الفهرس 
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Abstract
Due to the evolution of antimicrobial resistance in most pathogenic bacteria, including Staphylococcus and Salmonella strains, stimulated by the uncontrolled usage of antibiotics in both human and animals feed to enhance the growth of food animals, and their use in veterinary medicine for treating bacterial infections, extensive research will be needed that aim to discover new antibiotics and study their efficiency and applicability.
The present study tries to approve the ability of S. littoralis larvae to obtain an antimicrobial activity in their hemolymph, upon immunization (non-lethal infection) with bacterial pathogens and to test the role of AMPs as natural antibiotics against an otherwise lethal septic bacterial infection. Biochemical characterization of the immunized hemolymph to determine the biodiversity, functional properties and amino acid composition of AMPs have been also investigated.
Different inducers were used to enhance the synthesis of AMPs; including S. aureus, S. typhimurium, and a virulence factor (SEA), to study their different effects upon AMPs production and simultaneously the effect of the induced AMPs on different antibiotic resistant bacteria.
Spodoptera littoralis was the insect chosen in this research, since it had proved a great success in this kind of immunological studies a long time ago till now. This comes from the fact that they can be easily reared with inexpensive media and materials, can be maintained in large numbers, easily identified, have a fast lifecycle and they possess large blood volume.
The present work forms a part of a wide study aims to obtain more information about the probability of considering insect AMPs as promising class of therapeutics that can be alternative to antibiotics. Therefore, the results obtained from this study can be summarized in the following points:
1. dentification of the tested bacteria
Microscopic examination and Gram stain of S. aureus and E. faecalis showed G +ve cocci with purple color (retained the crystal violet stain). On the other hand, E. coli and S. typhimurium did not hold the crystal violet stain giving a red to pink color, being G -ve bacteria.
2. Toxin (SEA) purification
Staphylococcus Enterotoxin A (SEA) was purified from the culture supernatant of S. aureus by affinity chromatography. When the fraction obtained was analyzed by SDS-PAGE, and by using western blot technique a clear protein band with a dark purple color and with an apparent molecular mass 27 kDa was observed, which indicated that the purified band was the protein of interest and confirming the purified band was SEA protein.
3. Susceptibility of S. littoralis to bacterial pathogens
The 5th instar larvae of S. littoralis were more susceptible to G -ve Salmonella (LC50= 2.4×105 CFU/ml) than G +ve S. aureus (LC50 = 4.8×105 CFU/ml). This difference in pathogenicity came from the presence of an outer membrane in the cell wall of G -ve bacteria, made up of LPS and acted as an endotoxin, which was absent in G +ve ones.
Treatment with 0.6 μg/insect of SEA as a sub-lethal dose produced higher mortality rates to the larvae. The bodies of killed larvae turned black, suggesting the activation of prophenoloxidase that mediates melanization, since it inhibits totally many metabolic functions of the insect.
4. Antimicrobial susceptibility test
The normal insects exhibited a very weak antibacterial activity towards virulent bacteria without receiving any antigenic challenge, because of naturally occurring antimicrobial substances found in the food. Indeed, a weak antibacterial response to water challenge (control) was observed may be due to either a lower sensitivity or a higher induction-specificity of the larval immune system. The bacterial virulence factor (SEA) induced the strongest antimicrobial activity in larval hemolymph against all the studied bacterial species. This comes from the fact that SEA acts as a danger signal for the insect, leading to the activation of many proteolytic cascades in the insect hemolymph. This suggested that the SEA treatment induced a broader spectrum of AMPs and proteins in comparison with the live bacteria.
Larval plasma injected with Salmonella showed better results than that injected with S. aureus. This virulent effect comes from LPS associated to cell wall.
5. Effect of microbial injection on the hemolymph proteins of S. littoralis larvae
Hemolymph of bacteria-injected S. littoralis larvae recorded drastic changes in both the total protein content and the protein banding patterns following injections.
5.1. Effect on the total haemolymph proteins (THPs):
The THPs decreased significantly 48 h post-injection. This can be attributed to the intensive consumption of plasma proteins during multiplication and growth of bacteria. Also, some hemolymph sticky and soluble proteins may be involved in the attachment of the injected pathogens to the hemocytes or some native proteins might be converted into glycoproteins or lipoproteins after injection.
5.2. Effect on the hemolymph protein profile:
Great variations were detected in the number, kinds and percentage of protein fractions of larval plasma. Hemolymph of normal, control and bacteria-treated larvae with S. aureus, SEA and S. typhimurium were separated into 11, 13, 12, 12 and 14 bands, respectively. Bands with 350, 26 and 25 kDa are characteristic for the larval 5th instar.
The injected materials (water, bacteria and toxin) were differentially capable of changing the hemolymph proteins profile qualitatively through the induction of new proteins and the disappearance of others simultaneously, which may be attributed to their participation in the immune reactions. Protein bands with ~22 kDa (attacin) and ~15 kDa (lysozyme) were found to be common between the AMPs produced as a result of the three treatments, while an additional ~70 kDa, (PO) and ~14 kDa (gloverin) protein bands were found specific for SEA treatment suggesting the broader spectrum of AMPs and proteins in comparison with the live bacteria.
6. Amino acid analysis of the induced AMPs
The induced AMPs were subjected to further chromatographic analysis using HPLC to determine the amino acid content. Twenty types of amino acids were detected in both phenoloxidase, and lysozyme. They are listed as follow; Aspartatic acid, Serine, Threonine, Glutamatic acid, Glutamine, Glycine, Tryptophan, Alanine, Cysteine, Methionine, Valine, Isoleucine, Leucine, Phenylalanine, Tyrosine, Proline, Lysine, Histidine, Asparagine and Arginine with differences in their quantities and velocities. Cysteine amino acid was completely absent in attacin and gloverin AMPs. This variations in amino acids Content and structure may explain the observed difference in pathogenicity and virulence potential between immune elicitors.
In conclusion, the cotton leaf worm larvae gave different responses against different bacterial treatments. The virulence factor (SEA) induced the strongest antimicrobial activity in larval hemolymph with a broader spectrum of AMPs and proteins in comparison with the live bacteria. Salmonella (G-ve bacteria) provided the larvae with a higher activity than G +ve S. aureus. This finding offers a potential, novel mechanistic explanation for acquired resistance in insects. Each of bacterial agents requires specific pathway for induction of AMPs, suggesting systemic pathogen recognition via hemolymph.