ULCERATION OF THE PART OmOP8;4GEA IN Sm

AThmis -

Presentd to

The Faculty of Graduate Studies

of

The University of Guelph

In partial fullülment of requirements

for the degree of

Master of Science

August, 1999

6 Sergey Melnichouk, 1999 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 OaawaON KIAON4 Canada CaMda

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The author retains ownership of the L'auteur conseme la propriete du copyright in thk thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fhm it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced Mthout the author's ou autrement reproduits sans son permission. autorisation. ULCERATION OF THE PART OESOPEUGEA IN SHllYE

Sergey 1. Melnichouk Advisor:

University of Guelph Professor R M. Friendship

This thesis is an investigation of gastric ulcers @ms uesophagea) in pigs. Gastric ulceration was studied using both field and controiled trials. Based on necropsy findings, hemorrhage kom gastric ulceration was shown to be the most important cause of rnortality on a large Amencan swine fam. Helicdacfer heilndi-me organisms were identified by histology using silver staining techniques. An in situ urea test which resulted in a colour change in the presence of urease-producing bacteria appeared to be useful as a screening procedure forHelco6acter positive animals. 'ïratment with and azithromycin was not successfiil in preventing gastric lesions in pigs that had been fasted for 48 h during the treatment period. A high dosage of (40 mg) did appear to reduce gastric acidity for a 24 h period and result in decreased gastnc lesions. First and foremost, 1 would like to thankDr. Robert U Friendship, who has changed

my life, offered hïs bottomless patience, understanding and support during the past three

years, who helped me to find my place in my new Homdaod when I needed it the most. The

lessons 1learned fkom Robert I will carry for the rest of my We. Thank yoy Bob.

I would like to thank the other members of my advisory cornmittee, Dr. Cate E.

Dewey, who always gave fieely of her time and advice with continuous encouragement and

support, and Dr. Stephen Knith for his valuable advice.

Next 1 would like to thank the members of my examination comrnittee for their

participation in the completion of this work. Thanks to Dr. Scott McEwen, Dr. Robert

Friendship, Dr. Gaylan Josephson, and Dr. Ming Fan.

Special thanks are extended to Dr. Robert Bildfîell who was accepting of my limited knowledge of pathology and dedicated a lot of his tirne and expertise to help me run my project smoothly.

Many thanks to Dr. Nonie Smart for her help and advice that she freely gave to me even during her busiest time.

Many special thanks to Dr. Anne Deckert for her fnendship and valuable advice.

Many thanks are also extended to Dr. Luis Femandez for his fiiendship and assistance without which my trial would not have gone so smoothly.

Special thanks also goes to Dina Belais. for al1 her support. You encouraged me to take on new challenges and do my best. It is a pity that you could not stay with me to the end. 1missed your support veymuch And Sanjaya Kumar wherever you are, your help and fkiendship, especïaliy in our îirst year, was very much appreciated.

1am obliged to Bryan Bloomfield for ali his assistance throughout the trials and to my sister Evelyn that she found tirne to corne over and help me with one of my trials.

Thanks a lot Bryan and Evelyn-

Many thanks are dso extendeci to Dave Bamey and his staff at the Arkell Swine

Research Station paîticularly Doug Wey and to those who work in the meat laboratory at the

University for dl theu help and participation with the animal work.

Findy, I would Wce especidly to acknowledge my family, my Mom and Dad who are my life's ideal and who gave me a lot of support in my life. 1love both of you vety much.

This research could not have been undertaken without the financial support fiom the

Ontario PorkProducer's Marketing Board and the Ontario Ministry of Agriculture, Food and

Rural Affairs that was greatly appreciated. TABLE OF CONTENTS

ABSTRACT ACKNOWLEDGEMENTS ...... o...... o...... i TABLE OF CO~NTS~~~~~~~~~~~~~~m~m~om~~o~~~m~~~~~m~~o~o~~o~~~~~~m~~~~~~m~~~~~~~o~ooo~~~~~~...... O.iii .. GWETO TABLESmm..~-...~...mo~~~~~~~~~o~~~~~o~~m~~~~o~~s~~emo~a~mo~~~~e~~~~s~~ooo~~~~~~oo~~~~*~~*~~~~~~~~~œo~~~~~~t~ GUIDE TO FIGURES~~~~~m~~~~o~~~~~~~m~mo~~~~~~m~~~m~omo~œm~mœ~m~~mœo~~~o~~~~m~~~~~~o~oo~~~~~o~~~~*~~~o-**----..*..-o~

2.1, GENERAL REVIEW OF GASTRIC ULCERATION IN SWINE...*..000..3 2-1. 1. ETIOLOGY AND PATHOGENESIS...... O...... 3 2-1-2- EPIDEMIOLOGY...... 8 2-1.3. CLINICAL SIGNS AND DLAGNOSIS...... O...... 9 2-1-4- ~A~...~oI~~~~~~m~~~~~o~~~e~~~~~~~o~*o~~~~.me~~o~o ...... 11 2-1 -5 - P~VENTIONm..o.~~~~~m~~m~~mo~mm~~t~~~m~~~~~~~~~m~~m~m~~~~~~~~~~o~~~~*.~ooo12 2.2. GENERAL REVIEW OF HIELICOBALTER INFECTION...... o...... 13 2.2-1, INTRODUCTION AND HISTORY--...... -...013 2.2-2- TAXONOMY AND BACTERIAL CHARACTERISTICS ...... 15 2.2-3. EPIDEMIOLOGY...... ~~m~-m~~~~~-~~~moœm~mto~~~~m~rnmm*~~~.e~~~~~~~~o~~.~~~~~.~o~o~~.~~~l9 2-2-4-PATHOGENESISo...,..~~~~~~~mm~~m~~m~~~m~~~~-~mmm~~~o~~~~oooo~o~*~~~œ*~.o-~-**-~~oo-~.o-21

2-2-5-D~GNOSISo..om~...~~~~~~~mm~~~mo~mmmm~~~m~a~~~m~m~~~~~a~m~m~m~~~~~~~o~~o~~~~-~~*~~~~~m--.-o25

2-2-6-CLLNICALDISEASE.....-..o...~..-.m.ma---mooooomoo.omoooooooooo-o--o--.oo*ooo-o-o---28 2-2.7, TREATMENT..o.o....~~~~m~~mm~~o.~~m*~œmmm~~~to~-sm-~~~o~~~~o~o~~~~œo~~~*-*-*-*o~~~*~-*.-*30

2-2-8-PREVENTION..o.mmm..m~m~~~~~~mmm~~~~~ma~mom~~a~m~mmm~mm~~e.o~~.o~~~..*~o.~~~~-~~~~~*-~*oo32 2.3. ACID SECRETION IN THE STOWCH,...... 33 2.3. L. ANATOMIC-PHYSIOLOGICALC0NSIDERATI:ONS ...... 33 2.3 .2. GASTRIC SECRETION AND DEFENSIVE MECHANISMS ...... 36 2.3 -3- CONTROL OF GASTRIC ACID SECRETION...... 38 2.3.4. DRUGS INHIBITING ACID SECRETION AND ANTACIDS ..... 39 2-49 OBJE-Sm...... *...m..b..0bb...... b..43

Chanter 3 MORTALITY ASSOCIATED WITH GASTRTC ULCERATION- CASE STUDY...... 45

3.1. IMATERIALS AND METHODS ...... 45 3.1-1.CASE HiSTORY...... *...... ****.*...*..*...... o...b...... m..mm...... m..45 a) THE FARM ORGANIZATION...... 045 b) GROWER-FINISHER MANAGEMENT...... 46 3.1.2. SmYDESIGN,...... 47 3.1.3. STATISTICAL ANALYSIS...... 49 3.2 .RES-IiTLTS ...... 49 3.3. DISCUSSION ...... 59

Cha~ter4 H?ZLIcORA~RHEïL-I-LIKE BACTERIA IN THE STOlMACH OF PIGS WITH AND WITHOUT GASTRIC LESIONS ...... 64

4.1. INmODUCTLON...... 64 4-20 MATERIALS AND METBODS...... e....e.65 4.2.1. HERDS SUPPLYING PIGS ...... ,...... 66 4.2.2. GROSS EVALUATION OF STOMACH LESIONS...... 67 4.2.3, DIRECT AND INDIRECT ASSAYS FOR HELTCOBACZZZ-LIKE BACTEIUA TESTNG...... 67 Chapter 5 EVALUATION OF LANSOPRAZOLE AND AZXTHROMYCIN FOR CONTROL OF GASTRIC ULCERATION IN SWINE DüRING PERIODS OF FEED DEPRIVATIQN ...... m...... 82

5.1. INTRODUCmON...... 82 5.2. MATERIALS AND mTHODS...... 84 5.3. STATISTI:CAL.ANALYSIS...... 0.0..0..89 5.4. RESULTS...... 89 5.5. DIS~SION...... 97

Cha~ter6 THE USE OF OMEPRAZOLE TO ALLEVïATE STOMACH ULCERS IN SIrVINE DURING PERIODS OF FEED WITEDRAWAL ...... 102

Chapter 7 OVERALL CONCLUSIONS ...... ,...... m...... m.o.o...oooo.o.~o.o..oo.o.oooo ...O.111

REJOCRENCES ...... Il7

KAPPA FOR UREASE ASSAY ELISTOLOGY .m...... *.130 OUTPUT (THE SAS SYSTEM)...... 131

GUIDE TO TABLES

Table 2.1.2.1 : Abattoir surveys of woridwide previkace of lesions

Table 2.2. f : Tuonomy of Helicobocfp group (National Centu of Biotechnology Information (NCBr) June, 1999)...... ---*e~--~-oe----~e.L7

Table 2.2.2: Tuonomy of ClunpyIobucter group (Nationai Center for Biotechnology Information (NCBI) June, 1999: h~p:/~.ncbi.nlrn.nih.gov.)...... 18

Table 3.1.2:

Table 3-2.1 : Cause-specifie mortality, proportionai rates and invento y in grower-linisher groups by wceb basai on the site managers' records...... 5 1

Table 3.2.2: Mortality and inventoy in grower-finirher groups by quarters (pigs) ...... 52

Table 3 -2.3: Caustspecifîe and proportional mortility as determined by post-mortem evaluation of 283 grower-tinisher pigs ...... -53

Table 3 -2.4: Prevalence of gistric lesions @ors oesophagea) . detected at slaughter...... ~e~..~.e.e.~o.~~.....m.ee.e...... ~~.-e...-...... -....-...-----.54 Table 3.2.5: The prevalence of gastric lesions and urease reactivity ...... 59

Table 4.4.1 : Prevalence Helicobacter heilmannii-like bacteria ...... 75

Table 4.4.2: Prevalence of gastric lesions of thepcvs oesophagea and Helicobacter heilmmnii-1ike bacteria in 210 Ontario pigs ...... 77

Table 5.2-1: Pig allocation by week of treatment and by type ofdiet...... -..-.88

Table 5-2-2: Histological gcading ofthe lesions of thepors oesophagen by Mackin et ai! (1997) ...... 85

Table 5.3.1: Prevalence of gastric lesions by macroscopic and microscopie emminationl . ~.....,...... ~...~.~~.e....~...... ~...... -..-...... -...... ~9O Table 5.3 -2: Cornparison of stomach lesions betweea Fasted and Non-F'asted hogs, based on macroscopic/microscopicevriluation...... -92

Table 5.3.3 : Fasting or Non-Futing treatment and the presence of stomach laions (mieroscopic eumination).....m...... 94

Table 5.3 -4: Treatment Groups Ont, Two, and Controis and stomach scores (microscopic evaluation), stritificd by Fasting and Non-Fisting trutmentmw...m...... O...... 95

Table 5.3 -5: Treatment Groups One, Two, and Controls and stomach scores (macrosco pic waluation), stratifkd by Futing and Non-Fasting Treatment...... ,.96

Table 6.3 - 1: Pigs fed diibit.m and given either 20 mg omeprazole or no Treatment (controls) and esamincd 24 h later...... --106

Table 6-3-2: The gastric pH and ulcer scores of pigs fasted for 24 b and treated with 40 mg omeprszole, 20 mg omeprwle or untreated (controls)...... 107

Table 6.3.3 : The Gastric pH and Ulcer Scores of Pigs Fasted for 48 h and Tceated with 40 mg of Omeprazole (Sm)or Untreated (Con trols)...... 108

Table 1: The association between treatments and erosive lesions and ulceration, based on gross-evaluation...... 161

Table 2: The association betwetn treatments and erosive Iesions and ulceration, based on microscopie examinabian...... 162

Table 3: The association between treatments and erosive lesions and ulceration, based on grou evaluation and controiling for diet = F (Fasted)...... 163

Table 4: The association between treatmtnts and erosive lesions and ukeration, based on gross evaluation and controiling for diet = NF von-fasted) ...... 164 Table 5: The association between tratments and emsive lesions and ulcuition, based on microscopic esamination eontrolling for

diet = F ~t~d)m.mmmmm-mmmmœmmoœœœmmœmomoœ-œmœmœœœœmœœœmmmmoommmem-m~..~

Table 6: Association betwctn treatments and erosive lesions and ulceration, based on microscopic eumination controlling for diet =NF (N0n~f~td)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~~.~~~~~~~~--~~-~--~-----~~~66 GUIDE TO FIGURES

Figure 2.3-1: Comparative anatomy of hurnan and pig stomachs ...... 3S

Figure 3 -2-1: Hclcobllcfer heiintlu~nii-ükebacteri. in the pyloric region of the pig's stomachs (magnificrtion r 100, Warthin-Starry shrsain) ...... -...56

Figure 3 -2.2: The same bacteria arter enhancement by 200 ./O using cornputer technique ...... ~~...... 57

Figure 3 -2.3: Hdicobacter heifmnii-like bacteria deep in the crypts, dumped and partially Iysed (Warthin-Starry saver stain, magnification x 100) ...... 58

Figure 4.4.1: Hrlcobaeter heilmannii-like bacteria in pig stomachs from Ontario hvds (W-SsiIver stain, magnifirrtion x 100, cnhanced by 200% using eomputer technique) ...... 76 INTRODUCTION

Gastric ulcers are recognized worldwide as an important disease in swine. The majority of ulcerations occur in the non-giandularpms oesophagea. and relatively rarely in the findic and the pylonc regions of the stomach-

A high prevalence of ulceration of the pms oesophagea first occurred in North

America and Europe in the late 1950s. wherever confinement rea~gand the use of grain- based processed rations were introduced (Thoonen and Hoorens 1961, Curtin et ai- 1963,

Griffing 1963, Muggenburg et al. 1964, Nafktad et al. 1967). In certain herds, gastric ulceration ofthepars oesophaga can be a significant cause of death from acute intragastric hemorrhage or result in chronic il1 thrift. Mortality due to ulcers can commonly reach 1 % or greater, with additional culling losses of3-5 % (Nielsen 1995).

In a study by the Ministry of Agriculture and Forestry in Japan, the loss for 1971 that was attributed to thepars uesophagea ulceration on pig production was S 15-20 million (US)

(O'Brien 1992). Recent slaughter-house surveys indicate the prevalence of lesions in thepars oesophageu of the stomachs of intensively reared hogs ranges from 32 to 65 % (Ayles et al.

1996b) and may approach 100 % on some fms@riesen et al. 1987).

ULcerations of the glandular epithelium are usually associated with active septicemia in systemic diseases such as Afncan swine fever, hog choler% cchrnic SaIrnoneilo.sis and chronic Erysipel' and also as a reult of parasitic infestation by Hyostrongyliis nnrbihs, Asrcarops sfrongytimq Physocephhalus sexaGdrrs and Diperorms lanae (Penny and RiII

1973, O'Brien 1992, Straw et al_ 1992, Henxy 1996).

Gastric ulceration of the pms oesophrrgea is a disease of multiple etiologies with

many known nsk factors, but the etiology and pathophysiology of gastric ulceration is not

clearly defined (Davies 1993). Intrmption of feed intake has been implicated as a pcimary

cause ofuicer development @emy 1996). Researchers have shown that pigs fasted for 72 h

are prone to gastric lesions (Pocock et al. 1968)-

Helicohrpylorï are now accepted as being a major causal factor of human antral gastritis and gastric ulceration(0wen 1993)- It has been shown that the stomach of mine can

harbour the spiral bacterium Helicobacter heilmmnii (formerly Gastrospirillzm suis)

(Queiroz et ut- 1990, Mendes et al. 1991). In a study conducted by Queiroz et al. (19961, it was shown that an association exists between the occurrence of ulcers and preulcer Iesions of the pars oesaphagea in swine and the presence of Helicohcter heilmannii type 1

(Queiroz et ai. 1996). This bacterïum is the only Helicobacter that naturally infects several species of animals, as well as hurnans, and therefore it may be considered a zoonosis

(Wegmann et ai. 1991, Stolte et al. 1994). LITERATURE REVIEW

2-1.1. ETIOLOGY AND PATHOGENESIS

The most common site for gastrïc ulcenition in pigs is the pars oesophogea at the junction with the cardiac mucosa. Accordhg to Penny and Hiu (1973), the ulcers occur more

nequently at the left junction than at other sites.

The ability of the stratifieci squamous epithelium to protect itself against injury from noxious

lumuial agents is known as "tissue resistance" (Orlando 199 1). Stratifïed squamous epit helium

has the capacity to resist acid-peptic digestion for considerable periods of time. Studies have been shown that continuous perfusion of rabbit esophagus with 150 rnL HCI for up to 3 -5

h is often unassociated with either gros or histologie lesions of the epithelium (Salo et al.

1983). Argenzio and Eisernann (1996) demonstrated that organic acids, principally acetate and lactate, cm penetrate and damage gastroesophageal mucosa more rapidly than hydrochlonc acid.

Gastnc ufceration is defined as the "breakdown" of defensive factors which then allows damage from endogenous andor exogenous aggressive factors (Ziller and

Netchvolodoff 1993). Ulceration is a rapid process, and hyperkeratosis can occur in the space of a few hours (usuaiiy less than 24 h). Development of ulcers, from normal to fatal or even normal to non-fatal but total ulceration, is 1-2 days maximum (Henry 1996). Gross pathologicai changes include hyperpitfakeratosis, erosions and ulceration. The lesion may vary in size and shape, and may be single or multiple and may encompass the whole pars oesophugea-

Ulceration of thepsmmphagea is a disease ofmultiple etiology. There are many known etiological factors associated with ulceration of this region of the pig's stomach.

They can be grouped into three main categones: nutritional fhctors, housingl management factors and other factors, such as concurremt disease, parturition, season, genetics, histamine, somatotropin, and ascarid infection.

The following is a list of the most important nutdionai factors that are linked to ulcer development:

rnilling: rolling versus grinding, pelleting, heat processing;

particle size of feed;

type of grain;

lack of fibre;

vitamin E I Selenium deficiency;

rancidity of fat;

whey;

feed withdrawal (fasting).

Grain processing using a hammer mil1 tends to produce a large proportion of flour or "fines", while the use of a roller mil1 produces a more uniforni partide size (Wondra et al-

1995b). Flour undergoes microbial fermentation more readily than larger ground particles of grain, because fine particles present a far -ter surface area for microbial action than coarse particles. Therefore, finely ground grain provides a greater potentiat for organic acid

production, which Krakowka et 41. (1998) have shown to cause ulcers. Altematively, fine

particles lead to rapid transition the and more mirring.

Et is known that pigs fed a fïnely ground diet consume more water than pigs on a

coarse diet. Dnesen et al. (1987) suggest that this would increase motility of the stomach and

dow gastric juices to corne in contact with the psoesophagp- On the other hand, high water intake should dilute the gastncjuices and as a result increase pH and therefore decrease the noxious effect of hydrochlonc acid. In addition, high water intake contributes to rapid dilution of carbohydrates that encourage bacterial fermentation.

Cereal grains contain certain proportions of sugars (such as glucose and sucrose as opposed to starch), as well as a distinct proportion of fibre. The cereals that containless carbohydrates and more fibre such as oats and barley tend to be less ulcerogenic. Conversely, grains containing high concentrations of sugars and starch, and low levels of fibre, such as corn and wheat tend to be more ulcerogenic. Inclusion of coarse ground barley straw in the wheat diet at a 5-10 % level gives almost fùll protection (Stephens 1978). Potkins and

Lawrence (1989) have shown that when fibre is ground finely, there is no positive effect of fibre. They suggest that it is the coarse particle size not the quantity of the fibre that is important.

Heat processing called "gelatinization" is also associated with oesophageal lesions.

Corn is heated under pressure to ISOOC, with the rapid release of pressure resulting in an expanded grain kemal. Steam pelîeting raises the temperature of feed (to about 80°C) and as a result, also leads to megelatinization of starches, that augments microbial activity (Mahan et al, 1966, Moran 1982).

Feed withdrawal has been a consistent method ofexperimentdy producing gastnc

ulceration (Pocock et al 1968). Interruption of feed intake can occur as a result of

management negligence, or as a resdt of disease causing anorexia, or due to hot weather that

causes inappetence-

Gastrric lesions have also been seen in association with fat rancidity, as well as with

vitamin E and selenium deficiency (Ndstad et al. 1967). Possibly, the formation of active

peroxides advates the stress mechanism and causes an increase in gastric acid secretion.

The following are housing/management factors that are associated with ulcer

development:

confinement rearing;

herd sue;

mixing pigs;

over-crowding;

holding and transport.

Christensen and CuUinane (1 990) have shown that the prevalence of'rs oesophages ulceration increases as herd size increases. The association between stocking density and gastric ulceration is unclear. Picket et al. (1969) found an increase in prevalence and seventy ofgastric ulcers with increased stocking density, but Muggenburg et al. (1 967) and Handlin et al. (1972) did not find an association. A sparing effiof high density stocking on gastric lesions wes recorded by Driesen et al. (1987). Fnendship (1999) deems that factors such as environmental temperature, available foeder space, and number of available water nipples

hteract with pen space to cause these contradictory results.

Acute infectious disease and season are associated with ulcer development, thus pigs

with respiratory disease are 9-12 tirnes more likely to have lesions of pars oesophagea

compaied to pigs without respiratory disease (Senket al. 1994). Deen (1993) reported a nse

of mortality as a resuit of gastric hemorrhage fiom 0.5 % in winter to 2.5 % in summer. It

may be associated with a dramatic reduction of feed intake during hot weather.

Heretability may also play a role in ulcer development. There is an association

between fast growth rate and /or low fat and a high prevalence of gastric lesions (Berruecos

and Robinson 1972, Grondalen and Vangen 1974). Smith and Kasson (1991) have reported

that injection of pigs with porcine somatotropin increases the prevalence and severity of

gastric ulcers.

Experimental parasitic infestation of specific pathogen free (SPF) pigs with large

number of Ascms surrm eggs caused extensive ulceration of thepars oesophagea, however

when this experirnent was repeated on secondary SPF pigs, ulceration was not produced

(Nakamura et aï- 1975). Naturally occumng Ascaris sum or Hyostron~yizrsrubidus (the

red stomach wom of swine) infestation are not primary causative factors, however these factors affect mucosal integrïty and gastric secretion, therefore they might influence the formation of gastric lesions in some way (Friendship 1999). 2.1.2. EPIDEMIOLOGY

The prevaience and seventy ofpcas oesophogea ulceratÏon has varied with tirne and geographic area The table 2.1.2.1 presents the findings of various studies world-wi-de:

TabIe 2.1.2.1: Abattoir survqs ofwoddwide prevdence of lesions in the pars oaophagea (Friendship 1999)

References 1 Country LEumioed 1% with Lesions

Jensen and Frederic (1939) US A, 20,000 5

Curtin et al. (1963) USA, 443 86

GriBEuig (1963) USA, 610 91

kt* et al. (1974)

Kowacs (1974) Huagary 13,400 13

Szemeredi and Sola (1979) Cuba 2,457 20

Dnesen et al, (1987) Austraiia 5000 99

Christensen & Cullinane (1 990) New Zealand 2,66 1 32

Straw et al. (1992) U.S.A. 600 65

Elbers et al. (1995) The Netherlands 184 89

Ibert et al. (1995) The Netheriands 274 75

The data indicate gastric lesions (including parakeratosis, erosions and ulcers) vary fiorn 5 to 100 %. Recent studies fiom anas using modern feeding techniques record a prevdence of 65- 100 %- Pigs of any age may be affectecl by gastric ulcers, but the highest nsk groups are 3-6 month old pigs and sows at the theof parturition (ulceration has been reported as a cornmon cause ofsowmortality) (NakamuraetaL 1975). Frequently, sows' stomachs reveal extensive scar tissue indicating previous severe ulcerations (Chagnon et al. 199 1, Sadiord et al. 1994).

In abattoir surveys about 10-1 5 % ofthe sows are diagnoseci as having ulceration and overall about 60 % with stomach lesions (Hessïng et al. 1992, O7Suliivan et al. 1996). It is relatively rare to find any herd-associated clinical problems in mature boars, gestation sows or piglets fkom birth to 50 kg while gastnc ulcers may occur occasionaily in any individual

(Henry 1996).

