An ELISA For Differentiating Natural Infection From Vaccination In Foot-and-Mouth Disease

Foot-and-mouth disease (FMD) is a highly contagious disease that inflicts cloven-hoofed animals including cattle, sheep and pigs. The onset of FMD is very rapid and results in a drastic negative impact on the economic progress of animal industry, considerable losses in affected countries. When an outbreak of FMD occurs, many trade restrictions are placed upon,resulting in an dramatic reduction of susceptible animals and animal products. Approach to control and eradicate FMD is based either on a stamping out strategy or on systematic vaccination. Neutralizing antibodies against FMDV induce by vaccination. With low level of neutralizing antibody, subclinical infection, which can become persistent, the so called carrier state is established when vaccinated animals are exposured to infection. Animals of carrier state shed virus persistently and become a source of new outbreaks of FMD accompanying new viral variants and are not accepted in international export trades for wanting health centificates. Those infected animals with low level of neutralizing antibody that show no significant clinical symptoms have difficulties in differentiating from those vaccinated. Therefore, differentiating natural infection from vaccination may facilitate the control and eradication of FMD. Foot-and-mouth disease virus (FMDV) is the pathogen that causes FMD. FMDV has four structural proteins and eight non-structural single proteins (NSPs) besides two non-structural polyproteins 3AB and 3ABC. Eight non-structural single proteins include L, 2A, 2B, 2C, 3A, 3B, 3C and 3D protein. For genes of structural protein variating rapid, neutralizing antibodies against FMDV induced by structural proteins of a particular strain will bind that strain, but not recognize strains belonging to other serotypes. By contrast, Non-neutralizing antibodies induced by NSPs aren't serotype-specific.As we know, both infection and vaccination elict neutralizing antibodies and therefore virus neutralization test can not differentiate natural infection from vaccination. Instead, detecting antibodies against non-structural proteins (NSPs) is by now generally accepted as a promising means to differentiate natural infection from vaccination of FMDV vaccines irrespective of virus serotypes. Vaccines consisting of partly purified inactivated FMDV or recombinant structural proteins of FMDV induce antibodies theoretically to the structural proteins only, whereas infected animals develop antibodies to both structural and non-structural proteins. Amongst the NSPs, 3AB and 3ABC polyproteins are well documented as the most immunogenic and possess the longest duration of inducing antibodies response. Therefore, detecting antibodies against the polyprotein3AB and 3ABC has potentials to differentiate infected animals from vaccinated animals. In our study, 3AB protein was selected as coating antigen in indirect ELISA for differentiating natural infection from vaccination of FMDV.In this report, recombinant 3AB gene fragments were amplified from cDNA of FMDV strain AsiaI/JiangSu/China/2005 by RT-PCR using specific primers and subsequently cloned into prokaryotic expression vector pET-28a(+) to construct pET28a-3AB plasmids. The plasmids were transformed into competent E.coli cells BL21 (DE3), in which 3AB protein fusion with his-tag was successfully expressed. Nickel affinity chromatograph was used to purify recombinant 3AB protein whose immunogenecity was detected by Western blot. Using 3AB protein as coating antigen, an indirect ELISA to differentiating natural infection from vaccination of FMDV vaccine was developed. 40 sera from cows infected with FMDV and 30 sera from cows immunized were tested by the ELISA, with 20 Sera from healthy cow as negative controls. Eventually, different concentrations of coating antigen, coating buffers, blocking buffers and diluting buffers were evaluated in order to optimize the conditions for indirect ELISA.According to the Agarose Gel Electrophoresis and sequencing analysis, we obtained 3AB gene fragments of 672bp, and constructed recombinant pET28a-3AB plasmids. After transforming into E.coli cells BL21 (DE3), recombinant 3AB protein was expressed with a yield of more than 30% whole bacterial proteins, giving an apparent molecule mass of 34KD. The recombinant protein was purified by Nickel affinity chromatograph to 90% purity. Subsequently Western blot was conducted to comfirm the immunogenicity of recombinant 3AB protein. Result indicated that 3AB protein was immunogenic and recognized by sera from cow infected FMDV. Correspondingly, the result of ELISA for differentiating natural infected from vaccination of FMDV vaccines indicated that antibodies against FMDV non-structural protein 3AB were detected in sera of cows infected with FMDV (AsiaI, JMS) but not immunized with inactivated FMDV and A7. For further comfirming, we tested a panel of serum samples from bovine vaccinated with FMDV vaccines, normal and FMDV infected. Out of 40 sera from FMDV experimental infected cow, 37 sera were detected as positive, and out of 30 sera from immunized with vaccines cow, 29 were detected as negative. It was clear that the ELISA was capable of differentiating natural infection from vaccination of FMDV vaccines irrespective of serotype. The specificity and sensitivity of the ELISA were 96.7% and 92.5% respectively. Base on the results, we concluded indirect ELISA using 3AB protein as coating antigen was specific, sensitive and reproducible to differentiate natural infection from vaccination of FMDV. There were many parameters which influence the results obtained in an ELISA. Square matrix experiment was conducted to selected optimal antigen concentration and serum dilution. Recombinant 3AB protein should be diluted to 2μg/mL and the sera dilution of 1:100 was defined as the optimal sera dilution for the assay. Futhermore,after optimizing ELISA conditions, we selected PBS as coating buffer, 5% skimmed milk as blocking buffer and diluting buffer.Together, the ELISA we developed was simpler, faster and more practical for differentiating natural infection from vaccination of FMDV vaccines, fitting for large-scale screening comparing to liquid blocking ELISA.