INFECTION AND IMMUNITY, Jan. 1975, p. 171-179Copyright ® 1975 American Society for Microbiology

Vol. 11, No. 1Printed in U.S.A.

Pathophysiology of Infectious Hematopoietic Necrosis VirusDisease in Rainbow Trout: Hematological and Blood Chemical

Changes in Moribund FishDONALD F. AMEND* AND LYNNWOOD SMITH

Western Fish Disease Laboratory Naval Support Activity,* and College of Fisheries, University of Washington,Seattle, Washington 98115

Received for publication 5 June 1974

Infectious hematopoietic necrosis (IHN) is a rhabdoviral disease of rainbowtrout (Salmo gairdneri). Trout were injected with IHNV, and various hematolog-ical and biochemical measurements of clinically ill fish were compared touninfected controls. Infected fish had reduced corpuscular counts, hemoglobin,and packed cell volume, but normal mean corpuscular volume, mean corpuscularhemoglobin, and mean corpuscular hemoglobin concentration. The percentageof immature erythrocytes was increased, but the percentage of leukocytes was

unchanged. Differential leukocyte counts showed a significant decrease inneutrophils, increase in lymphocytes, but no change in monocytes. Unidentifia-ble necrobiotic cells were prevelant in blood smears and hematopoietic tissueimprints. Plasma bicarbonate, chloride, calcium, phosphorus, bilirubin, andosmolality were significantly reduced, but plasma glucose and anterior kidneyascorbate were unchanged. Plasma pH increased and the alpha fractions of theserum proteins were altered. No change was found in plasma enzymes, exceptthat a LDH isozyme was significantly increased. The alkali reserve wasdiminished and alterations in acid-base and fluid balance occurred. Deathprobably resulted from a severe electrolyte and fluid imbalance caused by renalfailure.

Infectious hematopoietic necrosis (IHN) vi-rus, prevalent along the Pacific Coast, infectssockeye salmon (Oncorhynchus nerka), chi-nook salmon (0. tshawytscha), and rainbowtrout (Salmo gairdneri). The virus has theproperties of rhabdoviruses (4, 11). The primarypathological lesion in diseased fish is extensivenecrosis of the hematopoietic tissues. In termi-nal cases necrosis of the liver, pancreas, andgranular cells of the lamina propria of the gut isalso found (3, 39). The potential for epizootics ishighest at 10 C and the disease does not occurnaturally above 15 C (2, 33). In cell culture,however, the virus replicates and causes cyto-pathic effects at temperatures up to 18 C (3,37).Although some information is available on

the properties of the virus and epizootiology ofthe disease (6), only limited data exist on thepathophysiology of the disease and the mech-anisms by which fish resist infection. Variousdifferential hematological, physiochemical, en-zyme, and organ function tests are used inmammalian medicine to aid in disease diagno-sis and to follow the pathogenesis and recovery

from disease. In this study various hematologi-cal and blood chemical tests were performed onclinically ill fish to determine if some tests maybe of diagnostic value, and to gain a betterunderstanding of the response of fish to thisviral infection.

(This paper is part of a thesis submitted tothe University of Washington School of Fisher-ies in partial fulfillment of the requirements forthe Ph.D. degree.)

MATERIALS AND METHODSRainbow trout (Kamloop variety) averaging about

25 g each were placed in 700-liter aquaria suppliedwith flowing 9 C dechlorinated municipal water.About 200 experimental and 200 control fish were inseparate aquaria. All fish were acclimated to thewater for 2 weeks before the experiment and weremaintained on a commercial dry diet. The water hada total hardness of about 20 Ag/ml (as CaCOs) and apH of about 6.6. Further characteristics of the watersupply were described by Amend (7).The fish were fasted for 24 h before injection. A

50-yg/ml Benzocaine solution was used to anesthetizethe fish, then 0.1 ml of IHN virus (107-5 mean tissueculture infective doses per ml) was injected intraperi-

