Antimicrobial plants of Australia have the potential to prevent lactic acidosis in ruminants

160 Pages·2008·1.02 MB·English

Antimicrobial plants of Australia have the
potential to prevent lactic acidosis in ruminants
by
Peter Hutton
B.Sc. (Animal Science, Hons I)

Eremophila glabra leaves contain secondary
metabolites that might pr event acidosis in ruminants

This thesis is presented for the degree of Doctor of Philosophy of
The University of Western Australia
Faculty of Natural and Agricultural Sciences
School of Animal Biology

2008
i
Summary

Antimicrobial growth promoters are added to feed to prevent lactic acidosis in
ruminant animals by selectively inhibiting rumen bacteria that produce lactic
acid. However, recently imposed or impending bans on the use of antimicrobial
growth promoters in animal production have lead to a critical need to find
practical alternatives that are safe for the animal and consumer and that obtain
similar production benefits. I investigated bioactive plants of Australia for their
potential to prevent lactic acidosis in ruminants. The unifying hypothesis tested
was that plants would be identified that selectively inhibit lactic acid-producing
bacteria and consequently protect against lactic acidosis. This hypothesis was
tested in a three phase process: phase 1, plant selection and collection; phase
2, a three stage protocol for screening plants and essential oils; phase 3, in vivo
experiments and chemical fractionation of the most promising plant.

I developed an in vitro bioassay that simulated acidosis by adding glucose to
rumen fluid in Bellco tubes and incubating for 5 h (Chapter 4). The pH and gas
production were used as indicators of acidosis and fermentation activity. I used
this bioassay to screen ninety-five plants (dried and ground material from 79
species) and ten essential oils and included a negative control (oaten chaff) and
a positive control (virginiamycin). One plant, Eremophila glabra, produced a
similar pH (5.63) to the positive control (5.43) although it inhibited gas
production to a moderate extent (P < 0.05). Four plants, Kennedia eximia,
Acacia saligna, Acacia decurrens and Kennedia prorepens also showed
potential to prevent acidosis.

The activity of the ethanolic extracts from these plants was tested against a
range of rumen bacteria (Chapter 5), including species that are known to be
involved in causing acidosis and others that are not. E. glabra selectively
inhibited the lactate-producers Streptococcus bovis and Lactobacillus spp. at
1.26 mg/mL. The remaining four plants selectively inhibited Lactobacillus spp. at
5 to 10 mg/mL, but showed no inhibition of S. bovis. The selective activity
provided additional support for the potential of these plants, in particular E.
glabra, as agents against acidosis.
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The in vitro bioassay used to screen the plants was used to demonstrate that
the ability of E. glabra and K. prorepens to prevent pH decline during a
carbohydrate challenge was due to a reduction in the accumulation of lactic
acid. Modifications to the in vitro bioassay included extending the incubation
period to 24 h and measuring the amount of cumulative D-lactate during that
incubation (Chapter 6). E. glabra prevented the final pH falling below 5 (5.15),
which was similar to the positive control (5.16). There was also less cumulative
D-lactate with the E. glabra treatments (7 mmol/L) than the negative control (47
mmol/L) without inhibiting fermentation (P < 0.05). There was more cumulative
D-lactate and the pH was higher after 24 h incubation with K. prorepens than
with E. glabra (P < 0.05). E. glabra was considered effective at preventing
acidosis in vitro because it limited the drop in pH, reduced the production of
lactic acid and did not significantly inhibit fermentation.

