U.S. patent application number 09/756102 was filed with the patent office on 2002-07-11 for easy read molecular tag.
Invention is credited to Krise, William F., Sternick, John L..
Application Number | 20020090656 09/756102 |
Document ID | / |
Family ID | 25042049 |
Filed Date | 2002-07-11 |
United States Patent
Application |
20020090656 |
Kind Code |
A1 |
Krise, William F. ; et
al. |
July 11, 2002 |
Easy read molecular tag
Abstract
Foreign proteins are placed into the blood of an animal so that
they can be recovered at a later time and used for identification.
Proteins are administered to the animal from a water bath via the
animal's gills (where appropriate), gut and, through the skin. The
foreign protein is detectable in small amounts using immune assays
which magnify the available signal or tags in an assay.
Inventors: |
Krise, William F.; (Bozeman,
MT) ; Sternick, John L.; (Mansfield, PA) |
Correspondence
Address: |
E. Philip Koltos
Division of General Law, Office of the Solicitor
Department of the Interior Mail Stop 6530
1849 C. Street NW
Washington
DC
20240
US
|
Family ID: |
25042049 |
Appl. No.: |
09/756102 |
Filed: |
January 9, 2001 |
Current U.S.
Class: |
435/7.5 ;
514/15.2 |
Current CPC
Class: |
A01K 61/90 20170101;
Y10S 435/968 20130101; Y10S 436/80 20130101 |
Class at
Publication: |
435/7.5 ;
514/12 |
International
Class: |
G01N 033/53; A61K
038/38 |
Claims
What is claimed is:
1. A method for marking an animal for subsequent identification
comprising exposing the animal to a protein that does not react
with the animal's immune system wherein said protein contains a
detectable marker.
2. The method according to claim 1 wherein the marker is
biotin.
3. The method according to claim 1 wherein the protein is bovine
serum albumin.
4. The method according to claim 1 wherein the animals are fish
which are exposed by immersion in a solution of the protein.
5. The method according to claim 1 wherein the marker is biotin and
the protein is bovine serum albumin.
6. The method according to claim 4 wherein the fish are salmonids.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a biochemical marker used
for tagging animals.
BACKGROUND OF THE INVENTION
[0002] For a variety of reasons, it is desirable to tag migratory
animals, such as fish, so that the animals can be followed or
tracked over a period of time. The conventional way of tagging
animals is with a visible implant tag or a fluorescent colorant.
For example, Haw et al., in U.S. Pat. No. 4,750,490, use visible
implant tags to identify fish. The tags are implanted into
transparent or semi-transparent tissue. When the fish is captured,
the location of the visible implant tags can be ascertained
visually and the tags can be excised and read or read through the
transparent to semitransparent tissue.
[0003] Sandstrom et al., in U.S. Pat. No. 4,392,236, disclose an
animal identification system using fluorescent implanted tags coded
with one or more higher atomic number chemical elements and an
automated method for reading the coded information.
[0004] Ekstrom, in U.S. Pat. No. 5,324,940, discloses an electronic
identification system for identifying fish by implanting tags
having identification codes of fluorescent colorants in the fish.
The information encoded in the tags is obtained from the tags by
measuring the spectrum of light emitted by the fluorescent
colorants.
[0005] Biegel et al., in U.S. Pat. No. 5,235,326, describe an
electronic identification system for identifying fish. The tags are
comprised of capacitors, inductors, transistors, and possibly other
solid-state devices packaged in a form adapted for attachment to or
implantation in a fish.
[0006] Horan et al., in U.S. Pat. No. 4,762,701, disclose a method
for tracking cells in vivo by labeling the cells with cyanine dyes
and detecting the cells by measuring fluorescence, absorbance, or
by detecting MRI probes included in the dyes.
[0007] Zohar et al., in U.S. Pat. No. 5,076,208, disclose a method
for administering compounds such as protein to fish in an aquatic
medium wherein the compound is added to the medium and ultrasound
is applied to the medium to enhance or effect uptake of the
compound by the animal. There is no indication that any of the
compounds administered is for purposes of tagging or otherwise
identifying the fish.
[0008] Conventional tags are either too large to be used with small
fish (2.5 cm or smaller), or they are too expensive and involved,
such as genetic markers, requiring samples from parents an progeny
with the need for a thoroughly trained geneticist for interpreting
the results.
[0009] However, it would be particularly useful to develop a method
for marking larvae or fry of salmonids or other fishes to provide a
practical means of stock identification over an extensive time
period. Development of a fry marking system will enable fishery
managers to determine fish origins, straying rates, and other
definitive marking needs for identifying groups of fish stocked as
fry.
[0010] Chemical marking methods typically have disadvantages of
attenuation as the fish grows, or accumulation in hard tissues,
requiring lethal sampling methods. Another drawback is less than
100% retention of the chemical mark.
