RETRACTED ARTICLE: Screening for the presence and prevalence of Edwardsiella tarda infection in fish harvested from Lakes Zeway and Langano, Southern Oromia, Ethiopia

A study was carried out from October 2009 to April 2010 with the objective of isolating Edwardsiella tarda an important fish pathogen from fish harvested for human consumption from Lake Zeway and Langanoo. A total of 372 tissue samples (three from each fish) comprising liver, intestine and kidney were collected from 124 fish (Clarias gariepinus and Oreochromis niloticus). Distribution of E. tarda infection among these three organs examined indicated that E. tarda was isolated most frequently from liver (6.5%) followed by intestine (2.4%) and kidney (0.8%) with significant difference. Statistical significant differences (p < 0.05) were found in E. tarda infection with respect to tissue samples. Fish from Lake Zeway was prevalently infected by E. tarda than Lake Langano. Male fish were more frequently harbor E. tarda than female fish and were not statistically significant (p > 0.05). The current study is signified that E. tarda infection is a potential threat to the fishery sector and public health. Therefore, awareness should be created on the hazardousness of E. tarda on public health significance and hence further studies have to be conducted in other lakes of Ethiopia that harbor fishes. *Corresponding author: Bedaso Kebede, Veterinary Drugs and Animal Feed Administration and Control Authority, Addis Ababa, Ethiopia E-mail: Kebede.bedaso@yahoo.com Reviewing editor: Fatih Yildiz, Middle East Technical University, Turkey Additional information is available at the end of the article

ABOUT THE AUTHORS Dr Teshome Habtamu and Dr Bedaso Kebede graduated from Addis Ababa University Faculty of Veterinary Medicine since July, 2010. Their research interests are focused on the animal diseases and public health. This paper focused on the impact of Edwardsiella tarda on fish sector and public health.

PUBLIC INTEREST STATEMENT
Edwardsiellosis is the most important bacterial diseases causing severe economic losses in fish farms of many countries. E. tarda is a health threat to other animals and humans apart from threat to fish that means it has a zoonotic significance. The predisposing risk factors were exposure to aquatic environment, pre-existing liver diseases, iron overload and raw sea food ingestion. Edwardsiellosis in humans usually cause diarrhea, gastroenteritis, wound infection and even death. There are reports of extra intestinal infection of the diseases with the clinical pictures including a typhoid like illness, peritonitis with sepsis and cellulites with occasionally liver abscess and meningitis. The practice of consuming partially cooked fish meals, manual handling of fish and unhygienic practice during filleting would expose the public to the higher risk of contracting the disease. Therefore, the disease deserves attention due to its impact on the fishery sector and its potential threat to public health.