An in vivo experiment was designed to determine if E. glabra was effective at
preventing acidosis when added with a wheat diet designed to induce acidosis
in fistulated wethers within 36 h (14 g of kibbled wheat/kg body weight) (Chapter
8). The E. glabra group (0.1 g dried and ground leaf/g of wheat, n=10) was
compared to a negative control group (oaten chaff at 0.1 g/g of wheat, n=16)
and a positive control group (virginiamycin at 40 µg/g of wheat, n=6). The wheat
and E. glabra were infused via rumen cannula, and samples were taken at
regular intervals for determination of pH, VFA and D-lactate. Sheep were
removed from the experiment and treated for acute acidosis when the rumen
pH fell below 5.0. All wethers that received the E. glabra developed low rumen
pH (< 5.0) within 12 h of the initial wheat dose. There was no significant
difference in rumen pH, D-lactate or osmotic pressure between the negative
control and the E. glabra groups. Virginiamycin prevented pH decline (5.63) and
D-lactate accumulation in the rumen relative to the E. glabra and negative
control groups (P < 0.05). E. glabra did not prevent acute acidosis in vivo, but
was only tested at a single dose rate although it may be effective at higher
doses.
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Collaboration with researchers at the School of Biomedical, Biomolecular and
Chemical Sciences at The University of Western Australia was developed to
isolate the major compounds of E. glabra and to determine if these were the
active secondary metabolites in the plant. Secondary metabolites were
extracted by acetone and fractionated using chromatography and nuclear
magnetic resonance (Chapter 9). Seven serrulatane diterpenes were identified
to be the major secondary metabolites in E. glabra. The metabolites were
screened using a broth dilution and microtitre spectrophotometry method and
were selective against S. bovis at between 320 and 1077 µg/ mL. The
serrulatanes from E. glabra were probably responsible for the activity against
acidosis that I observed in vitro, because they selectively inhibited lactate-
producing bacteria. It is also possible that a synergy between serrulatanes and
possibly other metabolites are responsible for the activity observed in vitro.

