Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans. Despite their importance, their modes of virulence have yet to be fully elucidated. Here, we present a previously unreported bacteriophage extracted from a toxin‐producing strain of V. harveyi isolated from moribund prawn larvae in tropical Australia. Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double‐stranded linear DNA). We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like). VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V. harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested). The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed.

Aims: To determine the complete nucleotide sequence of the bacteriophage VHML and establish a hypothesis for the virulence conversion caused by VHML infection of Vibrio harveyi. Methods and Results: The complete nucleotide sequence of VHML was determined (43 193 bp) and used to identify putative genes. The translated products of these genes were compared with reported sequences to assign hypothetical functions. All anticipated structural genes and putative genes for lysogeny were identified. In addition, we found a complete N6-adenine methyltransferase (Dam) gene that appeared to have an essential site for ADPribosylating toxins at the C-terminal of the translated product. Conclusions: Virulence conversion of V. harveyi by VHML may be associated with Dam transcriptional regulation. The Dam gene may also encode for a toxin component similar to ADP-ribosylating toxins. Significance and Impact of Study: This manuscript lays the foundation for understanding the virulence of toxin-producing V. harveyi. Further research into aspects discussed here will lead to a greater comprehension regarding the invertebrate disease vibriosis and its control in the farming of these animals.

Experimental infections indicate that Bohle iridovirus, a Ranavirus, is pathogenic for barramundi Lates calcarifer Bloch. Mortalities after bath-exposure or inoculation of barramundi in both freshwater a n d seawater were 100 % Symptoms of inoculated barramundi held in seawater and freshwater and bath-exposed barramundi in freshwater included cessation of feeding, shivering, loss of muscle coordination, spiral and often erratic swimming and decreased ventilation. Just pnor to death, the fish were incapable of any movement. In bath-exposed barramundi held in seawater, no symptoms were observed before death The infection was characterised by focal to diffuse necrosis of the haematopoletlc tissue of the kidney and spleen. In several BIV-infected barramundi, focal necrosis occurred in the llver. In bath-exposed and ~noculated barramundi, BIV was isolated on BF2 cell monolayers from muscle, llver, kidney and spleen tlssues, with recovery rates of 72.5, 58.5, 17.7 and 17.7 'Yo respectively. This is the first time that a virus isolated from a frog has been shown to cause mortalities in a flsh specles.

Studies on lymphoid organ spheroid (LOS) cells of Penaeus rnonodon were undertaken. Phenoloxidase and peroxidase assays showed that LOS cells have characteristics simllar to semi-granular and, in particular, large granular haemocytes. The mean percentage of LOS cells positive for phenoloxidase and peroxidase was 85 + 23 and 82 * 23%, respectively. There was no significant difference between the sites of phenoloxidase and peroxidase activity in LOS cells ( t = 1.617, df = 29, p > 0.05). The relative sectional area occupied by LOS cells relative to that of the strornal matrix cells from both laboratory-held and farmed prawns was not correlated to increasing weight or total length of the prawns (p > 0.05). An apoptosis detection assay showed that LOS cells were often apoptotic whilst stromal matrix cells were not. There was a significant difference (t = -5.533, df = 58, p < 0.05) in the mean percentage of apoptotic spheroid cells between laboratory-held prawns (52 * 24 % ) and farmed prawns with rnidcrop mortality syndrome (MCMS) (80 i 12%). In conclusion, LOS cells have the chdracteristics of exocytosed, granular haemocytes that have phagocytosed foreign material, particularly viruses, and probably constitute a major mechanism for penaeid antiviral defense.

Vibriosis is a major disease problem in prawn aquaculture. Until now there has been no clear explanation why some strains of Vibrio are pathogenic, while others are not. This study demon-

Streptococcus iniae, a bacterial infection in barramundi La tes calcarifer'~e p a r t m e n t of Microbiology and Immunology, James Cook University, Townsville, Queensland 4811, Australia '~e p a r t m e n t of Primary Industries, Oonoonba, Townsville, Queensland 4811, Australia ABSTRACT: The cause of ongoing mortality in barramundi Lates calcanfer (Bloch) in seawater culture was identified as Streptococcus iniae by biochemical and physiological tests. This is the first published record of this bacterial species in Australia and the first confirmed report of S. iniae causing mortality in barramundi. The bacterium was highly pathogenic for barramundi when challenged by bath exposure. The pathogen was found to have a LDS, of 2.5 X 105 and 3.2 X 104 colony-forming units at 48 h and 10 d respectively. Experimental challenge of barramundi resulted in high levels of mortality (>40%) within a 48 h period. Ten days after the challenge, S. iniae could not be isolated from kidney, spleen, liver or eye of surviving fish. However, the organism was easily isolated from the brain of both moribund and healthy fish, indicating that barramundi can carry the bacterium asymptomatically.

