Department of Natural Resources and Environmental Sciences, University of Illinois, Champaign, Illinois 61820, USA WILLIAM J. LOVE

Transcription

1 Joural of Aquatic Aimal Health 17: , 2005 Copyright by the America Fisheries Society 2005 DOI: /H [Article] Effects of Practices Related to Catch-ad-Release Aglig o Mortality ad Viral Trasmissio i Juveile Largemouth Bass Ifected with Largemouth Bass Virus EMILY C. GRANT AND KATE R. INENDINO Departmet of Natural Resources ad Evirometal Scieces, Uiversity of Illiois, Champaig, Illiois 61820, USA WILLIAM J. LOVE Departmet of Veteriary Pathobiology, College of Veteriary Medicie, Uiversity of Illiois, Urbaa, Illiois 61802, USA DAVID P. PHILIPP Ceter for Aquatic Ecology, Illiois Natural History Survey, ad Program i Ecology ad Evolutioary Biology, Uiversity of Illiois, Champaig, Illiois 61820, USA TONY L. GOLDBERG* Departmet of Veteriary Pathobiology, College of Veteriary Medicie, ad Program i Ecology ad Evolutioary Biology, Uiversity of Illiois, Urbaa, Illiois 61802, USA Abstract. Largemouth bass virus (LMBV; family Iridoviridae) has recetly emerged as a causative aget i fish kills of largemouth bass Micropterus salmoides. Little is kow about how the virus is trasmitted or what factors predispose fish to mortality subsequet to ifectio. Cocer has evertheless arise that activities related to recreatioal aglig may affect trasmissio dyamics ad may alter the susceptibility of ifected fish to cliical disease. This study examied the separate effects of two aglig-related factors o the susceptibility of juveile largemouth bass to mortality from LMBV ifectio ad o the trasmissio of LMBV from ifected to uifected fish. The first factor was hook-ad-lie aglig. Ifected fish that uderwet a simulated aglig treatmet did ot experiece higher mortality or have higher viral loads i their tissues tha those that were ot agled. The secod factor was direct cotact betwee ifected ad uifected fish, as would occur i live wells ad holdig taks. The LMBV was trasmitted from ifected to uifected fish through water, eve whe direct cotact was preveted. Trasmissio of LMBV betwee ifected ad uifected fish separated by a feestrated barrier was early as efficiet as LMBV trasmissio betwee ifected ad uifected fish that were allowed direct cotact. These results imply that aglig itself may have oly miimal effects o the survival of largemouth bass ifected with LMBV but that aglig-related practices that place ifected ad uifected fish together i a limited water volume may facilitate viral trasmissio. Partitioig or coolig of live wells ad holdig taks, as well as limitig their use, could reduce LMBV-associated mortality ad viral trasmissio. Largemouth bass virus (LMBV; family Iridoviridae; Plumb et al. 1996) is the oly virus kow to cause epidemic mortality i wild largemouth bass Micropterus salmoides (Grizzle ad Bruer 2003). The LMBV has bee associated with largescale fish kills i largemouth bass, particularly durig warm seasos (Plumb et al. 1996; Haso et al. 2001). While certai populatios of ifected * Correspodig author: tlgoldbe@uiuc.edu. Received September 8, 2004; accepted Jauary 2, 2005 Published olie August 24, 2005 largemouth bass have suffered fish kills, other ifected populatios suffer o discerible cliical sigs, suggestig that key evirometal factors may play a role i the pathogeesis of LMBV. Such factors may also play a role i the trasmissio of LMBV betwee fish ad betwee bodies of water (Goldberg 2002). Cocer has arise amog fisheries biologists ad aglers alike that hook-ad-lie aglig ad related activities may ehace LMBV-associated mortality or trasmissio (Goldberg 2002). Such effects could operate by reducig the immue ca- 315

