Caprine besnoitiosis: an emerging threat and its relationship to some other infections of ungulates by Besnoitia species.

Caprine besnoitiosis, caused by the cyst-forming protozoal apicomplexan Besnoitia caprae appears to be endemic in Kenya, Nigeria and Iran, but has yet to be detected in other parts of the world. The infection causes an important parasitic disease of goats in affected developing countries. Bovine besnoitiosis, is a widespread disease of cattle in Africa, Asia (but not Iran) and southern Europe. Recent epidemiological data confirm that the incidence and geographical range of bovine besnoitiosis in Europe is increasing, which is why growing attention has been given to the condition during the past decade. This paper reviews pertinent information on the biology, epidemiology, pathology, clinical signs, diagnosis and control of caprine besnoitiosis, together with its similarities to, and differences from, bovine besnoitiosis. The serious economic consequences of besnoitiosis on goat breeding and local meat and hide industries is also considered.

A long-term in vivo investigation on the effects of xenogenous based, electrospun, collagen implants on the healing of experimentally-induced large tendon defects.

AIM: This study was designed to investigate the effect of novel 3-dimensional (3-D) collagen implants on the healing of large, experimentally-induced, tendon-defects in rabbits. METHODS: Forty mature male white New Zealand rabbits were divided randomly into treated and control groups. Two cm of the left Achilles tendon was excised and the gap was spanned by Kessler suture. In the treated group, a novel 3-D collagen implant was inserted between the cut ends of the tendon. No implant was used in the control group. During the course of the experiment the bioelectrical characteristics of the healing and normal tendons of both groups were investigated weekly. At 120 days post injury (DPI), the tendons were dissected and inspected for gross pathology, examined by transmission and scanning electron microscopy, and their biomechanical properties, percentage dry matter and hydroxyproline concentration assessed. RESULTS: The collagen implant significantly improved the bioelectrical characteristics, gross appearance and tissue alignment of the healed, treated tendons, compared to the healed, control scars. It also significantly increased fibrillogenesis, diameter and density of the collagen fibrils, dry matter content, hydroxyproline concentration, maximum load, stiffness, stress and modulus of elasticity of the treated tendons, as compared to the control tendons. Treatment also significantly decreased peri-tendinous adhesions, and improved the hierarchical organization of the tendon from the collagen fibril to fibre-bundle level. 3-D xenogeneic-based collagen implants induced newly regenerated tissue that was ultrastructurally and biomechanically superior to tissue that was regenerated by natural unassisted healing. CONCLUSION: This type of bioimplant was biocompatible, biodegradable and appeared suitable for clinical use.

Fifty years of the British Equine Veterinary Association as a facilitator of progress in equine clinical science.

The British Equine Veterinary Association (BEVA) was established in 1961 and launched the Equine Veterinary Journal (EVJ) in 1968. This review outlines some of the major advances in equine science and practice that have occurred in that time and the role played by the Journal in facilitating those developments.

Histopathologic and ultrastructural studies on experimental caprine besnoitiosis.

The distribution pattern and associated tissue reactions with progressive changes in Besnoitia caprae cysts were investigated in 6 experimentally infected 16- to 20-month-old male goats. Each goat was subcutaneously inoculated with approximately 13 × 10(8) B caprae bradyzoites. The animals were examined daily for development of clinical besnoitiosis, and skin biopsies from distal parts of the limbs were taken at weekly intervals. At 15, 30, 60, 120, 180, and 365 days postinfection (DPI), 1 goat was euthanized. Samples were collected at autopsy from various organs for histologic and ultrastructural studies. No cysts were seen in tissue sections on 15, 30, and 365 DPI, but large numbers were present at 60, 120, and 180 DPI in the skin of the distal limbs, scrotum, and ears, with fewer in the tongue, palate, sclera, testicles, and spermatic cord. No cysts were seen in the lungs, liver, kidneys, spleen, central nervous system, or lymph nodes. Cyst numbers peaked at 60 DPI, then declined from 120 to 180 DPI. Degenerated cysts were relatively rare at 60 DPI but more numerous at 180 compared with 120. A granulomatous reaction--predominantly characterized by macrophages, lymphocytes, and plasma cells--surrounded each degenerated cyst. All goats showed testicular tubular degeneration with little or no spermatogenic activity. The sizes of cysts and their wall thickness, with the size of bradyzoites and some of their organelles, exhibited progressive chronologic changes.

