Effectiveness of systematic foot and mouth disease mass vaccination campaigns in Argentina.

The objective of this paper is to evaluate the effectiveness of systematic mass vaccination campaigns against foot and mouth disease in Argentina. The analysis was based on an estimation of the proportion of protected animals and protected farms in vaccinated populations, as reflected by levels of antibodies measured in liquid-phase enzyme-linked immunosorbent assay. The analysis was carried out in 49 animal health districts in Buenos Aires province, using data collected from four cross-sectional studies, in 2004, 2007, 2008 and 2011. Cattle were assigned to one of two categories on the basis of correlation between serological titres and expected percentage protection: non-adequately protected (expected protection < 75%) and adequately protected (expected protection ≥ 75%). The proportions of adequately protected cattle and significantly non-adequately protected farms were estimated and compared among sampled locations. Protection was variable among the districts; cattle aged one to two years showed higher levels of protection than cattle six to 12 months old, and the proportion of protected cattle was higher in the more recent studies. The results of the analysis will allow the national animal health service to investigate in depth those districts where protection was lower than the regional background protection. The authors propose that this methodology could be used to evaluate the effectiveness of vaccination campaigns in other countries or zones where systematic foot and mouth disease mass vaccination campaigns are undertaken.

Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA.

A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.

Measurement of psychological state changes at low dopamine transporter occupancy following a clinical dose of mazindol.

RATIONALE: The beneficial effects of psychostimulant drugs in the treatment of psychiatric disorders occur because they increase the extracellular dopamine concentration by inhibiting re-uptake of extracellular dopamine at dopamine transporters. However, the psychological effects at low dopamine transporter occupancy have not been well demonstrated. OBJECTIVES: The purpose of the study was to evaluate the psychological effects, dopamine transporter occupancy, and dopamine release induced by a single oral administration of a clinical dose of mazindol. METHODS: Ten healthy male volunteers were orally administered a placebo and a clinical dose of mazindol (1.5 mg) on separate days. The psychological effects of mazindol were assessed using a visual analogue scale to detect alterations in the state of consciousness. The amount of blockade of dopamine transporters was assessed using positron emission tomography with [18F]FE-PE2I and extracellular dopamine release was measured as the amount of change in [11C]raclopride binding. RESULTS: Following administration of a clinical dose of mazindol, the dopamine transporters were blocked by 24-25 %, and the binding potential of [11C]raclopride was reduced by 2.8-4.6 %. The differences of a score measuring derealisation and depersonalization associated with a positive basic mood were significantly correlated with the change in the [11C]raclopride binding in the limbic striatum. CONCLUSIONS: A subtle alteration in the state of consciousness was detected with a correlation to the changes in the [11C]raclopride binding, which implies that a subtle alteration in extracellular dopamine concentration in the limbic striatum by a small amount of dopamine transporter occupancy can affect the state of consciousness. TRIAL REGISTRATION HTTPS://UPLOAD.UMIN.AC.JP/CGI-OPEN-BIN/CTR_E/CTR_VIEW.CGI?RECPTNO=R000009703 : UMIN000008232.

Phospholipase A2 from Bothrops alternatus (víbora de la cruz) venom. Purification and some characteristic properties.

One single protein species with phospholipase activity has been isolated from Bothrops alternatus venom by a procedure involving gel-filtration on Sephadex G-50 (Step 1), chromatography on SP-Sephadex C-50 (Step 2) and gel-filtration on Sephadex G-75 (Step 3). The purified sample behaved as a homogeneous, monodisperse protein with a molecular weight of 15,000 and isoelectric point of 5.04. The yield in enzyme activity was 48% of the starting material and the apparent purification was 51-fold. When assayed on 1,2-diheptanoyl- or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, fatty acids and lysolecithins were the only reaction products, in accordance with the predicted stoichiometry. Studies on positional specificity suggested that the enzyme is a phospholipase A2. The enzyme requires Ca2+ ions for activity and exhibited stereochemical specificity, since the enantiomeric 2, 3-diheptanoyl-sn-glycero-1-phosphorylcholine was not hydrolyzed. Under the experimental conditions employed, reaction products representative of either phospholipase B or C activities could not be detected. After Step 1, the phospholipase activity recovered was higher than the total activity in the crude venom sample, which is explained by the separation of an inhibitor during enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with crude venom acting on aggregated substrates (lipoprotein, vesicles or micelles), while the rate of hydrolysis of monomeric lecithins was not affected.

The mechanism of inhibition of phospholipase activity of crotoxin B by crotoxin A.

