Articles

Quantitative Polymerase Chain Reaction for Transforming Growth Factor-nApplied to a Field Study of Fish Health in Chesapeake Bay TributariesCraig A. Harms,1 Christopher A. Ottinger,2 Vicki S. Blazer,2 Christine L. Densmore,2 Laurence H. Pieper, andSuzanne Kennedy-Stoskopf4'Environmental Medicine Consortium and Department of Clinical Sciences, College of Veterinary Medicine, North Carolina StateUniversity, Raleigh, North Carolina, USA; 2U.S. Geological Survey, Biological Resources Division, National Fish Health ResearchLaboratory, Kearneysville, West Virginia, USA; 3Maryland Department of Natural Resources, Stevensville, Maryland, USA;4Environmental Medicine Consortium and Department of Microbiology, Pathology and Parasitology, North Carolina State University,Raleigh, North Carolina, USA

Fish morbidity and mortality events in Chesapeake Bay tributaries have aroused concem over thehealth of this imprtant aquatic ecostem. We applied a recently described method for quanti-i mRNA of a immunosuppressie cyoine, trforming growhactor (TGF-j), by reversetrascnption quantitaie-competitive polymerase chain reacton,to a field sudy. of fish health inthe;C heseeBask,aand compa ithe rels to t e of a triioacelluar munoassayma phage b l aciv4 We slectd iewite perch (M. ose ena) as the sentinelfish speces becuse of its :abundance paal f the col rh were apled fromChesapeake Bay tributaries in June, Augut and October 1998.Spleni mononuclear cell TGF-[mRNA levels irsa ant d macrophage bactericidal ciitydeeased, pricula-ly in-easten shore 1O3tribut.ar June to August a Octoer. Te resut of the two aayscorrelatd inv ly (Kendall's x b _ -0.600; p = Q.0102). The resiuls indicated b temporal dspatial modulation of white prch immune sstems in the Chesapee Bsin, nd demonstrateldthe utility of quantitative:PCR for TGF- a amol biomarker for field asse t of celeosfish imm;unestatus. Ke word Chesapee Bay, fied study, acrophae btericid ityMoronc am'erica' quantitative PCR, transforming growth factor-[3, white perch. Environ HeatbPersect 108:447452(2000). [Online 28 March 2000]brtnp:/e/pnetl niehs.nih.go /docs/2000/108p447-452barm/abstrrahtml

Marine and freshwater models for environ-mental health research are receiving increas-ing attention (1), and fish immunology isone focus of this attention (2). The assess-ment of fish immune status in response toenvirontmental conditions has by necessitybeen dominated by assays not requiringspecies-specific or taxon-specific reagents.Examples include lymphoid organ weightand histology, hematology, serum proteinand protein fraction quantification, diseaseincidence or response to pathogen challenge,lymphocyte blastogenesis, and macrophageand granulocyte functional assays measuringchemotaxis, phagocytosis, respiratory burst,and bactericidal activity (2-5). Recent stud-ies in fish have focused on the detection ofcytokines, proteins that regulate cellular andimmune function. Evidence for the presenceof specific cytokines in fish was indirect (6,7)until the cloning and sequencing of trans-forming growth factor (TGF)-[ (8,9).

TGF-[s are cytokines with diverse func-tions that affect cell growth and differentia-tion, extracellular matrix regulation, woundhealing, and immune function (10-14).TGF-[ immunoregulatory properties areprimarily suppressive. Immune functionsdown-regulated by TGF-[B include the fol-lowing: major histocompatibility complexand Fc receptor expression, some cytokineproduction, thymocyte proliferation, T- and

B-cell proliferation, IgG and IgM production,IL-2 receptor expression, cytotoxic T-cell gen-eration and function, lymphokine-activatedkiller and natural killer cell activation andfunction, macrophage activation, macrophagerespiratory burst activity, neutrophil adhesionto endothelium, and hematopoiesis (13).Three isoforms of TGF-[ (-I3P -[32, and -[3)are expressed by mammals. In addition, one(-[34) has been identified from the chicken,and one (-[35) from the African clawed frog,Xenopus laevis. TGF-[4 and -[35 are consid-ered homologues of mammalian TGF-13I(15). TGF-jI is the major TGF-[ isoformwithin the context of the mammalianimmune system (10) .