The results from my experiments support the role that bioactive plants may
have to replace the antibiotics that are added to livestock feed. Australian plants
were identified containing compounds that were active against the bacterial
processes responsible for ruminant acidosis. To my knowledge this is the first
work undertaken to identify bioactive plants of Australia for their potential to
prevent acidosis. I developed in vitro screening bioassays that targeted key
indicators of acidosis. These bioassays enabled me to identify 5 plants from the
104 screened that could potentially control acidosis. One of these plants in
particular, E. glabra, showed a level of activity in vitro that was comparable to
antibiotic protection against acidosis. The exciting in vitro results were not
demonstrated in vivo but only one dose level of E. glabra was used, which was
based on the in vitro work. In contrast to the in vitro system the rumen is a
continuous flow system with greater complexity and it is possible that the
concentration of E. glabra that I used in vivo was not optimum. This places
importance on future dose response experiments to confirm the efficacy of E.
glabra in vivo.
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Table of Contents
Summary _____________________________________________________________i
Table of Contents_____________________________________________________iv
Statement of Contribution______________________________________________ix
Acknowledgements ___________________________________________________ x
Chapter 1: General introduction __________________________________ 1
Chapter 2: Review of the literature ________________________________ 3
2.1 Scope of the literature review________________________________________ 3
2.2 Introduction_______________________________________________________ 3
2.3 Antibiotic dependency in livestock production _________________________ 4
2.3.1 AGPs alter rumen fermentation to improve feed efficiency ________________ 4
2.3.2 Antibiotics have become essential in livestock production_________________ 5
2.3.3 Bans on the use of AGPs __________________________________________ 6
2.4 Plants as alternatives to AGPs_______________________________________ 7
2.4.1 Australian plants have evolved in isolation_____________________________ 8
2.4.2 Australian plants are specialised ____________________________________ 8
2.4.3 Secondary plant compounds with antibacterial activity ___________________ 9
2.4.4 Essential oils ___________________________________________________ 10
2.4.5 Saponins ______________________________________________________ 11
2.4.6 Condensed tannins ______________________________________________ 11
2.4.7 Potential for natural bioactive compounds ____________________________ 12
2.4.8 Antibiotics and antimicrobial plants: what is the difference? ______________ 13
2.4.9 The search for antimicrobial plants__________________________________ 14
2.5 Lactic acidosis is a ruminal disorder of microbial origin ________________ 14
2.5.1 Acidosis in ruminant livestock industries _____________________________ 14
2.5.2 Acidosis reduces animal production_________________________________ 15
2.5.3 Starch is a source of energy in the rumen ____________________________ 16
2.5.4 Too much rapidly fermentable carbohydrate causes acidosis_____________ 17
2.5.5 Acidosis is of microbial origin ______________________________________ 17
2.5.6 Subacute versus acute acidosis ____________________________________ 18
2.5.7 Lactate-users___________________________________________________ 19
2.5.8 Two stages of acute acidosis ______________________________________ 21
2.5.9 Systemic acidosis _______________________________________________ 22
2.5.10 Accumulation of solutes increases osmotic pressure____________________ 22
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2.5.11 Saliva buffers rumen pH __________________________________________ 24
2.5.12 Protozoa buffer against acidosis____________________________________ 25
2.5.13 Animal health is compromised by acidosis____________________________ 25
2.5.14 Difficulties in predicting in vivo outcomes_____________________________ 26
2.5.15 Measuring acidosis allows diagnosis and prevention____________________ 26
2.5.16 Which isomer of lactic acid is the best to measure?_____________________ 28
2.5.17 Prevention of acidosis____________________________________________ 29
2.5.18 Antibiotics protect against acidosis__________________________________ 31
2.5.19 Implications of control ____________________________________________ 32
2.6 Research objectives_______________________________________________ 32
2.7 Summary and hypotheses__________________________________________ 33
Chapter 3: General Materials and Methods ________________________ 35
3.1 Phase 1: Plant collection___________________________________________ 36
3.2 Phase 2: In vitro screening _________________________________________ 37
3.2.1 Simulated acidosis bioassay_______________________________________ 37
3.2.2 Selective inhibition of bacteria______________________________________ 41
3.3 Phase 3: Plant efficacy_____________________________________________ 43
3.3.1 Induced acidosis in vivo __________________________________________ 43
Chapter 4: A screening assay to identify Australian plants that have
potential to control acidosis in ruminants _________________________ 45
4.1 Introduction______________________________________________________ 45
4.2 Material and Methods______________________________________________ 46
4.2.1 Experimental design _____________________________________________ 46
4.2.2 Assumptions ___________________________________________________ 46
4.2.3 Analysis_______________________________________________________ 47
4.3 Results__________________________________________________________ 47
4.3.1 Effectiveness of the bioassay ______________________________________ 47
4.3.2 Plants screened_________________________________________________ 48
4.3.3 Essential oils ___________________________________________________ 52
4.4 Discussion_______________________________________________________ 55
Chapter 5: Australian plants selectively inhibit bacteria that cause
acidosis in ruminants__________________________________________ 60
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5.1 Introduction______________________________________________________ 60
5.2 Materials and methods_____________________________________________ 61