2 316 GRANT ET AL. pacity of fish or by icreasig cotact rates betwee ifected ad uifected idividuals. For example, catch-ad-release aglig has bee associated with physiological chages that affect homeostasis (Gustaveso et al. 1991; Suski et al. 2003, 2004; Cooke et al. 2004), ad these chages could result i immuosuppressio (Aderso et al. 1982; Aderso 1990). Regardless of the mechaisms ivolved, it would be importat to kow whether ad how aglig ad related activities alter the susceptibility of LMBV-ifected fish to cliical disease ad how such activities might affect viral trasmissio. Such iformatio would be critical for makig recommedatios aimed at limitig the impact of LMBV ad similar pathoges. The goal of this study was to examie the effects of two factors, both associated with hook-ad-lie aglig, o the survival of largemouth bass ifected with LMBV ad o the trasmissio of the virus from ifected to uifected idividuals. Both factors have bee widely implicated i LMBVrelated mortality ad i the trasmissio of the virus. The first factor examied was aglig itself. We ioculated juveile largemouth bass with LMBV ad subjected them to a aglig simulatio coducted at two differet temperatures. We the measured mortality rates ad viral loads i fish. The secod factor was direct cotact betwee ifected ad uifected idividuals as would occur i live wells or other settigs where fish are housed together at high desity. We housed uifected juveile fish i aquaria with ifected fish but separated them by a feestrated barrier that preveted direct cotact. We measured the efficiecy of trasmissio from ifected to uifected fish by quatifyig mortality rates ad viral loads that were the compared to those of uifected fish that were allowed direct cotact with ifected fish. We hypothesized that aglig would icrease mortality ad viral loads i ifected fish ad that trasmissio would be more efficiet betwee fish that were allowed direct cotact tha betwee fish that were kept physically separate. Methods Study orgaisms. The largemouth bass used for experimetatio were harvested from experimetal pods owed ad operated by the Illiois Natural History Survey s Ceter for Aquatic Ecology. This populatio of largemouth bass has bee maitaied i a isolated experimetal pod complex for over 7 years ad has ot bee exposed to LMBV. A sample of the populatio (N 15 adults) was iitially tested for LMBV by use of virus isolatio ad was foud to be LMBV-egative. The fish used for experimetatio were 4 9 moths of age; the mea SE legth of experimetal fish was mm, ad the mea weight was g. Although these fish were smaller ad youger tha fish typically agled i the wild, they were ameable to experimetal maipulatio at sufficiet umbers for meaigful statistical aalysis. Experimetal setup. After their removal from rearig pods, fish were housed i oe of twelve 75.6-L aquaria ( cm) i a cotrolled eviromet chamber. Each aquarium was equipped with a simple air-drive spoge filter ad was devoid of substrate or plat life. Aquaria were filled with tap water (ph 8.0) that was allowed to age for 3 d prior to itroductio of fish. A thermometer was placed i each aquarium, ad idividual glass tops covered all aquaria. Coditios iside the chamber were strictly cotrolled, icludig temperature (ambiet at 25 C), costat humidity (60%), ad costat photoperiod (12 h light: 12 h dark, provided by overhead fluorescet bulbs). Lights were o from 0700 to 1900 hours each day. Virus. The viral isolate used for each experimet was the Satee Cooper Reservoir isolate recovered from South Carolia i 1995 (Plumb et al. 1996). The virus used for ioculatio was a sixth cell culture passage isolate. The isolate was derived from a fifth passage virus ioculated at a multiplicity of ifectio of 1.0 oto a cofluet moolayer of fathead miow Pimephales promelas cells ad was grow, harvested, ad titered accordig to previously published methods (Goldberg et al. 2003). Exposure to LMBV. Each experimetal fish was ijected itraperitoeally with LMBV at the vetral midlie betwee the aal opeig ad the pelvic fis. Ijectios were made with a sterile, 1- cm 3 tuberculi syrige ad 1/2-i (1.27-cm) eedle cotaiig cultured LMBV diluted with Hak s balaced salt solutio (HBSS) (ijectio 0.1 ml/fish). The ifectious dose was tissue culture ifectious doses with 50% cytopathic edpoit of LMBV. Cotrol fish were ijected itraperitoeally with 0.1 ml of virus-free cell culture superatat. Simulated aglig experimet. Twelve fish per tak were acclimated to 25 C for 7 d i 10 aquaria iside the cotrolled eviromet chamber. Submersible 500-W heaters were used to heat five of the aquaria to 30 C. The fish were the give seve more days to acclimate to the elevated test tem-