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toneally into each experimental fish. The controlswere handled similarily except that only cell culturemedia was injected. The virus was originally isolatedfrom rainbow trout (laboratory stock 1274), fishpassed to test for virulency, and then reisolated frommoribund dish. The reisolated virus was neutralizedby IHN specific antisera.When the fish showed signs of the disease (3), they

were again anesthetized. After excess water wasremoved from the surface of the fish, the caudlepeduncle was severed and blood was collected intoheparinized capillary tubes from the caudal artery.Also, hemoglobin and erythrocyte (RBC) counts weredetermined and blood smears and anterior kidneyimprints were prepared (20). The capillary tubes weresealed and centrifuged at 1,000 x g for 10 min. Thepacked cell volume was recorded, and then theplasma pH, bicarbonate, and chloride levels weredetermined (36). The remaining plasma was frozen(-10 C) and the other biochemical tests were per-formed within 1 month. Control fish were randomlyselected and handled similarly except all fish wereprocessed on 1 day about the time mortality washighest in the infected group (7 days postinjection).All analyses were performed on individual fish.

Blood smears and tissue imprints were air-dried,fixed in methanol, and stained with Lieshman-Giem-sa. The cells were classified according to Klontz (19).Ascorbate analyses were performed as described byWedemeyer (34) using homogenized anterior kidneytissue, glucose was determined by the method ofDubowoski (13), and bilirubin was determined by themodified Nosslin method (35). Bicarbonate and cal-cium were determined with an Oxford titrator usingprepackaged reagents, and the plasma pH with aCorning model 12B blood pH meter at 37 C, then thepH values temperature was corrected to 10 C. Osmo-lality was measured with an Osmometer by thefreezing-point depression method and phosphorus wasdetermined photometrically using prepackaged rea-gents (American Monitor Corp.). The pCO2 wasdetermined mathematically by using the Henderson-Hasselbalch equation after the blood pH and bicar-bonate were determined. All data were statisticallycompared with the t test.Horizontal starch gel electrophoresis was used for

detecting alterations in plasma lactic dehydrogenase(LDH), esterase, peptidase, and glutamic oxalacetictransaminase isozyme concentrations (31). Using aphotometric analyzer, the optical density from pola-roid negatives of the stained gels was used to comparechanges in the LDH concentrations. A more quantita-tive measurement was attempted by using a densi-tometer, but the difference between peaks was toogreat to standardize on the machine. Disc acrylamidegel electrophoresis was used to separate plasma pro-teins, and the concentration of each peak was quan-titated by direct scanning of the gel with a densitome-ter equipped with an internal integrator. Plasmaprotein and enzymes analyses were determined on thesame fish.

Fluid balance was determined by weighing individ-ual fish to the nearest 5 mg. In this test 15 rainbow

trout averaging about 1 g were injected intraperitone-ally with 0.01 ml of IHN virus (106.0 mean tissueculture infected doses per ml) and 15 fish wereinjected with the same amount of cell culture media.Each fish was held in a separate container andweighed daily for 19 days. The fish were anesthetizedand excess water was removed by blotting on absorb-ent paper before each weighing. The fish were not fedduring this test and virus re-isolations were attemptedon all fish that died.

During these tests, IHN virus was grown on estab-lished epithelioid embryonic rainbow trout tissueculture cells. The cells were grown in Eagle minimalessential media (Earl salts) containing 10% agammafetal calf serum, 100 IU of penicillin G, and 100 mg ofstreptomycin per ml. Virus-infected cells were grownat 15 C and virus was harvested in about 6 days whenall cells showed cytopathic effects. The virus prepara-tion was centrifuged at 1,000 x g for 10 min to removecellular debris. The mean tissue culture infected dosewas calculated by the Reed-Muench method (29).

RESULTSHematology. The hemoglobin, hematocrit,

and RBC counts levels were significantly de-pressed in all moribund fish (Table 1). How-ever, the mean corpuscular volume, mean cor-puscular hemoglobin, and mean corpuscularhemoglobin concentration were normal. Thisindicates that the cellular concentration wasreduced but the RBCs were of normal size andhemoglobin content. The percentage of imma-ture RBCs was significantly increased (P =0.05) in the infected fish, but the differencebetween means of infected and control fish wasonly 2.2%. The percentage of leukocytes in thetotal blood smear remained unchanged, but inthe differential count, the percentage of lym-phocytes significantly increased and the per-centage of neutrophils decreased whereas nodifference in the percentage of monocytes wasfound (Table 1). The relative percentage ofincrease in lymphocytes was probably due tothe absolute reduction in neutrophils. It ap-pears that the neutrophil counts are the pre-dominant circulatory cell change being affectedby this disease. This perhaps is most apparentby comparing the control-moribund ratio inTable 1.The anterior kidney is the major site of

hemopoiesis in fish, and the imprints preparedfrom this area indicated areas of severe necrosis(Fig. 1). Although no attempt was made toquantitate the number of necrotic cells, thereappeared to be considerably more debris andnecrotic particles in the kidney imprint than inthe circulatory smears. Also, many of the blastcells were affected. A "vacuolated" cytoplasmcondition was seen in some macrophages, and