5.2.1 Experimental design _____________________________________________ 61
5.2.2 Treatments ____________________________________________________ 62
5.2.3 Bacterial species________________________________________________ 62
5.3 Results__________________________________________________________ 64
5.3.1 Essential oils ___________________________________________________ 65
5.3.2 Controls_______________________________________________________ 67
5.3.3 Ethanolic extracts of dried plant material _____________________________ 68
5.4 Discussion_______________________________________________________ 68
Chapter 6: E. glabra reduces the accumulation of lactic acid in a batch
culture system challenged with glucose __________________________ 74
6.1 Introduction______________________________________________________ 74
6.2 Materials and Methods_____________________________________________ 74
6.2.1 Experimental design _____________________________________________ 74
6.2.2 Treatments ____________________________________________________ 75
6.2.3 Inoculation and incubation ________________________________________ 75
6.2.4 Statistical analysis_______________________________________________ 76
6.3 Results__________________________________________________________ 76
6.3.1 pH ___________________________________________________________ 76
6.3.2 D-lactate ______________________________________________________ 78
6.3.3 Cumulative gas _________________________________________________ 78
6.3.4 VFAs _________________________________________________________ 79
6.4 Discussion_______________________________________________________ 80
Chapter 7: The concentration of E. glabra and the type of fermentable
carbohydrate affect the rate of pH decline in an in vitro assay for acidosis
____________________________________________________________ 85
7.1 Part 1. Establishing the level of E. glabra needed to protect against acute
acidosis ______________________________________________________ 85
7.2 Introduction______________________________________________________ 85
7.3 Materials and methods_____________________________________________ 86
7.3.1 Experimental design _____________________________________________ 86
7.3.2 Statistical analysis_______________________________________________ 87
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7.4 Results__________________________________________________________ 87
7.5 Discussion_______________________________________________________ 88
7.6 Part 2. The type of fermentable carbohydrate affects the rate of metabolism in
batch cultures_________________________________________________ 89
7.7 Introduction______________________________________________________ 89
7.8 Materials and methods_____________________________________________ 90
7.8.1 Experimental design _____________________________________________ 90
7.9 Results__________________________________________________________ 90
7.9.1 Substrate influence on the effectiveness of the additives ________________ 90
7.9.2 Comparison of effectiveness between the additives on final pH ___________ 93
7.9.3 The effect of substrate on gas production when E. glabra was added ______ 93
7.9.4 Comparison on gas production between the additives___________________ 94
7.10 Discussion______________________________________________________ 96
Chapter 8: E. glabra did not protect wethers against induced acidosis _ 99
8.1 Introduction______________________________________________________ 99
8.2 Material and methods_____________________________________________ 100
8.2.1 Experimental design ____________________________________________ 100
8.2.2 Establishing the level of kibbled wheat needed to induce acute acidosis ___ 101
8.2.3 Treatment of animals for acidosis__________________________________ 103
8.2.4 Methods for the primary experiment________________________________ 103
8.2.5 VFAs and D-Lactate ____________________________________________ 104
8.2.6 Osmolality ____________________________________________________ 104
8.2.7 Statistical analysis______________________________________________ 105
8.3 Results_________________________________________________________ 106
8.3.1 Rumen pH ____________________________________________________ 106
8.3.2 D-lactate _____________________________________________________ 107
8.3.3 Comparison of key indicators of acidosis at the pH nadir _______________ 110
8.3.4 UPE groups___________________________________________________ 111
8.3.5 The effect of E. glabra when comparing acidotic sheep_________________ 112
8.4 Discussion______________________________________________________ 113
Chapter 9: Serrulatane diterpenes from E. glabra selectively inhibit
lactate-producing bacteria_____________________________________ 119
9.1 Introduction_____________________________________________________ 119
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9.2 Materials and Methods____________________________________________ 120
9.2.1 Experimental design ____________________________________________ 120
9.2.2 Testing the ethanolic extracts of E. glabra in an acidotic environment _____ 120
9.2.3 Choosing the primary extracting solvent_____________________________ 121
9.2.4 Isolation of secondary metabolites _________________________________ 121
9.2.5 Testing the minimum inhibitory concentration of purified compounds______ 123
9.2.6 Statistical analysis______________________________________________ 123
9.3 Results_________________________________________________________ 124
9.3.1 Serrulatane diterpenes __________________________________________ 124
9.4 Discussion______________________________________________________ 125
Chapter 10: General discussion ________________________________ 127
References__________________________________________________ 134
ix
Statement of Contribution
The work presented in this thesis is the original work of the author. The
experimental work and manuscript preparation was carried out by myself after
discussions with my supervisors Dr Phil Vercoe and Dr Colin White. The work
described in Chapter 9 was developed in collaboration with Associate Professor
Emil Ghisalberti and Mr. Ryan Duncan the School of Biomedical, Biomolecular
and Chemical Sciences at The University of Western Australia who isolated and
purified the secondary metabolites from E. glabra. My contribution to this work
was the testing for biological activity of the metabolites on bacterial cultures.

Peter Hutton

February 2008