In the crotoxin complex isolated from Crotalus durissus terrificus venom, the component A inhibits the phospholipase A2 activity of crotoxin B only when the substrate is in the aggregated form, preventing the interaction of the enzyme with lecithin--water interfaces. In contrast, with similar rates of hydrolysis of dihexanoyllecithin monomers, the activity of the crotoxin complex is lower than that of crotoxin B when the substrate is aggregated into micelles. Crotoxin B readily hydrolyses dimyristoyllecithin vesicles, the rate being modulated by the physical state of the phospholipid, suggesting that the enzyme is tightly bound to the interface. With the crotoxin complex the rate of vesicle hydrolysis is much slower (about 1/10 that of crotoxin B) and is little affected by the physical state of the lecithin. Direct binding experiments demonstrate that, in contrast to crotoxin B, the crotoxin complex is unable to interact with lecithin--water interfaces. Together with the free accessibility of the enzyme active site in the crotoxin complex, this evidence suggests that a specific area on the enzyme surface, different from the active site and shielded by crotoxin A in the complex, is responsible for the interaction of crotoxin B with lipid--water interfaces.

Accessibility of the active site of crotoxin B in the crotoxin complex.

Basic phospholipases A and the crotoxin complex isolated from Crotalus durissus terrificus venom exhibited similar initial reaction rates, time course and degree of hydrolysis of synthetic short chain lecithins in the monomeric state. Although monomeric lecithins seem to promote dissociation of crotoxin up to a certain extent, this cannot explain the high activity observed with the complex. The crotoxin complex is able to bind the non-hydrolyzable analog D-diheptanoyllecithin, as demonstrated by equilibrium gel-filtration, with a dissociation constant of 0.12 mM. This value is similar to the dissociation constant of the crotoxin B-D-diheptanoyllecithin complex (about 0.13 mM), estimated from the protection against enzyme inactivation by p-bromophenacyl bromide, which further supports the free accessibility of the substrate to the enzyme active site in the crotoxin complex. The lack of enzyme inactivation when crotoxin is treated with p-bromophenacyl bromide may be interpreted in terms of the specific requirements of the reagent to react with the enzyme rather than protection of the active site. Crotoxin B inhibition by complex formation with crotoxin A, which is not apparent on monomeric substrates, seems not to involve the active site of the enzyme.

CBF change evoked by somatosensory activation measured by laser-Doppler flowmetry: independent evaluation of RBC velocity and RBC concentration.

The purpose of this study was to examine the timing and magnitude of cerebral blood flow (CBF) responses to neuronal activation. We measured the changes in local CBF (LCBF), red blood cell (RBC) velocity and RBC concentration by laser-Doppler flowmetry (LDF) as well as field potential recordings during activation of the somatosensory cortex of the rat in response to electrical stimulation of the hind paw. Electrical stimuli, 0.1 ms pulses of 1-1.5 mA for 5 s, were applied at 0.2, 0.5, 5, 10 and 50 Hz under alpha-chloralose anesthesia. LCBF showed the maximum increase at 5 Hz, and rose approximately 0.5 s after the onset of stimulation regardless of the frequency. The maximum frequency of the field potentials was also obtained at 5 Hz. During activation of the somatosensory cortex, the onset of rise in RBC concentration did not precede that of RBC velocity, and the peak RBC concentration was noted earlier than that of both LCBF and RBC velocity, suggesting that both arteriolar diameter and active changes in the capillary contributed to the LCBF response.

Application of a beta microprobe for quantification of regional cerebral blood flow with (15)O-water and PET in rhesus monkeys.

A beta microprobe was successfully applied to monitor arterial input function for quantification of regional cerebral blood flow (rCBF) in the monkey brain with (15)O-water and positron emission tomography (PET). The sensitivity of the probe was approximately 0.83 to 1.67 cps/kBq/ml depending on the studies. A preliminary study was performed to find a suitable use and to evaluate the performance of the system and data analysis procedure. The results showed that dispersion correction of measured input function was unnecessary if microprobes were connected directly to the arterial catheter. Then multiple CBF measurements were done in three monkeys under anesthesia. Identical regions of interest were placed with the aid of magnetic resonance imaging (MRI) of each monkey and rCBF values were estimated. Estimated rCBFs were reproducible for several measurements. The mean CBF value for a pentobarbital anesthetized monkey was 46.0 ml/min/100 g (PaCO2 = 46.3 mmHg). This shows that the use of the beta microprobe for quantification of rCBF with PET was validated. The lack of a need for dispersion correction of observed input function is an advantage with the beta microprobe system because the probes are small enough to be placed near the arterial sampling site.

Effect of tracer metabolism on PET measurement of [11C]pyrilamine binding to histamine H1 receptors.