Introduction
Aquaculture is growing rapidly worldwide with fish being the primary source of animal protein in many countries. The fishery sector plays a significant role in food security through supplementation of food for developing countries. As a whole fish currently makes up about 19% of the total protein consumption or just over the 5% of proteins from both plants and animals origin (Dugenci & Candan, 2003).
The major problems hampering production, development and expansion of the aquaculture industry is due to the fact that fish are possibly susceptible to microbial diseases. Microbial diseases are a global problem affecting fresh water, marine water, cultured, sport and ornamental fish. The problem is extremely important when fish are subjected to intensive culture practices (Trust, 1986).
The control of fish diseases is particularly difficult because fish are often farmed in system where production is dependent on natural environmental conditions. Changes or deterioration in the aquatic environment cause the occurrence of most fish diseases and also environmental effects play a great role in influencing the health status of fish. Therefore, understanding characteristics of potential pathogenic microorganisms of fish, aspects of the biology of fish as well as a better understanding of the environmental factors affecting such cultures will allow the application of adequate measures to prevent and control the diseases limiting fish production (Toranzo, Magarinos, & Romalde, 2005;WHO, 1999).
Edwardsiellosis is among the most important bacterial diseases causing severe economic losses in fish farms of many countries. The disease is caused by Edwardsiella tarda which is a gram negative, motile, facultative anaerobic, short rod shaped bacterium (1 μm in diameter and 2-3 μm long) pathogenic to a wide range of fish hosts such as Channel catfish (Ictaluri punctatus), Striped bass (Morone saxatili), eel (Anguilla anguilla), Nile Tilapia (Oreochromis niloticus), carp (Cyprinus cyrpio) and Flounder (Paralichthys olivaceus) (Plumb, 1999). These organisms found frequently in organically polluted and poor quality water which predispose fish to the disease by resulting stress in them (Novotony, Dvoska, Loremcova, Beran, & Pavil, 2004;Wei & Musa, 2008). Media used to isolate E. tarda were Edwardsiella Isolation Media (EIM), Brain Heart Infusion (BHI), Tryptic Soya Agar (TSA), Xylose Lysine Deoxycholate (XLD) and MacConkey agar. The culture of such bacteria is characterized as small, circular, raised whitish colonies with black center on XLD as well as appearing as pale colonies on MacConkey agar. Several factors hinders culture or grows of E. tarda on the medium are temperature ranges 25-37°C, pH ranges 7.0-8.0 and 0.5% NaCl concentration (Wei & Musa, 2008). Biochemical characteristics of E. tarda are catalase positive, cytochrome oxidase negative, glucose fermentative, indole positive, citrate negative, lysine positive, mannitol, dulcitol, sorbitol, inositol, xylose, rhamnose negative, produce hydrogen sulfide, alkaline slant and acid butt on Triple sugar iron Agar (Carter, 1984).
E. tarda is a health threat to other animals and humans apart from threat to fish that means it has a zoonotic significance (Clarridge, Musher, Fainstein, & Wallace, 1980). The predisposing risk factors were exposure to aquatic environment, pre-existing liver diseases, iron overload and raw sea food ingestion (Wang, Liu, Cheng, & Kao, 2008). Edwardsiellosis in humans usually cause diarrhea, gastroenteritis, wound infection and even death (Plumb, 1999;Vandamme & Vandepitte, 1980). There are reports of extra intestinal infection of the diseases with the clinical pictures including a typhoid like illness, peritonitis with sepsis and cellulites with occasionally liver abscess (Zighelboim, Williams, Bradshaw, & Harris, 1992) and meningitis (Plumb, 1999). The infection is more severe in immunocompromised individuals. The practice of consuming partially cooked fish meals, manual handling of fish and unhygienic practice during filleting would expose the public to the higher risk of contracting the disease. Therefore, the disease deserves attention due to its impact on the fishery sector and its potential threat to public health (FAO, 1995;WHO, 1999). In Ethiopia, the bacterium has been isolated from apparently healthy fish of Lake Zeway and Tana (Nuru, 2007;Yimer, 2000). However, there is no further work done in covering the different fish species and aquatic environments. Therefore, this study was conducted with the aim of screening for the presence and prevalence of E. tarda infection in Fish harvested from Lakes Zeway and Langano, Southern Oromia, Ethiopia.

Study animals, sample size and design
Study animals used in this study included African catfish (Clarias gariepinus, N = 30) and Nile Tilapia (O. niloticus; N = 94) which were harvested from Lake Zeway and Langanoo for human consumption. In this study area Nile Tilapia was more abundant than other species. Those harvested fish were kept under cold chain until reach necropsy room and further assessment take place. The fish were physically examined for any external lesions before necropsy and collecting tissue samples. The necropsy was undertaken at the necropsy room of Zeway Fishery Resource and Research Center, Batu via fish dissected in ventral approach to expose organs of study. The fish sample was cut along the midline of the abdomen starting from the anus up to the mouth using sterile dissecting scissor followed by another dissection from the anus to the lateral line and further along the lateral line up to the gills cover to remove the lateral side of the abdominal wall and expose the internal organs. Internal organs were examined for any gross pathology and the findings recorded. Tissue samples were then taken from intestine (N = 124), liver (N = 124) and kidney (N = 124) aseptically using sterile scalpel blade and forceps kept in sterile universal bottles of 100 ml capacities. The bottles containing the samples were then kept in ice box containing ice packs and processed for bacterial isolation and identification in Microbiology laboratory of College of Veterinary Medicine and Agriculture and National Veterinary Institute, Bishoftu.

