ISOLATION OF BACTERIA FROM TILAPIA FISH (Oreochromis nilotica)

ISOLATION OF BACTERIA FROM  TILAPIA FISH (O.nilotica)

Abstract

The present study was conducted to isolate, culture and identification (by gram staining) of bacteria from diseased fish Tilapia (  Oreochromis  niloticus).In the present study, monthly prevalence and abundance of the pathogenic bacteria were studied.The host Tilapia (Oreochromis niloticus) harbored different types of bacteria .Bacteria are found in different parts of the body. Such as: -ulcerative lesions, gills, intestine, kidney etc. The highest prevalence of bacteria in Tilapia (Oreochromis niloticus) was found in the month of March (80%) and lowest in the month of October (20%).The out break of EUS syndrome has been occurring by the bacteria during the period from November to March.  E. coli ;Aeromonas hydrophila ; Aeromonas fungatum ;   Aeromonas veronii ;Aeromonas salmonicida ;Aeromonas jandaei ;Seudomonas anguiliseticus ;Staphylococcus epidermatis ;Klebsiella sp are isolated from the ulcerative fish. But which is the causal agent of EUS can not be specifically identified. (Kar et al, 1999b).

Introduction

 Tilapia has recently been recognized as a global fish and described as one of the most important aquaculture species of the 21th century. Bacterial diseases constitute some of the major challenges facing sustainable tilapia production in Bangladesh. Diseased fishes were the vehicles for human infection and death by septicemia (Veennstra et.al; 1992, Woo and Bruno 1999). Aeromonas hydrophila is a primary (Esteve et al., 1993), secondary (Joice et al., 2002) and opportunistic pathogen (Dooley and Trust, 1988; Lio-Po et al., 1996) of a variety of aquatic (fish) and terrestrial animals, including humans. It is a ubiquitous,free living, gram-negative bacterium, mainly found in water and water-related environments and causes a wide variety of symptoms (Hazen et al., 1978a). The disease caused by A. hydrophila is called motile aeromonad septicaemia (MAS) and this pathogen is associated with number of other diseases in fish, for example, epizootic ulcerative syndrome (EUS) as a secondary pathogen (Roberts, 1993; Pathiratne et al., 1994; Lio-Po et al., 1998).

 The clinical signs in fish vary from tissue swelling, necrosis, ulceration and haemorrhagic septicaemia (Hazen etal., 1978a; Karunasagar et al., 1986; Angka, 1990; Aguilar et al., 1997; Azad et al., 2001). Aeromonas hydrophila is associated with disease in humans and domestic animals including sheep, dogs and cats, especially when exposed to periods of stress (Burke et al., 1984; Howard and Buckley, 1985a; Janda and Duffey, 1988; Ghenghesh et al., 1999; Ilhan et al., 2006).

 It is reported to contribute to intestinal and extra-intestinal infections including diarrhea in humans and other animals (Agarwal et al., 1998; Guimaraes et al., 2002). It has also been found in a variety of food products producing a range of toxins such as haemolysin, enterotoxin and cytotoxin (Callister and Agger, 1987: Yucel et al., 2005; Daskalov, 2006).

 Separation of a single bacterial species from a multi species colony is known as isolation of bacteria. In nature a single type of bacterial species is usually occurs as only one component of large and complex population containing many other organisms.

  To study the characteristics of one species, that must be isolated in pure culture. It is often helpful to use a selective method first. Such a method can increased the relative proportion of the desired species in the population so that it can be more easily isolated.

 A variety of techniques have been developed to isolation of bacterial colony. Some of these techniques are:-

The streak plate technique

  1.      Roll tube technique
  2.      The pour-plate and spread plate technique
  3.      Micromanipulator technique

Antibiotic means the chemical or substances origin from organism (mainly micro-organism) which acts as the inhibitor of other organisms. Some antibiotics kill the organism/microorganism directly and some inhibit their growth. The antibiotic sensitivity test implicates the degrees of effectiveness of any antibiotic as an inhibitor to any other organisms or microorganism.

