Method for diagnosing myofibrogranuloma (MFG) in walleye

An improved method for diagnosing myofibrogranuloma (MFG) in walleye without harming the fish is described. Blood samples are drawn from walleye and creatinine and calcium levels are then determined along with the sex and length of the fish. These data are then analyzed and compared to data from healthy walleye using discriminant analysis. The process is effective for identifying approximately 88% of MFG-positive walleye.

FIELD OF THE INVENTION 
The present invention relates to methods to diagnose disease, in 
particular, a method for diagnosing myofibrogranuloma (MFG) in walleye. 
BACKGROUND OF THE INVENTION 
Myofibrogranuloma (MFG) is a degenerative disease of skeletal muscle 
recognized only in walleye (Stizostedion vitreum). MFG is a unique form of 
skeletal degeneration. The disease is characterized by profound 
alterations of the trunk musculature produced by extensive hypertrophy of 
the muscle fibers. Two degenerative processes are involved. The first and 
most pronounced lesion consists of coagulation necrosis of muscle fibers 
accompanied by an inflammatory response and formation of granulomas 
(muscle tumors). The second is non-inflammatory and characterized by local 
areas of acute myolysis. Opaque, yellowish brown muscle with a sandy 
texture is seen in fish with advanced forms of the myopathy. Fishermen 
will usually discard fish with advanced MFG because the lateral trunk 
muscles (fillet) especially beneath the skin becomes sandy, discolored and 
somewhat jelly-like. 
MFG has been recognized by disease specialists to occur most frequently in 
older fish. Economon suggests MFG to be present in 1 of 10,000 walleye 4.5 
years or older. Economon, P. P., "Myofibrogranuloma, a Muscular 
Dystrophy-Like Anomaly of Walleye (Stizostedion vitreum), Minnesota Dep. 
Nat. Resour. Fish Wildl. Spec. Publ., 113: 1-11 (1975). The mean total 
length at capture for walleye five years of age or older is generally 
greater than or equal to 500 mm. 
The general nature of the muscle degeneration suggests MFG is of a 
hereditary nature. It has been suggested that MFG in walleye is apparently 
not sex-linked and that MFG represents a unique myopathy. Kelly, et al., 
"Chemical analysis of muscle from walleye (Stizostedion vitreum vitreum) 
with myofibrogranuloma, a chronic myopathy," Can. J. Fish. Aquat. Sci. 44: 
1425-1431 (1987). Further, it has been demonstrated that the disease is 
most likely not infectious. Holloway, et al., "A myopathy in North Dakota 
walleye, Stizostedion vitreum," J. Fish Dis. 5: 527-530 (1982) Thus, due 
to MFG's probable genetic transmission, shipment of potentially diseased 
eggs is a concern of fishery biologists. 
In addition, many lakes and reservoirs have increasingly used stocking as a 
management tool. For instance, at the Merritt Reservoir in north central 
Nebraska, the walleye population is maintained almost exclusively by 
stocking (Joel Klammer, Nebraska Game and Parks Commission, personal 
communication, Lincoln, Nebr. 68503-0370). Further, in 1990, over 6.2 
million walleye fingerlings were stocked into Lake Sakakawea, North 
Dakota, to enhance the population. In lower Lake Oahe 221,250(1986), 
299,500 (1987), 476,868 (1988) and 449,256 (1989) fingerling walleye were 
stocked. Fielder, D. G., "Evaluation of stocking walleye fry and 
fingerlings and factors affecting their success in lower Lake Oahe, South 
Dakota," North Am. J. Fish. Mgmt. 12: 336-345 (1992a); Fielder, D. G., 
"Relationship between walleye fingerling stocking density and recruitment 
in lower Lake Oahe, South Dakota," North Am. J. Fish. Mgmt. 12: 346-352 
(1992b). Moreover, multiple years of almost complete year class failure 
and angler demand on the reservoirs have led to the increased stocking of 
walleye to enhance and even establish year classes. 
MFG is of concern primarily in the Midwest, particularly in North and South 
Dakota, Minnesota, and Nebraska. Roe shipped from those states is sent 
throughout the country, wherever walleye is stocked for sport fishing. 
Therefore, a field test for this disease would be useful to remove 
diseased animals from the population and minimize the risk of spread via 
roe. 