Coding sequences for a TGF-[ fromhybrid striped bass [Morone saxatilis x Mchrysops, Genbank (16) accession no.AF140363 (9)] and rainbow trout [Oncor-hynchus mykiss, Genbank (16) accession no.AJ007836 (8,17)] grouping with XenopusTGF-15, chicken TGF-[4, and mammalianTGF-P1, were recently reported. Homologywith TGF-P1 suggests that the fish TGF-[isolates may also be important in regulatingimmune responses in these species. Evidencefor the conserved nature of TGF-[B activityin fish is provided by both biologic cross-reactivity and antigenic cross-reactivity.Bovine TGF-j31 enhanced respiratory burstactivity of resting rainbow trout macrophages

at low doses. At higher doses, however,bovine TGF-j1 inhibited activated rainbowtrout macrophages and countered the effectsof activating signals on resting macrophages(18). Chicken antiporcine TGF-f1 antibod-ies enhanced the ability of rainbow troutleukocyte-derived supernatants to stimulaterespiratory burst activity of anterior kidneymacrophages (19).

The ability to measure TGF-P mRNAproduction in fish could provide a means todetect dysfunctional responses to stressors inthe aquatic environment before the develop-ment of overt infectious disease, develop-mental abnormalities, or neoplasia. Harms etal. (20) used a quantitative polymerase chainreaction (PCR) technique to measure TGF-[3mRNA from lymphoid cells of teleost fish(9) in a controlled setting to demonstrate aninverse relationship between anterior kidneymacrophage bactericidal activity and splenicmononuclear cell TGF-,B mRNA levels inhybrid striped bass treated with a knownimmunomodulator. The application ofquantitative PCR for TGF-f to a field studyof fish health would help validate its use asan immunologic marker in more complexsettings. The opportunity for field applica-tion of the technique was presented byrecent fish mortality and ulcerative skinlesion events in Chesapeake Bay tributaries.

Atlantic menhaden (Brevoortia tyrannus)mortalities and ulcerative skin lesions with apredilection for the anal area occurred dur-ing August and September 1997 inMaryland tributaries of the Chesapeake Bay,including the Pocomoke River, Pocomoke

Address correspondence to C.A. Harms, Depart-ment of Clinical Sciences, North Carolina StateUniversity, 4700 Hillsborough Street, Raleigh, NC27606 USA. Telephone: (919) 515-8112. Fax:(919) 515-4237. E-mail: craigTharms@ncsu.eduWe thank T. Childers for technical assistance,

and C. Weedon and the Maryland Department ofNatural Resources for fish collection and site char-acterization.Funding sources include a U.S. EPA Science to

Achieve Results Fellowship (U-915209-01-0), aNCSU College of Veterinary Medicine StateResearch grant, and the USGS Place-Based StudiesProgram.Received 26 July 1999; accepted 7 December

1999.

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Sound, Chicamacomico River, and KingsCreek, a tributary of the Manokin River(21,22). Menhaden lesions were character-ized by necrotizing dermatitis and myositiswith severe chronic granulomatous inflam-mation containing aseptate fungal hyphae(23,24). Similar ulcerative mycosis lesionswere originally described in menhaden fromNorth Carolina in the early 1980s (25).More recently these lesions and mortalityevents of menhaden have been associatedwith blooms of the toxic dinoflagellate,Pfiesteria piscicida (26,27). Although acutedermonecrotic lesions of fish have beeninduced in laboratory studies of P. piscicida(28), the histologic appearance of chronicitysuggests caution in linking the skin lesions ofwild menhaden to the acute effects of thedinoflagellate toxin (23,24).

The morbidity and mortality exhibited byPocomoke-area menhaden, in addition to theincreased prevalence of lesions in other fishspecies, provided the impetus for a broad-based study of fish health in Chesapeake Baytributaries. The study was conducted by theU.S. Geological Survey's National FishHealth Research Laboratory (Kearneysville,WV) and the Maryland Department ofNatural Resources (Stevensville, MD).However, menhaden were not sufficientlyabundant at all of the sites scheduled formonitoring. Therefore, the white perch(Morone americana) was used as the sentinelfish species because of its abundance at all ofthe collection sites. In addition, methods forTGF-j reverse transcription quantitative-competitive PCR (RT-qcPCR) have been val-idated for white perch (9), and its position inthe middle to upper fraction of the water col-umn makes it less likely to be impacted bytoxins accumulated in the sediment.

