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5-2019

Humoral Immune Responses to Select BacterialPathogens in the American Alligator, AlligatormississippiensisBailey Marie AlstonClemson University, bmalsto@g.clemson.edu

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Recommended CitationAlston, Bailey Marie, "Humoral Immune Responses to Select Bacterial Pathogens in the American Alligator, Alligator mississippiensis"(2019). All Theses. 3135.https://tigerprints.clemson.edu/all_theses/3135

i

HUMORAL IMMUNE RESPONSES TO SELECT BACTERIAL PATHOGENS IN

THE AMERICAN ALLIGATOR, Alligator mississippiensis

A Thesis

Presented to

the Graduate School of

Clemson University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Environmental Toxicology

by

Bailey M. Alston

May 2019

Accepted by:

Dr. Charles D. Rice, committee chair

Dr. Thomas R. Rainwater

Dr. Thomas E. Schwedler

ii

Abstract

The American alligator, Alligator mississippiensis, is widely distributed and

abundant throughout the southeastern United States. Despite their abundance, these

reptiles have not been examined for their role in environmental pathogen distribution, as

a sentinel for the presence of various pathogens, or other humoral immune responses in

individuals from different habitats. This study investigates the humoral immune

responses of alligators to select bacterial pathogens. Immunoglobulin Y (IgY), an

immunoglobulin molecule found in amphibians, birds and reptiles, similar to IgG in

higher vertebrates. IgY was purified from pooled alligator serum collected in coastal

South Carolina. Mouse polyclonal antisera (pAb) was then generated against IgY to

develop a sensitive ELISA to quantify serum antibody responses and relative titers.

Serum samples from alligators collected from multiple localities in Florida were screened

for bacteria-specific antibodies to the following nine aquatic bacteria: Vibrio cholera,

Escherichia coli, V. anguillarum, V. vulnificus, V. parahaemolyticus, Brevundimonas

vesicularis, Mycobacterium marinum, Erysipelothrix rhusiopathiae and Streptococcus

agalactiae. C-reactive protein (CRP) is an ancient acute phase protein, participating in

complement activation and opsonisation of pathogens, and is usually indicative of

relative levels of systemic inflammation. Alligator CRP was purified and used to

generate a specific monoclonal antibody to develop ELISA-based approaches to semi-

quantifying circulating CRP in individuals. Finally, serum lysozyme enzymatic activity

was also quantified in individual samples. Humoral immune responses to various

pathogens, along with CRP and lysozyme activity, may be correlated with environmental

iii

health. This study provides the first baseline data and proof of concept, to compare

responses of wild alligators to pathogens throughout their distribution. This information

will allow A. mississippiensis to be used as a sentinel of pathogen occurrence and

environmental quality in future studies.

iv

Dedication

To the Academy of the Holy Names, Ms. Clancy and Mr. Triller, for instilling a

love of science in me at an early age. To my grandfather, “Daddy Max” for his

inspirational and lifelong pursuit of education.

v

Acknowledgements

First, I would like to thank Dr. Rice for providing me with such a wonderful

opportunity to get my Master’s in his lab and for being so supportive along the way.

Thank you for your mentorship and for setting me up for success. I came out of this

program having learned not only about research, but also important life lessons. Two of

the most important life lessons I have learned from Dr. Rice are that you shouldn’t take

yourself too seriously and that not every experimental error is the end of the world.

Thank you to Dr. Schwedler and Dr. Rainwater for serving on my committee. I really

appreciate your time, expertise, guidance and understanding throughout this process.

Thank you to Dr. Rainwater for providing samples and putting us in touch with Dr. John

Bowden at the University of Florida, and to Dr. Bowden for providing alligator samples.

To Kursten and Delia – the “angels on my shoulder”. Thank you for being such

supportive lab-mates and friends. I know I can always count on you two to have my

back, help clean up messes (both in my life and in the lab (i.e. the centrifuge incident))

and celebrate achievements. To my “ride or die”, Lindsey Louise Barnett Jr. - thank you

for always keeping it real and for hyping me up when I needed it (and when I don’t).

Natalie, thank you for being such a supportive and understanding roomie, cat mom and

friend this past year.

Last but not least, I would also like to thank my family. My parents, George and

Jennifer, for the opportunities and life experiences you’ve given me. For teaching me the

importance of education, always pushing me to do my best and the continuous support

and unconditional love over the past 25 years. Ashley, for showing me the importance of

vi

always being yourself. Grace, for being my voice of reason and best friend, and Will and

Christian for being the best little brothers (making me look good) and always having my

back.

vii

Table of Contents

Page

Title Page ...........................................................................................................................i

Abstract ............................................................................................................................ ii

Dedication ........................................................................................................................iv

Acknowledgements ........................................................................................................... v

List of Figures ............................................................................................................... viii

Chapters

I. Literature Review............................................................................................ 1

Alligator mississippiensis: Species background ............................................. 1

Effects of temperature ..................................................................................... 3

Threats to the American alligator ................................................................... 4

Alligator immunology ..................................................................................... 4

Reptilian innate immunity............................................................................... 5

Reptilian adaptive immunity ........................................................................... 8

Potential role as sentinel species and amplifier host ..................................... 13

II. Chapter One .................................................................................................. 15

Introduction ................................................................................................... 15

Materials and Methods .................................................................................. 20

Results ........................................................................................................... 28

Discussion ..................................................................................................... 41

References ....................................................................................................................... 45

viii

List of Figures

Figure Page

1. American Alligator Habitat Range ....................................................................... 2

2. IgY Structure ....................................................................................................... 12

3. Alligator serum average lysozyme activity ......................................................... 30

4. SDS PAGE Gel of American Alligator Purified IgY ......................................... 31

5. Western Blot of Bacterial Lysates Probed with Alligator Whole Serum ........... 32

6. E.coli titers in serum ........................................................................................... 33

7. M.marinum titers in serum .................................................................................. 34

8. S.agalactae titers in serum .................................................................................. 35

9. V.vulnificus titers in serum .................................................................................. 36

10. V.anguillarium titers in serum ............................................................................ 37

11. V.parahaemoliticus titers in serum ..................................................................... 38

12. V.cholerae titers in serum ................................................................................... 39

13. Alligator relative CRP levels in serum ............................................................... 40

1

Chapter I: Literature Review

Alligator mississippiensis: Species Background

The American alligator, Alligator mississippiensis, is the largest reptile and only

alligator native to the United States. These carnivorous reptiles are found primarily

throughout the Southeast U.S., from the Virginia-North Carolina border to South Florida

and as far west as Texas (Figure 1) (United States Geographical Services). Once

considered an endangered species, A. mississippiensis has made a full recovery as a result

of conservation efforts required by the Endangered Species Preservation Act (US Fish

and Wildlife Service).

Alligators inhabit freshwater marshes, swamps, rivers, and lakes and can

sometimes be found in brackish water. They play a large role in the ecosystem and are

considered to be both keystone species and ecosystem engineers (Schechter and Street

2000, Rice and Mazzotti 2004). As a keystone species and top predator, alligators affect

most aquatic life in their environment (Beard 1938). Using their snouts and tails, they

create ‘alligator holes’ which are used for shelter and hibernation. These burrows serve

an important role, especially in the everglades, as they retain water during the dry season,

providing refuge to other organisms (Craighead 1969).

2

Figure 1. Map of the southeastern U.S showing the geographic range of A.

mississippiensis (United States Geographical Services, 2000).

3

Effects of temperature

As poikilothermic ectotherms, alligators’ body temperatures adjust with the

environment and therefore must be regulated through behaviors. Basking in the sun

raises body temperature, while submerging underwater and allowing heat to be released

through the mouth lowers body temperature (Smith 1975). Physiological processes such

as immune function, feeding behavior, reproduction, growth and development are all

directly related to environmental temperature (Lance 2003, Lang and Andrews 1994). As

a result, there are seasonal changes in behavior that affect how resources are partitioned

(Zimmerman, Vogel, and Bowden 2010).