2.1.3. CLINICAL SIGNS AND DIAGNOSIS

Gastric ulcers are difficult to diagnose in the live pig. Otten diagnosis is accomplished on the basis of clinical history and post-mortem findings, and prevalence within the herd cmbe estimated by a slaughter-house examination. Diagnosis of more severe ulceration, when the epithelium is damaged and when blood vessels are exposed and bleeding is easier than diagnosis of subclinical lesions. Clinical signs tend to reflect the degree of blood loss. When blood loss occurs slowly, the presence of tany feces may be noted. Pigs show signs of anemia without fever, and rectal temperature is often below normal. Frequently, when blood loss occurs more rapidly, a pig that appeared to be healthy is found dead and the carcass is extremely pale.

On carefùl observation of individuai cases of disease the following signs may sometimes be noted: inappetence, vomiting (frothy bile-stained fluid), low rectal temperature, rigid back (indicative ofpain), pain in the xiphisternal area (pressure applied in

that region causes gninting), grindïng of teeth, and melena

Sporadic outbreaks of gastric uiceration tend to occur among grower-nnisher pigs.

Frequently, when one pig has suddenly died, others reveal signs of anemia The anirnals

appear to be in nonnai health, if ulceration occun without significant blood loss.

Post-mortem diagnosis is usuaiiy straight forward:

carws is pale, but in good body condition;

the stomach may contain clotted and unclotted blood and fibrinous exudate encloshg

a variable amount of food;

thepsoesophageal epithelium is broken, resulting in active ulceration and hemorrhage,

and the surface of the ulcer is generally deep and extensive;

a blood clot may be adhered to the surface of the ulcer.

Diagnosis can be supported by a slaughter-house examination for the presence of the gastnc ulceration in other pigs fiom the same herd. Ulcers can occur rapidly and heal quickly, therefore post-slaughter examination may reveal cicatrization and a reduction in size of the parsoesophagea, and in extreme cases, stenosis of the oesophagus at its entry to the stomach occurs.

Pigs af'fiicted with stenosis vomit shortly after eating, but are hungry and therefore will immediately begin to resume eating. Despite good appetites, these pigs tend to grow slower than their pen mates (Friendship 1999)-

Definitive diagnosis, when a post-mortem is not an option, requires endoscopy.

Therefore modern portable endoscopes can be usefûl tools in the diagnosis of gastnc ulcers in swine. At post-mortem early erosive lesions are commoniy overlooked due to the lack of

contrast, but are eady distinguished at endoswpy as rad lesions against the white

background of the pms oesophageal surface (Mackin et al. 1997).

2.1 -4, TREATMENT

Early intervention is needed to successtblly treat pigs with gastric ulceration.

Unfortunately, this is hampered by the dif16.cultyin diagnosing the disease in live animals and

its rapid onset. Pale and weak pigs should be segregated fiom pen mates ta avoid injury.

Euthanasia should be considered ifsevere blood loss has occurred.

Factors that lead to increased microbial fermentation in the stomach or its motility

should be minirnized. For instance, replacing finely ground pelleted feed with moderately

coarse mash feed results in heding (Ayles et al. 1996b). Aiso it has been show that when

pelleted feed is replaced by a 1: 1 ratio of pellets and meal mortality returned to nomial within

7 days of implementing these dietary changes (Kavanagh 1994).

Histamine Hz-receptor antagonists, including , , , and , reduce the acid production and have been considered potentially usefil dmgs.

Ranitidine and cimetidine in addition to inhibiting histamine-evoked gastric acid secretion appear to effectively control upper gastrointestinal bleeding in humans. These dmgs do not reduce the incidence or severity of ulcers associated with finely ground feed in pigs (Hedde et al. 1985).

E-type that promote mucus and reduce acid secretion are widely used in human medicine. They have not been found to be effective in treatment of gastric ulceration in swine. Cytoprotectants such as bismuth subcarbonate, kaolin, and pectin have been used

as protective coatings over the ulcerated area. Stapleton et al. (1989) have shown ulcer

prevention in bile duct-ligated pigs by using sucraitàte (an alumùuim sdt of suIfated sucrose) which adheres to gastric lesions and promotes healing while preventing fiirther attacks. The addition of suaalfate to a finely ground diet (1 or 4 mg per 3 kg) fed to somatotropin-treated swine failed to alleviate ulcers of the pars oesophgea

2.1- 5. PREVENTION

The cost-benefit of the prevention program is an important consideration in determining what action should be taken to reduce the losses fiom gastric ulcers on a particular fm. Many factors, if changed, wuld reduce ulcer prevalence but result in economic losses which far outweigh the benefit of reduced ulcer prevalence. For instance. decreasing stocking density, removing growth pmmotants fiom the ration, changing from fine diets to coarse may decrease gastric ulceration, but each will have a significant effect on growth rate and /or feed efficiency (Friendship 1999).

Feeding practices must be carefiilly monitored. Good management practices can rninimize the occurrence and influence factors that cause ulceration such as: heat stress, unpalatable feed, blocked waterers or feeders, an interruption of feed intake, the presence of vomitoxin in the feed- Feed providers, owners, production personnel, and veterinarians should coordinate their efforts in establishing feed preparation standards and management practices that will limit gastnc ulceration as a production problem without reducing swine performance

(Henry 1996)- 2.2- 1. INTRODUCTION AND HISTORY

In the past decade the field of gastric rnicrobiology has made rapid advances, as a result of the discovery that gastroduodenal ulceration in humans is pnmarily a result of a bacterial infection. Many trials have been done amund the world and have led to an inwease in Our knowledge of the curved or spiral bacteria that inhabit the mucus layer of the stomach of humans and various animal speci-es includhg the pig.

Spiral-shaped bacteria were first reported in the gastric mucosa of a dog in 188 1 by

Rappin (Ziller and Netchvolodoff 1993), and other mammalian species in 1896 by Salomon, but were neglected for a century. They were also recognized in the. stomachs of humans for at least 60 years (Doenges 1939), but little interest was shown in these mîcro-organismsuntil the 1970s when association between the presence of bactena and the occurrence of chronic ulcers in humans was suggested.

In April 1982 Barry Marshall and Robin Warren cultured C~obacrerpyIon'dis.

Three years later Marshal confirmed the association of this bacterial infection with peptic ulcers in 114 infected patients in Fremantle Hospital, Australia. In 1989 a new genus was created and the organkm was renamed as He&obacterpylori (Goodwin et al. L 997). This gram-negative bacterium is associated with nearly al1 duodenal and gastnc ulcen that are formed independently of the ingestion of non-stemidal anti-inflammatory dmgs (NSAID) or similar irritants (Marshall 1983, Warren 1983), and most Iikely plays a major role in causation ofthese ulcers in humans, in concert with a range of as yei un;uiown factors. The treatment ofgastroduodenal ulcers is accomplished with a short course of antacid drugs in combination with antibiotics. The success of this treatment regimen in humans is very high

(Blaser 1996)-

In 1989, during upper gastrointestinal endoscopy of human stomachs, spiral-shaped bacteria morphologically distinct from HelicobacterpyIori were found. They were named

Gastroqpirillum hominis (Mendes et al 1994). The fiagrnent 16s rRNA gene of

G~ospiriZIumhominis is 97-98.5 % similar to that ofHelicobacterfelisand therefore it has been suggested that "GmtrospirïlI~m"belongs in the Helicobacter genus (Soinick et aL

1992, Solnick et aL 1993). Helicobacter heilmmiii has been proposed as the name for this bacterium after the late Konrad Heilmann who was a pioneer in studying these bacteria

Helicobacter pyhi does not naturdy infect pigs, but gnotobiotic and conventional pigs have been successfblly colonized with experïmentai infections of Helicooacter pylorz

(Krakowka et al. 1987). Researchers have used the pig as a mode1 to facilitate the study of these bactena-

Naturally-occurring, spiral bacteria, morphologically distinct fiom Helicobacter pylori, have been identified in the gastric mucosa of cornmercially-reared pigs, and provisionally named Gustrospirillum suis (Mendes et al. 1990). Mendes et al. (1994) have shown that 16 sDNA sequence of Gastrospirillum suis obtained for the PCR amplified

DNA was 99.5 % similar to that for Helicobacfer heilnannii, fomerly Gastroqin*Ilrm hominis type 1. A strong association of Helicobacter heilmanrtii type 1 (fomerly

GQSfrospirillumsicis) with naturally ocairring preulcer lesion and gastric ulcers (pars oesophages) in swine has been shown (Queiroz et al. 1996). In this trial the bacteria were more cornmonly found in the stomachs with ulcers (100 % ofstomachs), and in those with

preulcer lesion (90 % ofstomachs), than in the stomachs with macroscopically normal pms

wsophagea (3 5 % of stomachs).

2-2.2-TAXONOMY AND BACTERIAL cwcmasms

Many spiral-shaped bacteria have now been isolated and have been demonstrated

to belong to the same genus, Helicoba~fer~In Table 2.2.1 five bacterial families that belong

to the Helicobacter group and that are closely related to Helicobacter pylon are shown.

Helicobacter species currently contairt 41 strains (Naùonal Center of Biotechnology

Information (NCBI) 1999). At present, Campyiobacters ( Table 2.2.2) are subdivided into

two families: CcnttpyIobacter and Acrobacter that currently contain 22 and 5 strains,

respectively-

Members of Helicobacter and CampyIobacter groups share many biologicai

characteristics, and therefore they are placed within Superfàmily VI (Garcia and Brooks

1993). Al1 members of this Superfamily are gram negative bacteria, and generally require micro-aerophilic (3- 15 % of 0%optimal 6 %) conditions for growth. They share some other common charactenstics, such as urease, catalase, and cytochrome-oxidase activity. They are not capable of producing volatile fatty acids (VFA) or neutral end products, because there is no oxidation or fermentation of carbohydrates. These bacteria have a respiratory metabolism. thus energy is obtained from arnino acids (glutamate, aspartate) or tricarboxylic acid (TCA) cycle intermediates (Srnibert 1983). Organisms in this Superfàmiiy are slender, spirally-curved rods (0.2-0.8 prn x 0.5-5

pm). They may have one or more spirais and can be as long as 8 pm. They also appear S-

shaped or seagull shaped when two bactena form a short chah They are motile, with a

characteristic corkscrew-üke motion by means of a single or multiple flagella at one or both

ends. Cells in old culhire rnay fom spherical or coccoid bodies. Helicobacters are non-spore

forming (Smibert 1983, Prescott 1984, Lee and O'Rourke 1993). Mendes et al. (1990)

descnbed helical-shaped bacteria in the pig's stornach, within the mucus and on the mucosal

surface of the pylonc pits. These bacteria had 3 to 8 spiral tums, fiattened and truncated ends

(approximately 0.6 x 4.0 pm), and had up to 6 flagella (up to 22 qm in diameter) at each pole. They named them Gt~~mqirilZurnsuis (now Helicobacter heilmunnii). These bacteria are more than twice as large as Relicooaclrpyl~n'~

Members of Superfamily VI tend to be difficult to culture and to define using wnventional laboratory tests. Molecular and DNA technology, such as PCR, made classification possible (Skirrow 1994). The method of determination of whether bacteria belong in the Heficobacter or Cmpyfobactergrmp is based on the sequence ofbases in their

16s ribosomal RNA or nuclear DNA (Lee and O'Rourke 1993, Mendes 1994).

Morphological structure of these bactena are remarkably different even within the same

Helicobacter group, but more than 90 % homology in ribosomal sequence indicates a close relationship, and therefore they are grouped together.

Some species of Superfamily M are pathogenic for man and animals, causing important reproductive, intestinal, and oral infections; others are saprophytic species. Table 2.2.1 : Tlixonomy of Helicobnctcr group (National Center of Biotechnology Information (NCBI) June, 1999)

Helicobacters: Gastrospirillum Fiexis~ira Thiovulum Wolinella 1 N. acinonvx 22 Helicobacter tvphlonicus G. hominis F.rappini Thiowlumsp. W.succinoaenes 2 H.bilis 23 Hçlicobacter ulmiensis

3 H. bimzeronii Heliibacter sp.: - -- 4 H,canis 24 H. sp. 'ATCC 43968' 5 H.cholecvstus 25 H. sp. 'Eaton 94-536' 6 H, cinaedi 26 H. SP. 'hamster B'

7 H. felis 27 . H. SP. 'MIT 95-20 1 1 ' 'MIT , 8 H. fennelliac 28 H. sp. 97-6 194-5' 9 H, heilmannii 29 H. sp. 'MZ640285' 10 H. hepaticus 30 H. sp. 'Robertson AN0 1' 1 1 H. mesocricetus 3 1 H. sp. 'Robertson AN02'

, 12 .H.muridanim 32 H. sp. Robertson AN03' 13 H,mustelae 33 H. sp. 'Robertson AN04'

14 H. nemestnnae 34 H. sp. ABHU3cae------~ 15 H. pullorum 35 H. sp. ABHU4stomsp 16 H. pvlori 26695 36 H,sp. CL03

1 7 . H. pvlori J99 37 H,sp. UNSW1.7col 18 H. rodentium 38 H,sp. LJNSW 1.7s~ UNSW3.l , 19 H. salomonis . 39 H. sp. lcae 20 H. suncus 40 H. sp. UNSW3SBsp

, 21 H.trogonturn 41 H. sp. UNSWMCSDI Table 2.2.2: Tuonomy of ~yfobacfergroup (National Center for Biotechnology Information (NCBI) June, 1999: http:/kww.nebi.nlm.nili.gov.~

Cam pylo bacter Acrobacter Campylobacter coli 1 Arcobacter buttleri Campylobacter concisus 2 Arcobacter cryaerophilus

Carnpylobacter cums 3 Arcobacter nitrofigilis

Campylobacter fetus 4 Arcobacter skirrowi

Campylobacter gracilis 5 Arcobacter sp- Campylobacter helveticus Campylobacter hominis Campylobacter hyoilei

C. hyointestinalis 1 - -- - - . .- - C. hyointestinalis subsp. Campylobacterjejuni Campylobacter jejuni-li ke Campylobacter lanienae Campylobacter lari Campylobacter mucosalis C. mucosalis-like bacterium Campylobacter rectus Carnpylobacter showae Campylobacter sputorum 1 1 Campylobacter upsaliensis Campylobacter sp. 2-2-3,EPIDEMIOLOGY

Infection with either HelicobacferpyIori or Heiicobacter heilmmnii causes gastritis in humans. Heiicooacferpyiori infection in humans is widespread, with up to 75 % ofthe world' s population carrying the bacteria (Taylor and Blaser 199 1, Mégraud 1993). However, fewer than 20 % of al1 infecteci people develop any clinical signs of disease (Blaser 1997).

Prevalence of infeaion rises with age so more than haif of al1 60-year-olds are infected, however, chüdren are rarely infecteci. Once acquired, HelicobacterpyIori infection persists for years and offen idected individuals remain carriers for Me, udess they are treated-

The incidence of infection in developed cwntries is low (up to 5 % per year). Studies have confirmed that the prevalence of Helicobacferpylori exists in higher proportions in saliva than in feces (L,i et al- 1996)- It is believed that the oral-oral route of infection is the most common rneans by which bactena are transmitted. In contrast, in developing countries prevalence of infection in the adult population is high (up to 66 %) and children are also commonly infected (Blaser 1996). General hygiene and sanitation are deemed to be important in the spread and control of Helicobacter pylori-

Helicobacter pylurx is almost exclusively a human pathogen, in that an animal reservoir as a source of Heiicobucterpyiori infection has not been found. Thus, person-to- person contact is considered the primary route oftransmission. Hilzenrat et al. (1995) have found He licobacter pylori and Helicobacîer heiimannii-like bacteria in biopsies fiom 9 12 endoscopies of humans stomachs. The histologie prevalence of these bacteria was 59 % and

0.5 %, respectively. It is believed that up to 1 % of the human population is infiected with Heficobacter heilmmniii (MazzuccheIii et al. 1993, Stoike et al- 1994)- Helicobacter

heilmannii seems to be widely distributed among pigs, cats, dogs, wild rats, ferrets and birds

(Seymour et al. 1994). Therefore, there is a strong possibility that humans can become

infected with Helicobacfer heilmamii niorn anirnals (Stoke et al. 1994, Meining et ai-

1998).

The prevdence ofHelicobacter heilnmiii in commercial pig fmsworld-wide is

unknown In an eariier study, Brazilian researchers reported the prevdence of this infection

to be about 11 % of 120 examined pig stomachs (Queiroz et al. 1990). In a study of85 pigs

from an Itaüan slaughter-house, a sidar result was obtained (9.4 % of stomachs) (Grasso

et al. 1996). More sensitive assays for detecting Heiicobucfer heilmannii are being

developed and it is certain that the prevalence rate wül be found to be much higher than in these initiai studies (Queiroz et a(. 1996). In a recent study, conducted in western France on the pig stomachs obtained fiom 8 dBerent fms, the distribution ofHelicobacerheiIm~71~1ii infection was found to be widespread on aii surveyed fanns. Organisms were found in 8 L % of sampled stornachs (65 out of 80) (Magras et al. 1999). At the sarne the, a PCR assay did not reveal Helicobacter pylon infection (Magras et al. 1999).

Transmission of Helicobacter heifmannii Ulfectionis likely via a fecal-orai or an ord-oral path from pig-to-pig. Animal vectors may be important in the introduction of this bacterium to a naive herd. Cats, dogs and a wild rats should be considered as potential carriers. In one study, 23 % of wild rats were found to carry a tightly-coiled spiral bacteria. morphologically similar to Helicobacter heiimmnii (Giusti et af- 199 8). In addition, transmission fiom humans-to-pigs need to be considered as a potential route of infection. 2.2.4, PATHOGENESIS

The members of SuperfdyVI are both pathogenic and saprophytic species. The

Cmnpylobacter group is the most important in vaerinary bacteriology and contains species

causing important genitai and intestha1 uifiions in animais and humans. In human medicine

the most important is the Nelicobacter group, specifidyWelicobocrerpylon, an important

species causing duodenal ulcers in humans.

CmpyIobacterjejuni is an important and common cause of acute diarrhea in man in

developed countries, and an important zoonosis, acquired directly or indirectly fkom al1

cornmon dornestic animals and birds. This bactenum causes diarrhea in young animais,

particularly in piglets, foals, lambs, and kittens, and causes enteritis in adult cattle and dogs

(Prescott 1984). Analysis and investigation of pathogenic properties of the CampyIobucter

group was beyond our interests in this research,

The success cf Helicobacter organisms in gastroduodenal lesions is related to both

pathogenic mechanisms, leading to disruption of the gastric mucosal barrier, and to unique

maintenance factors, aitowing the organism to persist ina hostile environment (Dunn 1993).

The characteristics ofHelicobacters which let them successfully colonize and survive in the

stomach are much better understood than the factors which lead to gastritis and ulceration

(Lee et al. 1993). The most important vinilence £horsUiclude motility, ability to adhere to

gastnc mucosa and mucus, and ability to produce adaptive enzymes and proteins.

Motility is achieved by the spiral shape and the polar flagella and is an essential

component of Helifobacter success in colonking the stomach. The bacteria are able to quickly burrow into the mucus and away fiom the acid environment of the stomach lumen. Motüity helps them avoid being flushed away during gastnc contractions. The strains of

Helicobacterpylorf with poor motility are unable to colonize the stomach of pigs dunng

experimental infection but highly motüe strains are successîùl (Eaton et al. 1989).

An important property ofHeIicoobcterpy!orf is its abiiity to adhere finnly to gastric

mucosa and as a result cause erosions and ulcers. In wntrast, adhesion of H. heilmmnii to

epithelial cells is Sequent, and the bacteria are mostly located at a distance fiom the

epithelium (Holck et al. 1997) and appear to rely on their very active motility to colonize

mucus and mucosal surfaces (Lee et al 1989). Stolte et al. (1997) in a matched control

study compared dBerences between Helicobacter heilmmnii and Helicobacter pylori

gastritis in humans. They found that colonization of Helicobacter heilmannii is mostly

circumscribed and maînly in the pyloric area, and induces a milder fonn of gastritis. The cases

of erosions and ulcerations Iinked with Helicobacter heilmmnii are rarer than in patients with Helicobacterpylon infection (Kolck et al. 1997). .

An enzyme, urease, that catalyzes the break down of urea to arnmonia and carbon dioxide, appears to be an important su~valfactor for Helicobacters. The strains exhibiting weak urease activity fail to colonize in pigs experimentally challengecf (Eaton et al. 199 1).

Arnmonia helps to neutdize the stomach acid in surrounding areas, once colonization has taken place and more Wendly alkaline environment is developed. Urease production may not be needed as an environmental modifier but rnay be an essential component in the pathogenic mechanism (Dum1993). Another important enzyme, catalase produced by Helicobacter species, protects the organisms fiom the toxic effect of reactive oxygen metabolites formed in neutrophiis from hydrogen peroxide (Lee et al. 1993). Pathogenk mechanisms inchde toxins and enzymes which act as mediaton of idammation or contribute to acidlpepsin activiv Hypochlorhydria is fiequently observed in humans patients recently Mected with Helicobacter pylori. Nelicobacfer spp. can inhibit gastric acid secretion fiom isolated rabbit parietal cells and it has been aiggested that specific inhibition of acid secretion may facilitate acute HelicokcterpyIon' infection.(Vargas et al.

1991).

Despite the fact that strong antibody response occurs as a result of Helicobacfer pyZori infection, bacteria fiequently persist for years, possibly for the life of the human host. unless treatment is initiated. Typically, the infiammatory readion ocnin deep in the gastric mucosa some distance fiom the bacteria, which may explain how the organism survives (Lee et al_ 1993).

Bacteria of Superfamily VI are chemo-organothroph, they derive their energy and carbon from organic compounds. Carbohydrates are neither fermented nor oxidized

(Smibert 1983). It is unlikely that they could persist for years relying on the food that the host ingests (Blaser 1996). Therefore, some regulated interaction between these organisms and host cells must exist. Possibly, Helicobacter pylori trigger inflammation as a means of attaining nutrients. Urease decomposition and production of arnmonia initiates imtation of the epithelia.

Cytotoxin-producing strains of Helicobacterpyiorî have been-identified and it has been demonstrated that they are directly associateci with the degree of gastritis in gnotobiotic pigs (Eaton et al. 1989) and humans. Two genes ofHelicobacterpyIon'responsible for toxin production have been recognized, identified and sequenced and named cagA and vacA- About 50 % of patients with chronic gastritis alone are infected with cagA strains, but

virtually all patients with duodenaiulcas are infiiedwith this strain. Toxh producing strains

induce production of vacuoles in tissue cultures, and they are 30-40 % over represented in

ulcer patients, compare to those with gastritis alone (Blaser 1996).

In humans, Helicobacfersinfiame the tissue that becornes ulcerated, therefbre one

can speculate that direct impact of the bacteria lads to ulcerative lesions. At least, this

appears to occur in certain patients in combination with other predisposing factors. In pigs,

ulceration is generally restricted to thepms oesophages, whereas, Helicobacter heilmannii

are observed mainly in the pyloric area at the lesser curvature region. Yeomans and Kolt

(1996) suggested that an infiited stomach over-secretes hydrochlonc acid, which causes

damage to the epithelium of the pars wsophogeu- In support of this theory, Moss and

Calam (1993) have found that hyperacid secretion due to increased gastnn release is

st imulated b y the ammonia produced as the result of Helicobacter pylori colonization. They

argue that shift in pH in the layer overlaying the gastnc epithelium, generated by the bacteria,

interferes with the normal inhibition of gastrin release by intramural acid. Eradication of

Helicobacterpylori infection genedy results in a decrease gastnn-mediated acid secretion

by two-thirds in patients with duodenal ulcers (El-Omar et al. 1993). Gnotobiotic swine

monoinfected with Helicobacter heiimannii and fed a high carbohydrate diet showed no difference in gastric pH value before or after infkction (Krakowka et al. 1998). 2-25DIAGNOSIS

A demand for a refiable test to detect the presence ofH. heilmmtni has been created by reports of an association between naturaliy occurring ulcers and preulcer lesions of the pms oesophages of swine and the presence of this organism (Queiroz et al. 1996). Ideally, such a test would have a high degree of sensitivity and specifie, would be easy to perforrn using routine equipment and techniques; would be minimally invasive, and would be inexpensive. Unfominately, such an ideai test has yet to be designed.

