Results. 354 Paul W. RENO et al. captoethanol, 2.7 ml M EDTA) (BEUTLER, 1975) Materials and Methods. and stored at -80 Ž until use.

Transcription

1 The effects of viral erythrocytic necrosis (VEN) on clinical blood parameters and erythrocyte me tabolism were investigated in Atlantic cod (Gadus morphua) and Atlantic herring (Clupea harengus h.). YEN-infected cod and herring exhibited lower hematocrits (HCT) and erythrocyte counts (E) in comparison to uninfected fish. In addition, YEN-infected cod, but not herring, had significantly lower hemoglobin concentrations (HB). No effects on plasma electrolyte or protein concentrations in either species were detected. Several metabolic changes were noted in YEN-infected erythrocytes. Infected cod erythrocytes had significantly lower lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PD) activities than erythrocytes from uninfected fish. No alterations in citrate synthetase (CS) activity were observed. No effects on LDH, G-6-PD, or CS activity were detected in YEN-infected herring. In VEN-infected cod erythrocytes the ATP levels were decreased approximately 50% compared to uninfected fish. In contrast, VEN infection in herring was associated with an elevation of ATP levels by approximately 33%. Introduction A variety of marine, anadromous and freshwater fishes have been found to be affected with viral ery throcytic necrosis (VEN) which is manifest by the presence of intracytoplasmic inclusion bodies and, in some instances, nuclear degeneration in.the erythrocytes (DOYLE, 1971; EVELYN and TRAXLER, 1978; JOHNSTON and DAVIES, 1972; LAIRD and BULLOCK, 1969; RENO et al., 1978; SHERBURNE 1973, 1977). Ultrastructural examination of affect ed erythrocytes of several, but not all, species has revealed the presence of large, icosahedral viruses, morphologically similar to viruses of the family Iridoviridae, associated with the inclusions. These viruses fall into three classes distinguished by size and morphology and are characteristic of the host species: nm in Atlantic herring (Clupea harengus harengus) (RENO et al., 1978), Onco rhynchus spp. (EVELYN and TRAXLER, 1978), and American eel (Anguilla rostrate) (DOYLE, 1971); nm in blenny (Blennius pholis) (JOHNSTON and DAVIES, 1973); and nm in Atlantic cod (Gadus morhua) (APPY et al., 1976; RENO and NICHOLSON, 1981; WALKER and SHERBURNE, 1977). In wild fishes, little evidence has been reported to indicate adverse physiological affects of these viral infections although the affected erythrocytes are morphologically either pre-necrotic or necrotic (EVELYN and TRAXLER, 1978; MACMILLAN and MULCAHY, 1979). MACMILLAN et al. (1980) dem onstrated a marked anemia and compromised homeostatic processes in experimentally VEN infected juvenile chum salmon (Oncorhynchus keta), but no overt mortalities attributable to infection occurred. In vitro studies have indicated that affect ed erythrocytes are more fragile than erythrocytes from unifected fish (RENO and NICHOLSON, 1980). In this report, we present evidence to indicate that feral adult Atlantic cod (Gadus morhua) and Atlantic herring (Clupea harengus h.) exhibit physiologic aberrations associated with VEN infection. No changes were seen in the alewife (Alosa pseudo harengus), an anadromous species; however, the levels of infection in this species were extremely low.

