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1986 Effect of Experimental Ostertagia Ostertagi Infection or Chronic Pentagastrin Treatment on Abomasal Pathophysiology and Morphology of Goats. Judit Eva Markovits Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Markovits, Judit Eva, "Effect of Experimental Ostertagia Ostertagi Infection or Chronic Pentagastrin Treatment on Abomasal Pathophysiology and Morphology of Goats." (1986). LSU Historical Dissertations and Theses. 4193. https://digitalcommons.lsu.edu/gradschool_disstheses/4193
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Markovits, Judit Eva
EFFECT OF EXPERIMENTAL OSTERTAGIA OSTERTAGI INFECTION OR CHRONIC PENTAGASTRIN TREATMENT ON ABOMASAL PATHOPHYSIOLOGY AND MORPHOLOGY OF GOATS
The Louisiana State University and Agricultural and Mechanical Ph.D. Col. 1986
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EFFECT OF EXPERIMENTAL OSTERTAGIA OSTERTAGI INFECTION OR CHRONIC PENTAGASTRIN TREATMENT ON ABOMASAL PATHOPHYSIOLOGY AND MORPHOLOGY OF GOATS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Interdepartmental Program in Veterinary Medical Sciences Veterinary Pathology
by Judit E. Markovits D.V.M., University of Veterinary Science, Hungary, 1978 August 1986 ACKNOWLEDGEMENTS
I wish to express my gratitude to Dr. T.G. Snider who awakened my interest in gastroenterology and was also instrumental to gain the support of Ayerst Laboratories.
Dr. Snider also gave guidance in the preparation of this dissertation. I am indebted to Dr. W.G. Henk for his suggestions and help in morphometric and electron microscopic techniques. I would also like to thank the other members of my graduate committee, Drs. H.W. Taylor,
J.C. Williams and K.A. Gossett for their advice and editorial comments.
A special thanks is due to Dr. H.W. Casey whose advice was invaluable throughout this study. I would like to thank' Dr. K.J. Wolfsheimer who gave excellent advice on the RIA technique. I also thank the support of Drs.
D.C. Baker and S.D. Gaunt throughout this experiment. Dr.
A.M. Saxton was helpful in the statistical evaluation of morphometry.
Terry Romaire shared with me the responsibilities of taking care of the animals. I am also very grateful for Terry's editorial comments. All my light microscopic tissues were processed by Cheryl Crowder, whom I have to thank for her patience and talent during the frustrating months of modification of special stains. Without the efforts of Dr. V.J. Banks and Pam Ellis the morphometry would have been impossible to complete.
Gary Bates. Rick Knight, Kenny Marbury, Rick Ramsey and Ted Vance helped on the farm. Serum chemistry and hematology was performed by Maureen Stockwell, Carol
Turnwald and Linda West. Sherry Gibson and Laura Tounger had some excellent practical suggestion on EM techniques.
I wish to thank Jason Osburn for his help with parasite recovery. Andy Smith made contact prints and developed film for me. Michael Broussard and Harry Cowgill were helpful with illustrations for the dissertation and seminars.
This work was supported by a grant from the School of Veterinary Medicine Organized Research Fund, Louisiana
State University. Additional support was provided by
Ayerst Laboratories, New York, NY.
iii Table of Contents Pa.fi e Acknowledgements ...... 11
Table of Contents...... iv
List of Figures...... v
List of Tables ...... ix
Abstract...... xi
Chapter I - Literature Review and Objectives ...... 1
Chapter II - Experimental Ostertagia ostertagi Infection or Chronic Pentagastrin Treatment of Goat Kids: Parasitological, Clinicopathologic and Macroscopic Observations Introduction...... 29 Materials and Methods...... 31 Results ...... 39 Discussion...... 43
Chapter III - Qualitative and Quantitaive Light Microscopic Studies Following Ostertagia ostertagi Infection or Chronic Pentagastrin Treatment of Goat Kids Introduction...... 66 Materials and Methods...... 67 Results...... 73 Discussion...... • 80
Chapter IV - U1trastructural Abomasal Changes Following Ostertagia ostertagi Infection or Chronic Pentagastrin Treatment of Goat Kids Introduction...... 107 Materials and Methods ...... 108 Results...... 113 Discussion...... 119
Chapter V - Summary and Conclusions...... 147
Bibliography...... 152
Vita...... 172
Approval Sheets
iv LIST OF FIGURES Page CHAPTER II
Fig. 1 Gastrin RIA kit validation, specificity . . 56
Fig. 2 Serum pepsinogen levels of Group I control goat kids ...... 57
Fig. 3 Serum pepsinogen levels of Group III goat k i d s ...... 58
Fig. A Serum pepsinogen levels of Group II goat k i d s ...... 59
Fig. 5 Gastrin immunoreactivity of Group I control goat k i d s ...... 60
Fig. 6 Gastrin immunoreactivity of Group III goat kids ...... 61
Fig. 7 Gastrin immunoreactivity of Group II goat k i d s ...... 62
Fig. 8 Abomasa of control and Ostertagia ostertagi-infected goat kids opened along the greater curvature...... 63
Fig. 9 Abomasum of an Ostertagia ostertagi- infected goat k i d ...... 64
Fig. 10 Abomasum of a pentagastrin-treated goat kid...... 65
CHAPTER III
Fig. 1 Schematic drawing of a caprine abomasum opened along the greater curvature...... 90
Fig. 2 Non-folded fundus of a control animal .. . 91
Fig. 3 Fundic fold of a control a n i m a l ...... 92
Fig. A Non-folded fundus of a pentagastrin- treated animal...... 93
Fig. 5 Fundic fold of a pentagastrin- treated animal...... 94
v CHAPTER III Con't
Fig. 6 Non-folded fundus of an 0 ostertaRi- infected animal ......
Fig. 7 Fundic fold of an 0 ostertaRi- infected animal ......
Fig. 8 OstertaRia ostertaRi larvae in the abomasal gland of an infected goat kid . . 97
Fig. 9 A dilated abomasal gland in an 0 ostertaRi-infected Roat kid ......
Fig. 10 Herniation of an abomasal gland in an 0 ostertaRi-infected Roat kid ...... 99
Fig. 11 Fundus of the abomasum of an 0 ostertaRi-infected Roat kid . . . . ;
Fig. 12 Mononuclear cellular infiltrate in the lamina propria of the abomasum of a goat kid infected with 0 ostertaRi ......
Fig. 13 Intestinal metaplasia in the pyloric antrum of an 0 ostertaRi-infected Roat kid . . . .102
Fig. 14 Fundus of the abomasum of a control goat kid......
Fig. 15 Fundus of the abomasum of a control goat kid......
Fig. 16 Fundus of the abomasum of an 0 ostertaRi-infected Roat kid ......
Fig. 17 Fundus of the abomasum of an 0 ostertagi-infected Roat kid .....
CHAPTER IV
Fig. 1 Transmission electron micrograph (TEM) of the abomasal fundus of a control kid. . . .126
Fig. 2 TEM of the parietal cells of a control goat kid...... 127
Fig. 3 TEM of the luminal opening of a parietal cell from a control goat kid...... 128
vi Page CHAPTER IV Con't
Fig. 4 TEM of a filamentous (brush) cell from a control goat k i d ...... 129
Fig. 5 TEM of parietal cells from a pentagastrin-treated goat k i d ...... 130
Fig. 6 TEM of the luminal opening of a parietal cell from a pentagastrin-treated goat kid ...... 131
Fig. 7 TEM of a young parietal cell from a pentagastrin-treated goat k i d ...... 132
Fig. 8 TEM of the luminal surface of mucous cells from a control goat k i d ...... 133
Fig. 9 TEM of the luminal surface of mucous cells from an () ostertagi-infected goat kid . . .134
Fig. 10 TEM of a degenerate mucous cell from an 0 ostertagi-infected goat k i d ...... 135
Fig. 11 TEM of an inactive parietal cell from an 0 ostertagi-infected goat k i d ...... 136
Fig. 12 TEM of a young mucous neck cell from an 0 ostertagi-infected goat kid ...... 137
Fig. 13 TEM of a young parietal cell from an 0 ostertagi-infected goat k i d ...... 138
Fig. 14 TEM of the inflammatory infiltrate from the lamina propria of an 0 ostertagi- infected goat kid ...... 139
Fig. 15 TEM of a globule leucocyte from an 0 ostertagi-infected goat k i d ...... 140
Fig. 16 Scanning electon micrograph (SEM) of the abomasum of a control goat kid...... 141
Fig. 17 SEM of surface epithelial cells from a control goat...... 142
Fig. 18 SEM of surface epithelial cells from a pentagastrin-treated animal ...... 143
vii CHAPTER IV Con't
Fig. 19 SEM of the abomasum of an 0 ostertagi-infected goat kid ...... 144
Fig. 20 SEM of an abomasal nodule from an 0 ostertagi-infected goat k i d ...... 145
Fig. 21 SEM of the abomasum from an () ostertagi-infected goat k i d ...... 146
viii LIST OF TABLES
CHAPTER I
Table 1 The actions of gastrin...... 27
Table 2 The factors Involved with the regulation of gastrin release ...... 28
CHAPTER II
Table 1 Gastrin RIA kit validation for caprine serumy precision...... 47
Table 2 Mean total plasma protein and serum albumin concentrations of goat kids following no treatment, ostertagi infection or pentagastrin treatment...... 48
Table 3 Serum calcium and phosphorus concentrations of goat kids following no treatment, 0 ostertagi infection or pentagastrin treatment...... 49
Table 4 Erythrocyte parameters at the beginning and at the end of experiment in three treatment groups ...... 50
Table 5 Leucocyte counts of Group I control goat kids...... 51
Table 6 Leucocyte counts of Group II goat kids receiving 75 ug/kg pentagastrin BID for 28 days...... 52
Table 7 Leucocyte counts of Group III goat kids infected with 15,000 L3 of 0 ostertagi...... 53
Table 8 Necropsy findings, abomasal pH and abomasal weight as a percentage corrected body weight in goat kids following no treatment, infection with 15,000 L3 of 0 ostertagi and 75 ug/kg pentagastrin BID for 28 d a y s ...... 54
Table 9 Worm recovery from goat kids experimentally infected with 15,000 third larval stage of () ostertagi ...... 55 Page CHAPTER III
Table 1 Distribution of mucosubstances in the abomasal fundus of control goats and following chronic pentagastrin treatment or experimental 0 ostertagi infection. . . 87
Table 2 Distribution of mucosubstances in the abomasal pyloric antrum of control goats and following pentagastrin treatment or experimental 0, ostertagi infection...... 88
Table 3 Quantitative light microscopic findings in the abomasal mucosa of control goat kids and following chronic pentagastrin treatment or experimental 0 ostertagi infection ...... 89
x ABSTRACT
European-type mixed-breed goat kids were infected
with Ostertagia ostertagi or were given chronic
pentagastrin treatment. Selected clinicopathological
parameters and fecal egg counts were monitored throughout
the study. At necropsy the abomasal physical parameters were recorded and tissue samples were saved for parasite
recovery, quantitative and qualitative light microscopy
and transmission and scanning electron microscopy.
Serum pepsinogen and gastrin immunoreactivity
increased in parasitized animals. The increase was
associated with the tissue phase of the parasitic
development. Pentagastrin did not alter pepsinogen or
gastrin immunoreactivity.
All 8 infected animals had patent infection, and approximately 5% establishment of the infective dose of
15,000 0 ostertagi was achieved. The majority of the recovered parasites were adult and approximately 60% of
them were female.
At the time of necropsy the 0 ostertagi-infected animals had increased abomasal pH and the abomasa of the parasitized and the pentagastrin-treated animals had slightly increased weight. The abomasal mucosa of infected animals had umbilicated, white,raised
xi nodules as has been described in bovine ostertagiasis.
Pentagastrin-treated animals had prominent fundic folds.
The light microscopic morphology of infected goats was similar to that of the bovine disease. There was a significant increase in the abomasal mucosal thickness, primarily due to the proliferation of mucous cells. The majority of the proliferating mucous cells contained sialo- or sulfomucins. In pentagastrin-treated animals only slight thickening of the mucosa occurred.
The transmission electron microscopic findings in parasitized animals were similar to bovine ostertagiasis, although there was no striking separation of the lateral plasmalemmata. In pentagastrin-treated animals the parietal cells appeared active and had abundant intracytoplasmic canaliculi lined by long microvilli.
Scanning electron microscopy revealed marked surface alterations in hypertrophic parasitized abomasa, while control and pentagastrin treated animals had morphology similar to other mammals.
Goats infected with 0 ostertagi developed a hyper trophic abomasitis. Hormonal factors appeared to be asso ciated with the development of abomasal changes. Penta gastrin did not have a significant effect on the caprine abomasum. Slight subcellular changes and slight hyper plastic changes occured.
xii CHAPTER I
Literature Review and Objectives
Introduction
Diseases of the alimentary tract are common in domestic animals. The alimentary tract is often regarded as a single tube, thus dysfunction at any level may have an effect on the whole organ. In veterinary practice special attention is paid to the stomach when animals are ill with the clinical signs of anorexia, cachexia, hypoproteinemia, diarrhea, regurgitation or vomiting, abdominal distention, hematemesis, melena or anemia. Some infectious diseases and systemic- conditions as toxemia or uremia can also produce gastric lesions in several species. The most often encountered gastric changes are inflammatory - acute or chronic gastritis. This latter category includes hypertrophic gastropathies of various etiologies.
The purpose of this study was to investigate a proliferative gastropathy. Morphological changes in the abomasa of goats infected with Ostertagia ostertagi were quantitatively and qualitatively evaluated and were correlated with selected pathophysiological changes. A short review of parasitic and other hypertrophic gastropathies of different species is provided. In
1 2 addition, some of the pertinent information on gastric enzymes, hormones and on the kinetics of gastric mucosa is summarized.
Morphology and life cycle of the parasite
Ostertagia ostertagi was first described by von Ostertag in 1890 as a parasite of cattle.*
This parasite belongs to the class Nematoda, subclass
Secernentea, order Strongylida, superfamily
Trichostrongyloidea, family Trichostrongylidae, and subfamily Trichostrongylinae.^ There are 4 species commonly identified from domestic ruminants: 0 ostertagi.
0 lyrata. 0 circumcincta. and 0 trifurcata.
The most significant of these nematodes is 0 ostertagi O which causes significant economical losses worldwide.
The morphology of this parasite has been reviewed in detail elsewhere.*»4|5,6, The adult male is 6-7 mm long while the female is 8-9 mm long and has a characteristic hood-like cuticular flap over the vulva. The eggs measure approximately 38-44 x 74-90 um. The bursa of the male has
2 large lateral and 1 small dorsal lobes. The spicules are equally short and terminate in 2-3 distal processes.
The life cycle of this nematode is direct and is similar to that of other Trichostongylidae. Eggs are passed in the feces and develop to the infective third larval stage (L3) on pasture. The developing parasites undergo 2 molts within 7-14 days and under favorable 3
conditions the L3 migrates onto grass in order to be
taken up by the host. For optimal survival on the pasture
Ostertagia sp. requires cool and moist weather with 5 cm
or more monthly precipitate and a monthly mean tempera
ture of 6-20 C.7 Following ingestion the L3 enters the
abomasal gland and molts into the fourth larval stage
(L4) within 4-6 days. In normal development the L4 grows
and molts into the fifth (adult) stage (L5) in the next
10-15 days, and young adults emerge to the surface of the
abomasal mucosa. Due to poorly understood mechanisms the
parasite can become inhibited at the early L4 stage and
remains in the abomasal mucosa for 3-6 months before
resuming development.
The cause of hypobiosis of 0 ostertagi has been the
subject of great controversy in the past. Host factors as
well as parasite factors and the environment were O implicated in past years. This diapause-like phenomenon
was generally considered to be primarily due to environ
mental factors, and could be experimentally induced by
chilling of larvae in northern temperate regions.^ In
subtropical climates, such as Louisiana and regions of
Australia larval inhibition was related to the increasing
temperature of the late spring months.*®’** Studies
utilizing different populations of 0 ostertagi
demonstrated that progeny of previously arrested worms
underwent hypobiosis more readily than those whose development had not been interrupted.^ Additionally, in animals with previous exposure to infection, an increased proportion of worms became inhibited in development.12,14
Bovine ostertagiasis
Bovine ostertagiasis is classified into 3 syndromes.
Type I ostertagiasis is an acute parasitic gastritis, most severely affecting yearling cattle. This form of the disease is characterized by high morbidity and low mortality, and is due to the normal development of the parasite with a prepatent period of 18-21 days. The major clinical manifestations are weight loss and severe diarrhea. It occurs most often in northern temperate climates in July-September, while in subtropical climates, with a winter rain fall,it occures in March-
May. Grossly it is characterized by oval or circular greyish white, raised, umbilicated nodules which persist for some weeks after larvae have emerged from the abomasal glands. In addition, the abomasal folds are congested and edematous in the Type I disease.
In pre-Type II disease animals harbour a large number of inhibited 0 ostertagi L4 and varying, usually low, numbers of other developmental stages. In some animals adult parasite can be present in sufficient numbers to produce mild clinical signs such as stunted growth or mild diarrhea, but in the majority of the cases animals 5 with this type of ostertagiasis are only subclinically affected.
