ml of LB (containing 50 g/ml kanamycin), and incubated at 37 ◦ C with shaking until the bacteria reached the midlog growth phase (OD600 = 0.6). For soluble protein expression, the
3.1. Production of the OppA recombinant protein The 1638 bp OppA gene was amplified using PCR, inserted into the plasmid pET-30a(+), and confirmed by DNA sequencing and double digestion with EcoRI and XhoI (Fig. 1). The recombinant plasmid was transformed into E. coli BL21 (DE3), and the OppA recombinant protein was successfully expressed. The expression condition of the OppA recombinant protein was optimized, and
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S. Chen et al. / Acta Tropica 148 (2015) 8–12
Fig. 1. (a) PCR amplification of the OppA gene of H. parasuis. Lane M, molecular standard DNA marker; lanes 1–3, DNA of H. parasuis Nagasaki strain; lane 4, negative control. (b) Restriction enzyme digestion of the recombinant plasmid pET-30a-OppA with EcoR1 and XhoI. Lane M, molecular standard DNA marker; lane 1, the recombinant plasmid pET-30a-OppA.
half of the OppA recombinant protein was successfully expressed as an insoluble form at 16 ◦ C for 16 h. The insoluble His-tagged fusion protein was purified using a Ni-NTA His Bind resin column, and identified to be approximately 66.8 kDa by SDS-PAGE analysis (Fig. 2a). The antigenicity of the recombinant OppA protein was confirmed by measuring its interaction with rabbit anti-H. parasuis positive polyclonal serum (Fig. 2b). 3.2. Development of IHA The recombinant protein OppA was used as antigen for sensitizing SRBCs to develop the IHA test. The optimal antigen concentration of the sensitized SRBCs was 0.005–0.01 mg/ml. In addition, the optimal temperature and time of sensitized SRBCs were 37 ◦ C and 30 min, respectively. The optimal sensitization buffer of 0.15 mol/l PBS (pH 6.4) was obtained using the titration
test. A total of 40 known negative pig serum (as determined by commercial ELISA) and 40 known positive pig serum samples were used to determine the cut-off level for IHA. The results showed that the IHA titer ≥1:8 and IHA titer ≤1:2 were positive and negative, respectively. The IHA titer of 1:4 was considered doubtful and should be retested because this dilution mainly appeared in H. parasuis positive serum using commercial ELISA, but occasionally appeared in H. parasuis negative serum. If the IHA titer retested was still 1:4, it was considered positive. The high sensitivity of the test was more important than a high specificity when the test was used as a screening assay to avoid the importation of seropositive pigs into a pig farm, and to adopt timely measures to control H. parasuis infection. In the specificity test, all antisera against 15 reference strains of H. parasuis and field strains were positive (IHA titer 1:128–1:1024), and antisera against six other types of swine bacteria were
Fig. 2. (a) Proteins were analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue. Lane M, molecular standard protein marker; lane 1, crude extract from induced cells with plasmid pET-30a-OppA; lane 2, purified OppA protein. (b) Western blot analysis of the purified OppA protein. Lane M, molecular standard protein marker; lane 1, purified OppA protein; lane 2, negative control of pET-30a(+) vector. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. Chen et al. / Acta Tropica 148 (2015) 8–12 Table 1 Antisera used in this study and the results of the IHA test. Antisera against strains
IHA titer
Result
H. parasuis field HS145 (serovar 1) H. parasuis reference SW140 (serovar 2) H. parasuis reference SW114 (serovar 3) H. parasuis reference SW124 (serovar 4) H. parasuis reference Nagasaki (serovar 5) H. parasuis reference 131 (serovar 6) H. parasuis reference 174 (serovar 7) H. parasuis reference C5 (serovar 8) H. parasuis reference D74 (serovar 9) H. parasuis reference H367 (serovar 10) H. parasuis reference H465 (serovar 11) H. parasuis reference H425 (serovar 12) H. parasuis reference 17975 (serovar 13) H. parasuis reference 22113 (serovar 14) H. parasuis reference 15995 (serovar 15) H. parasuis field Hps30 (serovar 4) H. parasuis field Hps31 (serovar 5) H. parasuis field Hps0818 (untypable) Actinobacillus pleuropneumoniae (serovar 1) Actinobacillus pleuropneumoniae (serovar 3) Actinobacillus pleuropneumoniae (serovar 7) Pasteurella multocida (serovar B1) Pasteurella multocida (serovar B2) Pasteurella multocida (serovar D2) Pasteurella multocida (serovar D3) Mycoplasma hyopneumoniae Streptococcus suis serotype 2 Bordetella bronchiseptica Escherichia coli BL21 (DE3)
1:512 1:1024 1:1024 1:512 1:1024 1:512 1:128 1:256 1:128 1:1024 1:1024 1:1024 1:256 1:1024 1:512 1:256 1:256 1:256 1:2 1:2 1:0 1:2 1:2 1:2 1:2 1:0 1:0 1:0 1:0
+ + + + + + + + + + + + + + + + + + − − − − − − − − − − −
A total of 325 clinical serum samples collected from pigs in herds with a history of fibrinous polyserositis and arthritis were tested with the IHA test. The results showed that the seroprevalence rate for H. parasuis antibodies ranged from 5.5% to 26.2% in all tested unvaccinated pig herds (Table 4). This result suggested different degrees of H. parasuis infection in pig herds in Hubei, China. Notably, the seroprevalence rates from 30- to 90-day-old pigs were significantly higher than those from replacement gilts and multiparous sows. Of the 325 serum samples, 57 samples were positive for H. parasuis antibodies, showing an overall seropositive rate of 17.5% (Table 4).