3 ANGLING AND LARGEMOUTH BASS VIRUS 317 peratures before exposure to LMBV. Fish were fed bloodworms (approximately 0.8 g/fish) at 0900 hours every morig except o the day of exposure to LMBV. Fish were moitored for morbidity ad mortality for 14 d subsequet to exposure. Fish at each temperature were experimetally agled 2 d after LMBV ijectio. The aglig simulatio cosisted of ettig out all the fish i a tak ad trasferrig them to a temporary holdig tak. Fifty percet of fish from each tak were hooked through the lower jaw o a sigle hook attached to a commercial moofilamet lie ad pulled i the opposite directio of atural motio. The lie was held cosistetly taut for 1 mi. Fish were the held out of the water ad suspeded from the mouth by the hook for 45 s before the hook was removed. After hook removal, each fish was give a partial fi clip for treatmet idetificatio ad was retured to its origial tak. The remaiig 50% of fish i each experimetal tak were ot agled. Twelve fish i each of two taks (oe 25 C tak ad oe 30 C tak) were subjected to the idetical aglig simulatio but were sham-ijected with virus-free cell culture superatat. These fish served as cotrols. This simulatio was desiged to be represetative of a catch-ad-release aglig evet i which the fish is reeled ito a boat after a sufficiet fightig sequece ad held out of the water prior to release. Applicatio times were based o physiological exhaustio ad recovery studies of agled largemouth bass (Cooke et al. 2004). The applicatio times of 1 mi of aglig ad 45 s of air exposure also caused obvious disequilibrium ad were adopted based o prelimiary trials idicatig that loger applicatio times caused immediate death. Trasmissio experimet. Eight aquaria, all maitaied at 25 C, were used i the trasmissio experimet. I four aquaria, a double feestrated barrier (two perforated Plexiglas sheets with a 1- i separatio betwee them) was placed i the ceter of the tak to divide the tak i half. The other four aquaria had idetical Plexiglas sheets placed 0.5 i from each ed of the tak, creatig a udivided water space equivalet to that i the divided taks. Te largemouth bass were placed i each of the eight taks. I three of the divided taks, five ifected fish were placed o oe side of the barrier ad five uifected fish were placed o the other side of the barrier. I three of the udivided taks, five ifected ad five uifected fish were placed together i the cetral space, thereby allowig direct cotact betwee ifected ad uifected idividuals. The remaiig two taks (oe divided ad oe udivided) housed cotrol fish; half of the fish i each tak were ioculated with virus-free cell culture superatat, while the remaiig fish were ot ioculated. Moitorig ad processig of fish. Fish were moitored for 14 d postijectio. Oce each day before feedig, all dead or moribud fish were removed from each aquarium ad immediately processed i the laboratory. Moribud fish were euthaatized by submersio i a solutio of clove oil ad ethaol (1:9; 0.25 L) added to 1.5 L of water (Taylor ad Roberts 1999). The date of death, state at death (dead or moribud), temperature or aglig treatmet ad tak, total legth (mm), total weight (g), ad the presece of gross exteral or iteral lesios were recorded for each fish durig processig. Body coditio was calculated as Fulto s idex (10 5 weight/legth 3 ), a commo metric for comparig body coditio of fish withi a populatio (Bolger ad Coolly 1989). Processig cosisted of removig visceral tissues (liver, splee, kideys, stomach, itesties, ad swim bladder) ad dilutig these tissues (1 g: 50 ml) i HBSS cotaiig atibiotics (100 uits of peicilli/ml, 100 g of streptomyci/ml, 0.25 g of amphoterici B/mL, ad 50 g of getamyci sulfate/ml). The tissue solutio was the homogeized with a Stomacher 80 Biomaster