When exposed to extreme temperatures, reptiles undergo a physiological stress

response. Studies have found that immunoglobulin production decreases in response to

cold weather (Zapata, Varas, and Torroba 1992). Water temperature has an effect on

haematological values in other crocodilians, further demonstrating how temperature can

play a role in immunosuppression (Turton et al. 1997). Phagocytic activity of

macrophages and the amount of histamine released by basophils can also be affected by

temperature (Mondal and Rai 2001, Zimmerman, Vogel, and Bowden 2010).

Alligators are active year-round, but most active during the warmer months. The

mating season begins in May and ends mid-July when females lay their eggs. They lay

one clutch of 20-60 eggs every one to two years (Brandt 1989). Alligators demonstrate

temperature dependent sex determination; the sex is determined by the incubation

temperature of the nest. There is variation in the temperature depending on where the nest

is built, as well as the position of the egg in the nest (Ferguson and Joanen 1982).

4

Temperatures of 32.2 oC – 33.9 oC produce mostly male alligators, while 27.8 oC - 30 oC

results in mostly females (Schechter & Street, 2000).

Threats to the American alligator

Although A. mississippiensis is no longer endangered, the Fish and Wildlife

Service classifies this species as ‘threatened’ because it resembles the endangered

American crocodile (US Fish and Wildlife Service, 2008). The biggest threat is habitat

loss from the development and drainage of wetland (National Wildlife Federation).

Global climate change may affect population dynamics because of the American

alligators’ reliance on temperature for sex determination; as temperature increases, the

male to female sex ratio may be skewed (Valenzuela et al. 2019).

Anthropogenic activities resulting in contamination of aquatic habitats pose

another serious threat to aquatic life. Reptiles commonly inhabit contaminated areas, and

because alligators have long lifespans, there is an increased likelihood that contaminants

will accumulate, increasing the risk of disease and other adverse effects (Guillette et al.

1994). Environmental contaminants are known to affect lymphocyte proliferation

specifically (Zimmerman, Vogel, and Bowden 2010).

Alligator immunology

The immune system protects against infection by microorganisms. Crocodilian

immune systems are similar to those of higher vertebrates in that the lymphoid tissues

include the thymus, spleen, gut associated lymphoid tissue (GALT) and bone marrow

(Kvell et al. 2007). The thymus is where T-cell maturation occurs. The spleen removes

5

old red blood cells and is the site of antibody synthesis. GALT traps and concentrates

antigens, allowing for interaction with lymphocytes. Bone marrow is the site of

hematopoiesis and B-cell maturation (Keller et al. 2005). Crocodilians and other reptiles

do not have lymph nodes, germinal centers or Peyer’s patches, as these structures are

only found in warm blooded vertebrates (Flajnik 2018).

Reptiles demonstrate species-specific, seasonal variation in lymphoid tissues,

analogous to that of hibernating mammals. The thymus and white pulp of the spleen

become less developed during the winter in reptiles and species-specific differences can

be seen in structures during the summer (Zimmerman, Vogel, and Bowden 2010). As the

thymus begins to redevelop in the spring, T-cell populations are reestablished, making T-

cell proliferation strongly dependent on seasonal cycles (Kruman 1992).

The reptilian immune system is comprised of both innate and adaptive immunity,

as seen in all gnathostomes (Dzik 2010). While the innate immune system can be traced

back to more primitive organisms, such as invertebrates, the adaptive immune system is

only found in vertebrates (Song, Sarrias, and Lambris 2000). The innate immune system

acts as the first line of defense with a non-specific response, in attempt to prevent the

spread of infection. Following the innate response, the adaptive response is activated.

The type of adaptive response, cell mediated or humoral, depends on both the type of

innate response and the pathogen (Zimmerman, Vogel, and Bowden 2010).

Reptilian innate immunity

The reptilian innate immune system includes physical barriers, antimicrobial

6

peptides (AMPs), enzymes, proteins (including complement) and non-specific leukocytes

(Finger 2012, Zimmerman, Vogel, and Bowden 2010). Physical barriers include skin and

gut mucosa, which act to keep microorganisms out (Finger 2012). Antimicrobial

peptides (AMPs) are similar in structure and function to beta defensins found in

mammals. Lysozymes, also found in mammals, are antimicrobial enzymes that

hydrolyze the membrane of both Gram-positive and Gram-negative bacteria, resulting in

osmotic lysis. Other antimicrobial enzymes found in crocodilians include phospholipase

A2 (PLA2) and dipeptidyl peptidase IV (DPP2, also known as CD26); both have been

confirmed in A. mississippiensis. PLA2 functions to destroy bacteria by increasing the

permeability of the cellular membrane (G Buckland and C Wilton 2000). The specific

function of DPP2/CD26 has not been determined in reptiles (Finger 2012). In mammals,

DPP2/CD26 plays an important role in numerous parts of humoral immunity, including

role in cellular development, migration and cytokine production (Yan et al. 2003).

The complement system plays a large role in regulating the adaptive immune

response through at least three different pathways: classical (CP), alternative (AP) and

lectin (LP) (Trouw and Daha 2011). It is comprised of around 30 proteins that either

directly lyse or opsonize bacteria in the plasma. Microbial opsonization results in

increased phagocytic efficiency by coating bacteria with proteins, allowing macrophages

to identify and phagocytose the foreign bacteria (Zimmerman, Vogel, and Bowden 2010).

The complement system is more diverse and well developed in poikilothermic species

than in higher vertebrates, yet it is not well characterized (Sunyer, Zarkadis, and Lambris

1998, Zarkadis, Mastellos, and Lambris 2001, Merchant et al. 2005). Studies have found

7

that serum complement from A.mississippiensis is effective against different strains of

Gram-positive bacteria, three Naegleria species and four Acanthamoeba species

(Merchant et al. 2003, Merchant et al. 2004).

Acute phase proteins (APP) are important in innate immunity of all animals

(Cray, Zaias, and Altman 2009). They are produced by hepatocytes as part of the acute

phase response (APR). APR is activated in response to many attacks on the body,

including infection, trauma, stress and inflammation, in attempts to prevent further

infection, initiate inflammation and reestablish homeostasis. C-reactive protein (CRP) is

an important APP in all vertebrates (Lee et al. 2017). CRP is produced by hepatocytes

and is made up of five monomers, each 25 kDa. Although the structure is evolutionarily

conserved, there are species dependent variations in the function of CRP and ligand

binding specificity. In humans, CRP is involved in many parts of the APR; the most

important function seems to be in defense against bacterial infection and clearance of

necrotic and apoptotic cells (Volanakis 2001). It is also used as a marker for many health

complications including infection, trauma and autoimmune diseases (Pepys and

Hirschfield 2003).

The leukocytes of the crocodilian innate immune system include heterophils,

basophils, eosinophils and monocytes. Heterophils are the first cells at the site of

infection (Jacobson 2007). They suppress invasion of microbes and are involved in the

inflammatory response, serving the same function as neutrophils in mammals (Montali

1988). These cells account for over 50% of peripheral leukocytes in A.mississippiensis

and can be found in damaged tissue within hours (Mateo, Roberts, and Enright 1984b, a).

8

This percentage may vary as a result of other factors, such as seasonal differences, age or

infection (Glassman, Bennett, and Hazen 1981, Mateo, Roberts, and Enright 1984b).

Basophils comprise ~12% and eosinophils make up ~10% of circulating leukocytes in

crocodilians (Mateo, Roberts, and Enright 1984b). Basophils are involved in allergic

reactions (Sullivan and Locksley 2009). They have antigen-specific immunoglobulins on

the surface and release histamines when triggered by an antigen. The amount of

histamine released is dependent on the concentration of the antigen and the temperature

(Zimmerman, Vogel, and Bowden 2010). The function of eosinophils is most likely

similar to the function in mammals: producing free radicals and peroxide in response to

parasites (Coico and Sunshine 2015). In A.mississippiensis, eosinophils have been

detected in response to infection by leeches (Glassman, Holbrook, and Bennett 1979).