Present methods for detecting MFG can not be accomplished without filleting 
the fish since walleye with MFG generally show no external symptoms or 
abnormal behavior. Kelly et al. chemically analyzed 15 cases of MFG in 
adult walleye from 1975 to 1985 from seven lakes in southern Manitoba and 
northwestern Ontario and from Lake Sakakawea, North Dakota. Kelly, R. K., 
et al., "Chemical Analysis of Muscle From Walleye with Myofibrogranuloma, 
a Chronic Myopathy," Can. J. Fish. Aquat. Sci. 45: 1425-1431 (1987). The 
inorganic analysis of representative samples revealed elevations or 
depressions of ions which were consistent with those of extensively 
damaged muscle membrane systems. One of the greatest differences was seen 
in calcium levels which averaged 55 times normal in muscle lesions where 
excessive calcium deposition was demonstrable by histochemical staining 
with nuclear fast red. Id. 
X-ray analysis has been utilized to show calcium deposits in the human 
kidney by Davidson, who stated that technicians are able to visualize 
various organs and systems radiographically because of differential 
absorption of X-rays, owing to differences in density of materials being 
examined. Davidson, A. J., "Excretory Urography", Computed Tomography, 
Ultrasound and X-Ray: An Integrated Approach, Masson Publishing U.S.A., 
Inc., New York, pp. 11-20 (1979). Thus, applicants hypothesized that 
muscles affected by MFG would have a higher density than normal muscle 
tissue, owing to the influx of calcium and subsequent calcification in 
advanced cases which could be detected with X-rays. The disadvantages of 
using X-rays, however, is that X-rays can be harmful to fish and would 
make subsequent genetic studies questionable. Therefore, there is a need 
in the art for a means of diagnosing MFG without filleting the fish or 
using X-rays. 
Vascular biochemistry seemed the most likely choice since human muscular 
dystrophy is diagnosed with blood chemistry values. Many specific 
diseases, whether caused by bacteria, viruses or metabolic dysfunctions, 
are characterized by specific changes in the composition of the blood. 
Shell, E. W., "Chemical Composition of Blood of Smallmouth Bass," Res. 
Rep. U.S. Fish Wildl., Serv. No. 57, U.S. Government Printing Office, 
Washington D.C. (1961). Diagnosis depends on the comparison of blood 
composition values of the animal with the suspected disease with blood 
composition of healthy animals of the same species. 
Oser states that the normal range for calcium is narrow and small 
variations indicate pathology such as bone abnormalities and muscle 
tumors. Oser, B. L., Hawk's Physiological Chemistry, McGrawHill Book 
Company, New York, New York (1965). If calcium is regulated in the 
walleye, fish exhibiting MFG should have significantly different serum 
calcium levels than healthy walleye due to the 55-fold increase in muscle 
calcium associated with MFG shown by Kelly et al. in 1987. It was 
hypothesized that MFG-diseased walleye would exhibit relatively higher 
serum calcium concentrations than healthy walleye. 
In 1993, it was discovered that there was a significant difference between 
mean serum calcium levels in spawning male and spawning female walleye. 
Serum calcium is higher in females due to the influence of estrogen which 
stimulates synthesis of yoke protein and an increase in protein bound 
plasma calcium. It was further found that the mean serum calcium levels of 
spawning male walleye was significantly different from the mean serum 
calcium levels of MFG-positive spawning male walleye. Similarly, there was 
a significant difference found between mean serum calcium levels of 
spawning female walleye and mean serum calcium levels of MFG-positive 
spawning female walleye. These elevations suggested that calcium was 
regulated in the walleye and that MFG was related to serum calcium levels. 
The calcium serum level test for MFG was not definitive, however, since 
there was an overlap of ranges between the healthy and MFG-positive 
spawning females and males. Therefore, it was determined that serum 
calcium levels, in and of themselves, could not be used to accurately 
diagnose MFG in walleye. 
According to the invention, creatinine is another vascular parameter which 
potentially may be used to assess muscle degeneration. Phosphocreatine in 
muscle spontaneously cycles to creatinine. The quantity of end product, 
creatinine, is proportional to muscle mass and thus may be utilized in 
evaluating muscle mass. It was hypothesized that creatinine concentration 
in walleye serum would reflect these muscle 
mass-phosphocreatine/creatinine relationships. It was further hypothesized 
that since muscle mass decreases in MFG-afflicted walleye, albeit up to 
20% in the most severe cases, that phosphocreatine may also decrease. A 
decrease in phosphocreatine means a reduction in the spontaneous 
cyclization to creatinine. If there is a reduction in creatinine levels in 
MFG-positive walleye, the values in the blood serum might reflect this. 
Therefore, a primary objective of the present invention is to utilize a 
method for diagnosing MFG in walleye which is accurate and is not harmful 
to the fish. This invention has as its primary objective the fulfillment 
of this need. 
