魚 病 研 究 Fish Pathology, 25 (3), 141-146, 1990. 9

Efficacy of Fumagillin Against Proliferative Kidney Disease and its Toxic Side Effects in Rainbow Trout (Oncorhynchusmykiss) Fingerlings

A. WISHKOVSKY1*, J. M. GROFF1, D. J. LAUREN1, R. J. TOTH2 and R. P. HEDRICK1**

Department of Medicine, School of1 Veterinary Medicine, University of California, Davis, California 95616, USA California Department of Fish and Game, Bishop, California 293514, USA

(Received April 13, 1990)

The potential of fumagillin DCH to prevent proliferative kidney disease (PKD) in rainbow

trout fingerlings was tested in field and laboratory studies. In a field study, PKX trophozoites

and accompanying inflammation (PKD) occurred in the kidneys of 92.7% of fish fed control diets and in only 7.9% of fish receiving 0.34 g fumagillin/kg of diet (fed at 6% biomass per

day) when examined 65 days after being placed in waters enzootic for the parasite. In a laboratory study, fumagillin treatment reduced the number of fish that developed

PKD following natural exposure to the parasite. In both the laboratory and the field studies, fish fed fumagillin demonstrated a loss of appetite beginning 2 to 4 weeks following feeding of

medicated diets. Cumulative mortalities of 92% and 33% for the field and laboratory studies were experienced by groups of fish continuously fed medicated diets.

The toxicity of fumagillin was evaluated in the laboratory following feeding at 0.1, 0.25,

0.5 or 1.0 g drug/kg feed (fed at 1.5-2.0% biomass daily) for 8 weeks at water temperature of 15•Ž. Fish administered 0.5 and 1.0 g drug/kg food exhibited anorexia beginning 4 weeks after initiation of feeding that lasted until the end of the experiment at 8 weeks. The cumu

lative mortality of fish fed the 1.0 g drug/kg reached 22% at the end of the study. The average weight of these fish was significantly decreased compared to fish receiving lower doses or no

fumagillin in the diet. The spleen and kidney of fish administered the two highest doses of fumagillin were grossly reduced in size. Microscopic examinations revealed a decrease in the

amount of interstitial hematopoietic tissue in the anterior and posterior kidney and splenic

lymphoid tissue. These effects were least apparent in the fish fed 0.1 g fumagillin/kg feed . These results indicate that fumagillin can have an adverse effect on the hematopoietic tissue

of fish when administered at increased concentrations over an extended period of time. How ever, these effects can be minimized when fish are fed low doses of fumagillin as a potential

treatment to prevent PKD.

The antibiotic, fumagillin DCH, has been and myxosporean diseases of fish has also been found to be effective in controlling diseases reported (KANO et al., 1982; MOLNAR et al., caused by certain microsporidia in insects 1987; HEDRICKet al., 1988a). BAILEY, 1953; LYNCH and LEWIS, 1971), and The myxosporean PKX, is the cause of primarily to control Nosema infection of honey proliferative kidney disease (PKD), one of the bees (KATZNELSON and JAMIESON, 1952). The most serious diseases of farm-reared salmonids potential of the drug for treating microsporidian in Europe and North America (CLIFTON * Current address: Ministry of Agriculture HADLEY et al., 1984; HEDRICK et al., 1986). , Fish eries Division, Laboratory for Research of Fish Recent reports have shown that treatments and Diseases, Nir David 19150, Israel. effective prophylaxis of this disease is possible. ** To whom correspondence should be directed . Periodic malachite green bath or flush treat 142 A. WISHKOVSKY, J. M. GROFF, D. J. LAUREN, R. J. TOTH and R. P. HEDRICK

