The role of laboratories in animal-related disasters and emergencies.

This paper outlines the role of laboratories in animal-health-related disasters and emergencies, with a particular focus on biological threats - intentional, accidental and natural. Whilst multisectoral coordination is increasingly recognised as necessary for effective preparedness and response to all kinds of disasters, the role of the laboratory is often overlooked. The laboratories' involvement, not just in the response, but across all phases of disaster management - mitigation, planning, response and recovery - is essential, not only for improved animal health but for preservation of livelihoods and for food security, social cohesion and economic stability.

Les auteurs examinent le rôle des laboratoires dans les catastrophes et les urgences affectant les animaux, en se centrant particulièrement sur les menaces biologiques (qu'elles soient intentionnelles, accidentelles ou naturelles). Si la nécessité d'une coordination multisectorielle pour une préparation et une intervention efficaces face aux catastrophes de toute nature est désormais mieux perçue, le rôle des laboratoires est souvent négligé. La participation des laboratoires est essentielle, non seulement dans les interventions mais aussi dans chacune des phases de la gestion des catastrophes, à savoir l'atténuation, la planification, l'intervention et le redressement et ce, non seulement pour améliorer la santé animale, mais aussi pour préserver les moyens de subsistance et la sécurité alimentaire, la cohésion sociale et la stabilité économique.

Los autores exponen a grandes líneas la función que cumplen los laboratorios frente a desastres o emergencias que guardan relación con el mundo animal, prestando especial atención a las amenazas biológicas, ya sean de origen natural, accidental o intencionado. Mientras que por un lado se asienta cada vez más la idea de que la coordinación multisectorial es indispensable para una eficaz labor de preparación y respuesta ante todo tipo de desastres, a menudo se pasa por alto la función que en la materia incumbe a los laboratorios. Es esencial que estos participen no solo en las labores de respuesta, sino en todas las fases del proceso de gestión de desastres (mitigación, planificación, respuesta y recuperación), y ello no solo para mejorar la situación zoosanitaria, sino también para preservar los medios de sustento y proteger la seguridad alimentaria, la cohesión social y la estabilidad económica.

Development and validation of a 3ABC antibody ELISA in Australia for foot and mouth disease.

OBJECTIVE: To measure the diagnostic performance of an Australian-developed ELISA for the detection of antibodies against the non-structural proteins (NSP) 3ABC of the foot and mouth disease (FMD) virus. DESIGN: Test development and validation study. METHODS: The diagnostic specificity was determined using 2535 sera from naïve animals and 1112 sera from vaccinated animals. Diagnostic sensitivity was calculated from the data for 995 sera from experimentally and field-infected animals from FMD-endemic countries in South East Asia. A commercial ELISA detecting antibodies against FMD virus NSP was used as the reference test to establish relative sensitivity and specificity. Bayesian latent class analysis was performed to corroborate results. The diagnostic window and rate of detection were determined at different times using sera from cattle, sheep and pigs before and after infection, and after vaccination and subsequent infection. Repeatability and reproducibility data were established. RESULTS: At 35% test cut-off, the 3ABC ELISA had an overall diagnostic sensitivity of 91.5% and diagnostic specificity of 96.4%. The diagnostic sensitivity in vaccinated and subsequently infected cattle was 68.4% and diagnostic specificity in vaccinated cattle was 98.0%. CONCLUSIONS: The 3ABC ELISA identified field and experimentally infected animals, as well as vaccinated and subsequently infected animals. Diagnostic sensitivity and specificity estimates for other FMD NSP tests are comparable with the results obtained in this study. This NSP ELISA was found to be 'fit for purpose' as a screening assay at the herd level to detect viral infection and also to substantiate absence of infection.

Modelling spatio-temporal patterns of long-distance Culicoides dispersal into northern Australia.

Novel arboviruses, including new serotypes of bluetongue virus, are isolated intermittently from cattle and insects in northern Australia. These viruses are thought to be introduced via windborne dispersal of Culicoides from neighbouring land masses to the north. We used the HYSPLIT particle dispersal model to simulate the spatio-temporal patterns of Culicoides dispersal into northern Australia from nine putative source sites across Indonesia, Timor-Leste and Papua New Guinea. Simulated dispersal was found to be possible from each site, with the islands of Timor and Sumba highlighted as the likely principal sources and February the predominant month of dispersal. The results of this study define the likely spatial extent of the source and arrival regions, the relative frequency of dispersal from the putative sources and the temporal nature of seasonal winds from source sites into arrival regions. Importantly, the methodology and results may be applicable to other insect and pathogen incursions into northern Australia.