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TABLE 1. Average hematological measurements, values and standard error of uninfected controls andmoribund rainbow trout infected with IHN virusa

Hematological test and unit of measure Controls Moribund Probability C/Mb

Whole bloodHemoglobin (g/100 ml) 9.4 4 0.2 (20) 7.1 ± .04 (20) P = 0.01 1.32RBC (x106/ml) 1.33 ± 0.13 (20) 0.97 ± 0.07 (20) P = 0.01 1.37Packed cell volume (%) 47 1 (19) 35 1 (20) P = 0.01 1.34Mean corpuscular volume (Mm3) 359 ± 11 (20) 347 ± 19 (20) NSc 1.04Mean corpuscular hemoglobin (ng) 72 ± 2 (20) 74 ± 2 (16) NS 0.97Mean corpuscular hemoglobin concentra- 20.1 ± 0.4 (19) 19.6 ± 1.7 (16) NS 1.03

tion (%)Blood smearSdMature RBC (%) 91.1 ± 0.7 (20) 87.8 ± 2.4 (18) NS 1.04Immature RBC (%) 7.0 ± 0.6 (20) 9.2 ± 1.1 (18) P = 0.05 0.76Total leukocytes (%) 2.0 ± 0.2 (20) 2.5 ± 1.3 (18) NS 0.80

DifferentialLeukocyte counteMonocytes (%) 5.3 ± 1.0 (20) 3.0 ± 1.0 (18) NS 1.7Lymphocytes (%) 90.7 ± 1.7 (20) 96.0 ± 1.0 (18) P = 0.05 0.94Neutrophils (%) 4.0 1.0 (20) 0.7 ± 0.3 (18) P = 0.05 5.7

a Sample size in parentheses.b C/M, control-moribund ratio.c NS, Not significant.d Immature RBC and leukocytes are expressed as percentage of a total of about 500 cells per fish counted,

including mature RBC.e Monocytes, lymphocytes, and neutrophils are differential percentages of about 100 leukocytes counted per

this appeared to be more prominent in fishwhere necrosis was less evident. As in thecirculatory smears, there appeared to be a lackof neutrophils, but differential counts were notmade in these imprints.

It appeared that the fish underwent a nor-mocytic aplastic anemia. Although hematopoi-etic tissue appeared to be responding by anincrease in percentage of immature cells, thedegree of response was less than expected.Perhaps the hematopoietic tissue was not func-tioning normally because of the extensive cellu-lar necrosis seen in the kidney imprints. Severalof the most anemic fish had hemoglobin valuesas low as 4.0 g/100 ml and a hematocrit of 7%but showed no evidence of increased hemopoie-sis. However, the mean values for hemoglobin,hematocrit, and RBC, though significantlylower than control values, were still in thenormal range (23; G. Wedemeyer and N. Nel-son, J. Fish. Res. Board Can., in press).Biochemical changes. The most severe

changes resulted in a reduction of the plasmaelectrolytes (Table 2). Calcium, phosphorus,osmolality, and bicarbonate levels were greatlyreduced and plasma chlorides decreased signifi-cantly. The bilirubin level was also reduced, butno significant change in plasma glucose or inanterior kidney ascorbate levels occurred. The

mean glucose values differed considerably be-tween test 1 and the replicate test 2. The valuesin test 1 are normal values; the values in test 2are high and may be due to excessive handlingstress (34), but the trend of both tests is thesame.

Tests for total protein indicated no significantdifference between moribund and control fish.However, using disc electrophoresis to detectwhether any of the various proteins fractionswere altered, we found that the alpha-2 fractionincreased significantly and the alpha-3 fractiondecreased significantly in moribund fish (Table3). In the same serum samples, no differencecould be detected in the plasma concentrationof esterase, glutamic oxalacetic transaminase,or peptidase isozymes, but there was a signifi-cant increase in the B" LDH isozyme, i.e., thehomotetramer of the common form of the B2locus (31) (Table 4). Two major regions of LDHactivity were evident in both control and mori-bund fish (Fig. 2), but the change was limited tothe B"' isozyme. Utter and Hodgins (31)showed that genetic variation occurs with LDHisozymes, but the B2 gene frequency is mostprevalent in serum. LDH isozyme patterns werealso compared in muscle, anterior kidney, pos-terior kidney, liver, spleen, and gill, but nodifferences were detected.

