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Gastric Ulceration in Horses the Role of Bacteria and Lactic Acid

Gastric Ulceration in Horses the Role of Bacteria and Lactic Acid

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Gastric Ulceration in Horses The role of bacteria and lactic acid

RIRDC Publication No. 08/033

HORSES

RIRDC

Innovation for rural Australia

Gastric Ulceration in Horses
The role of bacteria and lactic acid

By Dr Rafat Al Jassim, Dr Thomas McGowan, Prof Frank Andrews and Dr Catherine McGowan

October 2008 RIRDC Publication No 08/033 RIRDC Project No UQ-115A

© 2008 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 1 74151 622 6 ISSN 1440-6845 Gastric Ulceration in Horses: The role of bacteria and lactic acid Publication No. 08/033 Project No. UQ-115A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact Details Dr Rafat Al Jassim c/o School of Animal Studies The University of Queensland Gatton 4343 Phone: 07 5460 1521 Fax: 07 5460 1444 Email: r.aljassim@uq.edu.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web: 02 6271 4100 02 6271 4199 rirdc@rirdc.gov.au. http://www.rirdc.gov.au

Published in October 2008 by Union Offset

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Foreword
Gastric ulceration is widespread and a very common problem in horses in training. When it occurs in horses, gastric ulceration is a potential insidious cause of poor athletic performance or, when severe, an animal welfare concern. It is widely accepted that this is a problem resulting from feeding and management practices, especially in racehorses where the prevalence is extremely high. Racehorses are fed large meals of grain rich diets and with extended periods of fasting between meals. This combined with increased gastric acid production during exercise, reduction in saliva production due to a low fibre diet, and indoor confinement, is likely to contribute to the development of stomach ulcers. Recent work has indicated the presence of diverse microbial populations that survive the stomach environment of both starved and fed horses. Recent work in the USA has shown that volatile fatty acids produced by bacteria in the stomach of horses were associated with increased ulcer severity (Nadeau et al., 2000) and have a potent in vitro effect on reducing mucosal integrity, an effect even greater than that by normal gastric acid. A theory was developed: bacteria and their products, especially lactic acid and volatile fatty acids by bacteria in the non-glandular region of the stomach, play a vital role in the development and progression of gastric ulcers in horses. Identification of the key acid producing bacteria and dietary regimens that minimise their multiplication will aid in the development of strategies to control gastric ulcers in horses. This will undoubtedly help in reducing cost of medical treatment of gastric ulcers in racehorses and improve their health, welfare and performance. The results of this project have confirmed the diverse population of bacteria that live within the normal equine stomach. Importantly, it was found that the diversity of bacteria adherent to the stomach lining decreases during ulceration, which implies the potential development of a dominant population of pathogenic organisms. Lactic acid, produced in the stomach of horses increases the permeability of the equine stomach and this may be important in the pathogenesis of gastric ulcers (worsening or causing gastric ulceration). The project went further to both develop a model of dietary induced gastric ulceration and monitor natural recovery at pasture and with treatments aimed at microbial populations. The results indicated that gastric ulceration could be induced in stabled horses rapidly by increasing the concentrate grain based portion of the ration to 5 kilograms per day, and that the severity worsened when the roughage was restricted to three kilograms per day, similar to what is common practice amongst racehorse trainers in Australia. Lastly, treatment of gastric ulceration with oral antibiotics decreased the severity of gastric ulceration. There was a trend for the same effect with administration of a live bacterial culture ‘probiotic’ as a treatment. In conclusion, the role of bacteria in the pathogenesis of gastric ulceration in horses has been established through laboratory experiments and studying gastric ulceration in the live horse. A role for modification of microbial populations in the future treatment of gastric ulceration has been shown and the future use of probiotics and other non-medical manipulations of microbial populations in horse predisposed to gastric ulceration is a promising prospect. This project was funded from industry revenue that is matched by funds provided by the Australian Government. This report, an addition to RIRDC’s diverse range of over 1800 research publications, forms part of our Horses R&D program, which aims to assist in developing the Australian horse industry and enhancing its export potential. Most of our publications are available for viewing, downloading or purchasing online through our website: • • downloads at www.rirdc.gov.au/fullreports/index.html purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Acknowledgments
The authors would like to acknowledge the help of our research assistants Lillian Jedski and Kelly Jamieson for their organisation, excellent care and love for the horses and interest in the project.

Abbreviations
ADF Ash CP DE DGGE DM G HCl H2 Isc LA LAB LRS mM NDF NG NRS OM PD R TB VFA acid detergent fibre mineral ash crude protein digestible energy denaturing gradient gel electrophoresis dry matter conductance hydrochloric acid histamine type 2 short-circuit current lactate lactic acid producing bacteria lactated Ringer’s solution millimolar neutral detergent fibre non-glandular mucosa of the stomach in normal Ringer’s solution organic matter potential difference electrical resistance Thoroughbred volatile fatty acids

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Contents
Foreword ............................................................................................................................................... iii Acknowledgments................................................................................................................................. iv Executive Summary ............................................................................................................................ vii Introduction ........................................................................................................................................... 1 Animal welfare implications of gastric ulceration .............................................................................. 1 The stomach of the horse..................................................................................................................... 1 Microbiology of the stomach of the horse........................................................................................... 2 Pharmaceutical treatment of gastric ulcers.......................................................................................... 3 Objectives ............................................................................................................................................... 4 Methodology .......................................................................................................................................... 5 Study 1. The bacterial community of the horse stomach, .................................................................... 5 Protocol ............................................................................................................................................... 5 Study 2: In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa................................................................................................................................ 6 Specific Objectives.............................................................................................................................. 6 Techniques used .................................................................................................................................. 6 Analytical methods.............................................................................................................................. 7 Study 3: Induction and recovery of dietary induced gastric ulcers in horses ...................................... 7 Protocol ............................................................................................................................................... 7 Study 4: Treatment of dietary induced gastric ulcers in horses......................................................... 10 Results .................................................................................................................................................. 12 Study 1. The bacterial community of the horse stomach .................................................................. 12 Study 2: In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa.............................................................................................................................. 14 Study 3: Induction and recovery of dietary induced gastric ulcers in horses .................................... 15 Study 4: Treatment of dietary induced gastric ulcers in horses......................................................... 18 Analysis of the Severity of gastric ulceration................................................................................ 19 Analysis of the Number of gastric ulceration................................................................................ 19 Discussion of results ............................................................................................................................ 20 Bacterial Community of the equine stomach .................................................................................... 20 The role of lactic acid........................................................................................................................ 20 Induction of gastric ulcers ................................................................................................................. 21 Treatment of gastric ulcers ................................................................................................................ 21 Implications.......................................................................................................................................... 22 Recommendations ............................................................................................................................... 23 References ............................................................................................................................................ 24 Appendices ........................................................................................................................................... 26 List of presentations .......................................................................................................................... 26 Publications ....................................................................................................................................... 26

List of Figures
Figure 1. Gastroscopy examination procedure. From the left: Dr Rafat Al Jassim, Dr Thomas McGowan, Horse 4, and Dr Catherine McGowan Figure 2. Phylogenetic relationship of the derived sequences from 16S rDNA of cultured bacteria Figure 3. Clones derived from denaturing gradient gel electrophoresis (DGGE) bands identified using the V3 region of 16S rDNA. Genomic DNA was extracted from stomach contents and stomach mucosa Figure 4. This graph represents the mean body weight for each week of trial 1 (95% confidence interval error bars) Figure 5. mean gastric ulcer score for 13 horses fed concentrate ration and being confined during weeks 0 – 10 and subsequently during pasture recovery during weeks 10-16. Number and severity are shown separately as blue and purple respectively Figure 6. Endoscopic images of severity grade 3 ulceration in 2 horses. Figure 7. Graph representing the mean number scores for the 3 different groups in the study (Control, Pro-biotic, and Antibiotic) for each week during trial 2 (95% CI error bars) Figure 8. Graph representing the mean severity score for each of the 3 groups (Control, Probiotic and Antibiotic) for each week during the study

List of Tables
Table 1. Chemical composition of the hay and concentrate mixture fed to horses Table 2. composition of the concentrate mix Table 3. Grading system for gastric ulcers based on MacAllister et al., 1997

