A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections

Abstract

Mycoplasma gallisepticum is the primary agent of chronic respiratory disease causing important economic losses in the poultry industry. Serological monitoring is essential to maintain mycoplasma-free breeder flocks and often complicated by the cross-reactions between different mycoplasma species. To overcome serological cross-reactions, a large fragment of the M. gallisepticum PvpA cytadhesin, species-specific surface-exposed protein, was produced in E. coli as a recombinant protein (rPvpA336) and used as a potential diagnostic antigen. The rPvpA336 protein possesses 336 mycoplasma-specific amino acids with relative molecular weight of 44 kDa. A deletion region of 37 amino acids was identified when compared to the wild-type PvpA protein. Immunoreactivity of the rPvpA336 protein has been demonstrated by Western blot analysis with M. gallisepticum-positive and -negative chicken sera. Furthermore, an enzymatic rapid immunofiltration assay (ERIFA) prototype based on the rPvpA336 protein has been developed and its species-specific detection capability has been demonstrated by using M. gallisepticum and/or M. synoviae-positive and -negative chicken sera. In addition to its species-specificity, the ERIFA prototype presents certain advantages such as rapidity, field-applicability and cost-effectiveness. Therefore, these advantages would make the prototype a species-specific rapid diagnostic tool of choice in the field and limited laboratory conditions for screening M. gallisepticum infections.

Bi-Antigenic Immunoassay Models Based on the Recombinant PvpA Proteins for Mycoplasma Gallisepticum Diagnosis in Chickens

“…8 Escherichia coli DH5a and pCold-I bacterial expression vector b were used to produce recombinant protein.…”

“…h Polymerase chain reaction (PCR) amplification and amplicon purification protocols were essentially the same as described earlier. 8 Briefly, the purified amplicon of 402 base pairs in length was digested by SacI and XbaI, according to the manufacturer's instructions. i Cohesive-end ligation of the restricted amplicon pvpA134 into pCold-I vector was performed with the aid of T4 DNA ligase.…”

“…8 A single colony of the transformant containing pCold-pvpA134 vector was cultured until it reached 0.3-0.4 absorbance at 600 nm. Expression of the recombinant PvpA134 protein was induced by the addition of isopropyl beta-D-thiogalactoside (IPTG) i at a final concentration of 0.5 mM to the culture.…”

“…8 Briefly, 0.5 ml (1 mg/ml) of highly purified recombinant proteins were separately adsorbed onto the NC membrane as target antigens. After wetting and saturation of the NC membrane with 100 ml of PBST/FG, 50 ml of serum sample was added and flowed through the membrane.…”

“…Although WB is widely used as a confirmatory test, the use of rapid immunofiltration assay is rare in the veterinary diagnostic field. 8,19,22 However, bi-antigenic immunofiltration assay presents a promising individual field diagnostic model by virtue of its simplicity and the rapidity with which it can determine the reactor animals (in less than 5 min in the field and under limited laboratory conditions). In addition, this diagnostic model allows the evaluation of the results by visual inspection.…”