[0011] A useful fry marking method should include non-lethal
sampling procedures and development of simple field analysis
techniques. Using protein as a marker in the blood stream could
eliminate some of the disadvantages of attenuation because the
signal in blood can be magnified with immunoassay, such as ELISA
methods. The protein is not deposited in hard tissue, but in the
blood, providing a non-lethal sampling method.
[0012] Fish traditionally have been exposed to biologically active
proteins either via an injection or by immersion in a bath of
treatment. Comparisons of blood titer from both methods gives an
indication of the efficiency of protein delivery to the fish. To
deliver proteins to fish too small for injections, a bath treatment
is needed.
SUMMARY OF THE INVENTION
[0013] It is an object of the present invention to overcome the
aforesaid deficiencies of the prior art.
[0014] It is another objection of the present invention to provide
a method for marking fish fry that can be used for very small
fish.
[0015] It is a further object of the present invention to provide a
marker for fish and other animals that can be sampled in a
non-lethal method.
[0016] According to the present invention, foreign proteins are
placed into the blood, skin, or other surface tissues of an animal
so that they can be recovered at a later time and used for
identification. Proteins are administered to the animal from a
water bath via the animal's gills (where appropriate), gut, and
through the skin. The protein used, such as bovine serum albumin,
is similar to fish serum albumin, is tolerated by the fish's immune
system and is not entirely cleared from the blood stream. The
foreign protein in the blood is detectable in small amounts using
immune assays which "magnify" the available signal or tags or
layers of proteins bound together (avidin/biotin binding) in an
assay. The mark does not disappear in hard tissue, or require
killing of the animal to remove otoliths or other tissues.
[0017] Other proteins that can be used are those which are
tolerated by the immune system of the animal to be tested, e.g.,
avidin or avidin/biotin complexed bovine serum albumin.
[0018] The protein or other marker can be placed in the skin or
body scales. The protein's presence is recognized by a nontoxic tag
(fluorescent, biotin, or other dye) or the basic protein itself.
Tags are important additions to the basic protein because they
allow for increased numbers of identification by using different
tags on the same protein.
[0019] While there is no limit to the type of sample that can be
used, blood samples are easily used for detecting the proteins or
tags. The proteins provide a long lasting mark in the animals as
they grow.
[0020] While generally foreign proteins are not tolerated by the
immune system and are cleared from the animal within 90 days, the
proteins used in the present invention are still detectable in
animals after two years. The molecular mark of the present
invention resists attenuation or loss inside the aminal's
tissues.
[0021] The marking system of the present invention will enable, for
example, fishery managers to determine fish origins, straying
rates, and other definitive marking needs for identifying groups of
fish stocked as fry. The marking system of the present invention
includes non-lethal sampling procedures and simple field analysis
techniques. Non-toxic, slow-clearing, tagged proteins to tag
batches of groups of small fish or other animals are used so that
the molecular marks can be read as the fish or other animals grow.
Tagged proteins, such as biotinylated bovine serum albumin, can
form a chemical complex with avidin or other complex-former in
blood or serum of the animal that is detectable at low levels.
[0022] Bath marking provides advantages such as non-lethal
sampling, a simple and quick method for exposure, and a simple,
inexpensive procedure for distinguishing stocks which does not
require either sophisticated equipment or interpretation as with
genetics differentiation (Nielsen, 1992). This method can use used
for marking fry or larvae after hatching and before they being
feeding, and can be used as a group mark rather than individual
marks such as those which can be applied to juveniles such as coded
wire tags, brands, or elastomer tags (Moffet et al., 1997, Dussault
and Rodriguez, 1997).
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a standard curve of avidin dilution and the
effects of Tween 20 in ELISAs on serial dilution of avidin in PBS
and Atlantic salmon serum.
[0024] FIG. 2 shows serial dilution of Atlantic salmon serum to
show the point of nonspecific protein binding of the ELISA
(1:10,000).
[0025] FIG. 3 shows the mean ELISA absorbances to a specific assay
for biotinylated bovine serum albumin in Atlantic salmon sea-run
adults from the Connecticut, Merrimack, and Penobscot rivers.
[0026] FIG. 4 shows serum titres of eight juvenile Atlantic salmon
15 months after exposure to biotinylated bovine serume albumin as
fry. Negative control serum absorbance was 0.022.
[0027] FIG. 5 shows mean ELISA absorbances (+SEM) of Atlantic
salmon serum for the presnce of biotinylated bovine serum albumin
baths 24, 27, and 30 months after exposrue. Salmon fry were exposed
8 and 1 days before yolk absorption.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Animals traditionally have been exposed to biologically
active proteins either via an injection, or by immersion in a bath
treatment. Comparisons of blood titer from both methods give an
indication of the efficiency of protein delivery to the animal. For
delivery of protein markers to fish which are too small for
injections, it is necessary to use a bath treatment. Successful
bath exposures of salmonids exposed at optimum water temperature
result in high antibody concentrations about 30 days past exposure,
with high immune titers lasting about 75 days (Anderson et al.,
1979). Comparison of the two exposure methods, intra peritoneal
injection and bath immersion, through 90 days post-treatment, gives
an indication of effectiveness of exposure treatments.