2 354 Paul W. RENO et al. Materials and Methods Collection and holding offish. Atlantic cod were caught in lobster traps near Boothbay Harbor, Maine and held in tanks with circulating seawater at the.state of Maine Department of Marine Resources, Boothbay Harbor, Maine until trans ported to the University of Maine at Orono where they were kept in tanks with recirculating artifical seawater (Instant Ocean, Fisher Chemical, Med ford, MA.) (specific gravity , temper ature 10 } 2 Ž). Atlantic herring wer collected in purse seines near Prospect Harbor, Owl's Head, and Matinicus Island, Maine during the summer and fall, and blood samples were taken immedi ately. Alewives were collected from the weir at Orland, Maine during their upstream migration in spring, and were held in tanks of recirculating artifical seawater (temperature Ž, specific gravity ). Collection of blood and estimation of hematologic values. Blood was collected from all fishes by caudal venipuncture and dispensed into tubes with anticoagulent (ammonium geparin, Fisher Scientific Medford, MA). Hematocrits and hemoglobulin concentrations were estimated using standard hematological tech niques (BEUTLER, 1975). Erythrocyte counts were performed on a Hycel model 300 counter (Hycel, Inc. Houston, TX) at a threshold voltage of 6.9. Plasma electrolytes were determined on a flame pho tometer (Instrumentation Lab., Lexington, MA). Chloride concentrations were assayed by the titra metric method of SCHALES and SCHALES (1941). Plasma proteins. Plasma protein components were estimated by cellulose acetate membrane elec trophoresis (Gelman, Ann Arbor, MI) in high resolution Tris-barbital buffer (0.06 M ionic strength, ph 8.8); at constant voltage. The protein bands were stained with Ponceau S (Eastman Kodak, Rochester, N.Y.), peak intensities and lo cations read on a scanning densitometer (Transdyne General, Ann Arbor, MI) and albumin: globulin ratios determined. Enzyme and ATP assays. For enzyme assays, hemolysates of packed cells were prepared by lysing the cells in a hemolysing solution (0.063 M 2-mer captoethanol, 2.7 ml M EDTA) (BEUTLER, 1975) and stored at -80 Ž until use. Lactate dehy drogenase (LDH) activities were determined by the method of BEUTLER (1975) except that the assays were carried out at 20 Ž, the temperature de termined to be optimal for LDH for these species (SERREZE, unpublished observations). Glucose-6 phosphate dehydrogenase (G-6-PD) assays - were also performed by the method of Beutler (1975) with the exception of the temperature (20 Ž). cit rate synthetase (CS) activity at 20 Ž was estimated by the method of SRERE (1963). In all instances, since the enzyme activities for erythrocytes of the species studied here had not been previously re ported, optimal substrate concentrations were de termined from standard curves prepared for each assay system using uninfected fish. For estimation of ATP concentrations of eryth rocytes, the triose phosphate was extracted from whole blood by precipitation with cold perchloric acid and assayed with the hexokinase system de scribed by BEUTLER (1975). Results Effect of VEN infection on hematological values. Comparisons of hematological parameters were made between YEN-infected and uninfected cod, herring and alewives. The VEN infection rates were between % for cod, % for herring, and from < % for alewives; in all cases the fish appeared grossly normal. The results of these comparisons are presented in Table 1. The three primary hematological values, hema tocrit, erythrocyte count, hemoglobin concentra tion (HCT, E, HB respectively) of VEN-infected Atlantic cod were significantly diminished when compared to uninfected animals. In all instances, however, the ratios of these values (mean corpus cular volume, MCV, mean corpuscular hemoglobin, MCH, and mean corpuscular hemoglobin concen tration, MCHC) were not significantly altered. The depression of HCT, E, and HB amounted to 16, 17, and 21% respectively. Similarly, there was a signi ficant decrease in HCT and E in VEN-infected herring when compared to uninfected fish; the decreases were 10% for HCT and 21% for E. No significant change was noted in the HB levels be tween infected and uninfected fish. Again the sec ondary hematologic values remained unchanged.

3 VEN in Atlantic Cod and Herring 355 Table 1. Effect o f VEN infection on mean values of selected hematological parameters Table 2. Pearson correlation coefficients of VEN in fection and hematological components of cod and herring Table 3. Effect of VEN infection on mean values of plasma electrolytes No significant alterations were found in the blood constituents of VEN-infected alewives when compared to uninfected animals. In order to determine if the magnitude of alter ation in hematological values in cod and herring was related to the intensity of VEN infection, Pearson correlation coefficients were calculated and the results of the analyses are presented in Table 2. There were significant negative correlations between VEN infection rates and E of both species, sig nificant positive correlations between infection rate and MCV of both species and a significant negative correlation betwen HB and MCHC and infection rate in atlantic cod. Plasma electrolyte and protein levels in infected and uninfected fishes. Evidence has been presented to indicate that VEN infection has an adverse impact upon the osmoregulatory capacity of ju venile Pacific salmon, Oncorhyncus keta (MACMILLAN et al., 1980). To determine if any major alteration occurred in osmoregulatory capa citites of these YEN-infected marine and anadro mous fishes, plasma electrolytes were screened. As can be seen in Table 3, no significant differences were found.in plasma electrolytes of VEN-infected and uninfected cod or alewives; because of the inherent lability of herring erythrocytes, too few unlysed samples were obtained for accurate analysis of Na + in this species. Plasma proteins were seperated by cellulose ace tate electrophoresis to determine if any effect was generated by VEN infection, especially with respect to immunoglobulin levels. Electrophoretic profiles of Atlantic herring indicated that the plasma of this species had a relatively high concentration of al bumin and identifiable ć-globulin peaks (Table 4),

