VIBRIOSIS IN FARM REARED WHITE SHRIMP, LITOPENAEUS VANNAMEI IN ANDHRA PRADESH-NATURAL OCCURRENCE AND ARTIFICIAL CHALLENGE

In the present study, a total of five species of Vibrio bacteria were isolated from diseased shrimp, Litopenaeus vannamei, collected from commercial shrimp cultured ponds of Eethamukkala, Chinaganjam and Pedaganjam areas, Prakasam district, Andhra Pradesh. The isolated bacterial species were identified as Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio mimicus and Vibrio vulnificus. The symptoms shown by diseased shrimps include loss of appetite, red coloration of the body and pleopods, gills often appear red to brown in colour, reduced feeding, empty gut and general septicemia. In diseased shrimp, hepatopancrocytes may appear poorly vacuolated, indicating low lipid and glycogen reserve. In affected shrimps, localized lesions were also observed in the cuticle. Experimental infection trials reveals that V. parahaemolyticus is highly pathogenic to L. vannamei while V. harveyi found to be moderate pathogenic to challenged shrimp and remaining three bacterial species namely V.alginolyticus, V.mimicus and V.vulnificus were less pathogenic in nature.


Introduction
The intensification of the shrimp culture and the transfer of aquatic organisms worldwide have been accompanied over the last twenty years by an increased incidence of microbial infectious pathogens. In this regard, bacterial diseases due to Vibrio species are often associated with low survival rates in hatchery or grow out conditions (Denis Saulnier et al., 2000). Larval mortalities associated with the presence of V. harveyi have been reported in P. monodon and P. vannamei in Indonesia (Sunaryanto and Mariam, 1986), Thailand (Jiravanichpaisal et al., 1994), India (Karunasagar et al., 1994), Philippines , Australia (Pizzutto and Hirst, 1995), Taiwan (Song and Lee, 1993;Liu et al., 1996) and Ecuador (Robertson et al., 1998). Disease outbreaks attributed to other Vibrio species such as V. alginolyticus, V. damsela, V. parahaemolyticus, V. vulnificus and V. penaeicida have been observed in nursery or grow out ponds of P. vannamei, P. monodon, P. japonicus and P. stylirostris in Ecuador (Lightner, 1992) Malaysia (Anderson et al., 1988) Taiwan (Song et al., 1993Lee et al., 1996), Philippines (Alapide-Tendencia and Dureza, 1997), Japan (de la Pena et al., 1993) andNew Caledonia (Costa et al., 1998).
Vibriosis is a bacterial infection responsible for mortality of commercial shrimp culture system worldwide (Lightner and Lewis, 1975;Overstreet, 1978;Sidermann, 1990;Lightner et al., 1992;Lavilla-Pitogo et al., 1996;Lavilla-Pitogo et al., 1998;Chen et al., 2000). Vibrio species are widely distributed in aquaculture facilitates throughout the world. Vibrio-related infections also occur in hatcheries, but epizootics also commonly occur in pond reared shrimp species. This disease is caused by gram-negative bacteria of the family Vibrionaceae. Outbreaks may occur when environmental factors trigger the rapid multiplication of bacteria already tolerated at low levels within shrimp body (Sizemore and Davis, 1985) or by bacterial penetration of host barriers. Vibrio spp., are among the chitinoclastic bacteria associated with shell disease (Cook and Lofton 1973) and may enter through wounds in the exoskeleton or pores (Jiravanichpaisal and Miyazaki, 1994). The gills may appear susceptible to bacterial penetration because they are covered by a thin exoskeleton (Taylor and Taylor, 1992), but their surfaces are cleaned by the seto-branchs (Bauer, 1998). The mid gut, composed of the digestive gland (DG) and the mid gut trunk (MGT, often referred to as the intestine, (Lovett and Felder, 1990), is not lined by an exoskeleton and therefore seems to be a likely site for penetration of pathogens carried in the water, food and sediment (Ruby et al., 1980;Denis Saulnier et al., 2000 andJayasree et al., 2006). The present paper communicates incidences of Vibriosis in farm reared shrimp, L. vannamei Research Article in Andhra Pradesh-Natural occurrence and artificial challenges.

Collection of Diseased Shrimp Samples
In the present study, a total of 250 diseased alive shrimp (weight between 16-18 gm) samples were collected from commercial cultured ponds of Eethamukkala, Chinaganjam and Pedaganjam areas of Prakasam district, Andhra Pradesh. The diseased shrimp samples were brought to laboratory under sterilized conditions. Affected shrimps were observed for gross symptoms by keeping them in glass aquaria. Morphological and behavioral symptoms of affected shrimps were recorded. For the isolation of bacteria from affected shrimps, standard methods described by Lightner (1995) were fallowed. Haemolymph drawn from affected shrimp and plated onto Trytone Soy Agar (TSA) and Thiosulphate Citrate Bile Sucrose (TCBS). Samples were taken aseptically from different tissues such as hepatopancreas, intestine and haemolymph and inoculated onto the surface of TSA and TCBS agar plates. Inoculated plates were incubated at 30±2ºC for 24h. Bacterial colonies were observed in incubated plated from 24 to 96 h. The purification of bacteria was done by subsequent culturing of bacterial cultures. Identification and characterization of bacteria was done on the basis of their biochemical tests as per the methods of Buchanan and Gibbons (1974). Biochemical tests such as Gram's staining, Catalase, Oxidase, MR-VP test, Urase, Oxidative/Fermentative test, and Citrate utilization tests were carried out in the laboratory condition.