Evaluation of long-distance dispersal of Culicoides midges into northern Australia using a migration model.

The introduction of novel bluetongue serotypes and genotypes into northern Australia is considered possible via the long-distance windborne dispersal of Culicoides (Diptera: Ceratopogonidae) vectors from Southeast Asia. Initial findings from simulation modelling of potential dispersal over a 15-year period revealed that the greatest risk for incursion of windborne Culicoides from the island of Timor into northern Australia occurs during December-March. The regions at greatest risk for incursion include the top end of the Northern Territory and the Kimberley region in Western Australia, but there is potential for more widespread dispersal into northern Australia based on Timor as the putative source. The establishment of a more pathogenic strain of the virus, or of a novel Culicoides vector introduced by such inter-continental dispersal events, could dramatically alter Australia's current bluetongue disease status.

H5N1 highly pathogenic avian influenza in Southeast Asia.

Since the first H5N1 highly pathogenic avian influenza virus (HPAIV) infection in the region in August 2003, Cambodia, Laos, Malaysia, Myanmar, Indonesia, Thailand and Vietnam have all recorded outbreaks of the disease. The HPAIV continues to occur in some countries in Southeast Asia despite control programmes encompassing surveillance, vaccination and stamping out strategies. A number of strains have been circulating in the region since the first outbreaks in 2003, and although the source of the initial outbreaks in domestic poultry is not known, the continuing propagation of disease in the region is primarily the result of the movement of domestic poultry and poultry products, and people. A comprehensive approach using all the strategies available to break the chain of transmission of the virus in poultry will be needed to achieve lasting disease control.

A light and transmission electron microscope study of the distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the zebrafish eye.

The distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the eyes of the zebrafish, Danio rerio (Hamilton), were investigated using light and transmission electron microscopy. A comparative study of the quality of preservation provided by three different fixation procedures revealed no consistently striking general differences. However, somewhat subjectively, the fixative containing Millonig's buffer did consistently provide better fixation of myelin. Overall, nerve processes, depending on the site studied, were distributed as either (1) bundles (in the choroid near the optic nerve head and in the choroid adjacent to the limbus), (2) linear arrays (in the junction between the sclera and cartilage and in the choroid adjacent to the retina) or (3) individual units (in the choroid under the cartilage or in the sclera). Both myelinated and unmyelinated processes were identified in these locations. Myelinated processes usually contained both neurofilaments and neurotubules, but a few apparently contained only neurofilaments. Unmyelinated processes usually contained mainly neurotubules, but a few apparently contained only neurofilaments. Taken together, these findings indicate innervation of extra-retinal structures, as seen in zebrafish, is highly conserved among vertebrates, further supporting the use of zebrafish as a model for the vertebrate visual system.

A light and transmission electron microscope study of hepatic portal tracts in the rhesus monkey (Macacus rhesus).

This study reports on morphological features of hepatic portal tracts in the liver of a rhesus monkey. The light microscope shows that the number of each type of principal component comprising a portal tract varies but that there are usually one to five lymphatics, one bile ductule, one bile duct, one arteriolar and one arterial branch of the hepatic artery, and one hepatic portal vein. Bile ductules, in cross section, have 6-10 cells (mostly low pyramidal, but with a few cuboidal) bordering the lumen, an outside diameter of from about 20 to 25 microm, and a luminal diameter of from 2 to 10 microm. Bile ducts, in cross section, have more than 10 cells (about equal numbers of low pyramidal and cuboidal) bordering the lumen, an outside diameter greater than 25 microm and a luminal diameter of greater than 10 microm. The term "pyramidal" has not previously been applied to the cells of the ductules and ducts. The monkey tracts show several cytological features previously undescribed, viz., abortive cilia and basal bodies in the duct cells, abortive cilia in the ductule cells, and an occasional aggregation of ribosomes in arterial endothelial cells. They also show a major histological feature previously mentioned but not illustrated, viz., bundles of nerve processes which exhibit a preferential location, i.e., proximity to the arterioles and arteries.

Ultrastructural features of Glisson's capsule and the overlying mesothelium in rat, monkey and pike liver.