Lastly, monocytes make up ~1% (1.5+SD 0.3) of peripheral leukocytes in

A.mississippiensis (Mateo, Roberts, and Enright 1984b). In mammals, monocytes can

further differentiate into macrophages and myeloid dendritic cells (DCs) (Owen 2013).

All of these phagocytic cells release cytokines and serve an important function as antigen

presenting cells (APCs). APCs process then present antigens to T cells, serving as a link

between the innate and adaptive immune responses (Coico and Sunshine 2015, Finger

2012).

Reptilian adaptive immunity

As previously mentioned, the adaptive immune response depends on the innate

response. The initial, primary adaptive response ultimately results in clearance of the

9

pathogens and formation of an immunological memory. Memory cells are important in

the secondary response that occurs in the case of re-exposure to the same pathogen

(Owen et al., p 17). The two parts of the adaptive immune system are cell mediated

immunity and humoral immunity.

Reptilian adaptive immunity: cell-mediated

Reptiles are known to have functional T cells, the lymphocytes involved in cell-

mediated immunity (Burnham et al., 2005). T cells are divided into two major types:

cytotoxic T cells (TC) and T helper cells (TH) (Owen et al., p 40). Cytotoxic-like T cells

have been identified in reptiles; they function to kill infected host cells and abnormal

cells (Pitchappan & Muthukkaruppan, 1977; Coico et al., 2003). Studies have suggested

the presence of regulator TH cells in reptiles, however the exact function is unknown

(Pitchappan & Muthukkaruppan, 1977). In mammals, TH cells secrete cytokines which

recruit B cells (Owen et al., p 40). Reptiles do not have germinal centers and lymph

nodes, which provide a location for TH immune cell interactions, so these cells may have

a different function than seen in mammals (Zimmerman et al., 2009). Variation in the

strength of cell-mediated responses are seen between the sexes; females typically have a

stronger response (Ahmed et al., 1985).

Reptilian adaptive immunity: humoral

B cells, another type of lymphocyte, are responsible for antibody production

(Zimmerman, Vogel, and Bowden 2010). B cells are able to recognize antigens in their

10

natural state. When an antigen binds to a B cell, highly specific antibodies, with antigen

binding sites identical to the ones on the surface of the B cell, are secreted (Owen 2013).

The general antibody structure is the same in all jawed vertebrates, consisting of two

identical heavy chains joined to two identical light chains by a disulfide bond. The heavy

chain and the light chain both contain a constant region and a variable region. Antigen

binding sites are found on the variable regions of both the heavy and light chains and are

therefore important for protection against different pathogens (Zimmerman, Vogel, and

Bowden 2010). The variable region contains different gene segments that undergo

rearrangement, allowing for tremendous genetic diversity. The variable (V), diversity (D)

and joining (J) gene segments rearrange to make different variable regions on the heavy

chain; the V and D gene segments rearrange on the light chain (Owen 2013).

Crocodilians produce at least four immunoglobulins (Ig): IgM, IgD, IgY and IgA.

IgM is found in all jawed vertebrates (Pettinello and Dooley 2014). Found in high

concentrations in the blood and spleen, it is the first antibody produced by B cells and

activates complement (Pettinello and Dooley 2014, Zhang et al. 2017). IgM, IgA and

IgD are thought to be involved in mucosal immunity in reptiles, as in mammals (Iwata et

al. 2002, Pettinello and Dooley 2014). IgY is the main antibody of secondary defense and

is believed to be the precursor to IgG and IgE found in mammals (Flajnik 2018). IgY is

produced in larger quantities than IgM and is the main defense against infection (Warr,

Magor, and Higgins 1995).

There are two isoforms found in crocodilians (and some other reptiles): IgY and a

truncated form, IgY(∆Fc) (Figure 2) (Zhang et al. 2017). Both forms have two light

11

chain domains, with a molecular weight of 23.1kDa. IgY heavy chains have one variable

region and four constant regions, with a total molecular weight of 63kDa. IgY(∆Fc) has

one variable region and two constant regions, with a total molecular weight of 33.8kDa.

The total molecular weight of IgY is 180kDa and IgY(∆Fc) is 120kDa. The two isoforms

do not always serve the same function and not all organisms that have IgY have

IgY(∆Fc) (Warr, Magor, and Higgins 1995). IgY(∆Fc) lacks the receptor binding sites;

evidence suggests it is able to induce pathogen neutralization without inflammation (Li et

al. 2012).

12

Figure 2. The structure of the two isoforms of IgY. IgY(∆Fc) is a truncated form, with

only two constant regions on the heavy chain, versus the four constant regions on the IgY

heavy chain. Image taken from Warr, Magor, and Higgins 1995.

13

Potential role as sentinel species and amplifier host

The American alligator serves as a sentinel for habitat quality, specifically related

to environmental pollution in areas affected by anthropogenic activities (Tipton et al.

2017). Previous studies have confirmed that certain crocodilian responses directly relate

to environmental conditions (Mazzotti et al. 2009, Rice and Mazzotti 2004). Alligator

indicators rely on relative density determined by encounter rate, body condition and rate

of occupancy in alligator holes (Mazzotti et al. 2009).

Alligators have not been examined for their role in environmental pathogen

distribution or as a sentinel for the presence of various pathogens. They inhabit

environments known to harbor opportunistic pathogenic bacteria, yet even serious

injuries, including deep wounds and missing limbs, heal without signs of infections

(Merchant et al. 2003). Studies have found that crocodilians are able to live with

microbial infections with no physiological effects (Madsen 1993, Madsen et al. 1998,

Manolis et al. 1991).

There is also evidence that the American alligator may serve as an amplifying

host for West Nile Virus (WNV) (Jacobson et al. 2005). This is significant because there

have been multiple confirmed WNV outbreaks in farmed crocodilians worldwide

(Steinman et al. 2003, Debra et al. 2003, Jacobson et al. 2005). Most mammals serve as

dead end hosts, meaning the virus in the blood will not reach high enough levels to be

picked back up by other mosquitos (CDC, n.d.). However, samples from infected

alligators have had viremic levels above the infectious threshold for Culex pipiens and C.

quinquefasciatus mosquitoes; Culex mosquitos being the main disease vector (Jacobson

14

et al. 2005, Turell, O'Guinn, and Oliver 2000, Sardelis et al. 2001). Further studies are

necessary to fully understand the American alligator’s role in the spread of both bacterial

and viral diseases.

15

Chapter II: Humoral immune responses in the American alligator samples near

NASA, Florida

Introduction

Although the American alligator is the most studied of the extant crocodilians, the

reptilian immune system is not well understood (Brisbin 1986). Alligators have not been

examined for their role in environmental pathogen distribution, or as a sentinel for the

presence of various pathogens. They inhabit environments known to harbor opportunistic

pathogenic bacteria, yet they are often times able to heal serious injuries, including deep

wounds and missing limbs, without signs of infections (Merchant et al. 2003). Studies

have found that crocodilians living in their natural environment are able to live with

microbial infections with no physiological effects (Madsen 1993, Madsen et al. 1998,

Manolis et al. 1991)

This study investigates humoral immune responses in the Alligator

mississippiensis as an environmental monitor for emerging infectious diseases. The intent

is to correlate immune responses to various pathogens with the health of the environment

and establish a baseline of antibody titers against pathogens. This baseline will be used

to compare with responses in alligators throughout their distribution. This information

will allow A.mississippiensis to be used as a sentinel, for not only the overall health of the

environment, but also as a sentinel for the presence of various pathogens.

Lysozymes are antimicrobial enzymes that hydrolyze the membrane of both

Gram-positive and Gram-negative bacteria, resulting in osmotic lysis (G Buckland and C

Wilton 2000). Lysozyme activity can be measured through an enzymatic assay of

16

lysozyme activity. In this assay, Micrococcus lysodeikticus is added to the serum

samples. The lysozymes in the serum will lyse the bacterial cells, resulting in decreased

absorbance. The higher the lysozyme activity, the faster the absorbance will decrease.