Type II ostertagiasis is due to the sequential deve lopment and emergence of large numbers of inhibited larvae.
Clinically^ this syndrome is characterized by weight loss, severe diarrhea and submandibular edema. In Type II disease syndrome the abomasum can exhibit all the macroscopic lesions of Type I ostertagiasis simultaneous ly. Type II ostertagiasis is characterized by low morbi dity and high mortality in March-May in Great Britain and in September-Noveraber in Louisiana.*®’*® Epidemiologic studies conducted in Australia showed similar patterns to those of Louisiana. Inhibited larval development in
Australia and Louisiana occurs in the spring which precedes less favorable climate for the parasite.
In Type I ostertagiasis the first microscopic changes are characterized by dilation of glands containing larvae.*® The epithelium of the affected gland loses its mature differentiation and is lined by undifferen tiated cells. Large numbers of parietal cells are lost in this process. Hyperplastic changes are also present in neighboring, non-parasitized glands.*^ Simultaneously with the glandular changes, a mild cellular inflammatory reaction of mononuclear cells, neutrophils and eosinophils develops focally around infected glands. The inflammatory changes are associated with the development of edema.^ After maturation of larvae the parasitized glands can be recognized by flattened epithelium or hyperplastic epithelial papillary projections which are occasionally formed in cyst-like glands. Globule leuco cytes appear at this stage of the disease primarily in superficial areas of the glands between epithelial cells.
Globule leucocytes and plasma cell foci remain in the abomasal mucosa even when glandular changes are no 1 6 longer evident.
Histochemical studies in calves following a single inoculation with £ ostertagi demonstrated decreased carbonic anhydrase activity suggestive of a loss of
I Q parietal cells or parietal cell function. In addition, chief cells had decreased numbers of zymogen granules.
Another finding in nonenzymic histochemistry was an increase of structural fibers around parasitized glands.
Increased collagen accompanied by fibroblasts and an increased amount of reticulin fibers were demonstrated by 20 special stains. w
Intramucosal migration of larvae has been described in the pre-Type II syndrome.Nematodes located in the stroma of the mucosa attracted only a mild inflammatory reaction.
In the Type II syndrome irregularity in mucosal thickness was produced by variation of the length of the abomasal glands.While individual glands showed 7 similar changes in Type II disease as in Type I ostertagiasis, the inflammatory response was somewhat different. Usually in the outer third of the lamina propria large numbers of plasma cells and lymphocytes were present. Lymphocytes formed numerous lymphoid follicles and replaced glandular structures. Hyperplasia of globule leukocytes was also prominent. Congestion, edema and in some cases fibrosis also contributed to the increased wall thickness of the abomasum.
U1 trastructurally in Type I as well as in Type II' ostertagiasis the most significant change was the separa tion of the lateral plasmalemmata which form the zonula occludens.^*^ The widened intercellular zone contained an electron opaque amorphous material which was consi dered extravascular plasma protein. Parasitized glands were lined by tall mucus-secreting cells and some mature differentiated cells which exhibited degenerative changes.
Parietal cells had lost their intracellular canaliculi or had dilated canaliculi with short microvil 1 i.23,24
Zymogen cells had shrunken nuclei and a reduced number and smaller granules or granules that had lost their membrane.*^'^’^ In addition, the microcirculation of IQ 2*? 9L the abomasal mucosa was also damaged. Junctional complexes of endothelial cells of postcapillary venules
OO were separated. J
A proposed pathogenesis of ostertagiasis was based on ultrastructural and light microscopic studies. 15,16,23
After infection large numbers of parietal cells are lost in parasitized glands. The parietal cells incorporated in the stretched epithelium lose their microvilli and are unable to secrete HC1. Due to the increased abomasal pH pepsinogen is not activated and as a consequence dietary proteins are not denatured for digestion. There is also an increase in the bacterial flora due to the decreased bactericidal effects of the abomasal juice. In addition, the junctional complexes of undifferentiated and hyper plastic cells are not fully developed and remain partial ly or completely open.^’^ Increased permeability of the abomasal wall permits macromolecules, such as pepsinogen, to enter into the circulation from abomasal lumen. At the same time circulating plasma proteins, primarily albumin, are lost via the same route. The exchange of macromolecules produces the characteristic clinical pathological abnormalities of ostertagiasis: increased plasma pepsinogen levels and hypoalbuminemia. Maldiges tion is probably due to increased abomasal pH and the altered bacterial flora which cause the development of diarrhea. Maldigestion and diarrhea accompanied by the loss of appetite of unknown reasons are responsible for the weight loss of infected animals. A possible mechanism for the development of anorexia is an increase in the circulating levels of one of the gastrointestinal hor- 9
mones of the cholecystokinin family. In addition,
duodenal osmoreceptors can be important in the develop
ment of satiety signals.2^ In an interesting experiment a
cimetidine-like affect of an 0 ostertagi extract was 9 demonstrated in rat stomach. J Decreased HC1 secretion in
the stomachs of rats intramuscularly injected with the
extract was suggestive of a parasite factor which could
inhibit acid secretion by parietal cells and would thus
enable the parasite to survive in the abomasum. In
contrast, a 70% reduction in the adult ,0 circumcincta
population in sheep occurred following the intra-abomasal
administration of cimetidine, which increased abomasal
pH.26
Ostertagiasis of small ruminants
The parasitic infections of sheep and goats are often
discussed as one, although there are ample examples of
differences between parasitic diseases of the two species.
Naturally occuring incidental infections with
0 ostertagi were reported in the sheep as well as in
the goat.2^ Experimental inoculation of 0 ostertagi 28 failed to produce patent infection in sheep. ° In a more
recent study a surprisingly high level of establishment was attributed to the adaptation of the strain to sheep, and was explained by a common practice of mixed grazing of
cattle and sheep in New Zealand.2^ In another experiment
exposure of lambs to 0 ostertagi provided some protection 10 against challenge with 0 circumcincta in that the worm
counts were markedly reduced without an increase in the number of arrested larvae.Similarly there are conflicting data about caprine ostertagiasis, while
the Angora goat was found to be a suitable host for 0 ostertagi. only negligible establishment of 0 ostertagi occurred in mixed-breed goat.^’^*Experiments with
European-type mixed-breed goats supported the observa
tions that the goat can serve as a model of bovine
2 7 ostertagiasis.
In some geographic locations eg. Scotland,
Northwest US, primarily where the winter is wet and cold,
0 circumcincta is the most important abomasal parasite of sheep.33,34 Qstertagia circumcincta produces similar oc disease syndromes as _0 ostertagi in the cattle.
Morphological changes were similar to those of bovine ostertagiasis.^ The ovine form differed from the bovine disease in that degeneration of parietal cells and their replacement by proliferative cells occurred in a wider zone around the parasitized gland, and was probably due to the greater pressure exerted by the larger ovine q q species. J
The importance of naturally occurring i0 circumcincta infections prompted some elegant experimental work with much significance to the basic understanding of parasitic diseases. Investigations in Scotland have focused on the 11 local immune response of sheep infected with Ostertaeia circumcincta.*^"^ A surgical technique was developed for chronic cannulation of a major lymph duct of the abomasum.^ Increased IgA production, an increased out flow of lymphoblasts and eosinophils were found in experimentally infected animals.38*39 increase(j immUne response and resistance to the parasite were found in older animals after challenge with a higher inoculum. ^
In Australia the endocrine aspect of the Ostertaeia sp. infection was investigated. An early increase in gast rin was associated with the presence of parasites in the abomasum and a later increase was attributed to increased abomasal p H . ^ * ^ » ^ Animals were equipped with abomasal cannulae and cannulated abomasal pouches which allowed the opportunity to compare acid secretion in parasitized and non-parasitized areas of the abomasa. The hyper- gastrinemia produced increased acid secretion and parietal cell hyperplasia in the pouch, while parietal cells in the parasitized "abomasum proper" were inactive.
It was assumed that a locally-produced, probably parasite factor was responsible for the decreased acid secretion in the parasitized region of the abomasum. The relationship of hypergastrinemia and abomasal pH was further investi gated when adult Ostertagia spp. were transferred into the abomasa of sheep.In this experiment the physical 12 presence of parasites produced hypergastrinemia and increased pepsinogen levels before changes in the abomasal pH. Changes in plasma pancreatic polypeptide were also evaluated, which varied widely, but had the general tendency to be decreased following in fection. This latter gastrointestinal hormone was of interest because it is stimulated by similar factors as is gastrin.The extra-abomasal effects of ovine ostertagiasis were also investigated. Osteoporosis in sheep was attributed to secondary deficiencies of Ca and of energy and protein.^
Pepsinogen
Pepsinogen is the inactive form of the gastric acid protease, pepsin. It is secreted by the chief cells of the fundus of the glandular stomachs of animals. Pepsinogen is also produced in the mucosa of the antrum and the proximal duodenum.^ Pepsinogen is activated by the cleavage of
40-50 residues from the amino end at or below pH 2 or by pepsin.
There are 4 immunochemically distinct carboxy proteases in man and animals.48,49,50 These proteases are characterized by 2 aspartic acid residues in their active center hence the new name, aspartic protease. Pepsinogen A in animals is homologous with pep sinogen I in man and is considered to be the major pepsi nogen. Pepsinogen C of animals is the equivalent of pepsi- 13 nogen II of man or gastricsinogen and progastricsin. The non-pepsinogen acid protease or slow-moving protease in man probably corresponds to pepsinogen B in animals. The fourth group of acid proteases is the chymosin or rennin group, which is present in neonatal mammals with postnatal uptake of immunoglobulins. In addition to the 4 major groups several isoenzymes have been identified.
It is of interest that members of the aspartic protease
"superfamily" have been found in almost every eukaryotic cell and they share about 10% of their aminoacid residues. Moreover, when the single peptide chain of pepsinogens and prochymosins of different animal species were compared about 70-80% of the approximately 375 amino acid residues were found at identical positions in different species. Between different groups only about
45% of the residues were located at identical positions, within the same animal species.
Several neurogenic and hormonal factors have been identified which stimulate pepsinogen secretion.
Pepsinogens are secreted into the gastric lumen and are also released into the circulation. Vagal stimulation, histamine, gastrin and secretin stimulate gastric pepsin secretion, while somatostatin is inhibitory of both exocrine and endocrine secretion. In addition, it has ei co been-demonstrated in vivo and in vitro that synthesis and secretion of pepsinogens can be inhibited or stimu- 1A lated selectively.
Over the years several techniques have been developed to determine pepsinogen in bloody from simple digestion methods"*^'**^ through an agar gel diffusion technique'*'* to radioimmunoassay (RIA).^ The determina tion of serum or plasma pepsinogen is a useful diagnostic tool in human and veterinary medicine, although it is still not fully understood how pepsinogen enters the circulation. Pepsinogen levels in serum and plasma were found to be the same.~*^ Pepsinogen levels in the human have been correlated with different types of gastritis,
CO CO C7 gastric metaplasia and malignancies. The hyperpepsinogenemia in the Zollinger-Ellison syndrome was attributed to the continuous stimulation by endogenous gastrin.^ While a good correlation was found between serum pepsinogen I and gastric acid secretion capacity of the stomach in man, in bovine ostertagiasis increased pepsinogen levels were found in animals with increased abomasal pH.^»^®»^ In ostertagiasis an inverse correlation was found between pepsinogen levels and weight gain as well as a positive correlation between pasture contamination and pepsinogen levels.59,60
Gastrin
Gastrointestinal hormones are produced by the amine- precursor-uptake decarboxylase (APUD) cells, which originate in the neural crest of the embryo. Gastrin is 15
produced by 6 cells which are primarily located in the
pyloric antrum.
Gastrin shares a common carboxy terminal pentapeptide with cholecystokinin (CCK), and these hormones are
considered to belong to the same family of peptides. CCK is
found in the intestine, ranging in size from 8 to 39
residues, and is considered to be the more primitive pep
tide of the 2 hormones, evolving from a common ancestral
gene.*’3-
Gastrin exists in different amino acid chain lengths
and each form can be sulfated or non-sulfated at a single
tyrosine residue. Gastrin peptides range in size from
14 to 3A amino acid residues. The most active and common
peptides have 17 or 34 residues, but 2 larger molecules big
big gastrin (G60) and component I (G43) have been identi
fied. The active site of the molecule is the C terminal
pentapeptide. The minor changes found in the 2 major
gastrin molecules of different mammalian species are
located in the non-biologically active parts of the mole cules and represent single base changes in the DNA.^*
The primary function of gastrin is the stimulation of
gastric acid secretion, but in large doses it acts as a
growth stimulating hormone and causes an increase in RNA,
DNA and protein synthesis of target cel Is.62,63,64
Gastrin has multiple effects on the major secretory, absorptive and smooth muscle activity of the digestive 16 tract with trophic effect on the gastric and intestinal mucosa and pancreas (Table 1). The target cell in the stomach is the parietal cell which is the source of HC1.
The high affinity gastrin receptors were found on the parietal cell surface and were copurified with the adenylate cyclase system which in turn is an important factor in acid secretion.^ All the mechanisms to inhibit or stimulate the release of gastrin operate on G cells (Table 2). Gastrin release is inhibited by a simple negative feed-back mechanism. For maximal suppression a pH of 1 is needed. Long-term hypoacidity causes G cell hyperplasia as well as increased basal gastrin levels.
Hypergastrinemia was achieved in human subjects when 66 gastric pH was maintaned at pH 6-7 for 5 hours. °
Recently the possible mechanism of meal-induced gastrin release was reported. It was proposed that dietary aminoacids stimulate gastrin release after they are transported into G cells and converted into amines by 66 the decarboxylase enzymes of the cell.0 In this system monoamine oxidase type A was found to have a regulatory role. It was suggested that this mechanism can also serve as an intracellular regulator of hormone secretion in other APUD cells.^
Another interesting aspect of the stimulation of gastrin release was discovered with the isolation of a number of related peptides from the skin of various araphi- bians.®® This family of peptides was named bombesin
after the first tetradekapeptide isolated from a European
dicoglossid frog, Bombina bombina.^® Bombesin stimulated
gastric acid secretion in dogs through the release of
gastrin. Hyperplasia of 6 cells was produced in rats
by the administration of bombesin.®^ Bombesin-like
peptides have been isolated from the gastro-intestinal
extracts of mammals and birds. In addition, relatively
large concentrations of bombesin-like immunoreactivity
was demonstrated in the hypothalamus of several
mammals.^® Most of the gastrin is metabolized by the
kidneys, and human patients with extensive kidney damage
have increased basal gastrin levels.
Pentagastrin
Pentagastrin is the synthetic butyl oxycarbonyl
derivative of the active site of gastrin molecule with
7 9 qualitatively identical physiological activity.
Hypertrophic gastritis with parietal cell hyperplasia was
produced by the chronic administration of pharmacological
levels of pentagastrin to rats.^® In quantitative
electron microscopic studies the mean parietal cell
volume increased following treatment, but the subcellular morphology was similar in treated and in control animals.^ In a similar experiment the trophic effect of
pentagastrin was eveluated on normal and regenerating
parietal cells of rats.^®*^® Long-term treatment with 18 pentagastrin at doses that maximally stimulated DNA synthesis did not affect the ultrastructure of parietal cells in the normal mucosa. It was inferred that pentagastrin and gastrin increased the proportion of 76 77 progenitor cells to develop into parietal cells. ’ In an interesting study on the effect of chronic pentagast rin treatment on rat pancreas an increased pancreatic mass was due to increased protein content without a
7fi corresponding increase in specific enzyme activity.
Increased gastrin immunoreactivity was found in mice following chronic high dose treatment with pentagast rin.™
Hypertrophic gastropathies
Hypertrophic gastritises of various etiologies are the most commonly encountered chronic gastritises in human and veterinary medicine. An increase in circulating gastrin levels has been described in human and in canine patients with non-beta-cell tumors arising in the pancreas.®®’®* The Zollinger-Ellison syndrome or gastrinoma is characterized by gastric acid hypersecre tion with hypertrophic gastritis secondary to the hyper- gastrinemia. In these patients an increase in the parie tal cell population was described. Hypertrophic gastritis of unknown etiology has been also reported in several animal species. A syndrome was described in Basenji and
Boxer dogs 82-86 was similar morphologically to 19
Menetrler's disease of man. Affected stomachs were
characterized by superficial gastritis, hypertrophy and
hyperplasia of the mucous glands and the presence of
cystic glands in the submucosa. Clinically this disease
was characterized by hyposecretion or hypersecretion of
acid and protein loss into the gut. In dogs as in human
beings males appear to be affected more
frequently.®^*®^’®^ In a human patient hypergastrinemia
and transient appearance of antibodies to parietal cells
were found.U1trastructural studies revealed widening
of gastric tight junctions in patients with Menetrier's
OO disease when compared to healthy controls. A morpho
logically similar gastric hyperplasia was also described
in mice. A number of factors were identified in the
DQ development of gastric mucosal hyperplasia in mice. 7
Certain strains of mice spontaneously develop similar
lesions with aging. In other cases long-term crowded
housing produced high incidence of gastropathies. Giant hypertrophic gastritis developed in some strains of mice
following thymectomy at an early age.^O It was also
suggested that the nutritional state of animals could be
important. Affected stomachs had an increase in the
sulfomucin secretion suggesting an incomplete maturation
QQ of mucous cells. 7
Other parasitic gastropathies
In addition to ostertagiasis other parasites in 20 various animals have been associated with gastric lesions.
