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Mycoplasma synoviae, a major worldwide pathogen in poultry, causes respiratory tract infection and arthritis in chickens and turkeys. Two major surface antigens of M. synoviae are encoded by a single gene, vlhA (variably expressed lipoprotein and hemagglutinin). The gene product is cleaved post-translationally to yield the lipoprotein major surface protein (MSP) B (MSPB) and the hemagglutinin MSPA. The availability of MSPA as an antigen for serodiagnosis was studied by means of a protein chip based on surface plasmon resonance imaging (SPRi). The diagnostic potential of SPRi for measurement of levels of antibody to MSPA was compared with that of a conventional enzyme-linked immunosorbent assay (ELISA) kit. The results from SPRi, a process that took only 1 h, were similar to those from ELISA. Therefore, MSPA can be used as an antigen for serologic studies, and SPRi, a label-free and high-throughput method, may be a valuable tool in avian serodiagnostic studies.
Mycoplasma synoviae est un agent pathogène de la volaille mondialement répandu, qui cause des infections du tractus respiratoire et de l’arthrite chez les poulets et les dindons. Deux antigènes de surface majeurs de M. synoviae sont codés par un gène unique, vlhA (lipoprotéine et hémagglutinine à expression variable). Le produit du gène est clivé post-traduction pour générer la lipoprotéine dénommée protéine majeure de surface (MSP) B (MSPB) et l’hémagglutinine MSPA. La disponibilité de MSPA comme antigène pour le sérodiagnostic a été étudiée grâce à une biopuce protéinique basée sur l’imagerie par résonance de plasmon de surface (SPRi). Le potentiel diagnostique de SPRi pour mesurer les niveaux d’anticorps contre MSPA a été comparé avec celui d’une trousse immuno-enzymatique (ELISA) conventionnelle. Les résultats de SPRi, un processus qui n’a pris que 1 h, étaient similaires à ceux de l’ELISA. Ainsi, MSPA peut être utilisé comme un antigène pour des études sérologiques, et SPRi, une méthode sans marqueur et à haut débit, comme un outil utile dans des études en sérodiagnostic aviaire.
(Traduit par Docteur Serge Messier)
Mycoplasma synoviae is a major worldwide poultry pathogen that causes respiratory tract infection and arthritis in chickens and turkeys. It is an important cause of chronic respiratory disease and synovitis in chickens and causes serious economic losses in the worldwide poultry industry (1). Two of the major surface antigens in M. synoviae are encoded by a single gene, vlhA (variably expressed lipoprotein and hemagglutinin). The gene product is cleaved post-translationally to yield the lipoprotein major surface protein (MSP) B (MSPB) and the hemagglutinin MSPA (2,3). A previous study revealed that the immunodominant 45- to 50-kDa cluster of membrane proteins in the WVU-1853 strain of M. synoviae consists of 2 groups of membrane antigens, MSPA and MSPB, and that MSPB was expressed in all strains tested (3).
Control of M. synoviae infection is highly dependent on diagnosis by serologic assays, including rapid slide agglutination tests to detect infections within the flock. However, these assays have limited specificity and sensitivity, primarily because of cross-reactions (4). Several enzyme-linked immunosorbent assays (ELISAs) have been developed to detect antibodies against M. synoviae but have generally been based on poorly defined membrane components (5–9). An ELISA based on recombinant MSPB (rMSPB) has been described (10). Use of an ELISA based on rMSPB from M. synoviae strain H improved serologic detection in vaccinated birds relative to the use of an ELISA based on recombinant protein from a heterologous strain (4). However, serodiagnostic studies evaluating MSPA had not been performed until the present work.
Methods using ELISA are quite reliable but also time- and labor-intensive. Most of the currently used protein arrays rely on detection by means of enzymatic or fluorescent tags. In contrast, surface plasmon resonance imaging (SPRi) is a rapid, label-free, surface-sensitive spectroscopic technique used to examine bioaffinity interactions on thin gold films (11). It detects changes in the refractive index within a short distance from the surface of a thin metal film as variations in light intensity reflected from the back of the film and does not require labeling (12–14). This technique has been successfully used to screen various bioaffinity interactions using proteins (15,16). This article describes the use of rMSPA to develop a protein chip based on SPRi to detect antibodies to M. synoviae in chicken serum and reports the diagnostic efficacy of SPRi compared with that of conventional ELISA in detecting M. synoviae infections.
The M. synoviae strain WVU-1853 (American Type Culture Collection [ATCC] no. 25204) was obtained from the National Veterinary Research and Quarantine Service (NVRQS), Anyang, Korea, and DNA from this strain was used as a template for polymerase chain reaction (PCR) amplification. Recombinant MSPA was prepared with the use of a prokaryotic expression system (pRSET A vector; Invitrogen, Carlsbad, California, USA). Briefly, the MSPA gene (GenBank no. AF035624) was amplified by PCR with 2 primers, MSPA-F (5′-GGCC GGATCC ATG GATGAAGTTAGATTTTCTAA-3′, from nucleotides 1590 to 1609) and MSPA-R (5′-GGCC AAGCTT TCAACTATTGCTTGCTATTG-3′, from nucleotides 2227 to 2246), containing sites (underlined) for 2 restriction enzymes, BamHI and HindIII (B + H). The PCR products were cloned into the B + K-digested pRSET A (17), and the recombinant DNA was transformed into BL21(DE3)pLysS (Invitrogen) host cells. The transformants were grown in 25 mL of Luria–Bertani medium with ampicillin and chloramphenicol to an optical density (OD) of 0.5. Protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (final concentration 1 mM). The proteins were purified under denaturing conditions by means of an affinity purification system (Probond, Invitrogen).