C-reactive protein (CRP) is an important APP in all vertebrates (Lee et al. 2017). It is an

ancient acute phase protein, participating in complement activation and opsonisation of

pathogens, and is usually indicative of relative levels of systemic inflammation. CRP

levels can be semi-quantified using an ELISA based approach. Both lysozymes and

CRPs play an important role in immune response to bacteria. This study provides

baseline data that can be used to compare with levels in alligators throughout their

distribution.

IgY is the main immunoglobulin involved in defense against infection (Warr,

Magor, and Higgins 1995) in birds and reptiles. There are two isoforms found in

crocodilians (and some other reptiles) and birds: IgY and a truncated form, IgY(∆Fc)

(Figure 2) (Zhang et al. 2017). Both forms have two light chain domains, with a

molecular weight of 23.1kDa. IgY heavy chains have one variable region and four

constant regions, with a total molecular weight of 63kDa. IgY(∆Fc) has one variable

region and two constant regions, with a total molecular weight of 33.8kDa. The total

molecular weight of IgY is 180kDa and IgY(∆Fc) is 120kDa. The two isoforms do not

always serve the same function and not all organisms that have IgY, have IgY(∆Fc)

(Warr, Magor, and Higgins 1995). ELISAs can be used to quantify the level of

circulating antibodies in serum against pathogens. If the alligator has come into contact

with any of the pathogens that are immunogenic, there will be memory immune cells

17

against the pathogen from the adaptive immune response. Serum samples from 110

alligators collected throughout Florida, were screened for bacteria-specific antibodies to

select, ubiquitous, aquatic bacteria. It is important to look at serum titer levels for

potential correlation between location, gender, and size/age.

The nine bacteria used in this study were Vibrio cholera, V. vulnificus, V.

parahaemolyticus, V. anguillarum, Escherichia coli, Brevundimonas vesicularis,

Mycobacterium marinum, Erysipelothrix rhusiopathiae and Streptococcus agalactiae.

These bacteria were selected because they are abundant in aquatic environments and are

known human pathogens. V.cholera is the most infectious of the vibrio species.

V.cholera and V.parahaemolyticus attach copepods; one copepod can carry an infectious

dose V.cholera (Percival and Williams 2014, Huq et al. 1983). V.parahaemolyticus is

found in temperate coastal waters. It is the leading cause of foodborne illnesses and is

associated with raw shellfish and contaminated seafood consumption (Joseph, Colwell,

and Kaper 1982). V.anguillarum causes vibrosis in organisms inhabiting aquatic

environments (Frans et al. 2011). E.coli are a diverse group of bacteria that can be found

in aquatic environments; only some strains are pathogenic. Both E.coli and

V.parahaemolyticus have been isolated from A.mississippiensis (Russell and Herman

1970, Brown et al. 2001). B. vesicularis causes infection in immunocompromised

humans and is multi drug resistant (Shang et al. 2012).

M.marinum is able to infect all poikilothermic species and has been isolated from

crocodilians (Clark and Shepard 1963, Huchzermeyer and Van Wyk 2003). Many

reptiles are extremely susceptible to M.marinum infection, which often times become

18

systemic. Studies have found that the rate of infection is temperature dependent (Clark

and Shepard 1963). In humans, M.marinum causes a skin infection called “fish tank

granuloma” (Bouricha et al. 2014). E. rhusiopathiae can be a commensal or pathogenic

organism found in humans, domestic and wild animals (Wang, Chang, and Riley 2010).

It is possibly an environmental pathogen of concern near slaughterhouses and processing

plants on the water. S.agalactiae causes septicemia in a variety of aquatic organisms,

including crocodilians (Amborski et al. 1983, Bishop et al. 2007, Evans, Klesius, and

Shoemaker 2006, A. Plumb et al. 1974).

The objectives of this study were as follows:

1. Quantify lysozyme enzymatic activity in individual samples

2. Purify IgY from pooled alligator serum

3. Generate mouse polyclonal antisera against IgY

4. Western blot analysis to determine if whole serum recognized pathogen

antigens

5. Use enzyme linked immunosorbent assay (ELISA) to quantify the level of

circulating antibodies in serum against pathogens and relative titers

6. Purify CRP and generate mouse monoclonal antibody against CRP

7. Use ELISA based approach to semi-quantify circulating CRP in individual

samples

Humoral immune responses to various pathogens, along with CRP and lysozyme

activity can be correlated with the health of the environment. This study provides the

19

first baseline data and a proof of concept, to then compare responses throughout their

distribution. This information will allow A. mississippiensis to be used as a sentinel in

future studies.

20

Materials and Methods

Alligators serum samples

Blood samples were collected from 142 American alligators from sites in South

Carolina, including Yawkey Island. These samples were sent from Dr. Thomas

Rainwater and used for IgY purification. Blood samples were collected from 110

American alligators from sites near NASA, including Cape Canaveral and Merritt Island

National Wildlife Refuge in Florida. These samples were sent from Dr. John Bowden at

University of Florida. The samples used in this study were collected from various month

between 2009 and 2011 and include 74 males and 36 females.

Serum lysozyme activity

Modified, previously published methods were used to determine serum lysozyme

activity (Parry 1965, Burton et al. 2002). Micrococcus lysodeikticus solutions were

prepared immediately prior to use, using 50 mg of lyophilized cells and 100 mL of 0.1M

phosphate buffer (pH 5.9). Next, 25 μL of each plasma sample was added in

quadruplicates to a 96-well plate and 175 μL of Micrococcus lysodeikticus was added to

each sample. Absorbance was measured at 450nm immediately and after a 5 minute

incubation at room temperature. The change in absorbance was quantified by subtracting

the absorbance at 5 minutes from the immediate absorbance. It was previously

determined that 1 unit of activity is a change in optical density (OD) of 0.001 per minute

(Parry 1965).

21

Purification of IgY

Previous studies have indicated that IgY can be purified using the Protein-G from

streptococcal bacteria, which work by binding immunoglobulins (Work et al. 2015,

Rodgers, Toline, and Rice 2018). One Protein-G columns (Thermo-Fisher) were used to

purify the alligator serum in this study. The columns were equilibrated by running 10mL

of 0.10M tris (pH 8.0) through, followed by 10mL of 0.01M tris (pH 8.0). Serum

samples from each of the 12 individual alligators were compiled into one 15 mL tube

(1000 μL from each individual) followed by the addition of 1.2 ml of of 1 M tris (pH

8.0). The sample was centrifuged for 10 minutes at 1,000 x g then passed twice through

the Protein G column. The column was washed with 20 mL of 0.10 M tris (pH 8.0), then

20 mL of 0.01 M tris (pH 8.0). Lastly, 0.05 M glycine (pH 2.5) was used to elute IgY

into seven separate microfuge tubes containing 100 μL aliquots of 1.0 M tris (pH 8).

Generation of polyclonal antisera against immunoglobulin Y

Purified IgY was used to immunize six-week old female Balb/c mice, housed at

the Godley-Snell Animal Facility at Clemson University. All protocols used are in

accordance to Institutional Animal Care and Use Committee (IACUC) regulations. On

day 1, mice were administered a sub-cutaneous (s.c.) injection of 100 ug purified IgY in

0.9% saline containing TiterMax Gold adjuvant. Two weeks later, on day 14, the mice

received a second s.c. injection of 50 μg of purified IgY with Freud’s incomplete

adjuvant (FIA). Boosters of 50 μg of purified IgY were given at 21 day intervals - on

days 35, in saline via s.c. immunizations, on day 56 intraperitoneally. Five days after the

22

last immunization, slow lethal CO2 asphyxiation and bilaterial pneumothorax were used

to sacrifice the mice. After being sacrificed, the mice were bled to collect blood, which

was stored overnight at 4°C to allow time for clotting. The sample was then centrifuged

for 20 minutes at 10,000 x g, the serum was collected to be used as the crude polyclonal

antisera source. The specificity of the mouse antiserum against IgY was determined by

western blotting. Procedures previously described in other studies in our lab (Rodgers,

Toline, and Rice 2018).

