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. / Graduation date: 1997
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/34310 |
| Date | 11 September 1996 |
| Creators | Wu, Chen-Chi |
| Contributors | Matsumoto, Masakazu |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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