Histomorphology, ultrastructure and fatty acid composition of the adipose tissue in pansteatitis, the potentials in understanding the underlying mechanism and diagnosis of pansteatitis in the Nile crocodile.

BACKGROUND: In an effort to characterize the fat body and other adipose tissue in the Nile crocodile and the effects of pansteatitis on the structure and composition of the adipose tissue, we evaluated the regional variation in structure and fatty acid composition of healthy farmed crocodiles and those affected by pansteatitis. METHODS: Adipose tissue samples were collected from the subcutaneous, visceral and intramuscular fat and the abdominal fat body of ten 4-year old juvenile crocodiles from Izinthaba Crocodile Farm, Pretoria, South Africa while pansteatitis samples were collected from visceral and intramuscular fat of crocodiles that had died of pansteatitis at the Olifant River, Mpumalanga, also in South Africa. Histomorphology, ultrastrustucture and fatty acid composition by fatty acid methyl ester (FAME) analysis were conducted. RESULTS: Histological examination showed regional variations in the adipose tissue especially in the collagen content of the ECM, tissue perfusion and division into lobes and lobules by fibrous capsule. Considerable fibrosis, mononuclear cell infiltration especially by macrophages and lymphocytes and toxic changes in the nucleus were observed in the pansteatitis samples. Regional variation in lipid composition especially in Myristoleic (C14:1), Erucic acid (C22:1n9), and Docosadienoic acid (C22:2n6) was observed. Most of the saturated and trans fatty acids were found in significant quantities in the pansteatitis samples, but had very low levels of the cis fatty acid and the essential fatty acids with C18 backbone. CONCLUSION: This study demonstrates that there exists some regional variation in histomorphology and fatty acid composition in the healthy adipose tissue of the Nile crocodile. It also showed that pansteatitis in the Nile crocodile might have been triggered by sudden change in energy balance from consumption of dead fish; and probable exposure to toxic environmental conditions with the evidence of up scaled monounsaturated long chain fatty acids composition and toxic changes in the leucocytes observed in pansteatitis in the present study.

A fluorescent investigation of subcellular damage in H9c2 cells caused by pavetamine, a novel polyamine.

Gousiekte, which can be translated literally as "quick disease", is one of the six most important plant toxicoses that affect livestock in South Africa. It is a plant-induced cardiomyopathy of domestic ruminants characterised by the sudden death of animals within a period of 4-8weeks after the initial ingestion of the toxic plant. The main ultrastructural change in sheep hearts is degradation of myofibres. In this study, fluorescent probes were used to investigate subcellular changes induced by pavetamine, the toxic compound that causes gousiekte, in H9c2 cells. The sarcoplasmic reticula (SR) and mitochondria showed abnormalities that were not present in the control cells. The lysosomes of treated cells were more abundant and enlarged than those of the control cells. There was increased activity of cytosolic hexosaminidase and acid phosphatase, indicating increased lysosomal membrane permeability. Lysosomes play an important role in both necrosis and apoptosis. The degradation of the myofibres may be a consequence of the increased lysosomal membrane permeability. Pavetamine was also found to cause alterations in the organisation of F-actin. F-actin in the nucleus is a transcription regulator and can therefore influence protein synthesis. Actin filament organisation also regulates the cardiac L-type Ca(2+) channels. Fluorescent staining demonstrated that pavetamine may damage a number of organelles, all of which can influence the proper functioning of the heart.

Damage to some contractile and cytoskeleton proteins of the sarcomere in rat neonatal cardiomyocytes after exposure to pavetamine.

Pavetamine, a cationic polyamine, is a cardiotoxin that affects ruminants. The animals die of heart failure after a period of four to eight weeks following ingestion of the plants that contain pavetamine. This immunofluorescent study was undertaken in rat neonatal cardiomyocytes (RNCM) to label some of the contractile and cytoskeleton proteins after exposure to pavetamine for 48 h. Myosin and titin were degraded in the RNCM treated with pavetamine and the morphology of alpha-actin was altered, when compared to the untreated cells, while those of beta-tubulin seemed to be unaffected. F-actin was degraded, or even absent, in some of the treated cells. On an ultrastructural level, the sarcomeres were disorganized or disengaged from the Z-lines. Thus, all three contractile proteins of the rat heart were affected by pavetamine treatment, as well as the F-actin of the cytoskeleton. It is possible that these proteins are being degraded by proteases like the calpains and/or cathepsins. The consequence of pavetamine exposure is literally a "broken heart".