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Executive Summary
What the report is about This report is about the role that bacteria and lactic acid plays in the development of gastric ulceration in horses. It also explores the use of contrasting diets to determine whether they contribute to the build up of lactic acid and volatile fatty acids. Identification of the key acid producing bacteria and dietary regimens that minimise their multiplication will aid in the development of strategies to control gastric ulcers in horses. This will undoubtedly help in reducing the cost of gastric ulcer treatment in racehorses and improve their health and performance. Background Gastric ulceration is widespread and a very common problem in horses in training. It is widely accepted that this is a problem resulting from feeding and management practices. Racehorses are fed large meals of grain rich diets and usually fasted for an extended period before exercise. It is the combination of increased gastric acid production during exercise, reduction in saliva production due to a low fibre diet, and indoor confinement that likely contributes to the development of stomach ulcers. Physiologically, it is claimed that sudden increases in blood sugar supply elevates gastrin production, which in turn increases gastric acid production. This leads to prolonged periods of exposure of the unprotected region of the stomach to acid, which is a major cause of ulceration. Recent work in the US has shown that volatile fatty acids (VFAs) produced in the stomach of horses are associated with increased ulcer severity (Nadeau et al., 2000) and have a potent in vitro effect on reducing mucosal integrity, an effect even greater than that by normal gastric acid (HCl). This reinforces the concept that bacterial fermentation products play a role in the pathogenesis and persistence of gastric ulceration in horses (Nadeau et al., 2003a and b). An early report on the microbiology of fermentative acidosis in grain fed animals by Al Jassim and Rowe (1999) showed that Streptococcus bovis and Streptococcus equinus are the key lactic acid producing bacteria of the equine gastrointestinal tract. Recent work in our laboratory indicated the presence of a diverse microbial population that survive in the stomach environment of both starved and fed horses. We therefore developed a theory that bacteria and their products, especially lactic acid and VFA’s by bacteria in the non-glandular region of the stomach, are vital in the development and progression of gastric ulcers in horses. Aim The major aims of the project were: 1. To establish involvement of lactic acid producing bacteria and their products (VFA & lactic acid) in the pathogenesis of gastric ulceration in horses. 2. To determine the effect of contrasting dietary regimens on the microbial contribution to the build-up of lactic acid and VFAs in the non-glandular portion of the stomach. Objectives In order to address these aims, we divided the research into four studies with the following objectives: Study 1: to determine the bacterial community of the normal horse stomach and compare that with the bacterial community of the ulcerated stomach Study 2: to determine the in vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa Study 3: To develop a model for induction and recovery of dietary induced gastric ulcers in horses Study 4: To investigate treatment of dietary induced gastric ulcers in horses using modification of bacterial populations. Key findings The results of this project have shown that there is a diverse population of bacteria that live within the normal equine stomach, but the diversity of bacteria adherent to the stomach epithelium decreases during ulceration that implies the potential development of a population of pathogenic organisms. Lactic acid, produced in the stomach of horses increases the permeability of the equine stomach (nonvii

glandular mucosal), and this may be important in the pathogenesis of gastric ulcers. The project went further to develop a model of dietary induced gastric ulceration, to monitor natural recovery on pasture and to use treatments aimed at microbial populations. The results indicated that we could induce gastric ulceration in stabled horses rapidly by increasing the concentrate grain based portion of the ration to five kilograms per day. Also the severity worsened when the roughage was restricted to three kilograms per day; similar to what is common practice amongst racehorse trainers in Australia. Lastly, treatment of gastric ulceration with oral antibiotics decreased the severity of gastric ulceration. There was a trend for the same effect with administration of a live bacterial culture ‘probiotic’ as a treatment. Implications The implications of this research are that we have established the role of bacteria in the pathogenesis of gastric ulceration in horses through laboratory experiments and studying gastric ulceration in the live horse. There is a great opportunity to further investigate the use of antibiotics/and or probiotics for treatment of ulcers in horses. A role for modification of microbial populations in the future treatment of gastric ulceration has been shown and the future use of probiotics and other non-medical manipulations of microbial populations in horse predisposed to gastric ulceration is a promising prospect. Recommendations The role of bacteria in the pathogenesis of gastric ulceration in horses has been established through laboratory experiments and studying gastric ulceration in the live horse. A role for modification of microbial populations in the future treatment of gastric ulceration has been shown and the future use of probiotics and other non-medical manipulations of microbial populations in horse predisposed to gastric ulceration is a promising prospect. It is recommended that further studies on the use of different live bacterial probiotics on the prevalence of gastric ulceration. It is also recommended that restriction of roughage in horses on a high grain diet should be avoided and that horse owners feeding their horses five kilograms of high grain based concentrate diet per day should be aware of the very high prevalence of gastric ulceration under this regimen, especially if the horse is concurrently confined to a stable.

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Introduction
Gastric ulceration is a widespread and very common problem in horses in training. Thoroughbred racehorses usually have the highest incidence of stomach ulceration, affecting more than 80% of horses in training (Vatistas et al., 1994). It is widely accepted that this is a problem resulting from feeding and management practices. Racehorses are fed twice daily of grain rich diets and usually fasted for an extended period before exercise. It is the combination of increased gastric acid production during exercise, reduction in saliva production due to low fibre content of the diet, and indoor confinement that likely contributes to the development of stomach ulcers. Physiologically, it is claimed that sudden increases in blood sugar supply elevates gastrin production, which in turn increases gastric acid production. This will lead to prolonged periods of exposure of the unprotected region of the stomach to acid, which is a major cause of ulceration. Gastric ulcers in the non-glandular region of the stomach are likely caused by exposure to organic acids (hydrochloric [HCl], volatile fatty [VFAs], and bile acids) and the inadequate barrier defences, including the lack of a thick mucus layer and bicarbonate (Nadeau et al., 2000; Berschneider et al., 1999; Bullimore et al., 2001; Nadeau et al., 2003a; Nadeau et al., 2003b). Horses with gastric ulceration may suffer weight loss and poor performance; some severe enough to result in retirement from racing. This clearly has significant economic and welfare implications and has serious cost implications to the equine industry. The exact economic impact of gastric ulcer disease in horses is not exactly known, however, gastric ulcer prevalence estimates range from 25 to 81%, of which approximately 50% of horses have clinical signs of poor performance, colic, and weight loss (Hammond et al., 1986). Furthermore, in a California study of racehorses gastric ulcer severity was correlated with poor performance (Vatistas et al., 1994). This cost is not only in lost days to training and racing as a result of poor performance or ill health, but also in lost days racing due to withholding periods of medications currently used to treat gastric ulceration. This may result in millions of dollars of lost revenue each year in training and racing days, and in the cost of treatment. Furthermore, in severe cases, gastric ulcer disease can result in acute death due to fatal haemorrhage and gastric rupture (Traub-Dagartz et al., 1985; Todhunter et al., 1986). If more were known about the dietary factors involved in gastric ulcer disease pathogenesis, perhaps feeding practices could be altered to decrease the incidence and prevalence of gastric ulcer disease in horses. Therefore, by decreasing the incidence and prevalence of gastric ulcer disease in horses, horse suffering and the economic loss to the racehorse industry could be minimized.

Animal welfare implications of gastric ulceration
The horse welfare issue has always been a key theme in RIRDC research programs. Previous programs promoted knowledge about effective nutritional and housing management and emphasised the needs to improve the health and welfare of the horse. As mentioned earlier, stomach ulceration is a man made problem often associated with concentrate feeding, stabling, intensive exercise and transport. Therefore, knowledge of the exact cause, will lead to the development of preventive measures, which undoubtedly improve the welfare of the horse.

The stomach of the horse
Horses are herbivores evolved to digest fibre. The digestive processes begin with enzymatic digestion in the stomach and small intestines followed by extensive fermentation in the caecal and colon. The stomach of the horse is relatively small comprising only 8-10% of the gastro-intestinal tract with a net capacity of 7.5 to 15 litters, depending on feed type. The stomach has two distinct regions, the upper half of the stomach consists of squamous epithelial cells that lack an apparent mucus layer, while the lower part is glandular gastric with secretory tissue that has a mucous membrane lining.

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Horses on pasture graze continuously and maintain a full stomach. Food entering the stomach is poorly mixed allowing a pH gradient between the entrance of the stomach, the cardiac region and the pyloric, glandular region. Under normal roughage feeding conditions saliva is continuously delivered to the stomach providing buffering to the region close to the entrance. The pH at the non-glandular region is around 5.4 while that of the pyloric region is around 1.8. As a result, horses maintained solely on pasture rarely develop digestive problems such as gastric ulcers. However, feeding grain or concentrate mixes rich in energy and feed deprivation for prolonged periods increases the exposure of the stratified squamous epithelial mucosa of the non-glandular region, resulting in the development of gastric ulcers (Murray and Eichorn, 1996).