automatic stomacher (Steward, Ltd.) for 1 mi at ormal speed, ad the homogeate was froze at 80 C for future aalysis. Quatificatio of viral load. The DNA was extracted from fish tissue homogeates usig a QIAGEN QIAamp DNA blood miikit (250) ad a QIAvac 6S vacuum maifold (QIAGEN, Ic.) accordig to the protocol provided by the maufacturer. Real-time quatitative polymerase chai reactio (RT-qPCR; Higuchi et al. 1992, 1993) was used to measure the cocetratio of viral geomes i each sample of tissue homogeate (viral geomes per gram of fish tissue). The RT-qPCR was performed accordig to previously published protocols (Goldberg et al. 2003). Statistical aalyses. Statistical aalyses were performed by use of versio 8.2 of the Statistical Aalysis System (SAS Istitute, Ic., Cary, North Carolia). Survival data were aalyzed with Cox proportioal hazard regressio. Studet s t-tests, aalyses of variace, ad multiple liear regressio aalyses were performed o viral load data. Statistical models icluded cotrasts betwee ijected ad uijected fish exposed to differet aglig treatmets, those with differet Fulto s i-

4 318 GRANT ET AL. FIGURE 1. Cumulative survival of juveile largemouth bass that were ijected with largemouth bass virus, either agled or ot agled, ad held at either 25 C or 30 C. No mortality was observed i cotrol (shamijected) fish from ay treatmet; therefore, cotrol data from all treatmets were pooled. dex values, ad those that differed i status at the time of samplig (i.e., dead, moribud, or survived to the ed of the experimet). Post hoc Tukey s ad least-sigificat-differece tests were performed to idetify sigificat differeces betwee specific treatmets i aalyses of variace. Associatios were cosidered sigificat at a level of Results Effects of Simulated Aglig Media survival times for both agled ad oagled fish were 4 d postioculatio for fish held at 30 C ad 6 d postioculatio for fish held at 25 C. Largemouth bass that were held at 25 C ad that uderwet aglig simulatio experieced 75.0% cumulative mortality at 14 d, while oagled fish held at 25 C experieced 79.2% cumulative mortality at 14 d (Figure 1). Agled fish held at 30 C experieced a cumulative mortality of 91.7% at 14 d, ad oagled fish that were held at 30 C also experieced 91.7% cumulative mortality at 14 d (Figure 1). At both 25 C ad 30 C, LMBV-ijected fish died sigificatly more quickly tha did sham-ijected cotrols (25 C: , P ; 30 C: , P ). Aglig did ot have a sigificat effect o survival at either temperature (25 C: , P 0.57; 30 C: , P 0.23; Figure 1). Temperature, however, did have a sigificat effect o survival. The LMBV-ijected fish held at 30 C died more quickly tha did LMBV-ijected fish held at 25 C ( , P ; hazard ratio 4.41). No sham-ijected fish died durig the observatio period. Fish that died developed cliical sigs of viral ifectio cosistet with those described by Plumb ad Zilberg (1999), such as spiral swimmig, abdomial distetio, ad lethargy. Exteral gross lesios icluded visibly disteded abdomes, iflammatio at the ijectio site, ad localized or geeralized hyperemia. Exteral lesios were preset i 33% of fish that died durig the observatio period but 0% of fish that survived to the ed of the experimet ( , P ). Iteral gross lesios icluded a thick, white exudate i the coelomic cavity, a whiteig ad thickeig of the swim bladder wall (suggestive of peumocystitis), ad color chages to various visceral orgas, especially the liver. Iteral lesios were preset i 55% of fish that died durig the observatio period but oly 2% of fish that survived to the ed of the experimet ( , P ). Neither exteral or iteral lesios were observed i ay sham-ijected cotrol fish. Fulto s idex for both experimetal ad cotrol fish was 1.2. Similarly, fish that were foud dead or moribud ad fish that survived to the ed of the trial also exhibited Fulto s idices equal to 1.2. Values of Fulto s idex were statistically idistiguishable across treatmets ad outcomes. There was o sigificat differece i viral load betwee dead ad moribud fish (t 0.12, P 0.90; Table 1). However, whe