BACTERIOLOGICAL STUDIES ON AEROMONAS INFECTIONS OF NILE TILAPIA (OREOCHROMIS NILOTICUS ) IN FISH FARMS IN ALEXANDRIA GOVERNORATE .

BACTERIOLOGICAL STUDIES ON AEROMONAS

INFECTIONS OF NILE TILAPIA (OREOCHROMIS NILOTICUS ) IN FISH FARMS IN ALEXANDRIA GOVERNORATE .

OLA .A . BASHA

ANIMAL HEALTH  RESEARCH INSTITUTE

ALEXANDRIA BRANCH .BACTERIOLOGY DEPT.

SUMMARY

       A history of sever mortalities and morbidities among cultured Oreochromis niloticus in many different  freshwater fish farms in Alexandria province were reported.                                                                                                                                     Clinical observation , postmortem changes as well as bacteriological isolation and identification revealed the presence of Aeromonas hydrophila in 60 % of the examined fish samples .

     Both intramuscular and intraperitoneal  inoculation of Aeromonas hydrophila in 20 apparently healthy Oreochromis niloticus fish induced the previously observed clinical signs and re-isolation of the injected pathogen were done . isolated Aeromonas hydrophila were found to be highly sensitive to Oxytetracyclin and sulphamerazine .

INTRODUCTION

     Aeromonas  species has long been recognized as a pathogen of fish and frogs ( Popoff,1984 , Son et al . , 1997 and Lehane and Rawlin , 2000 ) . Aeromonas hydrophila occurs widely in nature, in water (Picard et al  . , 1983 and Kompanets et al . , 1992 ) , in milk ( Nagah  , 1991 ) , in meat (Ozbas et al . , 1996 ) and in human . Aeromonas causes gastroenteritis with sever human diarrhea (Millership et al . , 1983   and  Buchanan and Palumbo , 1985 ) In fish Aeromonas hydrophila consists of a complex of disease organisms  associated with bacterial  haemorrhagic septicaemia ( Trust et al . , 1974)                                                                                   

     Aeromonas is one of the most serious and frequently encountered pathogens of fish  in warm-water aquacuture throughout the world. Aeromonas species have                

Implicated  as  the  causative  agents  of  red mouth disease of fish , intestinal dropsy of carp and septicaemia in warm water fish (Mayer ,1970 , Snieszko and Bullock , 1974 , Essa et al . , 1985 , Shahat and Hameda , 2000 and Samaha et al. , 2004 ) . McGary et al . (1990 ) isolated species of Aeromonas and Vibrio from ulcerative disease syndrome of  Florida fish in U. S. A .  .

      Several biochemical properties and virulence factors such as , protease , hemolysins , enterotoxins , dermonecrotic factors have been reported as

potential indicators of pathogenicity in Aeromonas hydrophila . Allan and Stevenson (1981 ) reported that the toxic fraction  of  extracellular products is associated with hemolytic activity , while Kanai and Wakabayashi (1984 ) considered that protease's were the main virulence factors implicated in the pathological effects produced by culture filtrates .  In addition , Norman (1988)

reported that severe mortalities , endotoxic shock and morbidities among cultured fish had been occurred due to lipopolysacharide of the Aeromonas hydrophila cell  wall . On  the other hand , Lallier et al . (1984 ) concluded that undefined dermo-necrotic factors present in the extracellular products of  Aeromonas hydrophila were responsible for fish mortality .

     The present study was carried out to explore and document Aeromonas infections among cultured Oreochromis niloticus from farms in Alexandria province . Moreover the biochemical character of organisms and trials to induce experimental infection  and detection of drug of choice for the treatment were considered .

MATERIALS  AND  METHODS

      Sample collection : A total numbers of 100 moribund Oreochromis niloticus (100 g mean weight ) were collected alive from fish farms near Mariout lake ,  placed in plastic bags filled with  water and air and transported to laboratory without delay . The samples were subjected to clinical observation and postmortem examination according to method described  by Amlacher (1970 ) and Schaperclaus et al . (1992 )for naturally and expermintally infected fish . Fish were observed for any gross lesions and abnormalities including  haemorrhages , skin ulceration , fin erosions and abdominal distension .

