Bioterrorism: intentional introduction of animal disease.

The possibility of the intentional introduction of animal disease as an act of bioterrorism adds a new dimension to the development of strategies for assessment, prevention, response and recovery from exotic diseases, including the zoonoses. The vulnerability of livestock operations, the likelihood of success, the possibility of the use of genetically engineered organisms and limited resources to handle multiple outbreaks place new pressures on policy-makers and emergency responders to make best use of limited resources. The methods for managing a natural occurrence or accidental introduction of high-consequence diseases are generally applicable to containment and recovery from outbreaks of intentionally introduced animal diseases. Zoonotic agents increase the complexity at both international and national levels. Modern biology provides both increased threat of new disease entities and methods for earlier and more effective detection and intervention. Improved methods are emerging for defining trade restrictions and animal movement and for determining when it is safe to resume normal trade.

Diagnostic specificity of a real-time RT-PCR in cattle for foot-and-mouth disease and swine for foot-and-mouth disease and classical swine fever based on non-invasive specimen collection.

Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) are highly contagious and can cause great economic losses when introduced into disease-free regions. Accurate estimates of diagnostic specificity (Sp) are important when considering the implementation of surveillance for these agents. The purpose of this study was to estimate diagnostic Sp of a real-time reverse-transcriptase PCR assay developed for detection of FMDV in cattle and domestic swine and CSFV in domestic swine based on non-invasive specimen collection. One thousand and eighty-eight range beef cattle were sampled from thirteen geographic locations throughout Texas. One thousand and one hundred market hogs and cull sows were sampled. Results for both FMDV and CSFV were considered positive if amplification occurred at or before 40 PCR cycles, inconclusive between 40 and 45 cycles and negative otherwise. Ten cattle had nonspecific PCR amplifications for FMDV, but none were classified as positive and only one as inconclusive. Specificity (95% confidence interval) was estimated as 100% (99.7, 100). There were 19 nonspecific PCR amplifications for FMDV in sampled swine with 1 classified as positive, 6 as inconclusive, and 12 as negative. Specificity (95% confidence interval) was estimated as 99.9% (99.5, 100). There were 21 nonspecific PCR amplifications for CSFV, and 1 was classified as positive. Specificity (95% confidence interval) was estimated as 99.9% (99.5, 100). These assays have high Sp, but nonspecific PCR amplifications can occur.

Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat.

Glutamatergic neurotransmission in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia is critical in the expression of basal ganglia function, and increased glutamate transmission in these nuclei has been implicated in the pathology of Parkinson's disease. In order to determine the precise spatial relationship of subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors to nerve terminals enriched in glutamate or gamma-aminobutyric acid (GABA) in one of the output nuclei, the entopeduncular nucleus (EP), and the STN, postembedding immunolabelling for glutamate receptor subunits and for glutamate and GABA was carried out in the rat. Immunolabelling for the AMPA glutamate receptor subunits 1, 2/3, and 4 (GluR1, GluR2/3, and GluR4) and the NMDA receptor subunit 1 (NR1) was localized predominantly within asymmetrical synapses in both the EP and STN. Quantitative analysis revealed that, on average for the whole population, each of the receptor subunits was evenly distributed along the synaptic specialization. Multiple AMPA receptor subunits and the GluR2/3 and NMDA (NR1) subunits were co-localized within individual synapses. The combination of immunolabelling for glutamate and GABA with the receptor immunolabelling revealed that the majority of axon terminals presynaptic to the receptor-immunoreactive synapses were enriched in glutamate immunoreactivity and were GABA-immunonegative. However, at some NR1- and GluR2/3-positive synapses, the level of glutamate immunoreactivity was low in the presynaptic terminal and, in the STN, some of them were GABA-immunopositive. It is concluded that glutamatergic transmission at individual synapses of different origins in the EP and STN is mediated by a combination ofAMPA and NMDA glutamate receptors.

Glutamate-enriched cholinergic synaptic terminals in the entopeduncular nucleus and subthalamic nucleus of the rat.

Several lines of evidence suggest that the cholinergic neurons of the mesopontine tegmentum contain elevated levels of glutamate and are the source of cholinergic terminals in the subthalamic nucleus and entopeduncular nucleus. The object of this study was to test whether cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, also express relatively high levels of glutamate. To address this, double immunocytochemistry was performed at the electron microscopic level. Perfuse-fixed sections of rat brain were immunolabelled to reveal choline acetyltransferase by the pre-embedding avidin-biotin-peroxidase method. Serial ultrathin sections of cholinergic terminals in both the entoped uncular nucleus and subthalamic nucleus were then subjected to post-embedding immunocytochemistry to reveal glutamate and GABA. Quantification of the immunogold labelling showed that choline acetyltransferase-immunopositive terminals and boutons in both regions were significantly enriched in glutamate immunoreactivity and had significantly lower levels of GABA immunoreactivity in comparison to identified GABAergic terminals. Furthermore, the presumed transmitter pool of glutamate i.e. that associated with synaptic vesicles, was significantly greater in the choline acetyltransferase-positive terminals than identified GABA terminals, albeit significantly lower than in established glutamatergic terminals. In the entopeduncular nucleus, a small proportion of cholinergic terminals displayed high levels of GABA immunoreactivity. Taken together with other immunocytochemical and tracing data, the elevated levels of glutamate in cholinergic terminals in the entopeduncular nucleus and subthalamic nucleus, is further evidence adding weight to the suggestion that acetylcholine and glutamate may be co-localized in both the perikarya and terminals of at least a proportion of neurons of the mesopontine tegmentum.