4. Discussion
Experiment Dilution of the serum 2−1 2−2 2−3 2−4 2−5 2−6 2−7 2−8 2−9 2−10 2−11 2−12 # # #
# # #
# # #
# # #
# # #
# # #
# # #
# # #
# # #
+++ +++ ++
+ + +
− − −
−, 0%; + 25%; ++, 50%; +++, 75%; #, 100% hemagglutination.
negative (IHA titer 1:0–1:2) (Table 1). In the sensitivity test, the dilutions of H. parasuis IHA positive control serum (from 2−10 to 2−1 ) were all above 50% hemagglutination. This result suggested that the sensitivity was 2−7 (2−10 /2−3 ) by the developed IHA (Table 2). Furthermore, the results of three parallel experiments of the IHA test were identical, which indicated that IHA had excellent reproducibility (Table 2). For the stability of IHA, similar sensitivity and specificity were achieved with the freeze-dried sensitized SRBC antigen stored at 4 ◦ C for six months and at −20 ◦ C for 12 months. 3.3. Validation of IHA A total of 160 serum samples selected from four different pig herds from four provinces (Sichuan, Henan, Jiangxi, and Guangxi) in China were tested by both IHA and commercial ELISA kits to evaluate IHA. As shown in Table 3, among the 80 ELISA-positive Table 3 Agreement ratio between the developed IHA and commercial ELISA for the detection of antibodies against H. parasuis. Serum samples
ELISA
80 Field samples 80 Field samples
Positive Negative
a Formula for the calculation (A + D/A + B + C + D) × 100.
Agreementa
IHA Positive
Negative
A (68) C (4)
B (12) D (76)
of
the
agreement:
90.0% %
serum samples, 68 were positive by IHA. In addition, among the 80 ELISA-negative serum samples, 76 were negative by IHA. The sensitivity of IHA was calculated to be 85.0%, and its specificity was 95.0%. The overall agreement between IHA and commercial ELISA kits was 90.0% (Table 3).
3.4. Application of IHA
Table 2 Sensitivity of the developed IHA to detect H. parasuis IHA positive control serum.
1 2 3
11
agreement =
The isolation of this organism is usually difficult, particularly after antibiotic treatment (Olvera et al., 2007). However, the isolation of H. parasuis is still the “gold standard” for the diagnosis of Glässer’s disease from diseased pigs, and further serotyping is necessary because it is part of the normal respiratory microbiota. The commercial indirect ELISA kit, which explored the oligopeptide permease A (OppA) as coat antigen, has been suggested to track antibodies against H. parasuis (Macedo et al., 2011). In the present study, an IHA test was developed using another ABC transporter protein, the oligopeptide permease ABC transporter membrane protein (only 29% amino identity with previous OppA). These results indicated that the developed IHA could specifically detect antiserum against H. parasuis (15 reference H. parasuis, nontypeable strains, and field isolates), and no cross-reaction was detected with other related bacterial antisera. This method showed excellent reproducibility and high sensitivity, and it was highly consistent with commercial ELISA. Moreover, it was simpler, much cheaper, and faster than commercial ELISA. Thus, this method is suitable for large-scale serological screening of H. parasuis antibodies in pig serum. Selecting an antigen is important to establish the IHA assay. Several studies demonstrated that IHA tests based on boiled or autoclaved extracts can be used for serotyping of H. parasuis, and such tests showed more sensitivity than the immunodiffusion test (the “gold standard”) (Del Rio et al., 2003; Tadjine et al., 2004). During the preliminary study, whole cells were used as the antigen to sensitize SRBCs, and extensive cross-reactions were observed with A. pleuropneumoniae and P. multocida polysera. The possibility of the OppA protein as an antigen was then examined. The results showed that OppA proteins owned 97–99% amino identity among H. parasuis strains, as well as a maximum of 74% amino identity with other bacterial pathogens examined (data not shown). Protein expression was optimized to obtain an active protein. Low temperature (16 ◦ C) and long time (16 h) could partly induce the expression of recombinant protein in soluble form. This study established recombinant OppA-based IHA for detecting species-specific antibodies against H. parasuis, and determined that it could be used to distinguish antibody against H. parasuis from other bacterial pathogens. However, OppA-IHA was unable to differentiate infection from vaccinated pigs, so further research should be carried out to develop a different serological method that differentiates infected animals from vaccinated animals.
