The Isolation And Antibacterial Activity Of Tissue And Cell Extracts From A Crocodile: Crocodylus Siamensis

In decades, antibacterial component, which plays an important role in immune systerm, is getting more and more attention of scientists. As a representation of reptile, crocodile is charactered with strong livability and great resistance againt infection. Up to now, only little researches about antibacterial components of crocodiles have been reported, although some study on that of a great variety of reptile were carried out. In present thesis, several methods by which antibacterial components were extracted from the blood, intestine, liver, spleen and kidney of Siamese crocodile (Crocodylus siamensis), antibacterial effect of the extracts, especially the effect of extracts from peripheral blood leukocytes against varieties of pathogenic bacteriums, for example Streptococcus iniae that infected in Kunming rats, as well as the physical and chemical properties of antibacterial components were described. This thesis includes four parts as follows:1. Isolation and distribution of antibacterial components from Crocodylus SiamensisAntibacterial components were extracted from varied tissues of Crocodylus Siamensis by three methods: the modified CH₃COOH method, the (NH₄)₂SO₄ method, and the CH₃COCH₃+HCHO method respectively, and their antibacterial effects were compared. Distribution of antibacterial components in crocodile tissues were analysied by detecting their antibacterial effects. The components extracted by the modified CH₃COOH method presented higher antibacterial activity than that by (NH₄)₂SO₄ method with 9.1 mm, no antibacterial activity in extracts by CH₃COCH₃+HCHO method was detected. Of three methods, the modified CH₃COOH method showed the highest efficiency to extract antibacterial components from tissues of crocodile and extrats from peripheral blood leukocyte by this method presented 9.1 mm of antibacterial circle in diameter by protein concentration of 0.256 mg/mL.The extracts from intestines, livers and peripheral blood leukocytes showed high antibacterial activity with 16.2mm, 8.5mm, 9.1 mm of antibacterial circle in diameter by 3.242, 2.973, 0.265 mg/mL expectively, while no antibacterial activity were detected in extracts from spleen and kidney. These results proved that the antibacterial components of Siamese crocodile exist mainly in their intestines, livers and peripheral blood leukocytes.2. The Antibacterial Activity of the Crocodylus Siamensis Antibacterial Components in VitroLeukocytes were isolated from whole blood of crocodiles by differential sedimentation. The leukocytes were disrupted in 10 % AcOH and by ultrasonic and its extracts were dissolved in0.1 % AcOH. The efficacy of this acid leukocytes extracts were detected to kill pathogenic bacterial or inhibit its growth. The results showed that extracts origined from C. siamensis leukocytes had significant effect on killing or restricting the growth of Eschenrichia coli,Streptococcus iniae,Pasteurella pneumotropica,Aeromonas sobria,Staphylococcus aureus,Aeromonas hydrophila,Vibrio parahemolyticus,Vibrio alginolyticus,Vibrio vulnificus,Vibrio cholerae. The inhibition zone against S. iniae, E. coli and V. cholerae was respectively 22.1 mm, 21.7 mm and 7 mm. The acid extracts of the crocodiles' leukocytes showed superior antibacterial activity with 21.3 mm, 5.3 mm, 0.67 mm in inhibition zone against S. iniae to the tortoise (Trachemys scripta elegans) and the chook (Coturnicops noveboracensis).3. Antibacterial Efficiency of Extracts from Leukocyte of Crocodylus Siamensis against Streptococcus Iniae Infection in VivoThe extracts were isolated from peripheral leukocytes of Crocodylus siamensis by breaking cell and differential sedimentation and were given to mice with three different ways in order to determine thedefending effect on Streptococcus iniae infection in mice. The results showed that after experimental mice were infected with S. iniae and presented clinic signs, they would gain the RPS of 77.8 %,55.6%,44.4% respectively if injected with leukocytes extracts of crocodiles with three different concentration. If experimental mice infected with S. iniae and at same time injected with leukocytes extracts at above different concentration, they would presented RPS of 77.8 %,66.7% > 44.4%. If leukocytes extracts were mixed with 5. iniae in vitro and then given to mice by injection, experimental mice would have RPS of 55.6%,88.9%,77.8% respectively. 4. The physical and chemical characters of the antibacterial componentsThe Crocodylus siamensis antibacterial components' physical and chemical characters were tested. The results showed that the leukocytes extracts of Crocodylus siamensis presented the sensitivity to EDTA, solubility in acid and heat-stability in inhibility to E. coli. Depended on its physical characteristics, we infered that effective components in the leukocytes of Crocodylus siamensis was a kind of antibacterial peptide.