Burdett, R. D.

Fujihara, M. P.

Strand, J. A.

UNT Digital Library

6. r    ,BNWL-SA-     5 2 3 7/   1 034)*41 -73-0 51 3-SUPPRESSION OF THE PRIMARY IMMUNERESPONSE IN RAINBOW TROUT, SALMO GAIRDNERI,SUBLETHALLY IRRADIATED DURING EMBRYOGENESISbyL_-J. A. Strand, M. P. Fujihara, and R. D. BurdettNOTICEreport was prepared as an account of workspunsored by the United States Government. Neitherthe United States nor the United States EnergyResearch and Development Administration, nor any oftheir employees, nor any of their contractors,March 1975 subcontractors, or their employees, makes anywarranty, express or implied, or assumes any legalliability or responsibility for the accuracy, completenessor usefulness of any information, apparatus, product orprocess disclosed, or represents that its use would notinfringe privately owned rights.Ecosystems and Biology DepartmentsBattellePacific Northwest Laboratories MASTERRichland, WA  99352DISTRIBUTION OF THIS COCUM...  i  UNLiMITED41%DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency Thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. DISCLAIMER Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. b,Eggs of rainbow trout were spawned artificially in the laboratory,fertilized, and immediately immersed in 0, 0.01, 0.1, 1.0, and 10.0uci/ml tritium (biological grade) contaminated spring water (pathogenfree).  Rearing through 20 days of embryogenesis at 10.5 + 0.2°C wasfacilitated within a recirculation drip incubation system of 150 litercapacity.  Exposure of the embryos to 0, 0.01, 0.1, 1.0, and 10.0 uCi/mltritium resulted in an estimated total dose of 0, 0.048, 0.470, 4.550,and 40.348 rads.At  20 days (eyed stage), the embryos were transferred  to a single-pass incubation system maintained on thermally controlled, 10.5 1 0.2°C,dechlorinated sanitary water.  Hatching occurred at 28.5 + 0.5 days.At 14 days post hatch, the fry were transferred to 10 ft x 2 ft x1 ft fiberglass rearing troughs also maintained on thermally controlleddechlorinated sanitary water.  Rearing temperature was adjusted to 20.010.3°C at a rate of 2.0 1 0.5°C per week.A final transfer of the fingerlings to permanent 10 ft x 4 ft x 3 ftconcrete rearing ponds also maintained on thermally controlled, 20 10.5°C dechlorinated sanitary water was affected 3 months post hatch.Rearing thereafter was in accordance with standard hatchery practice,At 5 months post hatch, control and irradiated test fish wereadministered intraperitoneally 0.1 cc of a heat killed antigen (1.8x 108 cells per ml, Chondrococcus columnaris) in 25% Freund's incompleteadjuvant.  A 0.1 cc sham vaccination containing saline and 25% Freund'sincomplete adjuvant was similarly administered to another group of controlfish.  At 3 weeks post vaccination and at weekly intervals thereafter, astandard tube agglutination test for the specific antigen of Vaccinationwas performed on the serum from each control and irradiated test group.Selected results of serum agglutination tests to assess thepotential for suppressed immune competence in juvenile rainbow troutsublethally exposed to 0, 0.048, 0.470, 4.550, and 40.435 rads oftritium irradiation during embryogenesis are reported in Table I.As in previous experiments, the percentages of fish withagglutinins and the magnitude of titers in both control and irradiatedtest groups increased following immunologic stimulation, Figure 1.However, control, 0.048, and 0.470 rad treatment groups demonstratedhigher agglutinin titers suggesting a suppression of immune competenceat the 4.550 and 40.348 rad levels.  A chi-square test of independenceof the titer distributions of each group verified this finding.To determine the potential for qualitative and quantitative changesin serum components which would account for reduced immune competencein irradiated fish, polyacrylamide gel electrophoresis was performed.Additionally, total serum protein analyses were conducted.Preliminary results with 5% acrylamide separation gel stained with0.25% amido black 10 B demonstrated 31 bands as reported in Figure 2.Each band is numbered following decreasing mobility.  The most rapidlymigrating bands, 1-5, were found to be slightly more mobile than humanserum albumins.  