Synaptic integration of functionally diverse pallidal information in the entopeduncular nucleus and subthalamic nucleus in the rat.

To determine the principles of synaptic innervation of neurons in the entopeduncular nucleus and subthalamic nucleus by neurons of functionally distinct regions of the pallidal complex, double anterograde labeling was carried out at both light and electron microscopic levels in the rat. Deposits of the anterograde tracers Phaseolus vulgaris-leucoagglutinin and biotinylated dextran amine were placed in different functional domains of the pallidal complex in the same animals. The tracer deposits in the ventral pallidum and the globus pallidus gave rise to GABA-immunopositive projections to the entopeduncular nucleus, the subthalamic nucleus, and the more medial lateral hypothalamus that were largely segregated but overlapped at the interface between the two fields of projection. In these regions the proximal parts of individual neurons in the entopeduncular nucleus, lateral hypothalamus, and subthalamic nucleus received synaptic input from terminals derived from both the ventral pallidum and the globus pallidus. Furthermore, the analysis of the afferent synaptic input to the dendrites of neurons in the subthalamic nucleus that cross functional boundaries of the nucleus defined by the pallidal inputs, revealed that terminals with the morphological and neurochemical characteristics of those derived from the pallidal complex make synaptic contact with all parts of the dendritic tree, including distal regions. It is concluded that functionally diverse information carried by the descending projections of the pallidal complex is synaptically integrated by neurons of the entopeduncular nucleus, lateral hypothalamus, and subthalamic nucleus by two mechanisms. First, neurons located at the interface between functionally distinct, but topographically adjacent, projections could integrate diverse information by means of the synaptic convergence at the level of the cell body and proximal dendrites. Second, because the distal dendrites of neurons in the subthalamic nucleus receive input from the pallidum, those that extend across two distinct domains of pallidal input could also provide the morphological basis of integration.

Glutamate-enriched inputs from the mesopontine tegmentum to the entopeduncular nucleus in the rat.

In order to clarify the origin and to examine the synaptology of the projection from the mesopontine tegmentum to the entopeduncular nucleus, rats received discrete deposits of anterograde tracers in different regions of the mesopontine tegmentum. Anterogradely labelled fibres in the entopeduncular nucleus were analysed at the light and electron microscopic levels. To determine the neurochemistry of the projection, the distributions of GABA and glutamate immunoreactivity in anterogradely labelled boutons in the entopenducular nucleus were studied by postembedding immunocytochemistry. The morphological characteristics of anterogradely labelled structures were compared to those of choline acetyltransferase-immunopositive structures. The anterograde tracing demonstrated that the projection to the entopeduncular nucleus arises from the area defined by the cholinergic neurons of the pedunculopontine region and from the more medial and largely non-cholinergic, midbrain extrapyramidal area. The anterogradely labelled terminals formed asymmetrical synaptic contacts with dendritic shafts, cell bodies and more rarely spines in the entopeduncular nucleus, and they were significantly enriched in glutamate immunoreactivity compared to identified GABAergic terminals in the same region. The morphology, trajectory and synaptology of the anterogradely labelled fibres showed similarities to those of choline acetyltransferase-immunopositive fibres and terminals, providing indirect evidence in support of previous suggestions that at least part of the projection is cholinergic. The structures postsynaptic to the anterogradely labelled boutons also received input from other classes of terminals that had the morphological and neurochemical characteristics of boutons derived from the neostriatum, globus pallidus and subthalamic nucleus. These findings imply that the mesopontine tegmentum sends a projection to the entopeduncular nucleus that is heterogeneous with respect to its origin and also possibly its neurochemistry. The synaptology of the projection underlies one route through which the mesopontine tegmentum can exert effects on movement by modulating the direct and indirect pathways of information flow through the basal ganglia.

Pharmacological activity of VUF 9153, an isothiourea histamine H3 receptor antagonist.

The pharmacological activity of the histamine H3 receptor antagonist VUF 9153 (S-[3-(4(5)-imidazolyl)]propyl-N-(4-chlorobenzyl)isothiourea) has been investigated in vitro and in vivo. VUF 9153 displaced [3H]N alpha-methylhistamine binding to rat cortex/hippocampal membranes (pKi = 9.77 +/- 0.03) and antagonised the inhibitory responses to (R)-alpha-methylhistamine against electrical field stimulation in the isolated longitudinal smooth muscle preparation of guinea-pig ileum (pKB = 9.95 +/- 0.07). In these assays, VUF 9153 was 10-50-fold more potent than the prototype H3 receptor antagonist thioperamide. VUF 9153 showed no or very weak activity in in vitro functional assays for histamine H1 or H2 receptors. Systemic administration of VUF 9153 (s.c. or p.o.) dose-dependently inhibited the ex vivo binding of [3H]N alpha-methylhistamine to rat cortex/hippocampal membranes and dipsogenic responses induced by (R)-alpha-methylhistamine. Calculation of ED50 values, at the 1 h pretreatment time used, revealed that VUF 9153 administered s.c. or p.o., was approximately 2-fold weaker than thioperamide. These data indicate that, like thioperamide, VUF 9153 is a potent and selective antagonist for histamine H3 receptors in vitro, possesses the ability to penetrate the blood-brain barrier to access central H3 receptors and can inhibit H3 receptor-mediated functional responses in vivo.