The use of µCT technology to identify skull fracture in a case involving blunt force trauma.

A 40-year-old man was admitted to hospital with a scalp wound but died 22 days later after unsuccessful treatment. Initial assessment of the cranial fragments removed during surgery revealed fine fracture lines on the endocranial surface, and a dark arcuate line on the ectocranial surface. To investigate the extent of the fractures a µCT scan of the fragments was taken, examined in 3D, and compared to plain radiographs. Some fractures were found to extend through the full thickness of the skull. This case presents a novel application of µCT technology to forensic radiology.

Transforming growth factor-betas in a rat model of neonatal posthaemorrhagic hydrocephalus.

Posthaemorrhagic ventricular dilatation (PHVD) is a common complication of intraventricular haemorrhage in premature infants. The aim of this study was to investigate the role of transforming growth factor-betas (TGF-betas), a family of polypeptides with potent desmoplastic properties, in the aetiology of PHVD in a newly developed neonatal rat model of this disorder. Pups were injected with citrated rat blood or artificial cerebrospinal fluid (ACSF) into alternate lateral ventricles on postnatal days 7 and 8. The brains were perfusion-fixed 14 days later and immunohistochemistry was performed for TGF-beta1, -beta2 and -beta3, p44/42 mitogen-activated protein (MAP) kinases, and the extracellular matrix proteins laminin, vitronectin and fibronectin. Ventricular dilatation occurred in 58.3% of animals injected with blood and 36.7% of those injected with ACSF. Periventricular immunoreactivity for TGF-beta1 and -beta2 increased in injected animals irrespective of the presence or absence of ventricular dilatation, although the levels of both isoforms tended to be higher in animals with hydrocephalus. TGF-beta3 immunoreactivity was elevated in hydrocephalic rats only. The immunolabelling for phosphorylated p44/42 MAP kinases rose in a pattern similar to that for TGF-beta1 and -beta2. Expression of TGF-betas was accompanied by deposition of the extracellular matrix proteins fibronectin, laminin and vitronectin. The changes caused by injection of ACSF were the same as those caused by injection of blood. Our results raise the possibility that expression of TGF-betas, together with extracellular matrix protein deposition, may be involved in the development and/or maintenance of hydrocephalus after ventricular distension due to haemorrhage in the neonate.

Tissue oxygen tension and brain sensitivity to hypoxia.

Mammalian brain is a highly oxidative organ and although it constitutes only a small fraction of total body weight it accounts for a disproportionately large percentage of bodily oxygen consumption (in humans about 2 and 20%, respectively). Yet, the partial pressure and concentration of oxygen in the brain are low and non-uniform. There is a large number of enzymes that use O(2) as a substrate, the most important of which is cytochrome c oxidase, the key to mitochondrial ATP production. The affinity of cytochrome c oxidase for oxygen is very high, which under normal conditions ensures undiminished activity of oxidative phosphorylation down to very low P(O(2)). By contrast, many other relevant enzymes have K(m) values for oxygen within, or above, the ambient cerebral gas tension, thus making their operations very dependent on oxygen level in the physiological range. Among its multiple, versatile functions, oxygen partial pressure and concentration control production of reactive oxygen species, expression of genes and functions of ion channels. Limitation of oxygen supply to the brain below a 'critical' level reduces, and eventually blocks oxidative phosphorylation, drastically decreases cellular (ATP) and leads to a collapse of ion gradients. Neuronal activity ceases and if oxygen is not re-introduced quickly, cells die. The object of this review is to discuss briefly the central oxygen-dependent processes in mammalian brain and the short-term consequences of O(2) deprivation, but not the mechanisms of long-term adaptation to chronic hypoxia. Particular emphasis is placed on issues which have been the focus of recent attention and/or controversy.

Effective selective head cooling during posthypoxic hypothermia in newborn piglets.

