Parasite Induced Vibriosis in Stolephorus commersonii

MATERIALS AND METHODS

Identification of Parasite
During the routine observation of S. commersonii in fish landings at Parangipettai (Lat. 11° 29'N; Long. 79° 46'E, India) an isopod parasite was observed on fish with an average length and weight of 11.6±0.21 cm and 9.68±0.14 g, respectively (Fig. 1). The parasite was identified as Nerocila phaiopleura Bleeker, 1857 (Crustacea; Isopoda; Cymothoidae). Incidence of isopods was maximal on the branchial and the body surface of the fish. At the site of parasitic settlement, skin lesions were observed in the infected fish.

Fig. 1:	Nerocila phaiopleura on Stolephorus commersonii

The damaged area was red coloured and devoid of scales. The work was carried out at CAS in marine Biology, Annamalai University, Parangipettai, Tamil Nadu, India in March, 2007.

Microbiological Analysis
Microbiological study was performed after removing 1 g of the injured skin from parasite infected and uninfected fishes. Tissues excised were separately homogenized with sterilized seawater and subjected for further microbiological investigations.

One milliliter of the serially diluted samples were used for the estimation of Total Heterotrophic Bacteria (THB) and Vibrio count (VC). Salmonella and Pseudomonas were also counted. THB was evaluated using Zobell marine agar, VC using thiosulphate citrate bile salts sucrose agar (TCBS), Salmonella in SS agar and Pseudomonas in centrimade agar. All mean counts were expressed as colony forming units g^-1 (cfu g^-1). Isolates were purified and stored in nutrient agar slants fewer than 4°C. Bacterial strains isolated from the aforementioned media were identified according to Bergey’s manual of determinative bacteriology (Buchanan and Gibbons, 1974) based on their biochemical and physiological characteristics.

RESULTS AND DISCUSSION

In the parasitized fishes, THB, Vibrio, Salmonella and Pseudomonas loads were higher than the uninfected fishes (Table 1).

In the branchial region of the parasitized fish, THB was found to be 7.2x10⁵ cfu g^-1 whereas in healthy fish it was 1.2x10³ cfu g^-1 and the Vibrio loads were 3.5x10³ and 1.2x10¹ cfu g^-1, respectively. Salmonella and Pseudomonas counts rose to 6.0x10³ and 7.0x10² cfu g^-1 in parasitized fish whereas they were 3.6x10¹ and 4.9x10¹ cfu g^-1, respectively in uninfected fishes.

The parasitized areas of the body surface of fish showed a THB value of 5.4x10⁵ cfu g^-1 whereas in uninfected specimens it was only 1.5x10³ cfu g^-1. The VC from the body surface was 2.3x10³ and 2.1x10² cfu g^-1 in infected and uninfected fish. The Salmonella counts were of 3.9x10³ and 1.03x10² cfu g^-1, for Pseudomonas 6.2x10² and 5.8x10¹ cfu g^-1, in parasite infected and uninfected fish, respectively.

The Vibrio isolates were comprised of V. angullarium, V. parahaemolyticus, V. cholerae and V. vulnificus. The two dominant species were V. angullarium and V. parahaemolyticus. Salmonella and Pseudomonas isolates showed only S. typhi and P. aeruginosa.

A number of contaminations were reported resulting from eating Vibrio contaminated raw seafoods (Sanjeev and Stephen, 1994). The major contaminants are V. parahaemolyticus, V. cholerae, V. vulnificus, V. fluvialis and V. alginolyticus. The presence of human enteric organisms on marine food products clearly indicates the contamination of terrigenous origin.

Table 1:	Total heterotrophic bacteria and Vibrio counts on infected and uninfected Stolephorus commersonii
cfu g-1: Colony forming units/gram

Ulcerations or external lesions in fish might have been caused by a number of factors other than bacterial infection induced by parasitic attack (Sindermann, 1979), such as polluted environment and other adverse influences. However, parasites also contribute significantly to the spread of microbial diseases. Parasitic crustaceans are the largest fish parasites, which cause considerable damages to their hosts. They were involved in the spread of Lymphocystis disease (Nigrelli, 1950), Icthyophorrus contamination (Mann, 1970) and other secondary microbial infections. Overstreet and Howse (1977) proposed that haemorrhagic lesions in the spotted gore parasitized by the cymothoid Anilocra acuta, were subjected to secondary infection. In the present study, cymothoid N. phaiopleura parasitizing S. commersonii paved the way for pathogenic germs.

