Alaa Eissa, Manal Zaki , Mohamed Moustafa, Ahmed Abdel Aziz, and Sherein Saeid (2010) :

1. Mycobacteriosis in the Dusky grouper (Epinephelus marginatus) from Mediterranean coastal shores of Marsa Matrouh province, Egypt Alaa Eissa, Manal Zaki , Mohamed Moustafa, Ahmed Abdel Aziz, and Sherein Saeid (2010): Biosecurity for Aquaculture Facilities, 2009

2. Back ground • High population densities together with the presence of high levels of excreted carbon and nitrogen compounds in the re-circulated water in hatcheries contribute to the growth, establishment and multiplication of aquatic fastidious organisms such as Mycobacterium

3. Dusky grouper • Dusky grouper (Epinephelus marginatus) is one of the grouper fishes that are native to the Mediterranean coastal zones. • Their range extends from France, Spain, Moroco, Tunisia, Egypt and Israel Mediterranean coastal shores. • This category of fishes is carnivorous in nature where they prey on fish, octopus, crab, lobster and dead carcasses in the lack of their natural food prey

5. Mycobacteriosis • Mycobacteriosis, caused by aquatic Mycobacterium species, is a common disease in a wide variety of fish species world-wide. • In aquatic environment, three species of Mycobacterium, M. marinum, M. fortuitum and M. chelonae, have often been reported as the cause of mycobacteriosis in wide variety of aquatic species. • Among aquatic mycobacteria, M. marinum is the most prevalent species in tropical freshwater and marine fishes. 9

6. Fish Mycobacteriosis is An Emerging Disease of Highly zoonotic and communicable importance

7. Transmission of Mycobacteriosis • Mycobacteriosis of fish can be possibly transmitted in the aquatic environment : • ingestion of contaminated food or aquatic debris • bacterial invasion through damaged skin or gill tissue • Untreated water. • Invasive spp as rabbit fish. • It appears unusual in being not only fish pathogen but also a human pathogen, causing chronic skin ulcerationscalled swimming-pool granulomas 9

8. Zoonosis

9. Pathology of piscine mycobacteria in fish and mammals

10. The aim of the study The current study presents a unique record of Mycobacteriosis as one of the most important emerging diseases among grouper populations at the coastal south Mediterranean zones of Egypt.

11. Materials and methods Biosecurity for Aquaculture Facilities, 2009

12. Sampling • A total of 5 representative dusky grouper fishes were collected during the event of mass mortalities among the grouper population from the Saloum bay and rocky coastal zones of Marsa Matrouh province, Egypt. • The groupers mortalities were at their peak during late summer and early fall of 2008. • Fish were clinically examined for any external abnormalities before being dissected

13. Bacteriological Examination • tissue samples were re-suspended in HBSS solution then 100 µl of the suspension were streaked onto Lowenstein - Jensen media slants supplemented with 5% Sodium Chloride. • Inoculated slants were incubated at 28 ºC and 37 ºC for 14 days. Slants were inspected on weekly base for any bacterial colonial growths. • Colonies were examined for acid-alcohol fastness by the Ziehl - Neelsen technique • The bacterium was identified by its rate of growth, colonial morphology, pigmentation at dark and light, and biochemical properties.

14. Histopathology • Tissues sample were fixed in 10% neutral buffered formalin solution, and were then processed and embedded in paraffin. • Five-micron sections of tissue were stained with hematoxylin and eosin (H & E).

15. Water analysis • Water samples from (1- 4 meter) depths were collected from the Saloum bay and rocky coastal zones of Marsa Matrouh province during the event of mortalities. • The water samples were analyzed for any changes in: • Water pH • Temperature • Dissolved oxygen • Toxic ammonia.

16. Results Biosecurity for Aquaculture Facilities, 2009

17. External Clinical finding: • skin discoloration • abnormal, irregular skin ulcerations • Hemorrhagic skin • apparent exophthalmia • mild ascitis

18. Internal finding: Different sizedgranulomatous nodules scattered within the liver and spleen Adhesion of the internal organs together with the mesentery has been noticed in two of the affected fishes.

19. Bacteriological results • Bacterial isolates retrieved from the pooled tissue samples for each individual of the 5 groupers were presumptively identified as M. marinum using both cultural characteristics and conventional biochemical tests Biosecurity for Aquaculture Facilities, 2009

20. Histopathology liver showing the presence of multiple focal granulomas

21. liver showing the ultra-details of the typical mycobacterium granulomas ( Necrotic center without caseation or calcification (N), Aggregations of macrophages or mononuclear cells (M) surrounded by fibrous connective capsule

22. liver exhibiting typical mycobacterium granulomas with few mycobacterium bacilli embedded in the core necrotic structure of the granulomas. The entire structure is surrounded by fibrous connective capsule

23. Water quality parameters

24. Conclusion • the deteriorated environmental pattern (increase pH, increase ammonia, decrease DO). • the natural behavior of the investigated fish (prey on dead carcuses). • static water lagoons between rocks act as natural reservoir to many environmental hazard (carbon, nitogen, phosphorus) due to sewege, shipping,industrial. • Poor nature of food web in this zone of Medet. • Co-habitation with other wild spp. as water reptiles, aquatic amphibians and migratory birds. • were highly incriminated as the main stimulus behind the emergent eruption of M. marinum outbreaks among the dusky grouper (Epinephlus marginatus) in the current study.