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Table 4 Positive rates of the anti-H. parasuis antibodies determined by the developed IHA in three herds in Hubei, China. Type of pigs
30 Days 60 Days 90 Days Replacement gilt Multiparous sows Subtotal Total
Suizhou
Wuxue
Yangxin
No. of positive/no. tested
Positive rate (%)
No. of positive/no. tested
Positive rate (%)
No. of positive/no. tested
Positive rate (%)
15/30 3/28 9/29 1/10 0/10 28/107 57/325, 17.5%
50.0 10.7 31.0 10.0 0 26.2
6/28 3/30 11/30 2/10 1/10 23/108
21.4 10.0 36.7 20.0 10.0 21.3
6/30 0/30 0/30 0/10 0/10 6/110
20.0 0 0 0 0 5.5
Aside from antigen, other factors, such as antigen concentration, sensitization temperature, time, and sensitization buffer, could affect the sensitivity of the IHA test. The optimized conditions for sensitized SRBCs were determined. The SRBC donors should also be selected carefully and tested simultaneously as non-sensitizing SRBC control when the IHA procedure was performed because some donors of SRBCs could agglutinate several types of bacterial antisera, such as P. multocida. This phenomenon may be due to the existence of heterophile antibodies, and it can be solved by incubating the diluted serum with glutaraldehyde-treated tanned SRBCs at 37 ◦ C for 30 min as previously described (Levieux et al., 1992). The seropositive rate of H. parasuis antibodies in tested clinical pig serum samples ranged from 5.5% to 26.2%, thereby suggesting that H. parasuis infection is endemic in Hubei, China. This result was consistent with that reported by Zhang et al. (2012). In the present study, 30- to 90-day old pigs were more likely to be seropositive than replacement gilts and multiparous sows. This age difference in the susceptibility of H. parasuis was also stated in reports elsewhere (Olvera et al., 2007). In conclusion, the developed OppA-IHA is a useful serological tool for epidemiological investigations and immunization surveillance of H. parasuis. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was funded by the Special Fund for Agro-scientific Research in the Public Interest (Grant no. 201303034) to LZX. References Angen, O., Oliveira, S., Ahrens, P., Svensmark, B., Leser, T.D., 2007. Development of an improved species specific PCR test for detection of Haemophilus parasuis. Vet. Microbiol. 119, 266–276. Cai, X., Chen, H., Blackall, P.J., Yin, Z., Wang, L., Liu, Z., Jin, M., 2005. Serological characterization of Haemophilus parasuis isolates from China. Vet. Microbiol. 111, 231–236. Calsamiglia, M., Pijoan, C., Solano, G., Rapp-Gabrielson, V., 1999. Development of an oligonucleotide-specific capture plate hybridization assay for detection of Haemophilus parasuis. J. Vet. Diagn. Invest. 11, 140–145. Chen, H.T., Chu, Y.F., Liu, Y.S., Zhang, J., Lu, Z.X., 2010. Loop-mediated isothermal amplification for the rapid detection of Haemophilus parasuis. FEMS Immunol. Med. Microbiol. 60, 283–285. Chu, Y.F., Gao, P.C., Zhao, P., He, Y., Zhang, N.Z., Liu, Y.S., Liu, J.X., Lu, Z.X., 2011. Genotyping of Haemophilus parasuis isolated from northwest China using PCRRFLP based on the ompA gene. J. Vet. Med. Sci. 73, 337–343.