Another objective of the present invention is to provide a method of 
diagnosing MFG in walleye which is more accurate than previously available 
methods. 
Yet another objective of the present invention is to provide a method for 
diagnosing MFG which is easy to perform and cost effective. 
The method and means of accomplishing these and further objectives of the 
invention will become apparent from the detailed description which follows 
hereinafter. 
SUMMARY OF THE INVENTION 
The present invention relates to a procedure for diagnosing 
myofibrogranuloma in walleye without impairing the fish for subsequent 
study or for use in spawning. The procedure involves the discriminant 
analysis of fish sex, total length, and a novel procedure measuring 
humoral concentrations of creatinine and calcium in blood serum to assess 
normal levels and the differences which are associated with and can be 
used as a marker for MFG. Tests are performed on walleye having a length 
greater than or equal to 500 mm and reveal that 88% of MFG-diseased 
walleye can be accurately identified.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the process of this invention, walleye having a total 
length of greater than or equal to 500 mm, since only walleye of at least 
this size (at least five years of age) show an incidence of MFG, are first 
collected. A blood sample is then collected via cardiac puncture, caudal 
artery, or other appropriate sites. Should cardiac puncture be used, an 
appropriate method uses a heparinized 21 gauge needle and 10 cc syringe. 
If samples are prepared in advance, they should be coagulated, clot milked 
and centrifuged within 24 hours of collection. Next, the samples are 
frozen until ready for analysis. The total length (mm), wet weight 
(grams), and sex (visual inspection) of the walleye are then determined. 
The fish may then be returned, unharmed, to their habitat. 
The walleye blood is next analyzed for creatinine and calcium 
concentrations. Any methods used by those of ordinary skill in the art for 
making these determinations are suitable so long as they give accurate 
results. Finally, these values along with the walleyes' sex and length are 
analyzed using any accurate form of discriminant analysis. 
Creatinine Determination 
A preferred method for determining serum creatinine levels utilizes the 
principle that creatinine with alkaline picrate produces a color which is 
destroyed at acid pH. The difference in color chemistry measured at or 
near 500 .mu.m before and after acidification is proportional to the 
creatinine concentration. 
Reagents 
(1) Sodium hydroxide solution, 1.0N, Sigma Diagnostics. Catalogue No. 
930-65; 
(2) Acid reagent, mixture of sulfuric acid and acetic acid. Catalogue No. 
555-2; 
(3) Creatinine color reagent, picric acid, approximately 0.6%, sodium 
borate and surfactant. Catalogue No. 555-1; 
(4) Creatinine standard, Creatinine, 3.0 mg/dL (0.26 mmol/L), in 
hydrochloric acid, 0.02N. Catalogue No. 925-3; 
(5) Creatinine standard. Creatinine, 15 mg/dL (1.32 mmol/L), in 
hydrochloric acid, 0.02N. Catalogue No. 925-15. 
Preparation: 
Alkaline picrate solution is prepared by mixing 5 volumes of creatinine 
color reagent (Catalogue No. 555-1) with 1 volume of sodium hydroxide 
solution (Catalogue No. 930-65, e.g., 50 mL creatinine color reagent plus 
10 mL sodium hydroxide solution). 
Procedure 
The described procedure is for a 3.4 mL reaction volume, requiring a 10-12 
mm cuvet. 
1) To cuvet labeled BLANK, add 0.3 mL water. To cuvet labeled STANDARD add 
0.3 mL creatinine standard (Catalogue No. 925-3). To cuvet labeled TEST 
add 0.3 mL sample. 
2) To all cuvets add 3.0 mL alkaline picrate solution. Mix and allow to 
stand at room temperature for 8-12 minutes. 
3) Read and record absorbance (A) of STANDARD and TEST vs BLANK as 
reference at or near 500 nm. This is initial A. 
4) To all cuvets add exactly 0.1 mL acid reagent (Catalogue No. 555-2). Mix 
immediately and thoroughly. Allow to stand 5 minutes at room temperature. 
NOTE: A precipitate may form upon addition of the acid reagent, but it 
dissolves after mixing. 
5) Read and record absorbance (A) of STANDARD and TEST vs BLANK as 
reference at same wavelength used for the initial readings. This is FINAL 
A. 
##EQU1## 
Calcium Determination 
A preferred method for determining serum calcium levels mixes the blood 
serum with murexide (ammonium purpurate) to form a red-colored calcium 
murexide complex which is in equilibrium with free calcium ions. Titrating 
the complex with EDTA chelates the free calcium ions, causing the murexide 
complex to release additional calcium ions to maintain the equilibrium. As 
the murexide ion/calcium murexide ratio increases there is a gradual shift 
of color from red to purple. The titration is followed photometrically at 
620 nm to the endpoint, where the increase in optical density is maximum. 