ments can reduce the severity and incidence of trout were fed at 6% biomass per day. of PKD in rainbow trout (Oncorhynchus mykiss), Cumulative mortalities were estimated by the but this compound can be toxic to fish and is number of survivors at the end of the study. considered unsafe for food fish due to its po After 65 days, the kidneys and spleens of 76 tential teratogenic properties (ALDERMAN and experimental and 82 control fish were removed, CLIFTON-HADLEY, 1988; ALDERMAN, 1985). fixed in DAVIDSON'S solution (HUMASON, 1979), Preliminary studies in our laboratory indicated embedded in paraffin, sectioned to 6 ƒÊm, and that chinook salmon (O. tshawytscha) finger stained with hematoxylin and eosin. The lings experimentally-infected with PKX were kidney sections were examined for PKX and protected from PKD when fumagillin was any pathological changes. administered in the diet. In contrast, 90-100% of the untreated fish developed PKD (HEDRICK Efficacy of Fumagillin on Naturally-Acquired et al.,1988a). Infection: Laboratory Study In this paper we report on field and labor Rainbow trout fingerlings (mean weight 39.7 atory studies conducted to examine the efficacy g) were obtained from a PKD enzootic area of extended oral administration of fumagillin to (State of California American River Hatchery, protect against PKD in naturally-infected Rancho Cordova, California) during an active fingerling rainbow trout. In addition, the ef infection. Fish (35.0 g) from the same stock fect of selected drug doses on the growth of were moved to the laboratory 3 mo earlier, a fish and possible toxic effects were examined time at which infectivity is absent from the in laboratory studies. hatchery water supply (FOOTT and HEDRICK, 1987), and were treated as uninfected controls.

Materials and Methods At the initiation of the experiment, 20 fish from each group were examined for PKD. Two

Medicated Diets groups of infected fish (70 fish each) and two Fumagillin DCH dissolved in 100% isopropyl control groups (50 fish each) were placed in ethanol in the field study or in 95% ethanol for 130 l aquaria supplied with flow through aerated subsequent experiments was sprayed onto a com well water (15•Ž). One group of the experi mercial pelleted feed (100 ml alcohol solution/ mental fish and one control group were fed kg feed). These pellets were then coated by fumagillin diet (1.0 g drug/kg diet fed at 1.5% spraying with a mixture of alcohol and cod body weight daily); the other two remaining liver oil (50 ml of each/kg feed) and allowed to groups were fed unmedicated diet. Feeding of dry. Medicated and control feeds were stored medicated diet began immediately after in at -20•Ž immediately after preparation. fected fish were transferred to the laboratory and was continued until week 5. Beginning at Efficacy of Fumagillin: Field Studies week 5 until the end of the study at 8 weeks, The field studies were conducted with rain all groups received unmedicated diets. Mor bow trout at the State of California Hot Creek talities were recorded daily. Kidneys from 10 Hatchery, Bishop, California. This site has a fish in each group were taken and processed water supply with an annual history of PKD for routine histopathological examination at 4 infectivity (HEDRICK et al., 1988b). Water and 8 weeks after the initiation of the temperature at this location is a constant experiment. 14.5•Ž.

Two groups of 1000 rainbow trout (mean Toxic Effects of Fumagillin: Laboratory Study weight of 2.0 g) were transferred to adjacent Because field studies indicated that increased compartments in a raceway receiving water doses of fumagillin resulted in an adverse toxic known to contain the infective stage of PKX. effect, a laboratory study was designed to One group was fed a commercial (unmedicated) determine the effects of selected concentrations diet and the second group fumagillin medicated of the drug on rainbow trout fingerlings. Fish feed (0.34 g fumagillin/kg feed). Both groups (mean weight of 3.89•}0.2 g, and mean fork Effect of Fumagillin on Rainbow Trout Fingerlings 143

length of 6.65•}0.11 cm) were obtained from a

commercial culture facility in California with

no history of PKD. The fish were fed a com-

mercial diet at 1.5-2% of their body weight

daily. Twenty groups of 18 fish each were

maintained in 10l aquaria supplied with flow

through aerated well water (15•Ž). Four groups

of fish (72 fish) were fed either 1.0, 0.5, 0.25,

or 0.1 g of fumagillin per kg of feed. A fifth

group received an unmedicated control diet. Mortalities were removed and recorded daily.