Our objectives were to apply the newly-described method for measuring TGF-PmRNA production in teleost fish (9) to afield study of fish health, and to relate con-stitutive TGF-1 transcription levels to astandard cellular immune function test,macrophage bactericidal activity, undercomplex field conditions.

Materials and MethodsFish and collection sites. In the summer andfall of 1998, we collected adult white perchfrom the lower Pocomoke [37°58' N,75°38'W (all coordinates + 1'); collected on16 June, 13 August, and 5 October], theWicomico (38°15' N, 75°48' W; collectedon 17 June, 4 August, and 14 October), andthe Choptank (38039' N, 75°58' W;collected on 24 June, 3 August, and 7October) Rivers on the Eastern Shore of theChesapeake Bay. Perch were also collectedfrom the Back River (39°15' N, 76°27' W;collected on 10 June, 12 August, and 8

October) on the western shore of the bay.We timed the collections to span the periodsbefore and after dates when menhaden mor-talities were recorded from the Pocomokethe previous August.

The Pocomoke and Wicomico Riverspass through rural areas of intense poultryrearing and are subject to high levels ofnutrient run-off (21). There were menhadenmortalities in the Pocomoke in August1997. The Choptank River also drains arural area but is further removed from theareas of reported fish mortality. However,Pfiesteria sp. was identified from sedimentsof Jenkins Creek of the Choptank River in1992 (29). The Back River runs through anindustrialized area of Baltimore, Maryland,and was thought likely to contain fish withaltered immune function. We recorded airtemperature, water depth, surface and bot-tom water temperature, dissolved oxygen,pH, and salinity at each sample site.

We collected fish by 4.9-m semiballoonotter trawl in 10-min tows starting between0715 and 0918 hr. Twenty-four white perch> 170 mm total length from each site werecollected and transported 15-30 min in alive well to shore for processing. Nonquan-titative observations of skin lesions weremade before the release of menhaden cap-tured incidentally.

Fish processing. White perch were eutha-nized with an overdose of tricaine methane-sulfonate. We recorded the weight and totallength and noted the presence of externallesions. Fish were bled from the caudal veinfor related hematology studies, and to reduceperipheral blood mononuclear cell contentof anterior kidneys and spleens. We deter-mined packed cell volume (PCV) and plas-ma total solids by refractometry on site.Spleens and anterior kidneys were asepticallyharvested for TGF-P mRNA quantificationand cellular immunoassays. Sex was deter-mined by internal examination.

TGF-P RT-qcPCR (spleen). We placedspleens from 10 white perch in sterile trans-port medium [RPMI 1640 plus 10% heat-inactivated fetal bovine serum (FBS), 100U/mL penicillin, 100 pg/mL streptomycin,2 mM EDTA; subsequently referred to ascomplete RPMI] at 40C. The perch weretransported on wet ice by overnight courierto the laboratory, where they were processedfor TGF-~ mRNA quantification.

We prepared mononudear cells for RT-qcPCR as previously described (9). Spleenswere minced finely, forced through a finewire mesh, and resuspended in completeRPMI. Spleen homogenates were centrifugedon two-step Percoll gradients (specific gravity1.053 and 1.066 g/mL in 0.15 M saline) at400gfor 5 min, then 800gfor 20 min at 4°C.Mononuclear cells were harvested from the

1.053/1.066 g/mL interface. Cells werewashed twice in complete RPMI. Viable cellcounts were performed with cells suspendedin 0.2% trypan blue, and differential countswere performed on cytospin preparationsstained with LeukoStat (Fisher Scientific,Pittsburgh, PA). Cell viability typicallyexceeded 95%.

Total RNA was isolated by the guanidinethiocyanate method (Tri Reagent; MolecularResearch Center, Cincinnati, OH). The RNApellet was resuspended in sterile diethyl pyro-carbonate-treated water at a concentration of5 x 104 cell equivalents/lL. Messenger RNAwas reverse transcribed (Superscript II RT;Gibco-BRL, Gaithersburg, MD) to cDNAwith oligo dT1 5 priming of 3 x 106 cellequivalents of total RNA. We stored samplesof cDNA at -20°C until their use. NegativeRT controls were run in parallel.