4 356 Paul W. RENO et al. Table 4. Effect of VEN infection on mean values of plasma proteins with ƒá-globulins comprising approximately 15% of the plasma protein. By contrast, cod serum had a Table 5. Effect of VEN infection on mean activities of erythrocyte enzymes proportionately lower albumin content, lacked a detectable ƒá-globulin fraction and had markedly higher ƒ 2 and ƒà-globulin levels than herring. No statistically significant effects of VEN infection were noted on the plasma proteins of either species. Effects of VEN infection on erythrocyte enzymes and energy production. To determine if the mor phological changes seen in VEN-infected eryth rocytes were associated with significant biochemical lesions in the cells, three key enzyme activities in glucose metabolism as well as ATP content of the erythrocytes were examined. The enzymes selected for analysis were associated with three major cata bolic pathways: glucose-6-phosphate dehydro genase (G6PD), hexose monophosphate shunt; lac tate dehydrogenase (LDH), glycolysis; citrate syn thetase (CS), tricarboxylic acid cycle. Preliminary experiments showed that the enzymatic activities of the much less numerous leukocytes were negligible when comared to the activities of the erythrocytes and consequently samples for assay were taken from packed cell pellets rather than from gradient purified preparations of erythrocytes. Since eryth rocyte enzyme activities have not been previously reported for these species, the G6PD and LDH activities of human erythrocytes were assayed in our system and found to be within normal range, thus confirming the accuracy of our assay system. When comparisons of enzyme activities were - made between VEN-infected and uninfected fish of the three species, some significant differences were found (Table 5). It is interesting to note that erythrocytes from cod had markedly greater LDH Table 6. Effect of VEN infection on mean values of erythrocyte ATP content and G6PD activities than did the two clupeid species, but that the CS activities of erythrocytes from all three species were comparable. The LDH activity of erythrocytes from VEN-infected cod (infectivity rang %) was significantly de pressed by about 25% when compared to uninfected

