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Anti-Microbial and Anti-Viral Potential


Emu Oil
Its Anti-Microbial and Anti-Viral Potential


A report for the Rural Industries Researchand Development Corporation
by Thomas V Riley and Christine F Carson
University of Western Australia
October 1999
RIRDC Publication No 99/132
RIRDC Project No UWA-37A

© 1999 Rural Industries Research and Development Corporation.
All rights reserved.
ISBN 0 642 57948 2
ISSN 1440-6845
Emu Oil – Its Anti-microbial and Anti-viral Potential
Publication no 99/132
Project no. UWA-37A
The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report.
This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details
Associate Professor Thomas V Riley
Department of Microbiology
Queen Elizabeth II Medical Centre
Nedlands WA 6009
Phone: (08) 9346 3690
Fax: (08) 9346 2912
Email: triley@cyllene.uwa.edu.au
RIRDC Contact Details
Rural Industries Research and Development Corporation
Level 1, AMA House
42 Macquarie Street
BARTON ACT 2600
PO Box 4776
KINGSTON ACT 2604
Phone: 02 6272 4539
Fax: 02 6272 5877
Email: rirdc@rirdc.gov.au
Website: http://www.rirdc.gov.au
Published in October 1999
Printed on environmentally friendly paper by Canprint

Foreword


The viability of the emu industry depends on the successful development of high value markets for the oil. These markets include meat and leather industries, however, the highest potential value and return to the Australian economy is in the area of natural medicines derived from emu oil. Anecdotal stories suggest that the anti-inflammatory properties of emu oil are certainly worth investigating. An
area that has received less interest is that of antimicrobial activity.

The purpose of this project was to:
  • investigate the ability of emu oil to kill or inhibit various bacteria, fungi and viruses,
  • compare the activity of emu oil with other natural products, and
  • to recommend, if appropriate, further areas for research.
This report, a new addition to RIRDC’s diverse range of almost 400 research publications, forms part of the Corporation’s New Animal Products R&D Program, which aims to accelerate the development of viable new animal industries.

Most of our publications are available for viewing, downloading or purchasing
online through our website:
  • downloads at www.rirdc.gov.au/reports/Index.htm
  • purchases at www.rirdc.gov.au/pub/cat/contents.html
Peter Core
Managing Director
Rural Industries Research and Development Corporation

Acknowledgments


The assistance of John Snowden and Peter O’Malley in providing samples of emu oil for testing was appreciated. The technical assistance of Ms Katherine Hammer is gratefully acknowledged, as is the provision of facilities by the Western Australian Centre for Pathology and Medical Research.

Contents


Foreword ........................................................................................................... iii
Acknowledgements ............................................................................................ iv
Executive Summary ........................................................................................ vi
1.0 INTRODUCTION 1
2.0 OBJECTIVES…………………………………………………………………… 2
3.0 METHODS………………………………………………………………………. 3
3.1 Oil samples…………….............................................................................. 3
3.2 Antimicrobial testing…………………………............................................. 3
3.3 Disc diffusion assay………………………………………………………... 3
3.4 Viability of organisms in 50% emu oil…………………………………….. 3
4.0 RESULTS 5
4.1 Oil set 1………………………………...................................................... 5
4.2 Oil set 2………………………………………........................................... 5
4.3 Oil set 3……………………………………………………………………. 5
4.4 Tea tree oil comparison group…………………………………………….. 6
4.5 Antiviral activity…………………………………………………………. 6
5.0 CONCLUSIONS AND RECOMMENDATIONS…………………………… 7
6.0 REFERENCES .. ........................................................................................... 8
7.0 RIRDC PUBLICATIONS .. ............................................................................ 9

Executive Summary


In a preliminary investigation of emu oil no consistent antibacterial or antifungal effect could be demonstrated. Using a disc diffusion assay to detect activity, only one sample of 19 collected at three different times and using different feeding and rendering schedules, showed any effect, and this was against a fungus Candida albicans. However, this effect could not be repeated with other oils.
By comparison, tea tree oil showed consistent activity against bacteria and fungi. Methods were developed for antiviral testing, however, due to technical difficulties, these assays will be completed at a later date.