Selective head cooling has been proposed as a neuroprotective intervention after hypoxia-ischemia in which the brain is cooled without subjecting the rest of the body to significant hypothermia, thus minimizing adverse systemic effects. There are little data showing it is possible to cool the brain more than the body. We have therefore applied selective head cooling to our hypoxia-ischemia piglet model to establish whether it is possible. Nine piglets were anesthetized, and brain temperature was measured at the surface and in the superficial (0.2 cm) and deep (1.7-2.0 cm) gray matter. Rectal (6-cm depth), skin, and scalp temperatures (T) were recorded continuously. Lowering T-rectal from normothermia (39 degrees C) to hypothermia (33.5-33.8 degrees C) using a head cap perfused with cold (6-24 degrees C) water was undertaken for up to 6 h. To assess the impact of the 45-min hypoxia-ischemia insult on the effectiveness of selective head cooling, four piglets were cooled both before and after the insult, and four, only afterward. During selective head cooling, it was possible to achieve a lower T-deep brain than T-rectal in all animals both before and after hypoxia. However, this was only possible when overhead body heating was used. The T-rectal to T-deep brain gradient was significantly smaller after the insult (median, 5.3 degrees C; range, 4.2-8.5 degrees C versus 3.0 degrees C; 1.7-7.4 degrees C; p = 0.008). During rewarming to normothermia, the gradient was maintained at 4.5 degrees C. We report for the first time a study, which by direct measurement of deep intracerebral temperatures, validates the cooling cap as an effective method of selective brain cooling in a newborn animal hypoxia-ischemia model.

Interactions of bioactive glasses with osteoblasts in vitro: effects of 45S5 Bioglass, and 58S and 77S bioactive glasses on metabolism, intracellular ion concentrations and cell viability.

In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass caused extra- and intracellular alkalinization, a rise in [Ca2+]i and [K+]i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H+] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca2+]i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.

Methylmalonate toxicity in primary neuronal cultures.

Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.

Glucose-induced intracellular ion changes in sugar-sensitive hypothalamic neurons.

In the lateral hypothalamic area (LHA) of rat brain, approximately 30% of cells showed sensitivity to small changes in local concentrations of glucose. These "glucose-sensitive" neurons demonstrated four types of behavior, three of which probably represent segments of a continuous spectrum of recruitment in response to ever more severe changes in blood sugar. Type I cells showed maximum activity

Effects of intrastriatal injection of quinolinic acid on electrical activity and extracellular ion concentrations in rat striatum in vivo.

Changes in neuronal activity and extracellular concentrations of ions were measured in rat striatum for 60-90 min after intrastriatal injection of quinolinic acid, an agonist of the N-methyl-D-aspartate receptor. The excitotoxin induced bursts of synchronous electrical activity which were accompanied by rises in [K+]e (to approximately 6 mM) and decreases in [Ca2+]e (by less than 0.1 mM); [H+]e usually increased (0.1-0.3 pH unit) after a short and small (< 0.1 pH unit) alkaline shift. The magnitude and frequency of these periodic changes decreased with time; after 90 min the amplitudes fell to 10-20% of the early values and the frequency to about one every 8 min as compared to one every 2-3 min immediately after quinolinate injection. By 90 min there was an increase in [K+]e from 3.3 mM to 4.2 mM and a decrease in [Ca2+]e from 1.34 mM to 1.30 mM. It is postulated that activation of the N-methyl-D-aspartate receptor causes disturbances in neuronal activity and ion gradients; restoration of the original ionic balances raises utilization of ATP and places an additional demand on energy-producing pathways. Increased influx of calcium into neurons may lead to an enhanced accumulation and subsequent overload of mitochondria with the cation. This, in turn, could result in dysfunction of the organelles and account for the decrease in respiration and [ATP]/[ADP] that have been observed previously in this model. The results of the present study lead to the conclusion that quinolinic acid produces early changes in activity of striatal neurons and movements of several cations which may contribute to subsequent abnormalities in energy metabolism and ultimately, cell death.

Energetic demands of the Na+/K+ ATPase in mammalian astrocytes.

