A macro- and light-microscopical study of the pathology of gousiekte in sheep

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INTRODUCTION

Gousiekte is characterised by a latent period of approximately four to eight weeks between exposure of animals to the plant material and natural death. Macroscopical lesions indicative of congestive heart failure are present in most cases. A diagnosis of gousiekte is traditionally confirmed by demonstrating the presence of “typical” microscopic lesions, namely necrosis, replacement fibrosis, and round cell infiltrates of varying intensity, especially in the sub- endocardial region of the apex and the left ventricular free wall (Theiler, Du Toit & Mitchell 1923; Newsholme & Coetzer 1984; Kellerman et al. 2005).

Dosing trial

Ten Merino sheep approximately 12 months old (ewes and wethers) were dosed per stomach tube with dried, milled Pachystigma pygmaeum plant material (table 3.1). P. pygmaeum (hairy gousiektebossie) plants were collected from Swartrand (260 17’S, 26048’E) in the North-West Province of South Africa where gousiekte is rife. The plant material was dried in the shade, milled to a coarse powder and stored at –100 C. P. pygmaeum was selected for the trial because it was the most readily obtainable of the gousiekte plants and farmers annually reported a high incidence of gousiekte in the area. It was therefore highly probable that the plants would be toxic. The South African National Biodiversity Institute in Pretoria verified the identification of the plants.

Pathology

All treated animals either died naturally or were euthanased with an overdose of pentobarbitone sodium when in extremis, between 31 and 51 days after the commencement of dosing (table 3.1). The control animals were euthanased at the time when the last experimental animal was necropsied (day 51). Animals were necropsied immediately after euthanasia. Animals that died naturally were necropsied as soon as possible after death but no later than two to three hours after death. At necropsy, for this study, three to four transmural blocks of tissue measuring approximately 1 cm3 were collected from the middle of the left free ventricular wall of all experimental and control animals and preserved in 10 % buffered formalin. Specimens from various organs, including the lungs, liver, spleen, kidney, gastrointestinal tract and brain, were also collected in 10 % buffered formalin from each case following a complete necropsy. The samples were routinely processed for histopathological examination and stained with haematoxylin and eosin (HE). Two transmural planes were sectioned from each myocardial block to allow examination of both the endo- and the epicardium. Selected sections were stained with Masson’s trichrome stain for collagen (Armed Forces Institute of Pathology 1968).

Imaging analysis

For imaging analysis, stained sections (HE and Masson’s trichrome) from two control animals (control group) and three of the treated animals (sheep 1, 6 and 10) were photographed with an Olympus BX 50 microscope using a CC12 soft imaging system. The scanned photomicrographs were imported to a drawing template of the 1TEM software imaging system and scaled to the original print of the photograph by using the “bar”. Measurements were taken with the 1TEM soft imaging system. The three treated animals were selected on the basis of their latent periods, namely 31, 42 and 51 days respectively, which represented the entire spectrum of the latent period (table 3.1). The following measurements were taken of not fewer than 15 randomly selected fibres that had full nuclear profiles in each animal in the subendocardial region of the left free ventricular
wall: myofibre diameter at the level of the centre of the nucleus (fig. 3.1), nucleus perimeter, and area.

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Dedication
Acknowledgements
Declaration
CHAPTER ONE: Introduction Objectives of this study
CHAPTER TWO: Literature review
2.1 The different plants that cause gousiekte
2.1.1 Pachystigma pygmaeum (Schltr.) Robyns (Rubiaceae)
2.1.2 Pachystigma thamnus Robyns (Rubiaceae)
2.1.3 Pachystigma latifolium Sond (Rubiaceae)
2.1.4 Fadogia homblei (= F. monticola) De Wild (Rubiaceae)
2.1.5 Pavetta harborii S. Moore (Rubiaceae)
2.1.6 Pavetta schumanniana F. Hoffm. (Rubiaceae)
2.2 Clinical signs
2.3 Macroscopical lesions
2.4 Light-microscopical lesions
2.5 Transmission electron microscopical lesions
2.6 Pathophysiology
2.7 Toxic principle in gousiekte plants
2.8 Pavetta harborii and pavetamine as a cardiotoxin in rats
2.9 Heart failure
2.9.1 Acute heart failure
2.9.2 Congestive heart failure
2.9.3 Intrinsic cardiac responses to reduced cardiac output
2.9.3.1 Dilated cardiomyopathy
2.9.3.2 Hypertrophic cardiomyopathy
2.9.3.3 Restrictive cardiomyopathy
2.10 Hypotheses
CHAPTER THREE: A macro- and light-microscopical study of the pathology of gousiekte in sheep
3.1 Introduction
3.2 Materials and methods
3.2.1 Dosing trial
3.2.2 Pathology
3.2.3 Imaging analysis
3.3 Results
3.3.1 Macropathology
3.3.2 Histopathology
3.3.3 Imaging analysis
3.3.3.1 Descriptive statics
3.4 Discussion
3.5 Conclusions
CHAPTER FOUR: A transmission electron microscopical study of the myocardial lesions in sheep with gousiekte
4.1 Introduction
4.2 Materials and methods
4.2.1 Dosing trial
4.2.2 Pathology
4.2.2.1 Light-microscopy
4.2.2.2 Transmission electron microscopy
4.3 Results
4.3.1 Light-microscopy
4.3.2 Transmission electron microscopy
4.4 Discussion
CHAPTER FIVE: A study of the pathological lesions in rats exposed to pavetamine
5.1 Introduction
5.2 Materials and methods
5.2.1 Pavetamine extraction
5.2.2 Experimental animals and dosing regimen
5.2.3 Pathology
5.2.3.1 Transmission electron microscopy (TEM)
5.2.3.2 Light-microscopy
5.2.3.3 Imaging analysis
5.2.3.4 Statistical analysis
5.3 Results
5.3.1 Clinical signs
5.3.2 Macroscopical examination
5.3.3 Light-microscopical examination
5.3.4 Transmission electron microscopical examination
5.3.5 Statistical analysis
5.4 Discussion
CHAPTER SIX: General discussion and conclusions on the pathogenesis of gousiekte
6.1 Introduction
6.2 Effect of pavetamine on heart muscle
6.3 Myocardial lesions
6.4 Evidence of ventricular failure
6.5 Compensatory mechanisms
6.6 Conclusions
6.7 Proposed future research areas
BIBLIOGRAPHY