A number of diagnostic tests are available to detect Helicobacter pylon infection

(Brown and Peura 1993, Goodwin et al. 1997), and they can be categorized as direct and indirect. Histologie demonstration of the bacteria or its identification by microbiologie rneans fiom cultured tissue constitutes direct evidence of its presence. Helicobacter heihnii (GastrospirilIum hominis) has been successfully cultured in vivo by using propagation in inoculated mice. For a long time in vitro culture was unsuccessfùl and only recentiy has the culture of Helicobucter felis and other bacteria structuralIy similar to cCGastr~spitiZZirmhominis" from dogs been achieved. The culture tirne is 5-10 days

(Haminen et al. 1995). Happonen et al. (1996) claim successfbl culhire ofHeZicobacter-like bacteria, resembling (under electron microscopy) human Helicobacter heiimcmnii, isolated from gastric biopsies of three dogs and one cat. The helical-shaped bacteria have been cultured eom the cat stomach on laboratory media (Lee et al. 1988). Holck et al. (1997) described the first culturable case of Helicobacter heilmannii-like organisms fiom human gastric biopsies in one of four cases assayed. Also, G~sfroqi.IZzîmhominis has been cultured in Australia fiom a human stomach (Lee unpublished). Indirect techniques rely on the response of the immune system to the bacteria present 6.e-, specific antibodies) or detecting a characteristic of the bacteria (e-g., the ability to hydrolyze urea). These rnethods of diagnosing infection include serology and the urea breath test. Serological tests for Helico&cterpyfori have been shown to be highly accurate with sensitivity and specincity of 95-100 % and are available in an ELISA kit system. Earlier problerns of cross-reaction with C'pyIoobucters and other gram negative bactena seem to be eliminated (VeItzhe-Schliehenir et al. 1992)-

The urea breath test is baseci on the ability of Helicobacfer qp. to decompose urea which is labeled with either 13C (a stable, non-radioactive isotope) or "C (a radioisotope with long half-life) to produce "labeled" carbon dioxide (CO2 in the stomach that later is exhaled fiom the lungs (Atherton and Spiller 1994). Exhaled air is sarnpled and the level of radioactivity measured as a means of determining whether urease producing bacteria were present in the stomach of the patient. The probe is highly sensitive and specific, easy to perform, and produces a rapid result. The test is cornmonly used in human medicine as a follow-up method of monitoring the success of Helicobacter pylon eradication treatment

(Caiiam 1997) and can be used as a screening procedure to ducethe necessity of invasive techniques (McColl et al. 1997).

Direct tests are cornmonly regarded as the "gold standard" with which the indirect methods are compared. However, the diagnostic accuracy of the direct methods is Iimited because the sensitivity is lus than ideal. Uneven distribution of Helicobacter throughout the gastric muwsa influences the histologie detection of organisms. A number of studies have addressed the so-called patchiness ofHelicobacfer colonization ofthe body and fundus of the human stomach Although the antnim appears to be more unifomily involved, there has been some concern that a single biopsy may lead to a fiilse negative result (Morris et al. 1989,

Wyatt et ai. 1988). AIthough only in a minority of cases (approximately 10 %) are any sections fkom a positive case wmpletely fiee oforganisms. Sarnpling error is thought to be a rninor difnailty in histologic diagnosis of Heiicobacferpylori, provided t hat biopsies are obtained from the antrum. Generaliy, two biopsies taken nom within 5 cm of the pylonis are considered to be sufficient for diagnosis. In addition to uneven distribution of the organism, a second factor that infiuences the accuracy of the test is the stain used. None of the dains used are specinc for Helicobucter pylwi or heilmrmnii. The appearance of the organism on histologic examination is neither conspicuous nor consistent. The conventional hematoxylin-and-eosin stain is regarded as unreliable for demonstrating

Helzcobacter pyluri and heiimannii (Gilman et ai. 1986, Bartel and Everett 1990). For instance, a large spiral bacteriurn was seen on carbol-ftchsin stained section but was not seen on hematoxylin-and-eosin (H&E) staining (Rocha et al. 1989). The sensitivity of this technique depends to a considerable extent on an experienced observer. The Warthin-Starry silver stain (W-S) tends to mapi@ the organisms, making them more prominent. This method has gained popu1arit.y despite its cost and technical demands. Carbol-fuchsin stain advocated by Rocha et al. (1989) tends to be simple, quick-, cheap and suitable for routine demonstration of spiral bactena in tissue section ofgastric mucosa. Positive sections show spiral bacteria as dark red bodies on gastnc mucus against a reddish background. The fluorescent stain acridine orange is comparable in effectiveness to the Warthin-Stany stain, but requires the availability of a fluorescent microscope. Serology may be a more accurate method of diagnosis than biopsy, since gastdis can be a patchy phenornenon, and the biopsy technique assesses only a smaii ara of the stomach. Consequently. indmect tests may iden* cases missed with direct methods. Thus,

"fdse-positivey7serology may, in fact, reflect falsely negative biopsy results- The main advantage of serologic assays is that they allow screening of large numbers of people rapidly and at low cost. The disadvantage of thÏs method is that antibody titers may take several months to becorne elevated and remain several months after bactena have been eradicated

(Frornrner et al. 1988, Moms et al- 199 1).

Rapid urease testing of biopsy materid is simple and less costly than histologie examination. A rnethod for mapping urease-positive areas directly on the gastnc mucosa was developed. This assay allows detection of al1 urease-positive areas on the gastnc mucosa and may be usefully utilized for culture attempts and for rnicroscopic examination (Grasso et al.

1995).

A polyrnerase chah rqction @CR) for specific detection of Helicobacter is another reliable approach that allows very precise determination to which species the organism belongs.

2.2-6. CLLNICAL DISEASE

It has been established that gastritis and gastroduodenal ulcers in humans are strongly associated with presence of HeIicobucter pylon These organisms have a more difise pattern of distribution compared to Heficokcter heiImannii that are distnbuted focdy and restricted to the pyloric region. Gastritîs caused by Helicobacter heiimannii is significantly dderthan the di- caused by Helicobocterpylon (Stolte et al. 199 1).

Helicobacterpylori naturally infect odyhumans, however, gnotobiotic pigs have been experimentaiiy ïnfècted with these microorganisms. Once infected, pigs do develop gastritis which can lead to erosions and ulceration in the glandular araof the stomach, generally in the junction of findus and pylonrs (Krakowka et UL 1995). Gnotobiotic pigs experimentally infected with Helicobacter heilmmnii and fed a high carbohydrate diet responded in a similar way to pigs infected with Helicobacter pylw, except that the idlamation was generally distributed in the findus not in the antrum and cardia. The piglets did not develop lesions in the purs oesophagea (Krakowka et al. 1998). It is important to note that most of the stomach ulcers in commercially reared pigs occur in the non-glandular pars oesophagea.

There is no mucus production in this region, the ara is Iined by stratified squamous epithelial cells, and is not suitable for Neiicobacter colonization. However, the pars oesophagea is normally inhabited by various fementative bacteria including Loclobacihs and BaciilusSIn the same study, Krakowka et al- (1998) experimentally infected gnotobiotic pigs with these fementative bacteria and fed them a carbohydrate enriched ration, and this resulted in epithelial damage of thepors oesophogea-Volatile fàtty acids produced by these bacteria on high dietary carbohydrate Ievels may be important interactive factors for ulcer development in the pars oesophagea.

In contradiction, several epidemiological surveys conducted in Brazil and Italy have observed the presence of Helicobacter heilmmmii in the glandular stomach to be associated with lesions of thepurs wsophaga (Mendes et ai- 1991, Grasse et ai.1996). Barbosa et al. (1995) performed a screening test with 32 pigs with chronic ulceration of the pms

oesophageu and another 32 pigs with grossly nodpmsoesophages They found that the

prevdence ofHelicobucterheilmannii in the group ofpigs with and without gastric lesions

was 84 and 41 %, respectively-The rate ofgastrïc Iesions inHelicobocter heiI'mnii Uifected

pigs was 7.9 times greater than the rate of gastric lesions in non-infected pigs. Queiroz et al.

(1996) exarnined the stomachs of 70 pigs with different degrees of Iesions. They found

bactena present in 100 % of stomachs with ulcers, in 90 % of stomachs with parakeratosis,

but in only 35 % of grossly normal stomachs. Therefore, they have suggested that there is a strong association between Helicobacter heilmonnii and gastric ulceration of the purs oesophgea

2.2.7, TREATMENT

Helicobacter pylorï is responsible for two of the rnost common gastrointestinal diseases: gastric and duodenal ulcers, and they play an important role in the development of gastrk cancer, the second most cornmon lethal neoplasm atfecting humankind (Axon 1996).

Therefore, eradication therapy is considered to be the most appropnate way of treating gastric and duodenal ulcers associated with Helicobacter infection,-However, treatment remaùis a controversial area. These bacteria are sensitive to a wide variety of antibiotics in vitro but monotherapy in vivo is rarely successful. Helicobacter pylon' have the ability to maintain a tolerable pH in their periplasmic space. They can survive in a penplasmic pH range between

4.0 to 8.0, but growth cm occur between a periplasmic pH of6.0 to 8.0; at the range 0f4.0 to 6.0, the bacteria wiii not divide. In the absence of division, antibiotics such as clarit hromycin and arnoxycillin are ineffective. Proton pump inhibitors, such as omeprazole

and lansoprazole, elevate the gastric pH and as a result increase the population of dividing

organisms, and hence cause a synergetic effect with these antibiotics (Scott et al 1998). At

present, the "Gold standard" for treatment of Helicobacferpylori infection is a one week -

course of twice daily dosing of either: clarithromycin 250 mg, omeprazole 2Omg and metronidazofe 500 mg or clarithromycin 500 mg, amoxiciliin 1000 mg, and omepratole 20 mg. These treatment regimes have eradication rates of more than 90 %, an easy dosing schedule, and few side effects. Once eradicated, re-infection is rare (less than 3 % in the first year and about 1 % in the second year) (Matisko and Thomson 1995). According to Jenkins and Basset (1997). ulcers recur in less than 2 % of patients after Helicobucter sp. are eradicated but in 50 % in whom the bacteria persist.

Infection in some patients would appear to be ineradicable.

Bismuth-based compounds are commonly included in multiple-drug protocols.

Bismuth accumulates beneath bacterid ce11 walls and causes ce11 lysis. At present, bismuth compounds are not vsed as a mono-treatment, because results are variable and side-effects signifiant.

Eradication of Helicobacter heilmmiii appears to be easier than Helicobacter pylori.

Lee and OYRourke(1993) described eradication ofthese bacteria in eight patients after four weeks of bismuth subsalicylate therapy.

Little information on the effectiveness of protocols for eradication Helicobacter infection in veterinary patients is avaiiable. A combination of metronidazole, amoxicilfïn, and bismuth is effective in elirninating infection in ferrets (Fox 1995). Marked improvernent in the

31 cihicai signs of Helicobactr infection is evident in 90 % of the cats and dogs treated with a combination of metronidazole, amoxicillin, and Eàmotidine (Novo and Magne 1995).

2-2-8.PREVENTION

The discovery of an association between Helicobuctee heihzmnii and the occurrence of ulcas @ars oempiaagea) may lead to a change in the focus of prevention in treatment of gastric uIceration in swine.

Research into the development of a human vaccine against pylorÏ using a mouse mode1 is proceeding. Reseacchers have shown that mice can be protected against infection with large doses of viable Helicobacter felis by oral immunization using sonicates of

Helicobacter felis or Helicobocter pyfon*or by recombinant Helicobacter pylori urease together with cholera toxin or cholera toxin-B subunit as the mucosal adjuvant (Lee 1995).

Parenteral injection of antigen or intragastric administration of high-dose antigen without adjuvant elicited senun IgG but no IgA antibodies and did not confer protection.

Irnmunization of dready infécted anunals results in eradication of infection (&ee 1995). This raises the intriguing possibility that therapeutic immunization might be a viable option in the management of Helicobacfee-associated disease in other species including swine. 2.3. AClD SECRETION IN TEfE STOIMACH

2.3.1. ANATOMIC-PHYSIOLOGICALCONSIDERATIONS

The structure ofthe stomach varies widely fkom species to species, depending on the nature of their dies, and semes to initiate digestion. The general fùnction of the stomach is to begh the digestive process by breaking down the physical htegrity of food so that digestive enzymes wül have easy access to substrates as the digesta passes through the intestines. This initial proteolysis is mostly accomplished by subjecting the food particles to a low pH. The acidic conditions of the stomach also helps to prevent bacterial overgrowth in the stomach and upper intestines.

Pigs are sùnilar to humans in that both species are omnivorous monogastrics and have a digestive system adapted to handling a wide range of feedstuffs. However, significant differences exkt in anatomy and therefore physiology of their stomachs. In pigs, motility is relatively weak and slow mhhg and emptying occurs. This situation favours gastric digestion and the greatest rnixing occurring at the distal end of the stomach and rnicrobiological fermentation taking place proximally (Moran 1982).

There are four functionaüy different zones in the pig stomach; each of these regions has a distinct lining, see Fig. 2.3.1.

the pors oesophagea (does not occur in humans) is an easily recognized and sharply

demarcated area surrounding the oesophageal opening. It is comprised of a stratified

squamous epithelial surface simüar to the oesophagus. Unlike the rest of the stomach, the

pms oesophagea does not produce glandular secretions. In addition to lacking the bicarbonate secreting mucous layer (which is a major barrier to acid insult in the oxyntic mu-) thepars oesophogea also lacks the abiiïty for rapid repair by of viable cells fkom beneath the injury (Argenzio and Eisemann, 1996). The normal pms oesophaga appears as a glistening, smooth, white sufice. In reaction to insult, the su~acebecomes thick and rough as a result of keratinization, and may appear yellow in colour as a result of bile staining- the cardiac region is adjacent to the pars wsophagea and represents about one-third of the total gastric area. An out-pocketing called the ventral diverticulum is located in the cardiac region. The cardiac mucosa does not contain acid secreting glands. Cardiac glands are located in the submucosa and secretea specialired mucus to protect the surface epithelia fiom digestion. Bicarbonate ion exchange for chioride ions occurs in this region helping to raise the surface pH up to 5 (Yeomans and Kolt 1996). In contrast, in humans most of the gastric epithelium is oxyntic with ody a small area of cardiac mucosa. the &ndic region contains glands for the digestion of food, as well as glands for the protection of the epithelium The sufice of the fùndic region is charactenzed by folds or rugae which increase the surface area of this region. There are three types of glands

(mucus neck cell, parietal cell, and chief cell), and each produces a separate secretion with a dEerent purpose. Parietal (oxyntic) cells produce HCl at the membrane surface.

Porcine parietal ce11 luminal membranes have been shown to have an ATPase that catalyzes an K+K electroneutral exchange (Moran 1982). Chief cells produce pepsinogen, an inactive protease form of pepsin. Activation of pepsinogen occurs at a low pH of about 2. the pyloric region does not contain acid secreting glands but mucus-produchg ceUs are

present, as weii as G ceils which produce the gastrk stimulatory hormone gastrin-

Figure 2.3.1: Comparative anatomy of hiiman and pig stomachs

s VentraC dherticulum Q -Thepars oesophagea i -Thecardiac region The fundic region The pyîoric region Humans Pigs

Gastnc motility involves peristaltic contractions ofthe circular smooth muscle layer that moves progressively fiom the oesophageal region tbughto the pylorïc sphincter. The greatest mWng occurs terminally in the pyloric ant- where peristaltic waves are strongest. if the feeding interval is short, then the most recent meal will senle on top of the remains of the earIier one. Small particlesof food (less than 2 mm)pass through the stomach in a 12 h period (72 %), whereas larger particles (2 cm) remain in the stomach for up to 60 h or longer (Clements 1975). 2-3-2-GASTRIC SECRETION AND DEFENSIVE MECKANISMS

The balance between aggressive and defensive factors in the stomach protect it against self innicted injury. There are several aggressive factors that may cause gastric lesions hydrochloric acid (HCI), pepsin, biie acid, varied bacferia, and abrasive foreign bodies.

Hydrochioric acid production results in the high concentration gradient of El? ions between gastric lumen and the epithelial ceUs in these areas. These ions are able to cause epithelial darnage ifthe defensive balance in the stomach is impairad

Normaily, pig's stomachs are rarely empty and have relatively weak motility, thus the relatively neutral proximal region is not exposeci to the acidic effect ofgastric juice (Rüs and

Jacobsen 1969). When pigs are fmed or féd a finely ground diet (in cornparison to a coanely- ground diet) t his pH gradient is lost. Schiessel et al. (1990) believed t hat a back flow of H+ ions can cause ulceration the pars oesophagea because it is not protected by a mucus covering. However, more recent work has shown that the stratified squamous epithelium lining the pms oesophagea is relatively resistant to hydrochloric acid and pepsin (Orlando

1991, Argenzio and Eisemann 1996).

Pepsin is not responsible for initial iesions of the pms oesophagea, because it is not active above a pH level of 3.5. However, pepsin is capable of causing visual tissue damage by digestion of the epithelial Iining (Schiessel et al- 1990). Reimann et al. (1 968) found that pepsin activity varies in the pig stomach in the range of 4 to 13 unitdml depending on particle size of feed. The finely-ground diet induces a higher pepsin secretion than a coarse diet. Bile acid may work in conjunction with gastric acid to cause the rapid destruction of epithelial tissue of the pars msophagea. Back flow of bile from the duodenurn increases the celi permeability for H? ions, favoring acid back diision (Schiessel et al. 1990).

Gastrin is released by glands located in the pylonc region in response to food intake in the stomach. It strongly stimulates secretion of gastric acid and pepsin and weakly stimulates gailbladder contraction and secretion ofpmcreatic enzymes (Davies 1993).

The pH of the digesta in the oesophageal region tends to be higher than the findic and pyloric areas. LactobaciIZus and BaciIIus, as well as other microbes, are normal inhabitants of the pms wsophqgea and are capable of adhering to the keratinited epithelium. Both

IActobaciilus and Bacilhs contribute to total gastric acid levels by producing organic acids such as lactic, acetic, and proprionic acids, as well as ethanol and hydrogen peroxide during fermentation (Krakowka et al. 1998). An absence of salivary amylase, which could augment microbial activity is thought to be a purposeful evolutionary adaptation to avoid excessive fermentation @forart 1982). Krakowka et al. (1995) have shown in their experiments that combinations of these bacteria and a carbohydrate-enrichecl Iiquid diet causes erosions and ulcerations in the pms oesophagea. Argenzio and Eisemann (1996) suggested that fermentativebacteria are capable ofcausing rapid destruction ofthepmsoesophagea at much higher pH levels than levels at which HCl acid alone causes injuries in the stomach-

Helicobacter heilmannii alrnost exclusively colonize the pyloric regions and may stimulate parietal or G cells to produce excessive amounts ofgastrk acid. Altematively, they may interfixe with acid secretion inhibitory mechanisms (Yeomans and Kolt 1996).

niegastric mucocus bamer, bymeans of lubrïcation, protects the underlying mucosal ceils from mechanical abrasion, and by means of bicarbonate secretion and restriction ofw ions provides mixing protection against the hostile effect ofhydrochloricacid, pepsin, and bile (Men and Garner 1980). A thin layer of muais encompasses the whole stomach, except the psoesophogeu- Gastric mucosa pH on the epithelial side of the mucus layer is near neutral (about 7.59) when luminal pH is about 2.36 (Williams and Turnberg 1980). The

H+/K'- kTPase cornplex, in the apical membrane, maintains the high intracellular pH that îs needed to prevent injury to mucosa nom H? ions.

2.3 -3-CONTROL OF GASTRIC ACID SECRETION

The control of gastric acid secretion by the stomach occurs at a variety of levels and is controlled by neural, hormonai, and paracrine mechanisms. There are three phases of gastric secretion and each phase involves nervous and hormonal components. The first, or cephalic stage, occurs when the sight, taste, or chewing of food triggers an increase in vagal nerve activity. Vagal stimulation can induce the secretion of hydrogen ions directly, and in addition, vagal stimulation of G aUs causes gastrin to be released into the blood Stream.

The second, or gastric stage, of acid secretion occurs as a result of stomach distension. The gastric wall contains int~sicneurons that are part of the enteric nervous system. Distension stimulates acid release via cholinergie pathways from receptors in the body and antrum of the stomach to the parietal cells. In addition to local stimulation, the central nervous system receives signals and acetylcholine is released at nerve terminais enervating both parietal and G ceils. Histamine also plays a role in regulating acid secretion.

The third phase of gastric secretion occurs when gastric acid enten the duodenum.

The duodenal hormones, secretin and cholecystokinin, act to inhibit secretion. Sirnilarly, 10w pH levels (near 1.0) in the anmirn cause the inhibition of gastrin release fiom the G cells. The mechanism by which the main neural (acetylcholine), hormonal (gastrin), and paracrine (histamine) secretory stimulants affect the patietal ceil is known (Schubert and

Sharnburek 1990). Ail three regdatory mediators converge to shulate an WK-ATPase located at the luminal surface of the parietai ceil, thus evobg hydrogen ion secretion.

Histamine causes an increase in adenylate cyclase actMty, and acetylcholine and gastrin are mediated via an increase in cytosolic levels of calcium. Histamine acts synergistically with either acetylcholine or gastrin because it uses a different pathway to create its effect.

Acetylcholine and gastrin are capable of stimulating the release of histamine fiom the gastnc mucosa. Somatostatin and E inhibit acid secretion by reducing adenylate cyclase activity. The knowledge of receptors and signal transduction mechanisms as well as neural and paracrine pathways has led to the development of drugs capable of inhibiting gastric acid secretion.

2.3.4. DRUGS INHlSITING ACID SECRETION AND ANTACIDS

Histamine Hz-receptor antagonists selectively and reversibly bind Hpxeptors on the basolateral membrane of the parietal cell, causing a reduction in cytosolic cyclic AMP production (Freston 1990). Examples ofthese drugs include cimetidine, ranitidine, famotidine, nizatidine, and . Histamine Hz-receptor antagonists inhibit secretion response to al1 modes of stimulation by eliminating the direct and synergistic influence of histamine on gastrin- and acetylcholine -stimulated acid secretion and by blocking the effect of histamine released by gastrin or acetylcholine (Schubert and Shamburek 1990)-

Unfortunately, histamine Hz-receptor antagonists have not been found to reduce the incidence or severity of ulcers of the pms uesophageu in mine associateci with finely-ground feed

(Hedde et al. 1985). Likewise, ranitidine (150 mg per day) administered by injection, three times per day Medto protect somatotropin-treated pigs from developinggastric ulcers (Baile et uL 1994)- htrarnuscular administration of cimetidine (4.3 mgkg) or ranitidine (0.75 mgkg) causes gastric pH to rise above 3.5 for only about 2 h in swine compared to a much longer tirne in humans (Sangiah et a/, 1990)-

Muscarinic antagonists such as and act on ganglionic muscarhic Mlreceptors to inhibit release of acetylchohe, and to a lesser extent on peripheral

M, recepton present on parietal ceUs (Schubert and Shamburek 1990). They also block the inhibitory effect of acetylcholine on somatostatin ceUs, allowing increased inhibition of - parietal cells by sornatostatin- Their clinical use has been somewhat restricted because of side-effects,

Inhibition of H$/KttATPase profoundly reduces acid secretion stimulated by any means because this proton pump is the final step of acid secretion. Benzimidazole derivatives such as omeprazole and lansoprazole are irreversible inhibitors ofthe luminal enzyme, H+/Kt-

ATPase. New enzymes need to be created before the effect can be reversed and this takes at least 24 h in humans- Omeprazole in doses of 20 mg or more, once daily, virtually abolishes acid secretion in human patients (Freston 1990). These benzirnidazole compounds have been shown to successfiilly reduce severity and prevalence of ulcers in swine treated with porcine somatotropin (Baile et ai. 1994).

Gastrin-receptor antagonists such as the benzodiazepine denvatives L364,7 18 and

L365,260 and , block the direct and synergistic effect of gastrin on acid secretion after meals (Schubert and Shamburek 1990). lh addition, they may block the atrophic effect

of gastrh on parietal celis. Somatostatin-reseptor agonists such as octreotide (a synthetic

analogue of somatostatin) inhibit histamine-stimulated secretion by suppressing cyclic AMP.

They inhibit acetytcholinesrgastrin-stimulated secretion via somatostatin recepton coupled

to other pathways. Their main disadvantage is that they require parenteral administration.

Prostaglandins, prirnady in the E series, have been used in human medicine to treat gastroduodenal ulcers for over 20 years. These analogues include: , rioprostil, arbaprostil, trimoprostil, enisoprostil, and . Acid secretion is reduced because prostaglandin analogues interact with inhibitory receptors ofparietal cells interferhg with the production of cyclic AMP. In mucus-secrethg cells, they interact with stimulatory receptors producing aikaline secretion and inhibit mucosal histamine release.

Antacids have been used for many years in human medicine to provide buffenng of gastric acid in an attempt to provide a suitable environment for gastnc ulcers to heal. In swine studies, sodium bicarbonate is the most comrnonly used antacid,. Early trials found no eEect on ulcer severity in pigs by feeding 3 % NaHC03 (~ambteet al. 1967) whereas in one recent study, slight improvement with inclusion of 1 % NaHC03 in the ration was observed

(Wondra et al. 1995a). In another trial, feeding the same concentration of sodium bicarbonate pigs receiving the antacid treatment had more severe ulcers than the controls (Southern el al.

1993). The ingestion of sodium bicarbonate in small amounts over time can result in an increase in acid secretion, and this may nullify the advantages of its buffering capacity- Non- absorbable antacids such as aluminum hydroxide and magnesium silicate may be preferable to sodium bicarbonate because they act slower and the eEect is prolonged. There are products which can enhance mucosal defense in the face of excess acid producbon. Suc-te is a oulfàted disaccharidecomplexwith aluminum hydreth heals ulcers by rnechanisms otha than decreasing gastnDcacidity Sucraif'ate binds to the base of the ulcer providing local protection and possibly stimulates prostaglandin synthesis. Its effeaiveness in preventing ulceration in bile duct-ligated pigs has been demonstrated

(Stapleton et a% 1989).

