Investigation of tail fan necrosis of live-held Southern Rock Lobsters

Abstract

Tail fan necrosis (TFN) is a disease that affects southern rock lobsters during live-holding. The damage to affected tail fan uropods is seen as a major constraint in the development of a live-holding industry. A previous study has demonstrated that various Vibrio species are associated with diseased tissue (May, 2002. B.Sc. Honours Thesis, University of Adelaide). However, that study was restricted to an examination of TFN lesions that formed 8 weeks post infection for lobsters held under optimal growth conditions. Although damage to tail fan tissue by instruments contaminated by organisms isolated from TFN affected tissue was shown to result in formation of TFN-like lesions, the microbial community of lesions associated with TFN over time in terms of both the cultivable and non-cultivable communities was not identified. The extent of damage to tail fan tissue by bacteria and the response of lobster immune cells to infection was also not determined. Furthermore, the presence of potentially pathogenic Vibrio spp. within the diseased tissue was identified as a potential public health risk, particularly in food preparation facilities where live lobsters are handled. The work described in this thesis specifically examined the development and effect of TFN on the overall health of affected lobsters, as well a confirmation that the Vibrio spp. involved in establishment of TFN may represent a public health risk. To answer these questions, a larger infection trial was set up. Uropod tissue of groups of lobsters were intentionally damaged with sterile instruments or instruments contaminated with a Vibrio spp. isolated from a TFN lesion. The lobsters were maintained in controlled environment aquaria and uropod tissue samples taken and subjected to microbiological, microscopic and molecular analysis. Microscopic analysis of developing lesions demonstrated that several morphologically different bacterial cell types colonise the surface of TFN lesions. Bacteria involved in infection are essentially restricted to the surface of the lesions, but where significant damage to the uropod tissue occurs, these bacteria may invade the damaged tissue and penetrate deeper underlying tissue. Infection of tail fan tissue results in inflammation and concomitant loss of internal structure of the carapace and deposition of fibrous material within the soft tissue underlying the chitinous exoskeleton. In cases of severe inflammation, a central core develops within the fibrous tissue consisting of a number of cell types, including hyaline cells, granulocytes and fibrocytes. However, there was no evidence of deep bacterial invasion into the underlying inflamed tissue. Viable counts and identification of the bacteria associated with the diseased tissue demonstrated that the bacterial population of TFN lesions is dominated by Vibrio species. Whilst there was no significant increase (P < 0.05) in the total viable bacterial counts associated with the diseased tissue compared with healthy tail fan tissue, Vibrio species were isolated more frequently from tissue samples from uropods subjected to simultaneous damage and infection. 8% of bacterial isolates recovered from lesions were identified as V. vulnificus and 27% of isolates were identified as V. parahemolyticus. Isolates of V. vulnificus displayed colony morphology consistent with pathogenic strains. Similarly, all isolates of V. parahaemolyticus were tdh negative, but 41% were trh positive. The majority of these species were able to express cytolysins capable of lysing CHO cells. This data indicated that vibrios responsible for establishment of TFN may have potential to cause human infections and therefore lobsters with TFN lesions should be regarded as a potential health risk to consumers. The majority of isolates of Vibrio spp. recovered from infected tissue expressed extracellular lipase and/or chitinase, and this indicated that these enzymes may enable Vibrio spp. to induce TFN in damaged tail fan tissue. The predominance of Vibrio spp. associated with lesions was confirmed by analysis of amplicons representative of genes encoding 16S rRNA prepared from lesion tissue DNA extracts. This was achieved by sequencing randomly selected clones of amplicons and by use of Denaturing Gradient Gel Electrophoresis to separate amplicons according to nucleotide sequence diversity. Unlike other crustacean shell diseases, TFN does not induce changes in serum protein levels, lead to significant bacteraemia or changes in the circulating haemocyte population. Furthermore, TFN has apparently little effect on the overall health of affected lobsters. This observation may explain the lack of mortality associated with this disease. Only a non-specific activation of lobster phenoloxidase in response to TFN was observed and only limited activation of phagocytosis of Vibrio spp. in vitro could be demonstrated. This data suggested that the lobster immune system is unable to respond to infection and may explain why the bacteria are able to induce persistent infection resulting in formation of TFN lesions. Nevertheless, localised melanisation surrounding the wound site induced by carapace degradation products is able to restrict bacterial invasion into the haemolymph. The impact of TFN on appearance and consumer acceptance is dependent on the extent of damage caused by TFN. Minor lesions are resolved during moulting, whereas more severe lesions are maintained across more than one moult cycle.