[0029] The present invention thus provides a marking method which
can be used for stock identification. The marking of fish fry less
than 25 mm long requires that the foreign proteins be delivered by
bath exposure rather than injections, primarily due to the size of
the fish. Conventional chemical marking methods typically attenuate
as the animal grows, or they accumulate in hard tissues, requiring
lethal sampling methods, and there is much less than 100% retention
of the chemical mark (Nielsen, 1992). By using a protein mark in
the blood stream, the disavantages of dilution of the marker as the
animal grows are reduced because the signal can be amplified in the
blood using ELISA or other immunoassay methods. The protein is not
deposited in hard tissue, but rather in the blood, which means that
non-lethal sampling methods can be used.
[0030] While other assay methods can be used to determine the
marking protein, ELISA has been found to be the simplest and the
most effective. Typical fluorescent tags such as Texas red, FITC,
etc. are not as effective in assaying fish, because there is a
natural fluorescence in fish blood over the ranges of these tag
absorbances or emittances, leaving high background values in serum
which makes detection more difficult. Thus, the tags used for fish
assays should be those which do not absorb or emit in the ranges of
the naturally occurring fluorescence in fish blood.
[0031] Materials and Methods
[0032] Atlantic salmon parr, mean length 104 mm and mean weight
13.0 g, were randomly separated into seven groups of 42 fish each,
and each group was exposed to biotinylated bovine serum albumin
either by intra peritoneal injection or by bath immersion. Four
intra peritoneal treatments were given with 10, 25, 50, and 100
.mu.g bovine serum albumin in 0.1 ml sterile distilled water. Thus,
the protein was administered at rates of 1.0, 2.5, 5.0, and 10.0
.mu.g per fish. The fish were anesthetized in tricaine
methanesulfonate, given the intra peritoneal injection with a 26
gauge needle, and placed into separate rearing units for each
treatment group.
[0033] Three groups of fish were immersed in bath treatments at one
of three bovine serum albumin concentrations: 5, 10, and 10
.mu.g/ml. The bath treatments were given in polypropylene tubs with
the fish held in nets within the tubs. The fish were lightly
anesthetized prior to immersion to reduce stress. The immersion
lasted for seven minutes, after which the fish were placed into
separate 43 L glass rearing units with a 4 L/minute flow of
9.degree. C. water
[0034] Fish given DMSO baths of 0, 1, 2, and 4% DMSO were lightly
anesthetized and placed into a polypropylene tub. The fish were
held in a net and immersed into protein bath for eight minutes.
Groups of 45 to 50 fish were marked in one of three ways:
[0035] 1. Group 1 was immersed in a 10 .mu.g/ml bovine serum
albumin bath, rinsed, and removed;
[0036] 2. Group 2 was immersed in a 10 .mu.g/ml bovine serum
albumin bath, rinsed 3-5 minutes, and immersed in a second bath of
3.3 .mu.g/ml FITC-avidin with DMSO used only in the first bath
(bovine serum albumin);
[0037] 3. Group 3 was given the same double bath treatments of
bovine serum albumin and avidin as the second group except that
DMSO was used in both the first and second baths.
[0038] Once the bovine serum albumin bath or double bath treatments
were complete, the fish were placed into rearing tanks as in the
above study, and held for 90 days. Blood was sampled on days 10,
30, 40 and 90 after exposure to the baths. Samples from both tests
were collected in the same manner.
[0039] Blood samples from fish in both tests were collected from
anesthetized fish (n=10 per treatment) 10, 30, 40, and 90 days
after administration of treatments. The blood was refrigerated 4.6
hours, centrifuged for 8 minutes at 13,000 rpm, the serum removed
and stored frozen until assayed for bovine serum albumin by ELISA.
Fluorescence assays for FITC on a Perkin Elmer HTS 7000 microplate
reader were inconclusive.
[0040] The ELISA procedure for all tests was for detection of
bovine serum albumin and included positive controls of phosphate
buffered saline (PBS), bovine serum albumin added at one or more of
five concentrations: 1.90, 1.00, 0.50, 0.25, and 0.10 mg/ml. Serum
or controls were adjusted to minimum volume of 100 .mu.L with
phosphate buffered saline, then diluted by half in phosphate
buffered saline, added to wells of a 96-well microplate (100
.mu.L/well) and incubated in a refrigerator overnight. Absorbances
were adjusted to the ratio of phosphate buffered saline added to
serum before the 1:1 dilution with phosphate buffered saline.