Bacterilogical examination :

    Samples were taken aseptically from skin lesion , muscles , kidney , spleen , liver and intestine, about one gram from tissues and saline was streaked on Rimler – shotts media , nutrient agar and MacConkey's agar.  After 24 hours of incubation at 30°C , the plates of Rimler –shotts were examined for  the presence of yellow orange  colonies ( the specific colonies of Aeromonas as stated by Rimler and shotts , 1973 ) . Colonies from nutrient agar were picked up and transferred to nutrient agar  slant for further identification . Gram stain and morphology of colonial appearance were reported with  identification by  biochemical  tests which   were done ( according  to Cruickshank et al . ( 1975) and Lennette et al . (1980 ) .

  Pathogenicity  test :

    The recovered  bacterial isolates were examined for its ability to produce the disease in apparently healthy Oreochromis niloticus  under experimental  condition .  40  fish with an average weight 50g ±(1%) were divided to four groups and each group was kept in a well prepared glass aquarium (110x 40x 50cm) equipped  with aeration and  supplied  with dechlorenated tap water . The water temperature in the aquarium was thermostatically adjusted to 28°C . Fish  were adapted for one week before starting the inoculation . The first group was inoculated intramuscularly (I /M ) below  the dorsal fin with 0.5 ml of

suspension of Aeromonas hydrophila  that contained 3x10 ⁷  cfu  this was done by djusting the optical density to 0.3 at a wave - length 435 um by using spectrophotometer (Spectronic 20 , Bausch &Lomb ), while the second group was inoculated intraperitoneally ( I / P ) with the same dose . The third and fourth groups were left as controls and injected with 0.5 ml sterile broth I / M and I / P  respectively. All groups were observed daily for 2 weeks for clinical signs and any pathologic lesions and abnormalities .

Reisolation of the injected bacteria were done on Rimler – shotts media

(shotts and Rimler , 1973 ) .

   Sensitivity test :

      The antibiograms of the isolated pathogen were done using the disc diffusion method of Bauer et al . (1966 ) . The interpretation of zones of inhibition were estimated according to the limits given by Finegold and Martin (1982 ) and Bio – Merieux (1984 ).  Different antibiotic discs were used such as Oxytetracyclin , Ampicilline, Streptomycin, Penicillin, Neomycin, Erythromycin , Sulphamerazine  and  Chloramphenicol .

RESULTS

      The clinical signs and postmortem  findings of diseased fish were characterized by erythema in the mouth , base of the fins and around the anus as well as exophthalmia . Affected fish showed congestion and petichial haemorrhage in the internal organs as well as erythema in the intestine with bloody mucous . The bacteriological examination  revealed that Aeromonas hydrophila could be isolated from 60 out of 100 diseased fish samples

examined ( 60 % ) . The organisms were a Gram negative , motile rod that gave round colonies ( 2 – 3 mm in diameter) , and yellow- orange in colour on Rimler–shotts medium . Isolates showed beta hemolytic activity on blood agar . The morphological and biochemical characteristics of Aeromonas isolates are illustrated in Table 1  .

   The I / M infected fish showed the same clinical signs and postmortem lesions that observed in natural infection with 70 % mortality rate ,while , the I / P infection  revealed erythema  at  the  site of  injection with congestion  in  the internal organs and  with  50 %  mortality . Aeromonas  hydrophila  were        re-isolated from liver , kidney and spleen of all freshly  dead and clinically diseased injected  fish .   No mortality and clinical signs were observed in control fish . The mortality rate , dose and route of inoculation , and time of death / day  post- inoculation are described in Table 2 .

   The result of drug sensitivity were summarized in  Table 3 .From such  table we can notice that Aeromonas hydrophila were sensitive to Oxytetracycline and Sulphamerazine .