Microbiology of the stomach of the horse
Although the organism has been detected in the equine stomach (Contreras et al., 2007), there is no evidence to suggest equine gastric ulcers are caused by Helicobacter pylori which is the bacterium that is the common cause of ulcers in humans (Pagan, 1997). In fact, the microbial community of the equine gastro-intestinal tract has received very little attention despite its importance to the health of the animal. Available information has dealt mainly with the fermentation processes in the hindgut, especially these related to fibre digestion (Lin and Stahl, 1995; Julliand et al., 1999; Daly et al., 2001) and fermentative acidosis (Al Jassim and Rowe, 1999). However, recent developments and use of molecular techniques have shown the bacterial diversity within the equine large intestine (Daly et al., 2001) and to a lesser extent that of the stomach (Scott et al., 2003). Bacterial fermentation starts in the stomach despite the mild acidic conditions of the nonglandular region of the stomach, with the production of VFA and lactic acid. Fermentation may cease at the more acidic glandular region of the stomach but lactic acid producing bacteria remain viable when horses are deprived of food for a period longer than 12 hours and seem to tolerate acid shock. An early report on the microbiology of fermentative acidosis in grain fed animals by Al Jassim and Rowe (1999) showed that Streptococcus bovis and Streptococcus equinus are the key lactic acid producing bacteria of the equine gastrointestinal tract. Previous work in our laboratory indicated the presence of a diverse microbial population that could survive the stomach environment of both starved and fed horses. More recently 25 bacterial isolates were selected on the basis of their dominance and lactate production from different parts of the gastrointestinal tract of healthy and laminitis induced horses. Sequence analysis of their 16S rDNA indicated that most of the isolates were very closely related to species of the genus Lactobacillus, including Lact. mucosae, Lact. delbrueckii, and Lact. salivarius (Al Jassim et al., 2005). Interestingly, some isolates were very closely related to Mitsuokella jalaludinii, a strong D-lactate producing bacterium. Little is known about other members of the lactic acid producing bacteria of the equine stomach and gastrointestinal tract. Aggressive lactic acid producing bacteria in the equine stomach have recently been identified– produce L and D-lactate which has previously been undiscovered/not identified in the horse GIT. Further, evidence has indicated build up of acids in the non-glandular region of the stomach due to microbial fermentation of soluble carbohydrates – leading to the production and accumulation of lactic acid. Recent work in our laboratories at Gatton showed that the concentration of lactic acids in the non-glandular region of the stomach of horses fed grass and grains (4 kg grass hay and 4 kg dry-rolled or steam-flaked sorghum) was higher than 40 mmol/l at 2 -6 h after feeding (Al Jassim 2006). The same study showed that all the lactic acid is absorbed from the gastrointestinal tract before the contents reaches the caecum. Absorption of lactic acid into the mucosa may cause some damage. As acid build up is suspected to be the main cause for stomach ulcers in horses, it is therefore important to identify and quantify the different contributors to acid build up. Recent knowledge has provided new insight into a bacterial involvement in gastric ulceration. Recent work in the US has shown that VFAs produced in the stomach of horses were associated with increased ulcer severity (Nadeau et al., 2000). These acids have a potent in vitro effect on reducing mucosal integrity, an effect even greater than that by normal gastric acid (HCl) reinforcing the concept that bacterial fermentation products play a role in the pathogenesis and persistence of gastric ulceration in horses (Nadeau et al., 2003a and b). 2

The hypothesis is that the production of acids, especially lactic acid, by these bacteria in the nonglandular region of the stomach is the vital pre-disposing cause for the development of gastric ulcers. Identification of the key lactic acid producing bacteria will aid the development of strategies to control gastric ulceration. This will undoubtedly help in reducing costs of maintaining racehorses and improve their health, welfare and performance.

Pharmaceutical treatment of gastric ulcers
While there are treatments available for gastric ulcers, they are expensive; unable to be used close to racing and do not target the specific pathogenesis nor prevent the development of gastric ulceration. Recent knowledge has provided new insight into a bacterial involvement in gastric ulceration. The use of ranitidine, a histamine type-2 receptor antagonist drug (Murray and Eichorn 1996), and omeprazole an acid pump inhibitor (Andrews et al., 1999) have proved to work well to treat stomach ulcers in horses. These drugs reduce the secretion of acid, but do not target microorganisms that colonise the lesions or those responsible for the build up of acid in the non-glandular part of the stomach. The cost of treatment is expensive and there is a high recurrence rate when treatment is withdrawn, thus dietary management in horses would reduce costs and decrease suffering in horses. Besides, horses continue to have ulcers even though they are maintained on acid suppressive doses of medications. A more comprehensive approach to ulcer management is surely indicated to reduce the cost of gastric ulcer disease in horses. The role of VFAs in causing acid injury may explain why current gastric acid secretory inhibiting agents are not completely effective in healing gastric ulcers in horses. For example, treatment with histamine type 2 (H2) receptor antagonists in 55 horses with gastric ulceration lead to endoscopically confirmed resolution of lesions or improvement in only 32 horses. Of 32 horses treated with ranitidine for gastric lesions, there was significant improvement in gastric lesion scores and in only 16 horses, complete healing. Omeprazole (0.7 mg/kg) given once daily via nasogastric tube to 8 horses with chronic gastric cannulae was found to inhibit basal and stimulated gastric acid output by 69% and 76% respectively and increased pH from 3.2 to 4.6 by the 5th day of treatment in basal gastric juice. In that same study, stimulated gastric juice pH increased from 1.7 to 4.6 by the 5th day of treatment. Gastric ulcers were healed in only 8 of 12 horses by 18 days and in 11 of 12 horses by 21 days of omeprazole treatment. Identification of the bacteria involved in the pathogenesis of gastric ulceration will provide the necessary information for the development and or selection of antibiotics for treatment of clinical cases. Therefore, we proposed that bacteria are involved in the pathogenesis and or progression of equine gastric ulcer signs and so specific treatment and prevention strategies can be developed to reduce the incidence of gastric ulcers in horses. The aims of this project were then: 1: To establish involvement of bacteria and their products (VFAs and lactic acid) in the pathogenesis of gastric ulceration in horses. 2: To determine the effect of contrasting dietary regimes on the microbial contribution to the build-up of lactic acid and VFAs in the non-glandular portion of the stomach.

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Objectives
Hypothesis: The production of acids, especially lactic acid, by bacteria in the non-glandular region of the stomach is the vital pre-disposing cause for the development and progression of gastric ulcers. Identification of the key lactic acid producing bacteria (LAB) and dietary regimens that minimise their multiplication will aid in the development of strategies to control gastric ulcers in horses. This will undoubtedly help in reducing cost of gastric ulcer treatment in racehorses and improve their health and performance. In order to address this hypothesis, we divided the research into 4 studies with the following objectives: Study 1: to determine the bacterial community of the normal horse stomach and compare that with the bacterial community of the ulcerated stomach. Study 2: to determine the in vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa. Study 3: To develop a model for induction and recovery of dietary induced gastric ulcers in horses. Study 4: To investigate treatment of dietary induced gastric ulcers in horses using modification of bacterial populations.

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Methodology
Studies were approved by the Institutional Animal Ethics Committee.

Study 1. The bacterial community of the horse stomach 1,2
This study involved the investigation of normal bacterial populations in the normal and abnormal equine stomach using material obtained from an abattoir.

Protocol
Samples of stomach contents and lining were obtained from twenty horses post mortem. Stomach contents were pooled into four groups of five horses each and transported immediately to the laboratory for processing. One group consisted of samples from five horses with ulcerated stomachs. In addition, mucosa from the glandular region and squamous tissue from the non-glandular region of the stomachs were taken from healthy and ulcerated horses for identification of bacteria associated with these tissues. Epithelial samples were washed with phosphate buffered saline (PBS) immediately after sampling, placed in a sterile container and kept on ice during transport to the laboratory. All samples were processed by: 1. Culturing and isolation of lactic acid producing bacteria. 2. Extraction of genomic DNA and analysis of bacterial diversity by denaturing gradient gel electrophoresis (DGGE). 1. Culture, isolation and identification of lactate-producing bacteria Stomach contents were cultured for enumeration of lactate-producing bacteria (bacteria that grew in MRS agar medium). Samples were processed under CO2 and serially diluted in tenfold increments up to a dilution of 1:107. Then, three dilutions (1:105, 1:106 and 1:107) were used to inoculate roll tubes containing MRS agar medium. A modified non-selective MRS roll tube medium, Oxoid, England (De Man et al., 1960) was used as described by Al Jassim et al., (2003). After incubation for 48 h at 39 °C, counts were carried out. Then 45 colonies were picked from the roll tubes, transferred into BM10 broth medium (Caldwell and Bryant, 1966) supplemented with glucose (0.3% w/v) and cultured in roll tubes again. The procedure was repeated twice. After another 48 h of incubation, the remaining cultures were examined under a microscope for purity, morphology and Gram staining. Genomic DNA was obtained from each of the pure isolates and the 16S rDNA was amplified by PCR. The diversity of pure isolates was initially determined by restriction fragment length polymorphism (RFLP) analysis, and sixteen selected isolates from each RFLP group were cloned and sequenced. A more definitive analysis of population diversity was undertaken by comparing the near-complete sequences of 16S rDNA with those found in public databases. 2. Denaturing gradient gel electrophoresis (DGGE) At the laboratory, the epithelial samples were placed in bag containing 10 mL of PBS and homogenized in a stomacher (Stomacher 400 Circulator, Seward Ltd., Thetford, UK) for two cycles of 30 s at 230 rpm. The samples were then strained through two layers of sterilized cheesecloth and the filtrate was retained for DNA extraction. The filtrate was centrifuged at 13,000 rpm for five min at
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R.A.M. Al Jassim, S. Denman, J.D. Hernandez, C. M. McGowan, F. M. Andrews, C. S. McSweeny The bacterial community of the horse stomach. RRI INRA Gut Microbiology: 5th Biennial Meeting: Research to Improve Health, Immune Response and Nutrition. Reprod. Nutr. Dev. 46 (2006) S7 P2 INRA, EDP Sciences, 2006. 2 R.A.M. Al Jassim, C.M. McGowan and F.M. Andrews. Bacterial diversity and role of lactic acid in the pathogenesis of acid injury in the non-glandular region of the equine stomach. Recent Advances in Animal Nutrition in Australia 2007.