1.0 Introduction

Recent reports on the emergence of antibiotic resistant bacteria highlight the growing necessity for the development or discovery of novel antimicrobial agents. Traditionally, antibiotics have been derived from microorganisms, however, other sources have included
plants and, more recently, animals. It has been suggested that emu oil may possess antimicrobial activity although there are no published confirmatory reports of this and evidence is of an anecdotal nature. Confirmation of this suggestion would not only enhance
our ability to inhibit the growth of microorganisms, but would contribute to the development of a new Australian industry.
For the successful development of a novel antimicrobial agent, work must proceed on the basis of sound scientific investigation. Ultimately, registration of emu oil under the Therapeutic Goods Act may be sought and appropriate preliminary data is required to guide further research. Initially, the in vitro antimicrobial activity of emu oil against a range of clinically relevant microorganisms should be assessed using methods that have been developed to study the antimicrobial activity of tea tree oil (Carson and Riley, 1993; Carson and Riley, 1994; Carson and Riley, 1995). The reason for this is that these methods have been developed for hydrophobic compounds like emu oil, however, some modification may be required. The organisms tested should include reference organisms and/or recent clinical isolates. The former allows other workers to reproduce the work while the latter are of relevance clinically.
The data generated in this type of investigation is critical in focusing the potential applications of emu oil and future work would be guided by the spectrum of activity demonstrated in this initial stage. Once sufficient data regarding the in vitro activity of emu
oil are assembled, and results indicated adequate activity, then product formulation and assessment could proceed. If positive outcomes were obtained from an in vitro evaluation of the products, work could proceed to in vivo clinical trials. It is likely, given its history of use, that emu oil would only be suitable for external use and therefore organisms and infections amenable to topical treatment should be targeted.


2.0 Objectives


The purpose of this project, carried out on behalf of the New Animal Products Sub-Program of the Rural Industries Research and Development Corporation, was to:
• investigate the ability of emu oil to kill or inhibit various bacteria, fungi and viruses,
• compare the activity of emu oil with other natural products, and
• to recommend, if appropriate, further areas for research.


3.0 Methods
3.1 Oil samples


Three sets of emu oil samples were tested: a set of seven oils, a set of four oils and a set of eight oils. These were provided by either Mr Peter O’Malley or Dr John Snowden at the Western Australian Department of Agriculture. Some activity was detected in one sample
from the first set (see below). This oil was rendered at low temperatures from body fat taken from animals fed a standard diet. The second set of four samples was from animals fed different diets and rendered at low temperatures.

3.2 Antimicrobial testing

Two different methods were used in the evaluation. The test organisms were
Staphylococcus aureus NCTC 6571, Escherichia coli ATCC 10418 and Candida albicans
ATCC 10231. For comparative purposes, several samples of commercially available tea
tree oil were evaluated using similar methodology.

3.3 Disc diffusion assay

• A standard suspension of each organism was spread evenly over the surface of a 20ml Mueller Hinton agar plate.
• After the inoculum had dried, a 12.5mm paper disc was placed in the centre of the plate, and 50ul of emu oil was placed on the paper disc.
• After the oils had soaked into each disc, plates were inverted and incubated at 35°C for 24h.
• After incubation, plates were observed for a zone of no growth around each disc. This is called the ‘zone of inhibition’.

3.4 Method 2 – Viability of test organisms in 50% emu oil

• A standard suspension of each organism was diluted into double-strength Mueller
Hinton broth.
• This mixture was then used to inoculate an equal volume of each emu oil.
• These organism/emu oil solutions were mixed thoroughly, then incubated at 35°C for 24h.
• After incubation, viable counts were performed on each of the organism/emu oil solutions, to determine the number of viable organisms contained in each solution. This was achieved by performing serial ten-fold dilutions and spot inoculating 10ul samples
from the dilutions onto blood agar plates.
• These samples were incubated at 35°C for 24h after which time, the numbers of colonies were counted.

4.0 Results

Oil sets 1 and 2 were tested with the screening disc diffusion method only.

4.1 Emu oil set 1

Of the seven oils tested (samples numbers 1-7), one emu oil sample (sample 5) demonstrated activity against C. albicans, i.e. antifungal activity.

