Environmental Health Contaminant Biology Program

Prepared in cooperation with Pennsylvania Department of Environmental Protection

Development of a Suite of Functional Immune Assays and 

Initial Assessment of Their Utility in Wild Smallmouth Bass 

Health Assessments

Open-File Report 2020–1077

U.S. Department of the Interior

U.S. Geological Survey
Cover. Photographs showing the collection and laboratory processing of smallmouth bass tissue samples. Photographs by Vicki Blazer, U.S. 

Geological Survey (USGS), Kay Briggs, USGS, Cheyenne Smith, West Virginia University, and Heather Walsh, USGS.
Development of a Suite of Functional 

Immune Assays and Initial Assessment 

of Their Utility in Wild Smallmouth Bass 

Health Assessments

By Cheyenne R. Smith, Christopher A. Ottinger, Heather L. Walsh, 

and Vicki S. Blazer

Environmental Health Contaminant Biology Program

Prepared in cooperation with 

Pennsylvania Department of Environmental Protection

Open-File Report 2020–1077

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

DAVID BERNHARDT, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2020

For more information on the USGS—the Federal source for science about the Earth, its natural and living resources, 

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Government.

Although this information product, for the most part, is in the public domain, it also may contain copyrighted materials 

as noted in the text. Permission to reproduce copyrighted items must be secured from the copyright owner.

Suggested citation:

Smith, C.R., Ottinger, C.A., Walsh, H.L., and Blazer, V.S., 2020, Development of a suite of functional immune assays 

and initial assessment of their utility in wild smallmouth bass health assessments: U.S. Geological Survey Open-File 

Report 2020–1077, 23 p., https://doi.org/10.3133/ofr20201077.

ISSN 2331-1258 (online)
iii

Acknowledgments

This research was funded, in part, by Pennsylvania Department of Environmental Protection (PA 

DEP; Agreement #4000020352). We thank Geoff Smith (PA Fish & Boat Commission) and Timothy 

Wertz (PA DEP) for providing fish collections and Adam Sperry, Megan Schall, Ryan Braham, and 

Kelsey Young for assistance with fish necropsies. We also thank Darlene Bowling (USGS) and 

Pam Whittington (USGS) for preparation of histopathological slides, Megan Schall (Penn State 

University) for statistical advice, and Stephanie Gordon (USGS) for production of maps and land 

cover information.

v

Contents

Acknowledgments........................................................................................................................................iii

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Materials and Methods.................................................................................................................................2

Immune Functional Assays .................................................................................................................2

Leukocyte Isolations and Plating ..............................................................................................3

Bactericidal Activity of Adherent Cells (Phagocytes) ...........................................................3

Respiratory Burst Potential of Phagocytes .............................................................................6

Mitogenesis Assay ......................................................................................................................7

IDEAS® Gating Strategy for Data Analysis ..............................................................................8

Field Methods........................................................................................................................................8

Statistical Analyses ..............................................................................................................................8

Biometric Data and Immune Function Results .......................................................................................11

Biometric Data.....................................................................................................................................11

Immune Function.................................................................................................................................11

Seasonal Differences in Bactericidal Activity ......................................................................11

Seasonal Differences in Reactive Oxygen Species and Respiratory Burst 

Potential .........................................................................................................................12

Mitogenesis ................................................................................................................................12

Factors Potentially Affecting Immune Response ..........................................................................14

Microscopic Observations .......................................................................................................14

Chemicals in Surface Water ....................................................................................................14

Summary of Findings...................................................................................................................................17

References Cited..........................................................................................................................................20

Figures

1. Workflow for immune cell functional assays ...........................................................................4

2. Image-based flow cytometry gating strategy for IDEAS software. A, Primary 

gating of cells in focus. B, Doublet discrimination identifies single cells. C, 

Representative images of smallmouth bass leukocytes with masks overlaid. 

D, Secondary gating of cell populations present. E, Percentage of replicating 

immune cells of the total gated population ..............................................................................9

3. Fish collection sites at Tionesta Lake, Pine Creek, Upper Juniata River, and 

West Branch Mahantango Creek, Pennsylvania, with major land uses ...........................10

4. Age distribution of smallmouth bass collected from Pine Creek, Tionesta Lake, 

Upper Juniata River, and West Branch Mahantango Creek, Pennsylvania, 2016–17 .....11

5. Bactericidal activity against A, Aeromonas veronii biovar (bv.) sobria and B, 

Yersinia ruckeri in spring and fall 2016 and 2017 ...................................................................12

6. Production of reactive oxygen species. A, untreated leukocytes and B, 

leukocytes treated with phorbol 12-myristate 13-acetate ...................................................13

7. Untreated background lymphocyte proliferation grouped by season for all 

sites, 2016–17 ...............................................................................................................................14
vi

8. Lymphocyte stimulation by mitogens. A, Concanavalin A, B, Phytohemagglutinin 

from Phaseolus vulgaris, and C, Lipopolysaccharide versus untreated 

background proliferation of lymphocytes for all sampling sites .........................................15

9. Number of fish from each site that were stimulated versus not stimulated A, 

Concanavalin A, B, Phytohemagglutinin-P, and C, Lipopolysaccharide ............................16

10. Microscopic pathology in smallmouth bass spleen and liver tissue. A, Section 

of spleen from a Tionesta Lake smallmouth bass. B, Section of spleen from a 

Tionesta Lake smallmouth bass. C, Section of liver from an Upper Juniata River 

smallmouth bass D, Section of liver from an Upper Juniata River smallmouth 

bass. E, Focal granuloma ...........................................................................................................18

11. Chemical concentrations at selected sites before fish sampling occurred for 

immune function .........................................................................................................................20

Tables

1. Bactericidal activity protocol .....................................................................................................5

2. Respiratory burst protocol ..........................................................................................................6

3. Mitogenesis protocol ...................................................................................................................7

4. Mean (± standard deviation) age of mitogen-stimulated and non-stimulated 

individual fish ...............................................................................................................................17

5. Frequency of parasites by tissue, year, season, and site ....................................................19

Conversion Factors

International System of Units to U.S. customary units

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

micrometer (μm) 0.00003937 inch (in.)

meter (m) 3.281 foot (ft)

kilometer (km) 0.6214 mile (mi)

Volume

milliliter (mL) 0.03382 ounce, fluid (fl. oz)

liter (L) 33.81402 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

liter (L) 61.02 cubic inch (in3)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

kilogram (kg) 2.205 pound avoirdupois (lb)
vii

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F = (1.8 × °C) + 32.

Abbreviations

AF647 Alexa Fluor 647

BrdU Bromo-deoxyuridine

BSA Bovine serum albumin

bv. Biovar

CFU Colony forming units

Con A Concanavalin A

DMSO Dimethyl sulfoxide

DPBS Dulbecco’s phosphate buffered saline

EdU 5-ethynyl-2’-deoxyuridine

ELISA Enzyme-linked immunosorbent assay

FBS Fetal bovine serum

L-15 Leibovitz L-15 medium

LPS Lipopolysaccharide

PHA Phytohemagglutinin

PHA-P Phytohemagglutinin from Phaseolus vulgaris

PMA Phorbol 12-myristate 13-acetate

P/S Penicillin-streptomycin

RCF Relative centrifugal field

ROS Reactive oxygen species

SI Stimulation index

TSA Tryptic soy agar

TSB Tryptic soy broth

Development of a Suite of Functional Immune Assays and 

Initial Assessment of Their Utility in Wild Smallmouth 

Bass Health Assessments

By Cheyenne R. Smith,1,2 Christopher A. Ottinger,1 Heather L. Walsh,1 and Vicki S. Blazer1

Abstract

Methods were developed for measuring immune function in Micropterus dolomieu (smallmouth bass). The ultimate objective is to monitor and evaluate changes over time 

in immune status and disease resistance in conjunction with 

other characteristics of fish health and environmental stressors. 

To test these methods for utility in ecotoxicological studies, 192 smallmouth bass, age 2 years and older, were collected from three sites within the Susquehanna River Basin 

and one site in the Ohio River Basin during spring and fall 

2016 and 2017. The anterior kidney was aseptically removed 

and homogenized for leukocyte isolation. Leukocytes were 

tested for bactericidal activity against two species of bacteria; respiratory burst activity when stimulated with phorbol 

12-myristate 13-acetate; and mitogenesis activity when 

stimulated with concanavalin A, phytohemagglutinin, and 

lipopolysaccharide. Tissues were preserved for histopathological analyses.