Artificial Infection Trials
In order to confirm the pathogenicity of isolated bacterial species from diseased shrimp and to verify the Koch's Postulates, pathogenicity experiments have been conducted in the laboratory condition. For this purpose a total of 500 alive healthy, diseased free shrimp were procured from local shrimp farm (Average weigh 8-10 gm and Length 5-8cm) used in this study and were acclimatized in the laboratory condition for one week. In each group 20 animal were used and kept in glass aquaria and filled with 20 litre of freshwater. The isolated bacterial species were cultured in TS broth and purified. The purified cultures were used to prepare the bacterial cell suspension in 0.85% saline solution to get appropriate cell number in the suspension. Then the suspension contains cell number from 10 4 -10 6 cfu/shrimp were injected intramuscularly (IM) to the challenged shrimp. Each experiment was conducted in triplicates.

Bacterial Count
Strains were grown for 24h in TS broth to count the colony forming units (CFU). Bacteria were centrifuged at 10,000g during 20min at room temperature and the cellular pellet was washed two times with sterile saline water (0.85% NaCl) and resuspended in 1mL of the same water. The bacterial suspension was then adjusted to an optical density of one in a Thermo Spectronic Genesys 2 Spectrophotometer at 580nm. To determine the CFU/mL of bacterial suspension, serial dilution method was adopted.

Results and Discussion
In this study, five species of bacteria were isolated from diseased shrimps of different cultured ponds of L. vannamei in Prakasam district. Andhra Pradesh. The diseased animals showed signs like localized cuticular lesions, red coloration of the body and pleopods, reduced feeding, empty gut. In some cases, red colour animals appeared in the corners of ponds. Similar symptoms were also reported by number of workers (Lightner, 1995;Jayasree et al., 2006;Denis Saulneir et al., 2000).The isolated bacteria were identified on the basis morphological and biochemical characters. The isolated bacteria were identified as Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio mimicus and Vibrio vulnificus. Among the five bacterial species V. parahaemolyticus and V. harveyi has dominated in all diseased shrimp samples. The morphological and biochemical characters were given in Table-1. All the species of bacteria isolated in the present study are gram native, rod shaped and fermentative bacteria. On agar plates V. parahaemolyticus cultures appear as smooth, motile, circular, opaque colonies with entire margins. It showed oxidative positive while catalase negative. While V. alginolyticus, V.fluvialis and V. mimicus showed oxidative, catalase positive and fermentative bacteria. By virtue of biochemical test the isolated bacteria were identified as V. parahaemolyticus, V. alginolyticus, V. fluvialis and V. mimicus. The same characters were described by Buchanan and Gibbons (1974). Vibrio species are part of the natural microflora of wild and cultured shrimps (Sinderman, 1990) and become opportunistic pathogens when natural defence mechanisms are suppressed (Brock and Lightner, 1990). They are usually associated with multiple etiological agents. However, some of species of Vibrio have been identified as primary pathogens (Owens and Hall-Mendelin, 1989;Owens et al, 1992;Lavilla-Pitogo et al., 1990;de la Peñaa et al., 1995). Some of the pathogens like V. parahaemolyticus, V. harveyi, and V. vulnificus have causes serious disease problems in Thailand (Nash et al., 1992) and the Philippines . In the present study; it has been observed that same species of Vibrio bacteria have associated with diseased shrimp. Harris (1995) reported that luminescent V. harveyi appears to release exotoxins (Liu et al., 1996) and may cause 80-100% mortality in P. monodon hatcheries. Species like V. anguillarum, V. campbelli, V. nereis, V. cholerae and V. splendidus have also been reported their association with disease outbreaks in shrimp culture systems by various workers in India and abroad (Chen, 1992;Lavilla-Pitoga, 1990;Esteve and Quijada, 1993;Sahul-Hameed et al., 1996).  In this study, an experimental infection trial indicates that all the bacteria isolated from diseased shrimps are pathogenic in nature. V. parahaemolyticus is highly pathogenic and it produced disease symptoms within 24 h after injection. While V. harveyi is moderately pathogenic to challenged animals. In most cases, a high inoculum was needed to reproduce the disease and to reisolate the inoculated bacteria from the experimentally infected shrimp (Lightner, 1988). However, pathogenic Vibrio isolates have also been detected in apparently healthy shrimp (Nakai et al., 1997;Vandenberghe et al., 1998) and in seawater samples from near-shore and estuary areas, where shrimp farms rearing water is pumped and from affected farms, (Lightner, 1992;Lavilla-Pitogo et al., 1990, 1998Moriarty, 1998), as well as in sediment (de la Pena et al., 1992). These observations lead researchers to consider Vibrio diseases as secondary infections due to opportunistic pathogens and occurring only in immunologically compromised shrimps. Primary causes could encompass other infectious agents, nutritional deficiencies or intoxication, environmental and management practices and induced stress.

Conclusion
In the present study, five species of bacterial viz., Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio mimicus and Vibrio vulnificus were isolated from Vibriosis affected diseased shrimp. All the species of bacteria were pathogenic in nature. Among the five species of bacteria, Vibrio parahaemolyticus was highly pathogenic in nature while V. harveyi was moderately pathogenic to challenged shrimps.