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FIG. 1. Cellular changes in blood of rainbow trout infected with IHN virus. (A) Monocyte in blood smearshowing vacuolated cytoplasms; (B) and (C) arrows indicate necrobiotic disintegrating particles in blood smear;(D) macrophage with vacuolated cytoplasm and early necrotic debris in anterior kidney imprint; and (E)extensive necrotic debris in anterior kidney imprint.

Acid-base and fluid balance. The reducedbicarbonate levels (Table 2) indicated thatacid-base regulations were being affected, per-haps associated with alteration of the elec-trolyte balance. As can be seen in Table 5, theacid-base balance was severely affected. Thetotal buffering capacity was greatly reducedand the pH was significantly increased. Thismeans that pCO2 (H2CO0) was reduced morethan the HCOc- level, thus increasing normalbase-acid ratio. The condition suggests a trendtowards an uncompensated alkalosis (higherpH) or a metabolic acidosis (lower alkali re-serve) or both.

The body fluid balance was estimated bydaily weighing individual fish. The weight ofeach fish was combined into 3-day composites,and the 3-day mean of all fish in each group wasthen determined. The data were then subjectedto linear regression analysis. Because all fishwere fasted over the 19-day test, the control fishlost weight at a rate of about 5 mg per day or atotal 6.5% weight loss (Fig. 3). This weight lossfor fasting fish is comparable to Brett's data (9)for starved sockeye salmon. In contrast, theinfected fish continued to gain weight until theydied. The average weight gain was about 15 mgper day or about a 20% total weight gain. This

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difference was highly significant and demon-strates that the infected fish were not able toregulate their fluid balance.

DISCUSSIONThe first pathological lesions in IHN disease

occur in the hematopoietic tissues of the spleenand anterior kidney (39). Anemia is a typicalsign of this disease (3). Watson (32) describedsome of the cellular changes, but the present

report is the first to characterize and quantitatethese changes.Watson (32) and Amend and Chambers (5)

reported the appearance of necrotic particles indiseased sockeye salmon and suggested that thedegenerate cells were probably lymphocytes.Parisot et al. (27) reviewed Watson's work andsurmised that these degenerate cells were im-mature erythrocytic nuclear fragments fromnecrotic cells in the kidney. Leukopenia was

TABLE 2. Biochemical comparison of plasma from uninfected controls with plasma from moribund rainbowtrout infected with IHN virusa

Clinical test and unit of measure Control Moribund Statisticalsignificance

Bicarbonate (meq/liter)5Test 1 9.8 ± 0.5 (20) 5.8 ± 0.8 (6) P = 0.01Test 2 10.5 ± 0.5 (20) 6.1 ± 0.4 (15) P = 0.01

Chloride (meq/liter)Test 1 115 1 (10) 107 ± 4 (12) P = 0.05Test 2 119 2 (20) 113 2 (16) P = 0.05

Glucose (mg/100 ml)Test 1 81.1 ± 6.3 (15) 136.0 i 43.4 (11) NSTest 2 429 85 (10) 381 ±81 (10) NS

Osmolality (mosmol) 315 ± 2 (7) 277 ± 6 (9) P = 0.05Phosphorus (mg/100 ml) 15.38 ± 0.57 (10) 12.11 ± 0.49 (10) P = 0.01Calcium (mg/100 ml) 10.8 ± 0.39 (10) 8.07 ± 0.58 (10) P = 0.01Bilirubin (mg/100 ml) 2.1 ± 0.2 (16) 1.2 ± 0.4 (4) P = 0.05Interrenal

Ascorbate (jg/g) 51.5 4 3.4 (4) 45.8 ± 2.1 (10) NS

a Mean values with standard error and sample size in parenthesis.' Milliequivalents per liter.