Growth and preparation of bacteria

Samples of Vibrio cholera, Escherichia coli, V. anguillarum, V. vulnificus, V.

parahaemolyticus, and Mycobacterium marinum, were acquired from the American Type

Culture Collection and grown according to recommended protocols. Streptococcus

agalactiae cultures were obtained from John Hawke (Louisiana State University) and

were also grown as directed. 20 μL of each of the nine bacteria were inoculated into 5

mL of their specific growth medium. V.cholera and V.parahaemolyticus were inoculated

in nutrient broth with 3% NaCl, E.coli in Luria broth, V.angullarum in marine broth,

M.marinum in Middlebrook broth, and S.agalactiae into tryptic soy broth. The bacteria

were grown in separate 25 mL tubes at 37oC then centrifuged at 2,500 x g for 15 minutes.

The supernatant was poured off and 25 mL of 0.01 M PBS was added to the pellets, to

allow for re-suspension. The samples were centrifuged again at 2,500 x g for 15 minutes,

then washed again by resuspension with 25 mL of 0.01 M PBS and centrifuged under the

same conditions. Cell pellets were re-suspended in 5mL of PBS a final time. Thirty μL

23

of 5× Laemmli sample buffer with 2-mercaptoethanol was added to 120 μL of each

bacterial suspension, then boiled for 8 min. This was done to create reducing conditions

and generate bacterial lysates.

Serum Y reactivity with bacterial antigens

Twenty μL of each lysate were subjected to western blot analysis. A 4-20% Mini-

PROTEAN® TGX Stain-FreeTM Protein Gel (BioRad) was used for the SDS-PAGE.

First, 13 μL of Precision Plus ProteinTM KaleidoscopeTM Prestained Protein Standards

molecular weight marker (BioRad) was added to well 1. Next, 20 μL of the lysate

samples were added to wells 2-10 in the following order: V.cholerae, E.coli,

V.anguillarum, V.vulnificus, V.parahaemolyticus, M.marinum, and S.agalactiae. The gel

was run at 200V for approximately 40 minutes. The proteins on the gel were transferred

to a Immun-Blot® PVDF membrane at 100V for 1 hour. The blots were blocked for 16

hours with 10% fetal calf serum in 0.01M PBS at 4°C. Next, 50 μL of serum from the

pooled serum resource, was put in a 50 mL tube and diluted 1:500, in 25 mL of PBS.

The blot was incubated for 1 hour at room temperature with the diluted alligator serum

then washed three times for 5 minutes with PBS-TW20. Mouse polyclonal IgY antisera

was diluted 1:1000. The blots incubated in this solution for 1 hour at room temperature

followed by three, 5 minutes washes with PBS-TW20. The final incubation was with

goat anti-mouse IgG- AP (1:1,000 in PBS; Thermo-Fisher), following the same

incubation and wash procedure as the previous two steps. The blot was developed with

BCIP and NBT in AP buffer, allowing the AP activity to be visualized and recorded.

24

Development of enzyme linked immunosorbent assays (ELISA) to quantify relative

pathogen specific immunoglobulin Y titers in individual alligators

The seven aquatic bacteria mentioned previously were grown under the specific

conditions explained above. The supernatant was removed from each tube and 25 mL of

0.01 M PBS was added to re-suspend the pellet, this was repeated a second time. Once

the bacteria were re-suspended, the samples were diluted with PBS to optimal ODs that

had been previously established for 15 mL of total bacterial suspension. The optimal ODs

were: 0.200 for V.cholerae, E.coli, V.anguillarum, V.vulnificus, S.agalactiae, 0.1000 for

V.parahaemolyticus, and 0.050 for M.marinum.

High bonding 96-well plates (Medi0sorb, ThermoFisher) were coated with 75 μL

of 1 mg/mL solution of poly-D-lysine in distilled water, for 16 hours at 4 oC, then washed

with PBS-TW20. The optimized bacterial suspensions were then added to the plates; 50

μL of one bacterium was added to every well of three plates. This was repeated for all

nine bacteria, resulting in 27 coated plates, each coated with a single species. The plates

were centrifuged at 2,000 x g for 5 minutes, 50 μL of 0.5% glutaraldehyde was carefully

added to each well, then spun down again under the same settings. The plates incubated

at room temperature for 15 minutes then carefully washed (submerged) in PBS two times.

Next, 100 μL of 10 % BSA blocking buffer was added to each of the wells, followed by a

two hour incubation at room temperature. Each plate was washed three more times with

PBS-TW20. The plates were left out to dry for two hours then stored in plastic wrap at

room temperature for 36 hours.

25

Serum suspensions were prepared using a 100-fold dilution of serum from each

individual alligator into PBS in individual 1.5 mL microfuge tubes. A reference sample

for calculating relative titers was made from a composite sample, comprised of 50 μL of

serum from 6 randomly selected individuals in 2.7 mL of PBS. The composite serum

was added in duplicate to each plate, as a reference for variation from plate-to-plate.

Diluted serums were added to the wells, 75 μL in duplicate. The plates incubated for 16

hours at 4 oC, then washed three times with PBS-TW20.

Mouse polyclonal anti-alligator IgY was added to the wells in 75 μL volumes and

incubated overnight at 4 degrees C. The plates were then washed three times with PBS-

TW20, then 75 μL of goat anti-mouse IgG-AP (1:1,000) was added to the respective

wells and incubated for 2 hours at room temperature. The plates were washed again, four

times, with PBS-TW20. Lastly, 75 μL of 1mg/mL of p-nitrophenol (Fisher) in AP buffer

was pipetted into the wells and left to develop for 30 minutes. The plates were read at 405

nm and the data for each plate was recorded.

Generation of monoclonal antibody against CRP

To isolate CRP, plasma samples from several alligators were pooled and

centrifuged at 1000 rpm at 4 oC for 10 min. The supernatant was centrifuged again at

12,000g at 4 oC for 15 min. Following the methods described by Robey et al., and

modified by Karsten and Rice, the supernatant was brought to room temperature and

passed though an AH-Sepharose 4B-PC column (equilibrated in a 0.15 M NaCl, 0.05 M

Tris, pH 7.4, 0.05 M CaCl2 buffer solution) (Robey, Tanaka, and Liu 1983, Karsten and

26

Rice 2004). Alligator CRP and serum amyloid-P component (SAP) were eluted from the

column using a 0.15 M NaCl, 0.05 M Tris, 0.1 M EDTA, pH 7.4, buffer solution. To

separate alligator CRP from SAP, the eluent was dialyzed against the 0.15 M NaCl, 0.05

M Tris, pH 7.4, CaCl2 buffer solution and applied to a Sepharose CL-4B column

(equilibrated in a 0.15 M NaCl, 0.05 M Tris, pH 7.4, 0.05 M CaCl2 buffer solution). CRP

was collected and subjected to SDS–PAGE and subsequent Coomassie blue staining to

check for purity. Coomassie blue staining of the gel showed a band corresponding to

alligator CRP (at 25 kDa) in the CRP purified sample. Balb/c mice were immunized with

the putative CRP sample over a 51-day period and bled for polyclonal anti-sera, as well

as to collect spleens for subsequent steps in fusion steps for making mAbs. Fusion of

splenic plasma cells with Sp02-14 myelomas, screening, cloning, purification and

isotyping of the antibodies was done according to methods described elsewhere

(Margiotta, Bain, and Rice 2017, Rice and Weeks 1989). Western plot analysis

confirmed that the mouse anti-alligator mAb recognizes and is specific to putative CRP.

Semi quantification of circulating CRP levels using ELISA based approach

Methods previously developed in the lab (a quantitative capture ELISA) were

used to quantify the circulating CRP in alligator serum (Karsten and Rice 2004). This

involved the use of purified CRP and mouse anti-alligator mAb.

27

Statistical analysis

Lysozyme enzymatic activity, antibody titers against bacteria and CRP data were

analyzed using Analysis of Variance (ANOVA), followed by Bonferroni post hoc,

multiple comparison test using GraphPad7.

28

Results

An increase in lysozyme activity was seen during summer months. There were

significant differences (P<0.05, P<0.01, P<0.001) in serum lysozyme activity between

samples collected in in March / April, May, June, July, August and September of 2010

(Figure 3).