The end point is reached when the murexide concentration is at its peak 
and all calcium ions have been bound by the chelating agent. 
Reagents required: 
1) 0.05N sodium hydroxide. Dissolve 4 g sodium hydroxide in distilled water 
and qs to a volume of 2000 mL. 
2) 1.4N sodium chloride. Dissolve 81.8 g of sodium chloride in distilled 
water and qs to 1000 mL. 
3) 0.14N sodium chloride. Dilute 10 mL of the 1.4N sodium chloride to 100 
ml with distilled water. 
4) 0.005M stock EDTA solution. Dissolve 1.85 g disodium dihydrogen 
ethylenediaminetetracetate in 100 mL 1.4N sodium chloride and add 
distilled water to make 1000 mL. 
5) Stock murexide. Dissolve 0.25 g of ammonium purpurate in 5 mL distilled 
water and 25 mL of 95% ethanol. Refrigerate in a brown bottle. 
6) Stock calcium standard 1%. Place 2.497 g of dry calcium carbonate into 
an evaporating dish. Dissolve in a minimal amount of 6N hydrochloric acid, 
and evaporate to dryness on a steam bath. Dissolve the residue (CaCl) in 
sufficient distilled water to make 100 mL. 
7) Working calcium standard. Dilute 1 mL of stock calcium standard to 100 
mL with 1.4N sodium chloride. This solution contains 0.1 mg calcium per 
mL. 
Procedure: 
1) Prepare the working murexide solution by adding the stock murexide 
solution to 100 mL of 0.05N sodium hydroxide until optical density is 0.48 
to 0.39 in a 1 cm light path as compared to water at 620 nm. 
2) The titrant is prepared by mixing 18 volumes of the working murexide 
solution, 1 volume of 0.14N sodium chloride and 1 volume of stock EDTA 
solution. 
3) Place 0.25 mL of 0.14N sodium chloride solution in the blank cuvet, and 
equal quantities of fresh, nonhemolyzed serum and the working calcium 
standard in the test and standard cuvettes, respectively. 
4) Add 2.25 mL of working murexide solution and 0.5 mL of titrant to each 
cuvet, and mix. 
5) Set the blank cuvet to read a density of 0.500 on the photometer at 620 
nm and read the test and standard cuvettes. 
6) Remove the cuvettes and add 0.25 mL of titrant to each, including the 
blank. Mix well. 
7) Rebalance the instrument to read 0.500 density at 620 nm with the blank 
and again read the test standard solutions. 
8) Repeat this procedure until the end point is passed and no further 
increase in density occurs. 
9) The end point is determined by preparing a plot of optical density 
versus titrant added on standard graph paper. A steep straight line is 
plotted, then there is a break, and a second straight line plateau is 
evident which intersects the first. 
Calculation: 
The ml required to give the end point is determined by finding the 
intersection of the two lines and reading the volume of titrant used at 
that point. 
##EQU2## 
Chemicals: 
1) sodium hydroxide 
2) sodium chloride 
3) disodium dihydrogen EDTA 
4) 95% Ethanol 
5) ammonium purpurate 
6) calcium carbonate 
7) 6N hydrochloric acid 
Discriminant Analysis 
After each separate parameter is analyzed, a discriminant analysis is 
carried out on diseased and healthy walleye in which the data for sex, 
length, creatinine and calcium are utilized. Discriminant analysis 
provides a mathematical rule, or discriminant function, for guessing which 
class (MFG-positive or healthy) a fish belongs to based on the knowledge 
of quantitative variables. A preferred discriminant analysis utilizes the 
DISCRIM procedure which is appropriate for approximately normal 
within-class distributions. SAS Institute Inc., SAS User's Guide: 
Statistics, Version 5 Edition, SAS Institute, Inc., Cary, N.C. (1985). 
Similar to the creatinine and calcium serum level determinations, other 
accurate discriminant analysis procedures may be utilized by those of 
ordinary skill in the art for practicing the present invention. 
The following example is offered to illustrate experimentation we conducted 
wherein creatine kinase was rejected as a parameter to distinguish 
MFG-positive from healthy walleye. It also demonstrates that by using 
serum creatinine and calcium levels, MFG-positive walleye can be 
classified with great accuracy. 