At 1, 2, 4, and 8 weeks after beginning the

experiment, 18 fish from each treatment were

euthanized in 0.5 g,/‚Œ tricaine methanesulfonate

(MS-222) and body weight and length measure- ments were recorded. Heart, intestine, kid-

ney, liver and spleen were taken from 10 ran-

domly sampled fish from each group on 4 and

8 weeks after initiation of the study and pro-

cessed for histopathological examinations.

Results Fig. 1. Proliferative kidney disease (PKD) and PKX trophozoites in rainbow trout receiv- Efficacy of Fumagillin ing unmedicated diets after natural expo- At the end of the field study (week 8) only sures to the parasite. (A) Interstitial in- 6 of 76 (7.9%) of the fumagillin-treated fish flammation in the kidney. Bar=1 mm. (B) contained PKX parasites and renal interstitial PKX trophozoites (arrows). Bar=10ƒÊm.

inflammation. In contrast 76,82 (92.7%) of the Hematoxylin and eosin stains. kidneys from control fish were infected (Fig. 1). Experimental fish exhibited a loss of appetite the control group fed unmedicated diet. Be- beginning at week 4. While only about 5% of cause of this mortality, feeding of the medi- the control group died over the 8 week period, cated feed was terminated at the end of week a high cumulative mortality (92%) occurred in 4, and all groups were fed control feed until the medicated group by the end of the study. the end of the study (week 8). The administra- The kidneys and spleens of the experimental tion of fumagillin to the infected fish resulted group were generally atrophied and pale. in resolution of PKD by week 8 (Table 1). In Microscopic examinations revealed a severe contrast, the number of infected fish fed a con- depletion of the renal interstitial hematopoietic trol diet at 8 weeks was similar to that detected and splenic lymphoid tissues. at the initiation of the study (Table 1). No At the initiation of the laboratory study PKX PKX trophozoites or PKD were found in the trophozoites were found in 8 of 20 fish (40%) other two uninfected control groups. Fish fed of the naturally-infected group and no parasites fumagillin in the diet showed depletion of the were found in the control fish. The control interstitial hematopoietic tissues of the kidney. and infected groups administered medicated feed (1.0 g drug, kg feed, fed at 1.5% daily) Toxicologic Effects of Fumagillin began to exhibit anorexia 2 weeks after the Fish administered the two highest concentra- initiation of the experiment followed by mor- tions of the drug (0.5 and 10.0 g fumagillin/kg tality which reached 30% at week 4. In con- food) exhibited anorexia beginning at week 4. trast, only one fish died from the infected There were no mortalities in groups of trout group receiving no drug and no fish died from fed the three lower doses or no fumagillin. In 144 A. WISHKOVSKY, J. M. GROFF, D. J. LAUREN, R. J. TOTH and R. P. HEDRICK

Table 1. Effect of fumagillin DCH on actively acquired proliferative kidney disease (PKD) among rainbow trout (Oncorhynchus mykiss) fingerlings

a Actively infected rainbow trout (mean weight 39.7 g) from an enzootic area were transferred to the laboratory (40% of the fish within the population at the initiation of the experiment were infected), divided into two groups, and fed a medicated feed (1.0 g fumagillin/kg of feed) or unmedicated diet at 1.5% body weight per day for 4 weeks. Two groups of control trout were fed similar diets.b Number of fish infected/number of fish examined. Fish were examined 4 and 8 weeks after the initiation of the study.

Table 2. The effect of extended oral administration of fumagillin DCH on weight and length of rainbow trout (Oncorhynchus mykiss)

a Grams of drug per kg food . The food was given to fish at a ratio of 1.5-2.0% body weight per day. b Each number represents an average weight of 18 fish•}S .D.c Significantly different from control (p <0.025, Student t-test). d Each number represents an average fork length of 18 fish•}S .D. contrast, 22.2% of the fish receiving 1.0 g drug/ at week 8 (Table 2). The weights of the other kg died in the final week of the experiment. three experimental groups were not significant Fish from the medicated groups increased in ly different than control fish (Table 2). Fish weight and length at the same rate as control length was not affected by fumagillin treatment fish during the first 4 weeks of the study (Table (Table 2). 2). However, the growth rate in fish fed the Pathological changes were restricted to the highest dose (1.0 g fumagillin/kg food) was re renal and splenic tissues of the fumagillin fed duced during the next 4 weeks of the experi fish. At week 4, there was an atrophy or ment, and the mean weight (9.72 g) was lower depletion of the renal hematopoietic tissue (p <0 .025 , Student t-test) than controls (12.11 g) that was especially prominent in the anterior Effect of Fumagillin on Rainbow Trout Fingerlings 145