Samples from the liver of a male rat (Sprague-Dawley), a monkey (Macacus rhesus), and a longnose gar pike (Lepisosteus osseus) were studied in a transmission electron microscope to provide cytological and histological information about structures previously poorly documented in the literature. Glisson's capsule consisted of dense, irregular connective tissue of typical Type-I collagen fibrils. The capsule was formed by a single stratum of fibroblasts in the rat and in the pike, but by one or two strata of fibroblasts in the monkey. In the rat, but not in the monkey or pike, fibroblast processes interdigitated with processes from the hepatocytes. In the pike, fibroblast processes extended toward both mesothelium and hepatocytes. In some areas of the rat and pike, mesothelial cells had desmosomal connections and microvillous projections into the peritoneal cavity. Marginated heterochromatin was more abundant in the rat and monkey. The mesothelium was discontinuous in the rat and monkey but, in areas of discontinuity, the capsular surface was covered by a basal lamina, often barely perceptible beneath mesothelial cells of the rat and monkey, but prominent in the pike. In the pike, the mesothelium had numerous pinocytotic vesicles on both peritoneal and capsular surfaces.

Duranta erecta poisoning in nine dogs and a cat.

Four incidents of Duranta erecta (golden dewdrop, Sheena's Gold, Geisha Girl) poisoning affecting nine dogs and a cat produced drowsiness, hyperaesthesia and tetanic seizures in all affected animals with evidence of alimentary tract irritation (vomiting, gastric and intestinal haemorrhage, diarrhoea, melaena) in five dogs and the cat. Fruits and leaves were seen to be eaten by affected animals. Therapy was successful in three of the dogs. Repeated diazepam doses and, in some cases, additional pentobarbitone or propofol anaesthesia, were successful in controlling seizures.

Comparison of the anticonvulsant efficacies and neurotoxic effects of valproic acid, phenytoin, and the ketogenic diet.

PURPOSE: The purpose of this study was to measure quantitatively the effectiveness of the ketogenic diet (KD) in comparison to two clinically important anticonvulsant drugs (AEDs), valproic acid (VPA) and phenytoin (PHT), and to evaluate possible associated neurotoxicity. METHODS: Rats were maintained on either a calorie-restricted, KD or calorie-restricted, rodent-chow diet for 3-5 weeks, after which neurobehavioral and seizure testing was completed. AEDs (either VPA or PHT) were injected acutely at the time to peak effect before neurotoxic and seizure assessment. Seizures were induced by timed infusion of pentylenetetrazole (PTZ) and maximal electroshock (MES). RESULTS: VPA protected from both MES- and PTZ-induced seizures, whereas the KD only elevated PTZ seizure threshold; PHT only attenuated MES-induced seizures. The KD was as effective as a high dose of VPA (i.e., 300 mg/kg) and combined treatment (i.e., KD + VPA) showed an additive increase in PTZ seizure threshold. No observed neurobehavioral deficits were associated with either diet treatment; however, drug-related side effects were noted with high doses of either VPA or PHT. CONCLUSIONS: These data suggest that the KD ranks among VPA and PHT as an effective treatment for seizures, without observed drug-associated neurobehavioral contraindications. In combination with AEDs, our results indicate that the KD plus VPA work synergistically to increase seizure threshold, whereas the KD plus PHT may be complementary, elevating seizure threshold (KD) and reducing seizure severity (PHT). These findings may provide insights into future directions for rational polytherapy; however, it is important to be aware that the KD has been shown to elevate VPA-induced hepatotoxicity.

Evoked epileptiform discharges in the rat anterior piriform cortex: generation and local propagation.

The purpose of this study was to identify cellular and synaptic properties of neurons in a small region within the anterior piriform cortex (aPC), termed the area tempestas (AT), responsible for triggering forebrain seizures in rats. Using a brain slice preparation, we performed whole-cell patch recordings from neurons in the regions overlapping the functionally defined AT. Local electrical stimulation activated synaptic inputs to neurons in these regions, collectively termed the deep aPC (daPC). Synaptic inputs were blocked by selective ionotropic glutamate receptor antagonists. Excitatory bursts were evoked from 59% of daPC neurons as the stimulus intensity was raised above a precise threshold. Secondary bursts (6-15 Hz) occurred in 34% of daPC neurons. Evoked bursts were synaptically driven, as they were blocked by TTX (1 microM) or 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, 1 microM), but not by inclusion of cesium and N-(2, 6-dimethylphenylcarbamoylmethyl) triethylammonium (QX-314) in the internal patch solution. Neither augmentation of excitatory nor suppression of inhibitory transmission were required to evoke bursts from daPC neurons. However, bicuculline (20 microM) lowered the threshold intensity for evoking discharges and increased the incidence and duration of evoked bursts, indicating active inhibitory control of daPC neurons. Stimulation in the daPC evoked epileptiform field potentials from layer II of the adjacent PC and bursts from layer II pyramidal neurons. This work demonstrates that synaptically dependent excitatory burst discharges can be evoked from daPC neurons without altering the balance between synaptic excitation and inhibition. Stimuli that trigger bursts in daPC neurons also generate epileptiform activity in layer II pyramidal cells, indicating that propagation of excitatory activity triggered from the daPC to the pyramidal neurons of the aPC can contribute to the initiation of seizures induced by disinhibition of the AT in vivo.