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Development of a recombinant OppA-based indirect hemagglutination test for the detection of antibodies against Haemophilus parasuis Shengli Chen, Yuefeng Chu, Ping Zhao, Ying He, Yingna Jian, Yongsheng Liu, Zhongxin Lu ∗ State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou 730046, Gansu, People’s Republic of China
a r t i c l e
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Article history: Received 20 January 2015 Received in revised form 28 March 2015 Accepted 11 April 2015 Available online 21 April 2015 Keywords: Haemophilus parasuis OppA Indirect hemagglutination test Antibody detection
a b s t r a c t An indirect hemagglutination (IHA) test that could detect antibodies against Haemophilus parasuis was developed. The full-length cDNA sequence of the oligopeptide permease ABC transporter membrane protein (OppA) gene was cloned, and inserted into the prokaryotic expression vector pET-30a(+) to construct recombinant plasmid pET-30a-OppA. The recombinant OppA protein was expressed partly in soluble form in Escherichia coli BL21 (DE3) and then purified by Ni2+ column. Furthermore, the recombinant OppA protein was used as an antigen to develop an IHA assay for detecting antibodies against H. parasuis. Results showed that this IHA test could detect species-specific antibodies against H. parasuis. Compared with currently available ELISA, the IHA test had a sensitivity of 85.0% and a specificity of 95.0%. The overall agreement between these two methods was 90.0%. The developed IHA test was used to evaluate the seroprevalence of H. parasuis in Hubei Province, China. The H. parasuis seroprevalence rate ranged from 5.5% to 26.2% in 325 tested clinical serum samples that were collected from three different pig farms in Hubei Province, China. The IHA test developed in this study will greatly contribute to the epidemiological surveys and immunization surveillance of H. parasuis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Haemophilus parasuis is the causative agent of Glässer’s disease, which is characterized by fibrinous polyserositis, arthritis, and meningitis. This disease has caused great economic loss in the pig industry worldwide, including China (Cai et al., 2005). This bacterium has great serovar diversity; 15 serovars of H. parasuis have been defined, and some strains cannot be typed by serotyping (Kielstein and Rapp-Gabrielson, 1992). Numerous field isolates have been identified in China (Cai et al., 2005; Chu et al., 2011; Zhang et al., 2012), and serovars 4, 5, and 12 were considered the most prevalent H. parasuis strains (Cai et al., 2005; Zhang et al., 2012). Given the serovar diversity and limited cross-serovar protection of this microorganism, diagnosing and controlling H. parasuis infection are difficult. The traditional diagnosis of H. parasuis infections is based on clinical signs, pathological findings, and bacteriologic culture (Oliveira and Pijoan, 2004). Several etiologic diagnosis methods, such as
∗ Corresponding author. Tel.: +86 931 8342676; fax: +86 931 8340977. E-mail address: [email protected] (Z. Lu). http://dx.doi.org/10.1016/j.actatropica.2015.04.009 0001-706X/© 2015 Elsevier B.V. All rights reserved.
immunohistochemistry (Segales et al., 1997), oligonucleotidespecific capture plate hybridization (OSCPH) assay (Calsamiglia et al., 1999), polymerase chain reaction (PCR) (Oliveira et al., 2001), in-situ hybridization (Jung and Chae, 2004), improved speciesspecific PCR (Angen et al., 2007), real-time PCR (Turni et al., 2010), loop-mediated isothermal amplification (Chen et al., 2010; Wang et al., 2010), and plate agglutination (Guo et al., 2010), were developed for rapid diagnosis of H. parasuis. Several serological methods, such as complement fixation (Takahashi et al., 2001), ELISA (Miniats et al., 1991; Solano-Aguilar et al., 1999), and indirect hemagglutination (IHA), were developed to detect antibodies against H. parasuis, but the results were often inconsistent and inaccurate (Miniats et al., 1991). The early developed IHA, which employed the supernatant of sonicated whole cells or boiled cells as coating antigens for sheep red blood cells (SRBCs), was unreliable for the assessment of protective immunity against H. parasuis (Miniats et al., 1991). IHA based on H. parasuis antigen (serovars 4, 5, and 12) has been applied to investigate the most prevalent serovars of H. parasuis infection in China (Zhang et al., 2014); however, it is unable to detect antibodies against all serovars of H. parasuis. The oligopeptide permease ABC transporter membrane protein (OppA), which belongs to the ATP-binding cassette (ABC) transporter family, has previously been identified as a target for the
S. Chen et al. / Acta Tropica 148 (2015) 8–12