We performed RT-qcPCR based on apreviously described procedure (30) that wasfurther developed using primers and compet-itive fragments specific for teleost fish TGF-1and ,B-actin (9t). Each competitive PCR con-tained 2.5 x 104 cell equivalents ofcDNA asprepared above. The final reaction volume ofthe competitive PCR was 25 pL, and wasreached by adding 20 pL of a PCR mastermix to 5 jIL of each competitive fragmentdilution (1.5 x 108 copies down to 6 copies)in a Thermowell 96-well thin-wall polycar-bonate plate (Costar, Acton, MA) on ice.The final reaction concentrations were. 1 xPCR buffer [10 mM Tris-HCl (pH 8.3), 1.5mM MgCl2, 50 mM KCI], 250 PsM eachdNTP, 0.375 PM each primer, 30 gU/jLTaq DNA polymerase. Reactions were cycledon a PTC-100 thermocycler (MJ Research,Inc., Watertown, MA) at 94°C for 1 min,(94°C, 30 sec denature; 55°C, 1.5 minanneal; 72°C, 2 min extend) x 35 cycles,with a final extension at 72°C for 7 min.

PCR products were separated in 2%agarose gels in TAE, stained with ethidiumbromide, and photographed on a 300-nmultraviolet transilluminator. The images weredigitized and analyzed with the Alphaimager2000 Documentation and Analysis System(Alpha Innotech Co., San Leandro, CA).We measured fluorescence of target andcompetitor bands in each lane; these wereexpressed as area under the curve. We com-pensated for fluorescence differences due tomolecular weight differences by the formula:

CFR = TF(area) x CS(bp)CF(area) ---rTS(bp)

where CFR = the corrected fluorescence ratio,TF = the target fluorescence, CS = competitorsize, CF = competitor fluorescence, and TS =target size. The log10 of the CFR was plottedagainst the log,0 of the number of copies of

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competitor in the sample, and the point ofmolecular equivalence (the point at whichthe copy number of target cDNA equals thecopy number of competitor DNA) is the x-intercept. Finally, to control for sample-to-sample variation in RNA isolation, reversetranscription, amplification, and gel loadingduring quantification, we normalized theTGF-P results to those of the housekeepinggene, [1-actin.

Macrophage bacteriidl assay (anteriorkidney). We isolated mononuclear cells fromthe anterior kidney using a modification ofthe procedures described by Sharp et al.(31). Cells were kept cold during processingexcept where noted. Aseptically harvestedanterior kidney tissues from individual whiteperch were homogenized in 10 ml of L-1 5medium supplemented with 2% FBS, 100U/mL penicillin, 100 jg/mL streptomycin,and 10 U/mL sodium heparin (L-15/2%FBS). Homogenates were produced at thesample sites and transported back to the lab-oratory on ice. Transportation time rangedfrom 3 to 5 hr.

Homogenized anterior kidney tissueswere pelleted by centrifugation at 40C for 10min at 500g. Tissues were washed by resus-pension in 5 mL of L-15/2% FBS followedby centrifugation. Washed cell suspensionswere resuspended in 6 mL of L-1 5/2% FBSand layered on a single step Percoll gradient[density 1.047 g/mL in Hank's balanced saltsolution (HBSS) without phenol red]. Cellsuspensions on Percoll were centrifuged at4°C for 20 min at 400g and then leukocyteswere removed from the media/Percoll inter-face. Leukocytes were pelleted at 500g,washed as described previously, and resus-pended in 3 ml L-1 5/2% for counting. Wedetermined the number of viable leukocytesisolated from each fish by trypan blue exclu-sion (0.1% trypan blue in L-15/2% FBS) andthe cells were pelleted at 500g as describedpreviously. Leukocytes were resuspended at 2x 107 viable cells/mL in L-1 5, supplementedwith 0.1% FBS, 100 U/mL penicillin, and100 pg/mL streptomycin (L-15/0.1% FBS).We loaded leukocytes in L-15/0.1% FBS into96-well tissue-culture plates at 100 pL/welland incubated them for 2 hr in a container ofhumidified air at 20°C. After incubation, weremoved the L-15/0.1% FBS containing non-adherent cells and replaced it with 100gL/well of L-15/5% FBS at 20°C. We evalu-ated the adherent cells for macrophage bacte-ricidal activity.