EXAMPLE 
Field Collection of Walleye 
Walleye were collected by trap nets (1.8.times.2.4 m) and experimental gill 
nets (1.8.times.75 m) with mesh sizes of 19, 25, 38, 51 and 64 mm bar 
measure. Merritt Reservoir walleye were sampled April 1991 and 1992. 
Sampling of Lake Oahe was carried out in April 1991 and August 1991. Lake 
Sakakawea was sampled summer 1990, May to September 1991 and May 1992. 
After the fish were captured, total length (mm), wet weight (grams) and 
sex (visual inspection) were recorded. Skeletal muscle samples of diseased 
fish and selected healthy walleye were collected and preserved in Bouin's 
solution or a 50:50 mixture of formaldehyde and 100% ethanol. Cardiac 
puncture was used to sample blood from live walleye using a heparinized 21 
gauge needle and 10 cc syringe. Samples were coagulated, clot milked, and 
centrifuged within 24 hours of collection. Sera were collected in 
microcentrifuge tubes, frozen on dry ice for transport to the laboratory 
and placed in a -80.degree. C. freezer until analyzed. 
Serum Biochemical Tests 
Creatine kinase (CK) was determined by Sigma's protocol for the 
quantitative, kinetic determination of creatine kinase activity in serum 
or plasma read in a spectrophotometer at 340 nm. Sigma Diagnostics, 
Creatine Kinase Procedure No. 47-UV, St. Louis, Mo. (1989). A Bausch and 
Lomb Spectronic 20, matched cuvets (12 mm) and voltage regulator were 
utilized in determining CK activity. Bausch and Lomb Spectronic 20's are 
wide-bandwidth instruments which do not emit truly monochromatic light at 
340 nm. The absorbance readings of these instruments, while reproducible 
are not necessarily "correct". Therefore, readings from the Spectronic 20 
were adjusted to obtain the "correct" absorbance values prior to use in 
calculations. Sigma's procedure number 30-UV was used as a simple 
calibration procedure for adjusting the absorbance readings of our 
wide-bandwidth instrument. The determinations were carried out at 
30.degree. C..+-.one degree with the use of a constant temperature 
waterbath. The reagent and sample were incubated at 30.degree. for three 
minutes. Absorbance readings were then taken at 30 second intervals for 
two minutes to assure linearity. Creatine kinase activity (U/L) was then 
determined by use of the manufacturer's formula. Dilutions of lyophilized 
rabbit muscle CK (2100 U/L) were used as controls for testing procedures. 
Some assays did not proceed in a linear fashion due to hemolysis, which 
caused high levels of adenylate kinase, ATP and glucose-6-phosphate to be 
released from red cells affecting the reaction. These assays were not 
considered in the analysis. A frequency distribution of the data was made 
and a graphical test for normality (Gaussian distribution) carried out. 
The data were found not to follow a Gaussian distribution, therefore, 
.+-.2 SD from the mean is not an acceptable method to express the health 
range of CK. Creatine kinase values were analyzed by a procedure utilized 
on non-normally distributed serum values of dogs. Brunden et al, "A 
General Method of Determining Normal Ranges Applied to Blood Values for 
Dogs," Am. J. Clin. Path., 53: 332-339 (1970). Sommerville's table was 
used to obtain the value of m (a table value that satisfies a formulated 
inequality) which was utilized to calculate the upper tolerance limit. 
Sommerville, P. N., "Tables for Obtaining Non-Parametric Tolerance 
Limits," Ann. Math. Stat., 29: 599-601. 
Creatinine was evaluated in the serum of MFG-positive walleye using the 
Sigma Diagnostic's protocol. 
The procedure to determine the concentration of calcium (Ca) in serum was 
modified from Fales (1953) by Oser (1965). The latter protocol was used. 
The volume of serum and reagents were halved due to the small amount of 
serum available for some fish. A Ca standard (0.1 mg/ml) was utilized. 
Oser suggested setting the spectrophotometer at 0.50 density with the 
blank before reading the standard and test. In the text, Oser refers to 
balancing the instrument at 0.50 absorbance and reading optical density, 
however, he plots % transmittance. We elected to follow Fales (1953) 
original procedure and set the spectrophotometer at 50% transmittance, not 
0.50 absorbance. The light sensitive stock murexide solution was 
refrigerated in a brown bottle. The working murexide and titrant were 
mixed prior to each set of determinations, kept in brown bottles and used 
within 1.5 hours. Because of light sensitivity the cuvets were kept in a 
dark cardboard box to avoid exposure to room light. 
The determination was based on the titration of Ca with disodium dihydrogen 
ethylene-diaminetetraacetic acid (EDTA). The titration was followed 
colorimetrically at 620 nm using a Bausch and Lomb Spectronic 20. Matched 
cuvets (12 mm) and voltage regulator standardized the procedure. 
