Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy.

The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) using a sonicated pure culture of Lawsonia intracellularis as the antigen (So-ELISA). A total of 332 serum samples, consisting of 232 experimentally infected animals and 100 animals naturally infected with L. intracellularis, were used to assess the diagnostic sensitivity. Three hundred and fifty-five sera from uninfected animals were used to determine the diagnostic specificity. The receiver operating characteristic and mean +3 standard deviation of optical density (OD) values from uninfected animals were used for selecting cut-off points. The diagnostic accuracy of So-ELISA was considered to be high as the area under the curve index was 0.991 with 0.0029 standard error. The optimal cut-off for So-ELISA was set at 0.45 OD with 89.8% sensitivity and 99.4% specificity based on a combination of good sensitivity and high specificity. No cross-reactivity was found in sera from pigs exposed to Brachyspira pilosicoli, B. hyodysenteriae, Campylobacter mucosalis, C. jejuni, or C. coli. Inter- and intracoefficient of variation of all control sera tested with So-ELISA was less than 10%. The observed agreements between So-ELISA and the immunoperoxidase monolayer assay tested with experimental challenge animals and field samples were 95.08% with 0.88 kappa and 90.65% with 0.74 kappa value, respectively. So-ELISA was able to detect the seroconversion of infected animals at 2 to 4 weeks after exposure to L. intracellularis. Based on the validation results, So-ELISA could be used as an alternative serology for proliferative enteropathy diagnosis.

Fluorescence assays for measuring fatty acid binding and transport through membranes.

The authors' laboratory has applied a series of different fluorescence assays for monitoring the binding and transport of fatty acids (FA) in model and biological membranes. The authors recently expanded their fluorescent assays for monitoring the adsorption of FA to membranes to a total of three probes that measure different aspects of FA binding: (1) an acrylodan-labeled FA-binding protein, which measures the partitioning of FA between membranes and the aqueous buffer; (2) the naturally occurring fluorescent cis-parinaric acid, which specifically measures the insertion of the FA acyl chain into the hydrophobic core of the phospholipid bilayer, and (3) a fluorescein-labeled phospholipid (N-fluorescein-5-thiocarbomoyl-1,2,dihexadecanoyl-sn-glycero-3-phosphoethanolamine), which specifically measures the arrival of the FA carboxyl at the outer leaflet of the membrane. None of these probes allow the transmembrane movement of FA to the inner leaflet to be measured. FA translocation (flip-flop) is typically measured directly, using a pH-sensitive fluorophore such as 8-hydroxypyrene-1.3.6-trisulfonic acid or 2',7'-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein. These probes detect the release of protons from unionized FA that have diffused through the membrane to the inner leaflet. Because adsorption of FA to the outer leaflet must occur before flip-flop, these probes measure the effects of the combined steps of adsorption and translocation. In this chapter, detailed methods are provided on how to monitor the transport of FA through protein-free model membranes, and some of the fluorescent artifacts that may arise with the use of these probes are addressed. Also, experiments designed to investigate such artifacts, and improve the reliability and interpretation of the data are described.

Effects of oral administration of N-acetyl-d-glucosamine on plasma and urine concentrations of glycosaminoglycans in cats with idiopathic cystitis.

OBJECTIVE: To determine the effects of once-daily oral administration of N-acetyl-d-glucosamine (NAG) on plasma and urine glycosaminoglycan (GAG) concentrations in cats with idiopathic cystitis (IC). ANIMALS: 19 cats with IC and 10 clinically normal cats. PROCEDURES: Cats with IC were randomly assigned to receive 250 mg of NAG in capsule form orally once daily for 28 days (n = 12) or a placebo (capsule containing cellulose) orally once daily for the same period (7). In cats with IC, plasma and urine GAG concentrations and urine creatinine concentration were measured on days 0 (immediately before first dose), 7, 14, 21, 28, and 56. For purposes of comparison, those variables were measured in 10 clinically normal cats on day 0. RESULTS: Mean ± SEM urine GAG-to-creatinine concentration ratios (day 0 data) for cats with IC and clinically normal cats differed significantly (3.11 ± 0.62 µg/mL and 14.23 ± 3.47 µg/mL, respectively). For cats with IC, mean plasma GAG concentration in NAG-treated cats (39.96 ± 5.34 µg/mL) was higher than that in placebo-treated cats (24.20 ± 3.35 µg/mL) on day 21. In the NAG-treated cats, plasma GAG concentration on days 21 (39.96 ± 5.34 µg/mL) and 28 (39.91 ± 6.74 µg/mL) differed significantly from the day 0 concentration (27.46 ± 3.90µg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Cats with IC have lower urinary GAG-to-creatinine concentration ratios than did clinically normal cats. Administration of NAG (250 mg, PO, q 24 h) significantly increased plasma GAG concentrations in cats with IC after 21 days of treatment.