Cultured astrocytes and cell lines derived therefrom maintain a high energy level ([ATP]/[ADP]) through operation of oxidative phosphorylation and glycolysis. The contribution from the latter to total ATP production is 25-32%. A powerful Na+/K+ pump maintains potassium, sodium, and calcium gradients out of equilibrium. [Na+]i is about 20 mM, [K+]i is 130 mM and [Ca2+]i is less than 100 nM. Under non-stimulated conditions, the Na+/K+ ATPase consumes 20% of astrocytic ATP production. Inhibition of the pump by ouabain decreases energy expenditure, raises [creatine phosphate]/[creatine], and leads to a leakage of sodium, potassium, and calcium ions. Decrease in the pump function via a fall in [ATP] also collapses ion gradients; the rate and extent of the fall correlates positively with cellular energy state. Under "normal" conditions (i.e., when ATP production pathways are not inhibited), there appears to be no preferential utilization of energy produced by either glycolysis or oxidative phosphorylation for the support of pump function.

Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells.

Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na+]i and [Ca2+]i rose while [K+]i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na+]i and [K+]i observed during energy depletion were gradual and progressive whereas those in [Ca2+]i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na+]i were usually smaller than reductions in [K+]i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.

Limitation of glycolysis by hexokinase in rat brain synaptosomes during intense ion pumping.

Incubation of rat brain synaptosomes under conditions of either increased energy utilization (addition of Na+ channel opener, veratridine, or ionophores, monensin and nigericin) or inhibition of oxidative phosphorylation (addition of rotenone), or a combination thereof, decreased [ATP], increased [ADP] and stimulated glycolysis. The rates of lactate generation were linear over a 15-min interval in the presence of rotenone alone but decreased in the other two conditions. During the first 5 min, the amount of lactate formed with veratridine, monensin or nigericin was as high or higher than with rotenone, but it was lower in the last 10 min. With a combination of one of the stimulators of ion movements and rotenone the rate of glycolysis was always markedly lower than with each compound added singly. The stimulated rates of lactate formation correlated positively with the synaptosomal content of [ATP]. After 15 min, [ATP] was 0.9-1.0 nmol/mg with rotenone, 0.5-0.9 nmol/mg with veratridine (or ionophores), and <0.3 nmol/mg with a combination of the two. Under the conditions used, calcium did not affect glycolytic activity directly. The Lineweaver-Burk plot of the rate of lactate formation against [ATP] yielded a straight line with a Km for ATP of about 0.1 mM, which is very similar to the Km for this nucleotide of brain hexokinase bound to mitochondria. In C6 cells glycolytic rate measured with a combination of an ionophore and rotenone was higher than with each of these compounds added singly while [ATP] never declined below about 9 nmol/mg prot. It is concluded that in synaptosomes, the high rate of energy utilization required for intense ion movement decreases [ATP] to a level that limits hexokinase activity kinetically. This may contribute to a reduction in the rate of glycolysis and hence energy production in brain hypoxia and ischemia.

Calcium handling by hippocampal neurons under physiologic and pathologic conditions.

Despite the complexity of the mechanisms that control free calcium concentration in neural cells, considerable advances have been made recently in the understanding of the entry and exit pathways of the ion through application of selective channel and receptor blockers. In addition, useful knowledge has been gained of the internal regulation of calcium movements and the factors that lead to mobilization of the ions into, or their sequestration from, the cytosol. It is clear that calcium homeostasis is crucial to cell metabolism and survival, and that relatively small deviations from the norm can have serious or lethal consequences. It appears that many of the experimental tools are now available to assist in the elucidation of the mechanisms controlling intracellular calcium concentration in vitro. Nevertheless, it has to be accepted that however valuable results from model systems may be, the final testing of any hypothesis concerning the importance of calcium homeostasis in the intact brain requires extensive experimental and clinical investigations in vivo. Information obtained in vitro, if convincingly confirmed by in vivo studies, may well be crucial in formulating strategies to combat a wide range of pathologic conditions.

The effect of pH on glycolysis and phosphofructokinase activity in cultured cells and synaptosomes.

The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK-21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [P(i)] ([ATP]i, [ADP]i, and [P(i)]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [P(i)], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] and lower [glucose-6-phosphate] and [fructose-6-phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [P(i)] rose under all pHe conditions. Simultaneously [glucose-6-phosphate] and [fructose-6-phosphate] decreased whereas [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)