 

Taxonomy:

Taxonomy of tilapia fish are given bellow: –

Phylum- Chordata

Group- Vertebrata

Subphylum- Gnathostomata

Series- Pisces

Class- Telostomi

Subclass- Actinopterygii

Order- Perciformes

Genus- Oreochromis

Species- Oreochromis  niloticus

  Objectives:

The objectives of this research works are given bellow:-

  1.   To isolate of the bacteria from the selected fish.
  2. To observe the organ wise distribution of bacteria.
  3. To investigates the effect of bacteria on the host body.
  4. To gram staining and test of its sensitivity.
  5. To identify the causative agent for the diseases.

        Review of Literature

  The genus Aeromonas was first described by Zimmermann (189o), who isolated the bacterium from the drinking water supply of Chemnitz in Germany using gelatine agar. He named the bacterium “Bacillus punctatus”. Sanarelli (1891) isolated a similar bacterium from the blood and lympsh of frogs, which he called “Bacillus hydrophilus fuscus”, but in 19o1 Chester proposed a name change to “Bacterium hydrophilium” (Caselitz, 1966). In the first edition of the Bergey´s manual, this species was erroneously designated as “Proteus hydrophilus”.

 However, in the Sixth Edition the genus Proteus was reclassified as Pseudomonas (Speck and Stark, 1942; Rustigan and Stuart, 1943). The genus Aeromonas was finally adopted in the Seventh Edition of Bergey´s manual (Stainer, 1943), and this particular organism was classified as A. hydrophila.According to molecular genetic studies, Messner and Sleytr (1992) proposed that the genus Aeromonas might be placed in a new family, the Aeromonadaceae.

 This genus was previously placed in the family, Vibrionaceae (Farmer, 1992) based on its phenotypic expression. Sakazaki & Shimada (1984) serotyped strains of Aeromonas on the basis of its O-antigen lipopolysaccharide (LPS). The genus Aeromonas has been shown to be antigenically diverse with over 9o established or possible serogroups described within this genus (O’Farrell, 1975; Frerichs, 1989).The family is sub-divided into psychrophilic and mesophilic species. The psychrophilic group is non-motile, does not grow at 37oC and is therefore Chapter 1 Page 4 unimportant to clinical microbiology. Members of the mesophilic group grow at 37oC and are motile using polar flagella. This group is  divided into three principal groups, A. hydrophila, A. caviae and A. sobria (Korbsrisate et al., 2oo2). Aeromonas hydrophila is a fermentative rod approximately o.8-1.o µm shape and 1.o-3.5 µm in size, that is motile via a single polar flagella (Austin and Austin, 1999). It is able to produce two distinct types of flagella; polar flagella for swimming in liquids and lateral flagella for swarming over surfaces (Altarriba et al., 2oo3). The bacterium can be isolated on non-selective media such as nutrient agar or tryptone soy agar (TSA), or on selective media such as Rimler-Shotts medium (Zimmermann, 189o) or peptone beef-extract glycogen agar (Sanarelli, 1891) by incubating at 2o-3ooC for 18-36 hours Colonies of A. hydrophila that are grown on TSA at 28oC for 18-24 hours usually appear round, creamy to light yellow in color, raised, and 2-3 mm in diameter.

Most selective media use carbohydrate and ampicillin or penicillin as the selective agent (Palumbo et al., 1985). Kay et al. (1985) recommended using sheep blood agar with 10 ?g /ml ampicillin proceeded by overnight enrichment in alkaline peptone water (APW) for the isolation of A. hydrophila from humans. Cepahlothin has been reported as the best enrichment agent in APW for A. hydrophila isolation owing to its greater selectivity and efficiency in recovering stressed or low concentrations of bacteria (Sachan and Agarwal, 2000).

  The culture environment plays a major role in the growth and virulence of the bacterium, especially with respect to available nutrients, temperature and pH.Chapter 1 Page 5 Sautour et al. (2003) proposed a model for describing the effects of temperature, water activity (aw) (ratio between vapour pressure of water and that of pure water at the same temperature) and pH on the growth of  A. hydrophila, and showed that temperature and aw are the main influences on the bacterium’s growth, while no significant influence of pH was seen.