SimiIarIy, colloida1 can bind protein in the base of an ulcer, inhibii pepsin activity, and possibly stimulate prostaglandin synthesis. It has been comrnonly used in conjunction with acid secretion inhibitors as a treatment for gasaic ulcen in humans. 2.4. OBJECTIVES

Gastric ulceration is an important and cornmon disease, recognized in the swine industry world-wide. Ulceration of thepars oesophogea has a high prevalence and can be a signincant cause of death in certain herds due to bldmg nom deep, erosive lesions. Many pig fdgopedons are sustainhg considerable economic losses fiom this disease. The approxhate annual mortality attributable to pms oesophagea ulceration has been estimated to be between 0.5 % and 0.75 % (Deen 1993) and can reach 1 %, with culling Iosses of 3-5

% in such herds (Nielsen 1995). In Ontario, death loss has been estimated to be about 0-25

% of grower-fhisher pigs or about 10,000 hogs per year. In additi~r~subclinical lesions may cause a decrease in average daily gain (ADG) of about 50-70 g per day, at least in another

10-20 % of grower-finishers. These numbers may increase significantly in the next few years as economic pressures force the swine industry to focus more and more on feed efficiency-

The high cost of feed demands reducing feed particle size. Because reduction of feed particle size results in improved nutrÏent digestibility and better feed utilization, this will be emphasized even it leads to a significant increase in gastric ulceration.

A strong association has been reported between the occurrence of ulcers and preulcer lesions in the pars oesophagea and the presence of a spiral bacterium HeIicobuctei- hezlmannii. This bactenum is the only Helicobacter sp. that naturally infects several species of animals as well as humans, and therefore it may be considered a zoonosis.

Gastric ulcers are an old problem, but many questions have been left unanswered. The objectives of our studies were:

1. To investigate the level of mortality and morbidity due to gastric ulceration in a

modern swine operation.

2. To detemine whether Heficobacter heiimanii -üke bacteria can be readily detected

in North Amencan commercialIy-raised pigs, and whether the presence of these

organisms is associated with the occurrence of gastnc ulcers ofthepsoesophages-

3. To evaluate efficacy of difEerent treatments on alleviation and prevention of gastric

ulceration associated with feed withdrawal- Cha~ter3

MORTALlTY ASSOCIATED WlTE GASTRSC WLCERATION - CASE STZTDY

The purpose of this study was to investigate the prevalence of subclinical gastric

lesions in pigs fished in a large, modem, pig fdng operation and to determine the

monality rate due to internai bleeding as a resuIt ofulceration ofthepurs oesophagea.

3.1. MATERIALS AND METHODS

3.1- 1. CASE HISTORY

A large agncultural corporation with a history of signincant economic loss in

grower-finisher pigs due to gastric ulcer mortdity was used in this study. The Company

produced pigs at two location, one in Missouri and one in Texas. No concems had been

raised with regard to this disease at the Texas location. Therefore, our investigation was limited to the Missouri operation.

a) THE FAR.'ORGANIZATION

Farrowing, breeding-gestation, and nursery stages of production at the Missouri enterprise are located at the same site. The sow inventory is approximately 100,000. At the gestation barns, sows are vaccinated against parvovirus and Ieptospirosis. Merfarrowing, sows are transferred to the breeding barns.

There were four fmowing-nurserycomplexes at theMissouri operation, that supplied piglets for eight grower-finisher complexes and three multiplier grower-finisher locations- There were in total 520 grower-finisher curtain-sided finishg barns, with a capacity to hold

1,150 pigs in eadL Barns are clustered together in groups of 8, with one person managing each cluster of buildings.

Piglets were weaned and moved to the nursery units at about 17 days of age, and their average weight was 5.5 kg. Pigs remains in the nursery for seven weeks until they weigh 20-

25 kg at which tirne nurseries were emptied and animais were transferred to company-owned grow-finishing barns or to contract facilities.

b) GROWER-FINTSHER MANAGEMENT

The grower-finisher clusters were operateci on an &-in / dl-out basis. The pigs were kept on totaliy slatted floors, and fed a finely ground pelieted ration composed of corn and soybeans fed dIibi~um. They gained 680-770 gm per day for about 15-16 weeks until they reached a slaughter weight of 115 kg. The entire barn was fasted for up to 24 h while the heaviest pigs are weighed and selected for marketing. Pigs not ready for market were fasted for one day per week for several weeks until they were finaliy shipped. Feed withdrawal was done to ensure that pigs shipped to the slaughter plant had an empty stomach. Pigs grown in Texas were shipped to slaughter in Missouri, but in contrast, they were lefi with feed available until they were loaded on the truck to begin the 17 h trip to the abattoir. The barns were thoroughly washed before restocking. Al1 pigs were slaughtered at the corporate-owned abattoir.

Mortaiity was recorded on a weekly basis and the causes of the death as detennined by the herdsman was also entered into the computerized record system. The causes of death were divided into three categones: digestive disorders Cicluding gastric ulceration and

gastro-intestinal torsion), respiratory diseeses, and other (ïmcluding unknown). Herdsmen

assigned animais to one group or another on the basis of observed cliical signs or the

appearance of the carcass. Dead pigs that look c'bleached" or pale were assigned to the

"digestive disorder" categocy. ANmals that had shown signs of respiratory disorders before death were assigned to the "death fiom pneumoniaS7categov-

3- 1.2, STUDY DESIGN

nie Missouri operation was visited twice in a 3 month period (Apd and June 1998).

Production records were examinai at aii grow-finishing sites during each visit. By means of purposive sampling, the four largest grower-nnisher complexes were selected for the study.

In total, 49 of the 65 units were examined. Each of the sites were visited twice (once on each visit). The prevalence of subclinical gastric lesions was assessed at the company-owned abattoir. In total, 1,097 stomachs were examined, 1021 fiom Missouri and 76 fiom the Texas operation.

The stomachs were randomly selected from the 3,706 stomachs obtained Eorn pigs delivered to the meat plant fkom 5 Missouri and 2 Texas complexes. Each stomach was opened dong the greater curvature Corn the pylorus to the diverticuium. Ingesta were removed and the mucosai surfaces were gently washed with tap water. Thepars oesophageen were examined and lesions assessed and scored using a similar scoring system to Machet al. (1997), Table 3.1.2. Table 3-1-2: Classification of stomach ksions

1 Grade 1 Description 1

Normal - shy, white, glistenhg, and smooth surface O I

Parakeratosis - roughened, thickened, comgated surface, yellow

2 Small crosion(s) - padcularly at the border of the cardiac regions

3 Active ulcer andlor cicatrization - deep or extensive erosions

An in situ urease assay was performed on 45 randomly selected stomachs according

to the method of Grasso et al. (1995). Each stomach was placed in an aluminum tray and

covered with a thin layer of gel-like medium consisting of 2 % urea, 0.0012 % phenol red,

0.3 % agar, 0.01 % yeast extract, 0.0091 % monopotassium phosphate and 0.00995 %

disodium phosphate @H 6.7-7.0). Al1 sarnples were visudly monîtored for L -5 h to detect a

colour change fkom yeilow-orange to deep pink (due to pH increase) on urease positive areas.

Biopsies were taken for microscopie evaluation fiom al1 10 stomachs that produced a positive or equivocal results. A 3x3 cm sample ofgastric wall was taken from these areas (pyloric

region at the lesser curvature), and fixed in 10 % buffered neutral formalin for histological

evaluation.

The cause of death indicated by the herdsmen was confinned by means ofgross post- mortem examination. Investigators spent one day on uich chosen complex performing necropsies on al1 recently dead pigs at each site.

48 On the first and second visit, postmortem examinations were conducted on 146 and

136 animals, respectively. This constituted 12 % and 10 % of the total weekiy loues,

respectively. The number of pig deaths due to gastric ulceration as determined by necroscopic examination was comparexi to the proportion of mortality due to " Digestive disorders" recordeci by herdsmen based on ciinïcal history and appearance of the carcass.

3.1.3. STATISTICAL ANALYSIS

Ail data were stored and analyzed in SAS (PC - SAS 6.12, SAS Institute, Cary, NC).

PROC FREQ procedure was employed to examine these binary and ranking (gastnc lesions scores) variables. Mantel-Haenszel Chi-square test was caiculated, results were considered significant if p-value < 0.05. In addition, results were tested by two-sided Fisher Exact Test.

3.2. RESULTS

The grower-fïnisher pig inventory for the company-owned units was 503,321 and

617,589 pigs on the first and second visits in April and June, respectively. The crude (ttue) weekly mortality for the entire complexes was 1,219 (0.24 % of al1 pigs at risk) in April, and 1,372 (0.22 % of al1 pigs at risk) in June, which is almost as twice low as the rnortality in Ianuary (crude mortality 0.4 %)).

The total death loss in the 23 week period nom January until the second visit in June was 36,8 12 pigs or an average of 1.600 pigs per week. The average weekly inventory of grower-finisher pigs during this period was 55 1,959. The length of time a pig remained in the grower4nisher barns was 15 - 16 weeks, thus the mortality rate for pigs during the grower-finisher penod was about 4.4 - 4.6 %. This mortality figure included animals culled and destroyed.

Mortality attributable to digestive disorders as classified by the herdsmen was 573 pigs for the week ofthe April visit and 540 pigs for the week in June, accounting for 47 % and 50 % of al1 deaths respectively. In Ianuary this proportion was much lower and wmprised only 38 % of the totd death loss (Tables 33.1 and 3.2.2).

Post-mortem examinations of 146 pigs in April reveaied that 39 pigs (27 %) died fiom hemorrhage due to gasaic ulceration. Similarly, during the June visit, 137 pigs examineci indicated that 37 animals (27 %) had died as a resdt of hemorrhage induced by gastric ulcen.

Case specifïc mortality attributable to gastric ulcers (of thepars oesophageal varïed between the sites ranging fiom 1 1 to 36 % in the first examination and 22 - 3 6 % in the second visit. Total death loss from gastric hemorrhage and gastrointestinal torsion at the time of investigation was 39 and 35 %, respectively (in the weeks of April and June), which was significantly lower (p-value < 0.05) than data reported by herdsmen (See Table 3 -2.1 and

Table 3 -2.3)-

Table 3.2.2: Mortrlity and inventory in grower-finisher groups by quarters (pigs)

Total Mortality YO Total Inventory

Fint Qunrter (week 1-13) 23,909 034

1 Second Qunrter (week 14-23)"

TOTAL on the dry of investigation 36,8 12 0,29 12,695,049 \ 1 Average (per week)

-

* Week 24-26 are not included in Second Quarter

The second most cornmon cause of death as determined by gross necropsy . . examuiation was pneurnonia, accounting for 11 % of deaths in Apd, and 28 O/O of deaths

in June. The ApriI findhgs are almost twice as low as the 20 % pneumonia death rate

reported by the herdsmen , and in June it was almost twice as high as reported by site

managers. Cause specific mortaiity attributable to pneumonia, varied arnong complexes in

the range of 5 -17 and 19- 36 % in April and June, respectively. In addition, mortali'ty nom

"pneumonia" reported by the site managers in the first week of January was almost two

times higher than that reported in Apd.

At the abattoir 49 % of the stomachs examined had erosive lesions of the pars

oesophagea and only 8 % were classifieci as normal. A difference was noticed in the

prevalence of gastric lesions in pig stomachs fkom the Missouri and the Texas operations.

Only 6 % ofthe stomachs fiom Missouri had normal stomachs, 43 % had minor to moderate

parakeratosis, and 5 1 % had minor to extensive ulcerations. In contrast, ody 10 % of the

stomachs nom Texas had ulcerations of the pars wsophagea, 53 % had oniy minor

parakeratosis and 37 % had grossly nomal pcas oesophagea (See Table 3-2.4).

Table 3.2.4: Prevalence of gastric tuions @am oesophagea) detected itslaughter

Classification Source of pigs I 1 i Normal Smaii or Extensive üiceraîions Parakeratosis Total (0) (1) (2 + 3) Missouri 63 (6%) 434 (43%) 524 (51%) 102 1 r Texas 28-(37%) 40 (53%) 8 (10%) 76 r An in situ urea assay revealed two stornachs with a urease positive reaction (4 %), eight others (18 %) demonstrateci an equivod result, and the rest 35 (78 %) stomachs exhibiteci a negative result. Out oftwo urease positive stomachs. one had severe ulcen ofthe pars oesophages (grade 3). and the other had minor parakeratosis (grade 1). In both stomachs spiral-shaped, tightly-coiled badena that had 3 to 11 spiral turns were found (many had 8 spiral tums). The microorgarüs~swere readily seen in Warthin-Stany silver stain, and measured up to 7 p long and about 0.6 p wide (See Figure 3.2.1 and Figure 3.2.2). In addition, in one biopsy curved rods were seen, but were not identified. Al1 bacteria were observed in mucous and in the crypts, and they were not attached to the epithelium. It was noted that deep in the crypts bacteria were clumped together and partially or completely digested (See Figure 3 -2.3). Spiral bacteria were Gram Brown and Brenn (G-B&B) - negative, and non-acid fast (stained blue with Ziehl-Neelsen (Z-N) carbol-fuchsin). Organisms were not visible in slides stained with hernatoxylin and eosin stain (H&E). Figure 3.2.1: Helicobacter heilmnaii-like bacteria in pyloric region of the pig's stomachs (magnification x 100, Warthin-Strrry silver stain)

Figure 3.2.3: Eecicobacter hdïmanii-ülre bacteria deep in the crypts, clumped and parti*llylysed (Warthin-Starry silver stain, magrdication x 100)

One of eight stomachs which reacted with urea to produce uncertain results, was classified as normal (grade 0)and harbored the same spiral-shaped bacteria as were observed in the pyloric region of urea-positive stomachs. Bec of the eight stomachs had parakeratosis (grade l),and spiral bacteria were found in two of them. Three stomachs were graded as grade 2 (minor ulceration), and ody one of these reveaied identical microorgaoisrns. Only one stomach in this group of eight had severe ulceration with cicatrization (grade 3), and this stomach was free ofspiral bacteria (See Table 3.2.5). Table 3-25: The prevaknce of gunic Iesions and urease reactivity

- Grade O Grade 1 Grde 2 Grade 3

H-heifmanii-like c4+'y O 1 O 1 2 ne8 'w' heifmunii-like"-" O O O O O H: her'fmanii-like'&+" L 2 1 O 4 urea''j=" . I;I, hwWf~ii-me"-" O 1 2 1 4 Urcl =-" da 9 8 10 8 35 Total IO 12 13 IO 45

3.3. DISCUSSION

Cnide mortality in the grower-finisher area during the first 23 weeks of 1998 of approximately 4.6 % is higher than industry targets. Death due to gastric ulceration appears to be a major contnbutor of this high moitaiity- In the two studies using necroscopic examination, more than 25 % of the mortality was attributable to severe blood loss caused by gastric ulceration. The annuai economic loss attributable to gastric ulceration for this company can be estimated to be over 2.3 miIlion douars (US).

It has been estimated that in North Carolina 2 % to 2.5 % of grower-finisher pigs placed during the summer months bleed to death fiom gastroesophageal ulceration (Deen

1993). The level of mortality from gastric ulceration appears to be lower on this farm than the North Carolina estimate, however, the two visits to investigate mortdity occurred before the hot summer months of July and August. It is possible that annual death loss attributable to ulceration rnight be higher than estimated by this study. The Ievei of subciïnical gastric Iesions is correspondingly high with over 50 % of the

pigs fiom the Missouri operation having evidence at slaughter of erosions or ulcers. This

lwel is comparable to the highest fjvm prevalence of gastric lesions reported by Guise et al.

(1997) in a recent British study of 15 fms.

The interpretaîion of the slaughter data must be done with caution, in that several

researchers have demonstrateci that feed withdrawal pnor to slaughter results in a

significantly higher prevalence of erosive Iesions than if the animals were daughtered with

feed in their stomachs (Straw et al. 1992, Davies et al- 1994). The fact that feed is withdrawn

nom di pens of pigs in the entire barn for one day each week at which tirne the largest pigs

are shipped to market may contribute to ulcer development in the pigs that remain in the

The diet fed on this fdngenterprise consisting of finely-ground pellets is also a

probable contributing factor to the high prevalence of gastric lesions and high mortality rate

I attributable to ulceration (Hedde et al. 1985). Although both pelleting and srnall particle size

of feed are proven factors contributing to gmric ulceration, a switch to coarsely-ground

mash feed would be uneconornid even if al1 gastric ulcer death loss would be eliminated by

adopting such a feed change. Tt has been estimated that pelleting improves ADG by 5 % and gaidfeed by 7 % (Wondra et ai- 1995~).Reducing average feed particle size from L ,000 Pm

to 400 Hm increases gainlfeed by 8 %. The added feed cost associated with a change fiom hely-ground pellets to a coarse mash fm outweighs the cost cf the monality associated with gastnc ulceration in this herd. However, a temporary change to a mash feed for a short period of time rnight help to heal ulcers and reduce losses without a sigrifkant Ioss in feed efficiency (Ayles et al. 1996b). Altematively, research has shown that the use of a relatively small amount ofcoarse fibre, such as sunfiower huUs, added to a fine diet can decrease the number and severity of oesophagogastnc lesions to the same extent as coarse milling of a whole diet, but minimize the detrimentai effi on gaidfeed (Dirkzwager et ai- 1998).

However, coarse particles in a peiieted ration wili have a negative effect on pellet quality.

The solution to solving the problern of gastric ulcers on this farrning operation requires some compromise, and a geat deal ofagence with respect to feed manufacture.

In order to feed a finely-gnwd product, it is necessary that the feed mil1 must endeavour to produce a pellet that contains grain particles of a unifonn and appropriate size, and prepared at proper temperature. Feed production practices that incorporate flour or '%es" in pellets increase the risk of ulcers. In addition, high temperatures during pelleting might result in gelatinization of starches and therefore be mon ulcerogenic.

Research has suggested that fasting improves carcass quality (Morrow et al. 1999), therefore this operation is unwilling to stop the practice of feed withdrawal at the time of weighing and selecting pigs for market. Accordingly, it would appear prudent that dunng these periods of interruption in feed intake that pigs remaining in the barns should be treated to prevent ulcers. Numerous pharmaceutical agents have been used expenmentdly to neutralize acid, reduce gastnc secretions and/or promote ulcer healing. In this circumstance, with pigs about to be shipped to market being present in the barn, mass would need to be lirnited to prcducts which would not require a withdrawal period. Therefore, antacid preparations are likely the most suitable products for investigation. The use of sodium bicarbonate in the feed has been tned with mixed results (Wondra et al- L99Sa. Southem et al. 1993), but ody iimited information regarding water medication during

periods of feed withdrawal is available,

Helicobacter heiimamtii-Iike bacteria have been found in 10 biopsies taken fiom pig stomachs at the abattoir with positive and equivocal reaction on urea in siru assay. The prevalence ofgastric lesions in these stomachs was consistent with the prevdence in the entire group examined at the abattoir (Sa table 3.2.5)- Microorganisms were found in both normal and ulcerated stomachs. These spiral-shaped bacteria were morphologically similar to the bactena described by Mendes et al. (1990), but the stauiing properties were dEerent.

Bacteria found in Brazil stained eady with carbol-fuchsin stain, thus showing acid-fast properties. Helicobacter heiïmannii-like bactena found on this fmwere non-acid fast

(culturing of these microorganisms, was not attempted because at the tirne of study no reports of successful techniques for growing the bacteria were available).

Helicobacter heiZmmnii-like bacteria were found in normal stomachs and stomachs with difEerent degrees of lesions seventy. Only 22 % of stomachs showed a positive urease reaction or an uncertain reaction, and among these, on the microscopie examination, bacteria were found only in 60 % of the stomachs (33 % with ulcers, 50 % with parakeratosis and 17

% with nomal pars oesoy>hogea). In this small sample, there is no indication that the presence of Heïicobouer heilmmnii-like bactena is associated with gastric ulceration @ms oesophugeu)).However, this result is in contrast to other researchers findings (Queiroz et al.

1996, Grasso et al. 1996, Mendes et al- 1994). The small sample size rnight be the reason for this contradictory result. The ievei ofmortality attributable to gastric ulceration of about a quarter of the total grow-finishing mortality, and the prevalence ofstomach lesions at slaughter of close to 100

% of aiI pigs examined iflustrates that g&c ulceration is an important problem in modem pig famiing and is an area that warrants additional research. Cba~ter4

RELICORACZER BACTERIA IN lWE STOMACH OF PIGS

WlTE AND \NITHOUT GASTRK LESIONS

4.1. INTRODUCTION

Helicobactet heilmannii, unlike Helicobacter pyIori, have been shown to naturally infect pigs. Experimental Uifection of pigs with UeIicobacterpyiorï results in development of gastritis, erosions and ulcerations in the glandular area of the stomach, but not in thepars wsophagea (Krakowka et al. 1995). Epidemiologic studies (Mendes et al. 1994, Barbosa et al. 1995, Queiroz et ai. 1996, Grasso et al. 1996) suggest a possible relationship between

Helicobacter heilmannii infection and gastric ulceration of the pars oesophages in pigs.

However, expenmental infection of gnotobiotic pigs with t hese bacteria (Krakowka et al.

1998) result in gastritis, generally in the fundus, and no evidence of lesions in the purs oesophagea. The presence of these organisms in pigs has been demonstrated in Brazil and

Italy, but not in North Arnenca. The bacterium can not be detected using conventional hematoxylin-eosin stainùig and culture has been unsuccessfiil.

The purpose of this study was to determine whether Helicobacter heilmmnii-like organisms could be readily detected in North American swine, and whether the presence of these bacteria was associated with gastric ulceration. 4.2. MATERIALS AND METHODS

In this study, 163 pigs nom 3 dinerat hswere examïned and in addition, 47 pigs were examined during a pre-test trial, The experimentaI protoc01 was approved by the

University of Guelph Animal Care Cornmittee and was carried out in accordance with the principles published in the Canadian Council on Animai Care "Guide to the Care and Use of

Experbnental Animais",

Pre-test: Initially a pre-test was pefiomed on two smaU randody selected groups of pigs. The first group of 37 pigs were obtained from a closed, high-health, University of

Guelph research facility, and the second group of 10 pigs was a mixed group of 8 pigs originating fiom the same herd as the firt group and 2 pigs purchased fiom a commercial fam. This latter group was housed together in the same pen for about a month at the

University of Guelph Ctinical Research unit. Pigs were shipped to the University of Guelph abattoir, where they were kified within 4 h. The animals in both groups were not fasted prior to slaughter. Merthe pigs were slaughtered, stomachs were examined. Biopsies were taken from the pyloric and cardiac regions. It is known that Helicobacters have a patchy distribution that may cause biopsy sarnpling problems. To overcame false-negative results, an indirect technique was used an in sita urease assay described by Grasso et al. (1995), and this was perfonned directly on the 10 porche gastric mucosa of the second group. Samples were taken fiom areas ofthe stomach that reacted positively to the urease test. Ifthe test was negative then the biopsy was taken fiom the analogous site. 4.2.1, HERDS SUPPLYING PIGS

Herd 1was a closed SpecificPathogen Free (SPF) University of Guelph research fm

that exercised rigid bioseairity. This was a 300 sow fmow-tefinish operation, using au- idail-out management at each stage ofproduction. Grower-finisher rooms were thoroughly cleaned between filis, and the flooring was partialiy slatted. Pigs were fed ad-Iibifma finely- ground pelleted ration that was corn-soybeanbased. A total of 84 pigs were selected fiom this facility and 42 were fasted 24 h before slaughter-

Herd II was a University of Gueiph owned grower-nnisher facility operated on an all- idaii-out bais by site- The flooring was sotid concrete without bedding. The same feed as for

Herd 1 was used, and pigs were also fed ad-iibifum. The pigs were obtained nom a commercial operation and housed at this unit nom 25 kg (55 Ib) until market weight. Fifty- seven pigs were examined from this facility, twenty-five of them were fasted for 24 h before slaughter.

Herd III was a privately owned 150 sow farrow-to-finish operation. The grower- fi~sherpigs were housed in a continuous flow barn with solid cernent floors. Pigs were fed twice daity by dispersing feed on the fioor of each pen. The ration was a coarse mash feed composed of high-moisture corn, soybean supplement, and a vitamin-mineral premix that was prepared on the fm. A group of twenty two pigs were used in this study. Ten pigs in this group were fasted for about 24 h before slaughter. Stomachs were examined macroswpically, tested with the urease assay, and biopsies were taken for rnicroscopic evaluation and cultu ring. 4-2.2- GROSS EVALUATION OF STOMACfI LESIONS

Pigs fiom al1 herds were slaughtered at the University of Guelph abattoir.

Immediately derthe carcasses were eviscerated, stomachs were separated f?om the rest of the alimentary tract, cut open dong the greater cunmture from the diverticulum to the pyloms, and the gastnc contents were discarded. The stomach mucosa was gently washed with tap water to remove ingesta The stomachs were grossly examined for the presence or absence of lesions in thepars oesophages- The lesions oftheparsoesophageu were graded using a classifkation scherne fiom O to 3 as described in Table 3.1.2 (Mackin et al. 1997).

