الفهرس 
Cover Page Title in EnglishOnly 14 pages are availabe for public view
 |  | from | 51 |  |  |
| | | from | 51 | | |
Abstract
Y. enterocolitica is a Gram-negative bacillus-shaped bacterium, belonging to the family Enterobacteriaceae. It is motile at temperatures of 22–29°C, but becomes nonmotile at normal body temperature . Y. enterocolitica infection causes the disease yersiniosis, which is animal-borne disease occurring in humans, as well as in a wide array of animals such as cattle, deer, pigs, and birds. Many of these animals recover from the disease and become carriers; these are potential sources of contagion despite showing no signs of disease.
In our study, a total of one hundred (100) of cultured brood stock of sea bass (Dicentrarchus labrax L.) of average body weight (4500 ± 300 g) were collected from rearing fiberglass tanks (30 cubic meters) during spawning season whereas there is water temperature ranged (17-18 °C) showing typical lesions and other signs of Yersiniosis, freshly dead or in the moribund state from private marine fish hatchery at Borg El-Arab, Mariut valley region, Alexandria governorate, Egypt. Examined fish were found to have typical signs and PM lesions of Yersiniasis disease .
1) Water samples were taken parallel to fish samples and subjected to complete water analysis. The dissolved oxygen, salinity, Temperatures, pH values, levels of unionized ammonia and nitrite nitrogen (N02-N) and analysis of the heavy metals in the water.
2) These fish were subjected to the followings :
2.1. Full clinical examination (clinical signs and PM lesions).
2.2. Bacteriological examination (Isolation and Identification of the causative agent), whereas bacterial inocula were taken from internal organs (Kidney, spleen, heart and liver) and subcultured on tryptic soya agar and selective Yersinia agar media at 20-25 0C for 24-48 hrs. Bacterial isolates were then subjected to full traditional biochemical tests and API 20E, 20 NE and VITEK2 Compact System for further identification. The identified bacterial isolates were confirmed using conventional PCR for detection of the virulence gene (Yrp1 gene) of the identified Y. enterocolitica .
3) The prevalence of the identified Y. enterocolitica isolates in the naturally examined fish was also determined.
4) Antibiogram test was applied for the identified Y. enterocolitica isolates using different antimicrobial discs.
5) Experimental infection of Y. enterocolitica in sea bass fingerlings :
5.1. Lethal dose fifty (LD50) determinations: A total number of seventy (70) apparently healthy sea bass weighting (30 ± 4 g) were selected after the period of acclimation of two weeks and then divided into 7 equal groups; each group contained of ten (10) fish. The first six groups were consistently inoculated intraperitoneally (I / P) with bacterial suspension of Y. enterocolitica adjusted to densities (2x106, 2x105, 2x104, 2x103, 2x102 and 2x101 CFU / ml) respectively, at a dose rate of 0.2 ml while the control group (group 7) was injected I/P with 0.1 ml of sterile saline (0.9% NaCl) which serve as a control group.
5.2. Evaluation of vaccination strategy and herbal medicine for control of Y. enterocolitica : Another same 125 apparently healthy sea bass were divided into 5 groups; each group contains 25 fish. First group was inoculated (I.P.) with 0.2 ml / fish of normal saline and served as a control group. The second group was inoculated (I.P.) with 0.2 ml of 1/10 LD50 of tested bacteria. The third groups were injected by bacterin of Y. enterocolitica by the dose of 103 CFU / 0.2 ml plus infection with virulent strain of Y. enterocolitica . The fourth group was supplemented with feed and 1% of extract oil of O. vulgare . The fifth group was supplemented with feed and 1% of extract oil of O. vulgare plus infection with virulent strain of Y. enterocolitica by the dose of 103 CFU / 0.2 ml. All infected and control fish were observed daily to record their general health condition, clinical signs and mortalities.
5.3. Samples were taken from internal organs of experimentally infected sea bass to determine the histopathological alterations occurred .
The results of naturally examined sea bass revealed that :
1) Physico-chemical analysis of water samples in rearing tanks of sea bass broodstock : shows significant increase of Un-ionized form of ammonia (N-NH3) both nitrite (N-NO2-) and nitrate (N-NO3-) in the water of infected broodstock of sea bass. The results of heavy metal concentrations revealed that the all heavy metals were lower than the permissible limits except iron was revealed that the levels were higher than the permissible limits.
2) Bacteriological examination of water samples for presence of Yersinia spp in rearing tanks of sea bass broodstock : The results revealed that the total bacterial counts were reported in all examined samples by 2.20 x 107 and 3.47 x 107 and Yersinia spp count were reported in six examined samples only by 1.00 x 103 and 1.21 x 103 during winter season.
3) Naturally examined sea bass has typical signs of Yersiniosis as Naturally examined sea bass broodstock have Yersiniasis is characterized by the presence of congestive or haemorrhagic zones in various tissues and organs, particularly around the mouth and the intestines . General hemorrhagic septicemia, subcutanaeous hemorrhages in mouth cavity, around the chin, fin bases and anus, External clinical examination revealed the presence of marked congestion to extensive hemorrhages along all fins, the dorsal musculature, and lips
4) Cultural, morphological and biochemical characters of the isolated bacteria have typical characters of Y. enterocolitica .
5) The isolated Y. enterocolitica was confirmed with conventional PCR, whereas the PCR product had 100% typical identity and Consistent size with Yrp1 gene of Y. enterocolitica .
6) It was found that a total of 18 isolates of Y. enterocolitica out of 100 fish were found to be clinically infected with an average prevalence of the higher incidences observed in liver (72.22%,) and spleen (11.11%). The lower incidences of Y. enterocolitica were observed in kidney (11.11%) and heart (5.56%).
7) It was found that the bacterial isolates were markedly sensitive to Ciprofloxacin and Sulphamethoxazole-Trimethoprim combination, moderately sensitive to Enrofloxacin, erythromycin and Doxycycline and less sensitive to other antibiotics.
8) In order to determine the virulence of the identified Y. enterocolitica, a pathogenicity test was applied
Results revealed that :
1) The LD50 was (10-4 CFU/ml) and there were generalized signs of septicemia appear as: erythema and petechial hemorrhages on the abdomen and corneal opacity with orbital hemorrhage.
2) The re-isolation of the bacterium causing these signs and PM lesions found to have the same culture and biochemical characteristics of the injected bacterium and this confirm the cause of mortalities, signs and PM lesions.
3) Results of sea bass immunization with both of bacterin and herbal treatment and challenged with Y. enterocolitica revealed that the relative level of protection (RLP) was improved by injection of FKC of Y. enterocolitica intraperitoneally and 1% feed supplemented of oil extracts of O. vulgare . On the other hand, the RLP in control group was recorded as 12.0%.
4) Histopathological results in experimentally infected sea bass fingerlings revealed that the gills of infected sea bass with Y. enterocolitica showing bacteria in a capillary of the gill filament. Liver shows massive leukocytic infiltration and hyperplasia of bile ducts. Spleen shows coagulative necrosis and kidney with degenerated tubules and a notable increase in melanomacrophages as well as reduction in the lymphoid cells of hematopoietic tissue in the kidney.