pooled, dead ad moribud fish had sigificatly higher viral loads tha did fish that survived to the ed of the experimet (t 11.98, P ). Fulto s idex (t 0.23, P 0.82) ad the presece of iteral gross lesios ( , P 0.44) or exteral gross lesios ( , P 0.72) were ot sigificatly associated with viral load i fish that died or were euthaatized durig the observatio period. s of experimetal fish held at 25 C did ot differ sigificatly from viral loads of experimetal fish held at 30 C (t 1.20, P 0.23). s of agled fish held at 25 C or30 Cdid ot differ sigificatly from viral loads of oagled fish held at 25 C or30 C (t 0.30, P 0.76). There was o sigificat first-order iteractio betwee temperature ad aglig (t 0.72, P 0.48). Mea viral loads were ot statistically differet betwee fish held at 30 C ad those held at 25 C (Table 1). The RT-qPCR was

5 ANGLING AND LARGEMOUTH BASS VIRUS 319 TABLE 1. Mea SE viral loads of juveile largemouth bass exposed to equal doses of largemouth bass virus at 25 C ad 30 C ad subjected to experimetal hook-ad-lie aglig. s are expressed as log 10 trasformed viral geomes per gram of tissue. Similar letters sigify o statistically sigificat differece at the 0.05 level (letters i the fial colum refer oly to comparisos amog values withi that colum). Treatmet Foud dead or moribud State of fish Survived to ed of experimet All fish combied Agled at 25 C z x z Not agled at 25 C z x z Agled at 30 C z x z Not agled at 30 C z y z also ru o the cotrol fish from each treatmet. s were foud to be below miimum detectio limits ad were statistically idistiguishable from egative cotrol RT-qPCRs. Effects of Direct Cotact o Trasmissio The media survival time of all fish ijected with LMBV was 6 d (Figure 2). Oly oe cotrol fish ad oe uijected experimetal fish died durig the course of the experimet (Figure 2). The differece i mortality rates betwee ijected ad uijected fish (both experimetal ad cotrols) was statistically sigificat ( , P ). Mortality rates did ot, however, differ sigificatly betwee uijected experimetal fish ad sham-ifected cotrols. Mortality rates did ot FIGURE 2. Cumulative survival of juveile largemouth bass that were ijected or ot ijected with largemouth bass virus ad held either i taks divided by a feestrated barrier (prevetig direct cotact betwee ifected ad uifected fish) or i udivided taks. Oly oe cotrol fish (sham-ijected) died; therefore, cotrol data from all treatmets were pooled. differ sigificatly betwee LMBV-ijected fish i divided taks ad those i udivided taks. Fish that died durig the observatio period displayed cliical sigs ad gross lesios cosistet with LMBV ifectio, as described above. Exteral lesios were preset i 48% of fish that died durig the observatio period but 0% of fish that survived to the ed of the experimet ( , P ). Iteral lesios were preset i 28% of fish that died durig the observatio period but oly 3% of fish that survived to the ed of the experimet ( , P 0.03). Neither exteral or iteral lesios were observed i ay shamijected cotrol fish. The mea SE Fulto s idex of LMBVijected fish ( ) was sigificatly lower tha that of the uijected fish housed with them ( ; t 3.393, P 0.002). However, the mea Fulto s idex of LMBV-ijected fish did ot differ sigificatly from that of sham-ijected cotrol fish ( ; t 0.142, P 0.889). Mea SE viral loads differed sigificatly betwee fish ijected with LMBV (divided taks: log 10 viral geomes [LVGs] per gram of tissue; udivided taks: LVGs/g) ad the uijected fish housed with them (divided taks: LVGs/g; udivided taks: LVGs/g; t , P 0.001; Table 2). Amog fish that survived to the ed of the experimet, uijected fish i divided taks had margially sigificatly lower viral loads ( LVGs/g) tha did uijected fish i udivided taks, where direct cotact with ifected idividuals was possible ( LVGs/g; t 2.211, P 0.036; Table 2). Although statistically sigificat, the absolute value of this differece (0.22 LVGs/g) was small. s of cotrol fish were below miimum detectio limits ad were statistically idistiguishable from egative cotrol RT-qPCRs.