4.2.3. DIRECT AND INDIRECT ASSAYS FORHEUCOBAC7ER-LIKE BACTERIA

TESTING

An in siru urease assay was penonned prior to sampling on each stomach according to the method of Grasso et al. (1995) as it was described above. Biopsies for microbiologicai evaluation, culturing, and histological examination were taken, from urea-positive areas or similar regions in those stomachs where no colour change occurred. Generally, the junction ofthe antrum and oxyntic region at the lesser cuwature was used. In addition, biopsies were taken from the pars oesophagea region.

The samples for culturing were placed in 0.85 % saline solution and were delivered to Animal Health Laboratones, University of Guelph. Time from sampling to plating varied fiom one to four hours. Al1 samples were maintaineci at 4OC for the time period between sarnpling and plating using cold packers and styrofoarn cooler. The tissue samples for histological examination were fixed in 10 % buffered neutrd formaiin, then trimrned and parafnn blocks were prepared. Six pn sections were cut, deparafEzed, hydrated with distilled water and stained in Eesh filtered Hds' hematoxylin and eosin (HM) solution (prepared as follows: 5.0 grn hematoxylin; 50.0 mL alcohol, 100

% ethyl; potassium 100.0 gm; 1000.0 mL distillecl water. 2,s gm mercuric oxide, red) for su min. Slides were washed in tap water for four minutes, dipped twice in 1 % acid alcohol for

Herentiation, then briefly washed in tap water. The &des were placed in weak arnmonia water until sections became bnght blue and then again washed in mnning tap water for ten minutes, and wunterstained in eosin-phloxine solution for two minutes. The slides were dehydrated and cleared through two changes each of 95 % ethyl alcohol, absolute ethyl alcohol and xylene, for two minutes each (Allen 1992)-

For enhancing visualkation ofHeiicubacter-like organisms the Warthin-Stany silver staining method was employed. Staining was done using the following procedure: sections were cut six pm thick, deparafnnized and hydrated to distilled water, impregnated in i % silver nitrate solution at 4s0 C for thiriy minutes. The slides were placed on a staining rack using glus rods, and flooded with the developing solution until colour developed to yellow- brown and then quickly and thoroughly washed in hot tap water to stop the reaction. Slides were then dehydrated and cleared through 95 % ethyl alcohol, absolute ethyl alcohol, and xylene (two changes each, and two minutes each).

In order to idente spiral-shaped bacteria, a Brown and Brenn Gram @&BG) staining technique was perfiormed, kngthe following procedures: sections were deparfinized and hydrated using dïstiiied water, and were placed on a staininb rack ushg glass rods, 20 drops (1 mL) of crystal violet solution were poured on and five drops of 5 % sodium bicarbonate

solution were added for one minute. Slides were rinsed in tap water, fiooded with Gram's

iodine solution for one minute, rinsed in tap water, and decolourized in acetone. After that

the slides were rinsed in tap water agah, flooded with basic fichsin working soiution for one

minute and rinsed and then placed in a Copün jar in tap water. To start the reaction, each süde

individuaüy was dipped in acetone. Each slide was differentiated immediately with picnc acid-

acetone solution until sections developed to a yellowish-pink colour and then the siides were

quickly and thoroughly ~sedin acetone-dene solution. Slides were cleared in two changes

of xylene and mounted with resinous medium (Mngton 1992).

Culture was done by using chowlate agar on which Helicobacter pyiori can usually

be grown in a microaerophilic environment at 37%. The tissue samples upon arriving at the

laboratoc$ were manually macerated and a mal1 amount of this suspension was used to

inoculate chocoiate agar plates. The plates were incubated for four days at 37OC. After this

the, all plates were examineci for the presence af colonies characteristic of Hekobacter pylon and Campyl~~cfet~Colonies were subcultured for further identification.

rii addition, ailturing was done using BJ medium, Camp, and Belo-Horizonte media

To prepare 500 mL ofBJ medium, trypticase soy agar (20 g), pig feces extracts (25 mL), and

distilled water (438 mL) was used. To prepare lOOOmi Camp media, tiypticase soy agar (40

g), and distilled water (1000 mL) was used, after autoclaving in Agamatic-3 for 15 Mnat

12 1°C. Dispensing temperature was set at 44OC. When ready to dispense, the next ingredients

were added: previously melted aesailin (1 5 mL) 1 % femc citrate, and sheep blood (50 mL).

Agar plates were incubated at 37°C in a microaerophilic atmosphere for a total of 10 days. Growth on the plates was fkst examuied at 48 h of innibation. AN colony morphologies were

fùrther investigated by Gnun stain. Plates were subsequently examined at 96 and 192 h and

again at 10 d incubation. Belo-Horizonte medium is dso known as the standard

bactenologid culture media ofHelicohcfer pyW (Zwet et al. 1993). The medium was

prepared and used in accordance with the protocol describeci by these authors: brain hem

inhion agar (35 g/mL), sheep blood (10 %), vancomycin (6 mgL), halicidic acid (20 mg/L),

amphotericin B (2 mg& and 2,3,5-triphenyltetrazoiium chloride (40 mg&). Plates were

incubated at 37°C and were first examined at 48 h of incubation, and were subsequently

examined at 96 h, 19 1 h, and at 10 d incubation

4.3, STATISTICAL ANALYSIS

This study was designed as a field trial, where the unit of concem was an individual

pig, and the initial sampling unit was a herd. Neither fms nor pigs were chosen randomly.

Chosen pigs represented an entire one day marketing group for herds 1, LI and III.

The data were stored and dyzedin SAS program. (PC-SAS 6.12, SAS Institute,

Cary, NC). To grade gastric lesions we created a rsnking variable t hat was later transformed

into a binomial variable. The other data urea reaction and presence or absence of

Helicobacter-like bacteria were also binomial,

PROC FREQ procedure (PC-SAS 6.12, SAS Institute, Cary, NC) was employed to

examine these data. The association between the presence of Heiicobucfer -like organisms

on histological examination and the presence of erosive lesions of theprsaesophagea were

caiculated using the Breslow-Day Chi-square test for Homogeneity of the Odds Ratio (OR). When OR was found homogenous a Common OR was calculateci and tested with Mantel-

Haensrd Chi-square test controlling for fènn effed. Results were wnsidered significant ifp-

value < 0.05- In addition, results were tested by two-sided Fisher Exact Test when cells

contauleci insufficient numbers (9.

The degree ofagreement between microscopie Wigsand urease mapping to detect

the presence ofHelicobacferwas determined by calculating Cohen's kappa in PROC FREQ

mode in SAS. The specimens were coifected and eduated independently. Microscopie

observations were performed blindly (without knowing the results of the urease assay). The

rating categories were mutually exclusive and exhaustive.

4.4. RESULTS

Pre-test: Spirsl bactena were not found in histological HIE stained sections nor were

they isolated by culture. Warthin-Starry silver stain also did not reveat any spiral

microorganisms, even though the prevalence of gastric lesions in the first group of 37 pigs

was 100 %, with 87 % ulceration-

The urease assay perfomed on the second group of pig stomachs was positive in 9

out of 10 cases. Grossly 80 % of stomachs had lesions fiom grade 1 to grade 3, and onty 60

% had ulcers (grade 2 and 3). Biopsies were taken fiom areas of colour change (indicating urease production). Tightly coiled bacteria were observed in 8 of 10 stomachs by histological

examination using Warthin-Starty stained sections. Spirai-shaped bacteria were not found in

one of the urease positive biopsies. -Test: The overail prevalence of gastric lesions of the pms oesophagea in the five groups was 88 %. The range of the herd predence of lesions varied nom herd to herd Eom

50 % in Herd II to 100 % in the pre-test ofHerd 1- Gastric ulcers (grade 2 and 3) were confirmexi in 53 % of the total animals exarnined. Ulceration ofthepars oesophagea varied

Eom 10 % in Herd III to 85 ./o in the pre-test group of Herd I, (Table 4.4.1.).

Helicobacter heiimannii-like organisms were observed by in situ urease assay and microscopic enmination in the stomachs ofthe pigs fiom two of the three herds, and in the rnixed group of swine ftom the pretest study. This mixed group incorporated pigs delivered fiom Herd I (8 pigs) and Herd III (2 pigs) but housed together for several weeks pnor to slaughter. No bacteria were found either by urease assay or by microscopic examination in

Herd 1, in both pretest and test studies. Bacteria found in Herd 4 Herd III, and in the mixed group were similar to each other. They were tightly spiraled and most had 5 spiral turns

(range was 3 to 5 spiral turns), and measured up to 5 p in length. and about 0.6 p wide

(Figure 4-44. These microorganism were not found in the biopsies nom the pars oesophogea, but only in the glandular pyloric region. They were readily observed in (W-S) silver stained slides, but not with H&E or carbol-fichsin (Z-N) stain. These spiral bactena were gram negative. These microorganism were found in the mucus, and they did not attached to the epitheliurn. Deep in the crypts H. heilmmnii-like bactena .were clumped together and partially or completely lysed. No Helicobacfer-likeorganisms were isolated in this experiment on any culture attempts. Overgrowth or excessive bacterial contamination was not a significant problem on the primaiy culture of the tissue specimens. The prevaience of H- her'Immnii-iikebacteria varied significantly from herd-to-herd

and was not associated with the presence of erosive lesions ofthepars oesophogea (Table

4.4.1 and Table 4.4.2). Of the 210 stomachs examineci, rnicroscopic evidence of

Uelicobocter-like bacteria was seen in 28 % of stomachs: 6 % with ulcerations of the pms

oesophogea (grade 2 or 3). 14 % with parakeratosis (grade l), and 8 % with normal

stomachs(grade 0). Twenty seven percent of the stomachs reacted positively to this indirect test. There was a signifïcant varÏabnityh the prevaience of stomachs with Helicobacter-Iïke organisms between the herds (p-value C 0.005). Tii the Herd ï, organisms were not noted in any of the 121 stomachs exarnined @retest and test). In the rnixed group of pigs on the pretest, 80 % of stomachs carried H. heilmannii-like bacteria, but 90 % were detected positive by the urease test. Similarly, microscopic examination in Herd KI revealed spiral bactena in 54 % of the stomachs, (14 % with ulceration and 7 % with normal stomachs).

Fifty-eight percent of the stomachs reacted positive to the urea test. In Herd III, 90 % of stomachs were infected by spiral microorganisms based on histological evaluation. but only

10 % had erosive lesions (gnide 2 and 3), 36 % had parakeratosis and the rest of the stomachs were normal. SUay eight percent of the stomachs reacted positive to the urea assay.

Substantial agreement was detected between the in situ urease assay and histological detection of Heiicobacter heilmannii-like bactena (Kappa = 68 -9 %). This is based on 173 stomach samples (37 stornachs were not tested with urea), see Appendix 1.

Overall 77 animals were fasted prior to slaughter in both pretest and test groups-

Erosive lesions (grade 2 and 3) were observed in 47 pigs (61 %). In the non-fasted group, erosive lesions (grade 2 or 3) were observed in 64 of 133 pigs (48 %). Fasted pigs had a higher prevdence of gastric ulcers of the pms oesopliagea than non-fasted pigs when data were analyzed wntroUing for fann effect (Mantel-Ha«inszel Chi-Square = 3.649, p-value

41E-04,1-677 PCI5 7-943), see AppendOr 3. The pdenceof lesions @ms oesophages) was also higher in the fii~tedgroup where 73 pigs of 77 (95 %) had lesions (including parakeratosis), wmpared to the non-fasted group, where 112 out of 133 pigs (84 %), which was confimeci by Fisher Exact Test (2-Tail) (Mantel-HlaenszeI Chi-Square = 3 -422. p-value

= 0.026,), see Appendx 3. The prevaience of Iesions in Herd 1 reached 100 % in the fàsted group and 98 % in the non-fasteci group. Therefore statisticd differences between these two groups in Herd I was not detected on the basis of presence of Iesions including parakeratosis, but was detected on the basis of erosions of the pms oesopliogea @-value = 0.028).After controiiing for fmeffect, the rate of ulcers in the fiisted group is 3.5 times greater than the rate of ulcers in the non-fasted group (Logit Odds Ratio=3.522, 1.590 ICI 2 7.99, p-value

= 1 1E-4) (Appendix 3).

The relationship between fasting and the presence ofHelicobacter-likeorganisms was analyzed only in Herds II and Herds m,because in the pretest study animals were not fasted, and in Herd 1 no bacteria were found. In total, 35 pigs were fmted for 24 h, and 44 were not fasted. The presence of bacteria was greater in the non-fasted group (75 %) compared to the fasted group (5 1 %) (Mantel-Haenszel Chi-Square = 4.65, p-value = 0.03 6). Table 4-4-1: Prevdtnct Kelicobacter iieilmunnii-likt bacteria

Test

Totai

Herd 1 MIxed Herd I Herd 11 Herd Ul

_I I 20 59

(9 1%) (28%) 13 -(68%) 2

(go/.)

ITotaï Pigs Eramined Figure 4.4.1 : Hellcobacter heilmannii-like bacteria in pig stomachs from Ontario herds (W-Ssilver stain, magnification x 100, enbanced by 200% using computer technique) Table 4.4.2: Pmdence of gutric tesions of thepam oaopkagea and Hdicobacter heilmamnii-like bacteria in 210 Ontario pigs

Tord

L Hdiwbada Herd 1

Pretest

Heliwbader "+" Herd 1 ECICO~O~CT I Hdicobader Test Herd II Heliwbader "-a

Hdicobader Herd III Helicobîtdcr

* Stomach Score: Noml Parakeratosis Small Erosions Deep or Extensive Ulcers 4.5. DISCUSSION

Previous slaughter-house studies wnducted in Brazil on 120 pig stomachs and in ltaly on 85 pigs stomachs, reveded the prevalence of Relicobacter heiImmnii and Helicodacter heiImmnii4ike bacteria in 10.8 and 9.4 % of the stomachs examined, respectively (Queiroz et al. 1990, Grass0 et ai. 1996). Recent surveys using more sensitive detection techniques indicate that prevalence of these bactaia rnay be up to 60-80 % (Bahosa et ai. 1995, Magras et al. 1999). These studies have tended to be srnall and have examined a convenience sample rather than a sample reflective ofthe population.

Among the three fmsin this trial, the prevalence of stomachs with Helicobacter heiZ'miÏ-like bacteria varks a great deal fkom herd-to-herd. The wide variation might be explained by the remarkable dEerence in management and housing systew between farms in this study. The herd with the highest prevalence of these micro-organisms (Herd III) utilied solid flooring, continuous flow animai movement, and floor feeding. These factors would ali contribute to fecal-oral spread. Transmission of H. heiimannii from pig-to-pig, or from pig-to-humans or vise versais not known, but it is assumed that the fecal route is the most Wcely method of infection (Friendship et al. 1999). The second highest rate of

Helicobacter infection in our survey was in the mked group of pigs, where eight pigs were obtained from Herd 1( where no spiral bacteria were detected). and 2 pigs were fiom Herd

III (a HeIicobacter positive herd with a high prevalence of positive pigs). These pigs were kept in the same pen, on solid concrete flooringfor about a month and presumably this would have been sufficient time for al1 anUnais to bewme infected if one of the pigs was carrying the bacteria at the time they were mixeci- Herd I, which is a closed-herd with strict biosecurity and a high health status,

appeared to be fiee of Helicohcfer--Oceorganîsms. This suggests that the sarne biosecurity

measwes used to maintain fieedom of other major mine pathogens may be successfùl in

preventing the introduction ofH. heihmnK This might prove to be an important finduig.

A recent survey of human patients with H. heilmmii gastritis has indicated that pig contact

is a risk factor@4eining et ril. 1998). Cats, dogs, and rats may play a role in the introduction

ofHeIicobacter q. hto swkherds (Giusti et al. 1998, Seymour et al. 1994) ALthough H. heilmannii is not regarded as a serious pubiic health risic, fieedom from even minor zoonotic organisms may becorne an important marketing issue in the near friture. Another possibility for the lack ofHelicobucferq. found in Herd I, may be that our diagnostic tests were not sensitive enough to detect these bacteria

Some diagnostic techniques have been shown to be more sensitive than others in detecting the presence of H'elicobacter ip-The mouse inoculation technique has been shown to be a better method of finding positive animals than histology or urease testing

(Queiroz et al.- 1996). It is possible that the findings of the present study are an under- estirnate of the prevalence of Helicobacter sp..

The use of in siru urease mapping appeared to be helpfùl in detecting positive animals and Localking the urease producing bactena. It has been suggested that Helicobacter heilmannii wlonize in clusters and therefore, a bbipsy sarnple may not reflect the tme status of the animal if it is taken from the wrong location. The urease test should improve the accuracy of histological examination(Grasso et al. 1995) ,and might be used as a screening procedure to determine head status. Both false posîtives and fdse negatives usùig the urease test were observed in this aial. Presumably ifthere are only a srnail number of bactena, they may not produce sufficïent urease to cause a colour change. On the other hand, other bactena can produce urease and therefore produce a colour change in the absence of

Helicohcter q..It is poasile that Nelicobacferp- are present to produce a colour change but the tissue examineci by histology did not contain bacteria. This type of error could be rectined by multiple sampling. In human medicine, H. pyIonn is so widespread that two biopsies are considered sunicient to detect Helr'cobucfer infection in greater than 95% of cases (Brown and Peura 1993).

Other researchers have reported a strong association between the presence of

Helicooacfer-lieorganisms and gastroesophageal ulceration (Barbosa et al. 1995, Queiroz et al. 1996, Grasso et al. 1996). It is diflticult to explain why the findings in the present study diner nom these previous studies. On the other hand, the mechanism by which Helicobacter heilmmii rnight cause ulceration ofthepswsophagea has not been well investigated. The organism colonizes the glandular region of the stomach and in humans, ulceration occurs near the site of colonization and intlammation. It has been suggested that in swine Helicobacter sp. stimulates parietal cells to over-secrete acid, either by direct imtation or by causing increased gastrin release (Yeomans and Kolt 1996). In humans, eradication of H. pylori generdy results in a decrease in basal and rneal-stimulateci gastnn secretion @-Omar et al.

1993). However, in experimental infection with either H. pylon' (Barbosa et al. 1995) or H. heilmannii (Krakowka et al. 1998). ulceration of thepmswsophugea has not been observed.

The results of this present study demonstrate that the role ofHelicobucter-like organisms in the stomach of dewith respect to the aeation ofgastroesophageal ulcers is unclear, but unlikely to be a major factor.

In this study, fdgwas associatecf with an increase in gastric ulceration, as it was reporteci in a number of other observational studies (Straw et al. 1992, Davies et al 1994).

Interestingly, fewer stomchs were found to contain WeIicobacfer-Lüreorganisms in the pigs which were fasted. Possibly the environment of an empty stomach is unsuitable for the

Helicobacter organisrns because of increased acidifïcation or lack of substrate to support bacteriai growth, or possibly Helicobacter are more readily flushed into the srnall intestine when ingesta is not present. This may have confounded oome of the results regarding individual herd prevalence - if more arümals were fasted, fewer pigs might be detected as

Helicobucter positive.

In conclusion, tiom this study, it seems likely apparent that Helicobacter-like organisms (presumably H. heilmanniii) can be readily found in commercial swine in Ontario using urease mapphg and histological examination with Warthin-Starry silver staining. Their role in gastric ulceration is unclear, but would appear to be relatively mi-nor. Helicobacter sp- idinfection of swine may become more of an issue in the fbture because of its zoonotic potential. This study suggests the management strategies used to control other swine pathogens rnight be useful in the contml of Helicokcter sp.. Cha~ter5

EVALUATION OF LANSOPRAZOLE AND AZITHROMYCIN FOR CONTROL

OF GASTRIC ULCERATION IN SWlBE DURXNG PERIODS OF FEED

DEPRIVATION

5.1. INTRODUrnON

Feed dismption, regardless of cause, is considered a major risk factor in gastric (pars

oesophages) ulceration of swine. It is generally believed that as the stomach empties the

normal pH gradient between the acidic distal portion and the more neutrai-proximal region

is lost. Increased fluidity and mWng resdts in exposure of the sensitivepars oesophagea to gastric acid and pepsin. However, recent work has shown that although theparsoesophugeu is not protected by a mucus wvering Uethe glandular regions of the stomach, the stratined squamous epithelium is relatively resistant to hydrochloric acid and pepsin (Orlando 1991,

Argenzio and Eisemann 1996). Severd factors such as bide back-flow and short chah fatty acids produced by fermentative bacteria rnay cause rapid damage to epithelial cells of the pars oesophogea (Schiessel et ~1.1990,Argenzio and Eisemann 1996). Fermentative bacteria such as LactobaciZlus and Bacillus may work in conjunction with gastric acid to cause the rapid destruction of the epithelial tissue of thepms oesophageu at pH Ievels that are much higher than levels at which hydrochlork acid alone causes ulceration (Argenzio and Eisemann

1996).

Wèiicobacterheilmanii-like bacteria as well as fementative bacteria have also been implicated as a significant nsk factor in ulceretion of the pms oesophagea (Mendes et ai- 199 1, Barbosa et al. 1995, Grass0 el al. 1996, Queiroz et al. 1996), even though these bacteria are not capable of producing volatile fatty acids and are mostly located in the pyloric area at a distance nom thepms wsophugea @IoIck et al. 1997).These microorganisrns may stimulate G celis or parietal ceiis leading to excessive acid production. Alternatively, they may interfere with acid secretion inhibitory mechanisms (Yeomans and Kolt 1996).

In human medicine, Helicobacter eradication programs have been very successfiil in a>rùig gastrïc utcers and preventing reouxrrence (Unge 1996, Hopkins et al. 1996, Blaser

1996). Commonly, these programs utilize an effective acid secretion suppressor such as an

H+/K'-ATPase inhibitor such as lamprazole and a broad speanim antibiotic.

Lansoprazole, a bennmidazole compound, has been shown to effectively suppress acid secretion in human adults for 24 h at a 30 mg dose (Verdu et al. 1994)- Lansoprazole, an H+f

K+-ATPase inhibitor suppresses gastric acid secretion by blocking the final step of acid production inhibithg both basal and stimulated gastric acid secretion irrespective of the stimulus (Physicians Genk-Drug Information 1996, Verdu et al. 1994). This drug has a rapid absorption rate and is 97 % plasma protein bound, does not accumulate and its pharmacokinetics are unaltered by multiple dosing. The dmg is not available in injectable fom. It is supplied in delayed-release capsules for oral administration.

Azithromycin is a new macrolide antibiotic derived ftom erythromycin. It has excellent tissue distribution and extended halGIi6e, and is far more stable than erythromycin in an acid environment (Girard et aï- 1987, Fiese and Steffen 1990). Azithromycin has been successfùlly used in conjunction with an ITK-ATPase inhibitor to eradicate Helicobacter p. in humans (Caselli et al. 1996). This drug is supplied in red opaque hard-gelatin capsules containhg azythmmycindihydrate equivalent to 250 mg aqthromycin. There is no injectable fom available. Rapid distribution ofazithromycin in tissues and high concentrations within cells result in signïficantfy higher anthromycin concentrations in tissues than in plasma or senim. Food deauises the absorption of azithromycin reducing the maximum plasma concuitration (CU) by 52 % and the area unda plasma concentration by 43 % (AUC - area under the curve). It is recommended in hwnan medicine that the antibiotic be given at Ieast one hour before or two hours &er a meal-

A little information on the effectiveness of Kelicobacter q-eradication protocol in small animal practice is avdable. Methods of eliatingthese bacteria have ken described in ferrets using a combination of metronidazole, amoxicillin, and bismuth, and in dogs medicated with metronidazole, amoxicillin, and famotidine (Fox 1995, Novo and Magne

1995). There are no published treatment protocols for eradication of Helicobacter sp. infection in pigs.

The purpose of this research trial was to detemhe whether an acid secretion inhibitor such as lansoprazole, either alone or in combination with the antibiotic azythromycin, could protect pigs hmgastric ulceration when they whey were subjected to a 48 h period of fasting.

5.2. MATEIUALS AND METaODS

Eighty-four, purebred Yorkshire, grower pigs housed at the closed, high-health

University of Guelph research facility were selesteci for the trial. Pigs were randody selected from a group of 129 pigs available. AU animais were in good health and were about 3-4

months old. The average weight of the group was 45.2 kg (range 30-60.4 kg).

The experimentai protocol was approved by the University of Guelph Animal Care

Cornmittee and was carried out in accordance with the p~ciplespublished in the Canadian

Council on Animal Can "Guide to the Care and Use ofExperimentaI Animds".

Pigs were randomly aliocated into three equal shed groups (28 animais in each

group). Du~gthe weighing pigs were Pllocated con~ecufivelyinto one of three groups: a

Control group (untreated), Treatment One group pigs were given 30 mg lansoprazole

(l?revacidTM,Abbott Laboratories) once per day orally for seven days and Treatment Two group in which pigs were given 30 mg of lansoprazole once per day orally for seven days and

500 mg azithromycin (2ithromaxTM,Pkr) orally once per day for the first three days.