Sheep infected with Camelostrongylus mentulatus. a gastric parasite of camels and gazelles, exhibited similar abomasal changes to those described in bovine ostertagiasis.^ This parasite produced a hypertrophic abomasitis with antral metaplasia and an increase in the abomasal pH. In addition, plasma pepsinogen was elevated in infected lambs.
A nodular abomasitis due to the trichostrongylid nematode Longistrongylus sable was identified in irapala lambs in the Republic of South Africa.^ This infection had minimal significance to impala herds. The nodular lesions macroscopically were larger than those of oster tagiasis in domestic ruminants. Microscopical changes were similar to that of ostertagiasis, although this parasite deposited eggs in the dilated abomasal glands.
01 lulanus tricuspis. a minute, viviparous gastric parasite of felines has been recognized in the US with a limited geographic distribution. This parasite was pri marily reported from catteries in the Pacific Northwest and Midwest.93,94 tp^g most important microscopic features of the infection were the marked fibrosis in the lamina propria, the development of lymphoid follicles and the proliferation of globule leucocytes. The life cycle of this parasite is unusual in that the primary route of infection is the consumption of vomitus containing
larvae.^
Nochtia nochti. a gastric nematode of primates
produced plaques of gastric hypertrophy, most often at
the border of the fundus and antrum. The gastric mucosa
exhibited papillomatous thickening with occasional
penetration and cystic dilation of glands through the muscularis mucosae.
Trichostrongylus axei infects the stomach of horses
in addition to the abomasa of ruminants. Infection
with this parasite is characterized by mucous cell meta
plasia and hyperplasia. An inflammatory component has
been also described following infection with T axei. ^
The inflammatory reaction was primarily found in the
superficial lamina propria and was composed of eosino
phils and lymphocytes. Animals had an increased abomasal
pH, alterations in the electrolyte concetrations of the
abomasal fluid and hypoalbuminemia.^ It was concluded
that the diarrhea of infected calves was due to the entry of the abnormal abomasal fluid into the small 07 intestine. '
Trichostrongylus colubriformis establishes itself mainly in the first 3 meters of the small intestine of
sheep. In addition to the villous atrophy in the small intestine this parasite also produces structural changes go the abomasa of infected sheep.70 Parietal cells 22
. appeared inactive and degenerate and although there was no
evidence of massive hyperplasia, the mucous neck cells
extended deeper into the abomasal fundic glands than in the
uninfected controls. Infected animals had an increased
abomasal pH and hyperpepsinogenemia. The pathogenesis of
these changes could have an endocrine etiology, but it
remains unidentified.^
Increased gastrin levels have been reported in
parasitic infections of species other than ruminants. In
intestinal parasitism due to Trichinella spiralis
in the rat and Strongyloides ransomi in the pig an acute
increase of gastrin was described.99,100 wag suggested
that intestinal parasites caused an increase in the
release of a bombesin-like peptide which in turn in
creased gastrin levels.^ Increased gastrin levels were
also found in rats after chronic infection with a liver
parasite, Taenia taeniaeformis.^* Proliferative changes
were noted in the mucosa of the stomach and small intes
tine of infected animals. The development of larvae not
only stimulated the mitotic activity of the stomach
mucosa, but also interfered with the normal differentia- 1 02 tion into chief and parietal cells. In the stomach
mucous cells replaced the parietal and the chief cells.
Most of the mucous epithelial cells in affected stomachs
produced neutral mucin.In addition to the increased
mucosal thickness in the intestine, an increased number 23 of mast cells and eosinophils was present in the lamina propria of the duodenum.
Gastric histology and kinetics.
In the glandular stomach of animals 2 major areas can be easily identified. The gastric body and fundus is covered by fundic mucosa, while antral-type mucosa is in the antrum and pylorus. In most species the cardia is represented by only a narrow band of mucosa. Mucus secreting cells line the surface of the gastric mucosa and these cells extend into foveolae. The convoluted gastric glands, which open into gastric pits, are lined by acid and intrinsic factor secreting parietal cells and pepsinogen producing chief cells. In the pyloric antrum beneath the surface mucous cells antral gland cells are present, which in addition to mucus granules also contain pepsinogens.A new cell type has been described recently in the fundus as well as in the pyloric antrum of several species. This cell has been named the fibrillovesicular, filament-containing or tuft cell and is characterized by short, bushy microvilli and intracytoplasmic filaments. 104,105,106 jn every level of the stomach several types of endocrine cells are present.107-111
The proliferative zone of the gastric mucosa is 112 located in the isthmus and neck regions. Cells migrate upward from this area to replace surface mucous cells. 24
The renewal of cells varies by location and species, but averages between 2-6 days. In normal conditions surface epithelial cells are extruded from the mucosa, while in stressed stomachs cells more frequently degenerate in situ.**^ The renewal of the glandular portion is slower and the origin of parietal and chief cells is controversial. Parietal cells most likely originate from undifferentiated stem cells and are not able to divide further. In contrast, chief cells may be able to divide at a slow rate, about once a month in mice.^®^ Experimental work with stomach grafts demonstrated totipotential cells which were able to differentiate into mature mucous, parietal, chief and argyrophil cells. 114,115
Objectives.
The objectives of this study were:
1. To produce and characterize 0 ostertagi infection in
goats.
Nematode free 2-3 month old European-type mixed-breed goats were inoculated with 15,000 third larval stage of
Ostertagia ostertagi. Selected clinicopathological parameters and fecal egg counts were monitored throughout the study. At the time of necropsy the abomasal pH was measured and abomasa were weighed. One half of each of the abomasa was saved for.parasite recovery, while the other half was used for light and electron microscopic studies. 25
A stratified random sampling technique was used for the
light microscopic morphometry. Selected sites were
embedded in plastic and stained with a modified Zimmerman
stain. Mucosal thickness and the number of mucous
epithelial, zymogen and parietal cells were determined.
Cell volumes were estimated by the perimeter of cellular
fragments. Furthermore a section from each abomasum was
taken from the folded and non-folded fundus and pyloric antrum. These paraffin embedded sections were stained with Mayer's hematoxylin-eosin and special stains for mucosubstances and argyrophil granules. Additional small
pieces were processed for transmission and scanning electron microscopy.
2. To determine whether or not hypergastrinemia
develops in 0 ostertagi infected goats.
A commercial gastrin radioimmunoassay kit was validated for goats. Gastrin immunoreactivity was determined semiweekly.
3. To characterize mucosal changes induced by exogenous
gastrin (pentagastrin) in goats.
Nematode free 2-3 month old European-type mixed-breed goats received 75 ug/kg pentagastrin for 28 days every 12 hours. For the characterization of the treatment effect the same techniques were utilized as described in objectives 1&2.
4. To compare changes observed in the abomasa of
parasitized and pentagastrin-treated goats. Proliferative changes in the 2 treatment groups were compared with qualitative and quantitative light microscopy. The cell populations of the 2 groups were also compared on the ultrastructural level. Table 1 - The actions of gastrin*
WATER & ELECTROLYTE SECRETION
Stomach, Pancreas, Liver
Small intestine, Brunner’s glands
ENZYME SECRETION
Stomach, Pancreas, Small intestine
INHIBITION OF WATER, ELECTROLYTE & GLUCOSE ABSORPTION
Small intestine
STIMULATION OF SMOOTH MUSCLE
Lower esophageal sphincter
Stomach, Small intestine, Colon
Gall bladder
INHIBITION OF SMOOTH MUSCLE
Pyloric sphincter, Ileo-cecal sphincter
Sphincter of Oddi
RELEASE OF HORMONES
Insulin, Calcitonin
INCREASE IN BLOOD FLOW
Stomach, Small intestine, Pancreas
TROPHIC ACTION
Gastric mucosa, Small bowel mucosa
Pancreas
* After J.H. Walsh63 Table 2 - The factors Involved with the regulation of gastrin release*
Stimulation
Luminal stimulants
PEPTIDES & AMINOACIDS**
DISTENTION (acting through reflexes)
Neural stimulants
VAGAL CHOLINERGIC (Initiated from head or
stomach)
Blood borne stimulants
Calcium, Epinephrln
Inhibition
Luminal inhibitors
ACID
Blood borne inhibitors
Secretin, Glucagon
Gastric inhibitory peptide
Vasoactive intestinal peptide
Calcitonin
* After J.H. Walsh63
** Factors thought to operate under physiologic
conditions are capitalized CHAPTER II
Experimental Ostertagia ostertagi Infection or
Chronic Pentagastrin Treatment of Goat Kids:
Parasitological, Clinicopathologic and Macroscopic
Observations
Introduction
Clinical signs, gross and selected clinico- pathological observations have been described for cattle and goats infected with _0 ostertagi and for sheep infected with () circumcincta.*^,^ ,^ ,^
Three clinicopathological syndromes of the disease were recognised in cattle and in. sheep. While in the pre-Type II syndrome animals had intermittent diarrhea, both the Type I and Type II syndromes were characterized by diarrhea and weight loss. Moderate anemia, hypoalbuminemia and submandibular edema were primarily D described in the Type II syndrome.
Type I ostertagiasis is a typical parasitic gastritis and was experimentally produced by a single dose of 0 ostertagi in cattle.*6,22 patent infections in cattle developed within 18-21 days postinfection. Goats infected with 0 ostertagi developed patency later than cattle, but passed eggs for an extended time.^ When goats of various ages were infected it was noted that patent infection
29 30 developed in 85% of the animals.
Selected clinicopathological parameters were also evaluated in 0, ostertagi infected animals. No change was found in the packed cell volume, hemoglobin concent ration, white and red blood cell counts, total serum protein or albumin concentration in calves following infection with a single dose of 100,000 0 ostertagi larvae.Ostertagiasis in all three ruminant species was characterized by increased plasma pepsinogen levels.24,27 jn addition, increased plasma gastrin was detected in sheep infected with 0 circumcincta. The first rise in the gastrin level was due to the presence of parasites in the abomasum while a second increase was attributed to abomasal achlohydria.^*^*^
Five main features of gross lesion have been described in Type I ostertagiasis in a field study*^ and in the experimental disease their development was followed from the 2nd to the 70th or 90th postinfection day.^*^ Nodules were most commonly found in the fundus, between or on the fundic folds. These nodules were oval to circular, greyish white and umbilicated. Especially in field cases when severe clinical signs were present, diffuse irregular hyperplasia produced the so called
"morocco leather" or "crazy paving effect". Another type of lesion in heavy infection was described as "thumbprint lesion" and was due to large areas of surface erosion. 31
Edema and congestion were also common findings. In addi tion to the abomasal lesions marked reactive changes were described in the regional lymph nodes.
Pentagastrin, the synthetic pentapeptide of the active C terminal of gastrin, did not have any effect on clinicopathological parameters in laboratory animals.^ Long-term treatment with pharmacological levels of pentagastrin produced gastric and pancreatic hyperplasia.73,78 Pentagastrin also caused an increase in gastrin levels in nude mice.^
The objectives of this study were to characterize the most important clinicopathological and gross changes in goats following a single inoculum of 0 ostertagi and chronic pentagastrin treatment and to compare the changes in the 2 treatment groups. This experiment also gave the opportunity to evaluate the goat as an inexpensive model for bovine ostertagiasis.
Materials and Methods.
Experimental animals— Experimental animals originated from a European type mixed-breed goat herd maintained on a parasite-contaminated pasture. Kids were born within a 1 month time period and were removed from the pasture with their does within 48 hours after birth. A brick building served as housing for kids and does. No other ruminants were kept in this building during the 32
experiment. The wood-shaving bedding was changed weekly.
Animals were fed with a commercial feed, composed of 54%
cotton seed hulls, 14% cotton seed meal, 24% cracked
corn, 5% cane molasses, 0.5% oyster shell flour, 0.4%
trace minerals. Alfalfa hay was also offered at feeding
time, and water and salt were available ad libitum. Kids
were weaned at five weeks of age.
Experimental Design— Eighteen goat kids free of
gastrointestinal parasites were selected for
the experiment one week before inoculation. The 2 to 3-
raonth-old mixed-breed goats were randomly divided into
three treatment groups. Five kids in Group I did not
receive treatment and five animals in Group II received
75 ug/kg pentagastrin twice daily for 28 days, starting
on the seventh experimental day. The third group
consisting of eight kids (Group III) were infected with
15,000 third larval stage (L3) of Ostertagia ostertagi.
Inoculation— The parasite inoculum originated from a
bovine isolate of 0 ostertagi in Louisiana and was cul
tured using standard techniques. Briefly, fresh feces was mixed with vermiculite to form a crumbly, moist mass, and cultures were maintained at room temperature for two weeks. A Baermann funnel was filled with tepid water and the feces-vermiculate mixture was immersed in the water.
This system was left undisturbed overnight. In the morning the larvae were collected in a large beaker and 33
were washed several times and stored in the refrigerator
at 4 C. Larvae were examined for viability and 15,000 0
ostertaei L3 were placed into separate tubes. Animals
were inoculated per os using disposable plastic pipettes.
Pentagastrin (Peptavlon, Ayerst Laboratories, New
York, NY) solution was prepared weekly with dimethyl
sulfoxide (DMSO) and deionized water. Animals were injec
ted subcutaneously with 75 ug/kg pentagastrin every 12
hours for 28 days, beginning on experimental day 7.
Necropsy procedures— Animals were euthanized b y .
electrocution on the 35th and 36th postinfection day.
The kids were weighed and the forestomachs and abomasa
were quickly removed from the carcasses. The corrected
body weight was determined by substracting the weight of
the forestomachs from the total body weight. A small
opening was cut in the wall of the abomasum and the pH of
the contents was measured. The abomasum was opened along
the greater curvature, its contents were removed and the mucosa was gently washed with phosphate buffered saline
solution (PBSS) pH 7.1 at room temperature to remove
adhered particles. Care was taken to use only a small amount of washing fluid which was saved with the abomasal
contents. The weight of the abomasum was recorded, and the abomasum was cut in half along the lesser curvature. One- half of the abomasum was processed for parasite recovery and the other half was slightly stretched and pinned to a 34 styrofoam board and placed into fixative. Abomasal lymph nodes were examined, removed and and saved in fixative.
All major visceral organs were examined with special attention to endocrine organs and tissue sections were saved in fixative. The brains of selected animals were also collected. All gross observations were recorded.
Clinical pathology— Blood samples were collected before feeding in the morning by jugular venipuncture.
Once-a-week samples were collected into tubes containing dipotassium ethylenediaminetetra-acetate (EDTA) and semiweekly into siliconized glass tubes without anticoagulant. A complete blood count (CBC) was deter mined on the first and last weeks of the experiment while white cell count (WBC), WBC differential, packed cell volume (PCV), and hemoglobin concentration (HGB) were determined weekly. Reticulocyte counts were performed on week 1 and week 6 of the experiment. Total plasma protein and serum albumin, calcium and phosphorus levels were also determined weekly, using standard methods.
Serum gastrin immunoreactivity and pepsinogen were determined semiweekly. Erythrocytes were counted with a Coulter ZBI and WBC and HGB were obtained using a
Coulter S790 (Coulter Electronics , Hialeah, FL). PCV was determined by the microhematocrit method. Total plasma protein was measured by refractometry. Serum albumin, calcium and phosphorus were determined with Baker Encore 35 reagents (Baker Chemicals, Pleasantville, NY).
Gastrin immunoreactivity was determined using a gastrin radioimmunoassay (RIA) kit (Cambridge Medical
Diagnostics Inc., Billerica, MA) which was based on a double antibody technique. Serum was stored at -70 C prior to analysis. All serum samples from any given animal were tested at the same time to eliminate inter assay variation. The principle of the test was based on the binding of hormone antibody with tracer antigen and the inhibition of this reaction by the native antigen of the sample. This test used two methods to separate the bound and free radiolabeled antigen. A second antibody was directed against the original antiserum and in addi tion, antigen-antibody complexes were chemically insolu bilized with polyethylene glycol. The quantity of unlabeled antigen in unknown samples was determined by coraparision of radioactivity of the precipitate with standards in the same assay system. The gastrin RIA kit
IOC contained rabbit antigastrin antiserum and labeled human synthetic gastrin, the second antibody was an anti rabbit IgG produced in goats. Test tubes were counted for one minute in an automatic gamma counting system (Searl
Analytic Inc., Des Plaines, 111). This kit was validated for the goat following the guidelines published earlier.Intra-assay precision was determined on 2 caprine serum samples in 10 replicates in the same assay 36 system. In the inter-assay precision test 3 goat samples were run on 10 test days. For the validation of the gastrin RIA kit, goat serum samples with normal and moderately elevated gastrin immunoreactivity levels were selected. Specificity of the kit was determined by the demonstration of dilutional parallelism between 3 goat samples and the synthetic human gastrin standards, using the buffer furnished in the kit (Fig. 1). The recovery rates of diluted caprine sera were 91%, 129% and 166% at
1:2, 1:4 and 1:8 dilutions respectively. Sensitivity was estimated by the 95% confidence interval of the zero standard and was calculated from 10 replicate runs of the zero standard. The kit had a sensitivity of 25.96 pg/ml of gastrin immunoreactivity. Results for precision validation are summarized (Table 1).