6 320 GRANT ET AL. TABLE 2. Mea SE viral loads of juveile largemouth bass either uijected or ijected with largemouth bass virus ad held i equal umbers i divided taks (prevetig cotact betwee ijected ad uijected fish) or udivided taks. See Table 1 for additioal details. Treatmet Foud dead or moribud State of fish Survived to ed of experimet All fish combied Divided tak, ijected z z z Divided tak, ot ijected y x y Udivided tak, ijected z z Udivided tak, ot ijected y y Discussio Effects of Simulated Aglig The LMBV-ijected largemouth bass that were experimetally agled experieced o higher mortality tha did LMBV-ijected fish that were ot agled. This result was cosistet both at 25 C ad 30 C. Largemouth bass ijected with LMBV ad held at 30 C, whether agled or ot, experieced higher mortality tha did bass held at 25 C (Figure 1), cofirmig that temperature elevatios approachig 30 C icrease the susceptibility of fish to the virus uder cotrolled experimetal coditios (Grat et al. 2003). Give that the survival of agled ad oagled fish did ot differ sigificatly, it is ot surprisig that the viral loads of agled fish did ot differ from those of oagled fish. The viral loads of fish that died durig the observatio period did ot differ sigificatly betwee fish held at 25 C ad those held at 30 C, eve though survival rates at these two temperatures were sigificatly differet. This was surprisig cosiderig previous results that did, i fact, demostrate such a tred (Grat et al. 2003). Experimetal exposure of fish to LMBV is kow to cause icosistet cliical outcomes (Grizzle ad Bruer 2003). We caot explai these icosistecies, but they could result from differeces amog fish ad fish populatios i levels of iate immuity, which i tur may be iflueced by temporally variable evirometal factors (Goldberg 2002). Withi each treatmet, fish that survived to the ed of the experimet had lower viral loads tha those foud dead or moribud. This observatio suggests that viral replicatio may have bee ihibited i some fish. It is ot kow why certai fish withi treatmets were able to withstad ifectio while others were ot. I both experimets, fish that died durig the observatio period were sigificatly more likely to have iteral ad exteral gross lesios tha fish that survived to the ed of the experimet. However, the presece of gross lesios was ot correlated with viral loads i fish that died or were foud moribud durig the observatio period. These results suggest that gross pathology may idicate LMBV ifectio but is a poor idicator of ifectio itesity. Cocer about the potetial effects of catchad-release aglig o the survival of LMBVifected fish has bee based o the assumptio that aglig is a stressful evet that ca icrease the sesitivity of fish to ifectio through immuosuppressio or similar effects. Although we did ot measure stress directly i this study, evidece idicates that catch-ad-release aglig does ideed icrease stress-related physiological characteristics i largemouth bass (Suski et al. 2003, 2004; Cooke et al. 2004). The fact that aglig did ot affect the subsequet survival of LMBVifected fish i our study suggests that ot all stressful evets are sufficiet to icrease mortality i fish ifected with a potetially lethal pathoge. We used oly a sigle, relatively short (approximately 2 mi) aglig protocol, however; perhaps repeated aglig episodes or more protracted stressors would have produced the predicted tred. The pricipal limitatio to the geeralizatio of our results is the size ad age of the fish used i the experimet. Juveile largemouth bass may respod differetly to aglig tha would larger fish, which are also more frequetly agled. Similarly, the effects of aglig may differ betwee the cotrolled experimetal settig of this study ad the complex ecosystems i which largemouth bass are typically agled. For example, icreased postrelease recovery times i LMBV-ifected fish would be of little cosequece for mortality i a eviromet devoid of predators. Clearly, future studies of the iteractios betwee aglig ad LMBV i larger fish ad i aturalistic settigs are warrated.