The present study was carried out to investigate the time course of [11C]pyrilamine metabolism and the degree of entry of metabolites into the brain. PET studies were performed in seven healthy volunteers and arterial plasma concentrations of [11C]pyrilamine and its labeled metabolites were determined. After intravenous injection, [11C]pyrilamine metabolized gradually in the human body, with less than 10% of plasma activity being original radioligand at 60 min. Tracer metabolism markedly affected the input function and the calculated impulse response function of the brain. Rat experiments demonstrated that although metabolites of [11C]pyrilamine might enter the brain, they were not retained for prolonged periods of time. At 30-90 min after injection of [11C]pyrilamine, less than 1% of the radioactivity in the brain was originating from metabolites of [11C]pyrilamine. Based on the rat data, the contribution of 11C-labeled metabolites to total [11C]pyrilamine radioactivity in the human brain was estimated and found to be negligible. These results suggest that the metabolites of [11C]pyrilamine do not accumulate within the cerebral extravascular space and that there is minimal metabolism of [11C]pyrilamine by brain tissue itself. Therefore, [11C]pyrilamine metabolites can be neglected in kinetic analysis, using either a compartmental or a noncompartmental model, of the [11C]pyrilamine binding to histamine H1 receptors.

[Development of a high resolution beta camera].

We have developed and tested a high resolution beta camera. The beta camera consists of thin CaF2(Eu) scintillator, tapered fiber optics plate, position sensitive photomultiplier tube (PSPMT). The output of the PSPMT is fed to position calculation circuit and accumulated in the memory. The data in the memory is fed to personal computer for display and analysis. We have developed two types of beta cameras. One is 20 mm diameter field of view (FOV) camera, and the other is 10 mm diameter camera. Intrinsic spatial resolutions were 0.8 mm FWHM and 0.5 mm FWHM for 20 mm and 10 mm FOV camera, respectively. We confirmed that developed beta cameras may overcome the limitation of the resolution of the PET camera.

[MRI, SPECT and MRS findings in a case of acute hemiplegia syndrome with a marked hemispheric brain edema].

Magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) and magnetic resonance spectroscopy (MRS) were successively recorded in a 3-year-old girl with the acute hemiplegia syndrome. She was admitted to our hospital with complaints of fever, loss of consciousness and right side dominant clonic convulsions evolving into status epilepticus, and then recovered with sequelae of aphasia and right hemiparesis. Electroencephalography showed a generalized slow rhythm at the onset, and very low activities on the left hemisphere in the follow-up records. Brain CT and MRI revealed edema of the left hemisphere initially, followed by left side dominant brain atrophy. No cerebral vascular lesion was detected by magnetic resonance angiography. N-Isopropyl-[123I]-iodoamphetamine SPECT showed marked hypoperfusion of the left hemisphere accompanied by crossed cerebellar diaschisis. MRS at the initial stage detected decreased N-acetyl-aspartic acid and increased lactic acid signals in the bilateral hemisphere, which subsequently normalized only on the right side. These findings suggested brain damage and neural cell death in the left cerebral hemisphere, caused by acute encephalopathy. SPECT and MRS are useful new techniques to study the pathophysiology of the acute hemiplegia syndrome.

[Supplementary motor area epilepsy associated with ADHD in an abused history].

A 6-year-old girl with attention-deficit hyperactivity disorder (ADHD) who had been abused by her mother in infancy developed supplementary motor area (SMA) epilepsy. The seizure was characterized by bilateral tonic seizure of the upper and lower extremities, speech arrest, preserved consciousness and a lack of postictal confusion. The duration of the seizure was usually 10-60 seconds. The seizures sometimes clustered. She was diagnosed as having SMA epilepsy based on the characteristic clinical symptoms, interictal EEG, ictal video-EEG and ictal SPECT. Though her seizure was initially improved by anti-epileptic drugs, the symptoms appeared again after discharge. Since her clinical course indicated that her seizure was aggravated by her mental state, treatment included both medication with anti-epileptic drugs and the adjustment of her living environment in cooperation with a child guidance clinic. Thereafter both her epileptic seizure and ADHD symptoms improved. These changes may be related to each other, because both conditions are associated with frontal lobe dysfunction. It was interesting that the adjustment of the environment improved frontal lobe epilepsy, which in turn ameliorated ADHD symptoms.

[The effect of sevoflurane on regional cerebral metabolism and cerebral blood flow in rhesus monkeys].

The effects of sevoflurane on cerebral metabolism and hemodynamics were studied in rhesus monkeys. Cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMR O2) as well as their regional changes were measured by means of positron emission tomography technique. After the measurement of regional CBFs and CMR O2s at 1.5% sevoflurane as control, the measurement was repeated at 3.0% sevoflurane and at the same sevoflurane concentration with the infusion of angiotensin II to restore mean arterial pressure. Regional CBFs and CMR O2s were compared among three different conditions. At 3.0% sevoflurane, regional CBFs increased significantly in response to the increase in the mean arterial pressure, suggesting the inhibition of autoregulation of CBF. However, regional CBF/CMR O2 ratio was not significantly different among the cerebral regions with each condition. It could be concluded that CBF during sevoflurane anesthesia up to 3.0% might become dependent on the cerebral perfusion pressure and the changes in regional CBFs varied among the regions. On the other hand, the ratio of oxygen consumption and delivery was well maintained throughout the brain regions.

