الفهرس 
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Abstract
Chapter I. Fish streptococcosis, a historical review The aquatic environment is a suitable habitat for bacteria to thrive even in absence of the hosts. Several pathogenic bacterial infections in fish had recorded, however, a relatively small number are responsible for the significant economic losses worldwide and streptococcosis is one of them. The presented chapter deals with fish streptococcosis from three main historical aspects; epidemiology, diagnosis, as well as prevention and control.
from historical and epidemiological points of view, the chapter nominated streptococci and lactococci different species pathogenic for fish, the more susceptible fish hosts, disease distribution worldwide, disease transmission and its zoonotic importance. In wider prospective, the most predisposing factors associated with the disease occurrence, pathogen (s) virulence, disease pathogenic picture and immune responses of diseased individuals were deeply exhibited through reported literatures. Sequentially, different technical tools used for the disease pattern investigations, laboratory diagnosis at traditional & advanced molecular levels and immunological parameter alterations were reviewed as long as several decades. from streptococcosis control points of view, the chapter magnified the risk of development of antimicrobial resistance (AMR) as a result of misusage of antibacterial agents. Besides, it gave an account on using of antibacterial alternatives of natural origin. On the other hand, the role of application of biosecurity standards was eventually focused as a standard of disease prevention in general and particularly in streptococcosis incidence. Moreover, the chapter attempted to cover different types of streptococcosis vaccine candidates at laboratory and/or commercial levels with considerations of their advantages and disadvantages.
Chapter II. Zone surveillance of marine streptococcosis with emphasis on Gilthead seabream, Sparus aurata L., and European seabass, Dicentrarchus labrax L. farms in Dibah triangle, Damietta Governorate, Lower Egypt
Dibah triangle is 43 thousand acres in the eastern north of the Nile Delta in the boundaries of Damietta governorate. A part of all area, 27 thousand acres designated as marine fish and shrimp farms. Gilthead seabream, European seabass, Meagre and Mullet are the main produced fish of concern and showed tremendously development in marine aquaculture sector. The total production of marine fish from this area represents more than 35-40% (more than 100 thousand tons) of the total country production including fisheries. Recently, chronic mass mortalities among several marine fish farms occurred at late spring and summer seasons with mortality pattern characteristic to streptococcosis. For the purpose, the represented zone surveillance study was conducted to screen the disease and associated biosecurity application flaws for elucidating a plate form of knowledge to cope the problem. Six different sites crowded with marine farms in Dibah triangle selected for the study. Such sites located at north, eastern north, west, western north, south and western south of the triangle. Investigation farm visits for more than 100 farms (108) performed for screening of streptococcosis surveillance. On pre-prepared 7-point checklist sheets, biosecurity application flaws recorded together with on spot water parameters analyses results. Besides, number of fish samples, species, healthy status and alterations patterns characteristic for streptococcosis also recorded on the same corresponding 7-point checklist sheet (s). Three hundred ninety fish samples; 200, 160 and 30 representing seabass, seabream and meagre, respectively, grossly investigated from infected, suspected and farms had a history of streptococcosis with number ranges of 2, 3 and 5 fish, respectively. Collected fish samples brought to laboratory as fresh as possible for post mortem examination, bacteriological isolation, biochemical and molecular identification of pathogen (s) incriminated in the disease. As a result, several biosecurity flaws were recorded either on the conceptual, structural, operational levels at the most of the investigated farms with mean percentages of 80.7 ± 4.38, 68.5 ± 9.02 and 70.7% ± 9.31%, respectively. Insufficient or absence of biosecurity standards application represented at more than 100-75% in several points such as absence of vaccination (100%), no ectoparasites control programs (94.4%), absence of water quality monitoring (92.6%), no veterinarian’s follow up (89.9%), absence of farm fences, unsecured water inlets (each, 86.1%), no periodical sanitary and cleaning programs (85.2%). In addition, absence of wild animal control (82.4%), unpaved roads (78.8%), farm design flaws (76.9%), absence of foot and car sanitary bathes (75%). Moreover, some biosecurity flaws represented at 50-75% of investigated farms such as, no geometric parameters in ponds construction (71.3%), ponds distribution flaws (69.4%), no fixed feeding programs, no hygienic disposal of dead fish carcasses (each, 65.7%), no employed human security (62%). Furthermore, some other biosecurity flaws were represented at 25-50% of investigated farms such as, flaws in constructing water inlet/outlet and no proper acclimation (each, 46.3%), trusted sources of fish seeds flaws (35.2%), flaws of fish seeds size and/or weight (32.4%). In addition, the results framed considerably bad water quality at all sites screened with exceptions. The