7 ANGLING AND LARGEMOUTH BASS VIRUS 321 Effects of Direct Cotact o Trasmissio The LMBV was trasmitted early as efficietly through water aloe as betwee ifected ad uifected fish that were allowed direct cotact with each other. Uifected fish i direct cotact with ifected fish had higher viral loads at the ed of the experimet tha did uifected fish separated from ifected fish by a feestrated barrier. Although this differece was statistically sigificat, it was evertheless small. Its biological sigificace is therefore ucertai, especially sice mortality rates of uifected fish i direct cotact with ifected fish were very low ad did ot differ sigificatly from mortality rates of uifected fish that were preveted from direct cotact with ifected fish. Values of Fulto s idex were lower i fish ijected with either LMBV or virus-free cell culture superatat tha i fish that were uijected. It would appear that the itraperitoeal ijectio of fluid, whether it cotaied LMBV or ot, was resposible for this reductio i Fulto s idex. The kow modes of trasmissio of LMBV iclude through water (Plumb ad Zilberg 1999) ad by igestio of experimetally ifected prey (Woodlad et al. 2002). The presece of LMBV i the mucus of ifected fish (Woodlad et al. 2002) provides a mechaism whereby direct cotact might be expected to facilitate trasmissio. Our study cofirms this hypothesis ad implies that keepig ifected fish physically separate from uifected fish could reduce trasmissio of the virus. Live wells ad holdig taks, which are commo features i recreatioal aglig settigs, may ehace viral trasmissio. Compartmetalizatio of such cotaiers or decreasig their usage could reduce trasmissio rates. We also suggest that reducig temperatures i live wells ad holdig taks might have beeficial effects. Our curret data demostratig a strog effect of temperature o the survival of LMBV-ifected fish ad our previous data demostratig that icreased temperatures may facilitate viral replicatio (Grat et al. 2003) support this maagemet recommedatio. We documeted a small effect of direct cotact o viral trasmissio that was evidet i the aalysis of viral loads but ot i the mortality rates of fish to which the virus was trasmitted. As i the experimetal aglig trial, these effects could differ i magitude for larger fish i actual live wells ad holdig taks. For example, the ratio of fish volume to water volume i a typical live well would be cosiderably higher tha that simulated i this study, such that the magitude of the effect of direct cotact o viral trasmissio might be correspodigly higher. Agai, experimets o larger fish i aturalistic settigs are warrated. Coclusios The results of our experimetal aglig study suggest that a sigle aglig evet may ot be sufficiet to icrease mortality rates i LMBVifected largemouth bass. However, the results of our trasmissio study suggest that certai aglig-related practices that place ifected ad uifected fish together i close proximity ca facilitate trasmissio of the virus. Alterig such practices (e.g., partitioig or coolig live wells ad holdig taks or discouragig their use) might decrease trasmissio of the virus ad thereby reduce its prevalece i ifected populatios. Verificatio of our fidigs will have to await future studies of aglig ad LMBV coducted o larger fish i aturalistic settigs. Ackowledgmets We thak Joh Grizzle ad Ady Noyes for providig the origial stock LMBV. Joh Epifaio, Patrick Weatherhead, ad Steve Cooke provided valuable discussios ad advice. Several of the cetral questios addressed i this study arose from LMBV workshops sposored by the Bass Aglers Sportsma Society. This research was supported by grats from the Illiois Coucil o Food ad Agriculture Research ad by the Coservatio Medicie Ceter of Chicago. Refereces Aderso, D. P Immuological idicators: effects of evirometal stress o immue protectio ad disease outbreaks. Pages i S. M. Adams, editor. Biological idicators of stress i fish. America Fisheries Society, Symposium 8, Bethesda, Marylad. Aderso, D. P., B. S. Robertso, ad O. W. Dixo Immuosuppressio iduced by a corticosteroid or a alkylatig aget i raibow trout (Salmo gairderi) admiistered a Yersiia ruckeri bacteri. Developmetal ad Comparative Immuology 2: Bolger, T., ad P. L. Coolly The selectio of suitable idices for the measuremet ad aalysis of fish coditio. Joural of Fish Biology 34: Cooke, S. J., C. M. But, K. G. Ostrad, D. P. Philipp, ad D. H. Wahl Aglig-iduced cardiac disturbace of free-swimmig largemouth bass (Micropterus salmoides) moitored with heart rate telemetry. Joural of Applied Ichthyology 20:28 36.

