A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM2), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM2/SDZ/SQX−bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T1, T2, and T3, respectively. With this method, the cutoff values for the three test lines were achieved at 80 μg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 μg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.

A rapid immunochromatographic assay (ICA) was developed and validated for the detection of sulfadiazine in eggs and chickens. Based on the competitive reaction mechanism, the competitor of sulfadiazine (sulfadiazine–BSA conjugate) was immobilized to the defined detection zone on a nitrocellulose membrane which acted as the capture reagent, and the monoclonal antibody against sulfadiazine was conjugated to colloidal gold particles which served as the detection reagent for the preparation of the immunochromatographic strips to test sulfadiazine. With this method, the semi-quantitative detection of sulfadiazine was accomplished in less than 15 min, with high sensitivity to sulfadiazine (5 ng/g) and low cross-reactivities with other sulfonamides. With experimental egg and chicken samples spiked with sulfadiazine at concentrations of 10, 20, and 100 ng/g, recoveries were demonstrated to be from 71% to 97% in egg samples and 71% to 95% in chicken samples. This method was compared with the enzyme-linked immunosorbent assay by testing 52 egg samples from the animal experiment, and compared with the high-performance liquid chromatographic method by testing 56 chicken samples, with an agreement rate of 100% for both comparisons, by using the maximum allowed residue of sulfadiazine (i.e. 100 ng/g) as the cut-off level as set by the European Union and China. The accuracy of ICA was also confirmed in an initial study with marketed egg and chicken samples. In conclusion, the method is rapid and accurate for the detection of sulfadiazine in eggs and chickens.

The S-layer protein CTC surface-display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying the protein of Mycoplasma gallisepticum (MG) agglutinin (pMGA) on the Bt cell surface. By fusing part of pmga1.2 (pmga1.2p) with the surface-anchoring motif of ctc, two recombinant plasmids, pCTC-PMGA1.2P and pCSPMGA1.2P, were constructed. They harboured the fusion genes ctc-pmga1.2p and csa-ctc-pmga1.2p (csa represents csaAB operon, important in anchoring the S-layer protein on the cell surface), respectively. Two recombinant Bt strains were constructed by electro-transferring recombinant plasmids to a Bt plasmid-free derivative strain BMB171. Strains obtained were BCCG (bearing pCTC-PMGA1.2P and the csaAB operon-carrying plasmid pMIL-CSA) and CG (pCSPMGA1.2P). The vegetative cells of both strains were used as antigens for haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA and HI assays showed that recombinant PMGA1.2P proteins were not only displayed on the cell surface of BCCG and CG, but also specific to MG-positive serum. After oral immunization of chickens with spores, both BCCG and CG elicited a humoral response to PMGA1.2P and exhibited immunogenicity, as indicated by serum plate agglutination (SPA) assays. This study suggests the possibility of generating heat-stable and oral vaccines against infectious diseases of fowl with Bt surface-display system.

A gold-immunochromatographic test-strip kit is used for the detection of IgG antibodies against the nucleocapsid protein of Avian Influenza Virus (AIV). Compared with the “gold standard”, i.e. hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) assays, the gold-immunochromatographic test strip has many advantages, such as high specificity, high sensitivity, convenience, is rapid and has low cost. The gold-immunochromatographic test strip provides a unique tool for the on-site surveillance and diagnosis of Avian Influenza.