TABLE 3. Comparison of plasma protein fractions following disc electrophoresis from six uninfected controlsand six moribund rainbow trout infected with IHN virusa

Fraction (% of total)Test

Albumin Alpha-1 Alpha-2 Alpha-3 Beta-1 Beta-2

Control 29.4 ± 1.3 3.1 ± 0.3 11.7 ± 1.0 27.8 ± 1.7 11.6 ± 1.1 16.5 ± 1.1Moribund 32.0 ± 1.7 3.9 ± 0.6 18.9 ± 2.3 17.6 ± 2.1 13.0 ± 0.5 14.7 ± 1.9Statistical significance NS NS P = 0.01 P = 0.01 NS NS

a Mean values with standard error.'NS, Not significant.

TABLE 4. Comparison of B' LDH isozyme concentration in plasma from control and moribund rainbow troutinfected with IHN virusa

Sample size Optical density StatisticalTest sgiiac

Control Infected Control Moribund significance

Test lb 4 4 2.51 ± .07 1.94 ± .10 P = 0.01Test 2c 9 9 0.851 ± .016 0.677 ± .021 P = 0.01

aValues are photometric measurements from photograph negatives of stained starch-gel electrophoreticdetermination. Mean values with standard error.

b Cumulative values of three readings taken at center and ± 1 mm of center of enzyme activity for each fish.c Reading taken at center of enzyme activity.

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(-) C. c2c3 C4 t2 13 'T'.XIFIG. 2. Lactic dehydrogenase isozyme from starch-gel electrophoresis. Abbreviations: C, Control serum from

four rainbow trout; T, serum from four moribund rainbow trout infected with infectious hematopoietic necrosisvirus.

TABLE 5. Comparison of acid-base balance between 15 uninfected controls and 15 moribund rainbow troutinfected with IHN virusa

Clinical test Control Moribund Statisticalsignificance

HCO.- (meq/liter) 10.54 4 0.48 6.14 ± 9.41 P = 0.01pH 7.454 + 0.019 7.594 ± 0.022 P = 0.01pCO2 (mm Hg) 15.56 ± 0.68 6.45 ± 0.33 P = 0.01H2COs (meq/liter) 0.47 i 0.02 0.20 ± 0.01 P = 0.01HCO3-/H2CO3 22.9 ± 1.0 31.5 4 1.6 P = 0.01Total CO2 (meq/liter) 11.01 ± 0.49 6.33 + 0.42 P = 0.01

a Mean values with standard error.bMillequivalents per liter.

also said to be characteristic of the disease (3);however, no quantitative data were presented inthe above papers. Although the data presentedhere do not resolve the identity of the necroticparticles, it disputes the idea of a generalizedleukopenia. Furthermore, there was no evidenceof a generalized leukopenia, but a neutropeniawas definitely associated with the disease. Neu-trophils and macrophages are common targetsof systemic viral infections and may serve as thesource for spread of the agent throughout thehost (14). Perhaps the necrobiotic particles, inpart, are debris from disintegrating neutrophils.Fluorescent antibody studies should help re-solve this question.Although the hematopoietic tissues of the

anterior kidney are the major site of necrosis,histological examination of the renal tubules

and glomeruli show little change (3, 39). Thisfinding, however, does not necessarily meanthat there is no physiological alteration of therenal functions, and the data presented heresuggest that kidney dysfunction may be theprimary cause of death.The telecost kidney functions primarily as a

regulator of body fluid and secondarily as anexcretory organ and electrolyte regulator (16).The freshwater fish is hypertonic to its environ-ment and water continually diffuses across thegills into the blood. The kidney eliminates thisexcess water and reabsorbs most of the bodyelectrolytes. The gill functions not only as arespiratory organ, but also as an excretory andion-regulating organ (10).The experiments described here indicated

that the normal physiology of the kidney was

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300- alkali reserve occurred. The alkali deficit couldbe due to metabolic acidosis, but the increased

200- pH indicates an over compensation, most likely1100 :O:0\56 ii-__ by respiratory mechanisms. The anemic condi-loo- __5-' Infected tion also must contribute because the PO2000 would be reduced, which in turn must affect theooo-_-

-b~-0003s pCO2 levels (28). Furthermore, the regulatory900- ~ ~ - mechanisms of the kidney may be reduced

Control which would contribute to uncompensated reg-.800 ulation of acid-base balance. The mechanisms