4.2 Emu oil set 2

Of the four oils tested (sample numbers 3-60°C, 7-60°C, 8-60°C, and canola-60°C), none demonstrated activity against any of the test organisms.

4.3 Emu oil set 3

• In the screening assay, no zones of inhibition were seen, which indicates that no oil inhibited the growth of any test organism.
• Using the second method (Table 1), starting concentrations of organisms were as follows (colony forming units per ml): Staph. aureus 4.1 x 105, E. coli 1.9 x 105, C.albicans 4.1 x 105.
• For three of eight oils, organism numbers decreased to below the detection limit.
• For three of eight oils, organism numbers remained approximately constant.
• For two of eight oils, organism numbers increased beyond the upper detection limit.
Table 1.
Organisms (cfu/ml) recovered from test organism/emu oil solutions after 24h
Emu oil # Staph. aureus E. coli C. albicans 5, 6, 10 not detected* not detected not detected
1 1.5 x 105 1.0 x 105 4.5 x 105
2 4.0 x 105 1.0 x 105 2.2 x 105
14 4.0 x 105 1.2 x 108 8.2 x 105
11, 12 too many to count too many to count too many to count
* Lower limit of detection is approximately 5 x 104 cfu/ml
Upper limit of detection is approximately 5 x 108 cfu/ml


4.4 Tea tree oil comparison group

A comparison with several samples of tea tree oil is shown in Table 2. All tea tree oil samples gave large zones of inhibition.
Table 2. Mean zones of inhibition for 9 commercially available of tea tree oils.
Organism Mean zone of inhibition (mm)
E. coli 33.9
Staph. aureus 25.4
C. albicans 20.3

4.5 Antiviral activity

Much effort was put into developing methods suitable for testing emu oil for antiviral activity. This was done in conjunction with another RIRDC funded project examining the antiviral activity of tea tree oil and many of the methodological problems are similar. At
present the following has been achieved:
• the production of suitable stocks of Herpes virus for assays (once virus titres reach high enough levels these stocks are frozen at minus 70 degrees for future work),
• the production of various tissue culture cell line for investigation (likewise these stocks are frozen for various assays),
• various solubilising agents have been assessed,
• a plaque reduction assay system has been developed and tested.
Unfortunately, due to a freezer breakdown and loss of stored tissue culture cell lines just before batch testing commenced, antiviral testing will have to be completed at a later date.


5.0 Conclusions and recommendations

Although the overall number of emu oil samples tested was small, and one showed some activity against C. albicans, it is difficult to conclude that emu oil has antimicrobial activity. The most likely explanation for activity in the one sample is either that the animal
consumed some sort of antimicrobial in its feed or that the sample was somehow contaminated in the rendering process. During the course of this project we have also tested several emu oil samples for various producers throughout Australia. None has shown any
antibacterial or antifungal activity.
Because of these negative results, an alternative additional testing plan was used for the last samples. In this, organisms were inoculated into a mixture of emu oil and broth and then sampled over a period of time. With this method, variable results were obtained, with three of eight samples not supporting the growth of test organisms. The effect was non-specific in that there was no difference between Gram positive Staph. aureus and Gram negative E.coli or the yeast C. albicans. One possible interpretation of this non-specific effect is that there may be an inhibitory tissue substance in the emu oil, in the same way that human
serum samples may be inhibitory to certain organisms. These substances are not true antibiotics and may be antibodies or even certain fatty acids. The latter would seem a distinct possibility given the rendering process. In any event, the minimum inhibitory
concentration (MIC) of emu oil could be calculated to be about 50%, far too great to be of commercial interest (cf. MICs of tea tree oil which are in the order of 0.25-0.5%).
It will be of interest to see if there is any antiviral activity, however, on the basis of the work completed, it is probably not worth pursuing antibacterial or antifungal activity any further.

6.0 References

  • Carson, C. F. and Riley, T. V. (1993) Antimicrobial activity of the essential oil of Melaleuca alternifolia. Letters in Applied Microbiology 16:49-55.
  • Carson, C. F. and Riley, T. V. (1994) The antimicrobial activity of tea tree oil. Medical Journal of Australia 160:236.
  • Carson, C. F. and Riley, T. V. (1995) Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. Journal of Applied Bacteriology 78:264-269.