Fig. 2. Microscopic comparisons of kidney and spleen of rainbow trout fed fumagillin DCH for 8 weeks to those of trout receiving control diets. (A) Anterior and (B) posterior kidney and (C) spleen of trout fed 1.0 g drug/kg of feed; (D) anterior and (E) posterior kidney and (F) spleen of trout fed control diets. Bars=100 pm. Hematoxylin and eosin stains. kidney. Depletion of the hematopoietic tissue 1.0 g fumagillin/ kg feed). After 8 weeks, the was most severe in the fish administered the spleen and anterior kidney were grossly re- two highest doses of the drug (i.e., 0.5 and duced in size in the fish administered the two 146 A. WISHKOVSKY, J. M. GROFF, D. J. LAUREN, R. J. TOTH and R. P. HEDRICK highest doses of fumagillin. Microscopic ex in initial rainbow trout trials. The effects of aminations revealed a decrease in the renal the drug following extended administration of and splenic hematopoietic tissue (Fig. 2). A fumagillin to rainbow trout (0.1 g to 1.0 g drug/ prominent accumulation of eosinophilic hyaline kg for 8 weeks) were evaluated by measure droplets within the renal tubular epithelial cells ments of survival, growth, gross and micro was often associated with tubular degeneration scopic pathological changes. The results sug and necrosis. Although all the groups admin gest that reduced concentrations of the drug istered fumagillin had a pronounced reduction can minimize toxic effects. Anorexia occurred in the renal and splenic hematopoietic tissue at only in the groups fed the two highest con the end of the experiment, the reduction was centrations (1.0 and 0.5 g fumagillin/kg feed) least apparent in the fish fed 0.1 g fumagillin/ and mortalities occurred only in the group fed kg feed. Histopathological changes were not the highest concentration (1.0 g/kg feed). All observed in the other tissues examined. mortalities occurred during the last week of the experiment which suggests a cumulative toxic effect with extended periods of feeding. Splenic Discussion and renal atrophy occurred in all the medicated The results of the efficacy studies, which groups. In other studies, administration of the were the first attempts in using an antibiotic drug to rainbow trout adults (0.25 or 1.0 g drug/ agent to prevent natural occurrence of PKD, kg feed, at 1% biomass per day) resulted in demonstrated that fumagillin in the diet is ef similar histopathological changes and caused a fective in reducing the incidence of PKD in significant decrease in the hematocrit values populations of rainbow trout fingerlings. Pro (LAURENet al., 1989). The effects of fumagillin tection was achieved in fish that were con on the hematopoietic tissues (kidney and tinuously exposed to the infectious stage of the spleen) may be due to concentration of the parasite in the field and among fish naturally drug or its metabolites in those sites. This exposed to the agent and subsequently moved may also explain why the drug has a simultane to the laboratory. A similar prophylactic effect ous effect against the PKX trophozoites that was observed in experimentally-infected are most abundant in these same tissues. chinook salmon fed 1.0 to 0.5 g drug/kg feed It is important to note that administration of (HEDRICK et al., 1988a). the lowest dose of fumagillin (0.1 g drug/kg The results of the efficacy studies reported feed) minimized the histopathological changes here, however, suggest that fumagillin can have in the kidney and spleen of the medicated fish. adverse toxic side effects, clinically manifested Concentrations of fumagillin of 0.1-0.2 g/kg as anorexia prior to an increased cumulative (fed at 1.5% biomass per day) have been shown mortality (up to 92% in the field study). Micro to protect rainbow trout from experimental in scopic examination of the fish revealed a severe fections and coho salmon (O. kisutch) from depletion of the renal and splenic hematopoietic naturally-acquired infections with PKX in the tissues. absence of serious toxic effects (unpublished The dose of fumagillin (1.0 g drug/kg feed) data). Additional studies are underway to used in our initial (HEDRICK et al., 1988a) and further define protective and safe doses and present studies was chosen because of the suc regimens of fumagillin treatments to control cessful long term treatments (74d) with the infections due to PKX and other important drug for renal sphaerosporosis in carp (Cyprinus myxosporean pathogens. carpio) as reported by MOLNAR et al. (1987). No toxic side effects of the drug were reported Acknowledgements in carp or in a study by KANO et al. (1982) using fumagillin to control Pleistophora infec We extend our appreciation to the histo tions in Japanese eels (Anguilla japonica). technicians, Veterinary Medical Teaching Laboratory experiments were conducted to Hospital, Davis, California, for the processing evaluate the toxicity of fumagillin as observed of tissues; to the State of California American Effect of Fumagillin on Rainbow Trout Fingerlings 147