Serum pepsinogen levels were determined twice-a-week using a modification of previously published techniques.^3,54 jn this test pepsinogen was activated by acid and the acid soluble tyrosine-like products of digestion were determined by a colorimetric technique, using diluted Folin-Ciocalteau reagent. From each serum sample 0.5 ml was placed into appropriately labeled centrifuge tubes and into a beaker marked ''pool". To this latter tube 2 ml of 10% trichloroacetic acid (TCA) was added and mixed well, making sure that the resultant precipitate did not adhere to the sides of the tube. 37
Hydrochloric acid (2.5 ml 0.06 N HCL) was added to all tubes and were incubated at 37 C for 3 hours. After incubation 2 ml of 10% TCA was added to all centrifuge tubes, except "pool" and were mixed carefully. All samples were centrifuged at 1000 g for 10 minutes. After centrifugation 2 ml of the supernatant from each sample and the "pool" were removed making sure that the precipitated protein did not mix with the supernatant.
Standard tyrosine solution was prepared adding 0.18 g
L-tyrosine to 100 ml 0.06 N HCL, from this solution 1 ml is added to a final volume of 100 ml deionized water. The
"standard curve" stock solution contained 0.2 g L-tyrosine in 100 ml 0.06 N HCL. From this solution 10, 20, 30, and
40 ug/rnl solutions were freshly prepared by adding 0.5,
1, 1.5,and 2 ml of stock solution respectively to final volumes of 100 ml with deionized water. At this point additional tubes were labeled and prepared as follows:
"blank" tube contained 2 ml of deionized water,
"standard" tube contained 2 ml of tyrosine standard, and
"standard curve" tubes were prepared by placing 2 ml of the appropriate standard curve solution into tubes. Four ml of 0.5 N sodium hydrozide was added to all tubes.
Finally 1 ml of freshly diluted Folin-Ciocalteau reagent was added to each tube and mixed well. The phenol reagent was prepared with equal volume of deionized water. All samples were read between 3 and 20 minutes in a linear 38 absorbance spectrophotometer (Coleman Instruments
Division, Oak Brook,111) at 560 nm. Using the following formula the international units of pepsinogen were calculated:
IU ° 5 .5(X-P) S
X a optical density of any particular sample
P a optical density of pooled samples
S a optical density of standard tyrosine solution
Parasitology— Rectal fecal samples were collected from the goats at weekly intervals. Fecal egg counts were performed by a centrifugation-floatation technique using sucrose solution (SP6R 1.2). One g of fecal material was used and all eggs were counted, several slides were prepared from each specimen to assure that all eggs present in samples were counted.
At the time of necropsy abomasal contents were saved and the mucosa was washed with PBSS. The contents were filtered through a 200 mesh screen and were diluted to 1 L. Later some of the supernatant was poured off and
150 ml of 10% formalin was added to the jars. One-half of the abomasum was soaked in 150 ml of water for 24 hours, then the mucosal surface was carefully massaged to release larvae. Fat particles were removed from the solution and approximately 20 ml of 10% formalin was added. Both solutions from each animal were 39 examined with a dissecting microscope and the nematodes were counted and collected in separate vials containing
70% alcohol. Parasites were characterized as to developmental stage and sex.
Results
Clinical observations— Animals in all 3 treatment groups appeared to be essentially normal during the experiment. Following the injection of pentagastrin local irritation was evident which was characterized by rolling, vocalization or laying in a corner for a very short time. Animals in the control and 0 ostertagi- infected groups appeared healthy and had good appetite throughout the study while animals in the pentagastrin- treated group had a slightly depressed appetite, but were otherwise healthy. While there was no evidence of anorexia in the Ostertagiasp. group 7 of the 8 kids had lost weight by the end of the experiment. In contrast, animals in the control group gained weight during the study. Occasional animals in all 3 treatment groups had experienced short time periods of mild diarrhea, related to slight coccidial infections.
Clinical pathology— There were only slight changes recorded in the selected serum chemistry and hematology parameters. Total plasma protein, albumin, phosphorus and calcium levels were not different in treatment groups AO
throughout the experiment (Table 2 & 3). Every animal developed slight anemia and no differences were found between treatment groups in hematology data (Table 4-7).
Reticulocytes were not found in any animal.
Increased pepsinogen levels were found in parasitized goats compared to values in the other two treatment groups.
The mean serum pepsinogen level remained below 0.5 IU in the control group during the experiment (Fig. 2). In the
0 ostertagi-infected group pepsinogen levels were above
0.5 IU frpm the 5th to the 36th experimental days, reaching the highest mean on the 8th experimental day
(Fig. 3). The highest individual value was 1.65 IU.
Pepsinogen levels in the pentagastrin-treated animals were similar to those in the control group (Fig. 4).
Differences were also noted in serum gastrin immunoreactivity of experimental groups. The gastrin immunoreactivity of control goats remained uniform throughout the experiment and no age related changes were detected (Fig. 5). One animal in the ostertagia infected group had an unusually high gastrin immunoreactivity level before infection. The other 7 animals had similar levels as the controls. A slight increase in gastrin immunoreactivity could be detected on the 5th experimental day in Group III 0 ostertagi-infected animals. Highest gastrin immunoreactivity levels were measured on the 8th experimental day. In most animals 41 gastrin immunoreactivity dropped suddenly within a week into a moderately elevated level which gradually decreased to near baseline by the end of the experiment
(Fig. 6). The mean gastrin immunoreactivity of the penta- gastrin-treated group was similar to that of the controls
(Fig. 7). Following treatment, individual animals reacted somewhat differently within the group. One kid had a slightly increased gastrin immunoreactivity, while another had a slightly lower gastrin immunoreactivity level. These individual values were not significantly different from controls.
Gross pathology— There were no significant differences (P>0.05) in the physical parameters of abomasa at the termination of the experiment. At necropsy the pH of the abomasal contents of the control and pentagastrin groups were similar, while in the Ostertagiasp. group it was slightly increased. In addition, there was a slight increase in the weight of the abomasa of 0 ostertagi- infected kids compared to the other treatment groups
(Table 8).
The most significant gross findings were in the abomasa. The mucosa of the abomasum in control animals was smooth, yellowish or greyish pink. The fundic folds were thin and in the transitional zone there were only a few, small, longitudinal rugae present (Fig. 8). The mucosa of 0 ostertagi-infected animals exhibited 42 multifocal, sometimes coalescing, white or grey, often umbilicated nodules, varying in size from 1 to 4 mm in diameter (Fig.9). Most of the parasitic nodules were found in the the fundus and the anterior transitional zone, some extended into the pyloric antrum. In addition, fundic folds were more prominent and an increased number of longitudinal and transverse rugae were present in the transitional zone and antrum (Fig. 8). The abomasal mucosa was slightly darker pink in infected animals than in controls. In two of the pentagastrin-treated animals fundic folds were more prominent than those of the controls and had thickened fundic folds (Fig. 10).
Gross changes were also noted in abomasal lymph nodes of infected kids. The abomasal lymph nodes were small in control and pentagastrin treated kids, and were moderately to markedly enlarged in parasitized animals.
A few incidental gross findings were also present.
In some animals from each treatment group the thymus was small or difficult to detect. The mesenteric lymph nodes of several animals were enlarged and wet. Some animals had a minimal amount of clear, pale yellow fluid in their abdomen. One animal in the 0 ostertagi-infected group had slight focal inhalation pneumonia and a mild, focal fibrinous pleuritis. No other gross lesions were evident.
Parasitology— Trichostrongylid-type eggs were found in the feces of every infected animal, while control and 43 pentagastrin-treated kids did not pass nematode eggs. By the 27th postinfection day all 8 animals in the (3 ostertagi group passed eggs in their feces. Four animals developed, patent infection by the 20th post infection day, and the egg counts increased weekly in each animal. Animals had a mean fecal egg count of 6, 39 and
113 on the 20th, 27th and 34th postinfection days with a range of 1-15, 4-86 and 14-251 respectively. Coccidial oocyst were found in every animal throughout the study.
0 ostertagi were recovered only from Group III infected animals, while abomasal contents and mucosa were negative for parasites in Group I control and Group II pentagastrin-treated animals. The mean nematode establishment' in parasitized goats was 5% with a range of
2-11%. The lowest level of establishment occurred in the smallest goat in the group. The majority of the recovered parasites were adults and approximately 60% of them were females. The results of parasite recovery are summarized (Table 9).
Discussion
In this study a group of 2 to 3-month-old goat kids were infected with 0 ostertagi and another group received chronic pentagastrin treatment.
Pentagastrin had a locally irritating effect similar to that reported in other species.^ In addition, animals 44 had a slightly decreased food intake as it had been observed in other ruminants.^ In this study, similar to a previous experiment with cattle, pentagastrin did not significantly affect serum calcium levels.In addition, serum gastrin immunoreactivity of pentagastrin treated animals did not change significantly, although slight increase or decrease was noted in individual animals. This finding was in contrast to recent observations where pentagastrin caused increased serum gastrin levels in mice.^ Serum pepsinogen levels in goats were unaffected.by pentagastrin. At the dose of
75ug/kg BID pentagastrin did not produce a significant increase in the abomasal weight, although some animals had slightly increased abomasal weights and prominent mucosal folds. In rats a marked increase in stomach weights was produced by 2000ug/kg pentagastrin.^® The pancreas of Group II animals appeared to be unaffected by the pentagastrin treatment. A marked increase in the pancreatic mass of rats was due to a much higher
(2000ug/kg) dose of pentagastrin.^®
All animals infected with 0 ostertagi developed patent infections. In another study, using goats with similar breeding history 85% of infected animals passed nematode eggs.®^ In addition, animals in that study reached patency later and had very low fecal egg counts. In contrast, in the present study 50% of the 45 goats had evidence of patency by the 20th experimental day which is similar to observations in bovine ostertagiasis.*^*^ Additionally, by another week, all 8 animals in Group III had patent infection and EPG counts were substantially higher than in the previous caprine 97 experiment.
No clinical signs were evident in ,0 ostertagi- infected animals, goat kids appeared to maintain their appetite, but with the exception of 1 animal they lost weight by the end of the study. The frequent collection of blood samples was probably a contibuting factor to the observed anemia. Goat kids often develop anemia, ii q primarily due to iron deficiency at 2-3 months of age.
Probably due to the relatively low level of infection no significant changes were found in selected serum constituents in goats infected with 0 ostertagi.
An increase of plasma or serum pepsinogen has been reported in bovine and ovine ostertagiasis. In these 2 ruminant species pepsinogen levels were usually higher than those of the present study. The differences were probably due to the differences in the worm burdens.
Hypergastrinemia was observed in sheep infected with 0 circumcincta. An early rise was due to the presence of parasite and a second increase was attributed to increased abomasal pH.24»43,44 jn the present study increased gastrin immunoreactivity occured during the 46 tissue phase of the parasitic development.
There was a mild increase in the pH of the abomasal contents in Group III 0 ostertagi infected animals, which is a hallmark of the bovine infection.*”* Abomasal weights were not measured in previous studies and were slightly increased in this experiment. The abomasal mucosal changes in parasitized goats in this study were similar to those observed in bovine ostertagiasis, although individual nodules appeared to be larger than those of the cattle.*”*
Also, the abomasal lymph nodes of 0 ostertagi infected goats were enlarged similarly to those of the bovine.**^
Based on this experiment goat kids were susceptible to 0 ostertagi and developed patent infection. Goats, similar to other ruminants, developed hyperpepsinogenemia simultaniously with an increase in gastrin immunoreacti vity. This observation was suggestive that hyperpepsinogenemia, at least in part, was secondary to the stimulatory effect of gastrin.
Pentagastrin was administered in a relatively low dose in this experiment and no significant changes were associated with the treatment.
In conclusion 0 ostertagi infection in goat kids in this experiment was similar to the bovine disease and goat could serve as an excellent model system for bovine ostertagiasis. 47
Table 1 - Gastrin RIA kit validation for caprine serum, precision
Inter-assay precision
Goat
#5 #13 #22
Mean* 316 pg/ml 150 pg/ml 253 pg/ml
S.D. 46 pg/ml 22 pg/ml 40 pg/ml
C.V.** 14 % 14 Z 16 Z
Intra-assay precision
Goat
#22 #930
Mean* 254 pg/ml 116 pg/ml
S.D. 8 pg/ml 7 pg/ml
C.V.** . 3 % 6 Z
* Mean of 10 replicates in duplicate runs.
**Coefficient of Variation ® S.D. x 100 Mean 48
Table 2 - Mean total plasma protein and serum albumin concentations of goat kids following no treatment, 0 ostertagi Infection or chronic pentagastrin treatment
Total Plasma Protein (g/dl)
week Control Pentagastrin Ostertagia
1 5.9 + 0.1* 6.0 + 0.5 5.7 + 0.4
2 5.9 + 0.4 6.1 + 0.6 5.8 + 0.4
3 5.7 + 0.5 5.8 + 0.5 5.3 + 0.5
4 5.8 + 0.6 5.5 + 0.8 5.5 + 0.5
5 6.0 + 0.5 5.6 + 0.9 5.6 + 0.4
6 5.9 + 0.6 5.8 + 0.9 5.9 + 0.5
Serum albumin (g/dl)
week Control Pentagastrin Ostertagia
1 3.1 + 0.3 3.4 + 0.3 2.8 + 0.3
2 3.0 + 0.3 2.9 + 0.1 2.8 + 0.3
3 2.8 + 0.4 2.5 + 0.2 2.5 + 0.4
4 2.6 + 0.4 2.3 + 0.3 2.3 + 0.5
5 2.7 + 0.5 2.2 + 0.5 2.2 + 0.5
6 2.6 + 0.5 2.1 + 0.6 2.2 + 0.5
*Mean jhS.D., means are not significantly different
(P>0.05) 49
Table 3 - Serum calcium and phosphorus concentatlons of goat kids following no treatment, 0 ostertagi infection or pentagastrin treatment
Calcium (mg/dl)
week Control Pentagastrin Ostertagia
1 9.3 + 0.5* 9.8 ± 9.3 + 0.5 •(S' o 2 9.7 + 0.5 9.7 • 9.6 + 0.6 1+
3 9.6 + 0.3 9.1 + 0.5 8.9 + 0.6 o 00 4 9.0 + 0.6 8.9 • 8.6 + 0.6 1+
5 9.5 + 0.5 9.2 + 0.8 8.8 + 0.5
6 9.2 + 0.4 8.8 + 0.8 8.7 + 0.7
Phosphorus (mg/dl)
week Control Pentagastrin Ostertagia
1 6.8 + 1.8 6.4 + 2.5 6.3 + 1.1
2 8.5 + 1.1 7.2 + 1.6 8.0 + 1.7
3 6.1 + 1.2 6.7 + 2.0 7.5 + 3.0
4 6.1 + 0.9 5.7 + 1.9 7.2 + 2.0
5 6.4 + 1.7 5.8 + 1.1 6.7 + 2.5
6 6.9 + 0.8 5.9 + 1.9 6.6 + 2.6
*Mean +S.D., means are not significantly different
(P>0.05) 50
Table 4 - Erythrocyte parameters at the beginning and at the end of experiment in three treatment groups test Control Pentagastrin Ostertagia week
RBC 20.0 + 6.4* 14.8 + 2.0 19.9 + 3.4 1
15.4 + 3.1 14.3 + 2.1 17.0 + 2.1 6
Hgb 8.7 + 1 .6 9.8 + 1.8 9.5 + 8.0 1
7.7 + 1.3 6.8 + 1.3 8.0 + 1.5 6
PCV 25.6 + 5.5 29.6 + 6.8 27.3 + 4.2 1
23.4 + 4.2 19.4 + 3.7 23.6 + 5.0 6
MCV 13.7 ± 4.5 20.4 + 5.5 14.0 + 2.8 1
15.3 ± 1.8 13.8 + 2.9 13.9 + 1.8 6
MCH 4.7 ± 1.5 6.8 + 1.6 4.8 + 0.8 1
5.1 ± 0.5 4.8 + 0.9 4.7 + 0.5 6 r
MCHC 34.0 ± 1.2 33.4 + 1.5 34.6 + 1.2 1
33.1 ± 1.3 35.0 + 1.8 34.2 + 1.3 6
RBC (red cell count)xlO^/mm^, Hgb (hemoglobin) gm/dl
PCV (packed cell volume) %, MCV (mean corpuscular volume)fl
MCH (mean corpuscular hemoglobin) gm/dl
MCHC (mean corpuscular hemoglobin concentration) %
♦Mean +S.D., means are not significantly different
(P>0.05) 51
Table 5 - Leukocyte counts (xlO^/mm^) of Group I control goat kids
week Total S L MEB
1 14.5* 5.3 8.5 0.1 0.1 0.0
(1.0)** (1.3) (0.6) (0.1) (0.1) (0.1)
2 14.8 5.7 8.9 0.1 0.1 0.0
(0.9) (1.2) (1.7) (0.1) (0.1) (0.0)
3 13.2 5.9 7.1 0.0 0.1 0.1
(2.0) (1.9) (1.3) (0.0) (0.1) (0.2)
4 12.7 4.7 7.8 0.0 0.0 0.2
(1.8) (1.9) (2.4) (0.0) (0.1) (0.1)
5 14.5 6.1 8.1 0.0 0.2 0.1
(1.8) (1.6) (1.1) (0.0) (0.1) (0.1)
6 14.0 5.3 8.0 0.0 0.3 0.1
(1.8) (1.6) (2.5) (0.1) (0.2) (0.1)
Snneutrophils, L=lymphocytes, M«monocytes,
E=eosinophils, B«basophils
♦Mean
**( )«S.D. 52
Table 6 - Leukocyte counts (xlO^/mm^) of Group II goat kids receiving 75ug/kg pentagastrin
BID for 28 days
week Total S L M EB
1 16.6* 8.2 7.3 0.1 0.1 0.0
(4.9)** (3.7) (1.8) (0.3) (0.2) (0.1)
2 15.9 6.5 9.1 0.1 0.1 0.2
(3.6) (1.5) (2.6) (0.1) (0.1) (0.3)
3 15.0 4.7 9.9 0.1 0.2 0.2
(4.8) (1.0) (4.3) (0.2) (0.2) (0.2)
4 14.9 5.1 9.2 0.0 0.3 0.1
(5.8) (1.5) (3.6) (0.0) (0.3) (0.2)
5 16.6 6.6 9.6 0.1 0.1 0.2
(5.9) (2.6) (3.2) (0.1) (0.1) (0.3)
6 19.0 9.4 9.5 0.0 0.1 0.1
(7.5) (5.7) (2.8) (0.0) (0.1) (0.2)
Saneutrophils, L>lymphocytes, M=monocytes,
Eaeosinophils, B»basophils
♦Mean
**( )-S.D. 53
Table 7 - Leukocyte counts (xlO^/mm^)
of Group III goat kids Infected with 15,000 L3 of
0 ostertagi
week Total S L M E B
1 13.0* 3.9 8.3 0.1 0.1 0.0
(2.4)** (1.0) (2.2) (0.1) (0.1) (0.1)
2 14.7 5.3 9.0 0.1 0.1 0.3
(3.1) (1.6) (2.0) (0.1) (0.2) (0.3)
3 13.0 3.9 8.6 0.1 0.1 0.3
(2.6) (1.6) (2.6) (0.1) (0.3) (0.1)
4 13.4 4.7 8.5 0.1 0.1 0.1
(2.4) (2.5) (2.1) (0.1) (0.1) (0.1)
5 12.0 3.6 8.0 0.1 0.1 0.3
(2.8) (1.4) (2.0) (0.1) (0.1) (0.2)
6 14.4 5.5 8.5 0.1 0.1 0.3
(3.7) (2.5) (1.5) (0.1) (0.1) (0.3)
Saneutrophils, Lolymphocytes, M»monocytes,
Eaeoslnophils, B»basophils
♦Mean
**( )-S.D. 54
Table 8 - Necropsy findings
Abomasal pH and abomasal weight as a percentage corrected body weight in goat kids following no treatment, infection with 15,000 L3 of ,0 ostertagi or 75 ug/kg pentagastrin BID for 28 days
Abomasal Control Pentagastrin Ostertagia pH 2.43+0.2* 2.65+0.39 3.12+0.58
weight^ 0.70+0.1 0.85+0.12 1.04+0.14
♦Mean hhS.D.