Lake Zeway
It is located on the Eastern side of Batu town, 163 km Southeast of Addis Ababa it lies in northern part of the rift valley between 7°51′ N to 8°7′ N and 38°43′ E to 38°57′ E with an open water area of 422 km 2 and shoreline length of 137 km. The lake is fed by two major rivers Ketar and Meki Rivers and has one out flow in the South Bulbula river which flow into Abiyata (LFDP, 1993). Five bigger islands are situated in the lake Viz Tulu Gudo (4.8 km 2 ), Tsedecha (2.1 km 2 ), Funduro (0.4 km 2 ), Debresina (0.3 km 2 ) and Galila (0.2 km 2 ). While the latter two have few inhabitants, the three bigger ones are populated with several hundreds of people (Anonymous, 1999). A fish habitat in the lake consist almost Nile Tilapia (Oeochromis niloticus). Since recent years, however, Catfish (Carias gariepinus) and crucian carp (Carcasius gracius) have appeared in small amounts of the total catch (LFDP, 1994). There are a number of landing points around the lake from where fish is collected either by boat or trucks and brought to the major landing points adjoining Batu town.

Lake Langanoo
It is located 200 km South of Addis Ababa lying between 7°36′ N; 38°45′ E. It is 18 km long and 16 km wide with an open water area of 230 km 2 , 7.5 km shoreline and 1,600 km 2 catchments area (LFDP, 1993). The main fish species in the lake include Barbus species, Clarias species and Oreochromis niloticus (FAO, 1995) with the total annual catch of 1,000 tones.

Isolation of E. tarda
Tissue samples from kidney, liver and intestine of Catfish and Nile Tilapia were homogenized in physiological saline. The homogenate was then taken by sterile loop and streaked on XLD agar plate (Titan Biotech) and then incubated at 37°C for 24 h. Colonies showing or resembling with morphological characteristics of E. tarda were further subcultured on MacConkey agar plates and incubated at 37°C for 24 h. All lactose non-fermented colonies were further subcultured on TSA containing 0.5% NaCl and incubated at 37°C for 24 h. Presumptive identification of the resulting isolates (colonies) was done employing different tests which included primary bacteria identification techniques and biochemical identification tests (Baron, Peterson, & Fine Gold, 1994;Quinn, Carter, Markey, & Carter, 1999;Rowland, Walsh, Teel, & Carnahan, 1994;Woodland, 2006).

Data analysis
Descriptive statistics such as proportions and frequency were employed in summarizing the data. Chi-square test of independence was employed in comparing the prevalence/occurrence of E. tarda infection with respect to site, sex, fish species and isolated organ. A confidence interval of 95% was used to interpret the statistical association and significance was considered when p-value is less than 0.05 (Agrawa, 1996).

Results
A total of 372 tissue samples comprising kidney, liver and intestine collected from 124 fish. E. tarda was isolated from 12 tissue samples (8 from liver, 3 from intestine and 1 from kidney). The isolates appeared as small punctuate grayish white colonies on XLD agar after 24 h of incubation at 37°C. Except few of the isolates, most showed typical characteristics of E. tarda. In biochemical tests, these typical isolates were positive for indole, H 2 S production, and lysine decarboxylase and unable to utilize Simmon's citrate and the different sugars used in this study (Table 1). However, some of the isolates showed variation from the typical characteristics. One isolate was negative for indole and able to utilize Simmon's citrate while the remaining was able to ferment mannitol, rhaminose, xylose and inositol (Table 1).
Distribution of E. tarda infection among the three organs examined indicated that E. tarda was isolated most frequently from liver (6.5%) followed by intestine (2.4%) and kidney (0.8%) with statistical significant difference (p < 0.05) among organs (Table 2). Statistical significant differences (p < 0.05) were found in E. tarda infection with respect to site although the bacterium was isolated from fish originating from both lake Zeway and Langanoo with E. tarda being more prevalent in fish sampled from lake Zeway. E. tarda was isolated more frequently from male fish, the differences in the occurrence of E. tarda infection with respect to sex were not significant (p > 0.05) indicating that both sexes are equally susceptible (Table 3). There was no statistical significant difference (p > 0.05) in isolation of E. tarda from Catfish (C. gariepinus) and Nile Tilapia (O. niloticus) indicating that both fish species are susceptible to the infection (Table 4).

Discussion
The genus Edwardsiella consists of two species E. tarda and E. ictaluri. E. tarda infects fish and other animals including human, while E. Ictaluri is a pathogen of fish only (Woo & Bruno, 1999). In this study, E. tarda were isolated from intestine, kidney and liver of fish to screen presence and prevalence E. tarda which is a potential threat to aquaculture and public health importance. The organism has been previously isolated from different samples such as intestine of fish and humans stool with sporadic cases of diarrhea (Vandamme & Vandepitte, 1980) and dressed fish (Noga, 1990;Wyatt, Nickelson, & Vanderzant, 1979).

Organ
Results Total   The present study showed two isolates were found non motile and this fact is similar to report of Okuda et al. (2007). Most of the phenotypic characteristics of the isolates were similar as claimed by Holt (1997) and variation in some of the biochemical tests particularly in the utilization of sugars which included mannitol, rhamnose, xylose and inositol. Such variation contradicts the study of Baya et al. (1997) where no variation was observed with respect to these biochemical tests among forty-four E. tarda isolates studied. Hence, Variation among E. tarda isolates was reported with respect to utilization of rhaminose (Wei & Musa, 2008), mannitol (Stock & Wiedemann, 2001). The occurrence of variation in phenotypic characteristics among E. tarda isolates may be due to the presence or absence of plasmid that control metabolic activities. Generally, the significance for the prevalence of E. tarda in catfish and Nile Tilapia could not be substantiated by references to the phenotypic characteristics of E. tarda alone (Acharya et al., 2007). Other means of identification may prove necessary for clarifying such aspect.
The absence of significant differences in the occurrence of E. tarda between males and females indicates that both sexes are equally susceptible to the bacterium. This is in agreement with the works of Savan et al. (2005) and Yu et al. (2004). The significant differences in the rate of isolation of E. tarda between the study lakes may be attributed to differences in the nutritional status of the fish, the environmental condition and water quality, changes in temperature, pH and fluctuation in dissolved oxygen which affect the occurrence of E. tarda infection (Cahill, 1990;Nuru, 2007;Ringoa, Olsenb, Mayhew, & Myclebusted, 2003). In current study E. tarda affect intestine, liver and kidney of catfish and tilapia but the highest percentage of the pathogen was isolated from liver this could be due to the metabolic activities of the organs (Cahill, 1990).

Conclusion and recommendations
Edwardsiellosis is the most important bacterial disease causing severe economic loss and hindrance in aqua farming. Apart from veterinary health importance, E. tarda has also public health significance in people engaged in fishery industry and those depend on fish products for their annual income. The isolation of E. tarda from wild fish population of Lakes Zeway and Langanoo destined for human consumption indicates that E. tarda fish pathogen may prove to be a serious threat to the fishery sector and public health. The finding of certain isolates that divert in their biochemical characteristics warrants further investigation using more advanced methods of bacteria characterization. Therefore, assessments on factors such as environmental condition, management strategies for controlling fish pathogen as well as other stress factors that could enhance the prevalence, distribution and severity E. tarda infestation, are crucial in designing effective disease control and prevention. Since the current state of knowledge on E. tarda infection in fish and humans in Ethiopia is relatively unknown, further studies into the epidemiology of such pathogen in different hosts and environments as well as comprehensive profiling of E. tarda strains for controlling measures and maintaining overall public health, merits scientific pertinence.