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4°C. The pellet was then resuspended in 2 mL sterilized distilled water and vortexed. Genomic DNA was extracted from all samples (gut lining and stomach contents) using centrifugation, lysis buffer and bead beating. PCR was performed to amplify the V3 region of the 16S rDNA in preparation for denaturing gradient gel electrophoresis (DGGE) using primers 341F + GC clamp and 518R (Muyzer et al., 1993). Samples were then run under DGGE conditions for 18 h, after which the bands were visualized using silver staining. Twenty-five bands were chosen for recovery of DNA for further analysis. Plugs were taken from these bands for DNA analysis and sequencing. The DNA of the gel plugs was amplified by PCR using the primers previously described for amplification of the extracted DNA prior to DGGE analysis and then subjected to electrophoresis using a 2% agarose gel. Purified PCR products were then ligated into a pGEM-T Easy vector (Promega, Madison, USA) and transformed into E. coli Top10 cells. Sequencing was performed on five transformed colonies using the T7 primer found within the pGEM–T vector. Sequence data analysis DNA sequence data was edited using Bioedit software (Hall, 1999) and characterization for the most closely related sequence was performed by pairwise BLAST (Tatusova and Madden, 1999). The contiguous sequences were aligned in a 16S rDNA database using the ARB software package (http://www.arb-home.de/) and the phylogenetic tree was generated using the neighbourhood joining methods of the ARB software package. Bootstrap analysis using 2000 replicates was performed using Paup*4.0b 10 to ascertain the robustness of the tree topology.

Study 2: In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa 3
This study involved investigating the effect of lactic acid on the integrity of the lining of the horse stomach using an Ussing chamber. This work was carried out by our collaborator Professor Frank Andrews, at the University of Tennessee USA. The objectives of this study were to compare acute tissue injury induced by different concentrations of LA at different pH (1.5, 4.0, or 7.0). In vitro electrophysiological measurements will be correlated to histological evidence of cellular injury.

Specific Objectives
1. To expose the horse’s non-glandular stomach mucosa to varying concentrations of LA (0 [control], 5, 10, 20, 40 mmol) at different pH (1.5, 4.0, 7.0), using an in vitro Ussing chamber system and measure potential difference (PD) across the tissue and short circuit current (Isc) to determine cell viability and function. 2. To examine the non-glandular stomach mucosa in objective 1 using light microscopy to determine area of histopathologic changes, so that these changes can be correlated with alterations in PD across the tissue and Isc. 3. Mucosa from the non-glandular stomach of 15 horses not suffering from gastric disease was obtained immediately after euthanasia. Information regarding sex, breed, and age of animal, recent medical history and dietary history has been obtained. 4. Mucosal tissues were studied in the in vitro Ussing chamber system, exposed to the various concentrations of LA at a pH of 1.5, 4.0, or 7.0. 5. PD across the tissue and Isc were recorded every 15 minute for each of the treatment combination. 6. The tissue were examined under light microscopy for area of histopathologic changes using an image analyzer and these changes were correlated to electrical values, LA concentration and pH.

Techniques used

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F.M. Andrews, B.R. Buchanan, S.B. Elliott, R.A.M. Al Jassim, C.M. McGowan, and A.M. Saxton. In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa. Accepted EVJ special colic issue January 2008.

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Fresh gastric non-glandular mucosa from horses was studied using the in vitro Ussing chamber system. Once the tissues were mounted in the Ussing chamber system, spontaneous PD and Isc were measured using Ringer’s-agar bridges, whose composition was identical to the serosal solution bathing the surface. Tissue resistance was calculated from the open circuit PD and from the current necessary to nullify the PD, the Isc. The tissue was clamped at 100uA of current when the open circuit PD is < 1 mV, and the resulting PD was recoded. The mucosal surface of the stomach tissue was perfused either with Ringer’s solution (control) or Ringer’s solution with differing concentrations of LA added. The resulting PD and Isc were measured for each LA concentration and pH. Once measurements are completed, the tissues were removed from the chambers, and placed in 10% formalin. Formalin-fixed sections were embedded in paraffin, sectioned at a thickness of 5 um, and stained with H&E for examination by light microscopy.

Analytical methods
Means and pooled SEM were calculated. Data were analyzed using the mixed procedure of SAS. The model used for each tissue type and variable was a split-plot ANOVA with treatment (buffer conditions) in the main plot and time and time X treatment interaction in the subplot. In the presence of a significant time X treatment interaction, an ANOVA was performed at each point of test for differences between or among means. A Dunnett’s one-tailed test was used to determine whether any mean value differs from the control value. Variables were correlated with area of histopathologic change using a Pearson moment correlation coefficient. A P ≤ 0.05 was considered significant.

Study 3: Induction and recovery of dietary induced gastric ulcers in horses 4
Ulcers were induced in 12 Thoroughbred horses by simply using stable confinement, low level exercise and high concentrate diet similar to that used in many Australian racing establishments over a period of 10 weeks. Once ulcers were induced, the horses were turned out to pasture to investigate how long it took them to recover from ulceration naturally, before an intervention experiment was carried out (Study 4).

Protocol
There were 8 mares and 4 geldings, with a mean age of 7.7 ± 3.5 years and an average weight of 478 ± 37 kg. 1. Pre-trial pasture period Horses had an initial 4 months pasture rest from August to November, representing predominantly tropical spring pastures. Pasture size was approximately 10 ha and able to accommodate the horses without supplementary feeding. Pasture was typical SE QLD pasture with a predominance of kikuyu and tropical grasses. 2. Induction period This induction period was 10 weeks in duration and involved bringing the horses in and housing them in a stable, feeding concentrate based diet and allowing exercise 5 days a week on a 12 bay horse walker. Housing allowed horses to see and interact with other stabled horses. Exercise consisted of 10 minutes walking and 10 minutes trotting followed by 5 minutes walking to cool down. On days when horses were not exercised on the walker they were turned out into a round yard during the cleaning of their boxes in small groups of 2 to 4.
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C.M. McGowan, T.W. McGowan, F.M. Andrews and R.A.M. Al Jassim. Induction and recovery of dietary induced gastric ulcers in horses. Journal of Veterinary Internal Medicine, Volume: 21 Issue: 3 Pages: 603 Abstract: 115, 2007.

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Diet • All horses were fed the total diet as 2 equally divided meals twice daily • • • • • • Acclimation period: Acclimation (2 wks, during weeks 0 -2) Horses were fed lucerne (alfalfa) hay (6 kg) and oats (3 kg) High roughage diet: (4 wks, during weeks 2 - 6) Horses were fed lucerne hay (5 kg) + concentrate (4 kg) Low roughage diet: (4 wks, during weeks 6 - 10) lucerne hay (3 kg) + concentrate (5 kg)

Table 1. Chemical composition of the hay and concentrate mixture fed to horses Chemical composition (%DM basis) Feed DM% OM Ash NDF ADF CP DE MJ/kg DM Hay 90.4 92.5 7.5 34.4 22.3 16.2 10.40 Concentrate 79.1 94.7 5.3 29.6 10.7 13.8 14.47 Legend: DM = dry matter, OM = organic matter, Ash = mineral ash, NDF = neutral detergent fibre, ADF = Acid detergent fibre, CP = crude protein, DE = digestible energy Table 2. Composition of the concentrate mix Inclusion Ingredients AS FED% OATS (Whole or 35.70 Bruised) BARLEY (Steam flaked) 30.00 CORN (Cracked) 14.00 Wheat Bran 4.00 Sunflower Seeds (Black) 4.00 Soyabean Meal 45.0 5.00 Molasses 5.00 Limestone 1.20 Di Calcium Phosphate 0.25 Salt 0.75 Dairy Vit/Min Premix 0.10

3. Post trial Recovery period Following the endoscopy at week 10, horses were returned to pasture for a further 6 weeks (during weeks 10 – 16). This was during February to March and represented drier, Autumn pasture and so was supplemented with lucerne (alfalfa) hay.