development of vaccines against pathogenic bacteria (Garmory and Titball, 2004), such as Yersinia pestis (Tanabe et al., 2006). This study aimed to explore the possibility of using the OppA protein as the antigen to sensitize SRBCs, and to develop an IHA test for evaluating H. parasuis seroprevalence in China. 2. Materials and methods 2.1. Strains and serum samples The 15 reference strains of H. parasuis and a serovar 1 Australian field isolate, HS145, were kindly provided by Dr. Pat Blackall, Qld DPI, Animal Research Institute, Australia. These strains and three isolates of H. parasuis from China, Hps30, Hps31 and Hps0818, were used for hyperimmune antiserum production by rabbits as previously described (Rafiee and Blackall, 2000). Antisera against Actinobacillus pleuropneumoniae (serovars 1, 3, and 7), Pasteurella multocida (serovars B1, B2, D2, and D3), and Bordetella bronchiseptica, as well as the rabbit anti-OppA polyclonal serum, were prepared and stored in our laboratory. Antiserum against Mycoplasma hyopneumoniae was provided by Jiangsu Academy of Agricultural Sciences, Nanjing, China. Antiserum for Streptococcus suis serotype 2 was provided by Wuhan Keqian Animal Biological Products Co., Ltd. (Wuhan, China). Escherichia coli BL21 (DE3)lysate polyclonal antibody was a product of Proteintech Group Inc. (Chicago, IL, USA). A total of 325 clinical pig serum samples were collected from three different pig farms located in Suizhou, Yangxin, and Wuxue of Hubei, China. All animal studies were approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute. 2.2. Construction of recombinant plasmid containing the OppA gene of H. parasuis Based on the nucleotide sequence of H. parasuis strain SH0165 (GenBank accession no: NC 011852.1), the primers OppA-F (5 GGCGAATTCATGCAAACAACCTTTACC-3 , underlined is EcoRI site) and OppA-R (5 -CCCTCGAGTTACTGCTTAATGATATA-3 , underlined is XhoI site) were designed to amplify the 1638 bp full-length OppA gene. DNA of the Nagasaki strain (serovar 5 reference strain) was extracted from a culture in tryptic soy broth using a TIANamp bacterial DNA kit (TianGen Corporation, Beijing, China) and further used as the PCR template. The PCR conditions were as follows: 94 ◦ C for 5 min, followed by 35 cycles (94 ◦ C for 50 s, 51 ◦ C for 45 s, and 72 ◦ C for 2 min), and a final 10 min elongation step at 72 ◦ C. The PCR product was then inserted into the TA cloning vector (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. Plasmid DNA-containing OppA of H. parasuis was purified with a TIANprep Mini Plasmid Kit (TianGen Corporation, Beijing, China) and sequenced. Furthermore, the TA plasmid-containing OppA was digested with EcoR1 and XhoI, and inserted into similarly digested pET-30a(+) to construct the recombinant plasmid pET-30a-OppA.
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protein expression conditions were optimized at different temperatures (37 ◦ C, 25 ◦ C, 20 ◦ C, and 16 ◦ C) and various concentrations of isopropyl -d-thiogalactopyranoside (0.01–2 mmol/l). The expression level was tested by SDS-PAGE and stained with Coomassie brilliant R250. Expressed protein was further purified with a Ni2+ column in His-Bind Kits (Gen Script, Nanjing, China). Protein concentration was estimated using a Nano assay. An aliquot of the purified protein was stored at −70 ◦ C for later use. 2.4. SDS-PAGE and Western blot analysis The purity and identity of protein were analyzed by 12% SDSPAGE and then stained with Coomassie brilliant R250. The antigenic protein was determined by Western blot. In brief, purified protein was separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking at room temperature for 2 h with 10% skimmed milk (Becton, Dickinson and Company, New Jersey, USA) in TBST, the membrane was incubated with the 1:200 diluted rabbit anti-H. parasuis polyclonal serum at room temperature for 2 h. The membrane was then washed and incubated with 1:5000 diluted HRP-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China), which was used to detect the bound antibodies. Colorimetric reaction was observed using the diaminobenzidine enhanced liquid substrate system (TianGen Corporation, Beijing, China). 2.5. IHA test For the IHA test, the purified OppA protein was used as the antigen. Rabbit anti-OppA polyclonal serum, healthy rabbit serum, and PBS (0.15 mol/l, pH 7.2) were employed as positive control serum, negative control serum, and dilution control, respectively; an antigen control was also included. The optimal antigen concentration (1.25, 2.5, 5, 10, 20, 40, 60, 80, and 100 g/ml), temperature (56 ◦ C and 37 ◦ C), and time of sensitized SRBCs (15, 30, 45, and 60 min), as well as sensitization buffer pH (pH 6.4 and pH 7.2), were evaluated using the titration test. The sensitized SRBCs were washed three times with PBS (0.15 mol/l, pH 7.2) with 1% healthy rabbit serum, and centrifuged at 3000 r/min for 10 min. The cell pellet was then resuspended with PBS (0.15 mol/l, pH 7.2) with 1% healthy rabbit serum at 10% concentration, and diluted to a final 1% for the IHA test. The specificity, sensitivity, reproducibility, and stability of the established IHA assay were determined. 2.6. Comparison of IHA and commercial ELISA The developed IHA and commercial ELISA kit (BioChek) were compared. The sensitivity, specificity and the agreement ratio were determined by testing 160 pig serum samples. These samples contained 80 positive samples and 80 negative samples for anti-H. parasuis antibodies, which were detected by ELISA. 2.7. Application of IHA IHA was used to evaluate the seroprevalence of H. parasuis in three different pig farms from three cities in Hubei Province, China.
