الفهرس 
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Abstract
Vaccines and Nano-technology approaches for protection of cultured freshwater fish against edwardsiellosis in United States and Egypt were investigated. Live attenuated Edwardsiella piscicida (E. piscicida) mutants from E. piscicida wild type (WT) strain C07-087 were constructed and their potential as live vaccine candidates to prevent edwardsiellosis in I. punctatus and I. furcatus (the major cultured fish species in east-southern part of USA) was evaluated. Also, chitosan/vitC, silver and zinc nanoparticles were tested for prevention of Edwardsiella tarda (E. tarda) infection in O. niloticus (the major cultured fish species in Egypt).
Live attenuated E. piscicida vaccines were constructed by in-frame deletion of some genes encoding two component systems through chromosomal cross-over and allelic exchange. Consequently, EpphoP, EpphoQ, EpbasR, EpbasS, EpcpxA, EpcpxR, EpcusR, EprstA and EpqseC vaccine candidates were formed. On the other hand, chitosan/vit C nanoparticles with average 247.3 ± 5.39 nm were prepared by ionotropic gelation. Silver and zinc nanoparticles were commercially obtained.
For in vitro evaluation of the live attenuated E. piscicida vaccines, growths in normal and under stress conditions were tested. Growth kinetics of the live attenuated E. piscicida vaccines appeared to be not greatly different from WT E. piscicida. Under stress conditions, the live attenuated E. piscicida vaccines were able to adapt to oxidative and acidic stress environments as well as WT after 24 hours (hrs), but they did not adapt as quickly as the WT. Similarly, survivability of the live attenuated E. piscicida vaccines were significantly decreased compared to E. piscicida WT when incubated with non-immunized serum and/or blood of non-immunized I. punctatus. Moreover, significant reduction in biofilm formation of the live attenuated E. piscicida vaccines were occurred after 48 and 72 hrs compared to E. piscicida WT strain.
Concerning the evaluation of the prepared chitosan/vit C nanoparticles in vitro, high ascorbic acid encapsulation efficiency (86 ± 0.4%) was obtained. Furthermore, transmission electron microscope analysis had shown the spherical shape as well as smooth surface of the prepared chitosan/vitC nanoparticles.
Regarding in vivo evaluation, the constructed live attenuated E. piscicida vaccines were tested in both I. punctatus and I. furcatus. In I. punctatus, they showed significant attenuation in intra-peroteneally injected I. punctatus. Mortalities were 6.63%, 6.25%, 6.00%, 4.25% and 4.17% in EpΔphoP, EpΔCpxR, EpΔCusR, EpBasR and EpΔphoQ respectively. Oppositely, EpBasS, EpRstA, EpΔQscA and EpΔCpxA had 4 % mortalities. Additionally, the relative percent survival (RPS) of the intra-peroteneally vaccinated I. punctatus was significantly higher than that of the non-vaccinated group (18.82%). RPS of I. punctatus intra-peroteneally vaccinated with EpΔphoQ, EpΔphoP, EpΔCpxA, EpΔCpxR, EpBasR, EpBasS, EpRstA, EpΔQscA and EpΔCusR were 97.92%, 96.25%, 96.15%, 95.83%, 95.65%, 94.23%, 93.88%, 93.75% and 93.25% respectively. The vaccinated I. punctatus showed high mean antibody titer at 14 and 21 day post vaccination. In addition, immune related genes IL-1β, INF-γ, CD4-1, MHC class I and MHC class II transcription profile were up-regulated expressed in the haemopoietic organs i.e. anterior kidney and spleen of I. punctatus immunized with EpΔphoQ, EpΔphoP, EpΔCpxA, EpΔCpxR, EpBasR, EpBasS at day 14 and day 21 post vaccination. Furthermore, there were no pathological alterations in liver, spleen and anterior kidney of the vaccinated I. punctatus.
On contrary, in I. furcatus, the live attenuated E. piscicida vaccines showed mortality decreasing (5 % - 40 %) except live attenuated EpΔcusR and E. piscicida WT gave 90 % and 100 % mortalities respectively. Regarding the protective efficacy, EpqscA and EpcpxR provided high protection (79.45 % and 75.21 % RPS) in the I. furcatus.
For in vivo evaluation of the organic and inorganic nanoparticles, LD50 of E. tarda wild strain was determined as 1.5 × 108 CFU/fish. Alongside, the LC50 of silver and zinc nanoparticles in O. niloticus was 8 mg/L and 50 mg/L. Oral administration of chitosan/vit C nanoparticle (300, 400 and 500 mg/kg diet for 45 days), were able to provoke the non-specific immune responses in a dose dependent manner accompanied with protection varied from 53.33 % to 80 % RPS in O. niloticus after IP challenge with 3 × 108 CFU/fish of E. tarda wild strain.
On the other side, oral administration of 0.4 mg silver and 2.5 mg zinc nanoparticles / kg diet for 6 weeks could provide 33.33 % and 60 % RPS in O. niloticus after i.p. challenge with 3 × 108 CFU/fish E. tarda strain. However, silver and zinc nanoparticles were able to stimulate the non-specific immune parameters such as nitric oxide, lysozyme activity and IgM production but they could not have provided reliable protection against E. tarda infection in O. niloticus.