Changes in Immunity, Expression of some Immune-Related Genes of Shabot Fish, Tor grypus, Following Experimental Infection with Aeromonas hydrophila: Effects of Autochthonous Probiotics.

In this study, the effects of orally administrated two native probiotics (Lactobacillus plantarum and Lactobacillus delbrueckii ssp. bulguricus), isolated from the intestine of Shabot fish, Tor grypus, on some immune response parameters and immune-related genes expression against Aeromonas hydrophila in T. grypus were evaluated. Four hundred and eighty juveniles weighing 45 ± 10 g were randomly divided into four groups (with three replications) and fed with the experimental diet containing 5 × 107 cfu g-1 of L. plantarum (G1), Lactobacillus bulgaricus (G2), Lactobacillus casei (G3), and a control diet (without probiotics) for 60 continuous days. At the end of the dietary treatments, fish were challenged with a lethal concentration of A. hydrophila (5 × 108 CFU ml-1) via intra peritoneal (i.p) injection. Blood and head kidney samples were taken from six fish in each treatment before challenging and 6, 12, 24, and 48 h and also 7 days after injection. The results showed that lysozyme, complement, bactericidal, and NBT activity of probiotic-treated groups were significantly elevated (P < 0.05). The IL-8, IL-1ß, and TNF-α gene expressions were significantly higher in all probiotic-treated groups (P < 0.05). Meanwhile, a high direct correlation was observed between serum immune parameters and expression of immune-related genes (P < 0.0001); furthermore, the highest correlation (R 2 = 0.634, P < 0.0001) was recorded between IL-1ß expression and NBT activity. It can be concluded that not only two native probiotics strains stimulate serum immune responses parameters and immune-related gene expression in T. grypus, but also a high correlation was seen among these indices. The study suggests that gastrointestinal colonization is preferred for host specificity as the strain previously derived from shabot fish displayed better colonization than the non-indigenous bacteria strain such as L. casei. Therefore, these native probiotics bacteria can be accounted as suitable candidates to immune stimulation in fish.

Molecular Cloning, Expression and Peroxidase Conjugation of Staphylococcus aureus Protein A.

BACKGROUND: Staphylococcal protein A (SPA) is a cell wall component of Staphylococcus aureus that binds to different IgG subclasses of human and several animal species. This bacterial protein can be used as an antibody detector in various immunological assays or as an isolation reagent for the purification of antibody molecules via immuno-chromatography procedures. OBJECTIVES: Molecular cloning and expression of SPA followed by the purification and conjugation of the recombinant protein to peroxidase enzyme. MATERIAL AND METHODS: Encoding DNA fragment of SPA was amplified and inserted into a prokaryotic plasmid vector for the expression of recombinant SPA fused to a maltose binding protein (MBP). The recombinant protein was purified using amylose resin column chromatography and conjugated to horseradish peroxidase (HRP) enzyme. Finally, the reactivity of the recombinant SPA was examined against human IgG molecules in ELISA. RESULTS: The results indicated that the recombinant peroxidase-conjugated SPA has a good recognition capacity for human IgG molecules and it was able to produce significant OD values after reacting with human IgG molecules at a concentration up to 0.06 µg.well-1. CONCLUSIONS: This recombinant protein can be very useful in all research laboratories and may decrease some of the expenses, e.g. those for preparing conjugated anti-antibodies.