♦♦ % corrected body weight ( body weight - forestomach weight) 55
Table 9 - Worm recovery from goat kids experimentally infected with 15,000 third larval stage of Ostertagia ostertagi
Animal Adult Fourth larval stage Female Male Developing Early
#927 375 143 40 0
#929 978 564 3 0
#930 635 287 31 0
#935 476 233 110 29
#942 326 180 4 0
#943 212 91 0 0
#944 396 215 6 0
#945 429 232 7 0 i. —GsrnRAkt aiain specificity. validation, kit RIA Gastrin 1— Fig. Parallel dilution curves of synthetic human gastrin and gastrin human synthetic of curves dilution Parallel arn sera. caprine Percentage bound 90 30 50 70 Gastrin Immunoreactivity pg/ml Immunoreactivity Gastrin \\s Standard 1200 56 57
3 1 .0- Z IU o oz CO Q. IU Q. s oc HI CO
f
-5 -2 1 5 8 12 15 19 22 26 29 33 36 Experimental Days
Fig. 2— Serum pepsinogen levels of Group I control goats Means +S.D. 58
3 1.0 z UJ O zo
-6 -2^ 1 6 8 12 15 19 22 28 29 83 38 Experimental Days
Fig. 3— Serum pepsinogen levels of Group III goat kids. Animals were infected with 15,000 L3 of 0 ostertagi on day 0 (arrow). Means +S.D. 59
3 1.0 - Z LU o zo (0 CL UJ 0. .5 s 3cc UI CO
•5-2 1 5^8 12 15 18 22 26 28 33 36 Experimental Days
Fig. 4— Serum pepsinogen levels of Group II goat kids. Animals received 75 ug/kg pentagastrin BID from day 7 (arrow). Means ,+S.D. 60
900*
700 o>a > I- > EP 500 O < lii cc oz 3s 300- s
cc I- 0) 100- < <3
-5 -2 1 6 8 12 16 19 22 28 29 33 38 Experimental Days
Fig. 5— Gastrin iramunoreactivity of group I control goat kids. Means HhS.D. i. —Gsrn rruoeciiy f ru II ot kid goat III iraraunoreactivity Group Gastrin of 6— Fig. Animals were infected with 15,000 L3 of 0 ostertagi 0 of L3 15,000 with infected were Animals on day 0 (arrow). Means +S.D. Means (arrow). 0 day on
GASTRIN IMMUNOREACTIVITY (pg/ml) 500- 300- 700- 9001 100 - 6 1 1 1 2 2 2 3 36 33 29 26 22 19 16 12 8 6 1 2 - 5 - if xeietl Days Experimental 900 *
^ 700- o> a
>• £ P 500- O < UJ oc 0 z 1 300- s
cc 4rli r h <2 100H f i f o
- 5 - 2 1 6 ^ 8 12 16 18 22 26 28 33 36 Experimental Days
Fig. 7— Gastrin immunoreactivity of Group II goat kids Animals received 75 ug/kg pentagastrin BID from day 7 (arrow). Means + S.D. Fig. 8— Abomasa of control and 0 ostertaei-infected goat kids opened along the greater curvature. The control abomasum (left) has thin fundic folds and only a few longitudinal rugae in the transitional zone. In the parasitized abomasum (right) fundic folds are thickened and an increased number of longitudinal and transverse rugae are present in the transitional zone and pyloric antrum. Fig. 9— Abomasum of an 0 ostertagi-infected goat kid. The multifocal white, raised nodules are often umbilicated. 65
m^iiii|iuij|iiii|mi£"'|
Fig. 10— Abomasum of a pentagastrin-treated goat kid. The fundic folds are prominent. CHAPTER III
Qualitative and Quantitative Light Microscopic
Studies Following Ostertaaia ostertaai Infection or
Chronic Pentagastrin Treatment of Goat Kids
Introduction
Abomasal lesions in cattle following a single dose of 0 ostertaai have been reported at several levels of inoculum. 16*17,22 development of the lesions was found to be similar and was monitored up to 90 days postinfection. Three phases were identified in the Type I disease process.^ The first phase was associated with the development of the parasite in mucosal glands and was characterized by the loss of mature differentiated cells, which were replaced first by flattened epithelium and later by tall undifferentiated mucus secreting cells. In the second phase large areas of the abomasal mucosa were lined by rapidly dividing mucous epithelial cells, especially in the neck region of the glands. This phase was associated with the maturation and emergence of the parasite into the abomasal lumen. In this second phase of the disease a mixed cellular inflammatory reaction was also evident in the mucosa. Lymphoid follicles developed mainly perivascularly and the lamina propria was infiltrated by eosinophils. The proliferation of globule 67 leucocytes was also evident in this phase. The third phase of the disease was associated with the loss of adult parasites. There was a gradual reappearance of parietal cells and zymogen cells in abomasal glands, but globule leucocytes were still present.
The effect of long term, high dose pentagastrin treatment has been evaluated with morphometric methods.^
The increase in the thickness of the gastric mucosa of rats was due to an increase in parietal cell mass with simultaneous increase in the parietal-chief cell ratio.
Although goats have been experimentally infected with £ ost£rt-agjL,^»29»^»^ the histological changes have not been reported.
The purpose of this study was to characterize the light microscopic changes in goats, corresponding to the second phase of the Type I ostertagiasis in cattle, following a single dose of inoculum. Proliferative changes were evaluated by morphometric methods and the mucus production of the proliferative cells was also characterized. In addition, the effect of chronic penta gastrin treatment was determined in a ruminant species.
The proliferative mucosal changes in parasitized abomasa were compared to those associated with the pentagastrin treatment.
Materials and Methods.
Experimental animals— Experimental animals 68
originated from a European-type mixed-breed goat herd maintained on a parasite contaminated pasture. Kids were born within a 1 month time period and were removed from the pasture with their does within 48 hours after birth.
A brick building served as housing for kids and does. No other ruminants were kept in this building during the experiment. The wood-shaving bedding was changed weekly.
Animals were fed with a commercial feed, composed of 54% cotton seed hulls, 14% cotton seed meal, 24% cracked corn, 5% cane molasses, 0.5% oyster shell flour, 0.4% trace minerals. Alfalfa hay was also offered at feeding time, and water and salt were available ad libitum. Kids were weaned at five weeks of age.
Experimental Design— Eighteen goat kids free of gastrointestinal parasites were selected for the experiment one week before inoculation. The 2 to 3- month-old mixed breed goats were randomly divided.into three treatment groups. Five kids in Group I did not receive treatment and five animals in Group II received
75 ug/kg pentagastrin twice daily for 28 days, starting on the seventh experimental day. The third group consisting of eight kids (Group III) were infected with
15,000 third larval stage (L3) of Ostertagia ostertagi.
Inoculation— The parasite inoculum originated from a bovine isolate of 0 ostertagi in Louisiana and was cultured using standard techniques. Briefly, fresh feces 69 was mixed with vermiculite to form a crumbly, moist mass, and cultures were maintained at room temperature for two weeks. A Baermann funnel was filled with tepid water and the feces-vermiculate mixture was immersed in the water.
This system was left undisturbed overnight. In the morning the larvae were collected in a large beaker and were washed several times and stored in the refrigerator at 4 C. Larvae were examined for viability and 15,000 0 ostertagi L3 were placed into separate tubes. Animals were inoculated per os using disposable plastic pipettes.
Pentagastrin (Peptavlon,Ayerst Laboratories, New
York,NY) solution was prepared weekly with dimethyl sulfoxide (DMSO) and deionized water. Animals were injected subcutaneously with 75 ug/kg pentagastrin every
12 hours for 28 days, beginning on experimental day 7.
Necropsy procedures— Animals were euthanized by electrocution on the 35th. and 36th postinfection day.
The kids were weighed and the forestomachs and abomasa were quickly removed from the carcasses. The corrected body weight was determined by substracting the weight of the forestomachs from the total body weight. A small opening was cut in the wall of the abomasum and the pH of the contents was measured. The abomasum was opened along the greater curvature, its contents were removed and the mucosa was gently washed with phosphate buffered saline solution (PBSS) pH 7.1 at room temperature to 70 remove adhered particles. Care was taken to use only a small amount of washing fluid which was processed with the abomasal contents. The weight of the abomasum was recorded, and the abomasum was cut in half along the lesser curvature. One-half of the abomasum was processed for parasite recovery and the other half was slightly stretched and pinned to a styrofoam board and placed into fixative. Abomasal lymph nodes were examined, removed and sectioned for microscopic evaluation. All major visceral organs were examined with special attention to endocrine organs and tissue sections were saved in fixative. The brains of selected animals were also collected. All gross observations were recorded and were presented in Chapter
II.
Tissue Processing— Tissue samples were fixed in a buffered mixture of 4 % formalin and 1 % of glutaraldehyde (4F1G). Abomasa were slightly stretched and pinned out for 24 hours immersed in fixative. At this time sites were selected for morphometry, light and electron microscopy.
Sections from the following tissues were processed routinely for light microscopy, embedded in paraffin, cut
6 urn thick and stained with Mayer's hematoxylin and eosin: abomasal fundus and pyloric antrum, forestomachs, lung, liver, kidney, heart, thymus, abomasal and mesenteric lymph nodes, spleen, pancreas, thyroid, adrenal, small 71 and large intestine, and brain. Paraffin embedded sec tions of abomasa were stained with Grimelius* argyrophi- lic technique for the demonstration of the endocrine cell population in the pyloric antrum. To characterize the mucosubstances secreted by the abomasal mucous cells two biphasic stains, periodic acid-Schiff and alcian blue stain (PAS-AB) at pH 2.5 and the high iron diamine-alcian blue stain (HID-AB) were applied to paraffin sections as previously described.121-124 These biphasic stains distinguished PAS-positive (red) neutral from AB-positive
(blue) acid mucosubstances and HID-positive (black) sulfated acid mucosubstances from non-sulfated AB- positive (blue) acid mucosubstances (sialomucins).
Morphometry— Five animals with increased abomasal weight and pH were selected from the 8 experimentally infected goats. One of the 5 selected animals had an increased gastrin immunoreactivity before inoculation and was excluded from the quantitative evaluation. One of the control abomasa became unacceptable for morphometry.
A stratified random sampling technique was selected due to the known structural differences in different regions of the abomasa.A grid composed of numbered 1 cm^ units was placed over the abomasa and areas were selected using a randomization table. A total of 9 areas were selected, 5 from folded fundic mucosa, 3 from the non folded transitional zone and pyloric antrum, and the last one from the distal antrum (Fig. 1). From these 1 cm^ areas an approximately 2 mm stripe was trimmed for processing. These stripes were embedded in a methacrylate plastic medium (Sorvall Embedding Medium, Du Pont
Comp.,Wilmington, DE), and 3 urn sections were cut at right angle. Plastic sections for light microscopic morphometry were stained with a modification of
Zimmermann's method as described elsewhere.126,127
Measurements and calculations were made using a morphometry software package (Bioquant, R&M Biometrics,
Nashville, TN), an IBM personal computer (IBM Corp., Boca
Raton,FL), attached to a digitizing pad (Hipad Digitizer,
Houston Instruments, Austin,TX) , connected to a microscope equipped with a TV camera which was also connected with a monochrome monitor. Mucosal thickness was determined on 10 measurements per site, measuring the mucosa from lumen to muscularis mucosae. Abomasal epithelial cells were counted in 5 randomly selected stripes of 0.07 mm width from each site and the perimeter of each cell was traced using a 100 x objective. The area of cytoplasmic fraction was generated for each cell from the measured perimeter and the calculated area was used as an indicator of the cell volume. Each cell with identifiable cytoplasm was measured even when the nucleus could not be visualized, and binucleated cells were counted as 2 mononuclear epithelial cells. The following 3 cell types were counted: parietal cell, chief cell and
mucous cell. Surface epithelium, mucous neck cell, antral
gland cell and the proliferating, poorly differentiated
cells in effected abomasa were counted as mucous cells.
Cell counts were expressed as cells per unit width (0.1
mm) of abomasal mucosa, calculated from the 0.35 mm
mucosal bands (5x0.07 mm) which were measured. The
parietal to chief cell ratio was generated from the
number of parietal and chief cells counted.
Statistics— Morphometry results are presented as
means of the means ± SEM and were evaluated by ANOVA and
LSD.
Results
Histopathology— The mucosa of the abomasal fundus
in contol Group I animals had a smooth luminal surface and
was composed of straight and narrow glands of equal
length (Fig. 2). The apical region of the surface mucous
cells was filled with mucus granules and the cells were
slender. The cytoplasm of parietal cells stained
homogenously pale pink (eosinophilic) with the H&E stain
and pale yellow with the modified Zimmerman stain. The
cytoplasm of chief cells was slightly foamy and blue
(basophilic) on H&E and greyish blue, finely foamy on
Zimmerman's. Only a few, small, discrete mononuclear cellular aggregates were present in the outer one-third 74 of the mucosa. Eosinophils and globule leucocytes were not observed in the abomasa of group I animals. Fundic folds in the control animals were thin (Fig. 3). While the stromal elements of the abomasal fundus in control animals was relatively inconspicuous, the lamina propria was more prominent in the pyloric antrum. The glands in the pyloric antrum were convoluted and opened into narrow pits.
In group II animals a multifocal, slight nodularity of the fundic mucosa was detected (Fig. 4). In sections from folded and non-folded areas of the fundus long, slender, linear glands were present. In one animal fundic glands were slightly dilated and were lined by slightly flattened cells . The fundic folds of pentagastrin-treated animals were also similar to those of the controls (Fig. 5). A few, small, discrete mono nuclear cellular aggregates were present in the lamina propria in group II animals. Eosinophils or globule leucocytes were not present in abomasa of pentagastrin treated animals.