Calculations were carried out as described by Oser (1965). A t-test was 
utilized to compare the mean serum calcium levels of healthy male and 
female walleye. Due to small sample size and differences in variance, mean 
serum Ca levels of healthy male and female spawning walleye were compared 
to mean serum Ca values of MFG-positive fish of the same sex using an 
unequal variance t-test. Sokal, R. R., and F. J. Rolf, Biometry, W. H. 
Freemand and Co., New York, N.Y. (1981). The critical level of 
significance utilized was also different for tests between male and female 
walleye (P&lt;0.05) and between MFG-positive and healthy male walleye and 
MFG-positive and healthy female walleye (P&lt;0.10). Different levels were 
used because of the numbers of MFG-positive fish (four females and six 
males). 
Discriminant Analysis 
After each separate parameter was analyzed, a discriminant analysis was 
carried out on diseased and healthy walleye in which the data for sex, 
length, creatine kinase, creatinine, and calcium were available (28 total; 
6 disease and 22 healthy). The procedure utilized was the DISCRIM 
procedure (SAS Institute Inc. 1985) which is appropriate for approximately 
normal within-class distributions. However, before the DISCRIM procedure 
was carried out, a stepwise discriminant analysis, STEPDISC procedure, 
(SAS Institute Inc. 1985) was performed to select a subset of variables 
for use in the DISCRIM procedure. Of the variables stated above (sex, 
length, creatine kinase, creatinine and calcium) none could be removed by 
STEPDISC based on: (1) the significance level of an F-test from an 
analysis of covariance, where the variables already chosen act as 
covariates and variable under consideration is the dependent variable or 
(2) the squared partial correlation for predicting the variable under 
consideration from the class variable. This second criterion allows for 
control of the effects of variables already selected for the model. Thus, 
all variables were included in the discriminant analysis. Another 
discriminant analysis using sex, length, creatinine and calcium was 
carried out. This enabled us to increase the number of walleye in the 
analysis to 33 (23 healthy and 10 diseased). 
Results 
Creatine kinase values of 91 healthy walleye ranged from 131 to 951 U/L. 
FIG. 1 represents the frequency distribution of CK values for walleye from 
lakes Sakakawea, Oahe, and Merritt Reservoir. Our desired upper 90--90 
tolerance limit (i.e., 90% probability of covering 90% of the population) 
was found to be the 95th ranked variate which had a value of 885 U/L CK 
activity (this is represented by the dashed line on FIG. 1 in which 
MFG-positive walleye are represented in black). According to the procedure 
by Brunden et al. (1970), on average (or more often), all CK values less 
than or equal to 885 U/L include 90% of the population of healthy walleye. 
Three (1082, 1902 and 2952 U/L) of the nine MFG-positive CK values (Table 
5 and FIG. 1) fell above the calculated upper limit, one value (787 U/L) 
was close to the established upper limit and five values (197, 295, 328, 
328 and 349 U/L) were below the established upper tolerance limit. 
Creatinine values for MFG-positive fish are presented in Table 1. The mean 
creatinine value for all MFG-positive walleye (Merritt and Lake Sakakawea) 
was compared to the mean value of 45 healthy walleye and no significant 
difference (P&gt;0.10) was found between the mean creatinine values of 
healthy (0.283 mg/dl) and MFG-positive walleye (0.34 mg/dl). 
TABLE 1 
______________________________________ 
Mean, standard deviation and range of 
creatinine in mg/dl for MFG-positive walleye. 
Sample Mean +/- Range 
Location size mg/dl SD mg/dl 
______________________________________ 
Lake Sakakawea 
n = 7 0.327 .167 .15-.59 
Merritt Reservoir 
n = 7 0.353 .110 .24-.51 
Combined n = 14 0.340 .137 .15-.59 
______________________________________ 
Selected calcium titration curves [standard, spawning female (NB201), 
spawning male (0066), MFG-positive spawning female (0043) and MFG-positive 
spawning male (0252)] are shown in FIG. 2. The titration curves show a 
steep straight line followed by the plateau at the bottom of the curve; 
their intersection indicates the amount of titrant (EDTA) required to 
titrate serum Ca. 
Table 2 shows serum Ca values of: (1) spawning female walleye, (2) spawning 
male walleye, (3) MFG-positive spawning male walleye, and (4) MFG-positive 
spawning female walleye. A significant difference t.sub.s =6.81, df=36, 
p&lt;0.05) was found between mean serum Ca levels of spawning male (X=11.68 
mg/100 ml serum) and spawning female walleye (X=15.36 mg/100 ml serum). 