All bands were generally well delimited.  Mobilities didnot change, but large dissimilarities in staining intensity were observedfrom one animal to another.  A lowered total serum protein level did notinfluence the relative position on the number of bands and generally onlyaffected stain intensity.Inconstant bands were encountered in certain patterns.  The mainvari ations occurred in bands 29, 30, and 31.  These bands were not observed.-Iin non-immunized fish, either irradiated or control; however, thepresence of these bands was found to consistently coincide with detectionof ahtibody titers of at least 1:40 in nearly all immunized test fish,either irradiated or control.It is presumptive that bands 29, 30, and 31 respresented specifichumoral antibody to the immunogen, Chondrococcus columnaris.  It followsthat in the greater proportion of fish irradiated at the 4.550 and 40.348rad levels, bands 29, 30, and 31 were significantly reduced, and undoubtedlyaccounted for the lowered antibody titers in these test fish demonstratedby agglutination assay.Total serum protein levels of all test fish throughout the testperiod remained the same; however, the Biuret technique here applied maynot be sufficiently sensitive to detect the changes observed in bands29, 30, and 31 by gel electrophoresis.M   ..TABLE I.  Serum Agglutinins to Chondrococcus columnaris in Juvenile Rainbow Trout,Salmo gairdneri Exposed to 0.048, 0.470, 4.550, and 40.348 rad ofTritium Irradiation During Embryogenesis  (Pond I, Compartment A-E).Weeks RangePost No. % Positive Positive . MeanTreatment Vaccination Fish Titers Titers TitersControl             0             6             0                              03            22            95          1:20 -40 1:285            22            95          1:20 -640 1:1967            21 100 1:160-1280 1:10749           20 100 1:640-1280 1 :102411            20 100 1:640-2560 1 :15680.048 rad         0           10            0                            03           22 100 1:20 -40 1:295            20 100 1:20 -320 1:1177            20 100 1:160-2560 1  : 12189           20 100 1:320-1280 1 : 70411            20 100 1:640-2560 1:11200.470 rad          0             7             0                             03            25            76          1:20 -40 1:225            24 100 1 : 20 -320 1:1287           22 100 1:320-1280 1:7209            20 100 1:640-1280 1:89611            20 100 1:320-2560 1 :9124.550 rad          0             8             0               -             03            24            63          1:20 -40 1:185           24 100 1 :20 -640 1 :1327            21 100 1:160-1280 1:7209            20 100 1:160-1280 1 : 58411            20 100 1:320-2560 1:81640.348 rad '0 6 0     03            24            88          1:20 -40 1:205           21 100 1:20 -320 1:BO7           21 100 1:80 -640 1:5189           20 100 1:160-640 1 : 50411            20 100 1:320-1280 1  : 704' , ~' J ... -<Z (...) 0 a:: 0.. -(...) LI.J $ a:: LI.J 1-t= z ~ ~Figure 1. The primary immune response t o a single intraperitoneal injection of heat killed Chondrococcus col umnaris antigen and 25% Fruend's incomplete adjuvant in control, 0.048, 0.470, 4.550, and 40.348 · rad treatment groups (Pond I, Compartm~nt A-E). 1600~---------------------------------------------------. 1400 1000 800 600 3 WEEKS CONTROL 0.048 rad 0.470 rad 4.550 rad 40.348 rad 5 WEEKS POND I 7 WEEKS 9 WEEKS 11 WEEKS .. . . : .. Figure 2. Electrophoretic pattern of rain bow trout serum proteins using 5% acrylamide separation gel. Bands a1~e numbered following de-·creasing mobility. Principal inconstant bands are indicated by dashed lines. The pattern presented is representative for band thickness and relative mobjlity, but does not account precisely for relative concentration. · ( +) 

Suppression of the primary immune response in rainbow trout, Salmo gairdneri, sublethally irradiated during embryogenesis

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Suppression of the primary immune response in rainbow trout, Salmo gairdneri, sublethally irradiated during embryogenesis

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