Study of non-halophilic Vibrio in Australian freshwater crayfish farms: distribution, epidemiology and virulence

Wong, Frank Yet Kheong. (2002). Study of non-halophilic Vibrio in Australian freshwater crayfish farms: distribution, epidemiology and virulence PhD Thesis, School of Population Health, The University of Queensland.

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Author Wong, Frank Yet Kheong.
Thesis Title Study of non-halophilic Vibrio in Australian freshwater crayfish farms: distribution, epidemiology and virulence
School, Centre or Institute School of Population Health
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Total pages 215
Language eng
Subjects 1199 Other Medical and Health Sciences
Formatted abstract
The closely related, non-halophilic species, Vibrio cholerae and V. mimicus are present autochthonously in Australian riverine and coastal environments. V. mimicus has been implicated in fatal systemic infections of freshwater crayfish, where stock mortalities associated with the bacteraemia have been independently reported for commercial crayfish farms in both the east and west coasts of Australia. The incidence and distribution of V. cholerae and V mimicus in three Australian freshwater crayfish farms were investigated in this study. One crayfish farm located in Western Australia (Farm A) and two farms in southeast Queensland (Farms B and C) were systematically surveyed for non-halophilic Vibrio over periods of six to 13 months. Both Vibrio species were found to be ubiquitous bacteria in these farms, indicating a widespread distribution in these environments. V cholerae non-O1 and V. mimicus were recovered from water in crayfish culture ponds and post-harvest purging tanks at the respective farms. V. cholerae serotype O1 was not detected in any of the farms. Vibrio cells were generally present at low concentrations in the crayfish aquaculture environments, with most samples containing less than four cells per 100 ml. However, maximum counts of up to 46 MPN V cholerae cells/100 ml in pond water, and 24 MPN V mimicus cells/100 ml in purging tank water were detected from two respective farms. A slight seasonal increase in Vibrio distribution was observed over the warmer months of the year. The effects of environmental factors such as water temperature, salinity, pH and dissolved oxygen (DO) were investigated in Farm B. Increased Vibrio incidence coincided with pond water temperatures of 21-31 °C, which lie within the optimal range for the distribution of V. cholerae in environmental waters. Moderately alkaline conditions (mean maximum pH of 8.7) and eufrophic DO levels indicating possible elevated organic nutrient concentrations, were also observed during this period. These conditions are known to promote Vibrio distribution in aquatic environments with an absent salinity gradient, as were the case in the crayfish culture ponds. Bacteria were also isolated from the haemolymph of crayfish collected from both ponds and purging tanks, with 11.6% of sampled crayfish internally contaminated with non-halophilic Vibrio cells. PFGE typing of chromosomal NotI-restriction fragments was used to examine the relationships among crayfish farm isolates. Thirty seven PFGE patterns were obtained, with 18 different patterns from 47 V. cholerae non-01 isolates and 19 patterns from 72 V mimicus isolates. Although the two Vibrio species did not share similar PFGE fingerprints, isolates of the same species with indistinguishable PFGE patterns were detected at different times separated by 1 to 15 months, and in different ponds within a farm. This indicated that certain strains were widely distributed and formed stable, persisting populations within the farm environment. Certain crayfish haemolymph isolates were also genotypically identical to isolates from pond or purging tank water, confirming interaction between the contaminated crayfish and the surrounding environment. In addition, some isolates shared closely related but distinguishable PFGE patterns. The presence of these clonally related isolates supported the long term existence of certain strains at the respective farms. In particular, V. mimicus crayfish farm isolates exhibited a high degree of clonal relatedness, with 81% of isolates genotypically related to one of five different clonal groups. There was also evidence that clonally related V. mimicus strains may either be widely distributed in southeast Queensland environments, or had previously been introduced into geographically separated Farms B and C from a common source. The potential for V cholerae non-O1 and V mimicus farm isolates to express cholera toxin (CT) or heat-stable enterotoxin (NAG-ST) were assessed by PCR screening for the ctxB and stn genes respectively. None of the Vibrio isolates possessed the CT subunit B gene, while only one (1%) V mimicus isolate was stn positive. Possession of the toxin gene did not correlate with the extent of clonal relatedness among isolates, since the stn positive isolate shared identical or closely related PFGE chromosomal genotypes with gene negative isolates. The potential for representative non-halophilic Vibrio isolates to infect apparently healthy crayfish was investigated using inoculation studies with two commercially cultured freshwater crayfish species, Cherax destructor albidus and C. quadricarinatus. V cholerae and V. mimicus farm isolates were confirmed to produce fatal systemic infection in crayfish inoculated with ca. 105 viable cells by intramuscular injection, although there were differences in the extent of virulence among isolates. In general, non-halophilic Vibrio isolates had moderate levels of infectivity for inoculated crayfish. Of the farm isolates tested, V. mimicus was shown to have greater infectivity for crayfish with a mean 50% lethal dose of 1.5X105 CFU per crayfish. Histopathology of hepatopancrea from moribund and dead crayfish due to Vibrio infection showed tissue disruption and haemocytic response typical of systemic bacteraemia. Inoculation of heat attenuated cells and culture supernatants of the stn positive V mimicus isolate provided no evidence of toxigenic effects in crayfish due to possible expression of NAG-ST. Infection by non-halophilic Vibrio cells was attributed to strains able to rapidly proliferate within contaminated crayfish. The haemolymph of 17% of crayfish were contaminated after four days co-immersion in tank microcosms with ca. 104 CFU/ml V mimicus cells. Application of non-halophihc Vibrio specific 16S rRNA gene targeted PCR primers and RNA hybridization probe for the rapid identification and detection of environmental strains of V cholerae non-01 and V mimicus was also examined in this study. A multiplex PCR incorporating both 16S rRNA and ctxB gene primers was able to specifically amplify all tested strains of non-halophilic Vibrio to the exclusion of halophilic Vibrio species and non-Vibrio bacteria, and was apphed to identity and CT screening of farm isolates. In addition, a Vibrio genus specific monoclonal antibody was applied to the detection of non-halophihc Vibrio cells from culture, and directly from haemolymph of infected crayfish.
Keyword Crayfish fisheries -- Australia
Crayfish culture -- Australia

Document type: Thesis
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Created: Wed, 27 Apr 2011, 18:27:53 EST by Ling Yan on behalf of The University of Queensland Library