Mean serum Ca levels of spawning male walleye (X=11.68 mg/100 ml serum) 
differed significantly (t.sub.s =2.088, df=17,5, p&lt;0.10) from mean serum 
levels of MFG-positive spawning male walleye (X=13.62 mg/100 ml serum). A 
significant difference (t.sub.s =2.561, df=19,3, p&lt;0.10) was found between 
mean serum Ca levels of healthy spawning female walleye (X=15.36 mg/100 ml 
serum) and MFG-positive spawning female walleye (X=20.20 mg/100 ml serum). 
TABLE 2 
______________________________________ 
Serum Ca (mg/100 ml) of spawning reservoir 
walleye (healthy male and female) with values of 
MFG-positive fish (male and female). 
Sample Mean 
Size Mean +/- SD Range Age 
______________________________________ 
Healthy female 
20 15.36 1.78 11.90-19.47 
8.7 
Healthy male 
18 11.68 1.51 9.09-14.61 
8.6 
MFG-Positive 
6 13.62 2.10 11.49-16.80 
8.8 
male 
MFG-positive 
4 20.20 3.70 17.12-25.35 
11.5 
female 
______________________________________ 
Discriminant Analysis 
The descriptive statistics from the discriminant analysis are presented in 
Table 3. Twenty-two healthy walleye and six MFG-positive walleye were 
included in the analysis. Five of six (83%) diseased fish were correctly 
classified as MFG-positive and 20 of 22 (90%) healthy walleye were 
correctly classified. Three of 28 fish (11%) were misclassified using the 
variables sex, length, creatine kinase, creatinine and calcium. Table 4 
presents descriptive statistics of the analysis which did not include 
creatine kinase as a variable. Twenty-three healthy and ten MFG-positive 
walleye were utilized in the analysis. Eight of ten (80%) diseased fish 
were correctly classified as MFG-positive and 21 of 23 (91%) healthy 
walleye were correctly classified. Four of the total 33 fish (12%) were 
misclassified using the variables sex, length, creatinine, and calcium. 
TABLE 3 
______________________________________ 
Descriptive statistics of discriminant 
analysis using the variables sex, length, 
creatinine, calcium and creatine kinase. 
Variable N Sum Mean Variance 
STD Dev 
______________________________________ 
Total-sample 
sex 28 12 0.43 0.25 0.50 
length 28 17051 609 5127 71.60 
creatinine* 
28 6.82 0.24 0.026 0.16 
calcium 28 391 13.98 13.3 3.64 
creatine kinase 
28 12590 450 54177 233 
Healthy-sample 
sex 22 9 0.40 0.25 0.50 
length 22 13189 600 4719 68.69 
creatinine* 
22 5.10 0.23 0.026 0.16 
calcium 22 286 13.01 7.0 2.64 
creatine kinase 
22 9519 432 39963 200 
MFG-positive-sample 
sex 6 3 0.50 0.30 0.55 
length 6 3862 643 6027 77.63 
creatinine* 
6 1.72 0.29 0.025 0.16 
calcium 6 105 17.55 23.0 4.80 
creatine kinase 
6 3071 512 118806 344 
______________________________________ 
TABLE 4 
______________________________________ 
Descriptive statistics of discriminant 
analysis using the variables sex, length, creatinine 
and calcium. 
Variable N Sum Mean Variance 
STD Dev 
______________________________________ 
Total-sample 
sex 33 13 0.39 0.25 0.50 
length 33 20092 609 4950 70.36 
creatinine* 
33 8.71 0.26 0.026 0.16 
calcium 33 464 14.06 11.9 3.45 
Healthy-sample 
sex 23 9 0.39 0.25 0.50 
length 23 13754 598 4556 67.50 
creatinine* 
23 5.58 0.24 0.028 0.17 
calcium 23 299 12.99 6.70 2.58 
MFG-positive-sample 
sex 10 4 0.40 0.27 0.52 
length 10 6338 634 5472 74.00 
creatinine* 
10 3.13 0.31 0.020 0.14 
calcium 10 165 16.55 16.2 4.03 
______________________________________ 
Only three of nine MFG-positive walleye showed elevated CK levels (i.e. 
above the 90--90 tolerance interval of 885 U/L). This suggests that the 
degree of muscle degeneration in most MFG-positive specimens was not great 
enough (.ltoreq.20% by visual inspection in most severe cases) to affect 
circulating CK levels. The muscle degeneration may have been progressing 
at such a slow rate that the elevations may have leveled out and only by 
sampling when the muscle was actively degenerating would an elevation of 
CK be detected. Even then elevated CK levels may not be detected. It may 
be necessary to use a micromethod at sites close to degenerating muscles 
because of dilution in the circulating blood. Another possible explanation 
may be a result of the activity patterns of the walleye. Economon (1978) 
suggests walleye are passive swimmers and are not even very active when 
captured by hook and line. Thus, CK may be present in low levels in 
walleye due to the fishes behavior (i.e., low activity hence little ATP 
required). Thus only a small amount of CK may be needed to phosphorylate 
ADP by dephosphorylating phosphocreatine to form the needed ATP. 
