Accelerated solvent extraction of animal feedingstuffs for microbial growth inhibition screening for the presence of antimicrobial feed additives.

Three plate systems (combinations of indicator organism and growth medium) were evaluated for the detection of analytical standards of the banned feed additives avoparcin, bacitracin zinc, spiramycin, tylosin and virginiamycin. When authorized in the EU, the previously recommended minimum inclusion rate (MIR) for each compound was 5 mg kg(-1). One of the plate systems (Micrococcus luteus ATCC 10240, nutrient agar) detected all five additives. This plate was used in a further study that evaluated the suitability of accelerated solvent extraction (ASE) as a first step in the development of a rapid single-plate screening assay. A drug-free (negative control) feedingstuff was fortified with the compounds (0-50 mg kg(-1)), extracted by ASE and the extracts applied to the plate at each of three pH ranges - unadjusted extract (pH 5.7-5.9), pH 6.5 and 8.0. At pH 6.5, sub-MIR concentrations of virginiamycin and tylosin were detectable. Avoparcin was detectable at 6.3 mg kg(-1). The detection of zinc bacitracin was#10; pH-independent (10 mg kg(-1)). At pH 8.0, spiramycin was detectable at 5.4 mg kg(-1). Mean +/- SD analytical recoveries from fortified feedingstuffs (n = 10) ranged from 57 +/- 1.5% for avoparcin to 96 +/- 4% for virginiamycin. The five additives were also detectable following ASE extraction from a range of different feedingstuffs fortified with each of the drugs. A further 24 compounds permitted for use in animal feeds were tested. Of these, nine were detectable at their recommended MIR. It is concluded that ASE is a versatile technique suitable for the automated extraction of a range of antimicrobials from animal feedingstuffs. Employing ASE with this single-plate detection system permits the rapid antimicrobial screening of animal feedingstuffs and allows the detection of the banned additives. Whilst the method is applicable as a screening test, more specific postscreening methods would be necessary for subsequent identification (and quantification) of antimicrobials in screening-positive samples.

Rehabilitation outcomes following percutaneous coronary interventions (PCI).

This prospective study evaluated the effect of an individualized, comprehensive, home-based cardiac rehabilitation program combining exercise training with risk factor modification and psychosocial counseling on risk factors, psychological well-being, functional capacity, and work resumption in 99 post-percutaneous coronary interventions (PCI) patients randomized to control (standard care plus telephone follow-up, n=49) or intervention (individualized, comprehensive, home-based cardiac rehabilitation, n=50) groups. Data were collected at time 1 (T(1)) during hospital admission, time 2 (T(2)) approximately 2 months post-PCI, and time 3 (T(3)) approximately 12 months post-PCI. Results suggest that the allocation to an individualized, comprehensive, home-based cardiac rehabilitation program provided more advantageous outcomes. At both follow-ups, the intervention group showed within-group improvement in serum cholesterol levels (P<0.02; P<0.01) and exercise participation (P<0.001; P<0.001) with differences in exercise participation favoring the intervention group (P<0.01) at T(2). Repeated measures ANOVA showed significant improvements over time in body mass index (BMI) (P<0.01), psychological well-being (P<0.001), and functional capacity (P<0.001) for both groups. More patients in the intervention group had returned to work at T(2) (P<0.001) and did so more quickly (P<0.01). These findings suggest that an individualized, comprehensive, home-based cardiac rehabilitation program improves risk factor profiles and work resumption patterns for patients following PCI.

Transfer of chlortetracycline from contaminated feedingstuff to cows' milk.

Three groups of four Friesian cows in mid-lactation were fed a compound feedingstuff contaminated with 2, 10 or 300 mg chlortetracycline/kg for 21 days, and were then fed an uncontaminated diet for seven days. A fourth group of four cows was fed an uncontaminated diet throughout the study. Daily pooled milk samples from each cow were analysed by high performance liquid chromatography (HPLC) with a detection limit of 50 microg chlortetracycline/litre. Chlortetracycline was detected in only two milk samples taken from one of the animals fed feed containing 300 mg 300 mg chlortetracycline/kg, and both contained less than the maximum residue limit (MRL) specified by the European Union (100 microg/litre). All the milk samples were also analysed by the Delvotest SP microbiological assay, which has a detection limit of 300 microg chlortetracycline/litre. During the treatment period, this method gave four presumptive false-positive results, because they were not confirmed by HPLC. Selected daily pooled samples from each treatment group were also analysed by the semi-quantitative Charm II radioreceptor assay with a detection limit of 10 microg chlortetracycline/litre. Immunoreactive chlortetracycline was detected only in the animals fed feed containing 300 mg chlortetracycline/kg and several of the results exceeded the EU MRL during the treatment period. No significant treatment effects on animal performance were observed. However, there was a trend towards a higher milk fat concentration (P<0.09) and a lower milk protein concentration (P<0.07) with increasing concentration of chlortetracycline in the diet.