We determined bactericidal activity usinga modification of the assay described byGraham et al. (32). The temperature of allsolutions was adjusted to 200C before use.Forty-eight-hour cultures of Yersinia ruckeri(Hagerman strain; National Fish HealthResearch Laboratory no. 11.40) were washed

3 times in HBSS and the suspension densitywas adjusted to 0.15 OD600 in HBSS.

After 36 hr incubation in a humidifiedcontainer at 20C, the L-15/5% wasremoved from the wells of macrophageplates and replaced with 100 pL/well ofunsupplemented L-1 5. The L-1 5 was thenreplaced with an equal volume of L-15/5%without antibiotics and K ruckeri suspensionwas added at 25 1L/well. Plates were incu-bated for 4 hr at 200C in a humidified con-tainer. After incubation, we removed themedia from the wells and replaced it with 50pL/well of 0.2% Tween 20 in tissue-culturegrade water. The resulting well contentswere serially diluted (logl0) in tryptic soybroth. Dilutions were plated as 10 ,uL dropson tryptic soy agar plates and incubated for24 hr at room temperature. We determinedthe number of colony-forming units (CFUs)for each well at the 10-4 or 10-5 dilution.Bactericidal activity was expressed as % CFUreduction = (1 - CFU treated/CFU control)x 100, where CFU treated = the replicatemean number of CFUs in wells containingadherent leukocytes (n = 3) and CFU con-trol = the replicate mean number of CFUsfrom wells with media only (n = 6). Thenumber of CFU treated was replicated on aper-fish (cell source) basis. The number ofCFU control was replicated on a per-platebasis (i.e., all of the CFU treated mean val-ues from a given plate were compared to asingle CFU control mean value).

Statstical analyses. We used nonparamet-ric statistical tests for comparisons due tononnormal distribution of TGF-P transcrip-tion and macrophage bactericidal activity datafrom some sample sites (Shapiro-Wilk test;JMP statistical software, SAS Institute, Inc.,Cary, NC). Values for splenic mononuclearcell TGF-J transcription, anterior kidneymacrophage bactericidal activity, and fishlength and weight were grouped according tosample (month and locality) and analyzed bythe Kruskal-Wallis test against the nullhypothesis that sample medians did not differ(33). We performed Dunn's multiple com-parison test after significant Kruskal-Wallistests to detect which pairs of sample mediansdiffered (33). We used Kendall's t-b to testfor correlation of variables according to sam-plings (MP). We performed least-squares lin-ear regression UMP) using sample medians toillustrate the inverse association betweenTGF-P transcription and macrophage bacteri-cidal activity. Sex distribution was comparedbetween samplings by x2. Significance was setatp < 0.05 in all cases.

ResultsWhite perch length (overall mean ± SD - 197± 17.7 mm) and weight (112 ± 30.8 g) didnot differ significantly between sample sites

and months (Kruskal-Wallis, p > 0.05). Therewas a predominance of females in the samplepopulation (65.5%, range 37.5-90.1%females), but sex distribution was notsignificantly different between samplings(x2 p > 0.05).

Menhaden with deep ulcerative skinlesions were collected incidentally from thePocomoke and Wicomico Rivers in Augustand the Wicomico and Choptank Rivers inOctober, although no mass mortalities werereported in the summer of 1998. Externallesions of white perch were minor, mostlyconsistent with capture and transport, andincluded foci of erythematous skin and red-dened or frayed fins in 23% (range 0-60%) offish sampled for TGF-f determination (Table1). Mean PCVs ranged from 29.1 to 49.3%,and plasma total solids from 4.3-6.1 g/dL(Table 1). Physical measurement ranges for airtemperature, surface water temperature, sur-face dissolved oxygen, surface pH, and surfacesalinity were 18-31.80C, 18-28.1°C, 4.7-8.1mg/L, 7.1-8.8, and 0.0-9.9 gIL, respectively;bottom-water temperature, bottom dissolvedoxygen, bottom pH, bottom salinity, and bot-tom sample depths were 18.0-27.8°C,2.4-6.9 mg/L, 7.1-8.8, 0.0-9.9 g/L, and1.2-9.0 m, respectively (Table 2).