[0041] The serum and controls were warmed to room temperature the
next day, and the serum and controls were removed and the plates
washed with PBS five times. The plates were then blocked witih 280
.mu.L per well of casein blocking buffer (Pierce, Rockford, Ill.)
and incubated for 45 minutes. The previous wash procedure was
repeated with 50 .mu.L Tween 20 added to the PBS. After washing, 2
drops (80 .mu.L) of ABC complex (Pierce, Rockford, Ill.) were added
to each well and the plates were incubated for 20 minutes. The
plates were washed again five times with BPS-T233n, substrate was
added (TMB Substrate, Pierce, Rockford, Ill.), and the plates were
read at five minute intervals. The 20-minute reading was selected
for providing peak readings. Absorbance was read on a Dynatech MR
580 (Chantilly, Va.) microplate reader.
[0042] Before statistical tests were conducted, results from the
different ELISAs were standardized to remove differences, which
could occur among plates run at different times. To do this, posive
control absorbance values were set at 1.000, and all absorbances
were algebraically adjusted the same amount needed to set the
negative control serum absorbance to zero. An absorbance ratio was
then calculated by dividing the absorbance value of each data point
(mean of duplicate wells) by the positive control value. SAS
statistical procedures were used for all analyses (SAS Institute,
1987).
[0043] For the first study, of bath and injection methods, two-way
analysis of variance was used to determine differences among
treatment means using bath or injection treatment level, and sample
data as variables For the DMSO data set, three-way analysis of
variance was used using percentage of DMSO, single/double bath
treatments, and sample data as variables. The Duncan's Multiple
Range Test was used to determine significant differences among
treatments for the two-way analysis, and the
Ryan-Einot-Gabriel-Welsch Multiple F Test was used for the
three-way analysis. All tests were performed at the 0.05 level of
significance.
[0044] Bath/Injection Study
[0045] ELISA absorbance from serum samples collected 10 days post
exposure showed high variability within treatment groups, and mean
absorbances were statistically the same for fish given either
injection or bath treatments of bovine serum albumin, as show in
FIG. 1. At this stage, several of the fish tested had low response
to the antigen regardless of its presentation or concentration.
[0046] Absorbance values 30 days post-exposure were also not
significantly different among any of the bath or injection
treatments (cf. FIG. 1). The 40-day sample showed that the mean
absorbance levels were either similar to or slightly lower than
those from fish 30 days post-exposure Relationships with treatments
remained the same as in previous treatments (FIG. 1).
[0047] One significant peak in absorbance occurred in the 10
.mu.g/ml bath group at 40 days post-treatment, as shown in FIG. 2.
The overall mean absorbances for all bath groups and all injection
groups were nearly the same, as shown in FIG. 2.
[0048] The 90 day sample yielded results similar to those of the
previous sample dates, except that it was at significantly lower
absorbance levels than either the 40 day bath group or the 30 and
40 day injection groups, as shown in FIG. 3. There were no
significant differences among treatments for either serum
concentration of bovine serum albumin in bath or injection
treatments. Concentrations from all levels of bovine serum albumin
yielded similar mean serum concentrations between 10 and 20 times
those of negative control serum. Further results showed that serum
levels were significantly higher on day 40 (0.492) than on days 10
(0.310) and 90 (0.127), lowest on day 90, and that day 30
absorbance ratios (0.426) were not significantly different from
those on days 10 or 40.
[0049] DMSO and Doouble Mark Study
[0050] There were no significant differences in the main effects
among any of the DMSO levels at any sampling date, as shown in
Table 1. One signifcant difference occurred among Atlantic salmon
of bovine serum albumin solution or bovine serum albumin/avidin
complex with DMSO treatment which was a higher absorbance ratio
where DMSO was used in both baths of the double bath group (2.275)
than in the bovine serum albumin group (1.701). The double mark
group with DMSO used only for the first bath (1.867) was not
different from either of the other treatments, as shown in FIG. 4.
This block includes data from all levels of DMSO from 0 to 4%.
There were also significant differences among sample times, due to
high readings on day 90 (4.004), which resulted from the use of
different negative control serum that gave high background
absorbance and altered the standardizaton procedure results. Mean
sample absorbances for the day 90 data were within the same range
as those from all other sample days prior to standardization (day
10, 0.578; day 20, 0.784; day 40, 0.642; day 90, 0.674). The range
of mean absorbance ratios for 10, 30 and 40 days were 0.794, 1.202
and 1.790, respectively, providing a similar pattern to those from
the bath/injection comparison. The standardization procedure
yielded mean day 90 absorbance ratios of 4.048, much higher than
those of earlier samples.
[0051] The results of this study indicate that juvenile Atlantic
salmon were able to achieve similar concentration of bovine serum
albumin over a range of injection rates and bath treatment
concentrations. Effective bath treatments for antigen entry into
fish have been given with as little as two minutes exposure time
(Anderson et al., 1979; Zapata et al., 1987) with varied dosages of
antigen from 1 to 1000 .mu.L. Anderson et al. (1979) found that a
10 .mu.g/mL concentration of Yersinia ruckeri O-antigen was
sufficient to produce a response. It has now been found that a 7-8
minute exposure time to bovine serum albumin was long enough for
sufficient bovine serum albumin to be taken up and remain in
Atlantic salmon.
