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A thesis presented in partial fulfilment of the requjrements for the degree of
Doctor of Philosophy in
Microbiology at :t;;assey University
CEPJSTOPrlER DENNIS FRE!�
1974
ii
ABSTRACT
A number of possible bases for a biological assay for the
aflatoxins v1ere examined, of these two were selected for further study.
An inhibition assay using Bacillus megaterium was developed in corebina
tion with thin layer chromatographic analysis. This cethod was capable of detecting 0.1 �g of aflatoxin B and of differentiating between
1 various mycotoxins.
An induction assay using a lysogenic strain of Bacillus megaterium was also developed. This assay has a sensitivity of 2-4 �g/ml aflatoxin B1 and is claimed to be more specific than the inhibition assay.
A nureber of other mycotoxins were examined to test the versatility and specificity of the assays. The inhibition assay was able to detect and differentiate between most of the toxins. The induction assay detected only those mycotoxins reported or suspected to be carcinogenic.
PREFACE
It has been known for many years that certain fungi prod�ce substances toxic to human beines and livestock. Toxic Basidiomycetes and ergotism have been responsible for the loss of many lives. It is only comparatively recently however that a�;tention has been directed to the imperfect fungi as sources of toxins. In particular, a dramatic outbreak of an unknown disease among poultry in the United Kingdom, called. Turkey X Disease, was eventually traced to a substance produced by .Aspergillus parasiticus. This active principle was named 'aflatoxin' after the Asperr,illus flavus-oryzae group to which Aspergillus parasiticus belongs.
'Aflatoxin' was soon shown to be in fact a number of related compounds which were designated B1, B2, G1 and G2 due to the blue or green colour of their fluorescence under ultra-violet light and the relative positions they reach on thin layer chro�atographic plates.
Not all the aflatoxins are of equal potency, B1 being the most potent and G2 the least.
The aflatoxins are of especial inteiest since they have been shown to be carcinogenic in very low concentrations in addition to their hepatotoxic effect at higher concentrations. Aflatoxins have been reported from a wide variety of fungi, chiefly Aspergillus and Penicillium species though other fungi, including a Rhizopus species, have been implicated. It was also shown that the toxins could be formed on a wide range of substrates and could easily occur in human food�tuffs if .the fungus was allowed to grow on them.
Chemicophysical methods of detection and quantitation have been developed, based mainly on extraction by an organic solvent followed by concentration, separation on thin layer chromatographic plates and fluorodensit ometric estimations of the spots.
Chemical assays are rapid, sensitive and very precise but may not always accurately reflect the biological activity of the substance under test. This is particularly the case where very small chemical differences
iv can make very large changes in activity. There have been shown to be at least nine different aflatoxins with biological activities varying from extreme toxicity to practically no effect . It is therefore desirable to have a biological assay to complement the results of th� chemical assay.
The ideal bioassay would be simple, speedy, sensitive, specific and, of course , reproducible.
Early
bioassay Glethods were based on thetoxicity of aflatoxin to vertebrates; the most sensitive and specific assay being that using ducklings. Fish larvae and fertile eggs have also been used. Later an inhibition-zone microbiological bioassay using
Bacillus me�terium was developed and put forward as a standard method in the Journal of the Association of Offici�l Analytical Chemists. There have been reports in the literature of other effects of aflatoxin which might be suitable for a bioassay system. Among these are tissue-culture abnormalities, induction of lysogenic bacteria and suppression of
chloroplast development in germinating cress seeds .
The assay based on ducklings is relatively specific but requires skill to perform and is not nearly as rapid or sensitive as chemical
methods. The inhibition assay using B. megaterium is more rapid and easier to perform but is not very specific and is also less sensitive than chemical assays though it requires much less material than does the duckling assay.
For these reasons it v;as considered worthwhile to look for an assay as specific as the duckling assay but with the advantages of a micro
biological assay. A nuober of possibilities had already been suggested by various authors and it was these that I proposed to investigate.
A CKNO','/LEDG E_,iENTS
I would like to thank the Dairy Division of the ��inistry of Agriculture and Fisheries, for giving me leave to undertake this work and the State Services Commission for supporting me financially over this period •
.Mrs Gradolf, my typist, for her unfaili!'lg speed and accuracy.