[The effect of cerebral perfusion pressure on cerebral blood flow in the rhesus monkey during sevoflurane anesthesia].

The effect of cerebral perfusion pressure on cerebral blood flow (CBF) was studied under the normocapnic condition in the rhesus monkey under sevoflurane anesthesia. CBF was measured by means of positron emission tomography technique. After the measurement of CBF at 0.5% sevoflurane as control, the measurement was repeated at 2.0% sevoflurane (1 MAC), when blood pressure was kept at a half of the control value. The measurement was also repeated at the same sevoflurane concentration, when the mean blood pressure was restored with the infusion of angiotensin II. Average CBF as well as regional CBFs were compared between two different mean blood pressures at 2.0% sevoflurane. Average CBF increased significantly (+35%), when the mean arterial pressure was increased by the angiotensin II infusion. All the regional CBFs except at frontal cortex increased significantly (+ about 30%) in response to the increase in the mean arterial pressure. The increase in occipital CBF was greatest (+52%). We conclude that CBF during sevoflurane anesthesia up to 2.0% might become dependent on the cerebral perfusion pressure, indicating the compromised autoregulation of CBF in the rhesus monkey.

Characterisation and epitope mapping of neutralising monoclonal antibodies to A24 Cruzeiro strain of FMDV.

Characterisation of seven neutralising monoclonal antibodies (mAbs) produced against foot-and-mouth disease virus A(24) Cruzeiro revealed three reactivity groups. Gr-I recognised linear epitopes where as Gr-II was conformation-dependent and trypsin-insensitive. The Gr-III was also conformation-dependent, but trypsin-sensitive. Mar (mAb neutralisation resistant)-mutants could only be produced against Gr-I and Gr-III mAbs. Capsid sequence comparison of Gr-I mar-mutants with parent virus revealed changes in the G-H loop of VP1 at positions 141, 143 and 147. Similarly, a Gr-III mar-mutant showed a change from a highly conserved glycine to a tryptophan at position 148 of VP1 along with three additional changes at the N-terminus of VP1, VP2 and VP4. This residue at 148 of VP1 is located at +2 position after "RGD" and is equivalent to the position identified by the mAb recognising site 5 in serotype O viruses. This site is probably formed because of the interaction of the G-H loop with other residues in different structural proteins.

Single dilution Avidity-Blocking ELISA as an alternative to the Bovine Viral Diarrhea Virus neutralization test.

This study describes the development and validation of a blocking ELISA that measures avidity of BVDV-specific immunoglobulins (Igs) as an alternative to the classic virus neutralization test. The assay comprises a recombinant soluble E2 glycoprotein as target antigen, a neutralizing serum as detector antibody and a washing-step with a chaotropic agent to determine BVDV-specific Igs avidity. Avidity-Blocking ELISA was validated with 100 negative and 87 positive BVDV-neutralization serum samples from either infected or vaccinated bovines (inactivated commercial vaccines). Specificity and sensitivity of the Avidity-Blocking ELISA were 100% and 98.8%, respectively. The assay was standardized to use a single dilution, so that 90 samples can be tested per plate. Results expressed as Avidity Index (AI) correlated with BVDV neutralizing titers (r=0.94). Unlike the virus neutralization test, the Avidity-Blocking ELISA could discriminate between infected and vaccinated animals (DIVA), suggesting that avidity measurement can be a valuable tool to achieve DIVA compliances. The data show that the avidity of anti BVDV antibodies is related to their capacity to block viral infection in vitro.

Development and validation of an ELISA for quantitation of bovine viral diarrhea virus antigen in the critical stages of vaccine production.

Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.

Dissociation of the crotoxin complex promoted by acetylcholine.

Neuromuscular blockage at the presynaptic level is the main biological effect exerted by crotoxin, the major toxin from Crotalus durissus terrificus venom. This effect requires the dissociation of this complex at the target membrane in spite of its tightness and stability under physiological conditions of pH, ionic composition and temperature. Complex dissociation should be determined by a specific set of physicochemical conditions prevailing at the neuromuscular junction. In this regard, we have studied the effect of acetylcholine on the stability of the crotoxin complex, since this effector is present at relatively high concentrations near the presynaptic membrane of the neuromuscular junction. Evidences arising from spectrofluorometric measurements, changes in enzymatic activity and crotoxin B inactivation by b-bromophenacyl bromide indicate that acetylcholine is indeed able to promote complex dissociation at neutral pH values, apparently by titration of 2-3 carboxylate groups in crotoxin A. This effect may, at least in part, contribute in determining the target specificity of this toxin.