The Protein G purification technique was successful in producing pure

A.mississippiensis IgY (Figure 4). Clear bands are present at approximately 63 kDa,

33.8 kDa and 23 kDa. The band at 63 kDa corresponds to the heavy chain of IgY, 7S

IgY, and the band at 33.8 kDa corresponds to 5.7S IgY, the heavy chain of IgY(∆Fc).

The band near 25 kDa corresponds to the light chain of both isoforms of IgY, which is

23.1 kDa. No other strong bands appear on the gel. This indicates that IgY was

successfully purified.

Using pooled serum samples as the probe and the pAb anti-IgY for detection

source of antibodies, distinct bands were seen for E.coli, V.vulnificus,

V.parahaemolyticus, and S.agalactiae (Figure 5). Minor bands of proteins were

recognized in other bacterial lysates as well. This indicates that there are circulating

antibodies against the bacteria in the American alligators used in this study.

Statistical analysis of the serum titer levels against seven bacteria indicate

significant differences between titer levels collected during different months. There was

a statistically significant difference (P <0.001) in the quantity of titers against E.coli in

the summer months compared to the winter and spring (Figure 6). Titer levels against

E.coli are increased in the summer and decrease in the winter. M.marinum titers

29

decreased in serum samples from August and September 2010. Significant differences

(P<0.05, P<0.001) were seen between samples collected in July 2010, August 2010,

September 2010, the Sum of 2009 and the rest of the samples collected earlier in 2010

(Figure 7). S.agalactae titers were highest in January 2010. There was a significant

decrease (p<0.01) in titer levels against this bacteria in months sampled between

February 2010 and August 2010 when compared to titer levels in January 2010 (Figure

8). There were no significant differences in titer levels against V. vulnificus and V.

anguillarium between sampling months (Figure 9, Figure 10). V.parahaemoliticus titers

also increased during summer months. There is a significant difference (P<0.001)

between titers in samples collected in March / April 2010, May 2010, June 2010, July

2010 and the rest of the sampling months (Figure 11). V.cholera titers were significantly

different (P<0.001) in June 2010, July 2010 and August 2010 compared to the rest of the

months sampled (Figure 12).

Analysis of Enzyme linked immunosorbent assay (ELISA) for relative putative

CRP levels for the American alligator indicates a significant increase (P<0.05) in samples

collected in June 2010 compared to those collected in March / April 2010 (Figure 13).

30

Figure 3. Comparison of American alligator average lysozyme activity between various

months of serum collection. Statistical differences were seen in March / April, May,

June, July, and August of 2010. *Indicates statistical significance of <0.05; ** indicates

statistical significance of p<0.01; ***indicates statistical significance of <0.001.

S e ru m L y s o z y m e A c t iv ity

M o n th /Y e a r S a m p lin g

Ly

so

zy

me

ac

tiv

ity

(U

nit

s)

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

2009

0

5

1 0

1 5

2 0

_ _ _ _ _ _ _ _ _ _ _ _*/**/****

31

Figure 4. SDS-PAGE gel with purified IgY from the American alligator. Lane 1 shows

the molecular weight marker (Kaleidoscope, BioRad), lane 2 is the sample. Clear bands

are seen around 63 kDa, 33.8 kDa and 23 kDa; 63 kDa corresponds to the IgY heavy

chain, 7S IgY. The band around 33.8 kDa corresponds to the heavy chain of IgY(∆Fc),

5.7S IgY. The band near 25 kDa corresponds to the light chain of both isoforms of IgY,

which is 23 kDa.

MW IgY

32

Figure 5. Western blot analysis showing A.mississippiensis whole serum (diluted 1:500)

activity against bacteria lysates using pAB anti-IgY. Lane 1 contains the molecular

weight marker (Kaleidoscope, BioRad). Lanes 2-9 contain the bacterial lysates in the

following order: V.cholerae, E.choli, V.anguillarum, V.vulnificus, V.parahaemolyticus,

M.marinum, E.rhusiopathiae, S.agalactiae, B.vesicularis.

MW Vc Ec Va Vv Vp Mm Er Sa Bv

33

Figure 6. Enzyme linked immunosorbent assay (ELISA) results against E.coli.

Statistical analysis indicates significant differences (p<0.001) between samples collected

in June 2010, July 2010, August 2010 and September 2010.

E c o li t ite r s

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3 _ _ _ _ _ _ _ _ _ _***

34

Figure 7. Enzyme linked immunosorbent assay (ELISA) results against M.marinum.

There is a significant difference between samples collected in July, August, September

from 2010 and the sum of 2009. *Indicates statistical significance of <0.05; ***indicates

statistical significance of <0.001.

M . m a r in u m t it e r s

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3

4

5

_ _ _ _ _ _ _ _*/***

35

Figure 8. Enzyme linked immunosorbent assay (ELISA) results against S.agalactae.

Statistical analysis indicates significant differences between serum titers against bacteria

collected in January, March / April, May, June, July and August 2010. *Indicates

statistical significance of <0.05; ** indicates statistical significance of <0.01.

S . a g a la c ta e t i te r s

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3

4_ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _

*

**

36

Figure 9. Enzyme linked immunosorbent assay (ELISA) results against V.vulnificus. No

significant differences were seen between months of collection.

V . v u ln if ic u s t ite rs

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3

4

5

37

Figure 10. Enzyme linked immunosorbent assay (ELISA) results against V.anguillarium.

No significant differences were seen between months of collection.

V . a n g u il la r iu m t i te r s

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3

4

5

38

Figure 11. Enzyme linked immunosorbent assay (ELISA) results against

V.parahaemoliticus. Statistical analysis indicates there are significant differences

(p<0.001) between samples collected in March / April, May, June, and July of 2010.

V . p a ra h a e m o li t ic u s t i te r s

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3

4

5

_ _ _ _ _ _ _ _ _ _***

39

Figure 12. Enzyme linked immunosorbent assay (ELISA) results against V.cholerae.

Statistical analysis indicates there are significant differences (p<0.001) between samples

collected in June, July, and August of 2010.

V . c h o le ra e t i te rs

M o n th /Y e a r S a m p lin g

Re

lati

ve

Tit

er x

10

2

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

Su

m 2

009

0

1

2

3

4 _ _ _ _ _ _ _ _***

40

Figure 13. Enzyme linked immunosorbent assay (ELISA) showing relative CRP levels

for the American alligator. Statistical analysis indicates significant differences (p< 0.05)

between samples collected in March / April, and May of 2010.

C R P

M o n th /Y e a r S a m p lin g

Re

lati

ve

CR

P l

ev

els

Jan

2010

Feb

2010

Mar

Ap

r 2010

May 2

010

Ju

ne 2

010

Ju

ly 2

010

Au

g 2

010

Sep

t 2010

2009

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

_ _ _ _ _ _*

41

Discussion

This study provides the first baseline data for serum parameters in the American

alligator. Lysozyme activity and CRP levels increase in alligators during summer months.

Seasonal variation is also seen between serum titers against the seven select pathogens

during the sampling months. Lysozyme activity, CRP levels and circulating serum titers

levels against bacteria can be used to compare responses in alligators throughout their

distribution. This information will allow A.mississippiensis to be used as a sentinel for

various pathogens in future studies.

Comparison of American alligator average lysozyme activity between various

months of serum collection indicated that there are seasonal differences. There was a

significant difference between lysozyme activity in summer months compared to the rest

of the year. During summer months, water temperatures increases with the increase in air

temperature, often resulting in increased pathogen loads in the environment. It is

possible that the increase in lysozyme activity is in response to the increase in bacteria in

their environment, as an increase in lysozyme activity is associated with increased ability

to lyse bacteria. Lysozymes play an important role in the innate response to bacteria, as

they are able to hydrolyze the membrane of both Gram-positive and Gram-negative

bacteria, resulting in osmotic lysis.

