Neumann_whole_thesis.pdf (2.5 MB)
Molecular detection of pathogens in southern bluefin tuna
thesisposted on 2023-05-27, 09:53 authored by Neumann, L
Within the Australian fish production industry the ranching of Southern bluefin tuna (Thunnus maccoyii) (SBT) continues to be one of the most profitable. As culture practices become more efficient, the requirement for efficient methods of pathogen detection and quantification are also of increasing importance. In an effort to better understand the impact of ranching on pathogen prevalence, as well as provide tools for rapid detection, this research aimed to elucidate variability in pathogen prevalence in wild versus ranched SBT through the development of quantitative PCR methods and application of 16S rRNA amplicon sequencing. The first three research chapters focus on the detection of blood flukes (Digena: Aporocotylidae) from genus Cardicola, specifically C. forsteri, C. orientalis and C. opisthorchis; including development and application of a qPCR assay to quantify each species from organ and environmental samples. The final research chapter details the bacterial diversity in SBT spleen using universal bacterial 16S rRNA primers to amplify the V1-3 region and amplicon pyrosequencing. The 2013 SBT ranching season marked the introduction of the anthelmintic called praziquantel (PZQ), which has also been used to mitigate C. orientalis and C. opisthorchis infections in juvenile Pacific bluefin tuna in Japanese culture systems. Both wild and ranched SBT (at harvest) were sampled annually for three consecutive years starting in July 2013. A hydrolysis probe-based qPCR assay was designed and validated to detect and quantify C. forsteri, C. orientalis, and C. opisthorchis ITS2 rDNA. The assay was applied to quantify C. forsteri and C. orientalis DNA in heart and gill, which documented a significant annual variability of C. forsteri infection in ranched SBT hearts. No C. orientalis was detected in 2014 and 2015 ranched SBT, whereas C. forsteri was found in at least 95% of fish examined in all three years. This was an important yet unexpected finding given C. orientalis was significantly more prevalent than C. forsteri in 2011/12 harvests. Lastly, this chapter reports the first detection of C. orientalis in wild SBT. The newly developed hydrolysis probe-based qPCR assay was utilized to examine the prevalence of C. forsteri and C. orientalis ITS2 rDNA in 1995 and 2004 archival formalin-fixed paraffin embedded (FFPE) SBT heart samples. Prior to the examination of archival samples, five FFPE DNA extraction methods were compared, consisting of with and without xylene deparaffinization, application of 1 h 90¬¨‚àûC incubation post proteinase K incubation, a QIAamp DNA mini kit¬¨vÜ (Qiagen) and a TRIzol reagent based protocol. In addition, extended proteinase K incubation at 37¬¨‚àûC, 72 h instead of 24 h, was examined and resulted in a significant increase in C. forsteri ITS2 rDNA. The inclusion of a 90¬¨‚àûC h sample incubation step post proteinase K incubation resulted in a 100-fold increase in DNA yield compared to without and was significantly higher than when using a Qiagen QIAamp FFPE kit. This retrospective examination documented the presence of C. orientalis in 1995 FFPE SBT heart, preceding previous published presence in SBT in 2008 samples. C. forsteri and C. orientalis ITS2 rDNA was detected in sea water samples from sea cages alongside commercial harvest and control locations north and south of the cages. Real-time qPCR methods using SYBR green nucleic acid dye was used in the first year, and the hydrolysis probe-based qPCR assay in the second two years. Of three consecutive sampling years, C. forsteri and C. orientalis ITS2 rDNA was only detected in the first, despite qPCR examination of SBT organs documenting the presence of both blood fluke species in the all the years examined. Samples from the control North location (3.5 km North of cages containing SBT) had significantly lower levels of C. forsteri and C. orientalis ITS2 rDNA in the water compared to cages and control South location (2 km South of cages containing SBT). Significantly more C. orientalis ITS2 rDNA (M = 4885.08, SD = 7328.71) was detected compared to C. forsteri (M = 232.67, SD = 337.82) in samples collected on 29th and 30th of August, 2012. Culture independent PCR was applied to spleen of wild (n = 10) and ranched (n = 10) SBT as part of sample preparation for an evaluation of bacterial diversity using 16S rDNA tag-encoded pyrosequencing. The most abundant phylum among wild and ranched fish was Proteobacteria (94.41% ¬¨¬±0.041) followed by Acidobacteria (4.71% ¬¨¬±0.035), Cyanobacteria (0.49% ¬¨¬±0.007), and Firmicutes (0.24% ¬¨¬±0.005). Both Acidobacteria and Cyanobacteria were significantly more abundant in ranched compared to wild SBT (p< 0.05) spleen. Bacteroidetes was significantly more abundant in wild SBT (p < 0.0001) and it was not detected in samples from ranched SBT. The top 18 most abundant genera constituted 99.2% of the spleen microbiota. These included Bosea (66.99%), Phyllobacterium (22.73%), Edaphobacter (2.74%), Methylobacterium (2.03%), Propionibacterium (2%), Bradyrhyzobium 0.69%), Ochrobactrum 0.52%), Mesorhizobium (0.31%), Pseudoalteromonas (0.21%), Corynebacterium (0.19%), Acinetobacter (0.19%), Chelatococcus (0.14%), Burkholderia (0.1%), Staphylococcus (0.1%), Psychrobacter (0.08%), Yersinia (0.06%), Enterobacter (0.06%), and Pseudomonas (0.05%). Although Edaphobacter and Pseudoalteromonas were present in both groups, they were significantly more abundant in ranched SBT spleen (p < 0.05). On average the SBT spleen microbiota had 148.9 (¬¨¬±35.4) operational taxonomic units (OTUs) (min = 109; max = 252) at 97% sequence similarity. No significant difference in alpha diversity indices was found. PCoA plot based on weighted UniFrac distance as a measure of beta diversity showed no visual difference between wild and ranched SBT spleen in the community structure. No distinct difference in bacterial communities between wild and ranched fish may suggest that the documented diversity described in this thesis represents the baseline for SBT. The molecular detection methods described and applied in this thesis as well as the insight to bacterial diversity in SBT spleen have given a unique understanding of the presence of pathogens and will play a significant role in the sustainable ranching and management of this critically endangered bluefin tuna species.
Rights statementCopyright 2017 the author Chapter 2 appears to be the equivalent of a pre-print version of an article published as: Neumann, L., Bridle, A., Leef, M., Nowak, B., 2018. Annual variability of infection with Cardicola forsteri and Cardicola orientalis in ranched and wild southern bluefin tuna (Thunnus maccoyii), Aquaculture, 487, 1-6