Dayi9101'3 16 18 20 of acid-base regulations by each organ is just0479lbDoys

I'3 20beginning to be understood; therefore, a more

realistic evaluation of the changes noted in thisFIG. 3. Comparison of mean weight of fasting rain-bow trout uninfected and infected with IHN virus at paper must await further information regarding10 C. the relative importance of gill and kidney in

regulating acid-base balance.

altered. The weight increase most clearly dem- Although pathological changes were also ob-onstratedthis.Much of the loss ofeserved in the liver and pancreas of fish termi-

onsadterhps.som of thehematolossof elctr es nally infected with IHN disease (3, 39), none ofand perhaps some of the hematological changes the..can be explained by hemodilution. The condi- the physiological tests indicated a change intion was also probably heightened by loss of function of these organs. Glucose regulation was

other renal regulatory functions. The alteration unchanged and bilirubin levels were lower inin acid-base balance could have been caused by infected fish. Enzymes can sometimes indicatethe physiological dysfunction of the renal ele- pathological changes due to viral infectionsments of the kidney. (24), and certain enzymes may be of diagnosticmetshofld value with some fish diseases (8). However, noIt should be noted that some of the valueswe.reported for acid-base balance are derived val- enzyme differences were noted here except for

.. . an lncrased olasma 2'LDH. Various patho-ues and do not necessarily represent normal an increased plasma B Lvalues. Garey (15) pointed out that different logical conditions can result in an increase inmethods for determining blood pH result in LDH (38). The LDH virus of mice inhibits thedifferent values. The pH of blood collected by reticular endothelial system from clearing LDH,s****1 1 1* r 1 1 rthus LDH accumulates in the serum (26). Incatherization is considerably higher than that of ther alcinfectio n thereasons for LDH

blood collected by cardiac puncture. Because . v

catherization is impractical in a 25-g fish, the increase are not clear, but tissue damage ispH values we reported are more comparable to usually suspected. The reasons for the specific

of Garey's cardiac puncture and Wede- B42' LDH increase in the present test were notthoseOcearey'scardiac puncture and wede-meyer's (personal communication) caudal ar- clearly evident. It is also not known whattery values. Furthermore, the values we re- change would occur in the few fish which do notported were calculated by the standard formula express this genetically variable enzyme (31);

usedfor humans: therefore, the value of the LDH test remains tobe determined.

pH = 6.1 + log (HCO 31 Diminished levels of serum proteins have0.03+cog been reported for several diseases of fish (18,

25). However, with IHN disease no apparentat 37 C. This, of course, is not temperature overall reduction in total plasma protein oc-corrected for pK and solubility constant (1, 28). curred, but specific alterations in some of theHowever, for comparison between moribund serum proteins was found. Klontz et al. (21)and control fish, the predictions derived from showed an increase in the beta-2 serum fractionthe data are still valid because all fish and of chinook salmon surviving an IHN infectionsamples were handled the same. and suggested it may be of immunologicalAs Wedemeyer and Chatterton noted (35), significance. The antibody activity of fish

the terms alkalosis and acidosis are relative to serum resides in the slowest migrating macro-temperature; consequently, the IHN-infected globulin (12, 17). In the present tests alterationsfish may not have suffered from alkalosis. In were observed in the alpha-2 and alpha-3 frac-fact, the pH was still within the normal range at tions. Although the reasons for this change are10 C, even though significant alteration of the not clear, tissue damage or some other hostacid-base ratio and severe depletion of the response may be responsible. The separation of

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control plasma was similar to that previouslyreported with disc electrophoresis (30).

In conclusion, the cause of death seems mostclosely correlated with the reduction in serumelectrolytes. The anemic conditions, raisedblood pH, and other changes found were notincompatible with survival. However, the verylow blood osmolality and plasma bicarbonatewere considerably below normal values (G.Wedemeyer and N. Nelson, J. Fish. Res. BoardCan., in press). The very low osmolality valueindicated a very poor prognosis (22) and alsosuggested a considerable loss of sodium. All ofthese results implicated acute kidney failure asthe most probable cause of death. Urine pH,HCO3-, water clearance, and osmolality wouldhave given much additional information, butthese tests were not possible on a 25-g fish.Future studies should include analysis of serumsodium and potassium.

LITERATURE CITED1. Alberts, C. 1970. Acid-Base Balance, p. 173-208. In W. S.