In contrast, in 0 ostertagi-infected goats the luminal surface of the abomasal mucosa was multifocally nodular (Fig. 6). Fundic folds were multifocally thickened due to submucosal edema and cellular accumulation (Fig. 7). Surface epithelial cells frequently had swollen nuclei and the cytoplasm was 75 devoid of mucus granules. In addition, a large number of bacteria were adhered to the surface mucous cells. Only occasional glands contained 0 ostertagi larvae, those present appeared to be viable and attracted only a minimal cellular infiltration, primarily composed of eosinophils. Other larvae were surrounded by proliferative cells, which formed short finger-like projections into the gland lumens (Fig. 8). A few glands were empty, markedly dilated and lined by markedly flattened mucous epithelial cells, but did not contain parasites (Fig. 9). In infected animals in the fundus as well as in the pyloric antrum occasional herniation of the mucosa was present. Small portions of abomasal glands extended through the muscularis mucosae into the sUbmucosa (Fig. 10). Another change in infected animals was the foveolar atrophy, which was characterized by the bridging of several glands by surface mucous cells and loss of foveolae (Fig. 11). Frequently abomasal glands were distorted with columnar cells in bizarre formations.
Irregularly shaped glands were formed by mucous cells of varying morphology. The majority of the undifferentiated cells were tall, columnar, but in some areas the cells were cuboidal. The proliferative cells stained deep blue (basophilic) with the H & E stain. The inflammatory cellular reaction in the abomasa of Group
III kids extended multifocally into the submucosa from 76 the mucosa and was composed of a mixture of lymphocytes, plasma cells and eosinophils . In the lamina propria subepithelial hypercellularity was often noted (Fig. 12).
Eosinophils were multifocally scattered in the lamina propria. In addition, intraepithelial globule leucocytes were present multifocally, mainly in the upper third of the glands. The thickening of the mucosa in infected animals was also due in part to slight multifocal edema in the lamina propria, mainly in the non-folded areas of the fundus, or less frequently it was due to a slight increase in the amount of fibrous connective tissue in the lamina propria. The epithelial cells of infected animals exhibited multifocal degenerative and dysplastic changes. Parietal cells in group III animals often had swollen nuclei and markedly granular cytoplasm. Chief cells appeared to have an increased amount of Cytoplasmic granules which stained yellow with the Zimmerman method.
Intestinal metaplasia was present in the pyloric antrum of 4 animals and one also had metaplastic changes in the fundus. In affected areas the normal glandular architecture was altered and the mucosa resembled intestine (Fig. 13). Cells similar to intestinal goblet cells were scattered multifocally and were more frequent in deeper areas of the mucosa. These cells contained clear vacuoles with the H & E stain which stained posi tive for sialomucins with AB stain. Another dysplastic 77 change, pseudopyloric metaplasia, was also present in infected abomasa. While in control animals the fundic glands extended far into the transitional zone of aboma sa, in parasitized, Group III animals the antral glands appeared more proximal. In contrast, the pentagastrin- treated animals had 1-2 parietal cells per 0.07 mm in the distal part of the pyloric antrum.
Stains for mucosubstances revealed a uniform cell population in the abomasa of control goats, While infected abomasa had an heterogenous cell population.
Control animals produced neutral mucosubstances on the surface and predominantly neutral mucus in the neck region of the fundus. In the neck region acid mucosubstances were sulfated. Mucous cells of Group I control abomasa had red cytoplasm on the luminal surface and were purple in the neck region with PAS-AB stain
(Fig. 14). With the HID-AB stain cells in the mucous neck region stained black in control fundic glands (Fig.15).
The parietal and chief cells in the deep regions of the glands were negative for mucus. In the antrum of Group I animals the surface epithelium had neutral mucus while foveolae and especially the antral glands were positive for sulfomucins. In group II animals an increased proportion of acid mucosubstances were found in the pyloric antrum as well as in the fundus. This increased staining characteristic primarily manifested as slight 78 lengthening of the neck region with a purplish staining with PAS-AB and grey to black staining with HID-AB. In parasitized glands mucous cells with acid mucus extended from the neck region into the deep glandular areas of the fundus. These foci stained blue with both mucus stains (Fig. 16 & 17). In addition, multifocal abomasal surface epithelial cells contained sulfomucins in the abomasa of Group III kids. The distribution of mucosubstances in the caprine abomasa is summarized
(Table 1 & 2).
The argyrophilic technique failed to demonstrate differences in the antral endocrine cell population of experimental animals.
The mitotic activity of abomasa was evaluated semiquantitatively. In control animals mitotic figures were found only rarely (approximately 1/9 slides), a slight increase could be appreciated in the abomasa of pentagastrin-treated goats. The increase in mitotic activity was especially striking in 0 ostertagi-infected animals. Mitotic figures were seen in the neck region and were most frequent (up to 5/slides) in the the antrum of parasitized animals.
The abomasal lymph nodes of group I and group II animals were similar, and were characterized by well developed lymphoid follicles and only a few cells in the medullary sinuses. The abomasal lymph nodes of group III animals were characterized by follicular hyperplasia with the expansion of the cortical zone and hypercellularity in medullary sinuses.
When the tinctorial characteristics and cellular details of the pancreas were evaluated in experimental animals no differences could be detected between treatment groups.
Infection with an Eimeria-like unidentified macroschizont was found in 2 animals as an incidental finding. Protozoal organisms were found in the abomasum of an animal in group III, without an associated cellular infiltration. A focal granulomatous lymphadeniti associated with a macroschizont protozoa was found in a mesenteric lymph node of an animal in .group II. No significant changes were found in other tissues examined.
Morphometry— The means of the abomasal mucosal thickness, cells per unit width, area of cytoplasmic frac tions and the parietal-chief cell ratio are summarized in
Table 3. Although no significant (P>0.05) differences were found in the mucosal thickness between control and pentagastrin treated animals, some pentagastrin treated animals had a slightly increased mucosal thickness. There was a significantly (P<0.05) increased mucosal thickness in group III animals compared to the control goat abomasa. The mean mucosal thickness of control goat abomasa was 338 urn while in parasitized animals the mean mucosal thickness was 524 urn. The cell population o£ the control and pentagastrln-treated animals was similar.
There was no significant (P>0.05) difference in the number of mucous epithelial cells, parietal cells or chief cells. When the parietal-chief cell ratio was evaluated in these 2 treatment groups, the ratio was found to be slightly increased in the pentagastrin treated animals. The area of the cytoplasmic fractions of the different cell types was similar in pentagastrin- treated animals to that of controls. The number of mucous epithelial cells of group III animals was increased signi ficantly (P<0.05) compared to control animals, while the number of the other 2 cell types were not significantly different in control and parasitized animals. The average number of mucous epithelial cells per 0.1mm was 16.4 in control goats and it was 28.3 in parasitized animals. In addition, there were no significant (P>0.05) differences between areas of cell fractions in group I and III animal
Discussion
The light microscopic morphology of the abomasa of control goats was similar to that of other domestic 1 Op mammalian species.Additionally the staining characteristics of epithelial cells with the modified
Zimmerman stain was also similar to that of other mammals.126,127 The following regions were identified in 81 the fundic glands: surface and foveolae, neck and deep gland. The neck region was determined as the part of the gland between the first parietal cell and the last mucous neck cell. In the deep gland region parietal and chief cells were found. In the pyloric antrum surface, foveolar and antral gland cell regions were identified.
The distribution of mucosubstances in the abomasa of control goats was in agreement with a comprehensive study of the gastro-intestinal mucosubstance histochemistry of 11 different species.While there were some species variations, in the majority of the species surface epithelial cells contained neutral mucosubstances. In the proliferative zone (in the neck region) of the fundic glands cells contained sialo and/or sulfomucins. In addition, in the deep gland area the parietal and chief cells of most species did not contain mucin. Similarly, in the antrum the surface and upper foveolar cells produced predominantly neutral mucus, while the lower foveolae and antral gland cells contained sialo- and sulfomucins.
While several sampling techniques have been published for the morphometric evaluation of the gastric mucosa of laboratory animals and man only limited information was found on the morphometry of the ruminant glandular stomach.73,77,80,129-133 was noted earlier that the cell populations of the anterior and posterior walls of the stomach were not significantly different.In 82
addition, from each goat the anterior abomasal wall was
selected for morphometry. A stratified random sampling
technique was selected due to the known structural
IOC differences in different regions of the abomasa.A J The
abomasa of goats in this study were similarly divided
into regions as it was described for sheep, with the
exception of the cardia which was not examined in this
study.No cardiac glands were identified in goats in
light microscopic sections. In the goat, as in guinea
pigs, parietal cells were more numerous than chief
cells.In other animal species examined, including man, chief cells were somewhat more common than parietal
cells.73,77,131,134 jn addition, when the regional
distribution of parietal and chief cellswas evaluated,
goats were again similar to guinea pigs. While in the other species examined, parietal cells occupied a smaller
percentage of the proximal mucosa of the stomach than in the distal part,*^* this uneven distribution was not 1 ^1 evident in guinea pigsA',J- or goats.
In the abomasa of goats receiving chronic pentagastrin treatment only slight morphological changes were observed with light microscopy. These changes included a slightly increased mucosal thickness (P>0.05) and a slightly increased parietal-chief cell ratio. Rats receiving much higher doses of pentagastrin (2000 ug/kg) had a significantly increased number of parietal cells and also had an Increased parietal-chief cell r a t i o . ^ It had been assumed that there was a threshold in the production of proliferative changes by pentagastrin. Pentagastrin initially caused increased cytoplasmic protein production resulting in increased cell volumes.^ In this study cells in the caprine abomasa did not have increased volumes as it was characterized by the average area of cytoplasmic fragments. A slightly increased number of parietal cells had a slightly decreased cell volume. In the proliferative zone of the abomasal glands of Group II animals had a slightly increased mitotic activity when it was evaluated semiquantitatively. In addition, goats in this treatment group had a slightly lengthened mucous neck region which was characterized by a slightly increased proportion of sialo- and sulfomucins.
Mucosubstances in the stomach of pentagastrin treated animals had not been studied previously by the 2 biphasic stains utilized in this experiment. Pentagastrin was thought to cause an increased rate of differentiation of progenitor cells rather than the increased rate of 7fi proliferation of progenitor cell.'0 It is also of interest, that morphometric studies on the gastric mucosa of patients with Zollinger-Ellison syndrome, revealed an increased number of mucous cells in addition to the QA marked increase in the number of parietal cells.00 It is also widely accepted that pentagastrin qualitatively has 84 the same effect as gastrin. In laboratory animals, following high doses (2000ug/kg) of chronic pentagastrin treatment, extragastric changes were observed.
These changes included a gross increase in the pancreatic mass with simultaneous tinctorial changes in the exocrine pancreas and were thought to be due to an increased production of structural proteins.^® In this experiment goats received a relatively low dose (75 ug/kg BID) of pentagastrin and there was no evidence of proliferative changes in the pancreas, although this organ was not evaluated by morphometric methods.
In the abomasa of Group III () ostertagi infected goats the observed inflammatory and proliferative changes were similar to those of Type I bovine ostertagiasis.*®*^ The inflammatory reaction in goats was primarily composed of lymphocytes, plasma cells and eosinophils. Similar to previous reports in cattle o o lymphoid follicles were formed multifocally in goats. *
The distribution of intraepithelial globule leucocytes in goats was also similar to that in cattle.*® The few dilated glands in parasitized caprine abomasa were lined by markedly flattened mucous cells as described for cattle.^ In addition, the proliferative changes in goat abomasa were similar in several aspects to adenomatous hyperplasia of mice and Menetrier's disease The abomasal mucosubstances in bovine ostertagiasis have not been characterized, but the changes in the abomasa of parasitized goats were similar to those of mice with, adenomatous hyperplasia.In the mouse stomach proliferating cells primarily produced sulfomucins, which was characteristic of poorly differentiated immature cells. These findings were contradictory to some recent
IOC studies in ovine £ circumcincta infection. In the sheep an increased proportion of neutral mucin production was found in parasitized abomasa. Intestinal metaplasia was not described in ostertagiasis before. In the rat intestinalization first developed in the antrum and later in the fundus. This metaplastic change was thought to be secondary to decreased gastric acidity. J
Morphometric studies have not been conducted on the abomasal mucosa in bovine ostertagiasis. An increased abomasal mucosal thickness was reported in sheep infected with 0 circumcincta.*^ The abomasal mucosa of parasitized goats in this study was significantly increased (P<0.05). The most important change in the cell population was the increase in the number of mucous epithelial cells. While in 0 circumcincta-infected sheep
1 0 7 marked loss of parietal cells was reportedAJ in £ ostertagi-infected goats only a slight decrease was noted in the number of parietal cells (P>0.05). Mitotic activity was especially high in the antrum in parasitized 86 goats as reported in other parasitic gastropathies.^ The proliferation of structural fibers as described in calves on was not a prominent feature in this study.
The reactive changes observed in the abomasal lymph nodes of 0 ostertagi infected goats were in agreement with previous observations in parasitized calves and sheep.
In conclusion, pentagastrin at 75 ug/kg BID for 28 days did not produce significant light microscopic changes in goat abomasa. Abomasal changes in 0 ostertagi- infected animals were similar to bovine ostertagiasis, therefore the goat could serve as a model for the bovine disease. The most important feature of this parasitic gastropathy was the proliferation of immature mucous epithelial cells which failed to differentiate in the normal fashion. 87
Table 1 -Distribution of mucosubstances in the abomasal fundus of control goats and following chronic pentagastrin treatment or experimental 0 ostertagi infection
Glandular region Control Pentagastrin Ostertagia
Stain surface/foveolae
PAS-AB R R/RP* RP/R
HID-AB 0/N 0/N N/NB/O neck
PAS-AB RP RP/BP RP/BP
HID-AB N N N/NB/B deep gland
PAS-AB 0 O/BP 0/RP/BP/B
HID-AB 0 . 0/N O/N/NB/B
R:PAS+; RP:PAS+predom; B:AB+; BP:AB+predom
N:HID+; NB:HID+predom; B:AB+; Otunstained
♦staining characteristics with decreasing frequency from left to right 88
Table 2 -Distribution of mucosubstances in the abomasal pyloric antrum of control goats and following chronic pentagastrin treatment or experimental 0 ostertaei infection
Glandular region Control Pentagastrin Ostertagia
Stain surface
PAS-AB R R RP
HID-AB 0 0 O/NB* foveolae
PAS-AB R/RP R/RP RP/R/BP
HID-AB N/0 N N/NB antral glands
PAS-AB RP RP/R/BP RP/BP/B
HID-AB N N N/NB
R:PAS+; RPsPAS+predom; B:AB+; BP:AB+predom
N:HID+; NB:HID+predom; B:AB+; 0:unstained
♦staining characteristic with decreasing frequency from left to right 89
Table 3 - Quantitative light microscopic findings in the
abomasal mucosa of control goat kids and following
chronic pentagastrin treatment or experimental 0
ostertagi infection
Control Pentagastrin Ostertagia
MUCOSAL THICKNESS (um)
338.0+69.7 345.0+73.2 524.4+69.9
AVERAGE NUMBER OF CELLS PER UNIT WIDTH (0.1mm)
Mucous 16.4+, 0.3 17.3_+ 2.3 28.3+ 6.0
Parietal 16.5+ 3.3 17.9+ 2.9 15.5+ 3.3
Chief 9.8+ 2.4 9.6+ 3.0 10.8+ 3.5
PARIETAL-CHIEF CELL RATIO
1.7+ 0.3 2.0+ 0.5 1 .6+ 0.6
AVERAGE AREA OF CYTOPLASMIC FRACTIONS (um2)
Mucous 89.2+17.4 72.2+ 6.8 78.6+ 8.0
Parietal 147.7+29.9 120.8+19.6 152.2+12.9
Chief 107.8+19.8 97.2+ 9.4 97.4+ 9.9 90
Fig. 1— Schematic drawing of a caprine abomasum opened along the greater curvature. The abomasum was cut into half along the lesser curvature (arrows) and 9 areas were randomly selected for morphometry. Fig. 2— Non-folded fundus of a control animal. The luminal surface is smooth and glands are straight and of equal length. H&E stain; x 200. 92
Fig. 3— Fundic fold of a control animal. The glands are straight and the fold is thin. H&E stain; x 79. Fig. 4— Non-folded fundus of a pentagastrin-treated animal. Abomasal glands are long and straight, the luminal surface exhibits a slight nodularity. H&E stain; x 79. 94
Fig. 5— Fundic fold of a pentagastrin-treated animal. The fold is thin, the glands of this animal are slightly dilated. Fig. 6— Non-folded fundus of an 0 ostertagi-infected animal. Marked nodularity and distortion of the glandular architecture with proliferation of columnar mucous cells are present. A coalescing multifocal hypercellularity is in the deep layers of the lamina propria. H&E stain; x 79. Fig. 7— Fundic fold of an 0 ostertagi-infected animal. The fold is thickened due to a prominent lymphoid follicle in the submucosa. H&E stain; x 79. 97
Fig. 8— Ostertagia ostertagi larvae in the abomasal gland of an infected goat kid. The larvae are in a gland lined by tall columnar cells. H&E stain; x 312.5. 98
Fig. 9— A dilated abomasal gland in an 0 ostertagi- infected goat kid. This gland in the transitional zone of the abomasum is lined by markedly flattened epithelial cells. A mononuclear cellular infiltrate is also present. H&E stain; x 200. Fig. 10— Herniation of an abomasal gland in an 0 ostertagi-infected goat kid. A portion of the gland extends into the submucosa. H&E stain; x 200. Fig. 11— Fundus of the abomasum of an 0 ostertagi- infected goat kid. Foveolar atrophy is characterized by the bridging of glands by surface epithelial cells. H&E stain; x 79. Fig. 12— Mononuclear cellular infiltrate in the lamina propria of the abomasum of a goat kid infected with ,0 ostertagi. The inflammatory cells are just below the surface epithelium. H&E stain; x 312.5. 102
Fig. 13— Intestinal metaplasia in the pyloric antrum of an 0 ostertagi-infected goat kid. The normal architecture is altered and mucous cells contain clear vacuoles. H&E stain x 312.5. 103
Fig. 14— Fundus of the abomasum of a control goat kid. Neck and surface mucous cells are positive for mucus while parietal and chief cells are negative. PAS-AB stain; x 312.5. Fig. 15— Fundus of the abomasum of a control goat kid. Sulfomucins are demonstrated only in the foveolar and neck region. HID-AB stain; x 200. Fig. 16— Fundus of the abomasum of an 0 ostertagi- infected goat kid. In the deep layers of the mucosa parietal cells are lost and are replaced by mucous epithelial cells. PAS-AB stain; x 79. 106
Fig. 17— Fundus of the abomasum of an 0 ostertagi- infected goat kid. An increased proportion of cells contain sulfomucins. Sialomucins are demonstrated focally in deeper parts of abomasal glands. HID-AB stain; x 79. CHAPTER IV
Ultrastructural Abomasal Changes Following
Ostertagia ostertagl Infection or Chronic Pentagastrin
Treatment of Goat Kids
Introduction
The ultrastructural changes in abomasa in Type I and
Type II bovine ostertagiasis have been reported.22,23 ^ g abomasa of infected animals exhibited degenerative, proliferative and inflammatory changes. The most important ultrastructural finding was the separation of the lateral plasmalemmata of epithelial cells by an electron-dense amorphous material which was thought to be plasma protein.^2 The pathogenesis of the infection was proposed based on the electron microscopic findings. Bovine ostertagiasis is characterized by abomasal hypoacidity, hypoalbuminemia and hyperpepsinogenemia.^** The changes in plasma constituents were explained by the development of a permeable abomasal wall. The poorly developed junctional complexes were incompletely closed between immature proliferative cells and enabled macromolecules to be 23 exchanged between abomasal lumen and circulation.