2.3. Expression and purification of recombinant OppA protein 3. Results Recombinant plasmid pET-30a-OppA was transformed into E. coli BL21 (DE3) competent cells to produce the recombinant strain E. coli BL21 (DE3). For protein expression, recombinant E. coli BL21 (DE3) cells were grown on LB agar containing 50 g/ml kanamycin. A 4 ml overnight LB culture (containing 50 g/ml kanamycin) originating from a single colony from the agar was inoculated into 400 ml of LB (containing 50 g/ml kanamycin), and incubated at 37 ◦ C with shaking until the bacteria reached the midlog growth phase (OD600 = 0.6). For soluble protein expression, the
3.1. Production of the OppA recombinant protein The 1638 bp OppA gene was amplified using PCR, inserted into the plasmid pET-30a(+), and confirmed by DNA sequencing and double digestion with EcoRI and XhoI (Fig. 1). The recombinant plasmid was transformed into E. coli BL21 (DE3), and the OppA recombinant protein was successfully expressed. The expression condition of the OppA recombinant protein was optimized, and
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Fig. 1. (a) PCR amplification of the OppA gene of H. parasuis. Lane M, molecular standard DNA marker; lanes 1–3, DNA of H. parasuis Nagasaki strain; lane 4, negative control. (b) Restriction enzyme digestion of the recombinant plasmid pET-30a-OppA with EcoR1 and XhoI. Lane M, molecular standard DNA marker; lane 1, the recombinant plasmid pET-30a-OppA.
half of the OppA recombinant protein was successfully expressed as an insoluble form at 16 ◦ C for 16 h. The insoluble His-tagged fusion protein was purified using a Ni-NTA His Bind resin column, and identified to be approximately 66.8 kDa by SDS-PAGE analysis (Fig. 2a). The antigenicity of the recombinant OppA protein was confirmed by measuring its interaction with rabbit anti-H. parasuis positive polyclonal serum (Fig. 2b). 3.2. Development of IHA The recombinant protein OppA was used as antigen for sensitizing SRBCs to develop the IHA test. The optimal antigen concentration of the sensitized SRBCs was 0.005–0.01 mg/ml. In addition, the optimal temperature and time of sensitized SRBCs were 37 ◦ C and 30 min, respectively. The optimal sensitization buffer of 0.15 mol/l PBS (pH 6.4) was obtained using the titration
test. A total of 40 known negative pig serum (as determined by commercial ELISA) and 40 known positive pig serum samples were used to determine the cut-off level for IHA. The results showed that the IHA titer ≥1:8 and IHA titer ≤1:2 were positive and negative, respectively. The IHA titer of 1:4 was considered doubtful and should be retested because this dilution mainly appeared in H. parasuis positive serum using commercial ELISA, but occasionally appeared in H. parasuis negative serum. If the IHA titer retested was still 1:4, it was considered positive. The high sensitivity of the test was more important than a high specificity when the test was used as a screening assay to avoid the importation of seropositive pigs into a pig farm, and to adopt timely measures to control H. parasuis infection. In the specificity test, all antisera against 15 reference strains of H. parasuis and field strains were positive (IHA titer 1:128–1:1024), and antisera against six other types of swine bacteria were
Fig. 2. (a) Proteins were analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue. Lane M, molecular standard protein marker; lane 1, crude extract from induced cells with plasmid pET-30a-OppA; lane 2, purified OppA protein. (b) Western blot analysis of the purified OppA protein. Lane M, molecular standard protein marker; lane 1, purified OppA protein; lane 2, negative control of pET-30a(+) vector. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. Chen et al. / Acta Tropica 148 (2015) 8–12 Table 1 Antisera used in this study and the results of the IHA test. Antisera against strains