Professor R. Hodges for the mass spectroeraphic analysis of a number of compounds.
Dr R. Brookes for atomic absorption and spectrographic analyses of agar extracts and ash.
Dr A. Tyree for mathematical analysis of the dynarr.ics of induction by aflatoxin.
Dr �'l. Haynes of the Northern Regional Research Laboratories for
cultures of Eacillus meeaterium strains KM, 899a and 1368.
Dr M. Chick, Miss S. Harvey and .Mr J. Shaw for samples of mycotoxins.
Professor· E. Carell, Dr V. Mayer and ?.�rs A. Jarvis for cultures of a number of micro-organisms.
My vlife for her uncritical acceptance of oy moods and her unceasing encouragement.
Finally I would like to record my very real gratitude to my Supervisors, Professor D. Bacon and Dr G. Pritchard for their kindness in helping me, their patience with my more unlikely hypotheses and their encouragement when things occasionally went ver-.J wrong.
To all these and to everyone else who helped in big or small ways by advice or technical assistance, I would like to acknowledge my debt.
vi
CO:NTENTS
Abstract ii
Preface
Acknowledgements Table of Contents List of Figures Introduction
Materials and Methods
iii
V
vi
Section I Examination of a number of biological systems as possible bases for bioassay
Section II Development of an inhibition bioassay system Section III Investigation of the properties of Bacillus
�zat� and its bacteriophage
Section IV Development of a bioassay for afl�toxin based on induction of megaphage 899a
Section V Application of the bioassays to extracts of aflatoxins
vii 1 14
22 45
57
86
and other mycotoxins 106
Discussion 11 6
Notes on Appendices 128
Appendix 1.! Modification by adenosine 3.' :5.' cyclic monophosphate
of megaphage 899a inuuction by aflatoxin B1 • 1 29
B
The
effect of age of culture on induction ofmegaphage 899a by aflatoxin B1 . 130
Appendix II Mathematical analysis of the dynamics of induction 133
References 135
LIST OF FIGURES
�
0.1 Structures of the aflatoxins 3
1 .1 A comparison of optical density and viable colalt of Sa.lrr.onella
typhir:mrium ( F22T) during gro\v.t� 29
1 .2 Cell numbers vs Klett units for Salmonella ty-;:>--i::lUrium
(P22T)
301 .3 Effect of ultra-violet light on Escherichi"'. coli PB240 36
1 .4 Growth of E. coli PB240 in the presence of 1 0 JJg/ml aflatoxin B1
2.1 Inhibition of Bacillus megaterium by aflatoxin B1 2.2 Effect of seed density on zone si?.e
2.3 Relationship between densitometric and iQhibition assays
2.4 Relationship between inhibition zone size end amount of aflatoxin B1 / spot
3.1 Maximum growth of Bacillus megateriurn
in
varying concentrations of glucose39
51 53 54
55
3.2 Cor�ection curve for optical density measurements 62 3.3 Relationship between optical density and bacterial protein 64
3.4 Effect of tryptone on growth rate of Bacillus me.'=:aterium 65 3.5 Adsorption of bacteriophage 899a to B. meeaterium strain ID� 68
3.6 Effect of agar extract on the phage lysis of B. megaterium strain KM
3.7 Effect of agar extract on phage production by B. �egaterium strain KM
70
71 3.8 Effect of agar ash extract on phage production 73 3.9 Effect of agar ash on plating efficiency 75 3.10 Effect of magnesium and copper ions on plating efficiency 78
3.11 Inhibition of plating efficiency by allliuiniL� and calcium ions
3.1 2 Megaphage 899a
3-13 Megaphage 899a adsorbed to B. megaterium strain KM
4·1 Growth of B. mega terium strains 899a and K:.: in the presence of aflatoxin B1
viii
79 82 83
90 4.2 Induction of bacteriophage 899a by aflatoxin B1 92 4.3 Optical density and phage counts after induction 94 4.4 Time course of phage production after induction by aflatoxin 95
4.5 Phage production after the initial burst 97
4.6 Collected induction assay results from seven experiments 1 05 5 .1 Structures of compounds used in bioa ssays · 111 6.1 The use of optical density to measure the ratio of lysed cells 121 7.1 Effect of age of culture on lag phase and lysis 132