Transfer of sulphamethazine from contaminated dairy feed to cows' milk.

Four groups of four healthy mid-lactation Friesian cows were fed a compound feeding stuff containing either 2, 10 or 250 mg sulphamethazine/kg, corresponding to 0, 2, 10 and 250 per cent of the therapeutic inclusion rate in rations for pigs, at a flat rate of 3 kg twice daily for 21 days, followed by a seven-day withdrawal period. The cows were machine-milked twice daily and pooled milk samples from each cow were analysed by a commercially available microbiological assay with a sensitivity of 100 micrograms/litre and by a high performance liquid chromatography (HPLC) procedure with a limit of detection of 10 micrograms/litre. No sulphamethazine was detected by HPLC in the milk samples taken from any of the cows fed the concentrate containing 2 or 10 mg/kg. The milk samples from all four cows fed the highest concentration of sulphamethazine contained from 21 to 120 micrograms/litre while they were being fed the contaminated concentrate. The cow with the highest concentrations of sulphamethazine was the only one which repeatedly tested positive by the microbiological assay. The concentration of sulphamethazine declined rapidly during the withdrawal period and the drug was not detectable by either method in samples taken from two days after the contaminated feed was withdrawn.

Evaluation of a single plate microbiological growth inhibition assay as a screening test for the presence of antimicrobial agents in compound animal feedingstuffs at therapeutic and contaminating concentrations.

The Inhibitory Substance Test (IST), a microbiological growth inhibition test, is used for screening animal feedingstuffs for the presence of (contaminating) antimicrobial compounds. The effectiveness of the IST was established for 33 compounds that may be incorporated in feedingstuffs. Minimum detectable concentrations (MDCs) for standard solutions were established and compared with those obtained following solvent extraction of an antimicrobial-free compound feedingstuff spiked with each compound at 0-20 mg/kg. Of the 33 standard solutions examined, the test organism was not sensitive to 11 and the MDC for one was greater than its maximum inclusion rate in complete feedingstuffs. Following routine extraction (25% acetone-phosphate buffer) of feedingstuffs spiked with each of the 22 compounds to which the organism was sensitive, 10 were not detected, 15 were detectable at both minimum and maximum feed-inclusion rates and four were only detectable at their maximum feed-inclusion rates. Extraction with methanol (25%) had a deleterious effect with 12 compounds not detected, nine detectable at both minimum and maximum feed-inclusion rates and five detectable at their maximum feed-inclusion rates. Increasing acetone and methanol concentrations to 40 and 55% respectively resulted in larger inhibitory zones for antibiotic-free feedingstuff (25.3 + 2.43 mm vs 21.1 + 1.02 mm) compared with both 25% acetone (11.3 + 0.22 mm) and 25% methanol (11.2 + 0.22 mm), requiring the establishment of greater threshold zone diameters and negating any advantage in increasing the solvent concentration under these test conditions. It is concluded that the IST may be particularly useful for detection of a number of the zootechnical feed-additives recently banned in the EU, which, if used illegally, may be present at sufficiently high inclusion rates to facilitate detection. Further alteration of extraction conditions may improve the scope of the assay.

Production and biological activity of murine monoclonal antibodies against GnRH.

Immunization against GnRH represents a nonsurgical means of castrating domestic species. However, clear target antibody titres for bioactivity have not been established. The aims of this study were to produce characterized anti-GnRH monoclonal antibodies and to determine a threshold titre. Three murine monoclonals were developed which produced IgG2a class immunoglobulins and bound 50% I(125)-GnRH at a 10(6) to 10(7) dilution. The antibodies were specific to GnRH, showed a strong affinity (Ka values from 1.99 to 2.60 x 10(10) litres/mole), and were directed towards the amino terminus. In female mice all 3 antibody clones interrupted ovarian cyclicity, causing an extension in diestrus followed by prolonged estrus/metestrus (12 to 30 d). Throughout this period circulating titres were greater than 15% I(125)-GnRH binding at a 5 x 10(4) dilution. In male mice, immunization with 0.2 ml of ascites significantly reduced testes (P < 0.05), epididymides (P < 0.001) and seminal vesicle (P < 0.01) weights. A 0.1 ml dose (61.4 +/- 18.6% binding at a 10(6) dilution) was ineffective. A serial dilution study indicated that a titre of 50% binding at 2 x 10(6) dilution (antigen binding capacity of 268 +/- 35 ng/ml) was required to completely block GnRH activity. This is a higher tire than threshold levels determined previously. Identification of factors determining the titre required for bioactivity is needed.

An algorithm for associating the features of two images.