The results of this study also show that it is possible to purify IgY from alligator

serum through a Protein G purification process. Our lab has previously purified IgY

from sea turtle serum with protein A/G columns (Rodgers, Toline, and Rice 2018). The

purity of the IgY was confirmed; the sample contained the heavy chain, and light chain of

42

both IgY and IgY(∆Fc). The Protein G purification technique is simple and inexpensive

compared to previous methods used to purify IgY, which involved purifying IgY from

eggs instead of from serum. The ability to be able to purify IgY from alligator serum is

important because it can be used to generate both polyclonal antisera and monoclonal

antibodies against these different parts of alligator IgY.

The alligator anti-IgY pAb was used as the probe in a western blot analysis of

alligator serum response to bacteria. The results indicate that the alligators produced

circulating antibodies against the seven different bacteria. This indicates that the

individuals were exposed to the bacteria at high enough levels to elicit an immune

response, at some point in their lifetime. The range of banding seen on the blot indicates

that the polyclonal antisera can successfully be used for detection.

Significant differences were observed in the serum titers against the bacteria

between sampling months, as indicated in the data from the ELISAs used to quantify the

levels. There was an increase in the quantity of serum titers in the spring and the summer

and a decrease in the winter in E.coli, V.parahaemoliticus and V.cholera. S.agalactae

titers were highest in January 2010; the titers remained relatively consistent in the other

months sampled. M.marinum titers decreased in serum samples from August and

September 2010. There were no significant differences in titer levels against V. vulnificus

and V. anguillarium from month to month. These results indicate that there are changes in

titers against various bacteria depending on the sampling month. This study serves as a

proof of concept, showing that that it is possible to successfully quantify circulating

antibodies in alligator serum using the reagents made in this lab.

43

The ability to generate monoclonal antibodies against alligator CRP and

polyclonal antisera against alligator IgY allows for immunological analyses, such as

Western blotting and ELISAs, to be performed on alligator serum samples. The

production of a mAb against CRP allows for an unlimited supply of antibodies that can

continue to be used in these immunological assays because the hybridoma will continue

to produce the mAbs indefinitely. This will allow for further studies to be done on

alligators in other laboratories.

Analysis of circulating CRP levels (using an ELISA) showed an increase during

summer months. There were significant differences between CRP levels in samples

collected in March / April 2010, May 2010 and June 2010. CRP is important in the acute

phase response, participating in complement activation and opsonisation of pathogens,

and is usually indicative of relative levels of systemic inflammation. Increased levels of

CRP during warmer months is likely in response to increased pathogen loads in the

warmer waters.

The observed seasonal differences in immune responses are likely a result of

temperature changes. Previous studies have found that immune function in reptiles is

directly related to environmental temperature (Lance 2003, Lang and Andrews 1994).

When exposed to extreme temperatures, reptiles undergo a physiological stress response.

Studies have found that immunoglobulin production decreases in response to cold

weather (Zapata, Varas, and Torroba 1992). Water temperature has an effect on

haematological values in other crocodilians, further demonstrating how temperature can

play a role in immunosuppression (Turton et al. 1997). Phagocytic activity of

44

macrophages and the amount of histamine released by basophils can also be affected by

temperature (Mondal and Rai 2001, Zimmerman, Vogel, and Bowden 2010).

These findings serve as a proof of concept that the generated reagents are

functional and the immunological assays can be used to determine CRP, lysozyme and

circulating serum titer levels against bacteria using serum samples. The results provide

the first baseline data for serum parameters in the American alligator. This study is

important because it adds to our understanding of the reptilian immune system, by

showing that there are seasonal changes in CRP, lysozyme and serum titers to various

bacteria in the A.mississippiensis. This study should be continued with a larger sample

size, including samples from various geographic locations and size / age groups. Once a

larger sample size is obtained, analysis of immune responses between location, time of

year collected, sex and size of organism (using snout vent length) should be further

investigated. This will provide enough data to determine set baseline parameters for

comparison throughout the alligator’s distribution in the United States. This information

will allow for better understanding of the reptilian immune system, which is essential in

understanding the evolution of vertebrate immune system function.

45

References

A. Plumb, J., J. H. Schachte, J. L. Gaines, W. Peltier, and B. Carroll. 1974. Streptococcus

sp. from Marine Fishes Along the Alabama and Northwest Florida Coast of the

Gulf of Mexico. Vol. 103.

Amborski, R. L., T. G. Snider, 3rd, R. L. Thune, and D. D. Culley, Jr. 1983. "A non-

hemolytic, group B Streptococcus infection of cultured bullfrogs, Rana

catesbeiana, in Brazil." J Wildl Dis 19 (3):180-4.

Beard, DB. 1938. "Everglades National Park Project: Wildlife Reconnaissance." US

Department of Interior, National Park Service. Washington, DC 106.

Bishop, E. J., C. Shilton, S. Benedict, F. Kong, G. L. Gilbert, D. Gal, D. Godoy, B. G.

Spratt, and B. J. Currie. 2007. "Necrotizing fasciitis in captive juvenile

Crocodylus porosus caused by Streptococcus agalactiae: an outbreak and review

of the animal and human literature." Epidemiology and infection 135 (8):1248-

1255. doi: 10.1017/S0950268807008515.

Bouricha, Mehdi, Bernard Castan, Elisabeth Duchene-Parisi, and Michel Drancourt.

2014. "Mycobacterium marinum infection following contact with reptiles:

vivarium granuloma." International Journal of Infectious Diseases 21:17-18. doi:

https://doi.org/10.1016/j.ijid.2013.11.020.

Brandt, Laura A. . 1989. "The status and ecology of the American alligator (Alligator

mississippiensis) in Par Pond, Savannah River site."Dissertation, Biological

Sciences, FIU.

Brisbin, I. Lehr. 1986. A bibliography of the American alligator (Alligator

mississippiensis). Edited by Savannah River Plant National Environmental

Research Park.

Brown, D R, J M Farley, L A Zacher, J M Carlton, T L Clippinger, J G Tully, and M B

Brown. 2001. "Mycoplasma alligatoris sp. nov., from American alligators."

International Journal of Systematic and Evolutionary Microbiology 51 (2):419-

424. doi: doi:10.1099/00207713-51-2-419.

Burton, J. E., I. R. Dorociak, T. E. Schwedler, and C. D. Rice. 2002. "Circulating

lysozyme and hepatic CYP1A activities during a chronic dietary exposure to

tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures in

channel catfish, Ictalurus punctatus." J Toxicol Environ Health A 65 (8):589-602.

doi: 10.1080/152873902317349745.

Clark, H. F., and C. C. Shepard. 1963. "EFFECT OF ENVIRONMENTAL

TEMPERATURES ON INFECTION WITH MYCOBACTERIUM MARINUM

46

(BALNEI) OF MICE AND A NUMBER OF POIKILOTHERMIC SPECIES." J

Bacteriol 86:1057-69.

Coico, R., and G. Sunshine. 2015. Immunology: A Short Course: Wiley.

Craighead, Frank Cooper. 1969. The role of the alligator in shaping plant communities

and maintaining wildlife in the southern Everglades: Florida Audubon Society.

Cray, Carolyn, Julia Zaias, and Norman H. Altman. 2009. "Acute phase response in

animals: a review." Comparative medicine 59 (6):517-526.

Debra, L. Miller, J. Mauel Michael, Baldwin Charles, Burtle Gary, Ingram Dallas, E.

Hines Murray, and S. Frazier Kendal. 2003. "West Nile Virus in Farmed

Alligators." Emerging Infectious Disease journal 9 (7):794. doi:

10.3201/eid0907.030085.

Dzik, J. M. 2010. "The ancestry and cumulative evolution of immune reactions." Acta

Biochim Pol 57 (4):443-66.

Evans, Joyce J., Phillip H. Klesius, and Craig A. Shoemaker. 2006. "Therapeutic and

prophylactic immunization against Streptococcus iniae infection in hybrid striped

bass (Morone chrysops x Morone saxatilis)." Aquaculture research 2006 v.37

no.7 (no. 7):pp. 742-750. doi: 10.1111/j.1365-2109.2006.01487.x.

Ferguson, Mark W. J., and Ted Joanen. 1982. "Temperature of egg incubation determines

sex in Alligator mississippiensis." Nature 296 (5860):850-853. doi:

10.1038/296850a0.