DISCUSSION

     Fish  farming for food has grown rapidly in the past 30 years . The incidence of disease outbreaks among cultured fish has also increased . One  of  the  common fish bacterial disease is the motile  Aeromonas Septicaemia  which  caused by Aeromonas  hydrophila .  It appears that once the infection is established , a rapid growth of the bacteria  and  elaboration  of  its toxic products  may  cause systemic damage  which  lead  to  death  (  Brenden  and  Huizinga , 1986) . This  conclusion is forcly supported by the present investigation as the infection in the cultured Oreochromis  niloticus spreads quickly and the mortality rate reach 60 % within few days . On the other hand , El – shaboury , (1998 )  recorded an outbreak of infection  in  Oreochromis  niloticus caused  by Aeromonas  hydrophila with 100 %  mortality . The differances  in  mortalities may be attributed  to the type of  fish  and  the culturing condition  of  fish , handling , crowding , as well as abrasion of surfaces  increasing the risk of damage.   The clinical signs and postmortem examination of the cultured naturally infected  Oreochromis niloticus in the present study nearly simulate the results given by Mayer, (1970 ) , Scheider and Nicholson , (1980 ) ,  Essa et al .,(1985 ), Grizzle and Kiryu , (1993 ) and Shahat and Hamoda , (2000) .

Snieszko and Bullock , (1974 ) and Boira (1996 ) recorded that the disease is not a simple result of contact between host and pathogen. It was a complex interaction between host , pathogen and environment  which played as an important stress factor for the occurance of infection by Aeromonas hydrophila. These enviromental factors  included  high temprature and humidity , waste products and organic matter, and long transportation  .  Nearly all these factors were present in the area of cultured fish  farms around Marout lake and these predisposing factors may increase the susceptability  of fish to infection. As shown in  Table 2,  the I / M inoculation of Aeromonas hydrophila in experimental  fishes  nearly  showed similar clinical  signs and postmortem lesions like those observed in natural infection. The mortalities were 50 and    70 % in Oreochromis niloticus followed I / P and I / M injection respectively as well as the re –isolation of the Aeromonas hydrophila from these fish indicating  that  these organisms caused the disease . The I/ M route was more effective than I / P route . Similar findings were observed by Essa et al . ( 1991 ) .       The pathogenicity of Aeromonas hydrophila to fish was attributed to many virulente factors such as proteases and haemolysin. Soluman , (1988 ) and Coles et al . , (1969 ) , reported that the virulent strains of Aeromonas hydrophila were able to produce enzyme capable of lysing Staphylococcus cells . while  Kou      ( 1972 ) found that the virulent strains produce proteolytic enzymes. Moreover , Wakabayashi et al . (1981 ) reported  that  Aeromonas  hydrophila  produce extracellular enzymes, cytotoxins and haemolysins . The result of drug sensitivity proved that , the isolated Aeromonas hydrophila  were highly  sensitive  to  Oxytetracycline  and  Sulphamerazine . These results are in                                                                

agreement with those reported by Neumann and Polger (1979 ) and Essa et al . (1991 ) . 

Table 1 : Morphological and  biochemical  characteristics  of

Aeromonas hydrophila isolated from Oreochromis niloticus .

Table 2 : Pathogenicity of isolated Aeromonas hydrophila in

healthy Oreochromis niloticus    .

Table 3 :Drugs sensitivity test of Aeromonas hydrophila  isolated From Oreochromis niloticus .

+   Low  sensitive

++   Moderate  sensitive

+++  Highly  sensitive

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الملخص العربى

دراسات بکتريولوجية عن الاصابة ببکتريا الايروموناس لاسماک البلطى المستزرعة فى

محافظة الاسکندرية

د/علا عبد العزيز محمد باشا

معهد بحوث صحة الحيوان

فرع الاسکندرية

إشتملت الدراسة على فحص عدد 100 سمکة بلطى مريضة تم جمعها من مزارع إستزراع أسماک البلطى المختلفة و المنتشرة بمحافظة الاسکندرية و التى تعانى من

إصابات مرضية شديدة مع نسبة نفوق عالية .

و قد تم فحص الاسماک فحصا ظاهريا إکلينيکيا و تم إجراء الصفات التشريحية ثم

بعد ذلک تم الزرع و الفحص البکتريولوجى و کانت نتيجة الفحص البکتريولوجى

عزل بکتريا ألايروموناس من ألاسماک المريضة بنسبة 60 %  .

کما تم إعادة  عزل هذه العترات من البکتريا بعد إجراء العدوى الصناعية فى المعمل لاسماک بلطى سليمة و ذلک عن طريق الحقن العضلى و البريتونى .

و وجد أن الميکروب مرضى و قادر على إحداث العدوى .

هذا و قد تم إجراء إختبار الحساسية الجرثومية للعترات المعزولة و وجد إنها حساسة

للمضادات ألاوکسى تتراسيکلين و السلفا ميرازين  .