Endoscopy and stomach sampling Gastroscopy examination using a 3 m Olympus endoscope was performed every 2 weeks from week 0.

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Horses were fed the evening before and had feed withheld the morning of the study. Water was withheld during the final 2 hours before examination. Horses were restrained in breeding stocks and sedated with xylazine and butorphanol to facilitate examination without use of other restraint (Figure 1).

Figure 1. Gastroscopy examination procedure. From the left: Dr Rafat Al Jassim, Dr Thomas McGowan, Horse 4, and Dr Catherine McGowan.

Complete gastroscopy examinations captured using computer digital video software and recorded. Ulcers graded using the Number/Severity system (MacAllister et al., 1997) (Table 3) independently by 2 people blinded to each other and the horse. The average grade was used for analysis. As well as documenting the increase in ulcer lesions endoscopically, small samples were taken throughout the study (every 2 weeks) of the gastric muscosa and gastric contents and analysed as per study 1 for bacterial populations, investigating the populations both in pastured horses, and those on high concentrate diets, and the bacterial involvement in gastric ulceration.

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Table 3. Grading system for gastric ulcers based on MacAllister et al., 1997 Number Scores 0 = none seen 1 = 1-2 localised lesions 2 = 3-5 localised lesions 3 = 6-10 lesions 4 = > 10 lesions or diffuse (or very large lesions) Severity Scores 0 = none seen 1 = Appears superficial (only mucosa missing) 2 = Deeper structures involved (greater depth than number 1) 3 = Multiple lesions and variable severity (1,2 and / or 4) 4 = same as 2 and has active = hyperaemic and/or darkened lesion crater 5 = same as 4 plus active haemorrhage or adherent blood clot

Study 4: Treatment of dietary induced gastric ulcers in horses
Involved re-induction of gastric ulceration using the method outlined above in study 3, yet once ulceration was confirmed in 12 horses; they were divided into 3 groups. Group 0 horses were simply control horses and were maintained on full rations with no treatment. Group 1 and 2 were also maintained on full ration, but group 1 received a probiotic, a live bacterial culture of “good” bacteria, based on the predominant lactic acid bacteria identified in study 1. The selected bacteria are known for their probiotic characteristics and have the ability to adhere to stomach lining. Group 2 were given an oral antibiotic. Samples as per study 1 for bacteriological analysis were also collected via endoscopy and following the conclusion of this study, a representative number of horses from each group were euthanased humanely and their stomachs harvested for full microbial analysis. There were initially 15 Thoroughbred horses, 10 from the previous trial that had been rested at pasture for 6 months, and 5 new horses that had an unknown period out of high intensity training. There were 8 mares and 7 geldings, with a mean age of 7.2 ± 4.5 years and weighing an average of 485 ± 39 kg. Induction period The induction period was 4 weeks in duration, following 1 week of acclimation where the concentrate diet was introduced. Horses were housed in a stable, feeding concentrate based diet and allowing exercise 5 days a week on a 12 bay horse walker. Exercise consisted of 10 minutes walking and 10 minutes trotting followed by 5 minutes walking to cool down. On days when horses were not exercised on the walker they were turned out into a round yard during the cleaning of their boxes in small groups of 2 to 4. Diet • All horses were fed the total diet as 2 equally divided meals twice daily. • • • • Acclimation period: Acclimation (1 wk, during weeks 0 -1) Horses were fed lucerne (alfalfa) hay (5 kg) and concentrate mix (3 kg) Induction diet: (8 wks, during weeks 1 - 9) Horses were fed lucerne hay (3 kg) + concentrate mix (5 kg)

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Endoscopy and stomach sampling Gastroscopy examination using a 3 m Olympus endoscope was performed in week 0, 3, 4, 7 and 9. Horses were fed the evening before and had feed withheld the morning of the study. Water was withheld during the final 2 hours before examination. Horses were restrained in breeding stocks and sedated with xylazine and butorphanol to facilitate examination without use of other restraint (Figure 1). Complete gastroscopy examinations captured using computer digital video software and recorded. Ulcers graded using the Number/Severity system (MacAllister et al., 1997) independently by 2 people blinded to each other and the horse. The average grade was used for analysis. As well as documenting the increase in ulcer lesions endoscopically, small samples were taken throughout the study (every 2 weeks) of the gastric muscosa and gastric contents and analysed as per study 1 for bacterial populations, investigating the populations both in pastured horses, and those on high concentrate diets, and the bacterial involvement in gastric ulceration. Treatments At week 4, endoscopy revealed 3 horses that did not develop gastric ulceration and so they were removed from the trial and the remaining 12 horses divided into 3 groups by RAJ so that neither of the veterinarians performing the endoscopy knew which treatment group horses were in. There were 6 mares and 6 geldings, with a mean age of 7.3 ± 3.8 years and weighing an average of 479 ± 41 kg. Treatment was started in week 5 and horses were examined after 2 and 4 weeks of treatment (weeks 7 and 9). 1. Probiotic preparation: Lactobacillus agilis Lactobacillus salivarius Lactobacillus equi Streptococcus equinus Streptococcus bovis Dose and concentration: Final concentration was between 109 and 1010 viable cell per ml Dose: 50 ml of the blend which provided 5 × 1010 to 5 × 1011 viable cells. 2. Oral antibiotic Trimethoprim suphadimidine (Trimidine powder Parnell laboratories (Aust) pty ltd) 15 mg/kg bid per os. 3. Control Horses continued with the diet, confinement and exercise as per study 1 for 4 weeks.

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Results
Study 1. The bacterial community of the horse stomach
The main findings of this study were 1.the diversity of bacteria adhering to the lining of the nonglandular region of the stomach decreased during ulceration which could results in opportunistic pathogenic organisms to colonise the ulcers, 2. Cultured bacteria clustered with common bacterial species belonging to the genera Lactobacillus, Streptococcus and Clostridium (Figure 2), while DGGE clones were more diverse with main groups clustering with the genera Propionibacterium, Clostridium, Lactobacillus, Prevotella, Pasteurella, , and Pseudomonas. Other fewer clones were closely related to Escherichia, Actinobacillus, Moraxella, Rhodococcus, Veillonella, Legionella and Eubacterium (Figure 3). Selected bacterial species on the basis of dominance and biochemical characteristics were selected for use as probiotics in study 4. 1. Culture, isolation and identification of lactate-producing bacteria The mean number of colony forming units in stomach contents cultured on modified MRS-agar medium was 2.3 × 107. Forty-five colonies were picked and transferred to a broth of BM10 medium supplemented with glucose (0.3%). Based on morphology, Gram stain reaction and RFLP profile, sixteen isolates were identified by 16S rDNA sequencing. The sixteen isolates belonged to three main groups closely related to the genera Lactobacillus, Streptococcus and Clostridium (Figure 2). Two isolates from ulcerated squamous tissue (UM21 and U31b) were closely related (98% identity) to Lact. agilis (M58803). Isolate G43, which was derived from the contents of healthy stomachs, was similar (99%) to Lact. equi (AB048833.1). Isolate G46 was closely related (99%) to Lact. salivarius strain RA2115 (AY389803.1, M59054) and isolate G1 was closely related (99.9%) to S. bovis (AF202263) and S. equinus (X58318) (98.7%). Isolate G113 clustered with S. equinus (X58314). Isolates G31, G34, G36, G315, G33, G310 and G32 originated from the stomach contents of healthy horses and were all closely related (≥ 99%) to C. perfringens (AB045283). Isolate U31 from ulcerated stomach contents clustered with C. butyricum (X68177) and G313 clustered with C. bifermentans (X75906). Similarities between the isolates and their closest known bacterial species were 90.8% and 84.9% for U31 and G313, respectively.

Figure 2. Phylogenetic relationship of the derived sequences from 16S rDNA of cultured bacteria 2. Denaturing gradient gel electrophoresis profiles (DGGE)

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Initial analysis of DGGE profiles indicated apparent differences in bacterial diversity between sample groups. Ulcerated tissues had less bacterial diversity than normal tissue from healthy stomachs. The bacteria represented by the bands must have the capacity to adhere to the stomach lining because the samples were washed with PBS. Samples of stomach contents containing residual feed produced more bands than samples free of residual feed, which indicates that the former samples had a more diverse bacterial community than the latter samples. The additional bands may represent bacteria associated with feed particles or bacteria ingested with the feed. Sequences generated from DGGE clones comprised a short region ( 200 bp; V3) of the 16S rRNA gene and were not used to determine phylogenetic identity. However, they were used to presumptively identify the different clones and relate them to database isolates. Eighty percent of the 56 clones (25 DGGE bands) belonged to five main genera: Prevotella (29%), Clostridium (14%), Pseudomonas (13%), Propionibacterium (13%) and Lactobacillus (11%) (Figure 5). Other clones (20%) clustered with Escherichia coli (n = 1), Legionella (n = 1), Voraxella (n = 2) and Pasteurella (n = 4) (Figure 3).