exceptions were the north and eastern north sites, which have acceptable water parameters unlike the south and western south that have the worst. At all levels tested, alteration of water parameters, particularly, dissolved oxygen, ammonia and salinity were extremely deviated from the acceptable limits. Consequently, streptococcosis prevalence percentage (s) was representing 18.5, 35.2 and 46.3% for northern, western and southern zones, respectively. On the other hand, behavioral alterations, clinical signs, postmortem and morbidity & morality patterns were closely related to the difference of investigated sites. Meanwhile, revealed results pointed to severe, moderate and mild infections at south, west and north sites, respectively. A part of 9 molecularly identified isolates (Streptococcus iniae, 3 isolates; Lactococcus garvieae, 6 isolates) 2 isolates of each species submitted in the GenBank with accession numbers of MT086601, MT086602 and MT158668, MT158669, respectively. Additionally, molecular sequencing and phylogenetic analyses results revealed high homologies and intraspecies similarities of the four isolates with the deposited sequences in the Genbank database with figuring out of phylogenetic close relationships. The presented chapter concluded that continuous monitoring of biosecurity standards is an essential issue in Dibah triangle, because of its harsh topographical nature itself extremely hindered aquaculture projects. Besides, employing periodical surveillance studies concerning disease episodes will give a clear map to overcome severe economic losses associated.
Chapter III. Vaccination with bacterial ghosts of Streptococcus iniae and Lactococcus garvieae originated from outbreak of marine fish streptococcosis, induces potential protection against the disease in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758).
Streptococcosis considered as an important disease affects fresh, brackish and marine fish aquaculture. Recently, Egyptian fish aquaculture suffered from severe economic losses, particularly mari-culture sector, and streptococcosis has accused. S. iniae and L. garvieae were the main causative agents isolated (Chapter II). Consequently, the represented chapter confined the preparation of bacterial ghost vaccine (BGV) candidates originated from such isolated pathogens of marine streptococcosis outbreaks using NaOH chemical approach. The 2 isolates, S. iniae (Genbank acc. MT086601) and L. garvieae (Genbank acc. MT158669), were selected for that mission. Firstly, selected isolates used for confirmation of their pathogenicity in Nile tilapia. Then, the fish selected as experimental fish model for evaluation of the prepared BGVs. Subsequently, the re-isolated S. iniae and L. garvieae from experimentally infected fish model purified, confirmatively identified and used for ghost preparations and fish challenges. A part of four experimental fish groups, 3 fish groups (60 individuals, 20 fish for each with 2 replicates) namely, A, B, C were designated for BGVs evaluations corresponding to their related immune responses and protective efficacies. The fourth group (D) designated as control and subdivided into subgroups (DA, DB, and DC) to be corresponding with the experimental groups (108 individuals, 36 fish for each subgroup). Vaccination experiments performed via intra-peritoneal (IP) injection of fish individuals of each designated groups with 0.1 mL (1.5x 108 CFU/mL) of their corresponding BGV twice with 2 weeks intervals. At the same levels, control subgroups treated like experimental ones, however, they received 0.1 mL of cold fish saline (CFS) instead of the vaccine (s). Blood, sera and tissues (anterior kidneys and spleens) samples were collected from each vaccinated and control groups at 2 and 4 weeks post immunization (PI). For vaccine (s) evaluation, hematological, innate and specific immunity parameters including PCV, TP, ALB, GLB, PP, SOD, CAT, MDA, C5, IL-1β, LZM, specific antibody titers and tissue immune related gene expressions, CD4 and MHC-II were estimated 2 and 4 weeks PI. To investigate the protective efficacies of designated BGVs, all remained fish individuals in immunized and control groups challenged with appropriated pathogen (s) and the relative percentage of survival was calculated. The results revealed that S. iniae and L. garvieae that originated from marine streptococcosis outbreaks exhibited high pathogenicity levels in Nile tilapia associated with LD50 of 3×105 and 3×107 CFU/mL, respectively. Three BGVs generated, one namely, SiG, in addition to two novels namely, LgG and SiLgG corresponding to S. iniae, L. garvieae (monovalent) and both former ghost mixtures (bivalent), respectively. Fish groups immunized with prepared BGVs revealed variable significant increases in PCV, GLB, PP, SOD, CAT, C5, IL-1β, LZM, specific antibody titers and CD4 and that was evident 2 and 4 weeks PI. On contrary, levels of TP, ALB showed no significant elevated levels. MDA levels decreased in all vaccinated groups and that was significantly obvious in the group C. Expressions of MHC-II showed significant elevation at 2 weeks PI, however, it significantly decreased at 4 weeks PI. Concerning challenge experiments, results showed relative percentage of survival of 90, 88.89 and 95.46% for immunized groups A, B and C, respectively. At all levels tested, obtained results proposed SiG, beside novel contributions of LgG and SiLgG as innovative vaccine candidates, which can protect cultured fish from being attacked by S. iniae, and/or L. garvieae.