The use of various challenge techniques has allowed the formation of a hypothesis for the mode of infection of Streptococcus iniae in barramundi. A bacterial dose of 1 × 10 3 colony forming units (cfu), corresponding to the LD 50 , delivered orally to barramundi could initiate the sub-acute form of the disease observed at the farms. The acute form of the disease could be initiated through bath exposure to the pathogen. S. iniae was equally as infective in freshwater, saltwater or when fish were subject to skin trauma prior to exposure, with LD 50 values of 3.2 × 10 4 , 2.0 × 10 4 , 3.2 × 10 4 cfu, respectively, when observed over a 10 d period. It is suggested that sub-acute infection occurs orally, with mass mortalities occurring through the increased presence of the bacterium in the environment. KEY WORDS: Barramundi · Streptococcus iniae · Route of infectionResale or republication not permitted without written consent of the publisher Dis Aquat Org 52: 199-205, 2002 bacterial exposure by immersion. Taniguchi (1982) suggested that bacterial infection in cultured yellowtail Seriola quinquinradiata was occurring orally, through being fed infected trash fish. More recently, Evans et al. (2000) suggested that nares infection might be important to the establishment of the disease in striped bass Morone saxtilis.These studies were all dependent on having high levels of Streptococcus iniae present in the environment for infection to take place. During preliminary research to determine the level of culturable S. iniae in the marine environment, it was found that the bacterium rarely reached numbers sufficient to become pathogenic to barramundi (Bromage 1997). This finding suggested that another mode of infection might be important. While barramundi culture relies on processed feeds, we hypothesized that the fish were becoming infected orally through the consumption of infected carrier fish or through the faecal-oral route. This study was devised to test the possible routes of entry of S. iniae into susceptible hosts in the marine environment, and to examine the potential of S. iniae as a pathogen of freshwater cultured barramundi. MATERIALS AND METHODSFish selection and maintenance. Barramundi of 100 ± 5 mm total length were obtained from a farm located outside the endemic area of the pathogen. All experimental trials were performed in 170 l glass aquaria. Each aquarium was filled with seawater (35 parts per thousand, unless otherwise stated) and heated to 27°C.Preparation of bacterial inoculum. All studies were conducted with isolate number 28-97-L-K, isolated in a previous study and known to be pathogenic to barramundi (Bromage et al. 1999). The bacterial culture was grown for 24 h in brain-heart infusion broth (BHI, Oxoid) with continual shaking to provide bacteria in the exponential growth phase. The culture was pelleted by centrifugation (4000 × g) and washed 3 times in phosphate buffered saline (PBS) before being resuspended in PBS until an optical density of 1.0 was obtained at A 600 (absorbance ...

Aims: There is growing awareness of the influence of the bacterial composition of the gut on the health and growth of the host. This study compared the bacterial flora from the digestive system of the wild and cultured prawn, Penaeus merguiensis. Methods and Results: Whole guts were dissected from wild and cultured prawns and divided into sections corresponding to the foregut, digestive gland, midgut and hindgut. Homogenates of these sections were plated onto seawater nutrient agar and the colonies identified to genus level and, in some cases, species. Quantitative and qualitative comparisons amongst gut regions for both wild and cultured prawns are presented. Conclusions: Both wild and cultured prawns supported remarkably similar bacterial floral compositions, which included members from the genera Aeromonas, Plesiomonas, Photobacterium, Pseudoalteromonas, Pseudomonas and Vibrio. Members of the genus Vibrio were quantitatively dominant. A number of Vibrio species were recovered solely from cultured prawns. Of these, Vibrio gazogenes was the most notable (numerically dominating in all but the midgut). The opportunistic pathogen V. parahaemolyticus was also recovered. Significance and Impact of the Study: The remarkable similarity of gut compositions between wild and cultured prawns, despite being drawn from very different habitats, suggests an influence of the host on the establishment of the gut flora. An understanding of host/gut floral interactions has significance in fostering conditions which promote the growth of cultivated hosts.