الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 158 |  |  |
| | | from | 158 | | |
Abstract
Enteric septicemia of channel catfish (ESC) is considered the number one disease problem and most common diseases facing the catfish industry (Freund et al. 1990). Because iron is essential for bacterial growth within the host, bacteria may use siderophore mediated iron acquisition or direct iron binding mechanisms to obtain iron from the host (Ratledge and Dover 2000). Although enteric septicemia is a serious disease affecting channel cat fish, the importance of the Fhu system during invasive infections of the host is therefore questionable. Virtually nothing is known regarding the role of fhu genes during infection.
The aim of this research is to study the role of ferric hydroxamate uptake genes in E. ictaluri virulence. The study was divided into three main parts:
1- Construct four E. ictaluri Δfhu mutants (EiΔfhuC, EiΔfhuD, EiΔfhuCD and EiΔfhuB) through in-frame deletion of ferric hydroxamate uptake genes.
2- Assessment the virulence and efficacy of the four E. ictaluri Δfhu mutants.
3- Test the ability of the four E. ictaluri mutants to grow in the presence of the iron chelator condition (2, 2’ Dipyridyl).
Four E. ictaluri Δfhu mutants (EiΔfhuC, EiΔfhuD, EiΔfhuCD and EiΔfhuB) were construct by in-frame deletion of fhu genes. In the EiΔfhuC mutant, a 717-bp nucleotide region was delete from a 774-bp fhuC gene length; in the EiΔfhuD mutant an 822-bp nucleotide region was deleted from a 933-bp the fhuD gene length; in the EiΔfhuCD mutant a 1635-bp nucleotide region was deleted from a 1707-bp of the fhuCD operon; and in the EiΔfhuB mutant an 1881-bp nucleotide region was deleted from 1995-bp the fhuB gene length.
Immersion challenge of catfish fingerlings using the four E. ictaluri Δfhu mutants and E. ictaluri wild type revealed that EiΔfhuB showed the highest attenuation (6.67% mortality), which was followed by EiΔfhuC (22.89% mortality). While, the EiΔfhuD showed (44.44% mortality) and EiΔfhuCD was (40.00% mortality) as compared to E. ictaluri wild type (46.91% mortality). No mortalities were observed in fish challenged with the Brain heart infusion (BHI) broth.
Evaluation of mutant’s efficacy of the channel catfish fingerlings challenged with four E. ictaluri Δfhu mutants (EiΔfhuC, EiΔfhuD, EiΔfhuCD and EiΔfhuB) by re-challenge with E. ictaluri WT indicated that relative percent survival values ( 21 days post re-challenge) among the four mutants were high (100%, 94.72%, 97.31% and 95.62% RPS) in (EiΔfhuD, EiΔfhuCD, EiΔfhuC and EiΔfhuB) respectively. The negative control group (previously challenged with E. ictaluri WT) was (92.54% RPS) and the positive control group (previously challenged with BHI) had low level of the RPS (41.70%). These results confirmed the ability of the four E. ictaluri Δfhu mutants to protect the channel catfish from ESC.
Based on the results of fingerling challenged experiments, in which E. ictaluri ΔfhuB mutant gave 6% mortalities only. This mutant was chosen to be challenged to channel catfish fry and the results revealed that E. ictaluri ΔfhuB mutant caused 48.55% mortality on fry, while E. ictaluri WT caused 82.06% mortality. Evaluation of the E. ictaluri ΔfhuB mutant’s efficacy by re-challenge with E. ictaluri WT after 21 days post challenge indicated that RPS in the EiΔfhuB previous challenged group was (93.1%), the negative control group (previously challenged with E. ictaluri WT) was (91.67%) and the positive control group (previously challenged with BHI) had low level of the RPS (8%).
In order to obtain iron depletion conditions, various concentrations of the iron chelator 2, 2’ Dipyridyl were added to the BHI broth. It was found that the minimum inhibitory concentration (MIC) that inhibited E. ictaluri growth was 120µM. This concentration was subsequently used for bacterial growth experiments for the presence or absent iron.
Neither in the normal conditions (BHI without the presence of iron chelator) nor in the presence of the iron chelator DPD (120µM), the growth rates of the four E. ictaluri Δfhu mutants (EiΔfhuB, EiΔfhuC, EiΔfhuD and EiΔfhuCD) did not showing significant different from the growth the E. ictaluri WT.