IHA titer
Result
H. parasuis field HS145 (serovar 1) H. parasuis reference SW140 (serovar 2) H. parasuis reference SW114 (serovar 3) H. parasuis reference SW124 (serovar 4) H. parasuis reference Nagasaki (serovar 5) H. parasuis reference 131 (serovar 6) H. parasuis reference 174 (serovar 7) H. parasuis reference C5 (serovar 8) H. parasuis reference D74 (serovar 9) H. parasuis reference H367 (serovar 10) H. parasuis reference H465 (serovar 11) H. parasuis reference H425 (serovar 12) H. parasuis reference 17975 (serovar 13) H. parasuis reference 22113 (serovar 14) H. parasuis reference 15995 (serovar 15) H. parasuis field Hps30 (serovar 4) H. parasuis field Hps31 (serovar 5) H. parasuis field Hps0818 (untypable) Actinobacillus pleuropneumoniae (serovar 1) Actinobacillus pleuropneumoniae (serovar 3) Actinobacillus pleuropneumoniae (serovar 7) Pasteurella multocida (serovar B1) Pasteurella multocida (serovar B2) Pasteurella multocida (serovar D2) Pasteurella multocida (serovar D3) Mycoplasma hyopneumoniae Streptococcus suis serotype 2 Bordetella bronchiseptica Escherichia coli BL21 (DE3)
1:512 1:1024 1:1024 1:512 1:1024 1:512 1:128 1:256 1:128 1:1024 1:1024 1:1024 1:256 1:1024 1:512 1:256 1:256 1:256 1:2 1:2 1:0 1:2 1:2 1:2 1:2 1:0 1:0 1:0 1:0
+ + + + + + + + + + + + + + + + + + − − − − − − − − − − −
A total of 325 clinical serum samples collected from pigs in herds with a history of fibrinous polyserositis and arthritis were tested with the IHA test. The results showed that the seroprevalence rate for H. parasuis antibodies ranged from 5.5% to 26.2% in all tested unvaccinated pig herds (Table 4). This result suggested different degrees of H. parasuis infection in pig herds in Hubei, China. Notably, the seroprevalence rates from 30- to 90-day-old pigs were significantly higher than those from replacement gilts and multiparous sows. Of the 325 serum samples, 57 samples were positive for H. parasuis antibodies, showing an overall seropositive rate of 17.5% (Table 4).
4. Discussion
Experiment Dilution of the serum 2−1 2−2 2−3 2−4 2−5 2−6 2−7 2−8 2−9 2−10 2−11 2−12 # # #
# # #
# # #
# # #
# # #
# # #
# # #
# # #
# # #
+++ +++ ++
+ + +
− − −
−, 0%; + 25%; ++, 50%; +++, 75%; #, 100% hemagglutination.
negative (IHA titer 1:0–1:2) (Table 1). In the sensitivity test, the dilutions of H. parasuis IHA positive control serum (from 2−10 to 2−1 ) were all above 50% hemagglutination. This result suggested that the sensitivity was 2−7 (2−10 /2−3 ) by the developed IHA (Table 2). Furthermore, the results of three parallel experiments of the IHA test were identical, which indicated that IHA had excellent reproducibility (Table 2). For the stability of IHA, similar sensitivity and specificity were achieved with the freeze-dried sensitized SRBC antigen stored at 4 ◦ C for six months and at −20 ◦ C for 12 months. 3.3. Validation of IHA A total of 160 serum samples selected from four different pig herds from four provinces (Sichuan, Henan, Jiangxi, and Guangxi) in China were tested by both IHA and commercial ELISA kits to evaluate IHA. As shown in Table 3, among the 80 ELISA-positive Table 3 Agreement ratio between the developed IHA and commercial ELISA for the detection of antibodies against H. parasuis. Serum samples
ELISA
80 Field samples 80 Field samples
Positive Negative
a Formula for the calculation (A + D/A + B + C + D) × 100.
Agreementa
IHA Positive
Negative
A (68) C (4)
B (12) D (76)
of
the
agreement:
90.0% %
serum samples, 68 were positive by IHA. In addition, among the 80 ELISA-negative serum samples, 76 were negative by IHA. The sensitivity of IHA was calculated to be 85.0%, and its specificity was 95.0%. The overall agreement between IHA and commercial ELISA kits was 90.0% (Table 3).
3.4. Application of IHA
Table 2 Sensitivity of the developed IHA to detect H. parasuis IHA positive control serum.