Incubation of A. hydrophila at different pH values, i.e. 6.0, 6.5, 7.0 and 7.5 did not significantly affect the growth rates, but lag phases were shorter at pH 6.0 than pH 7.0 (Buncic and Avery, 1995). Although A. hydrophila can be grown at a wide range of temperatures, many researchers claim that the most suitable temperature for culturing the bacterium is between 25 and 35oC, while some researchers have found 20oC as an optimal temperature for its growth (Popoff, 1984).

                However, Uddin et al. (1997) found that the optimum temperature for growth of A. hydrophila was 34.5±1.ooC, while protease production was greatest at 27.6 ±?4.9oC. It is neither salt (<5%) nor acid (min. pH~6) tolerant, and has the ability to grow at temperatures as low as –o.1oC for some strains (Daskalov, 2oo6).The growth of A. hydrophila at different temperatures ranging from 4 to 42oC and 5 to 35oC have been reported by Palumbo et al. (1985) and Callister and Agger (1987), respectively.

 

Materials and methods

3.1. Materials:

  1.     Fish samples.
  2.   Nicrom wire loop.
  3.    Spirit lamp.
  4.    Petri dish.
  5.     Conical flask.
  6.      Micro oven.
  7.     PH Meter.
  8.    Electric balance.
  9.    Auto clave machine.
  10.  Leminar air flow.

3.2. Reagents:

  1.  Peptone-1gm.
  2.   Yeast extract-0.5gm.
  3.   Nacl-0.5gm.
  4.  -100ml.
  5.   Agar-3gm.

3.3. Fish sample collection:

 Fifty tilapia fishes (Oreochromis niloticus) weighting between 200 to 300 gm were collected from the five fish ponds sampled between March and October 2010.The ponds were mostly derelict and sandy-muddy. Relevant water quality, parameters namely times dissolved oxygen level ammonia and organic load were measured. Bacteriological examination was carried out on samples taken from the intestines and gills of collected fish.

3.4. Culture media (Agar media):

     At first 100 ml DH2O water had taken in the flask. Then 0.5 gm yeast extract, 0.5 gm Nacl and 1 gm peptone were measured by electric balance and added them 100 ml DH2O water .After adding those reagent then had taken the PH of the medium by PH meter. At last 3 gm agar were added in this medium. After adding the agar in the medium of solution then it kept in the micro oven for mixing of that reagent .Then the solution with conical flask had kept to the autoclave machine (Temperature – 1260C, pressure-0.15MPa). After autoclave of the solution it had to use for bacteria culture.

 3.5. Bacteria isolation and culture:

  Bacteria were isolated from different   freshly anatomized fish organs like ulcerous lesions, gills, intestine and kidney by a streak plate technique. The Nicrom wire loop hitting(by the spirit lamp)it red hot and was touch in the respective ulcerative areas or anatomized organs and then streaking agar plate respectively. The agar plates were incubation at 20-24 hours for appropriate colony formation. After the incubation the single colony of each plate was selected for re-isolation to a pure culture.

 In this case of isolation, the loop was used to transfer a portion of culture from the culture Petri dish and was placed on a nutrient agar plate and streaked parallel from left to the right repeatedly. In this way, I streaked all the side of the plate and lastly streaked a line the middle of the plate.

 3.6. Gram staining:

 Gram staining was performed according to Gram (1884). The composition of the reagents used for Gram stain is described. A smear of bacteria was place Onto a slide to stain and heat fixed. Crystal violet was used to stain the smear for 1 minute and washed with tap water after or pouring of the stain gram iodine was then flooded onto the slides and left for 1 minute. This was powered off and the smear was distained with acetone for 2-3 second. The slides were washed with water then flooded with safranin for 2 minute. The slides were washed with tap water, air dried and examined under microscope.

3.7. Antibiotic sensitivity test:

(a)Requirements:

To complete the experiment the following equipments were used:  –

Cultured bacteria in test tube and Petri dish.

  1.    Nutrient broth.
  2.     Micropipette.
  3.      Petri dish and glass rod (spreader).
  4.    Antibiotic disc.
  5.    Forceps and
  6.    Oven.

