Improving the diagnosis of bovine tuberculosis using gold nanoparticles conjugated with purified protein derivative: special regard to staphylococcal protein A and streptococcal protein G.

Abstract

Different ancillary immunodiagnostic tests were traditionally-established for diagnosis of bovine tuberculosis (BTB) either cellular or humoral as tuberculin skin test (TST), gamma interferon (INF-γ), and indirect enzyme-linked immunosorbent assay (iELISA). These tests had been consumed more time and expensive, and needed sophisticated equipment. To dissolve these problems, serological diagnosis depending on humoral immunity is the aim of this work. Herein, slide-based agglutination test was chosen as a rapid and simple field test based on purified protein derivative (PPD) antigen in addition to some supplementation materials such as Staphylococcal protein A (SPA) and Streptococcal protein G (SPG) to improve detection of BTB antibody in serum samples. Gold nanoparticles (Au NPs) were synthesized by gamma ray, and after complete characterization, the synthesized Au NPs were spherical, small-sized, and stable without any impurities. Addition of such supplementation reagents for serodiagnosis of tuberculosis is of paramount important for the detection of serum antibodies against tuberculosis (TB) and it was considered an easily simple and possible way for improving TB diagnosis. In this work, 70 animals tested positive for TST as well as 20 animals tested negative for TST were used for the diagnosis of BTB depending on humoral immune response based on PPD slide agglutination test using reporter regents (SPA and/or SPG) either native or recombinant. The agglutination density was recorded and read in 4 degrees of positivity with scores ranging from negative (-) to very strong reaction (++++) occurred in different times of agglutination. Groups showed 100% positive reactivates employed in Exp. 1, 2, and 3 with differentiation of slide agglutination test density and was rated from moderate positivity (2+) to very strong (4+), with predominant positivity in density of (3+). Pink-colored intensity is associated with the strengthened reactions between PPD-conjugated Au NPs and serum antibody of each tested samples, which allows for visual rapid, simple, and effective attractive diagnosis of BTB. The specificity and sensitivity of the serological tests were characterized. TST offers the highest sensitivity (83.6%) among the other immunoassays, while the lowest specificity was recorded in TST (57.4%). SPA/SPG offers the best performance in term of combined sensitivity and specificity (performance index) of 175.4. Therefore, the development and uses of detection reagent (such as SPA and/or SPG) slide co-agglutination test (COAT), either native or recombinant (rSPA/SPG) for the detection of TB antibodies based on PPD antigen, as well as the uses of Au NPs rSPA/SPG as detection conjugate based on the same antigen, were also performed as a simple, rapid, sensitive, specific, eco-friendly, and low cost, which shows a great potential in field and lab diagnosis of BTB. So, high reduction in reagents that yields reactions similarly as traditional techniques was needed.