Propionibacterium spp 13% Eschirichia coli 2% Pasterell spp 7% Legionella londiriensis 2% Voraxella canis 4% Pseudomonas spp 13%

Rodococcus rhodnii 5% Prevotell ruminocola 29%

Clostridium spp 14% Lactobacillus spp 11%

Figure 3. Clones derived from denaturing gradient gel electrophoresis (DGGE) bands identified using the V3 region of 16S rDNA. Genomic DNA was extracted from stomach contents and stomach mucosa.

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Study 2: In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa
Animals and gastric tissues Thirteen horses aged from 2 to 33 years were recruited. 83% had low grade gastric ulcers in the nonglandular mucosa. Tissue specimens were collected from grossly normal nonglandular mucosa at or adjacent to the margo plicatus in the stomach of each horse, which is the region of the equine stomach where most ulcers develop (Begg and O’Sullivan 2003). Sixteen tissue specimens were collected from each of the 13 horses, making a total of 208 tissue specimens collected from this region. The 13 tissues (1 from each horse), immediately placed in neutral-buffered 10% formalin, did not have evidence of gross or histopathologic changes. Of the remaining 195 samples, 194 were analyzed in Ussing chambers. One tissue sample was discarded due to a chamber malfunction. The variables measured did not differ significantly between tissues from horses with ulcers and tissues from horses without ulcers. HCl exposure and recovered mucosa Decreasing the pH from 7.0 to 1.5 and 4.0 caused Isc and PD to decrease significantly indicating abnormal bioelectric properties. Mean Isc in tissues perfused with NRS at pH 7.0, 4.0 and 1.5 decreased 29%, 41%, and 41% from their initial values, respectively during the 270-minute exposure. Mean PD across the tissues decreased by 50% in tissues exposed to NRS at a pH of 1.5, compared with 5% from their initial values in tissues exposed to NRS at a pH of 7.0 and 4.0. Mean R or G did not change over the exposure time. After pH was adjusted to 7.0, mean Isc and PD immediately increased to near control values in tissues exposed to NRS at a pH of 1.5. Analysis of these data suggests that sodium transport in the nonglandular tissue and barrier function recovered once pH was increased to 7.0. Thus, the affect of HCl on sodium transport and barrier function is reversible; however prolonged exposure may lead to irreversible tissue damage and ulceration. Lactic acid exposure and recovered mucosa Mean Isc in tissues perfused with all concentrations of LRS at a pH of 4.0 and 7.0 did not significantly change from control tissues. Although there were no significant changes in tissue parameters during the study, there were trends toward and increase in tissue conductance and decrease in tissue resistance. It may be that LA requires a longer exposure than the 270 minutes to cause significant damage or may act synergistically with VFAs to cause significant acid injury. Mannitol fluxes Because tissue exposed to a 40 mM concentration of LRS showed a trend toward increased G and lower R, nonglandular tissues (n=2) were exposed to LRS (40 mM) in [14C] mannitol at pH 1.5. These tissues showed increased permeability to [14C] mannitol when compared to tissues exposed to the same level of LA at pHs 4.0 and 7.0. The increase in cell permeability and conductance with a corresponding decrease in tissue R may indicate that higher concentrations of LA at a low pH may cause damage to the nonglandular mucosa by disrupting paracellular spaces and allow leakage of acid between and into cells causing ulcers. LA and mucosal histopathologic changes Histologic examination of specimens of nonglandular mucosa exposed to NRS and LRS, at various concentrations, at pH of 4.0 and 7.0 were normal. On the other hand, mucosa exposed to NRS at a pH of 1.5 had multifocal cellular swelling and a mottled appearance in the superficial stratum corneum and stratum transitionale. Mucosa exposed to LRS at the various concentrations and pHs did not show cell swelling in the stratum transitionale and stratum spinosum other than was observed in tissues 14

exposed to NRS alone at the same pH. Thus, exposure of nonglandular mucosa to LRS (5, 10, 20, or 40 mM) a low pH did not result cell swelling consistent with damage to cellular sodium transport. The increased nonglandular mucosa permeability caused by LRS (40 mM) may be related to extracellular mechanisms.

Study 3: Induction and recovery of dietary induced gastric ulcers in horses
During the induction period 11 of 12 horses developed ulcers. The one horse that did not develop ulcers had ulcers at beginning and again 4 weeks on pasture, but not during the treatment period. Only one horse with ulcers (6) showed overt clinical signs (refusal of concentrate, and weight loss) but four others chose to eat roughage prior to concentrate. Also, after initial increase, there was a reduction in bodyweight on the high carbohydrate and restricted roughage diet, in weeks 6 and 8 (P<0.01) corresponding to the greatest severity of gastric ulceration. Then there was a rapid increase of bodyweight again from week 11 after the horses had been on pasture for one week following induction period (P<0.001) (Figure 4).

Figure 4. This graph represents the mean body weight for each week of trial 1 (95% confidence interval error bars) Gastric ulcer lesions were already greater than baseline by 2 weeks of concentrate feeding and increased in an almost linear fashion until they were turned out to pasture, where the ulcer scores decreased in a parallel manner (Figure 5).

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3.5 3 2.5 2 1.5 1 0.5 0 0 2 4 6 8 Week 10 12 14 16

Number Score Severity Score

Figure 5. Mean gastric ulcer score for 13 horses fed concentrate ration and being confined during weeks 0 – 10 and subsequently during pasture recovery during weeks 10-16. Number and severity are shown separately as blue and purple respectively The mean ulcer score of horses of horses in weeks 8 and 10 was approximately 3 (Figure 6, Table 3) demonstrating the severity of ulceration that would be common among similarly managed horses in the equine industry.

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Figure 6. Endoscopic images of severity grade 3 ulceration in two horses There were, however, a few surprising findings. Although they were mostly low grade, ulcers were found in 5 horses after 4 months pasture at the beginning the study (week 0). Severity: mean 0.54 ± 0.78; Number: mean 1.33 ± 1.74. Further, ulcer lesions were not resolved in 45% horses and new lesions occurred in 42% horses during 6 wks pasture recovery period. New lesions were diffuse superficial lesions but had a lot of inflammatory reaction associated with them. These results clearly demonstrate that confinement and feeding of high concentrate diets is sufficient to induce gastric ulceration in horses, without intense exercise. Gastric ulcers may not heal on pasture in a 6 week period in a proportion of horses and further that new, albeit low grade, ulcers can develop in pastured horses.

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Study 4: Treatment of dietary induced gastric ulcers in horses
During the induction period of Study 4, mean ulcer score increased similarly to study 3, to a peak score of 3.42±0.79 (number) and 2.42± 0.79 (severity) in week 4, following 4 weeks of high concentrate diet (Figures 7 (number score) and 8 (severity score)). During the treatment period, mean ulcer grade decreased from week 4 to week 9 overall to a mean of 2.00±1.6 (number) and 1.42±1.24 (severity). When groups were examined separately, ulcer scores were not significantly different at week 4. Means for groups 0 (control), 1 (Probiotic) and 2 (TMPS) were 3.75±0.50 (number) and 2.75± 0.50 (severity); 3.25±0.96 (number) and 2.25 ± 0.96 (severity); and 3.25±0.96 (number) and 2.25± 0.96 (severity) respectively. However in week 9 means for groups 0 (control), 1 (Probiotic) and 2 (TMPS) were 3.25±0.5 (number) and 2.25± 0.96 (severity); 2.25±1.50 (number) and 1.50± 1.29 (severity); and 0.50±1.00 (number) and 0.50± 1.00 (severity) respectively. Note that ulcers continued to increase in severity in comparative weeks (5-9) on the same diet and exercise regimen as study 3. This shows that oral antibiotics (TMPS) and to a lesser extent oral administration of probiotics reduce ulcer lesion number and severity compared to controls horses in a 4 week period.