In this paper we describe an algorithm that operates on the distances between features in the two related images and delivers a set of correspondences between them. The algorithm maximizes the inner product of two matrices, one of which is the desired 'pairing matrix' and the other a 'proximity matrix' with elements exp (-rij2/2 sigma 2), where rij is the distance between two features, one in each image, and sigma is an adjustable scale parameter. The output of the algorithm may be compared with the movements that people perceive when viewing two images in quick succession, and it is found that an increase in sigma affects the computed correspondences in much the same way as an increase in interstimulus interval alters the perceived displacements. Provided that sigma is not too small the algorithm will recover the feature mappings that result from image translation, expansion or shear deformation--transformations of common occurrence in image sequences--even when the displacements of individual features depart slightly from the general trend.

Visual motion ambiguity.

This paper discusses a number of points that arise in the visual interpretation of optic flow fields. Particular attention is given to the case in which a flow field is visually ambiguous, and the paper concludes with a detailed description of the case of the moving plane.

The visual ambiguity of a moving plane.

It is shown that the optic flow field arising from motion relative to a visually textured plane may be characterized by eight parameters that depend on the observer's linear and angular velocity and the coordinate vector of the plane. These three vectors are not, however, uniquely determined by the values of the eight parameters. First, the optic flow field does not supply independent values for the observer's speed and distance from the plane; it only gives the ratio of these two quantities. But more unexpectedly, the equations relating the observer's linear velocity and the plane's coordinate vector to the eight parameters are still satisfied if the two vectors are interchanged or reversed in direction, or both. So in addition to the veridical interpretation of the optic flow field there exist three spurious interpretations to be considered and if possible excluded. This purpose is served by the condition that an interpretation can be seriously entertained only if it attributes every image element to a light source in the observer's field of view. This condition immediately eliminates one of the spurious interpretations, and exhibits the other two as mutually inconsistent: one of them is tenable only if all the visible sources lie on the forward half of the plane (relative to the observer's linear velocity); the other only if they all lie on the backward half-plane. If the sources are distributed over both halves of the plane, only the veridical interpretation survives. Its computation involves solving a 3 X 3 eigenvalue problem derived from the flow field. If the upper two eigenvalues coincide, the observer must be moving directly towards the plane; if the lower two eigenvalues coincide, his motion must be directly away from it; in both cases the spurious interpretation merges with the veridical one. If all three eigenvalues are equal, it may be inferred that either the observer's linear velocity vanishes or the plane is infinitely distant.

A computational model of binocular depth perception.

We point out that the horizontal disparities between a pair of retinal images are inadequate for computing the three-dimensional structure of a scene unless supplemented by independent information about the distance and direction of the fixation point. We suggest that this supplementary information is derived not from non-visual sources, but from the vertical disparities of a few non-meridional image points. This hypothesis is shown to account quantitatively for Ogle's induced effect--the marked distortion of a scene by a vertically magnifying lens placed in front of one eye.

The role of the vertical dimension in stereoscopic vision.

It is shown how the full three-dimensional structure of a scene may in principle be computed from a correlated pair of retinal images, when all that is known about the orientations of the two eyes is that the planes of their horizontal meridians accurately coincide. The vertical dimension plays a crucial role in the theory; visible points which lie on the horizontal meridian supply no information about the angle of convergence or the direction of gaze. (i) If the scene contains three or more nonmeridional points, not all lying in a vertical plane, then their positions in space are fully determined by the horizontal and vertical coordinates of their images on the two retinas. (ii) If just two nonmeridional points are visible, or more than two, lying in a vertical plane, then their retinal images admit, in general, just two distinct three-dimensional interpretations. One of these is usually unrealistic; but a choice between them may be perceptually difficult if the vertical plane containing the points is nearly perpendicular to the interocular axis. These results suggest that vertical disparities may play an important role in the binocular perception of absolute depth. Elsewhere (Mayhew, this issue) this suggestion is found to provide a quantitative explanation of Ogle's 'induced effect'.

The interpretation of a moving retinal image.

It is shown that from a monocular view of a rigid, textured, curved surface it is possible, in principle, to determine the gradient of the surface at any point, and the motion of the eye relative to it, from the velocity field of the changing retinal image, and its first and second spatial derivatives. The relevant equations are redundant, thus providing a test of the rigidity assumption. They involve, among other observable quantities, the components of shear of the retinal velocity field, suggesting that the visual system may possess specialized channels for computing these components.

The perception of music.

A common but none the less remarkable human faculty is the ability to recognize and reproduce familiar pieces of music. No two performances of a given piece will ever be acoustically identical, but a listener can perceive, in both, the same rhythmic and tonal relationships, and can judge whether a particular note or phrase was played out of time or out of tune. The problem considered in this lecture is that of describing the conceptual structures by which we represent Western classical music and the processes by which these structures are created. Some new hypotheses about the perception of rhythm and tonality have been cast in the form of a computer program which will transcribe a live keyboard performance of a classical melody into the equivalent of standard musical notation.