The bacterial load involved in the microbial infection depends on the site infected. In the present study, a regional difference for bacterial proliferation was observed: THB, VC, Salmonella and Pseudomonas counts were higher in the host’s branchial region than in the body surface in both infected and uninfected fish. This may be due to the direct exposure of gills to the outer environment. The higher THB, VC, Salmonella and Pseudomonas counts in the branchial region could be attributable to the severity of the lesions at that level, as reported in the Creole fish parasitized by N. acuminata (Rand, 1986). Also, the recorded frequent contamination of this area may be due to the respiratory water current that carries bacteria along with food materials and could facilitate bacterial invasion. Besides, the increased level of bacterial population may also lead to further infections.

Pathogenic microbes and parasites could damage the physiological and reproductive activities of the host fish (Ranjit Singh and Padmalatha, 1997; Vismanis and Kondratovics, 1997). The bacterial invasion in the branchial region reduces the respiratory area injuring the gill lamellae and affects respiration as well as excretion of nitrogenous materials and disturbs the osmotic balance (Rand, 1986; Meenashi, 1997; Ravichandran et al., 2001). Lesions induced by parasitic attack become the sites for secondary infections by opportunistic bacterial and fungal pathogens that combined with the parasitosis impacts, which cause deleterious effects on the physiology of the animal and leads to death.

The attempt of the present study is to achieve an approach of the magnitude of such a secondary infection counting the Vibrio, Salmonella and Pseudomonas loads as well as the total heterotrophic bacteria using a battery of biochemical tests. THB, VC, Salmonella and Pseudomonas counts were higher in the infected area of fish, vulnerable to attack by the microorganisms. The two dominant isolated Vibrio species are V. angullarium and V. parahaemolyticus. V. angullarium has been earlier reported as a potential fish pathogen by different authors (Kusuda, 1966; Hacking and Budd, 1971; Parker and Smith, 1984; Singh et al., 1996). Vibriosis in fish caused by V. angullarium is known to affect a wide variety of fish species from brackish water to seawater (Loganathan, 1985). Lightner (1977) reported that V. parahaemolyticus is among the causative organisms of Vibriosis in prawn and also associated with outer secondary infections. This species was also reported from cultured marine fishes (Sindermann, 1970; Raghukumar, 1971; Ravichandran et al., 2001). V. parahaemolyticus has not been specifically identified as a pathogen of marine fish although Kusuda (1966) has reported Vibrios causing ulcerous diseases in cultured marine fish with close similarity to intestinal inflammation in man caused by V. parahaemolyticus. According to the above results, V. parahaemolyticus may be considered as a significant pathogen involved in secondary infection. Harrell et al. (1976) demonstrated that isolates of V. anguillarium act as the most important aetiological agent of vibriosis. In the case of uninfected fish, THB and Vibrio counts were very low. This may be due to the healthiness of the fish, which will interfere with the contact between the microbe and fish, as a result of continuous secretion of mucus from the skin of the fish.

Salmonella has been reported in the gut of Tilapia and carps (Iyer and Shrivastava, 1989; Ogbondeminu, 1993) and as a fish pathogen in salmon (Austin et al., 1982) as reposted in the present study. Occurrence of Pseudomonas in the infected fish was witnessed by Eddy and Jones (2002), Simidu and Tsukamoto (1985) and Salmonella by Karunasagar et al. (2004). Pseudomonas exists throughout the aquatic environment associated with both healthy (Evelyn and McDermott, 1961; Bullock and Sniesko, 1969) and unhealthy fish. Presence of Klebsiella pneumoniae in a badly damaged fin and tail has been recorded by Austin and Austin (1999). P. anguilliseptica and P. fluorescens were reported as a causative agent of red spot and generalized septicaemia in rainbow trout and most of the marine species (Austin and Austin, 1999). Statistical analysis of the results obtained from the present study showed a high significance of p<0.05.

ACKNOWLEDGMENTS

The authors are thankful to the authorities of Annamalai University for the facilities provided and to the Indian Council of Agricultural Research for the financial support.