Within each groups, pigs were subdivided into two groups: Fasted and Non-fmted, with every second pig in each group bcing allocated into the Fasted subgroup. AU animals were sorted according to their weight and housed in one room that had six pens on each side of the centered walkway. The Non-fasted subgroup was placed on one side of the room @ens number 1 to 6) and the Fasted subgroup on the other side (pens number 7 to 12). There were seven pigs in each pen. The heavier mimals weighing 47.6 to 59.4 kg and 48.2 to 60.4 kg were placed in pen 1 and 2 and number 7 and 8, respectively. The lighter weight pigs were subsequently placed in pen numbers 3-4 and 5-6 (weight 40.6 to 47.6 kg and 30 to 40.5 kg) and pen number 9-10 and 11-12 (weight 40.2 to 47.6 kg and 32 to 40.2 kg), respectively. Ail pigs were placed on a coarse mash feed for 21 days prior to the stan ofthe experiment in an attempt to ensure the stomachs of the pigs would be fiee of ulceration. Two weeks pnor to the treatment, the diet was changed and the finely ground

(ulcerogenic) pelletecf feed that usuaiiy has been used on this fm was fed to al1 pigs until slaughter. The treatment was done in three stages. In the first week heavier animals that occupied pens number 1 - 2 and 7 - 8, were treated. In the following week pens number 3 -

4 and 9 - 10 were treated, and in the third week pais number 5 - 6 and 1 1- 12 were treated

(Table 5-2-1).

Table 5.2.1: Pig allocation by week of treatment and by type of diet

Number of Pigs by Pen by Wetk of Treatment 1 First Week 1 Second Week

Fas ted

On the first day of the treatment, the Fasted subgroup (pens 7, 8, 9, 10, 1 1, and 12) underwent a 48 h fisting penod.

Two weeks after the completion of the treatments, animals corn Control and

Treatment One group were killed at the University of Guelph Abattoir and the animals from

Treatment Two group were euthanized at the Pathology Laboratory at OVC,the carcasses were eviscerated and the stomachs were studied Each stomach was separated fiom the rut ofthe alimentary tract and opened with an incision dong the greater curvature fiom the diverticulum to the pylorus. The contents were removed and the mucosai surface gently washed with =ter. AU neaopsy inspections were pedonned by r single pathologist who was blindeci to the treatrnent status of the pig. Gross

Iesions of thepcas oesophogeu were numericaiiy scored on a scale of O to 3 as described in

Table 3.1.2 @hckin et al. 1997).

An h-situ urease assay as described in Chapter 3.1.2 (Giasso et al- 1995) was performed immediately after the gross examination of the pars oesophagea- The stomachs were visually monitored for colour change for 1.5 h. A colour change fkom yellow to deep pink occurs as pH rises in the presence of urease producing bacteria due to ammonia accumulation. The protocol was such that if a colour change occurred two biopsies (one for culturing and one for rnicroscopic examination) would be taken nom that specific area.

Otherwise, two tissue biopsies (3 x 3 cm) were taken nom the pyloric and another two hm the cardiac region. At the same time, a linear stnp of epithelium (approximately 3 -5 x 6 cm) was collecteci fkom the area of the pars oesophagea for microscopic examination. These biopsies were collected across thepms oesophagea including thejunction with the glandular stomach. If an erosion or ulcer was detected grossly, the strip was oriented to include the lesion. Samples for histological examination were fixed in 10 % neutral fomaldehyde and processed routinely for parafiin-embedding and Iight microscopy. The samples for culturing were placed in 50 mL of thioglycolate broth and delivered immediately after collection to the

Animal Health Laboratones at the University of Guelph. Upon amving at the laboratory, the tissue samples were manually macerated and a small amount of the suspension was used to inoculate Belo-Horizonte media and chocolate 4%.The protocol for culturing and siaining techniques for spiral bactena detedon are deSccibed in Chapter 4.1.3.

To grade lesions ofthepms uesophagea histologidy, hematoxylin and eosin stain was used. The Iesions were gdedusing the classification scheme on a scale of O to 3.

Table 5.2.2: Hiitologicd grading of the lesions of the pcvs oesophages by Maclria et al (1997)

- f Score Microscopie Findnigs Macroscopic Findings 4 O No visibIe lesions No visible lesions

- - 1 Parakeratosis Parakeratosis

2 Erosions Focal, shallow erosions andor ulcerations

1 3 1 Ulcerations 1 Diffuse or deep ulcerations 1

Parakeratosis included any degree ofhyperplasia ofthe squamous epithelium with the retention of a thickened hypereosinophilic superficial layer. Partial epithelial loss classified as an erosion, was most commonly seen in the areas where the papillae of the lamina propna were close to the sufiace. Ulceration referred to loss of epithelium to and through the basement membrane, blood vessels were often exposed or in older ulcers much granulation tissue was present.

To detect spiral-shaped organisms sections were stained with the Warthin-Starry silver stain and with the Ziehl-Neelsen carbol-fichsin stain- 5.3. STATISTTCAL ANALYSIS

This study was designeci as a clinical triai, where the unit of concem was an individual

pig, and the initial sampling unit was a herd. The pigs for this trial were selected randody

among the grower pigs available at the tirne of the trial on this fm. They were randomly

docated into tiuee treatment groups and two subgroups. Both macroscopic and microscopic

scoring was done by creating a rankuig variable that later was transferred into a binomial.

These data were stored and anal@ with PC / SAS program (PC-SAS 6.12, SAS Institute,

cary, NC)-

The PROC FREQ procedure (PC-SAS 6.12, SAS Institute, Cary. NC) was used to

evaluate the effect of treatment on the basis of Fisher's Exact Test (2-Tail) and Mantel-

Haenszel Chi-square test (wntrolling for fsting category). If cells contained zero, Logit

(Odds Ratio) estimator was used as a correction for those cells in al1 tables (See Appendix

6 and Appendix 7). The lesions were wmpared in al1 treatment groups with and then without controlling for fasting.

An agreement between microscopic and gros scoring was determined by Weighted

Kappa in PROC FREQ mode based on ranking variables ftom zero to three, and McNemar's

Test Kappa was calculated for binary variables that were created for this purpose

(Appendix 5).

5.4. RESULTS

The overall prevalence of gastric lesions based on gross pathological examination, was 95 % (80 of84 pigs), and 99 % (83 of 84 pigs) based on histological evaluation, (Table 5.3.1). Twenty-eight pigs (33 %) demonstrated lesions ofparakeratosis grossly, and twenty-

four (29 %) were simiiariy dassined by histology* Prevalence of erosions and ulcers of the pms oesophagea, as shown in Table 5.3.1, was 62 and 70 % for macroscopic and

rniaoscopic grading, respectively. There was a moderate agreement between gross and histologie scores (Weighted Kappa = 58.9 %), and substantial agreement between these methods (McNemar's Test Kappa = 76.3 %), when grade zero and one were categorized together and grade two and three were grouped together (Appendix 5).

Table 5.3.1 : Previlence of gastric lesions by macroscopic and rnicroscopic esamination

STOMACH SCORESf EVALUATION TOTAL O 1 2 3

4 28 32 20 84 n!tACROScOPIc 4.76% 33,33% 38,1% 23,81% 100%

1 24 21 38 84 MxCROSCOPrc I 1 1.19% 1 28.57% 1 25% 1 45.24% 1 100%

* Stomach Score: O Normal 1 Parakeratosis 2 Small Erosions 3 Deep or Extensive Uiiirs

Ulceration of the pars wsophagea detected either on gross inspection or by rnicroscopic examination was more likely to ounir in pigs that had been fasted for 48 h compared to the pigs which had continuous access to feed @-value = 0.0006). Thirty-seven of 42 (88 %) of pigs in the Fasted subgroup were found to have stomach lesions categorized as erosions or ulcas (grade 2 or 3) based on histological examination, comparecl to only 22 of42 pigs (52 %) in the Non-fased subgroup. Similarly, gross lesions categorhed as erosions or ulcers (grade 2 or 3) wae detected in only 79 % of the pigs in Fasted subgroup and in 45

% of the Non-Wed group @hntel-Haenszel Chi-square = 4.439, p-value = 3-198-03).

Hence, the prevaience of aosive lesions ocairring in pigs from the htedsubgroup was found to be 6.7 times (macroscopic evaluation) or 4.4 times (microscopie examination) gregter than the prevaience of erosive lesions inNon-fâsted pigs (TsrbIe 5.3 -2)- Table 5.3.2: Cornparison of stomach lesions between Fasted and Non-Fasted hop, based on macroscopic / microscopie evaluation

LESIONS (Stomach Scores*: 1, or 2. or 3)

ut,, 66-66 Total Fisher's Exact Test (ZTiil), pV~lue

42 (100%) O 42

1 NON-FASTED Not Significant ( TOTAL

' 1 NON-FASTED Not Signifiant $ 7TOTAL ULCERS (Stomach Scores*: 2 or 3)

/ NON-FASTED U 1 TOTAL

e (TOTAL There was no dserence between the prevalence of gastric lesions in the Fasted and the Non-fasteci subgroups (99 % and 95 % based on microscopic examination and gros evaluation, respectivdy). Al1 anirnak but one showed evidence ofrnicroscopic lesions (Table

5.3 %and Appendix 6).

The differencein prevalence of lesions between the three treatment groups and lesions of the pors wsophgea (iicluding parakeratosis) and erosions/ulcerations of the pms oesophages were tested by both histologie examination and gross inspection. A difference was noted (on microscopic examamination)between the proportion of stomachs with erosions or ulcerations in Treatment Group Two and Treatment Group One (p-value = 0.044,

OR = 3.98, 1.178 s CI s 13.5 14). In the former group, treated with both azithromycin and lansoprazole, the prevaience of stomachs scored 2 or 3 reached 82 %, compared to the

Treatment Group One (lansoprazole only) where the prevaience of stomachs scored 2 or 3 was 54 %. There was a tendency for a higher proportion of the Non-fasted subgroup to have erosive lesions (grade 2 or 3) compared to the Fasted subgroup.(Fisher's Exact Test p-value

= 0.057, OR = 5.2, 1.348 < CI s 20.408, Table 5.3.3.). However, the lesions were more severe in the Fasted subgmup compared to the Non-fasted subgroup, in al1 three treatment groups. Of the pigs with erosive lesions, an ulcer score of 3 was detected in 10 out of 13 (77

%); 9 out of 11 (82 %) and 9 out of 13 (69 %) pigs in Control, Treatment Group One and

Treatrnent Group Two, respectively (see Table 5.3.4).

No differences were detected in the propomon of pigs with erosive lesions (grade 2 and grade 3) between Treatment Group One, Treatment Group Two and the Control Group

(Tables 1 - 6 in Appendix 7). Macroscopic grading did not reveal differences in gastnc ulcen (jws oesophgea) between Controi, Treatment Group One and Treatment Group Two

(Table 5 -3-5).

Table 5.3.3: Fisting or Non-Fasthg treatment and the presence of stomach tesions

Stomach Lesions4* Trcatnicnt* u+m CC-CC Total

2 IO 4 14 2vs. 1 Non-Fasted 1 4 10 14

1 Fisher Exact Test (2 Tail) p-value = 0.057, OR = 5.2 / 28

2 13 1 14 2 vs, 1 - Fas ted 1 11 3 14

*Treatment 1 - lansdprazole - 30 mg orally once per &y for 7 &ys Treatment 2 - lansoprazole - 30 mg ordly once perday for 7 days and azithromycin 500 mg orally once per day for 3 days.

**Stomach Score: "+' score 2 or 3 (2 - Small Erosions, 3 - Deep or Extensive Ulcers) cc-tc Normal Stomachs or Paraketatosis An in situ urease assay did not reveal urease positive areas in any of the stomachs

examineci. Spirai bacteria were not found in the biopsies stained with Warthin-Starry (W-S)

silver stain or with Ziehl-Neelsen (2-N)dot-fûchsin stah Culhiring did not reveal any

Helicobacfer--Onbacteria. There were rods and coccoid-shaped bacteria attached to the

epithelial surface of thepms oesophages in a fm stomach biopsies.

Table 5.3-4: Treatrntnt Groupa Ont, Two, and Controls and stomach scores (microscopie dÜation), strritilicd by Futing and Non-Fasting treatment

Stomach , Non-fmtcd subgroup Scores* ControP* 1 Treatment One** 1 Treatment Two*' 1 Total

Total 1 14 14 14 42 1 1 - 1 Fasted subgroup

* Lesion O = No visible lesions, 1 = Pafakeratosis, 2 = Erosions, 3 = Ulcerations **Treatment O = Control group - no treaûnent Treatment t = Treatment One - lansopirazole - 30 mg orally once per day for 7 days Treatment 2 = Trcatment Two - lansoprazole - 30 mg orally once per day for 7 days and azithromycin 500 mg orally once per day for 3 days- Table 5.3.5: Treatment Groups One, Two, md Controis and stomach scores (macroscopic evrluation), stratified by Fisting and Non-FPsting

Stomach Non-futed subgroup Scoresf Confiolf * 1 Tceatment One** 1 Treatment Twoef 1 Total

1 Total 1 1'4 1 14 1 14 1 42 1 S. Scons* 1 Fasted subgroup

* Lesion O = No visible lesions, 1 = Parakeratosis, 2 = Focal, shallow erosions ador ulcetations, 3 = Dfise or deep ulcerations. **Treatment O = Control group - no treatment Treatment 1 = Treatment One - iansop-le - 30 mg ;rdy once per day for 7 days Treatment 2 = Treatment Two - IansoprazoIe - 30 mg ordly once per day for 7hys and azïthromycin 500 mg orally once per &y for 3 days, 5.5, DISCUSSION

The moderate agreement between gross evaiuation and histologic examination grades

is sirnilar to preMous studies (Embaye et al. 1990, Mackin et al. 1997). Macroscopic

examination often fails to identay srnail or superficial ulcexations that are detected by

microscopie examination. In a study by Embaye et a1 (1990). of 155 pig stomachs observed

to be nomal macroscopically, 50 were found to have parakeratotic changes and 83 were

found to have minor to severe erosions on the basis of histologic examination On the other hand, because histologîcal examination was restricted to a small section of the pars oesophages, focal erosions might not be detected by this method. Methodical histologic exarnination of multiple serial sections ofthepms wsophagea would be necessary to ensure each stomach was appropriately classified. The higher percentage of ulcers found on histologic examination may also be because grossly abnormal sections were pre-selected for histologic exarnination. It should be noted that no attempt at morphoIogical quantification was made at the gros level so that minor lesions and severe erosions were scored the same.

This may have reduced the chances of detecting a difference in treatments.

A dismpted feeding pattern was associated with an increase in erosive and ulcerative lesions of the pars oesophagea. It should be noted that this finding was observed three weeks der48 h fasting had occurred. It is assumeâ that the finely ground ration fed during this penod prior to slaughter did not encourage healing during the post-treatment phase, as demonstrated in previous studies by using endoscopie exarnination (Ayles et al. 1996b).

Treatment with 30 mg lansoprazole daily for seven days with or without 500 mg azithromycindailyfor the first three days oftreatment did not appear to reduce the prevalence or severity of lesions of the pars oesophogeu at the thethe stomachs were examined two weeks aâer the completion of the treatment regimen 1t is possible that if the stomachs had been examineci immediately Pfta treatment, a ciiffierence mïghthave ben noted. It is known that dceration is a rapid proces, and hnerkeratosis may occur in the space ofa few hours.

Total ulceration ofthepms oesophvlgu may develop in 1-2 days (Henry 1996).

There was no sipnincant difference detected betwan either Treatment Group One or

Treatment Group Two and the ControI group and therefore the minor Merences found between Treatment Group One and Treatment Group Two are inconclusive and require firther investigati~nwith a larger group of animals and with a treatment protocol that will include higher orland more fiequent dosages ofthese drugs.

It was beyond the scope ofthis shidy to monitor acid secretion in the stomachs during the treatment. Therefore, there is no way of knowuig whether the dosage of 30 mg daily of lansoprazole was sufiïcient to inhibit gastric acid seaetion. Possibly a higher dosage would have been effective in preventing lesions, or possibly splitting the dosage so that it was given

2 or 3 tirnes during the day would have provided a more consistent reduction in acid secretion inhibition-

There are few studies which have examined the efficacy of benzimidazole compounds in controlling acid secretion in the pig. One triai proved both timoprazole and omeprazole at a dose of 20 mg twice daily were effective in preventing gastric ulceration in pigs injected with porcine somatotropin (Baile et al. 1994). Lower dosages oftimoprazole 5 mg twice daily were also shown to be effective @aile et al. 1994). Benzirnidazole compounds act by irreversibly inhibiting, HX+-Anaseand their effect can only be overcorne by the production of new enzyme. This process takes at lem 24 h to complete in humans (Freston 1990) and

therefore it was thought to be unnecessary to addminister the drug more than once per day.

However, Krakowka et al-(1998) suggest in order to achieve a constant low acidity in pigs a 12 h regimen is more effective than a 24 h protocol.

Certainiy, the lack of treatment effea myindicate that other factors besides gastric acid secretion are involved in the production of ulcers during a penod of fasting. Recent studies suggest that bile acîds act synergistically with hydrochlonc acid during perÏods of fasting to produce rapid destruction of the quamous epithelia of the pms oesophagea

(Lang et al. 1998). High concentrations of bile acids have ben shown to be associateci with gastrk ulcer severity (Ayles et al. 1996a). One would have expected that the administration of acid secretory inhibitors might have reduced the effkct of bile acids by maintaining a higher pH. The findings of this trial do not provide evidence that this occurred, but because gastnc pH was not monitored it is impossible to know what caused this lack of response to treatment,

The production oforganic acids by a proliferation of bactena in thepars oesophagea has been suggested as a mechankm causing gastric ulceration (Argenzio and Eisemann 1996,

Krakowka et al. 1998). One rnight have expected that the acid inhibitory treatment would possibly encourage this scenario. Generally, the low pH of the distal stomach would inhibit the growth of Locfubacillus and BuciIIus, but daily medication of lansoprazole and the sudden resumption of ad-lbitum feeding with finely-ground feed would be expected to be an ideal situation for the proliferation of fermentative bacteria. Likewise, one might expect the treatment with an antibiotic during the period when feed is reintroduced to reduce the effkts of bacteriai proliferation. However, this trial does not provide evidence that proliferation of bacteria producing organic aàds played a role in ulcers development in that, the pigs receiving antibiotic did not have féwer or less sever stomach lesions than controls, and pigs receiving only acid seaetoryùihibiton did not havemore sever stomach lesions than controls.

Helicobacfet-like organisms were not found in either silver or carbol-fuchsin stauis in any of the 84 pigs examuied niggestïng that this fm may be fkee ofthese bacteria This is a very important finding for fùture studies ofthis organism. Recent studies have suggested that Helicobacter heilmrmnii is widely distributed in the commercial swine population

(Mendes et al. 199 1, Barbosa et al. 1995, Grasso et al. 1996, Queiroz et al. L 996) and may be a source of infection for humans (Meining et al. 1998). The technique of it~-sittiurease mapping and rnicroscopic examination with Warthin-Starry silver stah is believed to be reliable in that this laboratory has readily demonstrated these spiral-shaped bacteria in stomach mucosa ofpigs from other herds (Meinichouketul. 1998). The lack ofHelicobacfer q.malces it impossible to judge the effectiveness of this combination of antibiotic and acid secretory inhibitor as an eradication program for swine. Therefore, in order to make a decision as to whether treatment with an antibiotic might be usefûf in reducing ulcers in herds

ùifected with Helicobacters, this trial must be repeated on a group ofpigs that is infected with Helicobocier-like bacteria. In addition, the fact that ulcers did occur in the absence of these organisms does suggest that if Helico~rsdo play a role in gastric ulceration in swine, they are iikely not the only factor involved. This study has shown that finely-ground peiieted rations and fdgare important risk factors in the development of gastnc ulcers and that the use of lansoprazole and azithromycin during a penod of feed deprivation was ineffective in preventing gahc lesions from dmloping. Cha~ter6

THE USE OF OMEPRAZOLE TO ALLEVIATE STOMACH ULCERS IN

SmDURING PERIODS OF FEED WITEDRAWAL

6.1. INTRODUCTION

Feed deprivation for a short period oftime (up to 24 h) is a common occurrence on grower-finisher operations. At certain times, such as during the weighing and sorting perÏod prior to shipping to market, feed may be purposely withdrawn, because fasting before slaughter hproves carcass quality (Morrow et al. 1999) and decreases the chance of carcass contamination during processing. Feed interruption may also be a result of other management factors including: overcrowding, Iack of feeder space, restricted access to water, muent moving and mixing, mistakenly running out of feed, or feeder malfunction. Similarly hot weather, the presence of mywtoxins in the feed, or acute disease can cause pigs to stop eating-

Fasting animals for 24 to 72 h has been shown to drarnatically increase the prevalence and severity of erosive lesions in thepms oesopkgeu (Chamberlain et al. 1967, Pocock et al. 1968, Straw et al. 1992, Davies et al. 1994, Henry 1996). Acute respiratory disease is associated with a 9 to 12 fold increase in the presence of ulcers (Senk et al. 1994). The reason for the relationship between a bief penod of féed disruption and gastric ulceration is not known but it has been hypothesized that without continuous feed intake, the normal pH gradient of the stomach is disturbed because of inaeased fluidity and mixing of gastrïc contents. In addition, there is a reduced pH associated with the absence of food buffers in the empty stomach (Lang et al- 1998 ). Presumably, ifacid secretion could be reduced during

fiisting periods, the acidic iasult to the unprotected tissues of thepms oesophageu could be

reduced.

BenPmidazole compoundssuch as omeprazole have been shown to be highly effective

in suppressing gastnc acid secretion (Adelstein et al. 1988, Mardh et al.1988). The

mechanism of action ofomeprazole is via inhibition ofEZV'K'-APase @roton pump) located

at the lumlnai sufice of the parietal cell. As this is the final step in evoking hydrogen ion

secretion, these proton-pump inhibitors block acid secretion caused by ail types of stimuli

(Schubert and Shamburek 1990). Omepratole is an irrevenible inhibitor and therefore elirninates acid secretion until new enzyme can be created (at least 24 h in humans).

Omeprazole in doses of 20 mg or more, once daily, virtually abolishes gastric acid secretion in human patients (Freston 1990).

The purpose of this trial was to determine whether omeprazole treatment would alleviate the lesions of gastnc ulceration associateci with fad disrupiion.

6.2. MATERIALS AND METHODS

A series of three controlled trials were conducted at the University of Guelph swine research facility. The pigs used in al1 three experiments were purebred Yorkshire pigs between 75 and 100 kg in weight. The animais on this trial were al1 in good health, fiee of

MycopIasma hycpneumoniae and other swine respiratory pathogens. Al1 pigs were individually identified and randomly allocated to treatment. They were housed in six pens in one room. Treatments were disperd arnong pens. Feed was a 16 % protein, corn-based,

pelleted grower ration-

In the fkst trial, five pigs were selected as controls and five pigs treated with 20 mg

omeprazole ordy ( LosezûB - 20 mg tablets, Astra Phanna hc., Mississauga, Ontario, L4H

lM4). Animals were Ieft in their orighd pens and allowed ad-lbiilum access to feed. Ail ten

pigs were euthanized 24 h PAer treatment via intravenous pentobarbitol injection. The

carcasses were placed in right laterd recumbency and opened via a deep incision along the

nb tine exposing the stomach. The pH of the gastric contents was measured with a portable

pH meter (Checke-, Hanna Instruments, Woonsocket, RI 02895, U.S.A)by incising a srnail

hole in the stomach wdin the hndic region and inserting the probe into the digesta. The pH

probe was calibrateci previously using standard solutions. The probe was washed wit h distilled

water &er each measurement.

After pH measurements were conducted the stomachs were opened along the greater

curvature and the contents emptied and the surface gently washed with tap water. Thepars

oesophaga was examined for evidence of fesions and a score was assigned based on a scale

fiom zero to three (Mackin et al. 1997), as described in Chapter 3.1.2.

In a second trial, three groups of pigs from the same larger group (five pigs in each

group) were assigned one of three treatments based on random allocation. Group one was

placed off feed for 24 h without medication, gmup two was placed off feed for 24 h and given 20 mg omeprazole oraliy at the beginning of the period of fasting, and group three was similarly fasted but medicated with 40 mg omeprazole. Al1 pigs were euthanized &et- the 24 h period of fasting and examined in a simiiar manner to the first trial. In a third triai, ten pigs were placed into two treatment groups (five hogs in each group). The first group was fasteci for 48 h without medication and the second group was fasted for 48 h and treated with 40 mg ornepramle et the beginning of the fésting period and

24 h later. Trial two and thbegan simultaneously with pigs on trial two being removed afta 24 h and pigs on trial three remaining one more day before being euthanked. Stomach pH and ulcer lesions were measured in a sirnïlar manner to that previously desccibed.

-Thedifferencein mean ulcer scores between treatments was evduated using Knrskal-

Wallis one-way non parametnc analysis of variance. DifEerences in pH means between treatments were tested using unweighted Least squares hear regression.

The experimental protocol was approved by the University of Guelph Animal Care

Colllfnittee and was carrieci out in accordance with the principles published in the Canadian

Council on Animal Care "Guide to the care and Use of Experirnental Animais".

