Effects of pre-treatment with waterborne selenium on redox homeostasis and humoral innate immune parameters in African catfish, Clarias gariepinus (Burchell, 1822), experimentally challenged with Serratia marcescens.

Bacterial infections have been associated with immune dysfunction and oxidative stress in cultured fish species while essential elements could boost immunity and exhibit antioxidant properties in fish. This study was therefore aimed at determining the effects of pre-treatment with waterborne selenium on humoral immunity and redox status of Clarias gariepinus experimentally challenged with Serratia marcescens. Juveniles C. gariepinus were pre-treated with 50 µg/L selenium for 14 days after which they were challenged with 5 × 103 CFU/mL of S. marcescens via oral gavage for 24 or 48 h. The control fish were not pre-treated with selenium and not challenged with bacteria. Thereafter, fish were sacrificed, blood collected into EDTA bottles for the determination of plasma nitric oxide levels and respiratory burst, and the liver excised for the determination of reduced glutathione, lipid peroxidation, and activities of catalase, superoxide dismutase, and glutathione peroxidase. Fish that were pre-treated with selenium prior to bacterial challenge (Sel + Bact) had decreased levels of nitric oxide and lipid peroxidation but a significant increase in the levels of reduced glutathione (at 48-h post-infection period only) compared to the fish challenged with bacteria without prior selenium pre-treatment (Bact). The respiratory burst and catalase activity decreased significantly in the Sel + Bact group especially at 48-h post-infection period while the activity of glutathione peroxidase increased significantly in the Sel + Bact group (at 24-h post-infection period only) compared to the Bact group. The results from this study showed that infection with S. marcescens is capable of disrupting the immune system and redox homeostasis in C. gariepinus, while pre-treatment with selenium has the ability to improve the physiological status of fish that were challenged with bacteria probably through its antioxidant properties. HIGHLIGHT: The pre-treatment of Clarias gariepinus to waterborne selenium for 14 days improved the redox homeostasis and innate immunity of fish that were experimentally challenged with the bacterium, Serratia marcescens.

Biochemical characterization of a surfactant-stable keratinase purified from Proteus vulgaris EMB-14 grown on low-cost feather meal.

OBJECTIVES: The bioaccumulation of keratinous wastes from poultry and dairy industries poses a danger of instability to the biosphere due to resistance to common proteolysis and as such, microbial- and enzyme-mediated biodegradation are discussed. RESULTS: In submerged fermentation medium, Proteus vulgaris EMB-14 utilized and efficiently degraded feather, fur and scales by secreting exogenous keratinase. The keratinase was purified 14-fold as a monomeric 49 kDa by DEAE-Sephadex A-50 anion exchange and Sephadex G-100 size-exclusion chromatography. It exhibited optimum activity at pH 9.0 and 60 °C and was alkaline thermostable (pH 7.0-11.0), retaining 87% of initial activity after 1 h pre-incubation at 60 °C. The Km and Vmax of the keratinase with keratin azure were respectively 0.283 mg/mL and 0.241 U/mL/min. Activity of P. vulgaris keratinase was stimulated by Ca2+, Mg2+, Zn2+, Na+ and maintained in the presence of some denaturing agents, except ß-mercaptoethanol while Cu2+ and Pb2+ showed competitive and non-competitive inhibition with Ki 6.5 mM and 17.5 mM, respectively. CONCLUSION: This purified P. vulgaris keratinase could be surveyed for the biotechnological transformation of bioorganic keratinous wastes into valuable products such as soluble peptides, cosmetics and biodegradable thermoplastics.

Cytotoxicity, redox and immune status in African catfish, Clarias gariepinus (Burchell, 1822) exposed to bisphenol A (BPA) and its analogues.

The objective of the study was to determine the comparative toxicities and immune dysfunction in the African catfish, Clarias gariepinus, exposed to bisphenol A (BPA) and its two analogues: bisphenol AP (BPAP) and bisphenol P (BPP). Juveniles of C. gariepinus were exposed to sublethal concentrations (70 and 140 µg/L) of BPA, BPAP and BPP for 7, 14 or 21 days after which various endpoints which are indicative of cytotoxicity, oxidative stress and haematological and innate immune parameters were determined in the liver homogenates or blood plasma. The exposure of C. gariepinus to BPA and its analogues caused significant increased activities of lactate dehydrogenase, catalase and superoxide dismutase. The exposed fish had increased levels of DNA fragmentation, lipid peroxidation, white blood cells, nitric oxide and respiratory burst, while the red blood cell counts and the percentage packed cell volume decreased significantly in the exposed fish compared to control. The toxic effects elicited by the bisphenols were both concentration- and duration-dependent. Generally, BPA exerted the most toxic effects on the fish, followed by BPAP, while BPP exerted the least toxic effects to C. gariepinus. Summarily, the findings indicated that BPA and its two analogues studied in the research are capable of causing cytotoxicity, oxidative stress and immune dysfunction in C. gariepinus.

Some physicochemical properties of two major soluble hepatic glutathione transferases of tilapia (Tilapia zilli).

Two distinct glutathione transferases from the liver of adult Tilapia zilli were identified and purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-150. These major GST forms labeled tzGST1 and tzGST2 accounted for approximately 42% of the activity detectable with 1-chloro-2,4-dinitrobenzene (CDNB) as a typical electrophilic substrate. Apparent subunit and molecular mass values, substrate specificities and sensitivity to inhibitors as well as kinetic studies were used to differentiate the GST forms. SDS/PAGE indicated subunit molecular masses of 22.0 kDa (tzGST1) and 26.1 kDa (tzGST2) while native molecular weight by gel-filtration on sephadex G-100 indicated native molecular masses of 46.8 kDa and 48.0 kDa for tzGST1 and tzGST2 respectively. They appeared to be homodimers. Inhibition studies showed that tzGST1 was more sensitive to ethacrynic acid (EA), hematin, tributyltinacetate (TBTA), triethyltinbromide (TETB), and triphenyltinchloride (TPTC) than tzGST2 with TPTC being the most potent inhibitor. T. zilli GSTs could conjugate CDNB, DCNB, ρ-NBC, and EA with GSH but displayed no observable conjugating activity with NBDCl. The K(m) and V(max) for tzGST1 and tzGST2 with CDNB were 0.56 ± 0.05 mM; 0.24 ± 0.03 µmol/min/ml and 0.91 ± 0.07 mM; 0.14 ± 0.05 µmol/min/ml respectively while K(m) and V(max) with GSH were 0.46 ± 0.02 mM; 0.19 ± 0.20 µmol/min/ml and 1.32 ± 0.15 mM; 0.21 ± 0.07 µmol/min/ml respectively. Denaturation and renaturation studies with guanidine hydrochloride (Gdn-HCl) revealed that concentration of 4.0 M Gdn-HCl completely denatured tzGST1 and the possible isoenzyme was able to renature to 92% of the original activity. The renaturation process was dependent on temperature. The outcome of this study indicated that tzGSTs are possible GST isoenzymes actively present and involve in the detoxification process in the liver of tilapia when the subject is exposed to chemical toxins. The wide range of chemical toxins encountered in the polluted environment may have directed the selection of multiple tilapia GST isoforms with broad substrate specificity via gene duplication. Consequently, tzGST1 has a better chemical toxin bio-transforming capacity than tzGST2 due to its higher affinity for its substrates--a form of adaption to the polluted environment.