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Tri al 2 Tri al 2 WK_0 WK_3 number number Trial 2 Tri al 2 Tri al 2 WK_4 WK_7 WK_9 number number number

C ontrol Group Pro-biotic Group Antibi oti c Group

Figure 7. Graph representing the mean number scores for the 3 different groups in the study (Control, Pro-biotic, and Antibiotic) for each week during trial 2 (95% CI error bars)

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5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Trial 2 Trial 2 Trial 2 Trial 2 Trial 2 WK_9 WK_7 WK_4 WK_3 WK_0 severity severity severity severity severity

C ontrol Group Pro-biotic Group Antibiotic Group

Figure 8. Graph representing the mean severity score for each of the 3 groups (Control, Probiotic and Antibiotic) for each week during the study

Analysis of the Severity of gastric ulceration
ANOVA was used to assess for differences in the mean severity scores between weeks 0 and 4 within each group and between weeks 4 and 9 within each group. The Control Group and the Pro-biotic Group mean severity scores were significantly higher in week 4 as compared to week 0 (p=0.003 and p=0.042, respectively), but not significantly different from week 4 to week 9. The antibiotic group mean severity score was significantly higher in week 4 as compared to week 0 (p=0.013) and week 9 was significantly lower than week 4 (p=0.027). There was no significant difference in mean scores of severity between week 3 and week 4 for any of the 3 groups. There was no significant difference of the mean severity of ulcer scores among the 3 groups in weeks 0, 3, or 4. There was a trend (p=0.067) for the antibiotic group to have a lower mean score of severity of gastric ulcers as compared to the control group in week 7. There was no significant difference between the mean severity scores of the Control Group compared to the Pro-biotic Group or between the Pro-biotic group and the antibiotic Group in week 7. The mean of the severity score of the Control Group was significantly higher than that of the antibiotic group (p=0.05) when analysis of the means of severity scores was performed across the 3 groups within week 9. There was no significant difference between the mean severity score of the Control Group and the probiotic Group, nor was there a significant difference in the mean severity scores between the Pro-biotic group and the Antibiotic Group.

Analysis of the Number of gastric ulceration
There was no significant difference in the means of numbers of gastric ulcers among the 3 groups (Control Group, Pro-biotic Group and antibiotic Group) when compared across the weeks 0, 3, and 4. There was a trend for the mean score of the antibiotic group to be lower than that of the Control Group (p=0.072). There were no other significant differences in week 7. In week 9, the mean score for the number of ulcers in the antibiotic Group was significantly lower than the mean score for the Control Group (p=0.006).

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Discussion of results
This study has achieved its aims by increasing the knowledge of gastric ulceration in horses, and highlighting how bacteria play a role by contributing to increased acidity of the stomach when horses fed concentrate diet, by identification of the different bacterial groups in ulcerated and healthy stomachs, by identification of bacteria in ulcers in vivo and by the response of horses to antibiotic and probiotics in markedly reducing gastric ulceration under proven ulcerogenic conditions.

Bacterial Community of the equine stomach
The results of study 1 have confirmed the diverse number of bacterial species that live in the equine stomach, many of which are acid tolerant and capable of fermenting starch and other readily fermentable carbohydrates and produce lactic acid which is along with the common short chain fatty acids produced could have a role in damaging the mucosal lining of the stomach. The culture-dependent technique provided evidence of the selectivity of the stomach environment for acid-tolerant bacteria of two main genera: Lactobacillus and Streptococcus, both are lactate producers. Other culturable bacteria were mainly spore-forming pathogenic clostridia, including C. perfringens and C. botulinum. These pathogenic bacteria are known as low-GC Gram positive and endospore forming bacteria that produce toxins responsible for gastroenteritis in humans and animals. The source of these clostridia was probably the collection yard because soil is their main habitat, where they live primarily in pockets rendered anoxic by other facultative organisms that metabolize organic compounds. Horses ingest a range of environmental microorganisms while searching for feed in the yards where they are held before slaughter. The presence of these bacteria in the stomach may be a temporary phenomenon and cause no harm to the horse when the microbial population of the gastrointestinal tract is balanced and the stomach and intestinal lining is healthy and colonised by the friendly bacteria. However, it is not known whether they have a functional role in the stomach or whether they produce toxins while resident in the stomach. The presence of high numbers of bacteria in the stomachs of fasted horses (2.3 × 107) has not been documented. The bacterial count in the stomach is generally expected to be very low because of its acidity, which acts as a chemical barrier to entry of microorganisms to the intestine. However, it is important to note that a pH gradient exists in the stomach of the horse; the non-glandular region is less acidic than the rest of the stomach and enables acid-tolerant bacteria to grow. The molecular techniques used in this study overcame the problem of media selectivity and revealed a diverse bacterial community. These techniques were based on analysis of genomic DNA, which was derived from all bacteria present in the sample regardless of whether they were an established community or were transient. In addition to the main bacterial groups that were identified by culture on MRS agar medium, bacteria belonging to the genera Pseudomonas, Prevotella and Propionibacterium were identified using DGGE analysis. Other genera identified were Escherichia, Legionella, Voraxella and Pasteurella. Although these were less prevalent than the other genera and were derived from fewer clones, they may play a role in causing disease under certain conditions. Further work is required to determine the roles of the various groups of bacteria, particularly the culturable species, in the pathogenesis of stomach ulceration in horses. Research with rats suggests that stomach bacteria may colonize gastric ulcers caused by acid damage and delay healing (Elliott et al., 1998). Work underway to complete the analysis of DNA samples that has been obtained from ulcers and healthy stomachs of horses used in ulcers induction trials. Preliminary data indicates that good bacteria may lose their ability to colonise the stomach lining when ulcerated. Most biopsies from ulcerated sites produced poor DNA after washing with saline solution compared with that from healthy region.

The role of lactic acid
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These results indicate that exposure of the non-glandular squamous mucosa of the stomach to lactic acid in the presence of HCl results in reduction of mucosal barrier integrity, which enables HCl to diffuse into tissue layers immediately deep to the stratum corneum of the non-glandular mucosa. This may undermine the superficial mucosa and cause ulceration. Although the damage observed in these in vitro experiments was mild compared to those observed after exposure to high concentrations of VFAs, a longer exposure may be needed to induce a similar extent of acid injury. Some horses may be more susceptible to the damaging effects of lactic acid than others, which would explain why some horses develop ulcers and others do not. The presence in gastric fluid of lactic acid, a by-product of bacterial fermentation, may contribute to gastric ulcer disease and may partly explain why diets high in soluble carbohydrates have been associated with the development of gastric ulcers. Further in vivo research is needed to determine the effects of lactic acid alone and in combination with other byproducts of fermentation such as VFAs to ascertain whether these compounds exhibit synergy in altering mucosal bioelectric properties.

Induction of gastric ulcers
Ulcers occurred in stabled horses on high concentrate, low roughage diet without intense exercise stimulus. It is not known how much of the stimulus could be related to housing as housing alone has been shown to have a role in the development of gastric ulceration in horses (Murray and Eichorn 1996). However, the increasing severity with increased concentrate and decreased roughage supports the contention that the diet had a large role in the development of the widespread severe ulceration in horses in this trial. The other interesting finding in this study was to observe the lesions over time. It appears that severity score 1 lesions also represent early lesions which contract and form a crater (severity 2) lesions over a number of days. Some of these ulcers will resolve while in other situations, especially in intensively managed horses, the ulcers progress to deeper lesions and do not resolve. In our research, we noted that ulcers did not resolve on pasture and further, that new ulcers developed. This is supported by recent research in New Zealand in racehorses trained from pasture (Bell et al., 2007). It seems that horses on pasture do have ulceration, but these tend to cycle through grade 1 to 2 severity and not progress. The results of this study support the hypothesis that the non-resolving lesions represent lesions colonised by bacteria where bacterial products further damage and allow progression rather than healing of gastric ulceration.

Treatment of gastric ulcers
Further supporting our original hypothesis of the involvement of bacteria in the progression and severity of gastric ulceration are the results of this final study. Despite horses being managed exactly the same way as in Study 3 where ulceration continued to increase in severity across the group, horses being treated with an oral antibiotic had a significant and dramatic reduction in their gastric ulcer severity score. The reduction was evident as a trend in just two weeks, but was fully significant as 4 weeks. These results are as good as or better than the results from antacid treatments. However, even more importantly was the indication that probiotics may do the same. The small volume of live bacteria given orally per day was able to reduce the severity of ulcers even in this small group. While these results were not statistically significant the trend is there, and in a small study of just four horses, is enough to warrant further research to test the possibility of a response in a larger number of horses in the natural situation. Use of a probiotic instead of an antibiotic has huge advantages in avoiding use of antiobiotics that can promote bacterial resistance, and avoiding expensive and potentially controlled medications for competition.

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Implications
The implications of this research are that we have established the role of bacteria in the pathogenesis of gastric ulceration in horses through laboratory experiments and studying gastric ulceration in the live horse. There is a great opportunity to further investigate the use of antibiotics/and or probiotics for treatment of ulcers in horses. A role for modification of microbial populations in the future treatment of gastric ulceration has been shown and the future use of probiotics and other non-medical manipulations of microbial populations in horse predisposed to gastric ulceration is a promising prospect.

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Recommendations
We recommend further studies on the use of different live bacterial probiotics on the prevalence of gastric ulceration. We also recommend that restriction of roughage in horses on a high grain diet should be avoided and that horse owners feeding their horses five kg high grain based concentrate diet per day should be aware of the very high prevalence of gastric ulceration under this regimen, especially if the horse is concurrently confined to a stable.

