الفهرس 
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Abstract
In the present study , we have analysed the enzymatic and virulence properties of six isolates of A.hyDROPhila previously isolated from apparently healthy and diseased O.niloticus and Clarias gareipinus collected from different localities in Kafr El sheikh Governorate. These isolates were identified by biochemical methods and PCR technique. These isolates were firstly tested for pathogenicity, for the aim of establishing possible relationships among some of the phenotypic characters of these isolates together with pathogenicity.
• Results of pathogenicity test revealed the presence of five highly pathogenic isolates causing 100 % mortalities within 24 hours of I/M inoculation of fish. The remaining isolate only was less virulent causing 60% mortalities.
• The production of the extracellular virulence factors was determined with respect to incubation time and temperature.
• Results of the haemolysin production, revealed that all the six tested isolates(100%)were able to produce B-haemolysins at 30 and 37ºc after 24 hours of incubation on 5% defibrinated sheep blood agar .While at 25 ºc, only 4 isolates (66.6%) produced haemolysins after 48 hours of incubation. However, at 4 ºc, none of the six isolates exhibited any haemolytic activity.
• The proteolytic assay of the six isolates was evaluated by both caseinase and gelatinase production tests using skimmed milk agar and gelatin agar, respectively. The two methods gave equal results. These results indicated that the five highly pathogenic isolates (83.3%) , produced caseinase after 24 hours of incubation at 37 ºc, while gelatinase was produced also by these five, but after 72 hours of incubation at 30 ºc.
• Detection of lipase production, was done by using 0.5% tributyrin agar, which revealed that all the isolates(100%) exhibited lipolytic activities within 24 hours incubation at 37ºc in the form of clear halo around the bacterial colonies.
• Concerning the quantification and heat stability of haemolysins, the results showed that all the six isolates exhibited haemoytic activity against human erythrocytes. The highest haemolytic titer was recorded in the samples incubated at 25°c for 30 minutes, and gradually decreasing on increasing the temperature till reach its minimum level in the 90°c heat treated samples, but not completely inactivated.
• The assay of cytotoxic activity of the six isolates using Vero cell line, demonstrated that the cell free filtrate of five isolates (83.3%) induced cytopathic effect on Vero cells in the form of cell rounding, elongation of the cells, and / or decrease in cell size and cell density. On studying the thermostability of the cytotoxins produced, we noticed that the cytotoxic titer gradually decreased with the increase of temperature, but it was retained in some samples without complete inactivation after 56 and 90°c heat treatment for 30 min. The cytotoxic effect was observed till 1:128 dilution of the isolates’cell free filtrate.
• The outer membrane proteins of these six isolates were extracted and characterized by SDS-PAGE, to identify the common proteins among these isolates. The profiling of these proteins revealed that these six local isolates shared five(5) common peptide bands together with the reference strain ATCC 7966. These bands were of 25-28KDa, 30-35 KDa, 39-43 KDa, 47-48 KDa and 65-71 KDa. The maximum molecular weight protein bands were detected at the 71 KDa, with minor variations in two isolates showing bands at the range of 107-112 KDa .
• In this study , we evaluated the antibacterial activity of three herbal plants(A.sativum, A.cepa and Z.officinale) in vitro, for the search of new bioactive materials to replace antibiotics in controlling A.hyDROPhila infections. They were tested by using agar disc diffusion method. The results demonstrated that only A.sativum (crude and aqueous extract) exhibited antibacterial activity against the six isolates of A.hyDROPhila . While the other two herbal plants did not show any antibacterial activity.
• The inhibition zone produced by the crude extract(100% concentration) of A.sativum ranged between 16-32 mm in diameter. While smaller zones of inhibition were produced by the other two aqueous concentrations(75% and 50%). No antibacterial activity was detected in the 25 % aqueous concentration.
• The thermostability of the crude extract of garlic was determined by incubating the crude garlic extract at four different temperatures(4 ºc, 25 ºc and -20ºc for 24 hours and 100ºc for five minutes), with one sample left as a control without any heat treatment, and tested directly after extraction by the agar disc diffusion method.
• The results revealed that the control freshly used extract exhibited nearly the same potent antibacterial activity as the extract stored at 4 ºc. While extracts kept at room temperature showed only moderate effect. On the other hand, crude extracts stored at - 20 ºc for 24 hours and those boiled for five minutes completely lost their antibacterial effect, and which may be attributed to physical and / or chemical changes which might take place during excessive heat treatment.
• In earlier studies, the inhibitory effect of garlic was suggested to be due to its main component allicin which interferes with RNA production, which in turn affects protein synthesis, and may be stopped at any stage. So, the present study demonstrated the effect of garlic on the total protein synthesis by the six isolates. The total soluble proteins were measured by spectrophotometer quantification in the cultured broth of the six isolates. The samples were classified into two groups (one was garlic treated, while the other was just a broth cultured with the isolates). The garlic was added directly after the inoculation of the bacterial isolates in broth, with incubation for 24 hours at 30 ºc .
• The results revealed that the protein synthesized by the six isolates was lower in the samples treated with garlic than the non treated group, indicating that garlic affected protein synthesis. The degree of affection of the six isolates was variable, ranging from 18-95% of the normal level.