(b)Methods of test:                                                                                                                          

At first 1 ml of supplied and cultured bacteria was taken in a Petri dish with the help of micropipette. Then the solution was spread through out the Petri disc with glass rod .If accidentally excess media (cultured bacteria) power into the   Petri     disc. Then the excess media with bacteria has been cast away from Petri disc and then it was covered and left for sometime. After then the supplied antibiotic disc were placed into the bacteria enrich medium on the Petri disc. To observe the effectiveness of the antibiotic and bacterial growth the Petri disc was left for 24 hours at 370 C.

  Result and observation

4.1. Isolation and pure culture:

 In the next day I had observed the Petri dish. I found that some colonies are formed then i separate that colony visually with the shape and size of the colony.

4.2. Antibiotic sensitivity test:

Finally it was observed that there were rounded zones around some of the antibiotic disc and on the other hand there were no zones around some antibiotic disc. So I can say that, the antibiotics which made quite 15 mm of zone were   susceptible to the supplied bacteria and the antibiotics which made the zone of   less than 15 mm or fail to make a zone were resistance to the bacteria.

4.3. Gram staining:

  The outer membrane is the outermost structure on the surface of Gram-negative bacteria surrounded by a. Generally, Gram-negative bacteria are characteristically double membrane: the cytoplasm or inner membrane, which is a phospholipids bilayer, and the asymmetrical outer membrane, which holds phospholipids and lip polysaccharides (LPS) in its inner and outer layers, respectively (Bos and Tommassen, 2004). Phospholipids are major structural and functional components of the cell envelop of all bacteria (Howard and Buckley, 1985b). The outer membrane assembly and the extracellular protein secretion are regulated by the expression of many genes such as exe genes (Howard et al., 1993). Proteins present in the outer membrane are composed of two classes’ lipoprotein, which are anchored into the outer membrane via an N-terminal lipid- tail and integral proteins that contain membrane-spanning regions. All proteins destined for the outer membrane are synthesized in the cytoplasm as precursors with N-terminal signal sequences, which are essential for translocation across the inner membrane (Bos and Tommassen, 2004).

  The OMP are believed to be a primary factor involved in the attachment (adhesion) of A. hydrophila to various host tissues, as this is a prerequisite to initiate infection (Del Corral et al., 1990). Quinn et al. (1994) isolated pore-forming OMP from A. hydrophila and suggested that it may be involved in the initial colonization of the bacterium on its host. Similarly, it was suggested that the 43 kDa porin may be an important adhesin with regard to entry into common carp epithelial cells because of an abundance of this particular receptor on the cell surface (Lee et al., 1997).

  The LPS is an important component of the outer membrane of Gram-negative bacteria (Howard and Buckley, 1985b), which consists of a hydrophobic membrane anchor, lipid A, substituted with an oligosaccharide core region that can be extended in some bacteria by a repeating oligosaccharide, the O-antigen(Bos and Tommassen, 2004). It plays an important role in the pathogenesis of the bacterium including having a role in adhesion and its ability to cause gastroenteritis (Merino et al., 1996a; Knirel et al., 2002). Shaw and Squires (1984) found an involvement of A. hydrophila LPS in bacteriophage attachment and other virulence properties such as serum resistance. It has been confirmed that A.hydrophila strains lacking a defined LPS are susceptible to killing by human serum (Janda et al., 1994a). Similarly, Mittal et al. (1980) reported that virulent strains of A. hydrophila express a unique O-antigen, and were able to differentiate between virulent and less virulent strains on the basis of serogrouping and cell surface characteristics. Many of the properties which facilitate the colonisation of the bacterium on its hostare associated with the cell surface of A. hydrophila, and are very important in host-pathogen infection (Dooley and Trust, 1988). Cell surface structures enable this pathogen to bind to a large number of cells and biomolecule in host tissues (Janda and Duffey, 1988; Ascencio et al., 1991a and 1998).