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References
Al Jassim, R.A.M. (2006). Supplementary feeding of horses with processed sorghum grains and oats. Animal Feed Science and Technology 125: 33–44. Al Jassim R.A.M. and Rowe J.B. (1999). Better understanding of fermentative acidosis and its control. Recent Advances in Animal Nutrition in Australia 12: 91-97. Al Jassim, R.A.M., Scott, P.T., Trebbin, A.L., Trott, D., and Pollitt, C.C. (2005). The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract. FEMS Microbiology Letters, FEMS Microbiology Letters 248: 75-81. Al Jassim, R.A.M., Gordon, G.L.R and Rowe, J.B. (2003). The effect of basal diet on lactateproducing bacteria and the susceptibility of sheep to lactic acidosis. Animal Science 77: 459-469. Andrews F.M., Sifferman R.L., Bernard W., Hughes F.E., Holste J.E., Daurio C.P., Alva R. and Cox J.L. (1999). Efficacy of omeprazole paste in the treatment and prevention of gastric ulcers in horses. Equine Vet J Suppl. 29:81-86. Bell R.J., Kingston J.K., Mogg T.D., Perkins N.R. (2007). The prevalence of gastric ulceration in racehorses in New Zealand. NZ Vet J. 2007 February 55(1): 13-8. Berschneider H.M., Blikslager A.T. and Roberts M.C. (1999). Role of duodenal reflux in nonglandular gastric ulcer disease of the mature horse. Eq. Vet. J. Suppl 29:24-29. Bullimore S.R., Corfield A.P., Hicks S.J., et al., (2001). Surface mucus in the non-glandular region of the equine stomach. Res. Vet. Sci. 70:149-155. Caldwell, D.R. and Bryant, M.P. (1966). Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Applied Microbiology 14: 794-801. Contreras M., Morales A., García-Amado M.A., De Vera M., Bermúdez V. and Gueneau P. (2007). Detection of Helicobacter-like DNA in the gastric mucosa of Thoroughbred horses. Lett Appl Microbiol. November 2007 45(5):553-7. Daly, K., Stewart, C.S., Flint, H.J. and Shirazi-Beechey, S.P. (2001). Bacterial diversity within the equine large intestine as revealed by molecular analysis of cloned 16S rRNA genes. FEMS Microbiology Ecology. 38: 141-151. De Man, J.C., Rogosa, M. and Sharp, M.E. 1960. A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23: 130-135. Hammond C.J., Mason D.K., Watkins K.L. (1986). Gastric ulceration in mature Thoroughbred horses. Equine Veterinary Journal. 18(4): 284-287. Julliand, V., de Vaux, A., Millet, L. and Fonty, G. (1999). Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine caecum. Applied and Environmental Microbiology. 65: 3738-3741. Lin, C. and Stahl, D.A. (1995). Taxon-specific probes for the cellulolytic genus Fibrobacter reveals abundant and novel equine-associated population. Applied and Environmental Microbiology. 61:13481351. MacAllister C.G., Andrews F.M., Deegan E. (1997). A scoring system for equine gastric ulcers. Equine Veterinary Journal 29: 430-433. 24

Murray, M.J. and Eichorn, E.S. (1996). Effect of intermittent feed deprivation, intermittent feed deprivation with ranitidine administration, and stall confinement with ad libitum access to hay on gastric ulceration in horses. American Journal of Veterinary Research. 57: 1599-1603. Nadeau J. A., Andrews F. M. Mathew A. G. (2000). Evaluation of diet as a cause of gastric ulcers in horses. American Journal of Veterinary Research. 61:784-790. Nadeau J. A., Andrews F. M., Patton C. S. Argenzio R. A., Mathew A. G. and Saxon A. M. (2003a). Effect of hydrochloric, acetic, butyric, and propionic acids on pathogenesis of ulcers in the nonglandular portion of the stomach of horses, American Journal of Veterinary Research. 4: 404-412. Nadeau J. A., Andrews F. M., Patton C. S. Argenzio R. A., Mathew A. G. and Saxon A. M. (2003b). Effect of hydrochloric, valeric, and other volatile fatty acids on pathogenesis of ulcers in the non nonglandular portion of the stomach of horses, American Journal of Veterinary Research. 4: 413-417. Pagan, J.D. (1997). Gastric ulcers in horses: A widespread but manageable disease. World Equine Veterinary Review. 2: 1-5 Accessed at: http://www.when.com/WEVR/v2n4/07/. Peat, Marwick, Mitchell, et al. (1987). The Economic Impact of the U.S. Horse Industry. Prepared by Policy and Economics Group, for the American Horse Council. (January, 1987). Todhunter R.J., Erb H.N., Roth L. (1986). Gastric rupture in horses: a review of 54 cases. Equine Veterinary Journal. 18(4): 288-293. Traub-Dagartz J., Bayly W., Riggs M., Thomas N., Pankowski R. (1985). Exsanguination due to gastric ulceration in a foal. Journal of the American Veterinary Medical Association. 186(3): 280-281. Vatistas N. J., Snyder J. R., Carlson G., Johnson B., Arthur R.M., Thurmond M., Lloyd K.C.K. (1994). Epidemiological study of gastric ulceration in the Thoroughbred race horses: 202 horses 19921993. 40th Annual Convention Proceedings of the American Association of Equine Practitioners: 125126.

25

Appendices
List of presentations
R. Al Jassim, An abstract/poster was presented at the RRI-INRA gut microbiology meeting in Aberdeen, Scotland 21-23 June 2006. R. Al Jassim, Seminar was presented at the College of Veterinary Medicine, University of Tennessee June 30, 2006. R. Al Jassim, Plenary talk at the Recent Advances in Animal Nutrition in Australia meeting, Armidale, Australia, July 9-11, 2007. R. Al Jassim, Plenary talk at The VII International Symposium on the Nutrition of the Herbivores, Beijing, China, September 17-22, 2007. C.M. McGowan, Induction and recovery of dietary induced gastric ulceration in Thoroughbreds, Australian College of Veterinary Scientists Science Week, Gold Coast, Australia, 2006. C.M. McGowan, Induction and recovery of dietary induced gastric ulcers in horses. ACVIM research abstracts Seattle Washington, June 2007.

Publications
R.A.M. Al Jassim, S. Denman, J.D. Hernandez, C. M. McGowan, F. M. Andrews, C. S. McSweeny The bacterial community of the horse stomach. RRI INRA Gut Microbiology: 5th Biennial Meeting: Research to Improve Health, Immune Response and Nutrition. Reprod. Nutr. Dev. 46 (2006) S7 P2 INRA, EDP Sciences, 2006. R.A.M. Al Jassim, C.M. McGowan and F.M. Andrews. Bacterial diversity and role of lactic acid in the pathogenesis of acid injury in the non-glandular region of the equine stomach. Recent Advances in Animal Nutrition in Australia 2007. C.M. McGowan, T.W. McGowan, F.M. Andrews and R.A.M. Al Jassim. Induction and recovery of dietary induced gastric ulcers in horses. Journal of Veterinary Internal Medicine 21(3): 603 Abstract: 115, 2007. F.M. Andrews, B.R. Buchanan, S.B. Elliott, R.A.M. Al Jassim, C.M. McGowan, and A.M. Saxton. In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa. Submitted Equine Veterinary Journal Special Colic Issue January 2008.

26

Gastric Ulceration in Horses
The role of bacteria and lactic acid
RIRDC Publication No. 08/033

Gastric ulceration is a very common problem in horses in training. It is widely accepted that this is a problem resulting from feeding and management practices. Racehorses are fed large meals of grain rich diets and usually fasted for an extended period before exercise. It is the combination of increased gastric acid production during exercise, reduction in saliva production due to a low fibre diet, and indoor confinement that most likely contributes to the development of stomach ulcers. This report is about the role that bacteria and lactic acid play in the development of gastric ulceration in horses. It also explores the use of contrasting diets to determine whether they contribute to the build up of lactic acid and volatile fatty acids. Identification of the key acid producing bacteria and dietary regimens that minimise their multiplication will aid in the development of strategies to control

gastric ulcers in horses. This will undoubtedly help in reducing the cost of gastric ulcer treatment in racehorses and improve their health and performance. Our business is about new products and services and better ways of producing them. Most of the information we produce can be downloaded for free from our website: www.rirdc.gov.au. RIRDC books can be purchased by phoning 02 6271 4100 or online at: www.rirdc.gov.au/eshop.

Contact RIRDC: Level 2 15 National Circuit Barton ACT 2600 PO Box 4776 Kingston ACT 2604 Ph: 02 6271 4100 Fax: 02 6271 4199 Email: rirdc@rirdc.gov.au web: www.rirdc.gov.au

This publication can be viewed at our website—www.rirdc.gov.au. All RIRDC books can bepurchased from:.

www.rirdc.gov.au/eshop

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