A ketogenic diet has different effects upon seizures induced by maximal electroshock and by pentylenetetrazole infusion.

The purpose of these experiments was to determine whether a ketogenic diet previously shown to elevate seizure threshold also reduced seizure severity. Seizure threshold was tested by intravenous infusion of pentylenetetrazole (PTZ) whereas seizure severity was determined from measuring the hindlimb extension to flexion (E/F) ratio after seizures were evoked by maximal electroshock stimulation (MES). Surprisingly, seizures evoked by MES were more severe in animals fed a calorie-restricted ketogenic diet. Controls fed an isocaloric, calorie-restricted normal diet also exhibited more severe seizures than did animals fed the same diet ad libitum. When seizure threshold was evaluated in the same animals, those animals fed a calorie-restricted ketogenic diet exhibited a significant increase in seizure resistance compared to animals fed a ketogenic diet ad libitum, a calorie-restricted normal diet or a normal diet ad libitum. These findings suggest that both the amount and type of food affect seizures in rats and show that diet-related seizure protection depends upon the method by which seizures are provoked.

Higher ketogenic diet ratios confer protection from seizures without neurotoxicity.

The present study was designed to establish a dose-response relationship for the efficacy of the ketogenic diet (KD). Sprague-Dawley rats were fed ketogenic diets containing varying ratios of fats; (carbohydrates + proteins) whereas control animals were fed rodent chow. Unless otherwise indicated, all animals were fed calorie-restricted, isocaloric diets beginning at P37 and ketonemia, seizure threshold and neurotoxic effects were determined. Despite being provided isocaloric quantities, animals fed lower ketogenic ratios gained weight relative to those fed diets having greater proportions of fats. A significantly increased metabolic rate was noted for animals fed a high-fat diet, suggesting a basis for the weight differences. Results also showed that the animals fed calorie-restricted high-fat diets exhibited significant ketonemia and protection from pentylenetetrazole (PTZ)-induced seizures. There were no detectable neurotoxic effects for any diet group. For animals of the same age, there was no correlation between beta-hydroxybutyrate (beta-OHB) and seizure threshold. These findings suggest that beta-OHB is not directly involved in the anticonvulsant mechanism of the diet. Also, data presented here show that the conventional 4:1 ketogenic diet does not confer the greatest level of seizure protection. We conclude that a 6:1 ketogenic diet, which shows no evidence of neurotoxicity, may be maximally efficacious in rats.

Path analysis shows that increasing ketogenic ratio, but not beta-hydroxybutyrate, elevates seizure threshold in the Rat.

Previous work has identified several criteria that may be important in determining the efficacy of the ketogenic diet as a treatment for intractable epilepsy in children. The present study was designed to investigate the influence of four major variables on seizure threshold, i.e. ketogenic ratio, body weight, age at diet onset and beta-hydroxybutyrate in rats. Path analysis was used to statistically model and quantify the causal relationships among variables. Results indicate that seizure threshold was significantly elevated with increasing ketogenic ratios (i.e. more fats vs. carbohydrates and proteins) and decreasing weight. Conversely, age at diet onset and plasma levels of beta-OHB showed no causal relation to seizure resistance. These results suggest that the efficacy of the ketogenic diet is independent of the level of ketonemia but is markedly influenced by diet and growth.

Seizure resistance is dependent upon age and calorie restriction in rats fed a ketogenic diet.

The present study was designed to evaluate the effects of age on the efficacy of the ketogenic diet in suppressing seizures evoked by tail-vein infusion of pentylenetetrazole (PTZ). Male rats of various ages were divided into three groups and fed one of three diets: (1) a calorie-restricted ketogenic diet, (2) a calorie-restricted normal (rodent chow) diet, or (3) a normal diet, ad libitum. After animals had been on experimental or control diets for more than 20 days, seizure threshold and blood levels of beta-hydroxybutyrate (beta-OHB) were determined. Animals fed a ketogenic diet exhibited significant elevations in levels of beta-OHB and seizure resistance compared to animals fed either a calorie-restricted normal diet or a normal diet, ad libitum. The levels of beta-OHB and seizure resistance were greatest for young pups. A surprising finding was that young animals fed a calorie-restricted rodent chow diet exhibited a significantly increased resistance to seizures compared to those fed the same diet, ad libitum. Results presented here demonstrate that the ketogenic diet produces the highest levels of ketonemia and seizure threshold in young animals. Collectively, these data suggest that age and caloric restriction are important considerations for implementing the ketogenic diet.