5 VEN in Atlantic Cod and Herring 357 fish. Similarly, the levels of G6PD activity of VEN infected cod were depressed by approximately - 33% of the level found in uninfected cohorts. Citrate synthetase activity was not significantly affected by VEN infection. The activity levels of all three erythrocyte enzymes studied in the clupeid fishes appeared to be unaffected by VEN infection (in fectivity range % in herring, % in alewives). ATP levels were determined in order to assess the overall energy composition of erythrocytes from both VEN-infected and uninfected cod and herring. As shown in Table 6, VEN infection had a signifi cant impact upon the ATP content of erythrocytes from both cod and herring. In VEN-infected cod erythrocytes, the ATP levels were found to be about half the level found in uninfected fish. By contrast, VEN infection in herring was associated with an elevation of the ATP level by approximately one third. Although the enzymatic activities of cod and the ATP levels of both cod and herring described above were found to be affected by VEN infection, there were no correlations betwen the VEN in fection rates and the activities as determined by linear regression analysis. Discussion The results of the studies presented here dem onstrated that viral erythrocytic necrosis (VEN) had detrimental effects upon two of three species (cod and herring) of fishes from the northern Atlantic Ocean. No effects were seen in VEN infected individivals of the anadromous - species Alosa pseudoharengus, which probably reflects the fact that the intensity of infection in this species has never been found to be greater than 0.1% in adult fish returning upstream to spawn in spring (SHERBURNE, 1977; RENO, unpublished obser vations). Consequently, it is not known whether hematologic or biochemical lesions would have been observed in alewives if the intensity of in fection were higher and it is, in fact, conjectural whether the low intensity of infection characteristi cally observed in spawning adults ever becomes elevated during the course of the infection process. The evidence presented here indicated that the lesions produced by VEN in Atlantic cod were more extensive than those in Atlantic herring in spite of the fact that the levels of infection commonly seen in feral herring were higher than in cod. Both hematologic and biochemical lesions were noted as a consequence of VEN infection in cod: depression of packed cell volume, erythrocyte numbers, and hemoglobin content of the blood; diminution of erythrocyte LDH and G6PD activity; and depres sion of ATP levels of erythrocytes. The indicators of blood homeostasis \HCT, E, and HB \were all equally depressed to approximately 80% of their normal values in VEN-infected fish, leading to a clinical state of moderate normocytic anemia. Normocytic anemia in higher animals is commonly a result of either hemolytic or aplastic disease and in view of the evidence that VEN-infected erythrocytes are extremely labile in vitro (RENO and NICHOLSON, 1980) and that hematopoisesis appears to occur during VEN infection in cod (RENO et al., un published data), it is probable that the cause of the anemia noted here is hemolysis. The character istic morphology of YEN-infected cod eryth rocytes also enforces this supposition since the presence of a high proportion of karyorrhectic nuclei in infected cells (APPY et al., 1976) \a microscopic manifestation of cell death--would indicate ultimate cell lysis. The anemia observed in VEN-infected herring was also of the normocytic type, even though no significant alteration was seen in the hemoglobin content of the blood. Since none of the secondary hematologic parameters (characterized as ratios of the primary parameters), indicators of the type of anemia present, were altered, it can be concluded that normocytic anemia was present. The anemia associated with VEN infection in herring was of approximately the same magnitude as that en countered in cod, an decline of 10-20%. The anemia associated with VEN infection in Atlantic cod and herring did not approach the magnitude or severity of that found in experimen tally infected fry chum salmon (Oncorhyncus keta) (MACMILLAN et al., 1980), where HCT levels were depressed by up to 90%. However, the earlier study differs from the present one in several respects which may account for the differences in the in tensities of the anemias associated with VEN in fection. First, the VEN infection in the prior study was induced experimentally, whereas the animals used in this study were naturally infected wild fish; the source of infectious virus and the mode of infection in these feral fish remains unknown at

6 358 Paul W. RENO et al. present. Thus, the experimental infection, designed to maximize the severity and level of disease, may have caused a more severe disease than the natural route of infection. Second, whereas the animals used in the experimental study were young fry and fingerlings and exhibited a negative age correlation with frequency and intensity of VEN infection, the fish used in this study were mature fish and may not have manifested lesions as severe as would be seen if juveniles had been obtained. Third, it is possible that, since these were feral fish, only individuals minimally affected by VEN would have survived into adulthood. Fourth, there may be inherent spe cies differences in the severity of the disease as seen elsewhere (APPY et al., 1976; SHERBURNE, 1973, 1977; EVELYN and TRAXLER, 1978). And lastly the virus associated with VEN in the Pacific salmon (EVELYN and TRAXLER, 1978) was clearly distinct morphologically and in size from the virus causing VEN in Atlantic cod (APPY et al., 1976) and may also be different from the virus in Atlantic herring (RENO et al., 1978). The enzymes chosen for study here wre selected because of their involvement in three major cata bolic pathways of hexose metabolism: lactate dehy drogenase (LDH)-glycolysis; glucose-6-phosphate dehydrogenase (G6PD)-hexose monophosphate shunt; and citrate synthetase (CS)-tricarboxylic acid cycle. Fish erythrocytes, in contrast to those of mammals, have mitochondria and an active TCA cycle. These pathways are responsible for the levels of triosephosphate production, especially adenosine triphosphate (ATP), from hexoses. Consequently, any decrease in these enzymatic activities would be expected to have detrimental effects on energy generation by the erythocytes. Atlantic cod infected with VEN exhibited a de pression in both LDH and G6PD activities in erythrocytes. This decrease in activity was reflected by a depression of erythrocyte ATP content in these animals. This could result in untoward con sequences for VEN-infected fish for two reasons. First, ATP is the primary energy currency of the cell and as such plays a crucial role in maintaining cellular integrity, especially via the sodium potassium pump (BREWER, 1974). A loss of avail able ATP would decrease the ability of the cell to maintain homeostasis and might make it more labile to lysis. Increased susceptibility to lysis has been previously detected in VEN-infected cod erythrocytes in vitro (RENO and NICHOLSON, 1980) and also may be operative in vivo. In order to con firm this, effective methods of seperating VEN infected from uninfected erythrocytes must - first be developed. Second, it has been demonstrated that ATP plays a role in fish as a primary allosteric modifier of hemoglobin-oxygen binding affinity (GREANY and POWERS, 1977). Therefore, higher levels of intracellular ATP would decrease the binding capacity of hemoglobin (with a conse quent release of oxygen at higher partial pressures of oxygen in the tissues) and lower levels of intra cellular ATP would result in a tighter binding of 02 to hemoglobin (with a consequent release of oxygen at lower partial pressures of oxygen in the tissues) (CHANUTIN and CURNISH, 1967). In physiologic terms, the 50% decrease in ATP con tent noted in erythrocytes from VEN-infected cod would result in a loss of available 02 for tissue respiratory activity since it would remain more tightly bound to hemoglobin. Under these circum stances it is evident that VEN-infected cod would likely have more difficulty in coping with stress ful situations than uninfected fish. This has been seen in severely affected Oncorhyncus keta finger lings in that VEN infection results in an inabil ity to adjust to osmotic changes or infectious di seases (MACMILLAN et al., 1980). Erythrocytes of herring, while generally exhibit ing higher VEN infection rates than cod, exhibited much less severe physiologic consequences of in fection. Indeed, there was a 33% increase in ATP content of infected erythrocytes. Since there were no significant changes in the activities of the enzymes tested, this increase of ATP may have occurred either as a result of changes of activities of other enzymes in the pathways or a decrease in the utilization of ATP by the cells. Since only single instantaneous samplings of activity were made, flux rates could not be determined and the possible mechanisms for this phenomenon remain conjec tural. The consequences of the increased ATP would be a greater availability of oxygen for tissue respiration at low partial pressures of oxygen. The differences noted in the VEN-induced ATP content between the two species may reflect an adaptive response to infection, perhaps influenced by different environmental pressures exerted on the animals. Cod are sedentary animals with a low tissue oxygen demand ( ml/g/hr at 10 Ž)

7 VEN in Atlantic Cod and Herring 359 (SAUNDERS, 1963) whereas herring are an active, mobile, schooling species and probably have a high tissue oxygen demand as do other active species ( ml/g/hr at 10 Ž) (COCHE, 1967). It is conceivable that herring generated elevated ATP to compensate for the loss of erythocytes from the National Heart, Lung, and Blood Institute of the National Institutes of Health, grant PCM from the National Science Foundation, and by the Maine Agriculture Experiment Station. caused by VEN-infection in order to maintain their high tissue normoxia, whereas cod, which do not Tequire as much oxygen, may be capable of sur viving at hypoxic levels. Separation of VEN infected erythrocytes from uninfected erythrocytes - and repeating these studies would be necessary if this question is to be resolved. To summarize the findings reported here, hema tological and physiological lesions were associated with VEN infection in Atlantic cod and herring. No effects were seen in alewives although the very low levels of infection probably precluded observing any effects in the species. The scope and severity of the lesions were more evident in cod than in herring, but neither species exhibited anemia as severe as in Pacific salmonids (MACMILLAN et al., 1980). It is interesting to note that the microscopic lesions seen in VEN-infected cod erythrocytes were more pro nounced than in herring, with pyknosis and karyor rhexis frequently evident in the former and chro matin margination, but not pyknosis or karyor rhexis, found in the latter (APPY et al., 1976; SHERBURNE, 1973). The presence of pyknosis and karyorrhexis are definitive indicators of cell necro sis, whereas chromatin margination is evidence of a potentially reversible pre-necrotic state. The dif ferences in the severity of the microscopic lesions between species may be a reflection of the differ ences in the physiologic lesions reported here. Although VEN infection in adults of these species may produce only a mild disease state, even a mild disease may significantly affect survival rates of the.species in the wild. Acknowledgements The authors would like to thank the personnel of Port Clyde Foods, Rockland, ME. for their assist ance in collecting the herring used in this study, and to Stuart Sherburne of the Maine Department of Marine Resources for providing holding facilities for the cod, and to the Town of Orland, Maine, for collection of alewives. Funds for this project were provided by grant 5-R01-OHL

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