 These cell surface receptors can bind with iron-containing proteins of the host and may be involved in the acquisition of iron by the bacterium (i.e. the siderophores mentioned earlier) (Ascencio et al., 1992). However, environmental factors such as temperature play a significant role in regulating virulent factors; including the biochemistry of the cell surface of A. hydrophila (Merino et al., 1992).The S-layer proteins are considered to play a major role in infection for a number of bacteria (Boulanger et al., 1977; Dooley and Trust, 1988; Messner and Sleytr, 1992). The S-layer or paracrystalline surface layer was first described in Spirillum using electron microscopy (EM) by Houwink in 1953. Thereafter it has been reported in nearly every taxonomic group of walled eubacteria, and is a feature of most archaebacterial cell envelops (Sleytr and Messner, 1983 and 1988; Messner and Sleytr, 1992). Pathogenic bacteria such as Clostridium botulinum, A.salmonicida, Campylobacter fetus subsp. fetus, C. rectus and Mycobacterium bovis have all been reported to possess S-layers (Boulanger et al., 1977; Messner and Sleytr, 1992).Using a laboratory challenge model, Thune et al. (1986) showed the Slayer of A. hydrophila to correlate with virulence. It is believed to influence the interaction between the bacterial cell and its environment (Austin and Austin, 1999). The localisation of the S-layer on the surface of the cell suggests it has an important role in the growth and survival of bacteria, and is the site of interaction between the bacteria and the external environment. It possesses anti-phagocytic activity which may aid in the systemic dissemination of bacteria once invasion through the gastrointestinal mucosa has occurred (Janda et al., 1994a). However, the degree of antigenicity of S-layer proteins varies among bacterial species (Kobayashi et al., 1993). The S-layer in conjunction with LPS has been shown to play an integral part in the overt resistance of A. salmonicida to complement-mediated lyses (Chang et al., 1992).

Table: 4.1: List of bacterial isolates and their sources.

 

Bacteria

Source

Remarks

Aeromonas hydrophila 1234

Veterinary Medical Aquatic Research Centre, Chulalongkorn University.

Isolate from carp kidney.

Aeromonas hydrophila 04082

Aquatic Animal Health Research Institute, Dept. of Fisheries Ministry of Agriculture.

Isolate from carp kidney.

Aeromonas hydrophila 2798

Dept. of Medical Science, Ministry of Public Health Thailand.

Isolate from stool.

Aeromonas sobria 12056

Nation Collection of Industrial Marine and Food Bacteria, U.K.

Isolate from stool.

Aeromonas sobria 12446

Dept. of Medical Science, Ministry of Public Health Thailand.

Isolate from stool.

Aeromonas caviae 13016

Nation Collection of Industrial Marine and Food Bacteria, U.K.

Isolate from stool.

Table.4.2: Sensitivity test of the isolated A. hydrophila to different antibiotics.

 

Antibiotic Symbol Sensitivity reaction
Ampicilin AM Resistance
Ciprofloxacin CIP Sensitive
Gentamycin GM Sensitive
Oxytetracyclin OT Sensitive
Streptomycin S Sensitive
Penicillin P Resistance
Erythromycin E Sensitive
Kanamycin K Resistance

 Discussion

        Lallier and Higgins (1988) showed all 65 A. hydrophila strains in their study isolated from either diseased mammals or healthy and diseased fish, to be positive for O-nitrophenyl-D-galactopyranoside, arginine dehydrolyase, glucose, mannitol and saccharose. They also reported that all these isolates were negative for ornithine decarboxylase, H2S, urea’s and inositol, and other biochemical characteristics were variable between the isolates. The biochemical results obtained with the API 20E microbial identification kit in this study were in agreement with the results of Lallier and Higgins (1988). However, minor differences were observed between the NCIMB reference strain and isolate T4, and with isolate T4 when it had been pass aged once and twice through a goldfish. For example, isolate T4 was positive for H2S before and after pass aging once through a fish, but negative after pass aging it a second time through fish. De Figueiredo and Plumb (1977) also observed variations in the biochemistry between the different isolates of A. hydrophila they screened.