8 322 GRANT ET AL. Goldberg, T. L Largemouth bass virus: a emergig problem for warmwater fisheries? Pages i D. P. Philipp ad M. S. Ridgway, editors. Black bass: ecology, coservatio, ad maagemet. America Fisheries Society, Symposium 31, Bethesda, Marylad. Goldberg, T. L., D. A. Colema, E. C. Grat, ad K. R. Iedio Strai variatio i a emergig iridovirus of warmwater fishes. Joural of Virology 77: Grat, E. C., D. P. Philipp, K. R. Iedio, ad T. L. Goldberg Effects of temperature o the susceptibility of largemouth bass to largemouth bass virus. Joural of Aquatic Aimal Health 15: Grizzle, J. M., ad C. J. Bruer Review of largemouth bass virus. Fisheries 28(11): Gustaveso, A. W., R. S. Wydoski, ad G. A. Wedemeyer Physiological respose of largemouth bass to aglig stress. Trasactios of the America Fisheries Society 120: Haso, L. A., L. Petrie-Haso, K. O. Meals, V. G. Chichar, ad M. Rudis Persistece of largemouth bass virus ifectio i a orther Mississippi reservoir after a die-off. Joural of Aquatic Aimal Health 13: Higuchi, R., G. Dolliger, P. S. Walsh, ad R. Griffith Simultaeous amplificatio ad detectio of specific DNA sequeces. Biotechology 10: Higuchi, R., C. Fockler, G. Dolliger, ad R. Watso Kietic PCR aalysis: real-time moitorig of DNA amplificatio reactios. Biotechology 11: Plumb, J. A., J. M. Grizzle, H. E. Youg, A. D. Noyes, ad S. Lamprecht A iridovirus isolated from wild largemouth bass. Joural of Aquatic Aimal Health 8: Plumb, J. A., ad D. Zilberg The lethal dose of largemouth bass virus i juveile largemouth bass ad the comparative susceptibility of striped bass. Joural of Aquatic Aimal Health 11: Suski, C. D., S. S. Kille, M. B. Morrissey, S. G. Lud, ad B. L. Tufts Physiological chages i the largemouth bass caused by live-release aglig touramets i southeaster Otario. North America Joural of Fisheries Maagemet 23: Suski, C. D., S. S. Kille, S. J. Cooke, J. D. Kieffer, D. P. Philipp, ad B. L. Tufts Physiological sigificace of the weigh-i durig live-release aglig touramets for largemouth bass. Trasactios of the America Fisheries Society 133: Taylor, P. W., ad S. D. Roberts Clove oil: a alterative aaesthetic for aquaculture. North America Joural of Aquaculture 61: Woodlad, J. E., C. J. Bruer, A. D. Noyes, ad J. M. Grizzle Experimetal oral trasmissio of largemouth bass virus. Joural of Fish Diseases 25: