First isolation and identification of Vibrio vulnificus (biotype 2) from cultured beluga, Huso huso in Iran

Authors

1 Research Organization of Caspian Sea Sari

2 Islamic Azad University of Sabzevar

3 University of Shahrekord

Abstract

By decreasing of sturgeon stocks in the Caspian Sea, the culture of different sturgeon species especially Huso huso was increased in Iran. Under stress conditions sturgeon can easily be infected by several opportunistic pathogens. In June 2011, mortality happened in 25-28°C water temperature, in one of the most important cultured sturgeon farms in Mazandaran province in the north of Iran. The mortality rate was 15%. The first clinical signs in moribund fish were lethargy and anorexia. A total of 20 moribund fish was transferred to a central laboratory for more bacteriological examination. Clinical signs including several deep ulcers on body surface, around the head, under operculum and the base of the pectoral fins, hemorrhage around of the anus, operculum, necrosis of the base of the pectoral fin and paleness gill was observed in moribund fish. The main internal signs were hepatomegally and spleenomegally, liquid accumulation in the intestine and diffuse visceral hemorrhage. The results of morphology and microscopic characterizations and also biochemical tests indicated that Vibrio vulnificus (biotype 2) was the etiological agent of mortality in infected fish. This study was the first report of V. vulnificus in cultured Huso huso in Iran.

Keywords


 [Short Communication]

First isolation and identification of Vibrio vulnificus (biotype 2) from cultured sturgeon (Huso huso) in Iran

R. Safari1, M. Adel1*, M. Ghiasi1, M. R. Saeidi Asl2, E. Khalili3

 

1- Dept. of Aquatic Animal Health and Diseases, Research Organization of Caspian Sea, Sari, Iran

2- Dept. of Food Science, Azad University of Sabzevar, Sabzevar, Iran

3- Dept. of Health and Food Quality Control, Faculty of Veterinary Medicine, University of Shahrekord, Shahrekord, Iran

*Corresponding author's E- mail: miladadel65@gmail.com

(Received: Dec. 12.2014 Accepted: May. 24.2015)

ABSTRACT

By decreasing of sturgeon stocks in the Caspian Sea, the culture of different sturgeon species especially Huso huso was increased in Iran. Under stress conditions sturgeon can easily be infected by several opportunistic pathogens. In June 2011, mortality happened in 25-28°C water temperature, in one of the most important cultured sturgeon farms in Mazandaran province in the north of Iran. The mortality rate was 15%. The first clinical signs in moribund fish were lethargy and anorexia. A total of 20 moribund fish was transferred to a central laboratory for more bacteriological examination. Clinical signs including several deep ulcers on body surface, around the head, under operculum and the base of the pectoral fins, hemorrhage around of the anus, operculum, necrosis of the base of the pectoral fin and paleness gill was observed in moribund fish. The main internal signs were hepatomegally and spleenomegally, liquid accumulation in the intestine and diffuse visceral hemorrhage. The results of morphology and microscopic characterizations and also biochemical tests indicated that Vibrio vulnificus (biotype 2) was the etiological agent of mortality in infected fish. This study was the first report of V. vulnificus in cultured Huso huso in Iran.

Key words:Vibrio vulnificus, first report, Huso huso, Iran


INTRODUCTION

Iran is one of the major exporters of sturgeon caviar in the world and Persian caviar is the most famous caviar, so that the production of meat and caviar were estimated about 363000 kg and 50192 kg in 2012 respectively (Iranian Fisheries Organization, 2012). By decreasing of sturgeon stocks in the Caspian Sea, the culture of different sturgeon species especially Huso huso was increased in Iran. Between the differente sturgeon species, H. huso and their hybrids have a good growth performance and survival rates in intensive culture conditions (Bagherzadeh Lakani et al., 2013; Kolman, 2002). Under stress conditions including poor water quality and high density of fish, sturgeon can easily be infected by several opportunistic pathogen bacteria especially Streptococcus sp., Aeromonas sp., Yersinia sp., Vibrio sp. and so on (Yanong et al., 2002; Kolman, 2002). V. vulnificus is a motile, gram-negative, curved rod- shaped bacterium with a single polar flagellum. This bacterium is a naturally occurring, free-living inhabitant of estuarine and marine environments throughout the world and may associate with zooplankton and other aquatic biological flora. It is taken up by filter-feeding molluscs such as oysters, clams, mussels and scallops and becomes concentrated in the gut and other tissues. Also, V. Vulnificus is commonly found in the intestines of variety of estuarine fish species and these fish may act as a reservoir and transport this bacteria to other organisms (Storm et al., 2000). Moreover, V. vulnificus has been described as an opportunistic human pathogen (Strom et al., 2000; Fouz et al., 2002). Currently, V. vulnificus is divided into three distinct biotypes based on phenotypic and host range differences. Biotype 1 strains are typically associated with shellfish colonization and human illness.

Biotype 2 has been implicated in infections of marine vertebrates, particularly in cultured eels, tilapia and shrimp and is increasingly recognized as a serious pathogen (Storm et al., 2000; Fouz et al., 2002). Biotype 3 has been isolated from wound infections in human in Israel (Buller, 2004).

The main objective of this study was the isolation and identification of the mortality agent in cultured sturgeon (H. huso) at the north of Iran.

 

MATERIALS AND METHODS

Sampling

Sampling was done randomly from cultured sturgeon (Huso huso) in the Mazandarn province in the north of Iran. A total of 20 moribund fish was randomly sampled and were individually weighed. Samples were transported to the Caspian Sea Ecology Research Center laboratory for bacteriological examination.

 

ISOLATION OF BACTERIUM AND BACTERIOLOGICAL EXAMINATION

Fish samples (kidney, liver, spleen and body ulcers) were cultured aseptically by streaking a loop onto blood agar (Merck) containing 1.5 % NaCl and Tryptic Soya Agar (TSA, Oxoid) supplemented with 2% NaCl and incubated at 28°C for 24-48 h. Suspected bacterial colonies were identified using the conventional biochemical system.

Specifically, growth at a wide range of Nacl (3%, 5%, 8%) were conducted at 28°C on TSA. In addition the following tests were also carried out: growth on MacConkey media, oxides, nitrate reduction, motility, sensitivity to O/129, fermentation of carbohydrate, indole, citrate, bile esculine hydrolysis, urease, gelatin liquefaction, methyl red and voges-proskauer (MacFaddin, 2000).

 

ANTIMICROBIAL SENSITIVITY TEST

Isolated bacterium was tested for antibiotic susceptibility using the disk diffusion method. The most important used antibiotics were sulfamethoxazole/trimetoprim (25μg.ml -1), oxytetracycline (20μg.ml -1), chlorotetracycline (20μg.ml -1), flumequine (30μg.ml -1) and oxolinic acid (10μg.ml -1).

 

RESULTS

 Mean weight and length of samples was 10 kg + 500g and 65 cm + 5 cm respectively. Water temperature, during sampling, was between 25–28°C. During the outbreaks, many Huso huso died (15% of total fish) with symptoms such as bleeding ulcers on ventral surface, base of pectoral fins, under opercula, hemorrhage in vent, necrosis in the base of the pectoral fin (Fig. 1,2,3), paleness of gill (Fig. 4). The main internal signs were hepatomegally, spleenomegally, liquid accumulation in the intestine and diffuse visceral petachiae.

The average size of isolated colonies in TSA medium and Blood agar was 2-3 mm in diameter, and was round, raised and opaque morphologicaly (Fig. 5,6).

The biochemical tests were showed that isolated bacteria were gram negative, motile rods, oxidase and catalase, nitrate reduction and lysine decarboxylase were positive, although indole, ornitine decarboxlyse, urease, arabinose, and inositole were negative. The isolate was sensitive to the vibriostatic agent O/129. Other biochemical tests have been shown in Table 1. The results of morphology and microscopic characterizations and also biochemical tests indicated that the isolate was V. vulnificus biotype 2. Also, this isolated has been confirmed by the Central Laboratory of Veterinary Medicine Faculty of Tehran University.

The results of antibiogram test showed that the isolate was sensitive to sulfamethoxazole/trimetoprim (25μg.m -1) and Oxytetracycline (20 μg.ml -1) but resistant to chlorotetracycline (20μg.ml -1), flumequine (30μg.ml -1) and oxolinic acid (10μg.ml -1) (Table. 2).

 

 

Table 1. Biochemical characteristics of isolated Vibrio vulnificus from infected Huso huso (KEY: G: Growth; V:

Tests

Response

Tests

Response

Catalase

+

Voges-Proskauer (VP)

-

Oxidase

+

O/129

S

MacCankey

G

Glucose

A

No3 reduction

+

Adonitol

-

Motility

+

Arabinose

-

Indole

-

Inositol

-

Citrate

V

Maltose

A

Esculin hydrolysis

V

Mannitol

V

Arginine dehydrolase

-

Mannose

A

Lysine decarboxylase

+

Raffinose

-

Ornitine decarboxylase

-

Rhamnose

-

Gelatin liquefaction,22 o C

V

Sorbitol

-

Urease

-

Xylose

-

Methyl Red (MR)

+

Trehalose

A

Variable reaction; S: Sensitive; a: Acid production)

 

 

Table 2. Results of antibiogram test on Vibrio vulnificus isolate from infected Huso huso (ten isolates were tested for each antibiotic)

N No

Antibiotic

Result

11

sulfamethoxazole/trimetoprim (25μg.ml -1)

Sensitive

22

Oxytetracycline (20μg.ml -1)

Sensitive

33

chlorotetracycline (20μg.ml -1)

Resistant

44

flumequine (30μg.ml -1)

Resistant

55

oxolinic acid (10μg.ml -1)

Resistant

 

 

 

 

Fig 1. Bleeding ulcers on the ventral surface and at the the anal fin.

 

 

Fig 2. Many ulcers under the opercula.

 

 

Fig 3. Necrosis of the base of pectoral fin.

 

 

                Fig 4.  Ulcers around of the gill.

 

 

Fig 5. Colonies of V. vulnificus on TCBS agar.

 

Fig 6. Colonies of V. vulnificus on Blood agar.

 

DISCUSSION

The results of different studies showed that various bacteria such as: Streptococcus dysgalactiae., Yersinia ruckeriAeromonas hydrophila and Flavobacterium hydatis can caused serious problems in sturgeon fish (Yang & Li, 2009; Vuillaume et al., 1987;Yanong & Floyd 2002; Kolman, 2002).

The present study proved the susceptibility of Huso huso to vibriosis that caused by V. vulnificus biotype 2. Biotype 2 has been implicated in infections of marine vertebrates, particularly in cultured eels, tilapia and shrimp and recognized as a serious pathogen (Storm et al., 2000; Fouz et al., 2002; Storm et al., 2000). Moreover, this organism has been described as an opportunistic human pathogen (Strom et al., 2000; Fouz et al., 2002).

V. vulnificus is responsible for important economic losses in intensive culture of European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus) (Fouz et al., 2002; Fouz et al., 2003). Other fish species, such as sea bass (Morone saxatilis), turbot (Scophthalmus maximus), sea bream (Sparus aurata) and Rainbow trout (Oncorhynchus mykiss) was more resistant than tilapia to V. vulnificus (Fouz et al., 2002).

The high temperature is one of the most important factors in the outbreak of vibriosis. The most infections occur during the warmer months of the year (Storm et al., 2000). In this study also, vibriosis was occurred on 26 June that water temperature was 25 - 28ºC.

Several studies have demonstrated that many virulence factors including polysaccharide capsule, Lipopolysaccharide (LPS), hemolysins,

 

degerdative toxins, enzymes and iron utilization which correlated with its pathogenesis. V. vulnificus produces enzymatic compounds such as hemolysin, elastase, collagenase, lipase, phospholipase, mucinase, chondroitin sulfatase, hyaloronidase, fibrinolysin and a protease with activity against native serum albumin (Strom et al., 2000; Baffon et al., 2001). Three enzymes including elastase, collagenase and chondroitin sulfatase possess elastolytic and collagenolytic activities. They are playing an important role to make lesions in skin and cartilage tissue (Linkous, 1999; Strom et al., 2000; Baffon et al., 2001). External lesions that observed in this study may be related to mentioned above enzymes activities. The presence of a capsular polysaccharide (CPS) is directly correlated with the virulence of V. vulnificus and is essential to its ability to initiate infection. All virulent strains include both encapsulated and non-ecapsulated isolates have an opaque colonial morphology whereas non–encapsulated isolates have translucent colonized and are less virulent or avirulent (Strom et al., 2000). The capsule protects the bacterium by conferring resistance to the bactericidal effects of serum and phagocytosis by macrophages (Linkous, 1999; Baffon et al., 2001). The morphology of our isolate was opaque and confirmed with capsule staining.

 

CONCLUSION

This study was the first report of V. vulnificus in cultured Huso huso at the north of Iran. H. huso could be considered as a susceptible V. vulnificus biotype 2 in cultured sturgeon farms in the north of Iran. The absence of reports on the isolation of biotype 2 from diseased fish in Iran could be due to fact that its occurrence seldom in the aquatic environment. More studies would be necessary in farms where there have been previous experience vibriosis outbreaks and the bacterium could be present in water, either associated with particulate material or with fish in a carrier state.

 

ACKNOWLEDGEMENTS

We thank the staff of the Department of Fish Disease at the Caspian Sea Ecology Research Center for microbial examination and staff of the Central Laboratory of Veterinary Medicine Faculty of Tehran University particularly, Dr. Soltani for final confirm of isolation.

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