Values of creatinine were obtained for 14 MFG-positive walleye (Table 1). 
In man, when muscle degenerates due to paralysis or muscular dystrophy the 
creatinine content of the blood falls (Emery 1988). The MFG-positive 
creatinine values were not significantly different than the mean value of 
the 45 healthy walleye and suggest creatinine may not be the variable to 
use in diagnosing muscle degeneration in walleye. The filtration of 
creatinine by the non-diseased kidney may be so precise that the small 
loss of muscle mass (.ltoreq.20%) does not affect the humoral systemic 
level of creatinine in MFG-positive walleye. 
Conclusion 
No single blood parameter examined seemed to clearly differentiate healthy 
walleye from MFG-positive walleye. Discriminant analysis allowed for the 
use of selected blood parameters, sex and length as variables. Because 
multiple parameters for some walleye were measured, a statistical 
procedure was utilized to classify fish as healthy or MFG-positive on the 
basis of the numeric variables (i.e. sex, length, creatine kinase, 
creatinine and calcium). All fish were not able to be correctly classified 
using these variables, but 89% (25 of 28 walleye) were classified 
correctly using all variables and 88% (29 of 33 walleye) were correctly 
classified without creatine kinase. If this procedure were utilized by 
fish managers, it would not "catch" all diseased fish. In the analysis 
using all variables as criteria 83% (5 of 6) of the diseased fish were 
recognized. Eighty-percent (8 of 10) of the diseased individuals were 
recognized in the analysis without creatine kinase as a variable. 
Discriminant analysis seems to provide a means of distinguishing most 
diseased walleye correctly. Perhaps, with an increased sample of diseased 
individuals, discriminant analysis would be more effective in 
distinguishing healthy walleye and MFG-positive walleye. Discriminant 
analysis would require a tagging procedure because data collection and 
analysis would take more time than spawning operations would allow. Thus, 
walleye classified as 
TABLE 5 
______________________________________ 
Descriptive values for serum parameters 
ascertained for MFG-positive walleye. 
(Cre = creatinine mg/dl; Ca = calcium mg/100 ml; 
Ck = creatine kinases U/L). 
Fish # Lake Sex Cre Ca CK Time 
______________________________________ 
0043 Sak! F@ 0.18 20.33 
787 SP# 
0063 Sak M& 0.40 13.83 
NV SP 
0065 Sak M 0.15 15.31 
1082 SP 
0139 Sak F 0.18 17.12 
NV SP 
0252 Sak M 0.47 16.80 
NV SP 
0278 Sak F 0.59 17.94 
197 SP 
1221 Sak M 0.32 12.23 
295 PS+ 
NB7 Mer* M NV% NV 1902 SP 
NB13 Mer M 0.30 NV NV SP 
NB18 Mer M 0.36 12.40 
NV SP 
NB40 Mer M 0.51 NV NV SP 
NB43 Mer M 0.50 NV 2952 SP 
NB212 Mer M 0.26 11.49 
328 SP 
NB214 Mer M 0.30 11.88 
328 SP 
NB235 Mer F 0.24 25.35 
349 SP 
______________________________________ 
! Sak = Lake Sakakawea 
* Mer = Merritt Reservoir 
@ F = Female 
& M = Male 
# SP = Spawning 
+ PS = Post spawn 
% NV = No value due to the volume of serum collected 
MFG-positive could be recognized and subsequently removed when recaptured. 
One of the greatest difficulties in studying MFG may be its incidence in 
adult walleye (one of 15 to 20 fish sampled). These walleye are the brood 
stock for future generations and fish managers are hesitant to allow 
researchers access to these individuals. Discriminant analysis will allow 
for the majority of the brood fish to be returned to the population being 
examined while most walleye with advanced cases of MFG can be removed for 
further study. 
The examples shown above are not intended to limit the described invention 
in any manner but are included for demonstrative purposes only. 
It is therefore seen that the present invention accomplishes at least all 
of its stated objectives.