Splenic mononuclear cell TGF-f:3-actinratios varied significantly by collection siteand month (Kruskal-Wallis test, p < 0.0001;Figure 1). Although TGF-, transcriptionwas greatest initially from Back River fish inJune, over the course of the sampling periodthe levels remained relatively constant whilethey increased in fish from the Eastern Shoretributaries. The increase was greatest for fishfrom the Wicomico River.

Anterior kidney macrophage bactericidalactivity also varied significantly by collectionsite and month (Kruskal-Wallis test, p

Table 1. Skin lesions, PCV, and plasma total solidsin white perch of Chesapeake Bay tributaries inJune, August, and October 1998.8

Skin Plasmalesions PCV (%) total solids

Month/river (%) + SD (g/dL) + SDJuneBack 20 39.2 ± 5.6 6.1 ± 0.5Choptank 60 29.9 ± 6.3 5.0 ± 1.2Pocomoke 40 30.1 ± 6.1 4.4 ± 0.9Wicomico 0 40.0 ± 5.7 5.1 ± 0.9

AugustBack 30 32.3 ± 6.2 4.7 ± 0.7Choptank 36 37.5 ± 4.7 5.3 ± 0.8Pocomoke 10 34.4 ± 4.9 4.8 ± 0.5Wicomico 0 35.5 ± 3.2 4.5 ± 0.6

OctoberBack 10 49.3 ± 7.3 5.3 ±0.8Choptank 20 39.1 ± 4.4 5.0 ± 1.2Pocomoke 20 45.5 ± 8.5 5.4 ± 1.2Wicomico 30 35.5 ± 3.1 4.5 ± 0.6

'n = 10 at each sample site and time, except for theChoptank River in August (n = 1 1).

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Table 2. Physical measures from Chesapeake Bay tributaries obtained during fish sampling in June,August, and October 1998.

Air temp Water temp ('C) pH Dissolved Salinity (g/L)Month/river (C) surface/bottom surface/bottom oxygen (mg/L) surface/bottom Depth (m)JuneBack 31.0 28.1/27.8 6.5/6.1 8.0/8.1 0.2/0.2 1.2Choptank 30.8 22.1/22.1 6.2/6.1 7.1/7.1 0.0/0.0 3.3Pocomoke 29.2 22.8/22.6 5.3/5.1 7.2/7.2 2.4/2.4 4.5Wicomico 29.9 24.7/24.6 6.8/6.9 7.3/7.3 5.5/6.8 3.1

AugustBack 31.2 27.8/27.6 8.1/5.3 8.4/8.4 2.1/3.0 2.3Choptank 29.6 26.1/26.1 5.5/2.4 7.3/7.3 4.3/4.4 7.4Pocomoke 31.8 27.8/27.8 4.7/4.7 7.3/7.3 8.4/9.0 3.9Wicomico 29.4 26.1/26.1 5.1/5.1 7.3/7.3 5.4/5.5 2.5

OctoberBack 18.0 18.0/18.0 6.4/6.4 8.8/8.8 5.4/5.4 5.0Choptank 19.0 19.0/19.0 6.8/6.8 7.6/7.6 8.1/8.1 7.0Pocomoke 21.1 20.0/20.0 5.7/5.7 7.4/7.4 7.5/7.5 6.0Wicomico 18.0 18.3/18.3 6.8/6.8 7.8/7.8 9.9/9.9 9.0

< 0.0001), but with a pattern inverse to thatof the TGF-P results (Figure 2). Similar tothe TGF-( results, bactericidal activity fromBack River fish varied least as compared tofish from the other sites throughout the sam-pling period. The general decline in bacterici-dal activity observed from June to Octoberwas greatest for the Eastern Shore tributaryfish as compared to fish from the Back River.

Sample medians for splenic mononuclearcell TGF-P:3-actin ratios correlated inverselywith anterior kidney macrophage bacterici-dal activity (Kendall's X b = -0.600, p =0.0102). This inverse correlation is illustrat-ed by least-squares linear regression (Figure3). There were no significant correlations ofsplenic mononuclear cell TGF-1 mRNAlevels with sex, weight, length, presence ofskin lesions, PCV, plasma total solids, airtemperature, surface or bottom tempera-ture, dissolved oxygen, or pH. There was apositive association of TGF-P mRNA levels

with both surface and bottom salinity(Kendall's X b = 0.504 and 0.546, p =0.0233 and 0.0136 for surface and bottom,respectively). Anterior kidney macrophagebactericidal activity correlated significantly(inversely) only with splenic mononuclearcell TGF-1 mRNA levels.