1 2 3
11
agreement =
The isolation of this organism is usually difficult, particularly after antibiotic treatment (Olvera et al., 2007). However, the isolation of H. parasuis is still the “gold standard” for the diagnosis of Glässer’s disease from diseased pigs, and further serotyping is necessary because it is part of the normal respiratory microbiota. The commercial indirect ELISA kit, which explored the oligopeptide permease A (OppA) as coat antigen, has been suggested to track antibodies against H. parasuis (Macedo et al., 2011). In the present study, an IHA test was developed using another ABC transporter protein, the oligopeptide permease ABC transporter membrane protein (only 29% amino identity with previous OppA). These results indicated that the developed IHA could specifically detect antiserum against H. parasuis (15 reference H. parasuis, nontypeable strains, and field isolates), and no cross-reaction was detected with other related bacterial antisera. This method showed excellent reproducibility and high sensitivity, and it was highly consistent with commercial ELISA. Moreover, it was simpler, much cheaper, and faster than commercial ELISA. Thus, this method is suitable for large-scale serological screening of H. parasuis antibodies in pig serum. Selecting an antigen is important to establish the IHA assay. Several studies demonstrated that IHA tests based on boiled or autoclaved extracts can be used for serotyping of H. parasuis, and such tests showed more sensitivity than the immunodiffusion test (the “gold standard”) (Del Rio et al., 2003; Tadjine et al., 2004). During the preliminary study, whole cells were used as the antigen to sensitize SRBCs, and extensive cross-reactions were observed with A. pleuropneumoniae and P. multocida polysera. The possibility of the OppA protein as an antigen was then examined. The results showed that OppA proteins owned 97–99% amino identity among H. parasuis strains, as well as a maximum of 74% amino identity with other bacterial pathogens examined (data not shown). Protein expression was optimized to obtain an active protein. Low temperature (16 ◦ C) and long time (16 h) could partly induce the expression of recombinant protein in soluble form. This study established recombinant OppA-based IHA for detecting species-specific antibodies against H. parasuis, and determined that it could be used to distinguish antibody against H. parasuis from other bacterial pathogens. However, OppA-IHA was unable to differentiate infection from vaccinated pigs, so further research should be carried out to develop a different serological method that differentiates infected animals from vaccinated animals.
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Table 4 Positive rates of the anti-H. parasuis antibodies determined by the developed IHA in three herds in Hubei, China. Type of pigs
30 Days 60 Days 90 Days Replacement gilt Multiparous sows Subtotal Total
Suizhou
Wuxue
Yangxin
No. of positive/no. tested
Positive rate (%)
No. of positive/no. tested
Positive rate (%)
No. of positive/no. tested
Positive rate (%)
15/30 3/28 9/29 1/10 0/10 28/107 57/325, 17.5%
50.0 10.7 31.0 10.0 0 26.2
6/28 3/30 11/30 2/10 1/10 23/108
21.4 10.0 36.7 20.0 10.0 21.3
6/30 0/30 0/30 0/10 0/10 6/110
20.0 0 0 0 0 5.5
Aside from antigen, other factors, such as antigen concentration, sensitization temperature, time, and sensitization buffer, could affect the sensitivity of the IHA test. The optimized conditions for sensitized SRBCs were determined. The SRBC donors should also be selected carefully and tested simultaneously as non-sensitizing SRBC control when the IHA procedure was performed because some donors of SRBCs could agglutinate several types of bacterial antisera, such as P. multocida. This phenomenon may be due to the existence of heterophile antibodies, and it can be solved by incubating the diluted serum with glutaraldehyde-treated tanned SRBCs at 37 ◦ C for 30 min as previously described (Levieux et al., 1992). The seropositive rate of H. parasuis antibodies in tested clinical pig serum samples ranged from 5.5% to 26.2%, thereby suggesting that H. parasuis infection is endemic in Hubei, China. This result was consistent with that reported by Zhang et al. (2012). In the present study, 30- to 90-day old pigs were more likely to be seropositive than replacement gilts and multiparous sows. This age difference in the susceptibility of H. parasuis was also stated in reports elsewhere (Olvera et al., 2007). In conclusion, the developed OppA-IHA is a useful serological tool for epidemiological investigations and immunization surveillance of H. parasuis. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was funded by the Special Fund for Agro-scientific Research in the Public Interest (Grant no. 201303034) to LZX. References Angen, O., Oliveira, S., Ahrens, P., Svensmark, B., Leser, T.D., 2007. Development of an improved species specific PCR test for detection of Haemophilus parasuis. Vet. Microbiol. 119, 266–276. Cai, X., Chen, H., Blackall, P.J., Yin, Z., Wang, L., Liu, Z., Jin, M., 2005. Serological characterization of Haemophilus parasuis isolates from China. Vet. Microbiol. 111, 231–236. Calsamiglia, M., Pijoan, C., Solano, G., Rapp-Gabrielson, V., 1999. Development of an oligonucleotide-specific capture plate hybridization assay for detection of Haemophilus parasuis. J. Vet. Diagn. Invest. 11, 140–145. Chen, H.T., Chu, Y.F., Liu, Y.S., Zhang, J., Lu, Z.X., 2010. Loop-mediated isothermal amplification for the rapid detection of Haemophilus parasuis. FEMS Immunol. Med. Microbiol. 60, 283–285. Chu, Y.F., Gao, P.C., Zhao, P., He, Y., Zhang, N.Z., Liu, Y.S., Liu, J.X., Lu, Z.X., 2011. Genotyping of Haemophilus parasuis isolated from northwest China using PCRRFLP based on the ompA gene. J. Vet. Med. Sci. 73, 337–343.
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