Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore-coated beads that mimic the behaviour of multi-liganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Reproductive failure in sows following experimental infection with a Belgian EMCV isolate.

In this study, a transplacental infection with fetal death was demonstrated following inoculation of pregnant sows with a Belgian encephalomyocarditis virus (EMCV) isolate. Eight multiparus sows were inoculated between 60 and 92 days of gestation with this EMCV-isolate to investigate its ability to cause reproductive failure in sows. Virus persistence and antibody titre in their offspring were also studied. Only the two sows inoculated at 60 days of gestation showed premature farrowing, but all sows seroconverted to EMCV. Virus was recovered from the offspring of all sows at the time of farrowing, but not from every piglet born. One month after birth EMCV could be isolated from all the piglets examined. These results can help in a better understanding of the spread of the disease in piggeries.

Simultaneous vaccination of piglets against foot-and-mouth disease and classical swine fever.

Six-week-old piglets, born of unvaccinated sows, were vaccinated against foot-and-mouth disease (FMD) with a trivalent, inactivated vaccine containing an adjuvant or vaccinated against classical swine fever (CSF) with a live attenuated vaccine or against both diseases simultaneously at two different sites. The antibody response to the FMD vaccine was not significantly influenced by the simultaneous vaccination against CSF. FMD vaccine administered simultaneously with the CSF vaccine produced a significantly higher antibody response to CSF than occurred with CSF vaccination only.

The serological response to foot-and-mouth disease vaccination is not affected by simultaneous infectious bovine rhinotracheitis/adenovirus/parainfluenza 3 vaccination.

Young calves were simultaneously vaccinated by subcutaneous route against foot-and-mouth disease (FMD) and against infectious bovine rhinotracheitis/adenovirus/parainfluenza-3 (IBR/Adeno/PI-3) by intranasal route. The serological response against the 3 FMD virus types of the FMD vaccine was clearly positive. There was no significant difference between results of simultaneous FMD and IBR/Adeno/PI-3 vaccination and FMD vaccination only.

Serological response to a booster foot-and-mouth disease vaccination with strains different from the primary vaccine.

Young calves were vaccinated with belgian foot-and-mouth disease (FMD) vaccine and revaccinated with either the same vaccine or with a foreign FMD vaccine. There was a significant serological response to the primary vaccine strains after the first vaccination which was greater following revaccination. At one and two months after revaccination there was no significant difference between the responses to revaccination with vaccine identical to the primary vaccine or with the foreign FMD vaccine. It was concluded that revaccination of young calves is effective even with an FMD vaccine different from the primary vaccine.

Reduction of animal usage in the control of foot-and-mouth disease vaccines.

FMD vaccines were evaluated by comparing the results of a series of assays made on virus suspensions and on the final product, to mean values derived from a data base containing information on more than 300 accepted vaccine lots. The potency was estimated from the average percentage characterizing the vaccine, using the general mean in vivo protective dose, also provided by the data base. The correlation between the in vitro estimated potency and the in vivo tested potency as made routinely, was relatively low. However the results obtained by both methods were always in good agreement. The estimation method appeared sufficient for the evaluation of the potency of FMD vaccines, at the stage of the production. This method could allow an important reduction in the animal usage for the control of FMD vaccines.

Use of short analytical ultracentrifugation runs for the isopycnic determination of foot-and-mouth disease virus concentration and density in autoformed caesium chloride gradients.

Short analytical ultracentrifugation runs of less than six hours with autoformed CsCl density gradients were used for routine FMDV density and concentration analysis. The values obtained after short runs were slightly higher for concentration and lower for density than after classical 22 h runs. These differences between the 6 and the 22 h values were related to virus strain and did not seem to result of the incomplete stabilisation of the CsCl gradient, but of the shorter virion/CsCl contact period.

Capacity of attenuated swine fever vaccines to prevent virus carriers in the vaccinated pigs, after contact with field virus.

Virus carriers have been detected in pigs vaccinated with attenuated Swine Fever vaccines after challenge with field virus. Virus carriers were found only if the vaccination was made with 20 PD50 or less. Clinical protection was obtained already with 8 PD50. In insufficiently vaccinated pigs, virus was detected in the tonsils as long as 6 weeks after the virus challenge, by Immunofluorescence. A minimal potency of 100 PD50 is proposed as requirement for the acceptance of Swine Fever vaccines.