DiscussionThis report marks the first time that quanti-tative measurement of specific cytokine pro-duction has been used in a field assessment offish health. There were significant differencesin splenic mononuclear cell TGF-43:-actinratios based on sample time and location.Constitutive TGF-, production generallyincreased from June to August and October,primarily in the Chesapeake Bay EasternShore tributaries. Based on its primarilyimmunosuppressive effects, elevated TGF-fproduction could be interpreted as an indi-cator of immunosuppression. However,

TGF-,B has some proinflammatory effects,including promotion of macrophage and neu-trophil chemotaxis (13). Whether TGF-, ispro- or antiinflammatory depends on its con-centration, the state of differentiation of targetcells, and the concentration of other proin-flammatory compounds (11). Therefore,TGF-f:f-actin ratios should be interpretedin the context of other health and immunefunction indicators. As compared to splenicmononudear cell TGF-,B transcription, ante-rior kidney macrophage bactericidal activitydeclined from June to August and October,and bactericidal activity correlated inverselywith TGF-,B mRNA levels. The inverse rela-tionship between TGF-, production andmacrophage bactericidal activity was expect-ed based on the known effects of TGF-3 onmacrophage activation and respiratory burst(13). This field-based finding of inverse cor-relation between the two assays also con-curred with a laboratory based study ofhybrid striped bass in response to adminis-tration of a known immunomodulator, tri-amcinolone (20). The agreement betweenthe quantitative PCR for TGF-P and thefunctional cellular immunoassay, conductedin two separate laboratories, indicates theoccurrence of immunomodulation consistentwith immunosuppression both spatially andtemporally in white perch of the ChesapeakeBasin. This immunomodulation took placein the absence of the widespread fish mortal-ities noted the previous year, and may indi-cate a seasonal and site predisposition todisease outbreaks under appropriate adverseconditions.

The immunomodulation detected (lowerbactericidal activity and higher TGF-,mRNA) coincided with the qualitativeobservations of deep ulcerative lesions in

~1(3b

June AUgUst

Mo., imuctober

Figure 1. TGF-f:rB-actin ratios varied significantly by month and river (Kruskal-Wallis test, p < 0.0001; n = 10 for all, except August in the Choptank River,where n = 11). Quantile boxes show nonparametric measures of dispersion:the 10th, 25th, 50th (median), 75th, and 90th quantiles; the horizontal line indi-cates the total response sample mean. Sample pairs that differed significantlyfrom each other are indicated by single versus double shared symbols (Dunn'smultiple comparison test, a < 0.05); all other sample pairs did not differsignificantly.

80

70

60

a 5040

a

'B 3

-10

.20-30

June August October

Month. 19!Figure 2. Macrophage bactericidal activity [CFU reduction (%)] varied signifi-cantly by month and river (Kruskal-Wallis test, p < 0.0001; n = 12 for all, exceptAugust in the Wicomico River, where n = 10). Quantile boxes show nonpara-metric measures of dispersion: the 10th, 25th, 50th (median), 75th, and 90thquantiles; the horizontal line indicates the total response sample mean. Therewere no bactericidal activity data for the Pocomoke River in June. Samplepairs that differed significantly from each other are indicated by single versusdouble shared symbols (Dunn's multiple comparison test, a < 0.05); all othersample pairs did not differ significantly.

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menhaden collected in the same areas. Theprevalence of skin and fin lesions in whiteperch did not correlate with TGF-f produc-tion or macrophage bactericidal activity, butthe white perch lesions were minor as com-pared to observed and previously reportedmenhaden lesions, and may have representedacute effects secondary to capture and trans-port. Two indicators of general health, PCVand plasma total solids, also varied accordingto sample site and time (Table 1, statisticalanalyses not shown), but did not correlatewith the more specific immunologic assess-ments of splenic mononuclear cell TGF-,production and anterior kidney macrophagebactericidal activity.