[0052] Atlantic salmon are immunocompetent at the end of the
yolk-sac absorption period with membrane IgM present on lymphocytes
(Ellis, 1977). Similarly, other salmonid species are
immunocompetent at the end of the yolk absorption period, such as
the chum salmon (Oncorhynchus keta, Nagae et al., 1993), or the
rainbow trout (Oncorhynchhus mykiss, Tatner, 1986). Immunological
tolerance at the pre-feeding stage in salmonids allows the use of
protein bath treatments for marking salmonid fry and allows the
marked fish group to be identified later. One skilled in the art
can, without undue experimentation, readily establish exposure
concentrations for marking fry.
[0053] The most important aspect of the functioning immune system
and the administration of a biochemical marker, such as a protein
such as bovine serum albumin, is the fact that the protein is
tolerated, or ignored, by the animal's immune system, so that the
protein remains within the blood stream for a significant amount of
time. Reasonable levels of bovine serum albumin were found in
salmon 90 days after exposure, which is what would have been the
end of the humoral immune response period (Anderson et al., 1979).
This may indicate that bovine serum albumin remains after the
humoral response. Other data shows this to be true for as long as
30 months in fish currently under a long term study.
[0054] One of the biggest advantages of the method of the present
invention over conventional methods is that the method can be used
over a prolonged period of time. As shown in FIG. 5, in 80 to 100%
(mean to 90% at 30 months) of the fish sampled, protein retention
in serum still occurs at 30 months after bath treatment. In this
case, salmon fry were exposed 8 and 1 days before yolk absorption.
Thus, it is clear that the present method can be used for stock
identification for up to about three years.
[0055] Because protein markers may enter fish through several
routes, it is believed that fry or alevins would be able to produce
an appropriate response to the challenge in a bath. Assuming that
the humoral immune response is functional in salmonid fry by the
time fish begin exogenous feeding [Ellis, 1977, Atlantic salmon,
Nagae et al., chum salmon; Fuda et al., 1991, masu salmon
(Oncorhynchus mason); Tatner 1986, and Castillo et al., 1993,
rainbow trout], then the routes of protein entry into a fry are
important in the fish's uptake and processing of a foreign protein.
Entry routes could include the gills, skin, and the
gastrointestinal tract. Antigen entry through the gills was shown
for juveinle Atlantic salmon in a two minute bath of Yersinia
ruckeri O-antigen (Zapata et al., 1987) and for rainbow trout in a
one hour bath of Flavobacterium branchiophilium. Ototake et al.
(1996) found that rainbow trout take up antigen through the skin,
using bovine serum albumin after exposing 15 juvenile trout to 2%
concentrations of bovine serum albumin in three minute baths. Last,
Robohm and Koch (1995) showed that a major route of uptake of
antigen also occurs through the gastrointestinal tract. The authors
plugged the esophagus of fish and showed greatly reduced uptake of
Clostridium botulinum toxin. In bath exposures as described herein,
Atlantic salmon could take up bovine serum albumin in any or all of
these routes, allowing for the partitioning of bovine serum albumin
within the fish.
[0056] Some variation in bovine serum albumin uptake and processing
experienced in the fish could be explained by the fact that the
fish were anesthetized to administer the mark, or that genetic
differences among fish could result in different protein retention
levels. Lobb (1987) found that channel catfish (Ictalurus
punctatus) had depressed antigen uptake due to anesthesia. Genetic
differences in hemolytic or lysozyme activity have been shown for
rainbow trout and Atlantic salmon (Roed et al., 1990; Roed et al.,
1992; Roed et al., 1993, and Lund et al., 1995). Differences among
genetics of individual fish studied could have led to their
variation in response. However, it appears that different
individual fish responses to anesthesia were probably the largest
cause of variability among treatment groups.
[0057] It was found that juvenile Atlantic salmon could take up and
store bovine serum albumin effectively through bath treatments
ranging from 5 to 20 .mu.g/ml beyond a 75-80 day humoral immune
response period. Concentrations of bovine serum albumin assimilated
by a wide range of bath concentrations (5-10 .mu.g/ml) were
statistically the same as those from fish given intra peritoneal
injections of bovine serum albumin. There appears to be large
variability in forgiveness in an effective protein dose rate for a
bath treatment. The use of DMSO in bath treatments only
significantly improved the uptake of proteins in a double bath
proceudre of two agglutinating proteins when DMSO was used in both
treatment baths. Exposure of Atlantic salmon to a foreign protein
bath thus provides an effective method of marking small fish for
stock identification.
[0058] A wide range of concentratons of proteins can be used in the
present invention, although concentrations of from 1 to 50 mg/mL of
bovine serum albumin are adequate. Different concentrations of the
protein are not significantly different in effectiveness from one
another.
[0059] The trays in which the fish are immersed in the protein bath
are preferably those to which the proteins do not stick. While
polypropylene is the preferred material for the pans, fiberglass
incubation trays can also be used.