Two of the sites were part of a monitoring program 

at which surface-water samples were collected monthly 

(bimonthly in spring) for chemical contaminants. Significant 

seasonal and (or) site differences in all three immune function tests were observed. Interpretations of seasonal trends 

in immune function of wild fish or correlations with environmental variables and other factors are difficult to make 

owing to the complex nature of the immune response and the 

environment. Differences in immune function could potentially be related to a variety of confounding factors; therefore, 

additional endpoints and repeated sampling over an extended 

period are essential to draw conclusions on the immune status 

of wild fish.

1U.S. Geological Survey.

2West Virginia University Division of Forestry and Natural Resources.

Introduction

Micropterus dolomieu (smallmouth bass) are an economically important sportfish throughout the Chesapeake Bay 

watershed. Large-scale episodic mortality and disease events 

have occurred since 2002 in the Potomac River and 2005 in 

the Susquehanna River, along with many of their tributaries 

(Blazer and others, 2007; Blazer and others, 2010; Smith and 

others, 2015; Walsh and others, 2018). Disease related mortality has mainly affected adults in the Potomac River (Blazer 

and others, 2010) and young-of-year in the Susquehanna River 

(Smith and others, 2015; Walsh and others, 2018). Over time, 

young-of-year smallmouth bass abundance in the Susquehanna 

River has declined, resulting in less recruitment, reduced 

adult populations, and interruptions to recreational fishing 

(Arway and Smith, 2013). Consequently, Federal and State 

wildlife management agencies in all four affected states—

Pennsylvania, Maryland, West Virginia, and Virginia—have 

reported large economic losses relating to the decline in smallmouth bass (Pelton and others, 2013).

No consistent cause or causes for disease or mortalities 

have been identified. Bacterial infections, heavy parasite loads, 

sometimes viral and fungal infections, and often co-infections 

of multiple parasites and pathogens have been observed. 

Pathogens isolated from smallmouth bass collected in the 

Potomac River have included Flavobacterium columnare, 

Enterobacter sp., Aeromonas hydrophila, and Aeromonas 

veronii biovar (bv.) sobria (Blazer and others, 2010; Starliper 

and others, 2013). In the Susquehanna River, researchers have 

observed or isolated bacteria (mainly Flavobacterium columnare and motile Aeromonas spp.), largemouth bass virus, and 

parasites, including trematode metacercariae, larval cestodes, 

and the myxozoan Myxobolus inornatus (Chaplin and others, 2009; Starliper and others, 2013; Smith and others, 2015; 

Walsh and others, 2018). The mixed infections and lack of any 

one consistent cause indicate that these fish are immunocompromised and hence susceptible to a variety of opportunistic 

pathogens.

Other effects on smallmouth bass health have been 

observed in the affected areas, including signs of estrogenic 

endocrine disruption, as evidenced by testicular oocytes 
2 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

(intersex) and vitellogenin in male fish (Blazer and others, 

2007; Iwanowicz and others, 2009; Blazer and others, 2014), 

and exposure to chemical contaminants, as evidenced by the 

increased presence of macrophage aggregates and oxidative 

damage in liver, spleen, and kidney tissues, and gill and skin 

lesions (Blazer and others, 2010). Numerous chemical contaminants linked to intersex, immunomodulation, and reduced 

disease resistance have been measured in affected locations, 

including up to 135 different compounds in the water at smallmouth bass nesting sites (Alvarez and others, 2009; Blazer and 

others, 2010; Reif and others, 2012; Kolpin and others, 2013; 

Walsh and others, 2018). Complex interactions between water 

quality, contaminants, pathogens, parasites, and other environmental stressors appear to be affecting smallmouth bass 

immunity, making it difficult to pinpoint any specific cause(s) 

for disease or death.

The immune system can be divided into two interacting 

components—innate and acquired immunity. Innate immunity 

is the first line of defense against infectious agents or other 

foreign material and is especially important in cold-blooded 

organisms such as fishes (Magnadóttir, 2006). It has also been 

the most studied aspect of fish immunity in terms of environmental stressors (Bols and others, 2001). Bactericidal activity 

and the respiratory burst of phagocytic cells, primary aspects 

of innate immunity, are closely related. Bactericidal activity 

assesses the ability of anterior kidney adherent phagocytes 

(macrophages, neutrophils) to kill bacteria; respiratory burst 

measures the phagocytic cells’ ability to produce reactive oxygen species (ROS) with and without stimulation.

Reactive oxygen species are essential for the activity of professional phagocytes, which in fishes includes 

macrophages, neutrophils, dendritic cells, monocytes, and 

potentially B cells (Rehberger and others, 2017; Biller and 

Takahashi, 2018). Upon ingestion of bacteria or other microscopic pathogens, phagocytic cells undergo respiratory burst 

(an increase in oxygen consumption of up to 20 times resting 

levels) to form potent bactericidal compounds (Slaninova and 

others, 2009; Biller and Takahashi, 2018). In a healthy organism, antioxidant enzymes produced in the host cell counteract 

damage caused by ROS (Biller and Takahashi, 2018). An overproduction of ROS, and thus an imbalance between ROS and 

antioxidant formation, is toxic to cells and leads to oxidative 

stress (Slaninova and others, 2009; Lushchak, 2016; Biller and 

Takahashi, 2018). High levels of oxidative stress over time 

can potentially have a profound effect on disease susceptibility 

(Tort, 2011). Phagocytosis may ultimately promote activation 

of the acquired immune response (Biller and Takahashi, 2018), 

which is measured by the mitogenesis assay.

Acquired immunity refers to learned cell- and humoralmediated responses capable of memory and building tolerance to specific antigens where the main cells involved are 

lymphocytes (Zelikoff, 1998). However, lymphocytes in fish 

can also behave as nonspecific immune cells resembling 

innate-like lymphocytes in humans (that is, γδT cells, mucosal 

associated invariant T cells, natural killer T cells, and B1-B 

cells), potentially performing activities such as phagocytosis, 

major histocompatibility complex independent direct killing 

by T-cells, and spontaneous antibody production in B-cells 

(Scapigliati and others, 2018). Mitogenesis tests the ability 

of lymphocytes (T- and B-cells) to proliferate in response to 

specific mitogens, proteins that induce cell division. Mitogens 

were chosen on the basis of evidence from previous studies 

that each is specific to either B- or T-cells. In fishes, this has 

been shown in Oncorhynchus mykiss (rainbow trout), where 

concanavalin A (Con A) and phytohemagglutinin (PHA) 

were specific to T-cells and lipopolysaccharide (LPS) was 

specific to B-cells (Boardman and others, 2012). Proliferation 

of anterior kidney lymphocytes occurring in the absence of 

mitogen-based induction in the experiment represents the level 

of background proliferation present at the time the fish were 

collected and will be referred to here as the untreated background response.

These immune characteristics can be affected by such 

factors as disease status (Magnadóttir, 2010), pesticides 

(Dunier and Siwicki, 1993; Slaninova and others, 2009; 

Lushchak, 2016), polycyclic aromatic hydrocarbons (Faisal 

and others, 1991; Lushchak, 2016), perfluoroalkyl acids (Lee 

and others, 2019), reproductive hormones or compounds 

mimicking their action (Wang and Belosevic, 1994; Harris 

and Bird, 2000; Szwejser and others, 2016), heavy metals 

(Zelikoff, 1993; O’Halloran and others, 1998; Lushchak, 

2016), and other external factors such as temperature and 

water quality (Makrinos and Bowden, 2016). To better 

understand the adverse effects of these multiple environmental 

stressors on the immune system of smallmouth bass, a suite 

of functional immune assays for bactericidal activity, respiratory burst activity, and mitogenesis were developed. This 

suite includes methods that have been previously used in other 

fish species, most often in laboratory studies. Each assay was 

optimized using 20 laboratory-held smallmouth bass (N = 60) 

fed with live minnows to best approximate natural conditions 

and immune response of wild fish (Ottinger and others, 2019), 

and then tested with 192 wild smallmouth bass from stream 

sites with varying land use in Pennsylvania. The optimization samples were used for method development and are not 

included in this report. Ultimately, understanding the effects 

of these complex mixtures of environmental stressors, as well 

as individual risk factors, on fish health will require a comprehensive assessment of immune function, along with identification of infectious agents, parasites, pathology, and physiological and molecular endpoints.

Materials and Methods

Immune Functional Assays

Immune functional assays were completed in the 

laboratory using isolated anterior kidney leukocytes from 

wild smallmouth bass. Following isolation and resuspension of leukocytes, cells were plated for three immune cell 
Materials and Methods 3

functional assays (fig. 1). Bactericidal and respiratory burst 

were plated simultaneously with cells suspended in adherence 

medium (L-15 media containing 290 micrograms (μg) per 

mL-1 L-glutamine, 100 units (U) per mL-1 penicillin, 100 μg 

per mL-1 streptomycin, and 0.1% fetal bovine serum (FBS); 

L-15/0.1% penicillin-streptomycin (P/S)). Remaining cells 

were resuspended in culture medium (L-15 media containing 290 μg per mL-1 L-glutamine, 100 units (U) per mL-1

penicillin, 100 μg per mL-1 streptomycin, and 5-percent FBS; 

L-15/5% P/S) and plated for mitogenesis assay.