 5.1. Pathogenecity:

Aeromonas hydrophila mainly causes motile aeromonad septicemia (MAS) and has also been reported to cause secondary infections associated with EUS outbreaks (Roberts, 1993). The disease caused by A. hydrophila has also been called ‘Red-sore’ disease (Huizinga et al., 1979). It does not usually cause problems in fish populations under normal conditions, but when fish are under environmental or physiological stress or infected by other pathogens, A. hydrophila is a potential pathogen (Plumb et al., 1976; Fang et al., 2000).

Generally, it has become an increasingly important pathogen in intensive fish culture due to increased environmental and physiological stress experienced by reared fish (Shaw and Squires, 1984). Under favorable environmental conditions, this pathogen seems to multiply and produce higher levels of ECP toxins in fish, which can cause sudden disease outbreaks and mortalities (Allan and Stevenson, 1981; Yadav et al., 1992; Vivas et al., 2004a). Several studies have described a wide variation in the pathogen city of A. hydrophila in different fish species. This is mainly due to the heterogenic city of strains and differences in the adhesive and enterotoxic mechanisms responsible for causing infection in fish (Fang et al., 2004). A natural infection model (immersion challenge) in a genetically stable inbred strain of southern platy fish, Xiphophorus maculates, was used to study the pathogenesis of the bacterium (Kawula et al., 1996). They showed that the mortality was dependent on the concentration of bacteria and the appearance of clinical signs in fish that eventually died. a major virulent factor, when its pathogen city was studied using a suckling mice model infection (Heuzenroeder et al., 1999).

 5.2. Clinical signs of infection:

The clinical signs of disease caused by A. hydrophila have been classified into four categories; acute, rapidly fatal septicemia, with few gross symptoms; an acute form with dropsy, blisters, abscesses and scale protrusion; chronic ulcerous form with furuncles and abscesses; and a latent form with no symptoms (Karunasagar et al., 1989). Clinical signs of infection have also been classified into three groups by other workers using an artificial challenge model in channel catfish: viz (1) MAS(Systemic infection and signs of disease), (2) cutaneous (infection limited to skin and the underlying muscle) and (3) latent (systemic infection but no external signs of disease) (Grizzle and Kiryu, 1993). The basic clinical signs of the pathogenesis of A. hydrophila are the presence of small surface lesions (which lead to the sloughing-off of scales), local hemorrhages particularly in the gills and vent, ulcers, abscesses, exophthalmia and abdominal distension, and moreover, it is often associated with abdominal edema or dropsy (Jeney and Jeney, 1995). Azad   et al. (2001) observed external signs, such as necrotic and edematous changes in tilapia, Oreochromis niloticus infected with A. hydrophila. As well as the external clinical signs described, various signs of disease have been reported in the internal organs of different fish species.

For example, liver and kidneys were found to be completely destroyed inLarge mouth bass, Micropterus salmoides infected with A. hydrophila (Huizinga et al., 1979) degeneration and mild necrosis of the kidney (Tafalla et al., 1999). Diffused necrosis in several internal organs and the presence of melanin-containing macrophages in the blood was also observed during a systemic A. hydrophila infection in channel catfish (Ventura and Grizzle, 1988). Apart from external and internal clinical signs, immunological and biochemical parameters are also affected by A. hydrophila infection. For example, septicemia in Indian major carp, catla  (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala) has been reported by Karunasagar et al. (1986 and 1989). Similarly, an increase in plasma glucose and leukocyte volume was noted in subordinate fish compared with dominant fish when juvenile rainbow trout were exposed to social stress following Challenge with A. hydrophila (Peters et al., 1988). Biochemical signs, especially accumulation of lipofuscin (a per oxidation product of lipid catabolism) together with tissue destruction in the fish, have also been observed (Ventura and Grizzle, 1988). Toxin induced changes in the host have been reported by Rodriguez et al. (1993), who demonstrated that the acetyl cholinesterase of A. hydrophila was found in the brain of infected rainbow trout which caused the fish to lose their “flight” reaction and then their equilibrium, leading to spasmodic swimming followed by death. The symptoms suggest that the toxin may be acting on the central nervous system. Endotoxins produced by A. hydrophila have been found to initiate febrile responses in the fish and other animals (Reynolds et al., 1978). According to Ko et al. (2005), mice artificially infected with A. hydrophila can evoke a pronounced proinflammatory cytokine response. In vitro assays have also been used as models to predict clinical changes in fish caused by A. hydrophila. In these experiments, the pathogen was seen to produce severe morphological changes in the first 2 h of incubating common carp epithelial monolayer in vitro with the bacterium, and changes could even been seen after 30 min incubation with one strain (Leung et al., 1996).