Chapter IV. Antibacterial activities of selected essential oils against S. iniae and L. garvieae
Streptococcosis considered as the most significant bacterial disease, causing fish farmers to suffer significant economic losses. The disease mostly controlled using antimicrobial agents. However, many negative aspects documented concerning antibiotics usage in aquatic environment. The most problematic issue is the explosion of AMR. In such respect, growing demand for the use of herbs and/or their EOs in aquaculture is becoming of significance. In this chapter, in vitro study conducted to investigate antibacterial activity of three commercial EOs namely; Thyme (TEO), Cinnamon (CEO), and Rosemary (REO) against selected virulent strains of S. iniae and L. garvieae. Therefore, two isolates of S. iniae (BNS 01718-BR, acc. MT086601) and L. garvieae (BNS 06718-BS, acc. MT158669) kept at – 80 °C selected and refreshed for further examinations. Original pure (98%) TEO, CEO and REO obtained, 10% of each dissolved in Dimethyl Sulfoxide (DMSO) and considered as original working solutions (OWS) for each EO. The bacteriostatic (MIC) assays performed using agar plate dilution method. In which, three replicates of serial double fold serial dilutions of each EO prepared to obtain dilutions from 10000 to 19.5μg/mL. Then, 1 mL of each diluted EO aspirated and poured in sterile perti-dish. Instantly, 9 mL of melted THA medium added. Two control plates were included in each plate patch, one containing DMSO and the other containing DW. After solidification, all plates streaked with appropriate selected bacterial suspensions. Inoculated plates incubated at 28 °C and bacterial growth monitored for 24 and 48h. For bactericidal (MBC) assays, microtiter-dilution method was used. Three microtiter plates, each designated for each EO. The 1st three rows designated for S. iniae as 3 replicates, while the following 3 rows designated for L. garvieae at the same manner. The 1st well dispensed with160μL of TH broth, whereas, the 2nd to the 12th well, received only 90μL. Then, 20μL of the WOS added to the 1st column to obtain 180μL of EO/THB mixture containing 1000μg/mL. Then, double fold serial dilutions (1000 to 2μg/mL) performed including all experimental rows. All microtiter-plates covered and incubated at 28 °C for 24 to 48h. Then, turbidity development monitored and estimated at optical density of 600 nm. Besides, viability tests carried out through picking up 10μL from all wells, streaked on freshly prepared THA plate, independently and incubated at 28 °C for 24 to 48h. Finally, The MBC/MIC ratio calculated. As a result, TEO showed MICs of 7.8:15.6 and 15.6:31.2μg/mL and CEO showed MICs of 31.2:62.5 and 62.5:125μg/mL against S. iniae and L. garvieae after 24:48h, respectively. However, REO’s MICs against S. iniae were 125 and 500μg/mL after 24 and 48h, respectively. Additionally, REO’s MIC against L. garvieae was 1000μg/mL after 24 and not detected after 48h. The MBCs of TEO, CEO, and REO against S. iniae were 15.6, 62.5, and 125μg/mL, respectively. Meanwhile, the MBCs of both TEO and CEO against L. garvieae recorded 125μg/mL. Whereas, none of REO concentrations tested showed bactericidal activities against L. garvieae. Ultimately, the MBC/MIC ratio of TEO, CEO and REO was at range of 2, except for that of TEO against L. garvieae recorded 4. In conclusion, the presented study gives a plate form of knowledge concerning TEO, CEO, and REO as cost-effective alternative antibacterial agents of natural origin with no detectable ecological effects against two major bacterial species incriminated in fresh, brackish and marine fish streptococcosis.