Leukocyte Isolations and Plating

Anterior kidney leukocytes were isolated following 

a procedure modified from Sharp and others (1991). Cell 

suspensions were pelleted by centrifugation at 500 relative centrifugal field (RCF) and 4 degrees Celsius (°C) for 

10 minutes (min), then washed 3 times by suspension in 

10 milliliters (mL) Leibovitz L-15 medium with 2-percent 

fetal bovine serum (L-15/2%), centrifuging as above between 

each wash. Following wash steps, cells were resuspended 

in 6 mL of L-15/2%, passed through a 70-micron (μm) cell 

strainer to remove clumps or clots, and layered onto 6 mL 

of 32-percent percoll concentration in Hanks’ Balanced Salt 

solution (Sigma-Aldrich, St. Louis, Mo.) without phenol red 

in 15 mL centrifuge tubes. Cells on percoll were centrifuged 

at 500 RCF at 4 °C for 45 min. Leukocytes were removed 

from the media/percoll interface and pelleted by centrifugation at 500 RCF at 4 °C for 10 min. Isolated leukocytes were 

resuspended in L-15/2% for counting. Total numbers of 

viable leukocytes were determined by trypan blue exclusion 

using the CountessTM II automated cell counter (ThermoFisher, Waltham, Mass.). CountessTM II viable-cell counts 

were optimized for smallmouth bass leukocytes prior to use 

in this study using laboratory fish. Final instrument settings 

were validated using parallel cell counts from a propidium 

iodide exclusion assay, with data acquisition performed using 

the Amnis FlowSight® imaging flow cytometer (Luminex 

Corporation, Austin, Tex.).

Following leukocyte counts, cells were pelleted by 

centrifugation, resuspended at 2×107 cells mL-1 in adherence medium and plated at 100 microliter (μL) well-1 for the 

bactericidal and respiratory burst assays or culture medium 

and plated at 50 μL well-1 for the mitogenesis assay. The 

bactericidal and respiratory burst assays were plated simultaneously. Remaining cells were concentrated by centrifugation 

(500 RCF at 4 °C for 10 min), and the adherence medium was 

replaced with culture medium before plating the mitogenesis 

assay. Isolated leukocytes were plated in 96-well CorningTM

CostarTM flat-bottom cell culture plates (Corning Cat. No. 

3596; ThermoFisher). Leukocyte incubations for all three 

assays occurred within a humidified container at 17 °C to 

provide the closest fit to the water temperatures from which 

the source fish were obtained. A minimum of 2.5 mL of 

isolated leukocytes resuspended at 2×107 cells per mL-1 were 

required to complete all immune function assays (1.1 mL for 

bactericidal assay against two different bacteria; 0.6 mL for 

respiratory burst assay; and 0.8 mL for mitogenesis assay). In 

some cases when leukocyte counts were low, not all assays 

were completed.

Bactericidal Activity of Adherent Cells 

(Phagocytes)

When adequate cell numbers were available to complete 

all immune function assays, the bactericidal activity against 

two different bacteria—Aeromonas veronii bv. sobria and 

Yersinia ruckeri—were tested. When adequate cell numbers 

were not available, only bactericidal activity against A.veronii

bv. sobria was tested. Aeromonas veronii bv. sobria has been 

isolated from affected adult and young-of-year smallmouth 

bass. Yersinia ruckeri is a cold-water pathogen (Kumar and 

others, 2015) to which smallmouth bass are less likely to be 

exposed; however, it is a widely distributed gram-negative 

bacterium that has been previously used in bactericidal assays.

Functional assessment of adherent anterior kidney leukocytes to kill bacteria was determined following a procedure 

from Harms and others (2000) with modifications (table 1).

Briefly, 2×107 viable cells mL-1 in AM (L-15/0.1% P/S) 

were added in quadruplicate to a 96-well tissue culture plate 

for the bacterial challenge and in triplicate on the same plate 

for adherent cell enumeration. One row on the same plate contained media-only control wells to measure bacterial growth in 

the absence of cells. After plating, leukocytes were incubated 

for 2 hours at 17 °C. After 2 hours, the media were removed 

from all wells to remove non-adherent cells and replaced with 

CM (L-15/5% P/S). Cells were cultured for 36 hours in a 

humidified container to allow activated leukocytes to reach a 

resting state before adding bacterial cultures.

Following 36-hour incubation, wells were washed to 

remove antibiotics prior to the addition of bacteria. Fortyeight hour cultures of bacteria grown in tryptic soy broth 

(TSB) were added to the treatment and cell-free control wells, 

leaving some wells bacteria free and available for adherent 

cell enumeration. After bacteria were added, the plate was 

incubated in a humidified container at 17 °C for 4 hours. 

After 4 hours, the media were removed from treatment and 

cell-free control wells (not bacteria-free rows) and replaced 

with a lysis buffer. Serial dilutions were performed for all 

treatments and controls, plated onto tryptic soy agar (TSA) 

plates, and colony-forming units (CFUs) were counted after 

24 hours. Cell counts were performed for bacteria-free rows 

using the CountessTM II automated cell counter (ThermoFisher, Waltham, Mass.). Bactericidal activity was expressed 

as the percentage CFU reduction corrected for mean adherent 

cell counts:

%CF Ur=

_

CF Utreated

CF Ucontrol)*100][

_1 A Cx ̄⁄1* 106], (1)

where

%CFUr is the percentage reduction in colony forming 
4 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

Plate for mitogenesis,

add mitogens

Plate cells for

bactericidal assay

Plate for respiratory

burst assay

Remove anterior kidney

Leukocyte isolation

Cell counts—resuspend

at 2 x 107

 cells

Expose to bacteria

Plate on TSA and count CFUs

Expose to PMA

Add CellROX Deep Red 

Reagent to detect ROS

Expose to EdU

Add reaction cocktail to

detect incorporated EdU

7.487

Ch02 Ch04 Ch11 Ch02/Ch11/Ch01

13.26

Ch02 Ch04

Ch11 Ch02/Ch11/Ch01

18272 7.487

Ch01 Ch02 Ch04 Ch11 Ch02/Ch11/Ch01

20 μm

12283 13.26

Ch01

Ch02 Ch04 Ch11 Ch02/Ch11/Ch01

20 μm

Figure 1. Workflow for immune cell functional assays. Following isolation and resuspension of leukocytes, cells were 

plated for three immune cell functional assays. Bactericidal and respiratory burst were plated simultaneously with cells 

suspended in adherence medium (L-15/0.1% P/S). Remaining cells were resuspended in culture medium (L-15/5% P/S) and 

plated for mitogenesis assay. (EdU, 5-ethynyl-2’-deoxyuridine; TSA, tryptic soy agar; CFUs, colony forming units; PMA, phorbol 

12-myristate 13-acetate: ROS, reactive oxygen species; L-15/0.1% P/S, L-15 media containing 290 micrograms (μg) per mL-1

L-glutamine, 100 units (U) per mL-1 penicillin, 100 μg per mL-1 streptomycin, and 0.1% fetal bovine serum (FBS); L-15/5% P/S, 

L-15 media containing 290 μg per mL-1 L-glutamine, 100 units (U) per mL-1 penicillin, 100 μg per mL-1 streptomycin, and 5-percent 

FBS)
Materials and Methods 5

Table 1. Bactericidal activity protocol.

[HBBS, Hanks’ balanced salt solution; P/S, penicillin-streptomycin; %, percent; L-15, Leibovitz L-15 media; OD, optical density; h, hour; min, minute; nm, 

nanometer; RCF, relative centrifugal field; mL, milliliter; NO P/S, no penicillin-streptomycin]

Task Step1

Day 1

Plating isolated leukocytes 1. Incubate bacterial cultures in tryptic soy broth (TSB) on shaker for 48 hours at room temperature.

2. Plate 2 × 107 cells ml-1 in adherence medium (L-15/0.1% P/S) and one row of media-only control wells.

3. Incubate for 2 hours.

4. Replace adherence medium in all wells with culture medium (L-15/5% P/S).

5. Incubate for 36 hours.

Day 3

Preparation of bacteria 1. After 48 h, prepare bacteria by centrifuging for 10 min at 2,000 RCF and wash 3 times2.

2. Determine the density of bacteria by absorbance at 600 nm on a microplate reader and adjust the stock 

solution to a working density of 0.15 OD600 by diluting the stock solution in HBSS to a final volume of 

10 mL.

Final preparation of 

macrophages and addition 

of bacteria

3. After 36 h and prior to adding bacteria, wash all wells3 in 36 h macrophage plates 1x to remove antibiotics, and add L-15/5% NO P/S. 

4. Add bacteria to all wells except wells being used for adherent cell counts

5. Incubate for 4 hours.

Macrophage lyses, bacteria 

dilution and plating on agar

6. Approximately 1 h before the end of 4-h incubation, prepare lysis buffer (0.2% Tween 20), load 180 μL 

of HBSS into rows A-F of serocluster plates (1 serocluster plate for each row of treated cells or bacteriaonly controls), and label one tryptic soy agar (TSA) plate for each fish plus three for bacteria-only 

controls.

7. At the end of the 4 h incubation, remove media from treated rows and bacteria-only control rows and 

replace with 50 μL of lysis buffer. 8. Mix well and transfer 20 μL from each row into row A of a corresponding serocluster plate for serial dilutions.

9. Perform serial dilutions by mixing and transferring 20 μL from row A to corresponding wells in row B.

10. Repeat the mixing and transferring process from row B to C, C to D, D to E, and E to F exchanging 

pipet tips in between.

11. After completing serial dilutions, plate 10 μL drops by column on TSA plates, mixing well contents 

prior to removal and changing tips between each column.

12. Plate 4 columns of dilutions per TSA plate to correspond to the 4 replicates for each fish and control.

13. Allow drops to air dry with plates partially uncovered, and then store covered plates inverted for 24 

hours.

Adherent cell counts 14. Following the plating of bacterial dilutions, count the number of adherent cells in the wells replicated 

for each fish using the CountessTM automated cell counter (combine 10 μL from wells with 10 μL of 

trypan blue, mix well, and add 10 μL to chamber slide for analysis).

Day 4

Count CFUs 1. Count colony forming units (CFUs) and record for each fish and bacteria-only control.

1Incubations should occur at species-appropriate temperature in a humidified container, and cells should be washed by pipetting up and down without resuspending the cells to minimize cell loss unless otherwise noted.

2Wash buffer: HBSS.

3Wash buffer: Unsupplemented L-15.
6 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

units by treated cells compared to controls 

(bacteria only),

CFUtreated is the number of colony forming units on agar 

plates with treated cells,

CFUcontrol is the number of colony forming units on agar 

plates with bacteria only, and

ACx ̄ is the mean adherent cell count.

Respiratory Burst Potential of Phagocytes

Respiratory burst was determined via procedures 

described by Coteur and others (2002) and Iwanowicz and 

others (2012) with modifications (table 2).

Leukocytes for respiratory burst were plated at the same 

time as the bactericidal assay so incubation times could run 

simultaneously. Briefly, isolated leukocytes at 2×107 viable 

cells per mL-1 in AM (L-15/0.1% P/S) were added in duplicate 

to a 96-well tissue culture plate for each treatment, including 

negative controls. Plates were incubated for 2 hours at 17 °C 

before media were removed from all wells and replaced with 

CM (L-15/5% P/S). Cells were then cultured for 36 hours in 

a humidified container to allow activated leukocytes to reach 

a resting state. Following 36-hour incubation, wells were 

washed to remove antibiotics, and plated leukocytes were 

treated with either phorbol 12-myristate 13-acetate (1 μg/mL 

PMA in dimethyl sulfoxide; DMSO), DMSO carrier control, or media only (negative control; L-15/5% NO P/S) for 

5 minutes at room temperature. After 5 minutes, CellROX®

Deep Red Reagent was added at a final concentration of 

5 micromolars (μM) to the cells and incubated for 30 minutes 

at 17 °C, protected from light. After 30 minutes, media were 

removed, cells were washed three times with Dulbecco’s 

phosphate-buffered saline (DPBS), and then resuspended 

in 1% bovine serum albumin (BSA) in DPBS before being 

analyzed for the fluorescent signal. The signal was analyzed 

on the Amnis FlowSight® imaging flow cytometer (Luminex 

Corporation, Austin, Tex.) within 2 hours, acquiring at least 

10,000 events for each sample. Instrument settings were 

Illumination, 642 laser at 100.0 millivolts (mV), 785 laser at 

5.00 mV; Fluidics, minimum flow speed.

Stimulation index (SI) values were calculated to compare 

the respiratory burst potential of treated leukocytes to control 

leukocytes treated with DMSO as a carrier control:

 SI

Ft

Fc

 and (2)

 F=

( count of positively fluoresced ces   ______________________

count of total gated cells (single and focused) 

)*100 (3)

where

SI is the stimulation index,

Ft is the percentage of positive cells in the 

treated replicate,

Fc is the percentage of positive cells in the 

DMSO-stimulated carrier control 

replicate, and

F is the percentage of positive cells in the total 

population.

Table 2. Respiratory burst protocol.

[mL, milliliter; L-15/0.1%; P/S, penicillin-streptomycin; h, hour; μg, microgram; DMSO; μM, micromolar; min, minute; °C, degrees Celsius; BSA, bovine 

serum albumin; DPBS, Dulbecco’s phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate] 

Task Step1

Day 1

Plating isolated leukocytes 1. Plate 2×107 cells mL-1 in adherence medium (L-15/0.1% P/S) in duplicate for each treatment.

2. Incubate for 2 hours.

3. Replace adherence medium in all wells with culture medium (L-15/5% P/S).

4. Incubate for 36 hours.

Day 3

CellROX® reagent staining 

and detection

1. After 36 h and prior to adding stimulant, wash all wells2 in 36 h plates 1× to remove antibiotics, and add 

L-15/5% NO P/S.

2. Add PMA (1 μg mL-1), DMSO carrier control or media only (L-15/5% NO P/S) to corresponding wells.

3. Incubate for 5 minutes.

4. Add the CellROX® Reagent (5 μM) and incubate for 30 min at 17 °C in the dark.

5. Remove medium, wash 3 times3, and resuspend in 1% BSA in DPBS.

6. Analyze on a FlowSight using 40× magnification within 2 hours.

1Incubations should occur at species-appropriate temperature in a humidified container, samples should be protected from light throughout protocol following addition of CellROX® Reagent, and cells should be washed by pipetting up and down without resuspending the cells to minimize cell loss unless otherwise 

noted.

2Wash buffer: Unsupplemented L-15.

3Wash buffer: DPBS.
Materials and Methods 7

The percentage of positive leukocytes in the total population from negative control samples provided a measure of 

background reactive oxygen species. SI values were calculated 

using data from the pooled replicate of leukocytes (two replicates per treatment for each fish) from the same fish.

Mitogenesis Assay

Mitogenesis was evaluated using mitogens obtained 

from Millipore Sigma (Burlington, Mass.) and following the 

bromo-deoxyuridine (BrdU) based enzyme-linked immunosorbent assay (ELISA) procedure from Gauthier and others 

(2003) with modifications. Ethynyl-deoxyuridine (EdU) was 

used instead of BrdU, and the Click-it® EdU (5-ethynyl2’-deoxyuridine) Flow Cytometry Assay Kit (Molecular 

Probes, Eugene, Ore.) was used as an alternative for labeling 

and detection (table 3).

Plated leukocytes were treated at 50 μL well-1 with either 

a mitogen or mitogen-free media (negative control wells). 

Mitogens used were concanavalin A (Con A) at 10 μg per mL-1

(0.25 μg well-1 final concentration), phytohemagglutinin from 

Phaseolus vulgaris (PHA-P) at 20 μg per mL-1 (0.5 μg well-1

final concentration), and lipopolysaccharide from Escherichia 

coli (E. coli) O111:B4 (LPS) at 100 μg per mL-1 (2.5 μg 

well-1 final concentration). Working solutions of mitogens 

were prepared in L-15/5%. Mitogen treated and control wells 

were duplicated in the same plate. Plates were incubated for 

24 hours before adding EdU. Following incubation with the 

mitogens, 25 μL well-1 of EdU in unsupplemented L-15 was 

added (EdU final concentration = 2.6 μM well-1) to all wells. 

Following an additional 18-hour incubation, incorporation of 

EdU/cell replication was detected by Alexa Fluor 647 (AF647) 

using the Click-iT® EdU (5-ethynyl-2’-deoxyuridine) Flow 

Cytometry Assay Kit (Molecular Probes, Eugene, Ore.), following the manufacturers protocol (MP 10419) for fixation, 

permeabilization, and labeling with volume adjustments; 

100 μL well-1 of wash permeabilization reagent, 25 μL well-1

of fixative, and 100 μL well-1 of reaction cocktail were used.

After labeling leukocytes with Alexa Fluor 647 (AF647), 

the cells were washed and resuspended in 200 μL of ClickiT® saponin-based permeabilization and wash reagent in 

Table 3. Mitogenesis protocol.

[EdU, 5-ethynyl-2’-deoxyuridine; μg, microgram; mL, milliliter; Con A, concanavalin A; PHA-P, phytohemagglutinin from Phaseolus vulgaris; L-15, Leibovitz 

L-15 media; %, percent; FBS, fetal bovine serum; h, hour; μM, micromolar; DNA, deoxyribonucleic acid; oC, degrees Celsius; DPBS, Dulbecco’s phosphate 

buffered saline] 

Task Step1

Day 1

Assay setup 1. Prepare working concentrations of mitogens (100 μg mL-1 LPS, 10 μg mL-1 Con A, 20 μg mL-1

PHA-P) in L-15 media with 5% FBS

2. Plate 2×107 cells ml-1 in L-15 media with 5% FBS and add working concentrations of mitogens or 

L-15 media with 5% FBS (no mitogen, non-treated cells)

3. Incubate overnight (24 h) to allow mitogens time to stimulate the cells

Day 2

Add EdU 1. After 24 h, add diluted EdU in unsupplemented L-15 to plates at 13 μM final concentration and 

incubate for an additional 18 h to allow incorporation of EdU into newly synthesized DNA

Day 3

Click-iT® reaction 1. Wash cells2 and remove supernatant without dislodging adherent cells/pelleting the cells

Fix and permeabilize cells 2. Fix and permeabilize the cells based on the manufacturer’s instructions for the Click-iT® EdU 

Flow Cytometry Assay Kit (Molecular Probes) for 15 minutes at room temperature

3. Wash cells2, remove supernatant (being careful not to dislodge pellet) and add 1X Click-iT®

saponin-based permeabilization/wash reagent (resuspend cells and mix well only if storing plate 

for later) 

4. At this point, fixed cells can be stored at 4 °C to finish the assay later (up to 1 week was investigated without signal loss) or proceed directly to click labeling

Detection of incorporated EdU 5. Perform click labeling following manufacturer’s instructions for 30 minutes at room temperature 

6. Wash cells3 and remove supernatant (do not dislodge pellet) then resuspend (dislodge pellet and 

mix well) in 1X Click-iT® saponin-based permeabilization/wash reagent

Analyze on FlowSight 7. Analyze on a FlowSight using 40x magnification immediately or store at 4 °C for up to 1 week

1Incubations should occur at species-appropriate temperature in a humidified container, samples should be protected from light throughout protocol following 

addition of EdU, and cells should be washed by pipetting up and down without resuspending the cells to minimize cell loss unless otherwise noted.

2Wash buffer: 1% BSA in DPBS.

3Wash buffer: 1X Click-iT® saponin-based permeabilization/wash reagent.
8 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

preparation for analysis using the Amnis FlowSight® imaging flow cytometer (Luminex Corporation, Austin, Tex.) or 

the SpectraMax M4 microplate reader (Molecular Devices, 

San Jose, Calif.) for method comparison. Cells from replicate 

wells (two wells per treatment for each fish) were pooled prior 

to data acquisition using the Amnis FlowSight®, and at least 

10,000 events were acquired for each sample. Instrument 

settings were Illumination, 642 laser at 100.0 mV, 785 laser 

at 5.00 mV; Fluidics, minimum flow speed. Cell analysis 

was performed using Image Data Exploration and Analysis 

Software (IDEAS® 6.2; Amnis/Luminex Corporation, 

Austin, Tex.).

IDEAS® Gating Strategy for Data Analysis

Primary gating of imaging flow cytometry data collected 

from 10,000 events isolated individual round cells that were in 

good focus by brightfield (fig. 2). Gating was optimized using 

data from laboratory and wild smallmouth bass.

The population of round cells in focus is labeled R1 

(fig. 2A). Doublet discrimination resulted in a population 

(R2) of single cells in focus (fig. 2B). Masking of brightfield 

images was performed using the adaptive erode function with 

89 erosion coefficient to generate custom masks to increase the 

accuracy of size specific analysis (fig. 2C). Secondary gating 

of masked images divided the R2 population into three cell 

populations (R3, R4, and R5) on the basis of cell diameter and 

side scatter, as a measure of cell density (fig. 2D), and positive 

staining for AF647/EdU was based on fluorescence intensity. 

The threshold for AF647 detection was set at an intensity of 

100 using visual inspection of the histograms and cell images 

(fig. 2E). Following initial analysis, a template was created 

containing the primary and secondary gating. This template 

was used to analyze all datasets to provide consistent analytics 

across samples.

Field Methods

Wild fish sampling was done in conjunction with ongoing fish health assessment and monitoring studies at four sites 

in Pennsylvania: Upper Juniata River (latitude 40.56082, 

longitude 78.0694), Pine Creek (41.282964, 77.321489), and 

West Branch Mahantango Creek (40.6478, 76.94296) within 

the Susquehanna River Basin and Tionesta Lake (41.4753121, 

79.438331) in the Ohio River Basin (fig. 3). Attempts were 

made to collect 20 adult smallmouth bass from each site by 

boat electrofishing in spring and fall 2016 and 2017. Captured 

fish were placed immediately in a live well and transported to 

shore to be necropsied on site following the protocol by Blazer 

and others (2018).

Fish were euthanized, according to the Leetown Science 

Center’s Institutional Animal Care and Use Committee 

guidelines, with a lethal dose (250 mg/mL) of tricaine 

methanesulfonate (MS-222; Western Chemical Laboratories, 

Ferndale, W.V.). The fish were measured (nearest millimeter), 

weighed (nearest gram) and bled from the caudal vessels. 

Fulton’s condition factors were determined for individual fish 

using the equation K = W/L3, where K = condition factor, 

W = weight of the fish, and L = length of the fish (Nash and 

others, 2006). Any visible abnormalities, external and internal, 

were recorded. The anterior kidney was aseptically excised 

and placed in 10 mL of cold L-15 media supplemented with 

290 μg per mL-1 L-Glutamine, 10 units mL-1 sodium heparin, 

100 units mL-1 penicillin, 100 μg per mL-1 streptomycin, and 

2 percent FBS (characterized, U.S. origin; Hyclone, GE Life 

Sciences). Supplemental media were designated subsequently 

as L-15/2%. Anterior kidney tissues in L-15/2% were homogenized into a single cell suspension using a sterile, manual 

Tenbroeck tissue grinder. The cell suspension for each fish was 

returned to the original sample tube and kept on ice. Samples 

collected in the field were transported to the lab for next 

day processing and kept at 4 °C overnight prior to leukocyte 

isolation (12–16 hours holding time). Unless otherwise stated, 

reagents were obtained from Sigma Aldrich (St. Louis, Mo.).

Following aseptic removal of the anterior kidney, a 

necropsy was performed; tissues were removed and fixed in 

Z-fixTM (Anatech LTD, Battle Creek, MI) for histopathological 

analyses and RNALaterTM (ThermoFisher, Waltham, Mass.) 

for gene expression analyses. Otoliths were removed for 

aging. Tissues for histopathology were routinely processed, 

embedded into paraffin, sectioned at 5 μm and stained with 

hematoxylin and eosin (Luna, 1992). Overall parasite intensity was rated on a scale of 0 to 4 for the sections of spleen 

and liver, and when possible the type of parasite was identified. The number of parasites associated with each rating was 

0 = 0 parasites; 1 = 1–4 parasites, 2 = 5–9 parasites, 3 = 10–14 

parasites, and 4 = 15+ parasites for spleen tissue and 0 = 0 

parasites, 1 = 1–19 parasites, 2 = 20–39 parasites, 3 = 40–59 

parasites, and 4 = 60+ parasites for liver tissue.

Statistical Analyses

Statistics were calculated using R 3.5.3 (R Core 

Team, 2019), ggplot2 (Wickham and others, 2016), ggpubr

(Kassambara, 2018), cowplot (Wilke, 2019), ggpmisc (Aphalo, 

2016), and plyr (Wickham, 2011) packages. Individual fish 

comparisons among seasons at each site were made using 

unpaired Wilcoxon rank-sum tests. The Kruskal-Wallis test 

was used for multiple comparisons. If significant, post hoc 

analysis was performed using the Dunn test (Dunn, 1964) for 

groups with unequal numbers of observations, adjusted with 

the Benjamini-Hochberg method. An α-level of 0.05 was used 

to indicate significant results.
Materials and Methods 9

BF Diameter (μm)

SSC Intensity (log)

6885

Ch11

20 μm

AF647 Intensity (log)

Count

9.95%

BF Focus Score

Count

78.6%

BF Aspect Ratio

R2

4903

Ch01

20 μm

7876

Ch01

20 μm

BF Area

4906

20 μm

3459

Ch01 Ch01 Ch06 Ch09 Ch11

20 μm

Ch01 Ch01 Ch06 Ch09 Ch11

Default mask Custom mask

A B

C

D E

Figure 2. Image-based flow cytometry gating strategy for IDEAS software. A, Primary gating of cells in focus (R1). B, 

Doublet discrimination identifies single cells (R2). C, Representative images of smallmouth bass leukocytes with masks 

(cyan) overlaid; default cell mask (Ch01) generated by IDEAS software versus custom mask (Ch01, Ch06, Ch09, Ch11) 

generated to increase the accuracy of cell diameter calculations. D, Secondary gating of cell populations (R3, R4, R5) 

present based on a density scatterplot of intensity versus cell diameter. E, Percentage of replicating immune cells (for 

example, positive for AF647/EdU staining) of the total gated population (R2). (μm, micron; BF, brightfield; SSC, side scatter;

AF647/EdU, Alexa Fluor 647 or 5-ethynyl-2’-deoxyuridine; %, percent)
10 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

78° 75°

42°

40°

PENNSYLVANIA

NEW YORK

MARYLAND

 ̄

Tionesta Lake Pine Creek

Upper Juniata River West Branch Mahantango Creek

Susquehanna 

River Basin

Ohio River 

Basin

EXPLANATION

Land use category

Wetland

Agriculature

Open water

Developed

Forest

Other

Major stream or river

Fish sampling site

0 5 10 15

0 5 10 15

MILES

KILOMETERS

0 5

0 5

MILES

KILOMETERS

0 5 10 15

0 5 10 15

MILES

KILOMETERS

0 5 10 15

0 5 10 15

MILES

KILOMETERS

0 20 40 60

0 20 40 60

MILES

KILOMETERS

Figure 3. Fish collection sites at Tionesta Lake, Pine Creek, Upper Juniata River, and West Branch Mahantango Creek, 

Pennsylvania, with major land uses.
Biometric Data and Immune Function Results 11

Biometric Data and Immune Function 

Results

Biometric Data

In spring and fall 2016, a total of 112 smallmouth bass 

were collected, age 2 and older (range = 2–13) from four 

sites—Tionesta Lake (n = 12, 2 females and 10 males), Pine 

Creek (n = 40, 17 females and 23 males), Upper Juniata 

River (n = 20, 5 females and 15 males) and West Branch 

Mahantango Creek (n = 40, 19 females and 21 males). In the 

spring and fall 2017, a total of 80 smallmouth bass were collected, age 2 and older (range = 2–12), from two sites—Pine 

Creek (n = 40, 23 females and 17 males) and West Branch 

Mahantango Creek (n = 40, 18 females and 22 males). The 

mean age was 4.6 ± 2.1 years in 2016 and 3.9 ± 2.0 years in 

2017 and was not significantly different among sites (fig. 4).

Immune Function

Comparisons among all sampling sites could be made 

only for spring 2016 when all four sites were sampled and 

only for the mitogenesis assay. Tionesta Lake and Upper 

Juniata River were sampled only in spring 2016; no data are 

available on bactericidal or respiratory burst activity for these 

two sites owing to optimization of the assays.

Seasonal Differences in Bactericidal Activity

In 2016, median bactericidal activity against A.veronii bv. 

sobria was significantly elevated at West Branch Mahantango 

during fall when compared to spring (fig. 5A; p < 0.001). 

In fact, the median bactericidal activity against A.veronii

bv. sobria was 0 percent during spring at West Branch 

Mahantango and during both seasons at Pine Creek in 2016. 

In 2017, seasonal differences were not significant at either 

EXPLANATION

Sampling season—

Vertical, dashed line 

indicates median age

Spring

Fall

(No samples collected

 in 2017)

(No samples collected

 in 2017)

14

12

10

8

6

4

2

0

0 2 4 6 8 10 12 14

14

12

10

8

6

4

2

0

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

Pine Creek Tionesta Lake Upper Juniata River West Branch Mahantango Creek

2017 count 2016 count

Age, in years

Figure 4. Age distribution of smallmouth bass collected from Pine Creek, Tionesta Lake, Upper Juniata River, and West Branch 

Mahantango Creek, Pennsylvania, 2016–17. Top row, age of the 112 smallmouth bass collected in spring and fall 2016; bottom row, age of 

the 80 smallmouth bass collected in spring and fall 2017. Vertical dashed lines indicate mean age for each season.
12 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

site; however, median bactericidal activity against A.veronii

bv. sobria was elevated in the fall at Pine Creek, the mostly 

forested site, but during spring at West Branch Mahantango 

Creek, the more agricultural site (fig. 5A).

No seasonal comparisons were made for bactericidal 

activity against Y. ruckeri in 2016 because the bacterium was 

not added to the assay until fall 2016. The only seasonal comparison within the same sampling year that could be made for 

bactericidal activity against Y. ruckeri was for fish collected 

at West Branch Mahantango Creek in 2017 where, although 

not significant, bactericidal activity was greater in the spring 

(fig. 5B). Bactericidal activity at Pine Creek against Y. ruckeri

was not tested during fall 2017 owing to low cell yields. 

Bactericidal activity against A. veronii bv. sobria, a known 

pathogen of smallmouth bass, was prioritized in cases of low 

cell yields.

Seasonal Differences in Reactive Oxygen 

Species and Respiratory Burst Potential

No seasonal comparisons were made for 2016 because 

the respiratory burst assay was completed only in fall of that 

year owing to optimization of the assay. No data on respiratory burst for Tionesta Lake or Upper Juniata River were 

available because those sites were sampled only in spring 

2016 when the respiratory burst assay was being optimized. 

In 2017, background levels of reactive oxygen species (ROS) 

were higher in fall compared to spring (fig. 6A) at Pine Creek 

(p < 0.001) and West Branch Mahantango Creek (p < 0.05). 

No, or exceptionally low, respiratory burst activity occurred in 

leukocytes from fish collected during fall after being stimulated with PMA (fig. 6B). Respiratory burst was greater during 

spring at Pine Creek and West Branch Mahantango Creek 

in 2017, although only significantly greater at Pine Creek 

(fig. 6B; p < 0.001).

Mitogenesis

Untreated background proliferation of anterior kidney 

lymphocytes differed seasonally with 2016 and 2017 data 

pooled. Background mitogenesis was significantly elevated at 

Pine Creek in spring when compared to fall (fig. 7; p < 0.001). 

The opposite was true for West Branch Mahantango Creek, 

although it was not significant (fig. 7). Seasonal comparisons 

could not be made for Tionesta Lake or Upper Juniata River.

Leukocytes were considered stimulated by mitogens if 

the stimulation index (SI) value was greater than or equal to 

1.25, meaning the treated leukocytes proliferated at least 1.25 

times more than untreated leukocytes. A cutoff of 1.25 was 

used because it allows room for errors with the data collection 

2016 2017

2016 2017

West Branch

Manhantango

Creek

Pine

Creek

(20) (17) (18) (12)

West Branch

Mahantango

Creek

Pine

Creek

(20) (15) (20) (20)

West Branch

Manhantango

Creek

Pine

Creek

(17) (12)

West Branch

Mahantango

Creek

Pine

Creek

(20) (20) (20)

A

B

50

40

30

20

10

0

Reduction of colony

forming units, in percent

50

40

30

20

10

0

Reduction of colony

forming units, in percent

EXPLANATION

(20)

Sampling season

 Spring

 Fall

Statistical outlier

Highest value within 1.5 times IQR of 75th percentile

Lowest value within 1.5 times IQR of 25th percentile

Indicates significant difference between seasons— 

Wilcoxon rank sum/Mann Whitney U test, p < 0.0001

75th percentile

Median

25th percentile

Interquartile range (IQR)

***

Number of samples

Figure 5. Bactericidal activity against A, Aeromonas veronii biovar (bv.) sobria and B, Yersinia ruckeri in spring and fall 

2016 and 2017. Median (line in box), interquartile range (box), and statistical outliers (black dots) are provided. (*** indicates 

significant differences between seasons [Wilcoxon rank sum/Mann Whitney U test, p < 0.001])
Biometric Data and Immune Function Results 13

and makes the analysis more robust. Individual fish that were 

stimulated by mitogens (SI ≥ 1.25) had lower levels of background proliferation than individuals that were not stimulated 

(SI < 1.25). At all sites, stimulated mitogenesis dropped 

below 1.25 as untreated background proliferation increased 

(fig. 8A-C).

The number of fish that were stimulated by mitogens 

(SI ≥ 1.25) was dependent upon site, season, and year. In 

spring 2016, more fish were not stimulated (SI < 1.25) 

than stimulated (SI ≥ 1.25) at all sites and for all mitogens 

(fig. 9A-C). In fall 2016, Con A was the only mitogen with 

more fish stimulated than not stimulated; this was true at 

two sites, Pine Creek and West Branch Mahantango Creek 

(fig. 9A). In spring 2017, no fish at Pine Creek were stimulated by PHA-P or LPS, and only five fish were stimulated by 

Con A. For West Branch Mahantango Creek in spring 2017, 

more fish were not stimulated than stimulated by all mitogens. 

In fall 2017 at Pine Creek, more fish were stimulated than not 

stimulated for mitogens when data from both seasons were 

collected (PHA-P and LPS), and at West Branch Mahantango 

Creek, the number of fish stimulated by Con A or LPS was 

equal to the number of fish which were not stimulated by 

either of those mitogens and more fish were not stimulated by 

PHA-P than stimulated.

Mean age varied among individual fish that were stimulated by mitogens and individuals that were not (table 4). 

During fall, stimulated individuals were younger than individuals not stimulated, except for PHA-P stimulated mitogenesis 

at West Branch Mahantango Creek where the average ages 

were the same for both groups. During spring, the relation 

between age and mitogenesis was not consistent. At some 

sites, stimulated individuals were younger, and at other sites 

they were older, than individuals that were not stimulated. Age 

also varied by mitogen. Correlations between age and mitogenesis were beyond the scope of this analysis, but the relation 

is worth investigating in a more robust statistical analysis.

*** *

***

West Branch

Manhantango

Creek

Pine

Creek

(14) (7)

(14) (7)

West Branch

Mahantango

Creek

Pine

Creek

(17) (7) (18) (18)

(18)

A 2016 2017

West Branch

Manhantango

Creek

Pine

Creek

West Branch

Mahantango

Creek

Pine

Creek

(17) (7) (18) (18)

B 2016 2017

90

70

50

30

10

80

60

40

20

0

Background reactive oxygen

species, in percent

4

3

1

2

0

Stimulated respiratory burst

EXPLANATION

Sampling season

 Spring

 Fall

Statistical outlier

Highest value within 1.5 times IQR of 75th percentile

Lowest value within 1.5 times IQR of 25th percentile

Significant difference between seasons—

Wilcoxon rank sum/Mann Whitney U test

p < 0.05

p < 0.0001

75th percentile

Median

25th percentile

Interquartile range (IQR)

***

*

Number of samples

Figure 6. Production of reactive oxygen species. A, untreated leukocytes and B, leukocytes treated with phorbol 

12-myristate 13-acetate (PMA). The dashed line at 1 represents the point where stimulated ROS production becomes 

greater than unstimulated/background ROS production. Median (line in box), interquartile range (box), and statistical 

outliers (black dots) are provided. (ROS, reactive oxygen species; %, percent; WB, West Branch. Asterisks indicate 

significant differences between seasons [Wilcoxon rank sum/Mann Whitney U test, * = p < 0.05; ** = p < 0.01; 

*** = p < 0.001])
14 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

Factors Potentially Affecting Immune Response

Microscopic Observations

Wild fish were infected by parasites and contained 

pathological lesions in spleen, liver, kidney, and other tissues. Microscopic lesions varied among individuals at a site 

and among sites and seasons. Some individuals had very few 

encysted areas containing killed and degenerating parasites 

(fig. 10A), whereas others had more intense infections with 

live and dead encysted parasites (fig. 10B). At some sites only 

trematode parasites were observed, whereas at other sites 

migrating cestodes (fig. 10C), encysted nematodes (fig. 10D), 

and myxozoan parasites were observed. At the Upper Juniata 

River site, smallmouth bass had focal granulomas (chronic 

inflammation) in the spleen and sometimes in the liver consistent with Mycobacterial infections (fig. 10E).

Parasite intensity varied among sites and between seasons. Pine Creek, the more forested site with low chemical 

contaminant concentrations had consistently higher parasite 

ratings than West Branch Mahantango Creek, except in fall 

2017 when ratings were similar (table 5). The opposite trend 

was observed for Tionesta Lake and Upper Juniata River; 

Tionesta Lake, the more forested site, had consistently lower 

parasite ratings than Upper Juniata River for spring 2016 when 

both sites were sampled (table 5). Unfortunately, chemical 

contaminant concentrations were not determined for these 

two sites.

Chemicals in Surface Water

Wild fish were potentially exposed to mixtures of organic 

compounds measured in surface water. Pine Creek and West 

Branch Mahantango Creek were part of a monitoring program, 

and surface-water grab samples were analyzed for a variety 

of organic compounds, including pesticides, phytoestrogens, 

hormones, pharmaceuticals, wastewater indicators, and mycotoxins bimonthly in late spring/early summer (April–June) or 

monthly the rest of the year (July–March) from 2012 to 2017 

(Williams and others, 2019). On average, water samples were 

collected 0 to 6 days before fish sampling occurred except in 

fall 2016, when water samples at Pine Creek and West Branch 

Mahantango Creek were collected 34 and 35 days before fish 

sampling, respectively.

More organic compounds and higher concentrations of 

mixtures were measured at West Branch Mahantango Creek 

than at Pine Creek (fig. 11). Herbicides (atrazine, metolachlor) 

and a fungicide (boscalid) were the only compounds detected 

at Pine Creek and only during spring 2016, whereas West 

Branch Mahantango Creek also had endocrine disrupting 

phytoestrogens (daidzein, equol, formononetin, genistein) and 

phenolic compounds (p-cresol). In 2016, higher concentrations 

of many chemicals were detected in spring than fall.

***

(30) (17) (11) (17) (13) (29)

Pine

Creek

Tionesta

Lake

Upper

Juniata

River

West Branch

Mahantango

Creek

50

40

30

20

10

0

Untreated background proliferation,

in percent positive

EXPLANATION

(20)

Sampling season

 Spring

 Fall

Statistical outlier

Highest value within 1.5 times IQR of 75th percentile

Lowest value within 1.5 times IQR of 25th percentile

Indicates significant difference between seasons— 

Wilcoxon rank sum/Mann Whitney U test, p < 0.0001

75th percentile

Median

25th percentile

Interquartile range (IQR)

***

Number of samples

Figure 7. Untreated background lymphocyte proliferation grouped by season for all sites, 2016–17. Median (line in box), interquartile 

range (box), and statistical outliers (black dots) are provided. (Asterisks indicate significant differences between seasons [Wilcoxon 

rank sum/Mann Whitney U test, *** = p < 0.001])
Biometric Data and Immune Function Results 15

8

6

4

2

0

Stimulation index

8

6

4

2

0

Stimulation index

8

6

4

2

0

0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40

Stimulation index

Untreated background proliferation, in percent positive

A

B

C

Pine

Creek

Tionesta

Lake

Upper Juniata

River

West Branch

Mahantango Creek

EXPLANATION

Spring sample

Fall sample

Figure 8. Lymphocyte stimulation by mitogens. A, Concanavalin A, B, Phytohemagglutinin from Phaseolus vulgaris, and C, 

Lipopolysaccharide versus untreated background proliferation of lymphocytes for all sampling sites. The dashed line at 1.25 

represents the point where stimulated mitogenesis becomes greater than untreated background proliferation. Seasons are 

differentiated by shape and color.
16 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

Pine Creek

Tionesta Lake

Upper Juniata River

WB Mahantango Creek

Pine Creek

Tionesta Lake

Upper Juniata River

WB Mahantango Creek

2016 count

A. Concanavalin A

20

15

10

5

0

2017 count

20

15

10

5

0

2016 count

B. Phytohemagglutinin from phaseolus vulgaris

20

15

10

5

0

2017 count

20

15

10

5

0

2016 count

C. Lipopolysaccharide

20

15

10

5

0

2017 count

20

15

10

5

0

Spring samples Fall samples

Spring samples Fall samples

Spring samples Fall samples

 EXPLANATION

Stimulation index

Less than 1.25

Greater than or equal to 1.25

 EXPLANATION

Stimulation index

Less than 1.25

Greater than or equal to 1.25

 EXPLANATION

Stimulation index

Less than 1.25

Greater than or equal to 1.25

Figure 9. Number of fish from each site that were stimulated (simulation index greater than or equal to 1.25) 

versus not stimulated (simulation index less than 1.25). A, Concanavalin A, B, Phytohemagglutinin-P, 

and C, Lipopolysaccharide.
Summary of Findings 17

Summary of Findings

The immune function findings for wild smallmouth bass 

at four Pennsylvania sites with differing land use provide 

evidence for seasonal changes as well as site differences. 