Routine titration of foot-and-mouth disease virus suspensions by analytical ultracentrifugation 2nd communication: sedimentation equilibrium method.

A routine method for the determination of the virus concentration in FMD virus cultures and vaccines was developed. This method was based on sedimentation equilibrium in the analytical ultraviolet scanning ultracentrifuge. The virus suspension was first clarified. The virions were then sedimented in a preparative ultracentrifuge. The resuspended virions were diluted in a Cesium chloride solution and brought to equilibrium in the density gradient generated in the analytical ultracentrifuge. The optical density of the virus band was measured by the UV scanning system. A calculation procedure was developed to compute the density at the limits and at the maximum of the virus band. The virus concentration expressed as weight, was calculated for the original virus suspension.

Studies about the adjuvant activity of saponin fractions in foot and mouth disease vaccine. III. Comparison of the irritant, adjuvant and hemolytic activities of six commercial saponins and their hemolytic fractions obtained by chromatography on sephadex G 100.

12 experimental vaccines were prepared to compare the irritant and adjuvant activity in cattle of 6 commercial saponin preparations and their hemolytic fractions. It is still not known if a single substance is responsible for the irritant, adjuvant and hemolytic activities of the saponin preparations. The quantities of saponin added were standardised on the base of a constant hemolytic activity rather than on a weight of powder per dose of vaccine base. A FMD vaccine was used to reveal the adjuvant activity. It was concluded that the irritation is related to the hemolytic activity and not to the weight of powder. Irritation is slightly reduced when a toxic effect appears. The adjuvant activity was higher for untreated saponin preparations with high hemolytic activity used at low dose and for one of the chromatographic saponin fractions. The adjuvant activity is reduced when toxic effect appear. Toxicity of less hemolytic saponins used at high dose is removed by chromatography. Highly hemolytic saponins used at low dose become toxic after chromatographic treatment.

Routine titration of foot and mouth disease virus suspensions by analytical ultracentrifugation. 1: Sedimentation method.

Infectivity and Complement Fixation (CF) tests are commonly used for the routine titration of Foot and Mouth Disease (FMD) virus suspensions. Only recently were techniques published for the routine determination of the virus concentration by the physical properties of the virions (Fayet et al., 1971; Barteling et al., 1974). These techniques are based on the separation of the virions from the culture fluid by sedimentation through a sucrose gradient, in a preparative ultracentrifuge. The ultraviolet absorption pattern of the tube content is recorded by a flow colorimeter. The virus concentration is estimated using either standard curves or direct caculation by the specific extinction coefficient (Bachrach et al., 1964). In our own attempts to develop a preparative ultracentrifugation technique for the routine titration of FMD virus suspensions, we had to deal with some problems such as remixing of the virus band at the end of the run. We therefore turned over to analytical ultracentrifugation methods. The manipulations are less complicated and the virus band is traced and measured while the rotor is spinning. Four samples are analyzed simultaneously and the scans are repeated to follow the move of the virus band. The sedimentation rate of the virus band, calculated from the repeated scans, helps to detect artifacts. The present paper describes the technique we developed for the routine titration of FMD virus suspensions, by the band sedimentation method, using an ultraviolet scanning analytical ultracentrifuge.

[Immunogenic quality of foot-and-mouth disease virus].

Routine analysis of suspensions of foot-and-mouth disease virus and eluates of vaccine by the isopycnic method in analytical ultracentrifuge demonstrates the important heterogeneity of the viral population. This heterogeneity increases during inactivation of the virus by formol. In view of this dispersion of the physical characteristics of viral particles it may be asked (a) whether the immunogenic value is linked to the total quantity of particles which, we know, are very different from each other and of which we can determine only the partial or total parameters or (b) whether, on the contrary, the immunogenic value is linked to the activity of marginal particles, very small in number, and perhaps even quantitatively indistinguishable by the physical methods available. However, the hypothesis of particles possessing different properties but converging in the final result cannot, a priori, be excluded. Consequently it cannot be hoped that a simple relation between the concentration in weight of a viral suspension and its immunogenic property may be determined.