A total of 302 field samples (from 14 farms) from chickens that had not been vaccinated against M. synoviae were screened by both SPRi and ELISA. Three positive samples from chickens with previously confirmed M. synoviae infection and 3 negative samples from specific-pathogen-free (SPF) chickens were used as controls. To rule out the cross-reactivity caused by antigens in common between M. synoviae and M. gallisepticum, 3 serum samples from SPF chickens experimentally infected with M. gallisepticum S6 (ATCC no. 15302; NVRQS) were used as species controls.
Surface modification of a patterned glass slide chip with a gold film (K-Mac, Daejeon, Korea) for the specific antigen binding was carried out as previously described (18). Briefly, the slides were cleaned with a freshly prepared piranha solution (3:1 mixture of concentrated H2SO4 and 30% H2O2), coated with ProLinker B (Proteogen, Seoul, Korea), washed in deionized water, and dried stream. The clean chips were soaked in 3 mM ProLinker B under a N2 solution for 1 h, then rinsed sequentially with chloroform, acetone, ethanol, and deionized water. The chips were dried and the antigens in phosphate-buffered saline (PBS), 100 μg/mL, spotted onto their surface by means of the ProteoChip (Proteogen), which has miniaturized wells for quantitative antibody assays that contain a minute amount of serum (1 μL per spot). The chips were then incubated for 20 min in 80% humidity at 37°C, rinsed 3 times with a mixture of PBS-T (PBS + 0.05% Tween-20, pH 7.4), and then rinsed with deionized water. Test samples of antibody or serum in a PBS buffer containing 0.1 mg/mL of bovine serum albumin (BSA), 5 to 10 nL per spot, were mixed with 1% BSA in PBS for 10 min to block the chip surface. The test samples were then applied to the chips for 15 min. The chips were dried and analyzed with the SPRi system (K-Mac).
The minimum detection threshold of the SPRi was determined by testing 8 serially diluted (1 to 1/5000) positive-control serum samples containing chicken polyclonal antibody against M. synoviae. The results were compared with those from a commercially available ELISA kit (FlockChek; IDEXX Laboratories, Westbrook, Maine, USA). The ELISA was performed according to the manufacturer’s instructions, and data from 2 separate experiments were compared; the results were expressed as the mean S/P ratio [and standard deviation (s)] of the end-point titers. The mean signal intensity (SPRi) and OD (ELISA) values of positive and negative samples were compared by means of Student’s t-test with the SPSS 14.0 program (SPSS, Chicago, Illinois, USA).
The rMSPA was produced and purified without contamination (data not shown) and used as an antigen for both SPRi and ELISA. The mean S/P ratio (and s) by ELISA was 0.044 (0.016) for the negative controls and 4.831 (1.404) for the positive controls; these values were significantly different (P < 0.005). The mean S/P ratio for the species controls (infected with M. gallisepticum) was 0.054 (0.029) (P < 0.005). All the tested field samples but 3 (99%) were positive [mean S/P ratio 2.800 (1.053)], which suggests that M. synoviae infection is quite common in Korea and highlights the need for a proper prevention program to protect chickens against M. synoviae infection.
Figure 1 shows that the signal intensity in the SPRi increased linearly with increasing antibody concentration; it also presents the original images for each control antibody dilution. The detection limits of the SPRi protein chip corresponded to a 1:500 antibody dilution and a signal intensity of 20. The mean signal intensity (and s) was 38.804 (3.177) for the positive controls and 0.714 (2.789) for the negative controls. An OD ≥ 9.080 (corresponding to 3 s above the negative-control mean) was considered positive. As a result, 281 of the field samples (93%) were considered positive by SPRi (Figure 2), which suggests that the protein chip based on SPRi diagnostics was as sensitive as the ELISA.
The mean signal intensity (and s) for the species controls was 4.518 (3.977) (P < 0.005). The antigenic relatedness of the 2 species used in this study makes it difficult to distinguish them with conventional serologic tests (19). In a recent study, M. synoviae ELISA kits showed a varying rate of false-positive reactions (due to cross-reactivity), resulting in a specificity ranging from 2% to 96% for serum samples infected with M. gallisepticum and from 33% to100% for uninfected samples (20). In this study, the species controls (infected with M. gallisepticum) had a low mean OD and a low mean signal intensity, which indicates that the cross-reactivity between the 2 species of Mycoplasma used in this study is not great enough to result in false-positive SPRi results, and there are some antigenic differences among M. gallisepticum and M. synoviae strains.
The newly developed SPRi was successfully used to detect antibodies against MSPA in chicken serum. This comparative study confirmed that ELISA is reliable but time-consuming. Many serodi-agnostic assays using MSPB as an antigen have been performed, and our data suggest that MSPA is a good candidate for development of the serodiagnostic method. Many SPR assays have recently been described for the detection of antibodies against pathogens (12–14). However, the SPR assay can be applied to only 2 to 4 samples simultaneously. Compared with the SPR assay, the newly developed SPRi protein chip with miniaturized wells proved to be rapid tool (assay time 1 h) with high throughput (up to 300 to 400 samples at a time) and value (up to 4 recycles) for the serodiagnosis of M. synoviae infection in chickens.
The authors acknowledge a graduate fellowship provided by the Korean Ministry of Education and Human Resources Development through the Brain Korea 21 project. The authors also thank Ms. Myeonghwa Kim, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea, for her excellent technical assistance.