Cytotoxicity and ultrastructural changes in H9c2(2-1) cells treated with pavetamine, a novel polyamine.

Intake of pavetamine, a novel polyamine, synthesized by certain rubiaceous plants, is the cause of gousiekte ("Quick disease") in ruminants. The disease is characterized by a latent period of 4-8 weeks, followed by heart failure. The aim of this study was to firstly investigate the cytotoxicity in H9c2(2-1) cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and LDH (lactate dehydrogenase) release assays. Maximum cell death occurred after pavetamine exposure of cells for 72h at a concentration of 200muM (55%+/-9.84), as measured by the MTT assay. LDH release was only observed after 72h exposure to pavetamine. Secondly, the ultrastructural changes induced by pavetamine in H9c2(2-1) cells were investigated. Changes in the mitochondria and sarcoplasmic reticula were observed. The nucleus was not affected during the first 48h exposure of cells to pavetamine and no chromatin condensation occurred. However, after 72h exposure to pavetamine, the nucleus became fragmented and membrane blebbing occurred. It was concluded that the ultimate cell death of H9c2(2-1) cells treated with pavetamine, was through necrosis and not apoptosis. Thirdly, the effect of pavetamine on the mitochondrial membrane potential (DeltaPsi) was evaluated by using the JC-1 (5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) and TMRM (tetramethylrhodamine methyl ester perchlorate) probes. Pavetamine treatment led to significant hyperpolarization of the mitochondrial membrane potential. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly, indicating that the MPTP (mitochondrial permeability transition pore) plays no role in the cytotoxicity of pavetamine.

A case-based, small-group cooperative learning course in preclinical veterinary science aimed at bridging basic science and clinical literacy.

In 1999 a dedicated problem-based learning course was introduced into the lecture-based preclinical veterinary curriculum of the University of Pretoria. The Introduction to Clinical Studies Course combines traditional lectures, practical sessions, student self-learning and guided tutorials. The self-directed component of the course utilises case-based, small-group cooperative learning as an educational vehicle to link basic science with clinical medicine. The aim of this article is to describe the objectives and structure of the course and to report the results of the assessment of the students' perceptions on some aspects of the course. Students reacted very positively to the ability of the course to equip them with problem-solving skills. Students indicated positive perceptions about the workload of the course. There were, however, significantly lower scores for the clarity of the course objectives. Although the study guide for the course is very comprehensive, the practice regarding the objectives is still uncertain. It is imperative to set clear objectives in non-traditional, student-centred courses. The objectives have to be explained at the outset and reiterated throughout the course. Tutors should also communicate the rationale behind problem-based learning as a pedagogical method to the students. Further research is needed to verify the effectiveness of this course in bridging the gap between basic science and clinical literacy in veterinary science. Ongoing feedback and assessment of the management and content are important to refine this model for integrating basic science with clinical literacy.

Toxicity, pathophysiology and pathology in sheep following dosing of the nephrotoxic plant Nolletia gariepina (DC) Mattf.

Ingestion of the plant Nolletia gariepina was confirmed as the cause of acute mortalities in cattle in the Kuruman area of the Northern Cape Province of South Africa. The aim of this trial was to investigate the toxic effects of this plant with respect to clinical signs, pathophysiology and pathology using the sheep as a model. At dosages of 1.5 g dried, milled plant material/kg body mass there were no detectable abnormal findings, while at dosages of 2.8-3.0 g/kg most of the animals died acutely. In subacutely affected sheep, depression, inappetance, teeth grinding, tachycardia, weak ruminal movements and recumbency were noticed. The most prominent pathophysiological changes observed, included a sharp rise in non-protein nitrogen substances in the plasma, remarkable decline in glomerular filtration rate, increase in sodium and potassium excretion, and a rise in urine gamma glutamyltransferase activity. Macroscopically a severe nephrosis was present in all the animals. The most important findings detected histologically were necrosis of the proximal convoluted tubular epithelium and large numbers of protein casts in the lumens.

Changes in the renal handling of urea in sheep on a low protein diet exposed to saline drinking water.