6.3. RESULTS

In trial one, the untreated and cd-lbitum fed control pigs rewrded a mean ulcer score of 0.6 * 0.9 and a mean gastric content pH reading of3 -3 * 1.O. One these pigs was observed to have a small erosion in thepars oesophageu,resulting ii a score of two. The pigs fed od-

Libitum and treated with 20 mg of omeprsu>!e 24 h before euthanasia were found to have a sùnilar mean ulcer score of 0.4 0.5 and a mean stomach content pH of3.9 0.5. There were no erosive lesions in the nomachs in this secand group. There were no statistical differences in ulcer scores or pH readings be~nthese two groups (Table 6.3.1). Table 6.3.1 : Pigs fddIibitrrm and gheither 20 mg omeprazole or no Trcatment (controls) and eumind 24 h Iater

Coatrol Pigs Treated Pigs

I Gastric pH Ulcer scoree* Gastric pH Ulcer scoree*

- - 2.6 1 4.2 1 3.3 * 1.0" 0.6 * 0.9* 3.9 * O.S* 0.4 * OS*

* Mean * standard devïation ** Ulcer score:O= nod, 1 = parakeratosis, 2 = erosions, 3 = extensive erosions and ulcers

In tria1 two involving a 24 h period offeed withdrawal, a significant difference in ulcer scores (p < 0.05) was noted between the pigs treated with 40 mg omeprazole and the other two groups (wntrols and 20 mg omeprazole). The mean ulcer scores for control, 20 mg omeprazole and 40 mg omeprazole were 2.0 0.8, 2.0 * 0.7 and 0.8 * 0.4, respectively. In each of the control and 20 mg groups, four out of five pigs demonstrated erosive lesions in the pars oesophages whereas no pigs in the 40 mg treatment group were noted to have erosions. The gastnc pH varied considerably within each treatment group with control, 20 mg omeprazole and 40 mg omeprazole groups recording mean pH readings of 2.8 * 0 .9,3- 1

* 0.9, and 4.1 * 1.6, respectively. There was no difference in the mean pH values for the three treatments (Table 6.3 -2). Table 6.3.2: The gastric pH and ulcer scores of pigs futrd for 24 h and treated with 40 mg oaieprazolc, 20 mg omeprazole or untruted (controls)

Control Pip 20 mg omcprazole 40 mg omeprazole GdcpK üicer score** Gdc pH Dker score** Guaie pH üïccr score" 1.9 3 2-5 2 1.8 1 2.0 2 3 *O 2 4.5 O 3.9 .L 2-6 3 6.3 1 2.8 2 2-6 1 3 -8 L 3 -5 2 4-7 2 4-0 L 2.8 0.9* 2.0 0.8* 3-1 0.9* 2.0 0.7* 4.1 * 1.6* 0.8 * 0.4*

* Mean * standard deviation ** Ulcer score: O =nod, 1 = parakeratosis, 2 =erosions, 3 = extensive erosioos and ulcers

For those pigs fasted for 48 h there was a dinerence in ulcer scores between the control group (mean score = 2.8 * 0.4) and the 40 mg omeprazole treatment group (mean score = 1.4 * 0.5, p-value c 0.05). The mean pH readings were 3.3 * 1. L for the control group, and 4.8 * 0.7 for the treatment group and these were not statistically dEerent

@ > O.OS), (Table 6.3.3). Table 6.3.3 : The gastric pH and ulcer scores of pigfutcd for 48 h and treated with 40 mg of omeprwk (SIDI or untreated (controis)

Control Pigs Treated Pigs 1 I Gastric pH Ulctr score* Gastric pH Uicer score+*

t Mean standard deviation

** Ulçer score: O = nomial, 1 = parakeratosis, 2 = erosions, 3 = extensive erosions and ulcers

When the data fiom al1 three tdswere combhed, it was noted that fasting was not

associated with a pH change in the stomach content of the pigs. However, the use ofeither

20 mg or 40 mg of omeprazole tended to result in trend toward a higher pH compared to untreated controls (p = 0.08).

Medication with 40 mg of ornepr-le resulted a higher gastnc pH compared to combined controls and 20 mg omepmle pigs @ = 0.01). The gastric pH was increased by

1-07(? = 0.23). Medicating with either 20 or 40 mg of omeprazole caused an increase in gastnc pH of 0.90 (p = 0.0 1, fi = 0.22). Fasting, for 24 h or 48 h, regardleu of treatment resulted in higher mean ulcer scores (1 -80 * 0.87) than pigs left on full feed (0.50 * 0.7 1) (p

= 0.004)- 6.4. DBCUSSION

The inhibition of acid secretion with 40 mg omeprazole daily during a penod of feed withdmwal proved to be effective in reducing the severity of erosive lesions of the pars uesophagea- Howmr, some tissue damage did ocair despite treatment, which suggests that other factors may be involveci in creating gastnc ulcenition in addition to gastric acid.

An omepramle dosage of 40 mg ddy resulted in higher gastric pH and less stomach lesions than 20 mg ofomeprazole although 20 mg daily is a common dosage level for adult humans (Verdu et al. 1994). Other researchers have reported that 40 mg once per day or 20 mg twice per day of omeprazole proved effective in reducing the incidence ofgastric ulcers in pigs receiving parenteral injections of porcine somatotropin (Baile et al. 1994).

Inthe present study, there was no düference in gastric pH between the fasted and ad- libitum fed controls. The meanirement of gastric pH was taken from the fùndic or distal region. Other workers have shown that as pigs are taken off feed the pH of the proximal stomach decreases suggesting increased mixing and fiuidity (Lang et al. 1998). This work supports the hypotheses that the deaease in pH in the proximal stomach is not caused by an increase in acid secretion during fasting but a fvnction of mixing occumng between the proximal and distal regions of the porcine stomach.

Studies have show that the stratified squamous epithelial tissue of the oesophagus and pors oesophogea can withstand acid-peptic digestion for a considerable penod of time

(Orlando 1991). This sîudy and others (Schubert and Shamburek 1990, Orlando 1991,

Argenzio and Eisemann 1996) suggest that although low pH appears to be an important factor in gssaic injury, the fact that fasteci animais whether treated or not had more lesions

than od-lbiiium fed pigs, suggest a combination of wmponents are involved.

Arge~0and Eisemann (1996) have speailated that short chah htty acids produced

by microbial fermentation couid be a contriutkg factor in cadgtissue damage to thepms

oesophrrgea and have dernonstrated this effect in vitro. Feeding a carbohydrate-enrïched

liquid diet and Uifecting gnotobiotic piglets with fennentative commensal bacteria

(Lact~bacin~sand Bacihs sp) causes gastroesophageal ulcers (Krakowka et al. 1998).

Bile acïds have ban shown to be associated with gastric ulcerations (Ayles et al.

1996a) and centstudies have dernonstrated that bii acids act synergistically and in a dose-

dependent marner with low pH to cause damage to thepms wsophageea ofpigs (Lang et al.

1998). This might explain why in this present study, although a relatively high pH was

achieved with omeprazole some damage still occumd. The pors wsophagea would

presumably have been exposed to high levels of bile acids for a considerable length of time

in the pigs fàsted for 48 h in this study and despite a relatively high pH some injury did occur.

Until researchers discover methods to control duodenal reflux or some way of neutralizing

bile acids, it would appear from this present study that the inhibition of acid secretion does

have merit as a control procedure for the control of gastric ulceration in pigs expenencing

feed withdrawal. However, because of the prohibitive cost of omeprazole treatment, this

technique may be oniy practical in the case of valuable breeding-stock at specifictimes of risk

I such as in transport to a new fm. The use of a coarse fæding program and ensuring feeding

schedule irregularities are kept to a minimum are more practicai solutions to this problem in

mOSt cases, OVERALL CONCLUSIONS

Uicecation of the pars wsophagea is an important, widespread, multi-etiologicai

disease of swi-ne. The peracute, hemoahagic fonn of this disease is associated with a high

mortality rate (Argenzio and Eisemann 1996). The prevaience of gastk lesions (ïnctuding

parakeratosis, erosions, and uIcers) can mach 100 % (Ito et al. 1974) under certain

circumstances, and often approaches 90 % when pigs are managed using modem

confinement husbandry practices @riesen et al. 1987).

In this present study of a large modem fhngoperation in Missouri, the prevalence

of gastnc lesions (ïmcluding parakeratosis) based on gross inspection at slaughter was 92 %.

In addition, erosive lesions were observed in 49 % of 1097 stomachs examined- At the same

tirne, very féw lesions were detected in the stomachs ofthe pigs delivered fkom the company's

Texas cornplex (only IO % of the stomachs had ulceration), where pigs are not fasted at any

tirne during production or pnor to shipment to the abattoir, and were fed a mash-feed. Fasting

and finely-ground pelleted rations are known to be important risk factors in the development

of gastnc ulcers. A change in the particle size of feed and the feeding regimen may reduce ulcer losses, but the subsequent economic losses due to poorer fad efficiency may be difficult to just@. However, the use of a coarse-ground mash feed during a period of planned disniption in the feeding schedule rnay be prudent.

The prevalence of gastric lesions on this Missouri fmis consistent with Our obsenations fiom three Ontario fms, where the overall prevalence of gastric lesions ofthe

11I pms oesophgea in five diierent groups of pigs wss 88 %. The range of the lesions vaiied fiom herd-ta-herd fiom 50 % to 100 % of stomachs showing some gross changes to the surface epitheiium ofthepars wsophrgea+Erosive changes of the pms oesophageu were found in 53 % of total animais examineci at the abattoir, and varied between herds fiom 10

% to 85 %. These findmgs are similar to previous findings of herd-to-herd variation in prevalence and severity of gastric lesions (O'Brien 1969, Guise et al. 1997).

Post mortem examination reveaied that the total death loss attniutable to gastrk hemorrhage on the Missouri fmwas 27 %. This wnfirmed that gastnc ulceration was the single most important cause ofmortality on this fm.The annual loss due to gastnc ulcers for this corporation can be estirnated to be over 2.3 million dollars (US). The level of mortaiity nom gastric bleeding on this fimi appears to be lower than reported previously for

North Carolinagrower-finisher pigs dunng the summermonths (Deen 1993), suggesting that continued monitoring of this operation 116ght reveai higher losses due to gastric ulceration, because the present study was conducted in the middle of spring and at the beginning of surnmer.

Feed deprivation was a cornmon practice at this operation and it was considered a major risk factor leading to a high prevalence of gastric ulceration; therefore, controlled trials, using a fasting regimen, were conducted. These studies confirmed that gastric lesions of the pars oesophageu are associated with fasting. This finding is consistent with several reports regiuding the effect of fasting in grower-finisher pigs on the occurrence of gastric lesions published by Chamberlain et al. (1967), Pocock et al (1968), Straw et al. (1992),

Davies et al- (1994). The effèct of 48 h of fasting was detectable even three weeks after fasting had occurred. Fasthg was not associateci with a pH change in the stomach contents

of the pigs as measured in the fiuidic region, but the absence of feed would have allowed the

fiuid contents ta mix and the pH gradient between the pars oesophgea and the diaal part

of the stomach would have been lost. It was found that fasted animais whether treated or not,

had more stomach lesions than adlibitum fed pigs. This finding suggests that possibly other

factors in addition to exponirr to the acid gastric juices are responsible for causing ulceration

of the pms oesophagea.

In human medicine, numerous pharmaceutical agents have been used to neutralize,

reduce or eliminate gastric acid secretion and promote ulcer healing. However, only a few

research studies have been done in pigs. The use of sodium bicarbonate in feed was tested by

Southern et al. (1993) and Wondra et al. (1995a), with mked results. However, only limited

information regarding water medication during fasting periods is avaiiable. Benzimidazole

compounds were studied to examine their efficacy in controlling acid secretion. These dmgs act by irreversibly inhibithg W/K+-ATPase and their effect can only be overcorne by the production of new enzyme. It was shown that this process takes at least 24 h to complete in humans. Omeprazole has been shown to be effective in a dose of 20 mg or more, once daily for treatment of peptic ulcers in humans (Freston 1990). Verdu et aI- (1994) descnbed lansoprazole as an effective drug that suppresses acid secretion in human adults for 24 h at a 30 mg dose. The be~midazoiecompounds omeprazole and timoprazole at a dose of 20 mg twice daily, have been shown to successhilly nduce severity and prevalence of ulcers in swine treated with porcine somatotropin (Baile et ai. 1994). Lowerdosages were also shown to be effective. Protowls for treatment of the gastric ulcers in humans was taken as a bais for our clinical trial with lansopnwle. The possible potentid of using this dmg with and without antibiotic (azithromycin) was examined- In another trial, the effect of omeprazole at low and high doses in fasted and non-fasted animais was uivestigated. Some possible benefit ofa high dose of omeprazole was detected. It is possible that the dosages of lansoprazole and omeprazole (20 mg) were indcient to cause the desved acid secretory inhibitory effect, or the fiequency of medication was inadequate.

The low, once daily dose of omeprazole did not reduce the severity of lesions ofthe pars oesophagea in pigs, and did not change the pH in their stornachs when measured 24 h after treatment. Krakowka et al. (1998) have suggested that omeprazole must be given at 12 h intervals to maintain a consistent high gastric pH. The double dose of omeprazole signincantly increased the pH in the pig stomachs and resulted in less severe stomach lesions, compared to untreated pigs and pigs treated with a low dose @-value = 0.0 1). Aithough the ulcer scores were lower in this group of pigs compared to control animals, epithelial damage still occurred. A possible explmation as to why gastric ulceration did occur in pigs treated with a high level of omeprazole is that the pms oesophagea would presumably have been exposed to high levels of bile acids for a considerable length of time in the pigs fasted for 48 h and (despite a relatively high pK) the combination ofbile and gastric juices would overcome epithelial defenses. Ayles et ai-(1996a) described an association between high concentrations of bile acids and the severity of gastric ulcerations in pigs. A recent study (Lang et al. 1998) suggested that bile acids act synergistidly and in a dose-dependent marner with low pH to cause damage to the pms oesophagea of pigs. Ifbile acids are the primary cause of tissue damage, then it is not surprising that even a high ievels of omeprazole was insufficient to prevent some tissue darnage nom oauning- At this the, there is no known method to control duodenai refluxr, or neutralire bile acid. These trials provide some evidence that acid secretory inhibitors might reduce the ulcerogenic effect of bile acid by maintaining a higher pKin that there was less tissue damage in pigs @en 40 mg omeprazole during a 48 h fasting period. It would appear fiom this present study that the inhibition of acid secretion does have some merit as a control procedure forthe prevention ofgastnk ulceration in pigs expexiencïng feed withdrawal. However, because these drugs are expensive and results are only moderately successftl, this approach would appear to be impracticai under most circumstances.

Arge~oand Eisemann (1996) demonstrated in an in-vifro study that the short chah fatty acids, pmduced by fementative bactena, can be a contributing factor in causing gastric ulceration of the pars oesophagea It has been shown that feeding a carbohydrate-enriched

Liquid diet and infecting gnotobiotic piglets with Lactobaciilus and Bacillus p causes gastroesophageal ulcers (Krakowka et al. 1998).

As well as fementative bacteria, Helicobacter heiImmnzi-Iike organisms have also been implicated as a significant risk factor in ulceration of thepms oesophageu (Mendes et al. 199 1, Barbosa et ai- 1995, Grasso et al. 1996, Queiroz et ai- 1996). These bacteria do not produce volatile fatty acids and are mostly located in the pylonc area at a distance fiom the pms oesophagea (Holck et (11. 1997).

An interesting finding of this study is that the prevalence ofHelicobacter heilmmtnii-

üke bactena might Vary a great deal nom herd-to-herd and might not be associated with the presence of erosive lesions of the pars oesophagea. This is in contrast to other studies. PreMous studies ofHeiïco&c~ërinfection in swine have ignod important ulcerogenic risk factors such as feed withdrawal and fine particle sue of feed. It is possible that an interaction between one of these fictors and the presence of Uelicobrrcter-like bacteria exist. The presence of Maoorganisms might only be a doundhg fàctor in causation of erosive lesions of thepms oesophgea~One ofthe three Ontario herds in this study appeared to be fiee ofNeiicobcïcfer heilmannii-like bacteria Even though, the prevalence of gastric erosive lesions was over 70 %. Spiral-shaped bacteria were present and readily identifïed ni the stomachs of pigs fiom the other two Ontario herds and fiom the Missouri herd.

The spiral-shaped bacteria are most likely spread by the fecal-oral route. The highest prevalence of these organisms was found on the fmthat utilized solid flooring with a combination of floor feeding and continuous flow animal movement. These factors would faciiitate fecal-oral spread of disease fiom pig-to-pig. Transmission of these microorganism fiom fm-to-fa- pig-to-pig or pig-to-hurnans or vise versa is not proven at this time.

Silver staining and an in siEu urease assay were found to be usefùl techniques in detecting and localking urease producing bacteria The latter method hproves the sensitivity of microscopie detection of these bacteria enabling one to biopsy in areas of colour change, thus improving the likelihood of selecting a tissue sample containing bacteria.

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The SAS System

TABLE OF HELBAC BY UREA HELBAC UREA 1 I I Frequency Y0 Row Pct Total Col Pct

103 11 114 59.54 6.36 65.9 O 90.35 9.65 88.79 19.30

13 46 59 7.5 L 26.59 31.1 1 22-03 77.97 11.21 80.70 Total 116 57 173 67.05 32.95 100

STATISTICS FOR TABLE OF HELBAC BY UREA

McNemrr's Test

Statistic = 0,167 DF = 1 Prob = 0,683

Simple Kappa Coefficient

95% Confidence Bounds

Kappa = 0.689 ASE = 0.059 0.574 0.804 OUTPUT (THE SAS SYSTEM)

/* This is the summary of al1 obtained &ta+/ /* ##1-84 treatment trial; Olcontrol, l-antacid,2-~dand antibiotic'V /* # 85-94-second pmtnal mixed grour of 10 pigs, fust tirnt urea &*/ /* # 95- 13 1 -fhst pretrïai group of 37 pigs attemt to find H. heiJmand*/ /* in this group we did not uscd urea media*/ /* 132-168 first post ttriai grwp Of37 pigs Tom's trial */ /* 169-188 second podt-trial20 pigs*/ /* 189-210 second pst-trial 22 pigW data thesis; input obs farm S pipbr pennbr grade diet S helbcter urea 1-10 treatmnt; if grade >1 then ulœr = "+"; if grade

1 AEKELL 161NFOOO 2 ARKELL 2 4 3 NF O O O

-..-) =; Proc print; OUTPUT: The SAS System

H T E R P P L E L IEG B AUE F G N RD CUTLS A N N A 1 T RMC 1 R B B DE E ENEO M R R ET RATR N ARKELL 161NFOOO-+ ARKELL 2 43NFOOO++ ARKELL 3 LlNFOOO-+ ARKELL 4 1 lNFOOO+ ARKELL 5 43NFOOO++ ARKELL ARKELL ARKELL ARKELL ARKEu ARKELL ARKELL ARKELL ARKELL ARKELL ARKEU ARKELL ARKEu ARKELL ARKELL, ARKELL ARKELL ARKELL ARKEU ARKEU ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELK, ARKELL ARUELL ARKELL A=L ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKEWI ARKELL ARKELL ARKELL ARKELL AwELL ARKELL ARKEIL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL PURELINE PURELN ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKELL ARKmJI ARKEU ARKELL ARKE. ARKELf. ARKELL ARKELL ARKELL ARKELL A. ARKELL ARKELL ARKELL ARKELL ARKELL ARKELt ARKELL ARKELL ARKELL ARI(ELL ARKELL ARKELL ARIax ARKELL PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY rnNS0NBY PONSONBY PONSONBY PONSONBY PONSOJSTBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY PONSONBY KINSONBY PONSONBY PONSONBY 02NF11++ PONSONBY OlNFlO- - + PONSONBY OONFll- - - PQNSONBY 01NF11- - + PONSONBY OLNF11- - + PONSONBY OLNFl1. - + PONSONBY 01NFlO- - + PONSONBY OONFLl- - - PONSONBY 02NFIl++ PONSONBY 01NFlI- - + PONSONBY OlFOO+ PONSONBY OlFOO*- + EQNSONBY OlFOO-+ PONSODBY OlFOl-+ PONSONBY O1FOO-+ PONSONBY OIF 11. - + PONSONBY OLFOO-+ rnNS0NBY OlFOO--+ PONSONBY O 1-F OO+ PONSONBY O2FOO++ PONSONBY OONFOI- - - PONSONBY OONFOO- - - PONSONBY 01NFOO- - + PONSONBY OLNFOO- - + PONSONBY OlNFOO- - + PONSONBY OONFOO*-- PONSONBY OONFOO- - - PONSONBY OONFO1- - - PONSONBY OONFOO- - - PONSONBY OONFOO- - - PURELINE OONFlO- - - PURELINE OONFlL---

PURELINE OONFIO- O - PURELINE OlNFIl-- + PURELINE OLNFll- - + PURELDE OONFlO- - - PURELINE OLNFll. - + PURELINE OONFl1- - - PURELINE OONF11- - - PURELINE OONF1lc- - PURELINE 01NFlL-+ PURELINE OlNFlO-- + PURELINE OLFOO+ PURELINE OOFOO--- PURELINE 02F IO++ PURELIN' 03F IL++ PURELINE OOF 11-- - PURELINE O1F lL+ PURELENE OlF11. * + PURELINE O1F 11-- + PURELINE OOF 11. - - PURELINE OOF 11-- - APPENDIX 3 PREVALENCE OF ULCERS AND LESIONS, CONTROLLING FOR FARM.EFFECT

p=neq; tables diet*ulcer f exact cmh masures; tables fàrm8diet*ulcer / exact cmh measUres; exact mr, nin; The SAS System

TABLE OF DIET BY ULCER

DIET ULCER I 1 Frequency

66+99 66-66 Row Pct Total Col Pct

Fasted

Total STATISTICS FOR TABLE OF DIET BY ULCER

Statistic DF Value Prob

Chi-squaze Likelihoud Ratio Chi-Square Continuity Adj. Chi-Square Mantel-BacnszelChilsquare Exact Tcrt (ï&) @gW - (2-T8iî) NOT SIGNIFICANT Phi coefficient Contingency Codlncient Cramer's V

Statistic Value ASE

Gamma Kendall's Tau-b Stuart's Tau* Somers' D ClR Somerst D NC Pearson Correlation Speafman Correlation Lambda Asymmetric Cp Lambda Asymmetric WC Lambda Symmetric Uncertainty Coenicient CF Uncertainty Coefficient WC Uncertainty Coefficient Symmetrk

Estimates of the Relative Risk (RowliRow2)

95% Confidence Bounds 95% Confidence Bounds Type of Study Vaiue (Asymptotic) (Emct)

Case-Controi 1.689 0,955 2,988 0,920 3-118 Cohort(C0llh-sic) 1.268 0,987 1,630 Cohort (Co12 Risk) 0.75 1 0.543 1,038

Sample Size = 210 SUMMARY STATISTICS FOR DIET BY ULCER

Cochraq-Mantel-Ehenszel Statistics (Based on Table Scores)

Smtistic AltcrnativeHypothesis DF Value Pmb

1 Nomm Correlation 1 3.251 0.071 2 Row Mean Scores DifEer 1 3,251 0.071 3 General Association 1 3.251 0.071

Estunates of the Common Relative Risk (Rowl/Row2) 95% TypeofStudy Methd Value Confidence Bounds casc-conttoi M8~td-H8a~~d1.689 0.955 2386 NOT SIGNIFICANT (Odds Ratio) Logit 1.689 0955 2.98%

The confidence bounds for the M-H estimates are test-based- The SAS System

SUMMARY STATISTICS FOR DIET BY ULCER CON'iROLLiNG FOR PARM

Cochran-ManteI-R;acnszel Statistics (Based on Table Scores)

1 Nonzero Correlation 1 10,644 llE-4 2 Row Mean Scores Diner 1 10,644 11E-4 3 General Association 1 10,644 11E4

Estimates of the Cotnmon Relative Risk (Rowl/Row2)

95% Type of Study Method Value Confidence Bounds

Case-Control ~antel-&&el 3.649 1.677 7.943 (Odds Ratio) Logit 3.522 L590 7.799 SIGNIFICANT

Cohort - Mantel-Hienszei 1.438 1,156 1.788 (hl1 Risk) Logit 1.309 1.097 1.562

Cohort Mantel-Hienszel 0,654 0.506 0,844 (Col2 Risk) Logit 0,757 0.614 0.933

The confidence bounds for the M-Hestimates are test-based-

denotes that the logit estunators use a correction of 0.5 in every ce11 of those tables that contain a zero.

