No abstract

Several quinolone and fluoroquinolone haptens have been used to raise polyclonal antibodies exhibiting both specific and generic properties for these classes of antimicrobial compounds. The antisera have been assessed in rapid enzyme immunoassays (ELISAs) designed to exploit the specificities obtained. A direct generic ELISA for both the quinolones and fluoroquinolones has been developed that uses the cross-reactivity of an antibody raised against norfloxacin (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid) linked to ovalbumin via a secondary amine group on the piperazinyl moiety to detect nine different drugs in these classes. Specific ELISAs to ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid), enrofloxacin (1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid), flumequin (9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo(ij)quinolizine-2-carboxylic acid) and nalidixic acid (1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid) have also been developed with a high degree of specificity to the individual compounds. The assays measure drug residues in bovine milk and ovine kidney with an interassay relative standard deviation (s(r)) of 10.5% or less and intra-assay s(r) of 11.2% or less. Sensitivity is less than 4 microg x kg(-1) for both the generic and specific assays for all but one of the compounds tested. (Pipemidic acid (8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)pyrido(2,3-d)pyrimidine-6-carboxylic acid) is detectable at 6 microg x kg(-1) in kidney.)

Scrapie of sheep and goats is the most common prion disease (or transmissible spongiform encephalopathy, TSE) of mammals and aggregates of abnormal, proteinase-resistant prion protein (PrP Sc) are found in all naturally occurring prion diseases. During active surveillance of British sheep for TSEs, 29 201 sheep brain stem samples were collected from abattoirs and analysed for the presence of PrP Sc. Of these samples, 54 were found to be positive by using an ELISA screening test, but 28 of these could not be confirmed initially by immunohistochemistry. These unconfirmed or atypical cases were generally found in PrP genotypes normally associated with relative resistance to clinical scrapie and further biochemical analysis revealed that they contained forms of PrP Sc with a relatively protease-sensitive amyloid core, some resembling those of Nor98 scrapie. The presence of these atypical forms of protease-resistant PrP raises concerns that some TSE disorders of PrP metabolism previously may have escaped identification in the British sheep population.

In the latter part of 1991 an unusual neurological disease was recognised on several farms in England. This report describes the case histories and clinical, biochemical and pathological findings in six calves and two lambs aged from two to 44 weeks obtained from five of these farms. Laminar cerebrocortical necrosis and severe bilateral necrosis of the thalamus and/or striatum progressing to cavitation were recognised in their brains. These changes are similar to those of experimental sulphate toxicity. Morbidity rates of 16 to 48 per cent and mortality rates of 0 to 8 per cent were recorded. The affected animals did not respond to vitamin B1 treatment; the erythrocyte transketolase levels of in-contact cattle and of one untreated affected calf and one untreated lamb were within the normal range. All five farms had recently introduced a proprietary concentrate ration containing ammonium bicarbonate. After this ration was withdrawn no new cases of nervous clinical disease were observed. It is suggested that, in at least some cases, the morphology and topography of lesions may distinguish sulphate induced polioencephalomalacia from that of sporadic thiamine-dependent cerebrocortical necrosis.

A novel CE-based noncompetitive immunoassay for prion protein (PrP) was established. Fluorescein isothiocyanate (FITC)-labeled protein A (FITC-PrA) was used as a fluorescent probe to tag monoclonal antibody through noncovalent binding of FITC-PrA to the Fc region of the antibody. The FITC-PrA-Ab was incubated with the analyte, prion protein, under optimized condition, forming the immunocomplex FITC-PrA-Ab-PrP. The complex was separated and analyzed by capillary zone electrophoresis. The addition of carboxymethyl-beta-cyclodextrin in the running buffer as dynamical coating reagent improved the reproducibility and the resolution. The complex was isolated in less than 1 min with theoretical plates of 3.8 x 10(4). Relative standard deviations of peak height and migration time for the complex were 3.46 and 1.48%, respectively. A linear relationship was established for the bovine recombinant prion protein (rPrP) concentration in the range from 0.2 to 2.0 mug/mL and the peak height. The correlation factor was r2 = 0.9969. The estimated detection limit for rPrP was approximately 6 ng/mL, which is 3 times the signal-to-noise ratio. The method was successfully applied for testing blood samples from scrapie-infected sheep.