[0060] The optimum temperature for treatment should be the same as
optimum temperatures of rearing of the species treated. For
example, temnperature ranges for salmonids would be from 5 to
18.degree. C., depending upon the species. The use of water
temperature on a species' outer range of tolerance can reduce
protein uptake.
[0061] Immunoassay Procedures
[0062] Several in vitro ELISAs were performed with Atlantic salmon
blood, serum, and with phosphate buffered saline (PBS) as control.
These media carried various concentrations of bovine serum albumin,
or avidin (Pierce, Rockford, Ill.). ELISA procedures are described
according to the type of result anticipated for each. Some
procedures are common to all assays and will be abbreviated after
their first description.
[0063] Determination of Bovine Serumn Albumin Coupled to Avidin in
Serum or Blood.
[0064] Three concentrations of bovine serum albumin (0.090 or 0.048
.mu.g/mL, 0.024, and 0.006 .mu.g/mL bovine serum albumin) were
added to whole blood or serum pooled from 12-20 juvenile Atlantic
salmon and used in an ELISA for bovine serum albumin. Two to three
mL of blood was collected from two year old Atlantic salmon
anesthetized in methane tricaine sulfonate from caudal puncture in
3 cc syringes, and pooled into 20 mL samples. One hundred .mu.L of
heparin, 250 units/mL, was added to whole blood to prevent
clotting, and blood used for serum collection (no heparin added)
was refrigerated overnight and centrifuged for ten minutes at
13,000 rpm to separate serum from blood cells. After separation,
the serum was drawn off and stored frozen until use.
[0065] In the assays described herein, 25 .mu.g/mL avidin was added
to the blood or serum and PBS control. After the addition of
avidin, three separate samples of 100 .mu.L of one of three
concentrations of bovine serum albumin, 1.900, 0.500 and 0.125
mg/mL, was added, giving a final bovine serum albumin concentration
of 90, 24, or 6 .mu.g/mL. The solutions were allowed to incubate at
room temperature for 15 minutes, then 100 .mu.L volumes were
transferred in triplicates to a 96 well microplate (Nunc, Immunolon
4 microplate). Following a 45 minute room temperature incubation,
the wells were washed five times with distilled water. Casein
blocker (Pierce, Rockford, Ill.) was then added to the wells in the
amount of 280 .mu.L/well, and blocking was allowed for 40 minutes.
The rinse procedure followed, then horseradish peroxidase
conjugated biotin was added. A 20 minute incubation followed, then
a rinse, and the addition of 3,3',5,5'-tetramethyl benidine (TMB
substrate, Pierce, Rockford, Ill.). Absorbances were read with a
Dynatech MR580 (Chantilly, Va.) microplate reader every 5 minutes
for 40 minutes. This was the standard ELISA for bovine serum
albumin.
[0066] A second test was conducted using the same procedure except
for an extended incubation time to 90 minutes after the addition of
blood or serum containing avidin and bovine serum albumin.
[0067] Serial Dilution of Avidin to Determine Lowest Levels
Detectable by ELISA.
[0068] To detect the lowest levels of avidin in serially diluted
samples of PBS, beginning with a stock solution of 7.5 .mu.L/ml
avidin, twelve dilutions were made from the stock solution, each
cutting the concentration of avidin in the previous solution by
half. The assay was run in duplicate sets of avidin dilutions in
PBS to test for differences between the use, or lack, of 50
.mu.L/ml Tween 20 (Sigma Chemical Company, St. Louis, Mo.) in the
wash buffer (PBS).
[0069] Serial Dilution of Avidin in Serum to Determine Nonspecific
Protein Binding Interference
[0070] A serial dilution of normal Atlantic serum was conducted
with 7.5 .mu.L/mL of avidin added in PBS. Normal serum was spiked
with avidin and diluted by a factor of ten from 1:10 to 1:10,000.
Each dilution was added to microplate wells in duplicate. After 100
.mu.L of solution was pipetted into the microplate wells, the
plates were incubated for 45 minutes at room temperature. The
plates were then washed five times with distilled water and blocked
with casein for 40 minutes. After incubation, 100 .mu.L of biotin
conjugated with horseradish peroxidase (HRP, Pierce, Rockford,
Ill.) as the second antibody, was added to each well either with or
without Tween 20 (50 .mu.L Tween/1.4 mL biotin-HRP), and the plate
was incubated for 20 minutes. The plate was then washed as before,
and TMB substrate was added. Absorbance readings were taken
immediately, and at five minute intervals for 40 minutes.
[0071] Molecular Marking of Atlantic Salmon Fry
[0072] To test for the natural presence of bovine serum albumin in
wild sea-run Atlantic salmon, serum was collected from 90 sea-run
adult fish during their migration into spawning rivers. The serum
was tested by the standard ELISA method for bovine serum albumin
from fish returning to one of three river systems: 45 fish in the
Connecticut River, 23 fish from the Penobscot Rvier, and 22 fish
from the Merrimack River.