A ketogenic diet increases the resistance to pentylenetetrazole-induced seizures in the rat.

PURPOSE: The purpose of this study was to test the hypothesis that a ketogenic diet would increase the resistance of rats to pentylenetetrazole (PTZ)-induced seizures and to understand the relation of ketonemia to seizure resistance. METHODS: A freely consumed, high-fat (ketogenic) diet was administered to male Sprague-Dawley rats for 5-10 weeks, while control animals were fed either rodent chow or a high-carbohydrate diet. Ketonemia was measured as plasma levels of beta-hydroxybutyric acid (beta-OHB). Seizures were induced by tail-vein infusion of pentylenetetrazole. RESULTS: The ketogenic diet produced a highly significant (p<0.01) increase in beta-OHB levels within 5 days. Induction of seizures by PTZ 35 days after animals were placed on their respective diets showed that ketogenic animals had a significantly (p<0.01) increased threshold for seizure induction compared with those fed an isocaloric diet of either high-carbohydrate or normal rodent chow. Ketogenic animals did not exhibit increased seizure severity relative to controls, despite receiving consistently higher doses of PTZ. CONCLUSIONS: The ketogenic diet resulted in an increased seizure threshold, confirming the hypothesis, and seizure threshold was found to be a direct function of the level of ketonemia.

Activation of NMDA and non-NMDA receptors by L-aspartate in the suprachiasmatic nucleus of the rat.

The receptors mediating the responses of neurons in the rat suprachiasmatic nucleus (SCN) to focal application of L-aspartate were studied in coronal slices by whole-cell patch clamp electrophysiology. Neurons in the SCN showed inward currents in response to both L-aspartate and L-glutamate. Responses to L-aspartate were only partially blocked by D-2-amino-5-phosphonovalerate (D-AP5) and by 10 mM Mg+2 at resting membrane potentials, suggesting an action upon-NMDA glutamate receptors. The remaining currents were blocked by the quinoxalines CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), DNQX (6,7-dinitroquinoxaline-2,3-dione), and NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline1), indicating an action of L-aspartate upon non-NMDA receptors. We conclude that cells in the SCN are among the relatively few types in which an action of L-aspartate upon non-NMDA receptors has been demonstrated.

Biodegradable polymer scaffolds for tissue engineering.

Synthetic polymer scaffolds designed for cell transplantation were reproducibly made on a large scale and studied with respect to biocompatibility, structure and biodegradation rate. Polyglycolic acid (PGA) was extruded and oriented to form 13 microns diameter fibers with desired tenacity. Textile processing techniques were used to produce fibrous scaffolds with a porosity of 97% and sufficient structural integrity to maintain their dimensions when seeded with isolated cartilage cells (chondrocytes) and cultured in vitro at 37 degrees C for 8 weeks. Cartilaginous tissue consisting of glycosaminoglycan and collagen was regenerated in the shape of the original PGA scaffold. The resulting cell-polymer constructs were the largest grown in vitro to date (1 cm diameter x 0.35 cm thick). Construct mass was accurately predicted by accounting for accumulation of tissue components and scaffold degradation. The scaffold induced chondrocyte differentiation with respect to morphology and phenotype and represents a model cell culture substrate that may be useful for a variety of tissue engineering applications.

Cardiac arrhythmias during arousal from hibernation in three species of rodents.

Cardiac activity was monitored during arousal from deep hibernation in three species of hibernators (Citellus tridecemlineatus, Marmota monax, and Mesocricetus auratus). Individuals of each species showed marked arrhythmias, and these were confined to a body temperature range of between 11 and 20 degrees C, measured orally or rectally. The arrhythmias consisted of intermittent periods of asystole and bradycardia and were sometimes marked by the appearance of premature ventricular contractions and elevated or depressed S-T segments. Surgical manipulation of euthermic M. monax, under pentobarbital sodium anesthesia, indicated that none of the arrhythmias observed was inducible by injections of cold saline into the vena cava.