Ultrastructural studies were also conducted following pentagastrin treatment of rats and mice.76,138 no subcellular changes were noted in rats, mice exhibited
107 108
marked ultrastructural changes following pentagastrin
treatment. Most of the parietal cells of mice had an
increase in the intracellular canalicular and microvillous
system.A membrane recycling hypothesis was proposed
following studies of porcine parietal cells, a dyna
mic equilibrium was evident between the cytoplasmic 1 smooth membrane system and intracellular canaliculi.
Active acid secretion by parietal cells was paralleled by
an increase in the secretory surface of the canalicular
system with a simultaneous decrease in tubulo-
vesicular structures.
The purpose of this investigation was to characterize
the ultrastructural changes in 0 ostertaei infection and
following pentagastrin treatment in goat kids. In this
study changes in the 2 treatment groups were compared and
the goat was evaluated as a possible model animal for bovine ostertagiasis.
Materials and Methods
Experimental animals— Experimental animals originated from a European-type mixed-breed goat herd maintained on a parasite contaminated pasture. Kids were born within a 1 month time period and were removed from the pasture with their does within 48 hours after birth. A brick building served as housing for kids and does. No other ruminants were kept in this building during the 109 experiment. The wood-shaving bedding was changed weekly.
Animals were fed with a commercial feed, composed of 54% cotton seed hulls, 14% cotton seed meal, 24% cracked corn, 5% cane molasses, 0.5% oyster shell flour, and 0.4% trace minerals. Alfalfa hay was also offered at feeding time, and water and salt were available ad libitum. Rids were weaned at five weeks of age.
Experimental Design— Eighteen goat kids free of gastrointestinal nematodes were selected for the experiment one week before inoculation. The 2 to 3- month-old mixed breed goats were randomly divided into three treatment groups. Five kids in Group I did not receive treatment and five animals in Group II received
75 ug/kg pentagastrin twice daily for 28 days, starting on the seventh experimental day. The third group consisting of eight kids (Group III) were infected with
15,000 third larval stage (L3) of Ostertagia ostertagi.
Inoculation— The parasite inoculum originated from a bovine isolate of 0 ostertagi in Louisiana and was cultured using standard techniques. Briefly, fresh feces was mixed with vermiculite to form a crumbly, moist mass, and cultures were maintained at room temperature for two weeks. A Baermann funnel was filled with tepid water and the feces-vermiculite mixture was immersed in the water.
This system was left undisturbed overnight. In the morning larvae were collected in a large beaker and 110 were washed several times and stored in the refrigerator at 4 C. Larvae were examined for viability and 15,000 ,0 ostertagi L3 were placed into separate tubes. Animals were inoculated per os using disposable plastic pipettes.
Pentagastrin (Peptavlon,Ayerst Laboratories, New
York,NY) solution was prepared weekly with dimethyl sulfoxide (DMS0) and deionized water. Animals were injected subcutaneously with 75 ug/kg pentagastrin every
12 hours for 28 days, beginning on experimental day 7.
Necropsy procedures— Animals were euthanized by electrocution on the 35th and 36th postinfection day.
The kids were weighed and the forestomachs and abomasa
i were quickly removed from the carcasses. The corrected body weight was determined by substracting the weight of the forestomachs from the total body weight. A small opening was cut in the wall of the abomasum and the pH of the contents was measured. The abomasum was opened along the greater curvature, its contents were removed and the mucosa was gently washed with phosphate buffered saline solution (PBSS) pH 7.1 at room temperature to remove adhered particles. Care was taken to use only a small amount of washing fluid which was saved with the abomasal contents. The weight of the abomasum was recorded, and the abomasum was cut in half along the lesser curvature. One-half of the abomasum was processed for parasite recovery and the other half was slightly Ill
stretched and pinned to a styrofoam board and placed Into
fixative. Abomasa! lymph nodes were examined, removed and
sectioned for microscopic evaluation. All major visceral
organs were examined with special attention to endocrine
' organs and tissue sections were saved in fixative. The
brains of selected animals were also collected. All gross
observations were recorded and were presented in Chapter H.
Tissue Processing— Tissue samples were fixed in a
buffered mixture of 4 % formalin and 1 % of
.glutaraldehyde (4F16). Abomasa were slightly stretched and
pinned out for 24 hours immersed in fixative. At this
time sites were selected for morphometry, light and
electron microscopy.
For transmission electron microscopy (TEM) the
abomasa were minced following 24 hours fixation and were
further fixed in 4F1G. Tissues were processed routinely:
following fixation tissues were washed with 0.1 M sodium
cacodylate (NaCac) with 5% sucrose at pH 7.4. Tissues
were postfixed with 1% osmium tetroxide in 0.1% NaCac.
Samples were washed in deionized water and stained en
block with 2% uranyl acetate in 0.2 M sodium acetate
buffer pH 3.5. Following another wash in deionized water
samples were dehydrated in an ethyl alcohol series.
Tissues were placed into propylene oxide and later into a
mixture of propylene oxide and epoxy resin (Epon 812, 112
Araldite 6005, and dodecenyl succinic anhydride) containing
increasing concentrations of the resin, in the final step
dimethyl-amino-ethyl phenol served as a catalyst. Tissues were embedded in flat embedding molds and polymerized.
The 70 nm sections were placed on collodian coated 50 mesh copper grids, and were stained with 7% uranyl acetate and Reynolds lead citrate. Sections were examined with a Zeiss EM 10 electron microscope (Carl Zeiss Inc.,
Oberkochen, Vest Germany).
Small cubes (0.5 cra^) of abomasa were processed
for scanning electron microscopy (SEM). Following fixation in 4F1G samples were washed in PBSS and incubated in 1% diastase for 10 minutes at room temperature. Following digestion specimens were washed again and postfixed in 1% osmium tetroxide in 0.1 M NaCac. Tissues were washed in a
0.1 M NaCac buffer containing 5% sucrose. Samples were placed into a 1% tannic acid solution containing 0.2 M
NaCac and 1 N NaOH. The specimens were washed again in
0.1 M NaCac with 5% sucrose and placed into the osmium tetroxide solution. Samples were washed in deionized water and dehydrated through an ethyl alcohol series. Samples were critical point dried with C02» mounted on tissue stubs and sputter coated with gold palladium alloy. Tissues were examined with a Cambridge
Stereoscan S-150 Scanning Electron microscope at 20 kV
(Cambridge Instruments, Cambridge, England). 113
Results
TEM— Tissue sections from 2 areas of the abomasa were examined ultrastructurally. In the mucosa of the fundus and the pyloric antrum several distinct cell types were identified (Fig. 1).
In Group I control animals surface epithelial cells were slender, columnar with a few microvilli on their luminal surface. The apical portion of the surface mucous cells was densely packed with mucus granules which were usually electron-lucent and often had a sraal1, electron dense core. These cells were also characterized by eleborate interdigitations on their lateral surfaces.
Nuclei were usually basally located and oval, the cytoplasm contained a few profiles of RER, elongated mitochondria and supranuclearly located Golgi complex.
The shape of mucous neck cells in control animals varied from columnar to flask-shaped; most often it had a tapered basal and a wide luminal surface. The mucus granules were located in the lateral and apical portions of the cell and their staining characteristics varied. Some mucous neck cells had granules of moderate electron density, but the majority of the cells had electron lucent granules which often contained an electron dense spherical structure. The lateral surface of these cells was simpler than that of the surface mucous cells and exhibited only a few interdigitations. Another 114 characteristic of mucous neck cells was the nucleus which usually had several prominent invaginations.
The most distinctive feature of the parietal cells was the intracellular canalicular system encircling the nucleus and opening into the lumen of the abomasal gland.
The canalicular system was well developed in most of the cells in Group I animals and was lined by slender micro villi (Fig. 2 & 3). Mitochondria in parietal cells were wider than those of the mucus producing cells and were closely packed in the cytoplasm. The cytoplasm of parietal cells also contained multivesicular bodies and a few tubulo-vesicular structures. While the lateral surface of these cells was usually straight, the basal plasmalemmata formed elaborate infoldings. Parietal cells had a large, round, centrally located nucleus.
The main morphological feature of the abomasal zymogen cells in control animals was the large number of
RER profiles. Cisternae were often arranged in parallel stacks and were especially numerous in the basal part of the cytoplasm. Zymogen granules were homogenous and of moderate electron density and most often were located in the supranuclear and lateral cytoplasm. The well developed Golgi complex was also supranuclear. The nucleus of chief cells was also round. Mitochondria of zymogen cells were similar to those of mucous cells. The lateral plasmalemmata of these cells were fairly straight. 115
The antral gland cells of Group I animals were
columnar or conical In shape. Their luminal surface
contained a few microvilli and the lateral plasmalemmata
formed only a very few Interdigitations. Nuclei were
round to oval with the long axis usually parallel to the
basement membrane. The mucous granules in these cells
were pleomorphic In size and staining characteristics.
Granules were electron lucent or moderately electron
dense and some smaller granules were electron dense.
These cells had well developed Golgi complexes, few
mitochondria and a few often slightly dilated RER
profiles.
Endocrine cells were scattered between the other
cell types of the abomasa and were usually located in the
basal portion of the glands. Most of the endocrine cells
were oval in shape and were wedged between adjacent
epithelial cells and only a few reached the gland lumen
and had microvilli on their apical surfaces. The most
important morphological feature of these cells were the
numerous small, electron dense cytoplasmic granules which
had a varying sized of electron-lucent halo around their
central densities.
In the abomasa of control animals filamentous cells were rare and were usually located in the upper part of the
glands between mucous cells. These cells had a
characteristic pear shape with short, stubby microvilli on 116 their luminal surfaces (Fig. 4). Microfilaraents were especially abundant in the apical cytoplasm where a large number of small vesicles were also present. The nuclei of filamentous (brush) cells were round to oval with slight indentations. The cytoplasm contained only a few mitochondria, free ribosomes and cisternae of RER.
In Group II animals ultrastructural changes were limited to the parietal cells. Although morphometric studies were not performed on the electron micrographs, the majority of the parietal cells appeared to have extremely well developed intracellular canaliculi which were lined by many slender microvilli (Fig. 5). The long microvilli were especially prominent at the luminal opening of canaliculi (Fig. 6). Young parietal cells were characterized by plump mitochondria, multlvesicular bodies, a few RER profiles, electron dense granules and poorly developed canalicular system or intracellular canaliculi were not evident. Some of the cells which were lacking intracellular canalicular system had long microvilli on their luminal surface (Fig. 7). One animal in this treatment grou)> had an elevated abomasal pH of 3.3 at the time of necropsy. The cytoplasm of parietal cells was electron-lucent in this animal and intracellular canaliculi were dilated and most of them lacked microvilli.
In 0 ostertagi-infected Group III animals degenerative and proliferative changes and inflammatory 117 cellular Infiltration were evident. A large number of bacteria was diffusely present on the luminal surface of mucous epithelial cells (Fig.8 & 9). Some of the epithelial cells were degenerate with electron-lucent cytoplasm and pyknotic nuclei. Other degenerated epithelial cells contained large cytoplasmic vacuoles which in turn contained electron-dense flocculent material or membranous structures (Fig. 10). Parietal cells often appeared inactive with dilated intracellular canaliculi and few short microvilli and/or numerous tubulo-vesicular structures (Fig. 11). Very rarely chief cells contained unusually large granules, but most of the chief cells were relatively unaffected.
Some of the proliferative cells were immature mucous cells and were characterized by RER profiles, slender mitochondria and clear vesicles of approximately 200 nm close to the apical surface of the cells, other small vacuoles contained electron-dense cores (Fig. 10). Other immature cells were similar to mucous neck cells, but contained only a few small, electron-dense granules
(Fig. 12). Many of the proliferative cells had large nuclei and a small amount of cytoplasm. These cells contained a large number of plump mitochondria, a few RER profiles and multivesicular bodies, but intracellular canaliculi were not visualized. Young parietal cells had a poorly developed canalicular system and also contained 118
electron-dense granules (Fig. 13). In samples of pyloric
antrum with Intestinal metaplasia an unusual type of mucous cell was found, which had remarkable similarities
to Intestinal goblet cells. The apical portion of these
cells was crowded with homogenous, slightly angular
granules which varied In size and in electron density.
The Inflammatory cellular Infiltration was composed
of plasma cells, lymphocytes and eosinophils in the
lamina propria (Fig. 1A). The intraepithelial globule
leucocytes had large drop shaped granules with a markedly electron-dense core and smaller round electron-dense granules (Fig. 15).
SEM— In the abomasa of Group I control animals regular surface convolutions were evident (Fig. 16).
Gastric pits were narrow and evenly spaced. Surface epithelial cells were concave and had a cobblestone appearance (Fig. 17). The abomasal surface of Group II pentagastrin-treated animals was similar to that of the control goats (Fig. 18). In contrast, marked surface alterations were noted in the abomasa of Group III animals. Multifocally the regular architecture with the evenly spaced surface convolutions and gastric pits was obliterated. The coalescing irregular nodularity of the surface of effected abomasa gave a cauliflower-like appearance to the surface (Fig. 19). The grossly described nodules had a central depression surrounded by 119 flattened epithelial cells (Fig. 20). Gastric pits in these areas varied in size and were unevenly distributed
(Fig. 21).
Discussion
Ultrastructurally the abomasa of Group I control animals had similar morphology to the gastric mucosa of monogastric animals and other ruminants. Studies in monogastric animals correlated functional activity with the morphology of parietal cells.*®®“*^® In these studies the cells secreting acid had well developed intracellular canaliculi lined by many microvilli.
Inactive parietal cells had dilated canaliculi with only short microvilli and the cytoplasm of these cells also contained tubulo-vesicular structures. Since ruminants secrete HC1 continously most of the parietal cells of goats appeared to be active. In an examination of the fine structure of the bovine abomasal epithelia, the majority of the parietal cells had well developed canalicular system*-^* as reported here in the goat.In addition to the commonly used markers for parietal cells
(plump mitochondria and intracellular canaliculi) in this study multivesicular bodies were also considered to be markers for parietal cells. Multivesicular bodies were thought to be involved in the activation or deactivation of parietal cell s e c r e t i o n * ® ® * a n d were often found in 120 cells with poorly developed canalicular system or were observed in cells when canaliculi could not be visualized.
The morphology of mucous and antral gland cells in goat abomasa was similar to those of other mammals. A slight difference in staining characteristics manifested as decreased electron density of mucus granules and was attributed to the use of general fixative instead of the routinely used glutaraldehyde.
In the abomasa of control goats filamentous cells were rarely found. In a comprehensive study of tuft cells of rats it was noted that the majority of this cell type was present in the cardia*®^ and these cells occured in decreasing frequency in the fundus and antrum.
In this study only the fundus and the pyloric antrum were evaluated due to the very narrow cardiac region in the , caprine abomasum. In goats as in rats most of the filamentous cells were found in the mucous neck region of the abomasal glands. The distribution of bovine filamentous cells was similar, although they appeared to be more common.While fIbrillo-vesicular cells of dogs*®^ and tuft cells of rats*®^ had similar morphology to the filamentous (brush) cells of goats in this study, bovine filamentous cells had a somewhat different morphology*®^ In the bovine these cells appeared to have a secretory function and contained small electron dense 121
granules. In other mammals these cells are thought to
have absorptive function and contain pinocytotic
vesicles.