[0073] Groups of 200 Atlantic salmon fry were exposed to 0.8, 1.6,
and 3.1 .mu.g/ml bovine serum albumin in baths, three bath
concentrations (0.08, 0.15, and 0.30 .mu.g/mL of bovine serum
albumin) of BSA Supercarrier (Pierce, Rockford, Ill.) with the
hapten DNP, or to avidin horseradish peroxidase complex (HRP-Av) at
0.60, 0.30, and 0.10 .mu.g/mL. Two groups of fish (three replicates
of each treatment) were exposed to each treatment, one each eight
days before, and one day before yolk absorption. The fish were
immersed in each bath for five minutes at 8.3.degree. C., using egg
incubation trays filled with five liters of water. After the bath
treatment, the fish were reared in 8-10.degree. C. water in 40
liter tanks (half tanks) for 16 months, then in 80 liter glass
tanks with flow through water suppled at four liters/minute. The
second group of Atlantic salmon swim up fry were randomly selected
into twelve groups of 300-400 fish each and placed into one of
three replicates each of four treatments, which were exposed by
bath immersion in either 20 .mu.g/mL bovine serum albumin in the
first bath and 3.0 .mu.g/mL or 1.5 .mu.g/mL avidin-fluorescein
complex (FITC-Av) in a second bath. The treatment combinations were
20 .mu.g/mL bovine serum albumin (BSA) and 3.0 .mu.g/mL FITC-Av; 20
.mu.g/mL BSA and 1.5 .mu.g/mL FITC-Av; 10 .mu.g/mL BSA and 3.0
.mu.g/mL FITC-Av; and 10 .mu.g/mL BSA and 1.5 .mu.g/mL FITC-Av.
Immersion lasted ten minutes for each bath with a two minute rinse
in between. The fish were then placed in rearing units.
[0074] At 15, 24, and 30 months exposure, 10 fish per treatment
replicate were anesthetized in tricaine methane sulfonate and blood
was drawn from the caudal vessels. The blood was refrigerated
overnight, centrifuged for eight minutes at 13,000 rpm the next
day, and the serum recovered was stored frozen. Serum samples were
measured by fluorometry for FITC-Av, and ELISA was used for BSA
determination. To perform the fourescence assay, 100 .mu.L/well of
serum were placed into 96-well black round bottom plates (Dynatex,
Chantilly, Va.) and fluorescence emittance was measured on an HTS
7000 fluorometer (Perkin Elmer, Norwalk, Conn.). Preliminary
standards were performed with black, white, and membrane unifilter
GF/B plates (Dynex Techcnologies, Chantilly, Va.) to determine the
lowest background interference for Atlantic salmon serum. Serial
dilutions of serum and FITC-Av were performed to determine the
sensitivity of the instrument with each type of plate. Black plates
had the lowest background interference and allowed the greatest
sensitivity. When compared to negative control Atlantic salmon
serum, 0.0006 .mu.g/ml FITC-Av in serum still showed a positive
result.
[0075] The standard ELISA procedure included the use of four
positive control groups of PBS with BSA added at one of four
concentrations: 1.0, 0.50, 0.25, and 0.10 mg/mL. One negative
control group of a 1:1 ratio of Atlantic salmon serum to PBS was
used. The controls and the serum samples were plated at 10 .mu.L
well and incubated in a refrigerator for 24 hours. The ELISA was
performed as above.
[0076] The assays differed for analysis of each chemical mark. In
every case, however, four groups of PBS with BSA added at one of
four concentrations: 1.0, 0.50, 0.25, 0.10 mg/ml, served as
positive controls. One negative control was used of a 1:1 ratio of
Atlantic salmon serum to PBS. The controls and the serum samples
were plated at 100 .mu.L well and incubated in a refrigerator for
24 hours. The BSA standard ELISA method as above was used. HRP-Av
samples required only blocking with casein, and addition of TMB
substrate to react with HRP-Av in the seurm. The absorbance was
measured in the ELISA reader. The assay procedure for BSA (BSA
Supercarrier and BSA/FITC-Av) included blocking with casein,
washing with PBS-Tween, then incubation for 45 minutes with
{fraction (1/1000)} monoclonal mouse anti-BSA antibody (MaBSA,
Sigma, St. Louis, Mo.). The plates were again washed with
PBS-Tween, biotinylated goat anti-mouse antibody (B-GaM, Pierce,
Rockford, Ill.) added and the plates were incubated for 45 minutes.
After washing with PBS-Tween, ABC complex was used to bind to
biotin on B-GaM for 20 minutes. The plates were washed, TMB
substrate added, and the absorbance was read.
[0077] Statistical Analysis
[0078] The statistical analysis was calculated from single mean
values derived from all duplicate pairs. The one way analysis of
variance was used to show significcant differences between
biotin-avidin formation in blood and in serum and the Friedman
ranked two-way analysis of variance to find differences among
response to the BSA/FITC-Av chemical complex formed in the fish.