Breslow-Day Test for Homogeneity of the Odds Ratios

Chi-Square = 0,975 DF= 2 Prob = 0.6 14

Total Sarnple Size = 210 TABLE OF DIET BYLESION

data thesis; input grade diet $ helbcter urea fatm S 1-10; if grade >l tben ulcer = "+"; if grade <2 tben uïcer = "-"; if grade >O thcn icsion = "+"; if grade <1 then lesion = "-"; -;

3 F O O ARKELC 3 F O O AR- 2 F O O ARKELL --- nui; prix: p~t; m; p=necl; tables diet*lesion / exact cmh measures; tables farm+diet8lesion/ exact cmh measures; exact rror; nui; The SAS System

Frequency '% Row Pct Total Col Pct l 73 4 77 Fas ted 34-76 1.90 36-67 94.8 1 5.19 39-46 16-00

112 21 133 Non-fas ted 53-33 10.00 63 -33 84.2 1 15.79 60-54 84-00 STATfSFCS FOR TABLE OF DIET BY LESfON

Statisîic DF Value hb

Chi-Square LIkcLibood Ratio Chi-Square Continuiiy Mj, chi-Sq& Mantel-Hacnszel Chl-Square Fishef s Exact Test (Left) (fi-gw (2-T&l) Phi Cocflticient Contingency Coefficient Cramer's V

Statistic VaIue ASE

Gamma Kendall's Tau-b Stuart's Tau* Somers' D Somers' D WC Pearson Correlation Spearman Correlation Lambda Asymmetcic Cm Lambda Asymmetric WC Lambda Symmetrïc

Statistic Value ASE

Uncertainty Cdcient CR 0038 0.029 Uncertainty Coefficient R[C 0,021 0,016 Uncertainty Coefficient Symmetrïc 0.027 0.021

Estimates of the Relative Ri& (Rowl/Row2)

95% Confidence Bounds 95% Confidence Bounds Type of Study Value (Asyrnptotic) (Exact)

Case-Control 3.422 1,129 10.374 1.087 14.199 Cohort (Col1 Ri&) 1-126 1.029 L.232 Cohort (Coi2 Risk) 0.329 0-1 17 0.923 SUMMARY STATISTICS FOR DIETBY LESION

Cochran-Mantel-Hkc~sztIStatistics (Based on Table Scores)

Statistic AlternativeHypothesis DF Value Prob

1 Nonzero Correlation 1 5.195 0.023 2 RowMeanScoresDiftcr 1 5.L95 0.023 3 Generai Association 1 5,195 0.023

Estimates of the Common RcIative RisL @owlhw2) 95% Type of Study Method Value CanfkknceBounds

Case-Control Mantel-Hhenszel 3,422 1,188 9.W6 (Odds Ratio) Logit 3,422 L-129 10.374

Cohort Mantel-Haenszel 1,126 1,017 1,247 (Coi1 Risk) Logit 1,126 1,029 1.232

Cohort Mantel-Ehenszel 0,329 0,126 0.856 (COU Risk) Logit 0,329 0.117 0,923

The confidence bounâs for the M-H estimates are test-based

Total Sample Size = 210 SUMMARY STATISTLCS FOR DIET BY IzESlON CONTROLLING FOR FARM

Cochraa-ManteI-HaemzeI Statisîics (Based on Table Scores)

Statwric AlteniativeEIypothesiS DF Value Pmb

1 Nonzero Correlation I 10.027 1SE4 2 Row Mean SccrresDüEer 1 10,027 ISE4 3 Generai Association 1 10,027 ISE4

Estùnates of the Common Relative Risk (Rowl/Row2) 95% TypeofStudy Method Value Confide~xBounds

Case-Control Mantel-Haemzel 7.476 2.152 25367 (Odds Ratio) Lagit * 3322 0.963 12.889

Cohort Uantel-Haenne1 1,164 1,060 1.279 (Col1 Risk) Logit 1.031 0,998 1.064

Cohort Mantel-HaenszeI 0.265 0,116 0-603 (Coi2 Risk) Logit * 0.543 0.241 1.222

The confidence bounds for the M-H estimates are test-based

denotes that the logit estimators use a correction of 0.5 in every ce11 of those tabIes that contain a zero-

Breslow-Day Test for Homogeneity of the Odds Ratios

Chi-Square = 4,410 DF= 2 Prob = O, 110

Total Sample Size = 210 APPENDIX 4

The SAS System data thesis; format fann S10.; input grade diet $ helbcter urra fPrm treaünn~ ifgraâe >i thcn ulœr = "+*; if grade <2 then ulcer = "-";

PONSONBY . PONSONBY - rnNS0NBY * rnNS0NBY * PONSONBY - PONSONBY - PIlNSONBY , PONSONBY - PONSONBY . PONSONBY . PONSONBY - PONSONBY -

PONSONBY * PONSONBY - PONSONBY - PONSONBY . FONSONBY .

PONSONBY * PONSONBY - PONSONBY - PONSONBY . rnNS0NBY .

PONSONBY * PONSONBY - PONSONBY - FONSONBY . mNS0NBY - PONSONBY - PONSONBY PONSONBY . PONSONBY . PONSONBY - PONSONBY * PONSONBY - PONSONBY - :mu 'Jeu m?xa lamw*wp sa~qm fbaq md 4 lld0 tldO 11dI Ild? llAT tld0 11dE O 1'd z OOAO OOdT 01m1 11T 1 Tm0 t lm0 I1dN O 1 lm 1 01mo 11m 1 1 lm 1 01mo T Tm O 01mo O om O O om O Tom O oom O O om O O om 1 O ON1 O 0- t O om O 1 OdN O OOdZ OOdI OOdl OOd1 tldl OOd1 IOdI OOdT 0031 OOdl 1 1dN 1 11m z TABLE DIET BY lPECICOBACZER

DIET KELBCTER Fre~uency % u-a Row Pct Total Col Pct

18 17 35 Fasted 22-78 21-52 44-3 51.43 48.57 35.29 60-71

33 11 44 Non-fasted 4 1-77 13-92 55.7 75.00 25-00 64-71 39.29

51 28 79 Total 100 64.56 35-44

STATISTICS FOR TABLE OF DIET BY HELBCTER

Chi-Square Likelihood Ratio Chi-!%pare Continuity Adj, Chi-Square Mantel-Hknszel Chi-Square Fisher's Exact Tcst (Leff) (Rwlt) (2-Tail) Phi Coefficient Contingency Coefficient Cramer's V

Statim-c Value ASE

Uncertainty Coefficient C@ 0.046 0.042 Uncertainty Coefficient WC 0,044 0.040 Uncertainty Coefficient Symmetric 0.045 0.04 L

Estimates of the Relative Risk (Rowl/Row2)

95% Confidence Bounds 95% Confidence Bounds Type of Study Value (Asymptotic) (Exact)

Case-Con trol 2.833 1.094 7.336 0,990 8,213 Cohort (Col1 Ri&) 1-943 1.050 3,593 Cohort (Co12 Risk) 0.686 0.476 0.987 SUMMARY STATISTICS FOR DIET BY BELBCTER

Cochran-Mantel-HaenszelStatlstics (Based on Table Scores)

Statistic Altemative Hypothesis DF Value Prob

1 Nonzero Conelation 1 4-674 0.031 2 RowM;ean Scores DSer 1 4.674 0.031 3 General Association 1 4,674 0.031

Estimates of the Common Relative Risk (RowlRow2)

95% Typeof Study Mettiod Value CoafidcnœBounds

Case-Control Mantel-Haenstel 2.833 1.102 7.284 SIGNIFLCANT (Odds Ratio) Logit 2.833 1,094 7,336

Cohort Mantel-&:nszel 1.943 1.064 3.548 (Coli Risk) Logit 1.943 1.050 3.593

Cohort Mîntel-Haenstel 0.686 0.487 0,%5 (Col2 Risk) Logit 0.686 0,476 0.987

The confidence bounds for the M-H estimates are test-based-

Totaï Sample Size = 79 APPENDIX 5

KAPPA AGREEMENT BETWEEN GROSS AND MICROSCOPIC EVALUATLON

options pF35 IF90 probsir2; /*&ta on aratmcnt trial in Arkeil84 pigs in totalf/ /*O+onttolL--nt with antacid 2-treatment with antacid & antiiioticf/

TABLE OF GROSS GRADING BY MICRO GRADXNG

GROSS MICRO Frequency, %, Row Pct, O 1 2 3 TOTAL Col Pct 1 3 O O O 1-19 3 -57 0.00 0.00 25-00 75,00 0.00 0.00 100,OO 12.50 0.00 0.00 O 20 8 O 1 0.00 23.8 1 9.32 0.00 0.00 7 1-43 28.57 0.00 0-00 83.33 38.10- 0.00

O 1 11 20 2 0.00 1-19 13-10 23 -81 0.00 3, L3 34.38 62.50 0.00 4-17 52.38 52.63 O O 2 18 0.00 0,OO 2.38 21-43 3 0,Oo 0-00 10.00 90.00 0.00 0.00 9.52 47-37 1 24 21 38 TOTAL 1.19 28.57 - 25 45.24 STATISTICS FOR TABLE OF CROSS BY MICRO

Test of Symmetry

Statistic = 23,172 DF = 6 Pd= 74E-5

Kappa Cdcicnts

Statistic Value ME 95% Confidence Bounds

Simple Kappa 0,423 0.073 0,280 0.565

WcigùtedKippr OS9 0.056 0.479 0,700

-pie Size = 84 KAPPA STATISTXCS MACRO BY MICRO BASED ON ULCER AND NO ULCER

/*data on treatment trial in Arkeii 84 pigs Zn total'/ /*OumtrolL- treatment with antacid 2-treatment with antacid & antr'biOtic*f /* "+" - ulœr, "2no dœr */

if gn,ss>f then mam ="+"; ifgross<2 then macro ="-"; ifmicn~lthen histo ="+"; ifmicnK2 then histo ="-"; TABLE OF MACRO BY HISTO

MACRO HISTO Frequency, "/4 RowPct , ULCER NO ULCER TOTAL col Pct, 51 1 52 ULCER 60-7 1 1-19 61-90 98.08 1-92 86-44 4.00 8 24 32 NO ULCER 9-32 28.57 38.10 25-00 75.00 13-56 96.00 TOTAL 59 25 84 70.24 29.76 100.00 ------

KAPPA STATISTICS FOR TABLE OF MACRO BY HXSTO

McNemarls Test

Statistic = 5.444 DF = 1 Prob = 0.020

Simple Kappa Coefficient

95% Confidence Bouncis

Kappa = 0.763 ASE = 0.073 0.619 0.907 PREVALENCE OF GASTRIC LESIONS AND ULCERS BASED ON GROSS AND MICROSCOPIC EXAMNATION

The SAS System options ps-35 ls-90 pmbsip2; /*&ta on treatment trial in Arkeli 84 pigs in totalV PO-control 1- treatmeat with antacid 2-tteatmcnt wîth antacid & antiiioticV PGROSS - gmss evaluation of gasîrïc lesions, MICRO-rnicmscopic evaluation */ &a Kappatrt; input tretmnt diet S gros micro; if gross >1 then dcer="+"; if gros <2 then iilœr="+; ifgross >O then lesion="+"; if gros l then ulcerl="+"; if micm <2 then ulcerl="-"; if >O then lesionl="+"; if micro cl then lesionl="-"; cards; OFll OF12

*.-, m; Pmfreq; tables diet*lesion / exact cmh measuces; tables diet*ulcer / exact cmh measures; tables diet*lesionl / exact cmh measures; tables diet*ulcerl/ exact cmh measutes; exact rror; =; TABLE OF DlET BY LESION CBASED ON GROSS EVALUATION)

Frequency, %, Row Pct, Total Col Pa ------42 O 5O.oo 0.00 42 Fasted 100.00 0.00 50 52.50 0.00

Total

STATISTICS FOR TABLE OF DLET BY LESION

Statisîk DF Value Prob

Chi-Square 1 4.200 0.040 Likelihoad Ratio Chi-Square 1 5.745 0-0 17 Continuity Adj- Chi-Square 1 2.363 O, 124 Uantel-Haenszel Chi-Square 1 4.150 0,042 Fisher's Exact Test ut) 1~000 @-lm) 0,058 (2-~d9 0.116 NOT SIGNIFfCANT Phi Coefficient 0.224 Contingency Coefficient 0.218 Cramer's V 0.224

Statistic Value ASE . Gamma Kendall's Tau4 Stuart's Taus Somers' D C(R Somers' D WC Pearson Correlation Spearman Correlation LamWa Asymmetric CF Lambda Asymmetric WC Lambda Symmetric Statisîic Value ASE

Uncertainty Coefficient Cm 0.179 0.034 Uncertainty Coefficient R[C 0.049 0,024 Uncertainty Coefficient Symmetrïc 0,077 0.033

Estimates of the Relative Risk ~I/Row2) 95% Type of Study Value Confidence Bounds

Cohort (Col1 Ri&) 1-105 1,002 1.219

One or more risk estimates not computed -zero ceU- WARNING: 50% of the ce& have expected counts les than 5- Chi-Square may not be a valid test

SuMMARY STATISTICS FOR DIET BY LESION

Cochran-UanteI-Haenszel Sîatistics @ascd on Table Scores)

Statistic Alternative Hypothesis DF Value Prob

1 Nonzero Correlation 1 4.150 0.042 2 Row Mean Scores DSer L 4,150 0.042 3 General Association 1 4,150 0,042

Estimates of the Common Relative Ri& (Elowl/Row2)

95% Type of Study Methoci Value Confidence Bounds

Case-Control Mantel-Haenszel - (Odds Ratio) Logit 9.935 0.518 190.600 NOT SIGNTFKCANT

Cohort Mantel-Haenszel 1.105 1.004 1-217 (Co11 Risk) Logit 1,105 1,002 1.219

Cohort Mlantel-Haenszel 0,000 - (Col2 Risk) Logit 0-111 0.006 2.001

The confidence bounds for the M-H estimates are test-based- To avoid undefined results, some estimates are not computed. * denotes that the logit estimators use a correction of 0.5 in every ce11 of those tables that amtain a zero- TABLE OF DIET BY ULCER lBASED ON GROSS EVALUATIOM

DlET ULCER Frequ-cy, %, U-a Row Pct, Total Col Pct

33 9 Fasted 39.29 10-71 42 78.57 21-43 50 63-46 28.13 19 23 Non-fristed 22-62 2738 42 45-24 54-76 50 36.54 71.88

Totaï 52 32 84 6 1-9 38.1 100

STATISTKS FOR TABLE OF DIET BY ULCER

Staîistic DF Value Prob Chi-auare Likelihood Ratio Chi-Square Continuity Adj. Chi-Square Mlantel-Raenszel Chi-Square Fiibcr's Exact Test (Left) m-gh) (2-TdI) SIGNIFICANT Phi Coefficient Contingency Cdcient Cramer's V

Statistic Value ASE

Gamma 0.632 Kendall's Tau-b 0.343 Stuart's Taus 0.333 Somers' D Cpt 0.333 Somers' D WC 0.353 Pearson Correlation 0.343 Spearman Correlation 0.343 Lambda Asymmetric CIR 0,125 Lambda Asymmelnc WC 0,333 Lamûda Symmetric 0,243 Statistic Value ASE

Estllnates of the Relative Risk (Rowl/Row2)

95% Confidence Bouads 95% Confidence Bounds Type of Study Valut (Asymptotic) @=CO

Cuc-Control 4.439 1.708 11.537 1.56û 13.082 SIGNIFICANT Cohort (Col1 Risk) 1.737 1.202 2.510 Co& (Coi2 Ri&) 0.391 0.206 0,743 Sample Size = 84

SUMMARY STATISTICS FOR DIET BY ULCER

Cochran-Mantel-HaenszI Statistics (Based on Table Scores)

Statistic Aitexnative Hypothesis DF Value Pmb

1 Noazero Correlation 1 9,776 18E-4 2 Ebw Mean Scores Diner 1 9.776 18E-4 3 Gened Association L 9,776 18E-4

Estimates of the Cornmon Relative Risk (Rowl/Row2)

95% Type of Smdy Method Vdue Corifidence Bounds

Case-Control Mantel-Haeaszcl 4.439 1.744 11.297 SIGMFICANT (Odds Ratio) Logit 4.439 1.708 11.537

Cohon Mantel-Haensix1 1,737 1.229 2,435 (Coli Risk) Logit 1.737 1.202 2.510

Coho rt Mantel-Haenszel 0.391 0.217 0,705 (Col2 Risk) hgit 0.391 0.206 0,743

The confidence bounds for the M-H estimates are test-based. TABLE OF DIET BY LESION 1 lBASED ON MECROSCOPIC EVALUATTON)

DIET LESION 1 Frequency, Percent, "-a Row Pcf, a+n Total Col Pct 42 O Fasted 50 0.00 42 100,00 0.00 50,Oo

Non-Fasted 48-81 1-19 42 97.62 2-38 I 50-00 49.40 100,OO

Total 83 1 84 98.8 1 1.19 100

STATISTICS FOR TABLE OF DIET BYLESIONI

Statistic DF Value Pd

Chi-Square 1 1,012 0.314 Likelihood Ratio Chi-Square 1 1.398 0.237 Continuity Adj, ChbSquare 1 0.000 1,000 Mantel-Haenszel Chi-Square 1 1.000 0.3 17 Fisher's Exact Test (Left) 1.000 (RW) 0.500 (2-Ttil) 1.000 NOT SIGNIFICANT Phi Coefficient 0.110 Contingency Coefficient 0.109 Cramer's V O, 110

Statistic Value ASE

Gamma Kendall's Tau-b Stuart's Tau* Somers' D Cp Somers' D WC Pearson Correlation Spearman Correlation Lambda Asymmetric CF Lambda Asymmetric WC Lambda Symmetric Statistïc Value ME

Unccrtainty Coef6citnt Cpt 0.129 0.030 Unccrtainty -&nt RJC 0,012 0,012 Uncertainty Coefficient Symmetric 0,022 0-020

Estimaies of the Relative Risk (Rowl/Row2) 95% Type of Snidy Value Confidenœ Bounds

Cohort (Col1 Risk) 1,024 0.977 1.074

Sample Sh= 84 One or more nsk estimates not computed -zero celL WARNING: ofthe aLls have expected counts less than 5, Chi-Square may not be a valid test.

SUMMARY STATISTICS FOR DIET BY LESION 1

Coçhran-Uantel-Haenszel StatistÏcs (Baseci on Table Scores)

Statistic Alternative Hypothesis DF Value Prob

1 Nonzem Correlation 1 1~000 0,317 2 RowMeanScotesDifEer 1 1,000 0.317 3 Generai Association 1 1.000 0.317

Estimates of the Common ReIative Risk (Rowl/Row2) 95% Type of Study Method Value Confidence Bounds

Cqdontrol Mantel-Haenszel . . (Odds Ratio) Logit 3.072 0.122 77.594 NOT SIGNIFICANT

Cohort Mantel-Hkenszel 1.024 0,977 1.074 (Coi 1 Risk) Logit 1.024 0,977 1.074

Cohort Mantel-Hacnszel 0.000 - (Col2 Risk) Logit * 0.333 0,014 7,956

The confidence bounds for the M-H estimates are test-based- To avoid undefined results, some estimates are not computed

denotes that the logit estimators use a codon of 0.5 in every ce11 of those tables that contain a zero.

Total Sample Size = 84 TABLE OF DIET BY ULCER1 (BASED ON MICROSCOPIC EVALUATION)

Brcqucncy, Oh, Ron Pct, Col Pct

Fasted

Total

STATISTICS FOR TABLE OF DIET BY ULCERl

Statistic DF Value Prob Chi-Square Likelihood Ratio Chi-Square Continuity Adj. Chi-Square Mantel-HaenszeI Chi-Square Fisher's Exact Test (Le@ WgW (2-Td) SIGNIFICANT Phi Coefficient Contingency Coefficient Cramer's V

Statistic Value ASE

Gamma Kendall's Tau-b Stuart's Taux Somers' D CIR Somers' D WC Pearson Correlation Spearman Correlation Lambda Asymmetnc Cm Lambda Asymmetnc I(IC Lambda Symmetfic Statistîc Value ASE

Estimates of the Relative Risk (RowI/Row2)

95% Confidence Bounds 95% Confidence Bounds Type of Smdy Vdue (Asymptotic) (Exact)

Case-Control 6.727 2310 20.475 2.021 25-676 Cohort (COU Ri*) 1.682 1335 2-291 Cohen (Col2 Risk) 0.250 0,104 0.604

SUMMARY STAnSTICS FOR DIET BY ULCERl Cochran-Mantel-HhellszelStatistks (Based on Table Scores) Statistic Aitemative Rypothesis DF Value Prob

1 Nonzcro Correlation 1 12.661 37E-5 2 RowMean Sams DNer 1 12.661 3755 3 Generai Association 1 12,661 37E-5

Estimates of the Common Relative Risk (Rowl/Row2) 95% Type of Study Method Value Confidence Bounds

Cohort Mantel-Haenszel 1.682 1.263 2.239 (Col1 Risk) Logit 1682 L.235 2.291

Cohort Mantel-Haenszel 0,250 0,116 0.537 (Col2 Risk) fiogit 0.250 0.104 0.604

The confidenœ bunds for the M-H estirnates are test-based- Total Sample Size = 84 ASSOCIATION BETWEEN TREATMENTS AND GASTRIC LESIONS

Summa y Tabla on SAS Output

Table 1: The association between trutmtnts and erosive lesions and utceration, bascd on gross evaiuation

LESIONS Fis htr's Exact M-H. Chi-square TREATMENTf Test (2 Tail), SC-SC or Logit (OR) P-Vdut

O vs. 2 111

TREATMENT* ULCERS Fisher's Exact M-H. ChEsquare Test (2 Tail), cc-cc or Logit (OR) cc+1* Total P-Value

1 vs. 2 0,785 2 18 10 28

'Treatment O = Control group - no treatment Treatment 1 = Treatment One - lansoprazote - 30 mg orally once per day for 7 days Treatment 2 = Treatment Two - lansoprazole - 30 mg orally once per day for 7days and arithromycïn 500 mg oralfy once per day for 3 days. Table 2: The association between trerrtmeats and erosive lesions and ulceration, bad on microseopic esamination

TREATMENTf FLher's-Euct LESXONS M-H. chi-square Test (2 Taii), or Logit (OR) u+n Totd P-Vdue

Not Signincant

1 27 1 28 1 vs. 2 1 Not Signifïcant 2 28 O 28

TREATMENT* ULCERS M-H- Chi-square - Test (2 Tail), cc-rc or Logit (OR) cc+py Total P-Value

*Treatment O = Control group - no treatment Treatment 1 = ~reatmentOne - lansoprazole - 30 mg orally once pet day for 7 days Treatment 2 = Treatment Two - lansoprazole - 30 mg orally once per day for 7days and azithromycin 500 mg ordy once per day for 3 days. Table 3: The association betwecn treritments and erosivt lesions and ulceratioq brwd on grou evrluation and conholling for diet = F (Fast&)

TREATMEI?l'T* Fuher'r LESIONS M-H.Chi-rq~ire Test (2 Taii), cep CC CL or Logit (OR) - Total P-Value I 1 O 14 O 14 ' 1 O vs-1 Not Significant 1 14 O 14

*Treatment O = Control group - no treatment Treatment 1 = Treatment One - lansoprazole - 30 mg ordy once per day for 7 days Treatment 2 = Treatment Two - lansoprazole - 30 mg ordy once per day for 7days and azithromycin 500 mg oraily once per day for 3 days. Table 4: The association bctwten treatments and erosive Iesions and ulceration, based on gross evduation and contrdling for diet = NF (Non-futed)

TREATMENTf LESIONS Firher's M-H. Cbilquare Test (2 Tail), =+" CC- 6& Total P-Vdue or Logit (OR)

O 13 1 14 OR = 3,545 O vs-1 0,596 1 11 3 14 0.321 s CI s 3936

O 13 1 14 O vs. 2 1 Not Significant 2 14 O 14

I 11 3 14 OR = 0,113 1vs.2 0.222 s 2 14 O 14 0,005 s CI 2-423

'Treatment O = Control group - no treatment Treatment 1 = Treatment One - lansoprazole - 30 mg orally once per day for 7 days Treatment 2 = Treatment Two - lansoprazole - 30 mg orally once per day for 7days and azithromycin 500 mg orally once per day for 3 days. Table 5: The assochtion between trcatments and erosive lesions and ulcemtion, bued on microscopie eumination contmlling for diet = F (F.stcd)

TREATMENT* Ficher's ULCERS *-HaChi-squrrc Test (2 Tail), Cs+97 cc cc or Logit (OR) - Total P-Value

1 vs. 2 0.596 2 II 3 14 1 0.233 r CI s 200.16

*Treatrnent O = Control gmup - no treatment Treatment 1 = Treatment One - Iansoprazole - 30 mg oraiiy once per day for 7 days Treatment 2 = Treatment Two - lansopramle - 30 mg oraliy once per day for 7days and azithromycin 500 mg oraiiy once per day for 3 days. Table 6: Association bttwecn accitments and emsive Iesions and ulceration, bued on mi~ro~~~picesaminition contmNing for diet = NF (Non-fasted)

1 13 1 14 1 vs. 2 1 Not Significant 2 14 O 14 l TREATMENT* ULCERS F*her's M-H. Chi-quare Test (2 Taiï), "+" cc-cc Total P-Value or Logit (OR)

O 8 6 14 OR = 0-616 O vs. 2 . 0.695 2 10 4 14 0-155 s CI s 2,444

*Treatment O = Controi group - no treatment Treatment 1 = Treatment One - lansoprazole - 30 mg orally once per day for 7 days Treatrnent 2 = Treatment Two - lansoprazole - 30 mg oraiiy once per day for 7days and azithromycin 500 mg oraliy once per day for 3 days.