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Development of a high pressure processing inactivation model for hepatitis A virus and its application in shellfish processing

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Grove, Stephen Francis (2008) Development of a high pressure processing inactivation model for hepatitis A virus and its application in shellfish processing. PhD thesis, University of Tasmania.

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Abstract

Hepatitis A virus (HAV) has been responsible for many large outbreaks of illness
throughout the world, often resulting from consuming raw or minimally cooked
filter-feeding shellfish contaminated with human faecal effluent.
High pressure processing (HPP) is an alternative food preservation technique to
heat, preserving the flavour, appearance and nutritional value of high quality
foods, including oysters, often with extended shelf life. In this study, the
effectiveness of HPP in inactivating HAV was assessed.
HAV, suspended in buffered tissue culture media containing either 15 parts per
thousand (ppt) or 30 ppt salt (NaCl), was treated with 300, 400 and 500 MPa for
between 60 and 600 s. A log-linear function was developed in Microsoft®Excel
to model the kinetic inactivation data with the effects of NaCl, pressure and
treatment time. The model can be used to predict HAV inactivation by
interpolation at processing parameters not actually tested for in the laboratory.
For the model to be validated in oysters contaminated with HAV, methods for
HAV extraction and purification from spiked oyster homogenate were first
evaluated. Methods evaluated included the crude extraction method, modified
from Kingsley and Richards (2003), and the PEG precipitation method, modified
from the glycine-polyethylene glycol (PEG)-Tri reagent-poly(dT) extraction
(GPTT) method described by Kingsley and Richards (2001). The PEG
precipitation method achieved a mean recovery of 12.6%. With modification, including the use of antibiotic/antimycotic treatment prior to assay, the recovery
was improved by up to 27.3%. In comparison, the crude extraction technique,
which did not include a virion concentration step, recovered on average more than
40% of the initial spiked titre, and was chosen as a reliable method to extract
HAV from oysters for cell culture quantitation.
Commercially grown and harvested Pacific oysters (Crassostrea gigas) were
contaminated with HAV by natural accumulation, when immersed for up to 24 h
in seawater contaminated with 1.1x107 TCID50/ml HAV. Infectious HAV was
detected in only two of the six oysters tested, and less than 1% of the initial
contaminating HAV titre was recovered in positive oysters, possibly due to the
association of viruses and microalgae with oyster shells and aquarium surfaces
throughout the trials.
A quantitative real-time reverse transcription PCR (qRT-PCR) method was
developed as an alternative method for HAV detection in contaminated oysters to
the infectivity assay. HAV was detected by qRT-PCR in all contaminated
oysters, including those negative by infectivity assay. An immuno-magnetic bead
separation technique was also developed, which additionally purified and
concentrated virions, improving the sensitivity of detection by qRT-PCR.
The log-linear inactivation model was validated in homogenised oyster meat
artificially inoculated with known titres of HAV. Salinity and temperature of
samples were adjusted to that of buffered samples, while intermediate times and
pressures were chosen for processing. The model tended to underpredict inactivation in homogenised oyster samples; i.e. it was fail-safe. Inactivation
tended to be greater in spiked oyster homogenate compared to pure culture in
treatments at higher pressures (400 — 500 MPa). The validated model may be a
useful reference for Australian oyster processors wishing to implement HPP into
their post-harvest processing regime.

Item Type: Thesis (PhD)
Keywords: Oysters, Food contamination, Food
Copyright Holders: The Author
Copyright Information:

Copyright 2008 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Available for library use only and copying in accordance with the Copyright Act 1968, as amended. Thesis (PhD)--University of Tasmania, 2008. Includes bibliographical references. Ch. 1. Literature review -- Ch. 2. General materials and methods -- Ch. 3. Evaluation and comparison of methods for high-throughput cultivation of hepatitis A virus -- Ch. 4. Development of a method for extraction and purification of hepatitis A virus from contaminated oysters -- Ch. 5. Accumulation of hepatitis A virus by oysters in a laboratory aquarium -- Ch. 6. Development of a high pressure processing inactivation model for hepatitis A virus -- Ch. 7. Rapid and quantitative detection of hepatitis A virus by quantitative real-time reverse-transcription PCR

Date Deposited: 09 Dec 2014 00:12
Last Modified: 27 Oct 2016 02:39
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