In this study several types of endocrine cells were
recognized, but in the lack of immunohistochemical and morphometric techniques they were not identified. There were no striking changes in the APUD cell population of
the treatment groups.
The subcellular morphology of parietal cells was
evaluated in laboratory rodents following pentagastrin
treatment. The quantitative ultrastructure of the
parietal cells of rats was not significantly different in 76 the pentagastrin group from that of the controls. ° In another study a strikingly homogenous parietal cell population was found in mice following the intraperitoneal injection of 125 ug/kg pentagastrin.^®®
These cells were characterized by extensive intracellular canaliculi which were occluded by the markedly elongated microvilli. Although morphometry was not performed in the present experiment on electon micrographs, animals in
Group II had extensively developed intracellular canaliculi and very long, fine microvilli. One pentagastrin-treated animal had an increased abomasal pH at the time of necropsy and parietal cells of this animal had the characteristic appearance of inactive cells.
In the 0 ostertagi-infected group the 122 ultrastructural changes were similar to those described in bovine ostertagiasis.^ A striking difference between the 2 experiments was in the separation of lateral plasmalemmata. While in the bovine disease the junctional complexes of the poorly differentiated proliferative cells were poorly developed and a moderately electron- dense material separated epithelial cells, this was not evident in the goats. This difference could be attributed to the severity of the disease. While in the bovine experiment animals had a heavy parasite burden and had 9 9 clinical signs of illness, in this experiment abomasal lesions were produced by a much lower parasite inoculum and clinical disease was not present. The majority of the proliferative cells in this study was more differentiated than those in affected cattle, which can be due to the time of termination of this experiment or to the fact that the goat abomasa were not as severely affected.
Moreover, when the separation of the lateral plasmalemmata was evaluated in Menetrier's disease in man, changes were slight and were appreciated following morphometry.®® The proliferative cells in goats were of an heterologous population as it was described in the bovine, although most of the cells in the goats appeared to be more differentiated and contained a larger number of cell organelles. In addition, immature mucous cells of goats contained small round or angular vesicles in the 123 apical cytoplasm, and similar vesicles in monogastric animals were demonstrated to produce sulfomucins. An increased proportion of sulfomucin production was described in ,0 ostertagi-infected goats in Chapter III,
Filamentous cells were found to proliferate in bovine ostertagiasis, but the filamentous (brush) cell remained a rare cell type in the goats following infection. There were no striking changes in the endocrine cell population of 0 ostertagi-infected animals.
In the infected goats mucous epithelial cells have undergone degeneration in situ more frequently than in controls. This was probably due to a non-specific effect of the parasite and was described in rat stomachs following stress.Another feature of degenerative changes in 0 ostertagi-infected animals was the formation of intracellular vacuoles containing flocculent or membranous material. These changes were also described in monogastric animals and were associated with cell death.The parietal cells of goats in Group III animals were often inactive with similar morphology as described in bovine ostertagiasis.^ The intracellular canaliculi were dilated and cells contained an increased number of tubulo-vesicular structures.
The inflammatory cell infiltrate in parasitized goats was similar to that of the cattle.^ Plasma cells, lymphocytes and eosinophils were identified in the lamina 124 propria. The globule leucocytes of goats had similar morphology to the ovine globule leucocyte.
During the preparation of specimens for SEM examination, similar difficulties were encountered as previously reported.While some of the mucus was not removed from the abomasal surface by enzyme digestion during gentle agitation of vials, other epithelial cells were damaged. Physical damage was characterized by irregularly broken areas on epithelial cells, similar as reported following brushing of the gastric mucosa of mice.^^ In addition, a varying number of cells in all three treatment groups exhibited a honeycomb appearance.
A similar morphology was observed in man in erosive gastritis and in sheep where it was attributed to the presence and feeding activity of the abomasal nematode,
Haemonchus con tortus.*^* The morphology of the abomasal mucosal surface in Group I and Group II goats was similar to other mammals.The SEM morphology of ostertagiasis in cattle or sheep has not been reported. The findings of the marked surface alterations of the abomasal mucosa in this study were in concert with gross and light microscopic observations.
When the parietal cell population of Group II and
Group III animals was compared, striking differences were noted. The majority of parietal cells in pentagastrin- treated animals were active and had abundant microvillous 125 lining in well-developed intracellular canaliculi. Most of the parietal cells following 0 ostertagi infection were inactive and contained tubulo-vesicles and the 'dilated canalicular system had only a few short microvilli. Young parietal cells in both treatment groups were primarily identified by the presence of a large number of plump mitochondria and multivesicular bodies. In Group III animals the young parietal cells were only a small percentage of the proliferative cell population. In contrast, the proliferative changes in pentagastrin treated animals were not a prominent feature of the abomasal morphology, but immature cells were mainly young parietal cells.
In conclusion, pentagastrin treatment of goats had similar effect to that of insulin or pentagastrin injection of mice. Ostertagia ostertagi infection in goats was ultrastructurally similar to bovine ostertagiasis in several aspects, therefore goat could serve as an inexpensive model of the bovine disease. Additionally, the immature proliferative cells in this infection were similar to the poorly differentiated cells of monogastric animals hence this parasitic infection could be also used to study proliferative gastropathies of unknown etiology. Fig. 1— Transmission electron micrograph (TEM) of the abomasal fundus of a control goat kid. In a deeper part of the abomasal gland chief cells (C), a parietal cell (P) and endocrine cells (E) are recognized. Uranyl acetate and lead citrate stain; bar«2um. 127
Fig. 2— TGM of the parietal cells of a control goat kid. The well developed intracellular canaliculi are lined by slender microvilli. The cytoplasm of parietal cells is crowded by plump mitochondria. Parietal cells are characterized by basal Infoldings (arrow head) and multivesicular body (arrow). Uranyl acetate and lead citrate stain; bar«lum. 128
Fig. 3— TEM of the luminal opening of a parietal cell from a control goat kid. The intracellular canaliculus is lined by many microvilli, mitochondria are plump. Several multivesicular bodies (arrowhead) are present. Uranyl acetate and lead citrate stain; bar»0.5um. 129
Fig. 4— TEM of a filamentous (brush) cell of a control goat kid. This cell is characterized by bushy microvilli, microfilaments (arrow) and vesicular structures (arrowhead). Uranyl acetate and lead citrate stain; bara0.5um. km1B * V ^
Fig. 5— TEM of parietal cells from a pentagastrin-treated goat kid. The extremely well developed intracellular canalicular system is lined by many slender microvilli. Uranyl acetate and lead citrate stain; bar«lura. Fig. 6— TGM of the luminal opening of a parietal cell from a pentagastrin-treated goat kid. Intracellular •canaliculi are lined by long microvilli. Uranyl acetate and lead citrate stain; bar«lum. 132
Fig. 7— TEM of a young parietal cell from a pentagastrin- treated goat kid. Intracellular canaliculi are not evident, but long microvilli are present on the luminalsurface. This cell also contains plump mitochondria and electron-dense granules. Uranyl acetate and lead citrate stain; bar^lum. 133
Fig. 8— TEM of the luminal surface of mucous cells from a control goat kid. The surface of mucous cells is void of organisms. Uranyl acetate and lead citrate stain; bar«lum. 134
Fig. 9— TEM of the luminal surface of mucous cells from an 0 ostertagi-infected goat kid. Bacteria are adhered to the surface of mucous cells. Uranyl acetate and lead citrate stain; bar^lum. 135
Fig. 10— TEM of a degenerate mucous cell from an 0 ostertagi-infected goat kid. A cytoplasmic vacuole contains flocculant material in this immature mucous cell. Just below the surface 200 nm vesicles are present (arrowhead). In a young mucous neck cell vacuoles contain electron-dense cores (arrow). The parietal cell has electron-lucent cytoplasm and dilated canaliculi. Uranyl acetate and lead citrate stain; bar**lum. 136
Fig. 11— TEM of an inactive parietal cell from an ,0 ostertagi-infected goat kid. The cytoplasmic canaliculi are dilated and have few short microvilli. The tubulo- vesicular structures (T) are numerous. Uranyl acetate and lead citrate; bar^lum. Fig. 12— TEM of a young mucous neck cell from an ,0 ostertagi-infected goat kid. The small electron-dense granules occasionally contain an electron-dense core (arrow). Uranyl acetate and lead citrate, bar«lura. 138
Fig. 13— TEM of a young parietal cell from an 0 ostertagi-infected goat kid. The intracellular canaliculi are poorly developed. An electron-dense granule is present. Uranyl acetate and lead citrate stain; bar»lum. Fig. 14— TEM of the inflammatory infiltrate from the lamina propria of an 0 ostertagi-infected goat kid. Several plasma cells T?) and an eosinophil (E) are present. Uranyl acetate and lead citrate; bar»2um. 140
Fig. 15— TEM of a globule leucocyte from an 0 ostertagi- infected goat kid. The granules vary in size and electron-density. Uranyl acetate and lead citrate stain; bar«luro. Fig. 16— Scanning electron micrograph (SEM) of the abomasum of a control goat kid. Regularly spaced surface convolutions and glandular openings are present (arrow). Several cells were damaged during processing. Bars25um. 142
Fig. 17— SEM of surface epithelial cells from a control goat kid. Epithelial cells are concave and have a cobblestone appearance. Bar-lOum. 143
Fig. 18— SEM of surface epithelial cells from a pentagastrin-treated goat kid. Cells are similar to that of controls. Bar«10um. 144
I
Fig. 19— SEM of the abomasum of an 0 ostertagi-infected goat kid. The normal architecture is obliterated due to coalescing irregular nodularity. Bar=50um. 145
Fig. 20— SEM of an abomasal nodule from an 0 ostertagi- infected goat kid. The nodule has a central depression. BaralOOum. 146
Fig. 21— SEM of the abomasum from an 0 ostertagi-infected goat kid. The focally flattened appearance of the mucosal surface is surrounded by proliferative changes. BarsSOum. CHAPTER V
Summary and Conclusions
,0 ostertagi produced patent infection in goats with increased serum pepsinogen and gastrin immunoreactivity.
This parasitic abomasitis was characterized by the proliferation of poorly differentiated cells which produced sialo- or sulfomucins. Following pentagastrin treatment goats showed only slight morphological changes. In this study the goat appeared to be a suitable host for£ ostertagi. Also, this study suggested that hormonal influences were important in the pathogenesis of ,0 ostertagi infection. In addition, similar to other proliferative gastropathies, the incomplete maturation of proliferative cells occurred.
The parallel increase of serum gastrin immunoreactivity and pepsinogen suggested that hyperpepsinogenemia could be due to the stimulating effect of gastrin and was not the result of a permeable abomasal wall as previously reported for bovine ostertagiasis.
Observations on gastric diseases and hyperpepsinogenemia in man also support this new hypothesis. The sharpest increase in gastrin immunoreactivity was measured at the tissue phase of the parasite and was probably due to the loss of parietal cell function secondary to the
147 148 presence of the parasite.
One animal had high gastrin immunoreactivity before inoculation with 0 ostertagi. During the experiment changes in gastrin immunoreactivity and pepsinogen were similar in this animal to those of the other 7 goats in the group.
This animal had a low fecal egg count and a low number of parasites were recovered from the abomasum. Moreover, while in other animals most of the parasites were adults, in this individual developing and early fourth larval stages were also found. At the time of necropsy this animal had the highest abomasal pH measured. Light microscopic examination revealed several Eimeria-like macroschizont in the abomasum. It appeares that decreased abomasal acidity was inhibitory to the development of 0 ostertagi in this animal. These findings are in agreement with a previous experiment where cimetidine caused increased abomasal pH and inhibited the development of the parasite.
The gross morphology of the goat abomasa following 0 ostertagi infection was similar to the gross lesions in bovine ostertagiasis, but individual nodules were larger than in cattle. The multifocal, white, raised, umbilicated nodules were more numerous in the fundus.
Parasitized abomasa showed marked nodularity with light microscopy as well as with SEM. Microscopically the parasitized abomasa exhibited proliferative and inflammatory changes. The most important morphological 149 feature of Infected abomasa was the proliferation of poorly differentiated mucous cells. Differences were noted in the distribution of mucosubstances in the abomasa of control and parasitized animals. An increase was found in the proportion of sialo- and sulfomucin containing cells. This observation was also supported with TEM; immature epithelial cells contained small vesicles or small secretory granules. The majority of the parietal cells in 0 ostertagi-infected animals appeared inactive with TEM as was expected based on the increased abomasal pH at necropsy.
The significant increase in the abomasal mucosal thickness of 0 ostertagi-infected goats was primarily due to a significantly increased number of mucous epithelial cells.
The coalescing multifocal inflammatory reaction in parasitized goat abomasa was similar to that of bovine ostertagiasis. Prominent aggregates were formed by lymphocytes, plasma cells, eosinophils and globule leucocytes. In addition, abomasal lymph nodes were hyperplastic in Group III animals as reported for bovine ostertagiasis.
In this experiment a relatively low dose of penta- gastrin was administered to goats. Similarly to previous report in other species no adverse reaction was found following treatment. At necropsy only slight changes were 150
appreciated. In this group the thickness of abomasal
mucosa was slightly Increased and the mucous neck region was lengthened and characterized by the slightly
Increased proportion of sialo- and sulfomucin producing
cells. The most striking morphological change in this
treatment group was the increase in the cytoplasmic
canalicular system of parietal cells which contained many
slender, long microvilli. Similar changes were reported in
mice following pentagastrin treatment.
It is widely accepted that pentagastrin has
qualitatively the same effect as gastrin and that both cause
parietal cell proliferation selectively without affecting
the other epithelial cell types in the stomach. In ()
ostertagi-infected goats young parietal cells represented
only a small fraction of proliferative cells. These
findings suggested that due to unknown reasons the maturation of proliferative cells was altered. Similar
observations were made in other hypergastrinemic
proliferative gastropathies in the absence of a parasitic agent. Moreover, while in Zollinger-Ellison syndrome the majority of the proliferative cells differentiated into
parietal cells, there was also an increase in the number of mucous epithelial cells. These observations suggest that in some pathologic conditions an unidentified factor modifies the effect of gastrin.
In conclusion, the goat infected with 0 ostertagi could serve as an excellent experimental model for bovine ostertagiasis. In addition, this host-parasite system may serve as an adequate model for proliferative gastropathies due to unknown etiologies of other mammalian species. BIBLIOGRAPHY
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New York 1974. Curriculum Vitae
Name: Judit E. Markovits
Birthdate: March 26,1953
Place of birth: Budapest, Hungary
Education:
D.V.M. University of Veterinary Science 1978 Budapest, Hungary
Ph.D. Louisiana State University 1986 School of Veterinary Medicine Department of Pathology
Doctoral Dissertation:
Effect of experimental Ostertagia ostertagi infection or chronic pentagastrin treatment on abomasal pathophysiology and morphology of goats.
Honors:
Charles L. Davis, D.V.M. Foundation 1985 Veterinary Pathology Scholarship Award
Major Professional Interests:
Comparative pathology Gastroenterology Disturbances of growth Endocrinology
Professional Experience:
1982-1986 Graduate assistant Department of Pathology School of Veterinary Medicine Louisiana State University Baton Rouge, LA
1982 January-July Diagnostic veterinarian VDACS Animal Health and Dairies Laboratory Harrisonburgh, VA
172 173
1981 Diagnostic veterinarian HDA Animal Health Laboratory College Park, MD
1978-1980 Researcher CNS Laboratory Pharmaceutical Research Institute Budapest, Hungary
Teaching Experience:
Diagnostic pathology to senior veterinary students (VM 5452)
Gross pathology to sophomore veterinary students (VM 5211 & 5221)
Professional Organizations:
American Veterinary Medical Association
Presentations:
J.E. Markovits, M.A.M. Turk, T. Hribernik, T.G. Snider: Chronic active hepatitis in a Doberman Pinscher, 11th Annual Southeastern Veterinary Pathology Conference; Tifton, GA, 1983.
J.E. Markovits, R.I. Miller: Nasal phaeohyphomycosis in a cat, 12th Annual Southeastern Veterinary Pathology Conference; Tifton, GA, 1984.
J.E. Markovits, T.G. Snider, et al: Experimental infection of goat kids with Ostertagia ostertagi. Annual Meeting of Animal Disease Research Workers in the Southern States; Gainesville, FL, 1986.
Publications:
S.M. Shane, J.E. Markovits, T.G. Snider, S. Harrington: Encephalitis attributed to dactylariasis in Japanese Quail chicks (Coturnix coturnix japonica). Avian Diseases 1985;29:822-828.
D.S. Greco, M.E. Petersen, D.Y. Cho, J.E. Markovits: Juvenile-onset hypothyroidism in a dog. J Am Vet Med Assoc 1985;187:948-950. 174
S. Barr, B.C. Baker, J.E. Markovits: Trypanosomiasis and laryngeal paralysis in a dog. J Am Vet Med Assoc 1986;188:1307-1309. I
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate: Judlt E. Markovits
Major Field: Veterinary Pathology
Title of Dissertation:Effect of Experimental Ostertagia ostertagi infection or Chronic Pentagastrin treatment on Abcmasal Pathophysiology and Morphology in Goats
:d:.
Chairman
Dean of 'th e Graduate School
EXAMINING COMMITTEE:
Date of Examination:
July 16, 1986