River and Hot Creek Hatcheries, California, gillin DCH protects chinook salmon (Oncorhynchus for providing fish; and to Chinoin Pharmaceu tshawytscha) from experimentally-induced prolifer tical and Chemical Works Ltd., Budapest, ative kidney disease. Dis. Aquat. Org., 4, 165-168. Hungary, for kindly supplying the fumagillin. HEDRICK, R. P., M. L. KENT, R. J. TOTH, and J. K. MORRISON (1988b): Fish infected with Sphaero spora spp. THELOHAN (Myxosporea) from waters References enzootic for proliferative kidney disease of salmon id fish. J. Protozool., 35, 13-18. ALDERMAN,D. J. (1985): Malachite green: a review. HUMASON, G. L. (1979): Animal tissue techniques. J. Fish Dis., 8, 289-298. W.H. Freeman Co., San Francisco., 661. ALDERMAN,D. J. and R. S. CLIFTON-HADLEY(1988): KANO, T., T. OKAUCHI,and H. FUKUI (1982): Studies Malachite green therapy of proliferative kidney on Pleistophora infection in eel, Anguilla Japonica disease in rainbow trout: Field trials. Vet. Rec., II. Preliminary tests for application of Fumagillin. 122, 103-106. Fish Pathol., 17, 107-114. BAILEY, L. (1953): The effect of Fumagillin upon KATZNELSON,H. and C. A. JAMIESON(1952): Control Nosema apis (ZANDER). Nature. London, 171, 212 of Nosema disease of honey bees with Fumagillin. 213. - Science, 115, 70-71. CLIFTON-HADLEY,R. S. and D. BUCKE, and R. H. LAUREN, D. J., A. WISHKOVSKY,J. M. GROFF, R. P. RICHARDS (1984): Proliferative kidney disease of HEDRICK and D. E. HINTON (1989): Toxicity and salmonid fish: a review. J. Fish Dis., 7, 363-367. pharmacokinetics of the antibiotic, fumagillin, in FOOTT, J. S. and R. P. HEDRICK (1987): Seasonal yearling rainbow trout (Salmo gairdneri). Toxicol. occurrence of the infectious stage of proliferative Appl. Pharmacol., 98, 444-453. kidney disease (PKD) and resistance of rainbow LYNCH, R. E. and L. C. LEWIS (1971): Reoccurrence trout, Salmo gairdneri RICHARDSON,to reinfection. of the microsporidian Perezia pyraustae in the J. Fish Biol., 30, 477-483. European corn borer, Ostrinia nubilialis, reared on HEDRICK, R. P., M. L. KENT and C. E. SMITH(1986): diet containing Fumadil B. J. Ingertebr. Pathol., Proliferative kidney disease in salmonid fishes. Fish 17, 234-236. Dis. Leaflet, 74, Department of Interior, U.S. Fish MOLNAR, K., F. BASKA,and C. SZEKELY(1987): Fu and Wildlife Service, Washington, D.C., 1-9. magillin, an efficacious drug against renal sphaero HEDRICK, R. P., J. M. GROFF, P. FOLEY and T. MC sporosis of the common carp Cyprinus carpio. Dis. DOWELL (1988a): Oral administration of fuma Aquat. Org., 2, 187-190.