Previous trials have demonstrated that sheep on a low protein diet and free access to water, and sheep dosed with boluses of NaCl intraruminally also with free access to water, showed decreases in urea loss via the urine compared to control animals. We monitored urea excretion in sheep on a relatively poor protein diet when they were exposed to saline drinking water, i.e. they were unable to vary their intake of NaCl:water. Sheep on isotonic saline drinking water (phase 3) excreted significantly more urea via the urine (284 mM/day) compared to phase 1 when they were on non-saline drinking water (urea excretion = 230 mM/day) and phase 2 when they were on half isotonic saline drinking water (urea excretion = 244 mM/day). This finding was explained by the high glomerular filtration rate (GFR) 91.9 l/day, compared to 82.4 l/day (phase 1) and 77.9 l/day (phase 2), together with a significantly raised fractional excretion of urea (FEurea) (51.1 %) during this phase, and was in spite of the significantly lower plasma concentrations of urea in phase 3 compared to phase 1. The FEurea probably results from the osmotic diuresis caused by the salt. There were indications of a raised plasma antidiuretic hormone (ADH) concentration and this would have opposed urea loss, as ADH promotes urea reabsorption. However, this ADH effect was probably counteracted to some extent by a low plasma angiotensin II concentration, for which again there were indications, inhibiting urea reabsorption during the phases of salt loading. As atrial natriuretic peptide both increases GFR and decrease sodium reabsorption from the tubule, it was probably instrumental in causing the increase in GFR and the increase in the fractional excretion of sodium (FE(Na)).

Neurophysiological correlates of affiliative behaviour between humans and dogs.

Few physiological parameters for positive human-companion animal contact have been identified and those that are established have all been in humans. The implication is that if the physiological reactions are mutual, dogs would experience the same psychological benefits from these neurophysiological changes as humans. Therefore, we have determined the role of certain neurochemicals during affiliation behaviour on an interspecies basis. Our results indicate that concentrations of beta-endorphin, oxytocin, prolactin, beta-phenylethylamine, and dopamine increased in both species after positive interspecies interaction, while that of cortisol decreased in the humans only. Indicators of mutual physiological changes during positive interaction between dog lovers and dogs may contribute to a better understanding of the human-animal bond in veterinary practice.

The effect of an angiotensin-converting enzyme inhibitor on water and electrolyte balance in water-restricted sheep.

The importance of angiotensin II in the regulation of water and electrolyte balance in sheep is questionable. In this trial the effects of an angiotensin-converting enzyme (ACE) inhibitor were quantified in sheep on restricted water intake. Comparing the phase of water restriction only with that of water restriction plus ACE inhibition, significant increases were observed during the latter phase in urine volume, sodium and potassium excretion via the urine, sodium concentration in the plasma and osmolar clearance. Urine osmolarity decreased with inhibition of angiotensin II formation while variables such as water, sodium and potassium loss via the faeces were unaffected. Most of the renal effects of ACE inhibition, except the increase in urinary potassium excretion, were explicable in terms of the established functions of angiotensin II. Furthermore, results of this trial indicate that angiotensin II has no significant effect on the intestine in regulating water and electrolyte excretion via the faeces.

Water and electrolyte intake and output in conditioned Thoroughbred horses transported by road.

The objectives of this trial were to measure the water and electrolyte intake and loss, of horses during road transportation in relatively hot environmental conditions. Six mature, Thoroughbred horses in full training were used in a balanced crossover design. The horses were conditioned on a treadmill for 6 weeks before the start of the trial in order to simulate the type of horse that is transported most often over long distances in South Africa. The horses were assigned randomly to one of 2 treatment groups. On a particular day 3 horses were transported, while the other horses served as controls. One month was allowed before the crossover. Horses in the 'transport group' were transported by tarred road in a circular route over 600 km. This allowed the standardisation of measurements and use of the same instruments in both groups. Data were collected in each treatment group during transportation that lasted for 8 h (transport phase) and for 6 h after travelling (recovery phase). The following data were collected or calculated: Water and electrolyte (sodium, potassium and chloride) intake and output, changes in bodyweight and feed consumption. Although water was always available, the transport group failed to drink during transit. Based on bodyweight, the transported horses were 3% dehydrated at the end of transit. This bodyweight loss was corrected within one hour after their return due to a significantly higher water intake compared to control horses. The feed intake in the transported horses was unaffected during travelling, but was decreased for 6 h following transportation. Urinary water loss was similar in the 2 treatment groups during and following transportation. The faecal water loss decreased in the transported horses and remained lower than the control group for 6 h following transit. Total sodium and chloride intake were unaffected, while the potassium intake was decreased during transportation. Sodium and potassium loss via the faeces and urine during the transport period were similar in the 2 treatment groups, whereas potassium output in the transport group was significantly decreased during the recovery period of the study. It was concluded that transportation by road affected the water and electrolyte balance of conditioned horses for a period up to 6 h after travelling.