Hoar and D. J. Randall (ed.), Fish Physiology, Vol. 4.Academic Press, Inc., New York.

2. Amend, D. F. 1970. Control of infectious hematopoieticnecrosis virus disease by elevating the water tempera-ture. J. Fish. Res. Board Can. 27:265-270.

3. Amend, D. F., W. T. Yasutake, and R. W. Mead. 1969. Ahematopoietic virus disease of rainbow trout and sock-eye salmon. Trans. Amer. Fish. Soc. 98:796-804.

4. Amend, D. F., and V. C. Chambers. 1970. Morphology ofcertain viruses of salmonid fishes. I. In vivo studies ofsome viruses causing hematopoietic necrosis. J. Fish.Res. Board Can. 27:1285-1293.

5. Amend, D. F., and V. C. Chambers. 1970. The morphol-ogy of certain viruses of salmonid fishes. II. In vivostudies of infectious hematopoietic necrosis (IHN)virus. J. Fish. Res. Board Can. 27:1385-1388.

6. Amend, D. F., W. T. Yasutake, J. F. Fryer, K. S. Pilcher,and W. H. Wingfield. 1973. Epizootiology of infectioushematopoitic necrosis virus in salmonids, p. 80-98.Food and Agriculture Organization of the UnitedNations technical paper no. 17 (Suppl. 2).

7. Amend, D. F., W. T. Yasutake, and R. Morgan. 1969.Some factors influencing susceptibility of rainbowtrout to the acute toxicity of an ethyl mercury phos-phate formulation (timian). Trans. Amer. Fish. Soc.98:419-425.

8. Bell, G. R. 1968. Distribution of transaminase (aminotransferases) in the tissues of pacific salmon (Oncor-hynchus), with emphasis on the properties and diag-nostic use of glutamic-oxalacetic transaminase. J. Fish.Res. Board Can. 25:1247-1268.

9. Brett, J. R. 1971. Energetic responses of salmon. A studyof some normal relations in the physiology and freshwa-ter ecology of sockeye salmon (Oncorhynchus nerka).Amer. Zool. 11:99-113.

10. Conte, F. P. 1969. Salt secretion. In W. S. Hoar and D. J.Randall (ed.), Fish physiology, vol. 1. Academic Press,Inc., New York.

11. Darlington, R. W., R. Trafford, and K. Wolf. 1972. Fishrhabdoviruses: morphology and ultrastructure of NorthAmerican salmonid isolates. Arch. Gesamte Virusfor-schung. 39:257-264.

12. Dorson, M. 1972. Some characteristics of antibodies inthe primary immune response of rainbow trout (Salmogairdneri). Symp. Zool. Soc. London 30:129-140.

13. Dubowski, K. M. 1962. An 0-toluidine method forbody-fluid glucose determination. Clin. Chem.8:215-235.

14. Fenner, F., and D. 0. White. 1967. Medical virology.Academic Press, Inc., New York.

15. Garey, W. F. 1972. Determination of the normal blood pHof fishes. Resp. Physiol. 14:180-182.

16. Hickman, C. P., and B. F. Trump. 1969. The kidney. InW. S. Hoar and D. J. Randall (ed.), p. 9-239, Fishphysiology, vol. 1. Academic Press, Inc., New York.

17. Hodgins, H. O., G. J. Ridgway, and F. M. Utter. 1965.Electrophoretic mobility of an immune globin fromrainbow trout serum. Nature (London) 208:1106-1107.

18. Hunn, J. 1964. Some patho-physiologic effects of kidneydisease in brook trout. Proc. Soc. Exp. Biol. Med.117:383-385.

19. Klontz, G. W. 1972. Hematological technique and theimmune response in rainbow trout. Symp. Zool. Soc.Cond. 30:89-99.

20. Klontz, G. W., and L. S. Smith. 1968. Methods of usingfish as biological research subjects, p. 323-325. In W. I.Gay, (ed.), Methods of animal experimentation, vol. 3.Academic Press, Inc., New York.

21. Klontz, G. W., W. T. Yasutake, and T. J. Parisot. 1965.Virus diseases of Salmonidae in Western U.S. III.Immunopathological Aspects. Ann. N. Y. Acad. Sci.126:531-542.

22. Lobdell, D. H. 1966. A practical guide to clinical osmome-try. St. Vincent Hospital Med. Bull. 8:1-14.

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