5.3. Diagnostics:

Diagnosing disease and identifying the infectious agents included are important for managing any disease .Visible internal and external clinical signs indicating disease caused by A. hydrophila. However, diagnosis can be very difficult, since the progress of infection by A. hydrophila is rapid in the fish under favorable Conditions for the bacterium (Jeney and Jeney1995), and other pathogens such A. salmonicida and Vibrio species could also possibly cause similar signs. Confirming the association of A. hydrophila with disease/infection is important before treating the disease. A number of methods have been reported for the detection and identification of this pathogen, including traditional (phenotypic and biochemical characteristics), immunological and molecular techniques. Traditional methods to detect and identify A. hydrophila include examination of the shape and colour of colonies on nutrient agar, Gram staining, morphology and motility of the bacterium and various biochemical analyses (Altwegg et al., 1990; Chaudhury et al., 1996; Yambot, 1998). A rapid method based on biochemical analysis using an API strip containing premixed chemicals has been routinely used for the identification of A. hydrophila (Dixon et al., 1990; Gatesoupe, 1991; Hettiarachchi and Cheong, 1994; Noterdaeme et al., 1991). However, traditional methods are not fully reliable for the identification of A. hydrophila to species level due to similarity in some phenotypic and biochemical characters with other species (A. caviae and A. sorbia). In addition, the biochemical characteristics expressed between different isolates of A. hydrophila are not always same (De Figueiredo and Plumb, 1977). Immunological detection methods such as enzyme linked immunosorbent assay (ELISA) were developed for the detection of A.hydrophila by Merino et al. (1993) and Sendra et al. (1997). Korbsrisate et al. (2002) produced polyclonal antibodies against A. hydrophila for use in a specific direct agglutination test to identify A.hydrophila. Monoclonal antibodies (Mabs) have also played a vital role for the identification of fish pathogens (Adams and Thompson, 2006), and Mabs that recognize a particular serotype O side-chain core oligosaccharide of A. hydrophila have been developed (Cartwright et al., 1994). Chanphong et al. (1999) developed Mabs against 41 a kDa protein of A. hydrophila, while Mabs were also produced against  A. hydrophila that recognized a 110 kDa polypeptide on the bacterium (Delamare et al., 2002). Although immunological methods have been useful in the identification of A. hydrophila, it might only be possible to identify particular isolates/serotypes with the specific antibodies developed due to heterogeneity of isolates (Merino et al., 1993). Molecular methods have been recommended for the identification A. hydrophila to overcome possible problems encountered with traditional or immunological methods. Sugita et al. (1994) suggested a deoxyribonucleic acid (DNA) based hybridization method for the identification of A. hydrophila, while amplification of specific genes (e.g. haemolysin) of A. hydrophila by polymerase chain reaction (PCR) has been recommended for detection of the bacterium (Xia et al., 2004).  A. hydrophila. A method combining immunological and molecular techniques (immuno-capture assay with PCR) has also been developed to provide a quick, sensitive and reproducible way of detecting A. hydrophila (Peng et al., 2002).A. hydrophila in goldfish has also been reported in differences to be insufficient to separate the isolates into virulent and avirulent groups. Several studies are reported in the literature relating to artificial challenge with various doses of A. hydrophila using different isolates in a range of fish species. The clinical signs which occurred were all similar at the time of mortality, although the time it took for these to appear varied depending on the concentration of A. hydrophila used. For example, when catfish (Clarias batrachus) were injected with 1 ×107 ml-1 A. hydrophila clinical signs became apparent by three days post injection, with petechiae and reddening of the abdomen, and similar clinical sign vitro (Shao et al., 2004)

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