Water and electrolyte homeostasis in sheep without functional colons.

The regulatory role of the colon in water and electrolyte balance and the renal compensation which follows impairment of colonic function were assessed using sheep with ileorectal anastomosis (ILRAN sheep) on restricted and free water intake as experimental models. Faecal electrolyte loss sustained by the ILRAN group was eight to nine times greater than that in the control animals. When water was available ad lib., ILRAN sheep lost 2.81 and 0.51 more water per day via the faeces and urine, respectively, than the controls. Urine volume in the ILRAN sheep comprised largely electrolyte-free water and the renal retention of water was entirely secondary to the high degree of sodium reabsorption in these animals compared with the controls. On restricted water intake, the urine volume of the ILRAN sheep declined due to retention of electrolyte-free water and even greater absorption of sodium (and hence water by osmosis) by the kidney tubules. The latter observation was substantiated by a decrease in fractional excretion of sodium from 0.29 to 0.08% when water intake was restricted. Plasma aldosterone concentration was markedly elevated in the ILRAN sheep as a result of the excessive loss of sodium and water via the faeces. Activation of the renin-angiotensin-aldosterone sequence is believed to underlie the increased sensation of thirst (ILRAN sheep drank on average about 2.51 more water per day than the controls), the homeostatic response by the kidneys and the relatively lower plasma potassium in the ILRAN sheep compared to controls.

Changes in kidney function and faecal excretion of water and electrolytes with sodium chloride loading in sheep.

The renal and faecal routes of water and electrolyte excretion in sheep were compared and changes in kidney function assessed when similar amounts of sodium chloride (NaCl) were dosed in free and fixed ratios in water. Sheep (n = 6) either had free access to fresh drinking water (control phase) or 0.9% saline drinking water, or NaCl was dosed intraruminally with free access to fresh drinking water. In the final phase of the investigation, sheep were dosed intraruminally with NaCl while water intake was restricted. Variables monitored included fresh or saline water intake, water, sodium and potassium loss via the urine and faeces, glomerular filtration rate (GFR), fractional excretions of sodium (FENa) and potassium (FEK) and solute free water clearance (CH2O). Results indicated that where NaCl intake was high, sodium excretion via the urine was of far greater importance than that via the faeces and that changes in kidney function which occurred in order to excrete excess sodium, included increases in GFR and FENa, and declines in FEK and CH2O. Where significant differences were obtained in variables between the phases of high salt intake, these were attributed to differences in sodium absorption from the gastrointestinal tract. It was concluded that when NaCl intake is high, sodium homeostasis is chiefly maintained by increasing the amount of sodium made available to the nephron tubule (increased GFR), by decreased tubular reabsorption of this sodium and by increasing solute free water reabsorption.

The effects of salt loading via two different routes on feed intake, body water turnover rate and electrolyte excretion in sheep.

The effect of dosing identical amounts of sodium chloride, via 2 different routes, on feed intake and water and electrolyte balance was investigated in sheep. Feed intake and plasma sodium concentrations were unaffected by salt loading, while water intake, fractional turnover of body water, plasma and urine potassium concentrations and urine sodium concentration changed significantly from control values (P less than 0.05). With a few exceptions, parameters were in general similar irrespective of the route whereby sodium chloride was administered.

Validation of the tritiated water dilution technique used to estimate fractional turnover rates of body water in sheep.

Daily fractional water turnover rate (i.e. that proportion of total body water which is exchanged daily) was determined in S.A. Mutton Merino sheep (n = 6) under thermoneutral conditions by direct measurement of total water intake and by the tritiated water dilution technique. No significant difference between direct and indirect methods was observed in any of the animals. Tritiated water space estimated from a plasma sample taken at 6 h after tritium administration in general overestimated this compartment when compared with values derived from the zero time intercept of the linear regression analysis obtained for each animal.