Graduate Thesis Or Dissertation
 

A chromogenic Limulus amebocyte lysate test for determination of endotoxin in the chicken plasma

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  • Endotoxin is a part of the cell wall of gram-negative bacteria, consisting of serotype-specific polysaccharide, core oligosaccharide, and lipid A. Lipid A is responsible for an array of pathophysiologic reactions in animal hosts. Amebocyte lysate originated from Limulus polyphemus (horseshoe crab) has been used extensively in various assay systems to detect endotoxin. One of the assays, a chromogenic Limulus amebocyte lysate (CLAL) test was developed in 1978 and has been used extensively in human clinical fields for its high sensitivity and ease in quantitation. The use of the CLAL test in veterinary fields has been limited to dogs, horses and cattle. The objective of the thesis research was to determine the level of endotoxin by the CLAL assay in broiler chickens. Since gram-negative septicemia is common in broiler chickens, the detection of endotoxemia would help in understanding the pathogenesis and in developing a new treatment or prophylactic mean. By the use of a kinetic method, the CLAL assay detected the standard endotoxin (phenol-water extract from Escherichia coli, 055:B5 strain) in the range between 100 ng and 10 pg/ml. The intra-assay variation was 1.2% and interassay variation was 18.8% based on 1.0 ng standard. The control showed spontaneous release of the chromophore starting around 40 min. after the start of the reaction. This spontaneous release was found not due to contamination of pyrogen-free water (PFW) or substrate by endotoxin. With chicken plasma, various non-specific reactions were detected. Plasma alone released the chromophore in a slow, steady manner, but this reaction was virtually eliminated by heating at 100 C. Chicken plasma contained both inhibitor(s) and enhancer(s) for the test. Endotoxin-free plasma samples were prepared by absorption and reconstituted with 1.0 ng/ml of endotoxin. After 1:10 dilution in PFW, heating (10 min.) at 100 C was found most adequate to inactivate these factors as compared with heating to 70 or 85 C. With plasma samples which had been diluted and heated at 100 C, however, still some nonspecific reaction was detected; the lysate, in the absence of substrate, caused some precipitates with chicken plasma in a nonspecific manner, making it difficult to interpret the OD readings. Because of these nonspecific reactions largely inherent to the state of lysate, sensitivity was judged only to 100 pg endotoxin/ml of chicken plasma. A commercial test kit also showed 100 pg/ml sensitivity with the end point method, but found unreliable since proper controls cannot be evaluated in a similar manner as the kinetic method. Thirty chicken plasma were collected from 3 local broiler farms and was judged to contain less than 100 pg/ml in 29 birds, while one bird showed 12.0 ng/ml of endotoxin. Twenty-three per cent of the chickens showed gram-negative bacteremia without detectable endotoxemia, and the bird with endotoxemia did not have bacteremia. One microgram of the standard endotoxin was injected intravenously to 20 broiler chickens raised in the laboratory, and 5 were sacrificed at 2, 30, 60, and 90 minutes after the injection. The endotoxin was found to be cleared from the blood at the rate of 152 pg/min. To increase the sensitivity and to decrease the cost of the CLAL test, future efforts should be made; 1) to significantly decrease the nonspecific reaction between the lysate and substrate; and 2) to block the precipitation or clotting reaction between the lysate and chicken plasma. If these nonspecific reactions be controlled, the CLAL test could be run in a simple end-point method and/or in an automated manner with chicken plasma.
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