Duncan's New Multiple Range Test was used to determine differences
among means. Absorbances in serial dilutions were plotted to show
differences among absorbances resulting from dilutions. The
decisions rule for all statistical tests was set at p=0.05.
[0079] As demonstrated above, the B-BSA-avidin-enzyme complex forms
a readable mark in Atlantic salmon or blood serum. By using this
method, one can produce a retrievable biochemical mark. When many
marks are applied, different groups of fish can be identified. The
lowest detectable levels of AV-B-BSA complex in vitro in serum
(1.88 .mu.g/mL) provided a basis for determining dosage for bath
treatments for fish, and defines some limits to sensitivity in in
vivo recognition of the stock identification marker. The method of
the present invention can be used with larval, or pre-feeding fish,
or those fish which are too small to accept juvenile fish tagging
methods such as fin clips, hot or cold brands, coded wire tags,
elastomer tags, or fluorescent sprays. (Bandow 1987, Negus et al.
1990, Yunk and Cook 1991, Thedinga and Johnson 1995, Moffett et al.
1997, Dussault and Rodriguez 1997).
[0080] The mark of the present invention is easily retrieved, and
there is no necessity for lethal sampling methods, as is the case
with otolith marks or rare earth element markers (Younk and Cook
1991, Nielsen 1992, Schroder et al. 1996). The methods for applying
the mark of the present invention, i.e., a five minute bath
exposure and rinse, are simpler than those for calcein fluorescent
markers, which may require exposures for up to 48 hours in a
recirculating chemical bath to provide a successful mark
(Gelsleichter et al. 1997, Mohler 1997). The biochemical marking
method of the present invention is useful as a batch marking
method. With different tags on the BSA, the method can be used to
mark several groups, such as families. While the marker of the
present invention may not be as useful as current genetic methods
which can be used for genetic analysis of families, the method of
the present invention does not require a search for suitable
alleles and sampling of both parents and progeny. Moreover,
genetics procedures require the use of sophisticated equipment and
genetics expertise to interpret results (Nielsen 1992), and
evaluation of genetic markers is costly. The biochemical batch
marking method of the present invention provides a very low cost
alternative to genetic procedures.
[0081] BSA remains in the blood of Atlantic salmon for over 30
months when a five minute bath at a concentration of at least 3.1
.mu.g/mL B-BSA is used. It was also found that lower concentrations
of "cationized" BSA in Supercarrier (Pierce, Rockford, Ill.) were
effective at very low concentrations (0.08-0.30 .mu.g/mL) with mark
retention up to 15 months (88%), but was less effective at 24
months (67%). These concentrations may be too low for continued
detection within the blood as the fish grows.
[0082] In forming biochemical complexes of B-BSA/FITC-avidin in
vivo, it was found that either the complex did not form well within
the fish or the immune system picked up the avidin and cleared it
from the system, resulting in lower retention rates than with the
use of BSA alone. The complete inability to find avidin after 15
months in all three groups of fish marked with avidin indicates
that the immune system may remove avidin from the blood stream.
However, Atlantic salmon tolerate BSA well, and the BSA remains for
periods of at least 30 months.
[0083] The humoral immune system is functioning by the time fish
begin feeding, so it is to be expected that pre-feeding larval
salmonid fish could take up and process an antigen tagged as a
stock identification marker. The Atlantic salmon has membrane IgM
at first feeding, indicating a functional humoral immune system
(Ellis 1977), and the chum salmon, Oncorhynchus keta, has rapidly
increasing IgM concentrations at first feeding (Nagae et al.,
1993). The rainbow trout, Oncorhynchus mykissis, is said to have
peak IgM at hatching (Castilo et al. 1993) and is able to produce a
titer at one month post-hatch (Tatner 1986). Because the immune
system is functional in salmonids at this age, it appears that the
tolerance to biological chemicals considered as foreign to the
immune systems indicates that the use of tolerant chemicals will
provide long lasting markers, even in fish marked at an older
age.
[0084] The in vitro tests conducted as described herein
demonstrated that the biotin-avidin complex will form in the serum
of salmonids and provides a method for biochemically tagging small
fish or other animals. It was found that 85-90% of Atlantic salmon
fry exposed to B-BSA prior to their first feeding can retain
recognition of their history of exposure to the protein for over
two years. The protein tags (biotin or fluorescence, etc.) make it
possible to find the protein readily within the fish's blood
stream. Since efficiency of tag recognition of less than 100% is a
characteristic of chemical tags (Nielsen 1992), 100% tag retention
is not expected for large groups of fish analyzed.
[0085] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without departing
from the generic concept, and, therefore, such adaptions and
modifications should and are intended to be comprehended within the
meaning and range of equivalents of the disclosed embodiments. It
is to be understood that the phraseology or terminology employed
herein is for the purpose of description and not of limitation.
* * * * *