Production of TGF-P varies duringdevelopment (12); therefore, it could varyaccording to age even in adult fish. In thisstudy, the age of white perch was assessedindirectly by length and weight, which corre-late with age in white perch (34). There wereno significant length and weight differencesbetween samplings, and TGF-,B mRNA lev-els did not correlate with either length orweight, suggesting that age was not a factorin TGF-P differences. Sex and reproductivestatus could also affect TGF-3 productionbecause estrogens induce the production ofboth TGF-I 1 and TGF-P2 in mammals (35).However, there were no significant differ-ences in sex distribution between samplings,and there was no correlation between TGF-[:3-actin ratios and sex. White perch spawnfrom late March to May in the Chesapeake(34). Outdoor laboratory-reared white perchfrom the Roanoke River in North Carolinaspawned in nearly the same time frame andhad basal concentrations of testosterone andestradiol from June through October (36).Reproductive status and sex therefore mostlikely had no effect on the immunologicassays in this study.

0.05

0.04

0.03

0.02

0.010 20 40 60 80

CFU rduction (%)Figure 3. Least-squares linear regression of TGF-0:3-actin ratios versus macrophage bactericidalactivity [CFU reduction (%)] sample medians toillustrate inverse correlation (Kendall's X b =-0.600, p = 0.0102).

Of the physical conditions measured atthe sample sites, none of the following cor-related with TGF-4:P-actin ratios or mac-rophage bactericidal activity: air temperature,water temperature, pH, depth, and dissolvedoxygen. Hypoxic conditions have been sug-gested as a possible factor in the develop-ment of menhaden ulcerative lesions (27), aswell as other fish morbidity and mortalityevents in estuarine environments. Danger-ously low dissolved oxygen (2.4 mg/L) wasdetected in a bottom-water sample from theChoptank River in August, although the sur-face water level was an acceptable 5.5 mg/L(36). The failure to detect associationsbetween these physical conditions and theimmune function indicators measured inthis study may mean that parameters werewithin a range tolerable by white perch, orthat adverse conditions were transient andnot detected with the sample schedule thatwe used. The estuarine environment is muta-ble with the tides and with river flow rates,and any of the parameters we measured couldvary considerably between sampling times. Apulsatile insult affecting immune functionmight no longer prevail at the time of sam-pling. Furthermore, freely moving fish col-lected during a field study might be recentimmigrants to the sample location and mayhave been subject to different conditionsbefore their capture.

Salinity measurements did correlate withTGF-P:4-actin ratios, but an inverse associa-tion with macrophage bactericidal activity wasnot statistically significant. Osmotic shockalters the synthesis of specific proteins byhemocytes of the American oyster (Crassostreavirginica), an estuarine osmoconformer (38).Osmoregulatory mechanisms could account,in part, for altered TGF-,B production byteleost fish responding to salinity changes inestuarine environments. Prolactin and cortisolboth play a role in regulating electrolyte fluxacross the gills (39), and sequential prolactinand cortisol surges are important for smoltifi-cation of anadromous fish as they make thetransition to seawater (40). In mammals, highblood prolactin concentrations correlate withTGF-P2 levels in rat milk (41), and human Tcells increase transcription of TGF-PI inresponse to the synthetic glucocorticoid dex-amethasone (42,43). Osmoregulatory mecha-nisms could, therefore, affect the immune sys-tems of estuarine fish as they move betweenwaters ofvarying salinity. Alternatively, differ-ences in salinity could be secondary to otherconditions, such as river flow rates, and haveno direct effect on TGF-, transcription. Theinteractions between salinity, osmoregulatorymechanisms, and TGF-, production ofteleost fish will require investigation undercontrolled conditions to verifr the field asso-ciation reported here.

We did not assess other conditions thatcould affect fish immune function inChesapeake Bay tributaries, such as nutrientload, toxic algae blooms, heavy metals, pesti-cides, and other toxins. Normal seasonalvariations in immune function could play arole in the observed immunomodulation.Seasonal cues such as photoperiod and tem-perature, acting through the neuroendocrinesystem, have been proposed as responsiblefor multiple changes in immune functionindicators in ectotherms (44). Irrespective ofthe proximate and ultimate causes, naturalor anthropogenic, the results presented hereindicate temporal and spatial immunomodu-lation in white perch of the ChesapeakeBasin in a pattern consistent with previouslyobserved fish morbidity and mortalityevents. The addition of a molecular-basedassay for a specific cytokine, TGF-0, applica-ble to a wide range of teleost species occupy-ing varied habitats promises to augment ourability to assess fi7sh immune systems inresponse to experimental and natural condi-tions of interest.

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