During spring, background levels of reactive oxygen species 

were low, and respiratory burst occurred when stimulated in 

vitro for smallmouth bass at all sites. However, during fall 

background levels of reactive oxygen species were high at all 

sites and respiratory burst after stimulation in vitro did not 

occur (fig. 6). Bactericidal activity and mitogenesis responses 

varied by sampling site and season. During spring, bactericidal 

activity was high at West Branch Mahantango Creek but low 

at Pine Creek (fig. 5). Untreated background mitogenesis was 

high at Pine Creek during spring (fig. 7) with little additional 

proliferation occurring after stimulation by mitogens (fig. 9). 

Spring was the only season when chemicals, specifically herbicides atrazine, boscalid, and metolachlor, were measured at 

Pine Creek (fig. 11).

For mitogenesis at each site, when untreated background 

proliferation was high, lymphocytes were no longer stimulated in vitro by mitogens (fig. 8). The level of untreated 

background proliferation for this to occur varied seasonally 

and geographically (by site). Untreated background proliferation tended to be higher in older smallmouth bass and these 

individuals were less likely to mount a proliferative response 

to mitogens in the laboratory. Some contaminants are known 

to accumulate with age (Brousseau and Fournier, 2013; Su and 

others, 2017). Such accumulations may be responsible for the 

increases in background lymphocyte proliferation observed in 

older individuals, but such a response has yet to be specifically 

demonstrated.

Differences observed in the immune response could 

potentially be related to a variety of factors, including land 

use and chemical inputs, but differences could be related to 

parasite loads and other infectious agents. Seasonal changes 

in immune function have been reported in many fish species 

(Bowden et al 2007; Magnadóttir, 2010; Szwejser and others, 

Table 4. Mean (± standard deviation) age of mitogen-stimulated and non-stimulated individual fish. The number of fish with a 

stimulation index greater than 1.25 of the total sample size and the average age of each group (stimulated or not) are listed by site, 

season, and mitogen.

[SI, simulation index; ≥, greater than or equal to; WB, West Branch; Con A, concanavalin A; PHA-P, phytohemagglutinin from Phaseolus vulgaris; LPS, lipopolysaccharide]

Mitogen Season Site Sample size SI≥1.25 Mean age stimulated

Mean age not 

stimulated

Con A Spring Pine Creek 30 6 4.2 ± 0.8 6.0 ± 2.5

Tionesta Lake 11 4 6.5 ± 1.7 5.9 ± 0.7

Upper Juniata River 16 3 7.7 ± 1.5 4.6 ± 1.6

WB Mahantango Creek 13 3 3.3 ± 0.6 4.7 ± 1.4

Fall Pine Creek 17 14 2.9 ± 0.7 3.7 ± 0.6

WB Mahantango Creek 29 15 2.7 ± 1.1 3.4 ± 2.2

PHA-P Spring Pine Creek 30 2 7.0 ± 5.7 5.5 ± 2.1

Tionesta Lake 11 5 6.4 ± 1.5 5.8 ± 0.8

Upper Juniata River 17 4 4.8 ± 2.4 5.5 ± 2.0

WB Mahantango Creek 13 4 5.5 ± 1.0 3.9 ± 1.3

Fall Pine Creek 17 8 2.9 ± 0.8 3.2 ± 0.7

WB Mahantango Creek 29 6 3.0 ± 1.7 3.0 ± 1.8

LPS Spring Pine Creek 29 6 4.7 ± 3.1 5.9 ± 2.1

Tionesta Lake 10 2 5.5 ± 0.7 6.3 ± 1.3

Upper Juniata River 15 4 6.5 ± 3.0 4.9 ± 1.4

WB Mahantango Creek 13 4 4.3 ± 1.3 4.4 ± 1.5

Fall Pine Creek 17 9 2.9 ± 0.8 3.3 ± 0.7

WB Mahantango Creek 29 12 2.4 ± 0.5 3.5 ± 2.1
18 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

A B

C D

E

Figure 10. Microscopic pathology in smallmouth bass spleen and liver tissue. 

A, Section of spleen from a Tionesta Lake smallmouth bass illustrating cyst 

(arrow) with remnants of a helminth parasite, probably trematode. Scale bar 

equals 50 microns. B, Section of spleen from a Tionesta Lake smallmouth bass 

with an encysted live trematode (arrow). Numerous encysted areas containing 

degenerating parasites (a) and yellowish brown ceroid or lipofuscin (b). Scale 

bar equals 50 microns. C, Section of liver from an Upper Juniata River smallmouth 

bass with a migrating cestode (a) and associated tissue disruption and necrosis 

(b); a macrophage aggregate (c) is also present. Scale bar equals 30 microns. 

D, Section of liver from an Upper Juniata River smallmouth bass with encysted 

nematodes (arrows). Scale bar equals 30 microns. E, Focal granuloma (a) typical of 

Mycobacterial infections and focal encysted areas (arrow) with inflammation (b). 

Scale bar equals 30 microns. Hematoxylin and eosin stain were used.
Summary of Findings 19

2016) relating to multiple factors, including fluctuations in 

water temperatures and dissolved oxygen; changes in photoperiod; changes in hormones, such as estrogens; and exposure 

to parasites and pathogens which also varies seasonally. In 

summary, it has been shown that confounding factors such as 

age, sex, reproductive status, exposure to contaminants, health 

status of the organism, and any other physical stressors, such 

as hypoxic conditions, could affect immune function in fish 

and must be considered in the overall analysis (Farkas and 

others, 2003).

Caution is needed when interpreting immune function in 

wild fish as either normal or suppressed owing to environmental stressors without ancillary data such as disease status or the 

presence of parasites that may have stimulated various aspects 

of the immune response. Many different characteristics will be 

required to establish baseline immune function for these wild 

smallmouth bass populations. The incorporation of additional 

endpoints such as plasma antibodies, hormone concentrations, 

and gene expression will be important in future studies as well 

as repeated sampling over a long period to establish seasonal 

trends and document patterns or changes in the data relating to 

environmental variables or other factors.

Table 5. Frequency of parasites by tissue, year, season, and site. The number of fish classified under each parasite rating of the total 

sample size for that site is listed.

[WB, West Branch; SD, standard deviation]

Tissue Year Season Sampling site Sample 

size

Parasite rating

0 1 2 3 4 Mean ± SD

Liver 2016 Fall Pine Creek 20 0 5 7 5 3 2.30 ± 1.03

WB Mahantango Creek 20 4 9 4 2 1 1.35 ± 1.09

Spring Pine Creek 20 1 17 1 1 0 1.10 ± 0.55

Tionesta Lake 12 7 5 0 0 0 0.58 ± 0.51

Upper Juniata River 20 1 15 4 0 0 1.15 ± 0.49

WB Mahantango Creek 20 6 14 0 0 0 0.70 ± 0.47

2017 Fall Pine Creek 20 0 7 10 0 3 1.95 ± 1.00

WB Mahantango Creek 20 0 12 5 2 1 1.60 ± 0.88

Spring Pine Creek 19 0 14 3 0 2 1.47 ± 0.96

WB Mahantango Creek 20 10 10 0 0 0 0.50 ± 0.51

Spleen 2016 Fall Pine Creek 20 2 7 7 2 2 1.75 ± 1.12

WB Mahantango Creek 20 6 11 2 0 1 0.95 ± 0.94

Spring Pine Creek 19 10 9 0 0 0 0.47 ± 0.51

Tionesta Lake 12 10 2 0 0 0 0.17 ± 0.39

Upper Juniata River 19 6 11 1 0 1 0.89 ± 0.94

WB Mahantango Creek 20 15 5 0 0 0 0.25 ± 0.44

2017 Fall Pine Creek 20 5 7 5 3 0 1.30 ± 1.03

WB Mahantango Creek 20 6 6 6 0 2 1.30 ± 1.22

Spring Pine Creek 20 5 10 3 1 1 1.15 ± 1.04

WB Mahantango Creek 19 18 1 0 0 0 0.05 ± 0.23
20 Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health

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Creek

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p.Cresol

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function. On average, water samples were collected 0 to 6 days before fish sampling occurred except in 

fall 2016, when water samples at Pine Creek and West Branch Mahantango Creek were collected 34 and 

35 days before fish sampling, respectively.
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For additional information, contact:

Director, Leetown Science Center

U.S. Geological Survey

11649 Leetown Road

Kearneysville, WV 25430

or visit our website at: 

https://www.usgs.gov/centers/lsc

Publishing support provided by the 

West Trenton Publishing Service Center
Smith and others—Development of Immune Assays and Initial Assessment of Wild Smallmouth Bass Health—OFR 2020–1077

ISSN 2331-1258 (online)

https://doi.org/10.3133/ofr20201077