Finger, J. 2012. "A review of innate immune functions in crocodilians." CAB Reviews:

Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources

7 (067). doi: 10.1079/pavsnnr20127067.

Flajnik, M. F. 2018. "A cold-blooded view of adaptive immunity." Nat Rev Immunol 18

(7):438-453. doi: 10.1038/s41577-018-0003-9.

Frans, I., C. W. Michiels, P. Bossier, K. A. Willems, B. Lievens, and H. Rediers. 2011.

"Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and

prevention." J Fish Dis 34 (9):643-61. doi: 10.1111/j.1365-2761.2011.01279.x.

G Buckland, Andrew, and David C Wilton. 2000. The antibacterial properties of secreted

phospholipases A2. Vol. 1488.

Glassman, A. B., T. W. Holbrook, and C. E. Bennett. 1979. "Correlation of leech

infestation and eosinophilia in alligators." J Parasitol 65 (2):323-4.

47

Glassman, Armand B., Carol E. Bennett, and Terry C. Hazen. 1981. "Peripheral Blood

Components in Alligator mississippiensis." Transactions of the American

Microscopical Society 100 (2):210-215. doi: 10.2307/3225803.

Guillette, L. J., Jr., T. S. Gross, G. R. Masson, J. M. Matter, H. F. Percival, and A. R.

Woodward. 1994. "Developmental abnormalities of the gonad and abnormal sex

hormone concentrations in juvenile alligators from contaminated and control

lakes in Florida." Environmental health perspectives 102 (8):680-688. doi:

10.1289/ehp.94102680.

Huchzermeyer, F., and W. Van Wyk. 2003. Crocodiles – Biology, husbandry and

diseases. Vol. 74.

Huq, A., E. B. Small, P. A. West, M. I. Huq, R. Rahman, and R. R. Colwell. 1983.

"Ecological relationships between Vibrio cholerae and planktonic crustacean

copepods." Appl Environ Microbiol 45 (1):275-83.

Iwata, A., T. Iwase, Y. Ogura, T. Takahashi, N. Matsumoto, T. Yoshida, N. Kamei, K.

Kobayashi, J. Mestecky, and I. Moro. 2002. "Cloning and expression of the turtle

(Trachemys scripta) immunoglobulin joining (J)-chain cDNA." Immunogenetics

54 (7):513-9. doi: 10.1007/s00251-002-0492-2.

Jacobson, E. R., P. E. Ginn, J. M. Troutman, L. Farina, L. Stark, K. Klenk, K. L.

Burkhalter, and N. Komar. 2005. "West Nile virus infection in farmed American

alligators (Alligator mississippiensis) in Florida." J Wildl Dis 41 (1):96-106. doi:

10.7589/0090-3558-41.1.96.

Jacobson, Elliott. 2007. Infectious Diseases and Pathology of Reptiles: A Color Atlas and

Text.

Joseph, Sam W., Rita R. Colwell, and James B. Kaper. 1982. "Vibrio Parahaemolyticus

and Related Halophilic Vibrios." CRC Critical Reviews in Microbiology 10

(1):77-124. doi: 10.3109/10408418209113506.

Karsten, A. H., and C. D. Rice. 2004. "c-Reactive protein levels as a biomarker of

inflammation and stress in the Atlantic sharpnose shark (Rhizoprionodon

terraenovae) from three southeastern USA estuaries." Mar Environ Res 58 (2-

5):747-51. doi: 10.1016/j.marenvres.2004.03.089.

Keller, J. M., P. D. McClellan-Green, A. M. Lee, M. D. Arendt, P. P. Maier, A. L.

Segars, J. D. Whitaker, D. E. Keil, and M. M. Peden-Adams. 2005. "Mitogen-

induced lymphocyte proliferation in loggerhead sea turtles: comparison of

methods and effects of gender, plasma testosterone concentration, and body

condition on immunity." Vet Immunol Immunopathol 103 (3-4):269-81. doi:

10.1016/j.vetimm.2004.09.029.

48

Kruman, II. 1992. "Comparative analysis of cell replacement in hibernators." Comp

Biochem Physiol A Comp Physiol 101 (1):11-8.

Kvell, K., E. L. Cooper, P. Engelmann, J. Bovari, and P. Nemeth. 2007. "Blurring

borders: innate immunity with adaptive features." Clin Dev Immunol

2007:83671. doi: 10.1155/2007/83671.

Lance, V. A. 2003. "Alligator physiology and life history: the importance of

temperature." Exp Gerontol 38 (7):801-5.

Lang, Jeffrey W., and Harry V. Andrews. 1994. "Temperature-dependent sex

determination in crocodilians." Journal of Experimental Zoology 270 (1):28-44.

doi: 10.1002/jez.1402700105.

Lee, P. T., S. Bird, J. Zou, and S. A. M. Martin. 2017. "Phylogeny and expression

analysis of C-reactive protein (CRP) and serum amyloid-P (SAP) like genes

reveal two distinct groups in fish." Fish Shellfish Immunol 65:42-51. doi:

10.1016/j.fsi.2017.03.037.

Li, Lingxiao, Wang Tao, Yi Sun, Gang Cheng, Hui Yang, Zhiguo Wei, Ping Wang,

Xiaoxiang hu, Liming Ren, Qingyong Meng, Ran Zhang, Ying Guo, Lennart

Hammarström, Ning Li, and Yaofeng Zhao. 2012. Extensive Diversification of

IgD-, IgY-, and Truncated IgY(Delta Fc)-Encoding Genes in the Red-Eared Turtle

(Trachemys scripta elegans). Vol. 189.

Madsen, M., P. Hangartner, K. West, and P. Kelly. 1998. "Recovery rates, serotypes, and

antimicrobial susceptibility patterns of salmonellae isolated from cloacal swabs of

wild Nile crocodiles (Crocodylus niloticus) in Zimbabwe." J Zoo Wildl Med 29

(1):31-4.

Madsen, Mogens. 1993. "Microbial flora of frozen tail meat from captive Nile crocodiles

(Crocodylus niloticus)." International Journal of Food Microbiology 18 (1):71-

76. doi: https://doi.org/10.1016/0168-1605(93)90009-6.

Manolis, S. C., G. J. Webb, D. Pinch, L. Melville, and G. Hollis. 1991. "Salmonella in

captive crocodiles (Crocodylus johnstoni and C. porosus)." Aust Vet J 68 (3):102-

5.

Margiotta, A. L., L. J. Bain, and C. D. Rice. 2017. "Expression of the Major Vault

Protein (MVP) and Cellular Vault Particles in Fish." Anat Rec (Hoboken) 300

(11):1981-1992. doi: 10.1002/ar.23645.

Mateo, M. R., E. D. Roberts, and F. M. Enright. 1984a. "Inflammation induced by

subcutaneous turpentine inoculation of young American alligators (Alligator

mississippiensis)." Am J Vet Res 45 (9):1870-5.

49

Mateo, M. R., E. D. Roberts, and F. M. Enright. 1984b. "Morphologic, cytochemical, and

functional studies of peripheral blood cells of young healthy American alligators

(Alligator mississippiensis)." Am J Vet Res 45 (5):1046-53.

Mazzotti, Frank J., G. Ronnie Best, Laura A. Brandt, Michael S. Cherkiss, Brian M.

Jeffery, and Kenneth G. Rice. 2009. "Alligators and crocodiles as indicators for

restoration of Everglades ecosystems." Ecological Indicators 9 (6,

Supplement):S137-S149. doi: https://doi.org/10.1016/j.ecolind.2008.06.008.

Merchant, M. E., C. M. Roche, D. Thibodeaux, and R. M. Elsey. 2005. "Identification of

alternative pathway serum complement activity in the blood of the American

alligator (Alligator mississippiensis)." Comp Biochem Physiol B Biochem Mol

Biol 141 (3):281-8. doi: 10.1016/j.cbpc.2005.03.009.

Merchant, M., D. Thibodeaux, K. Loubser, and R. M. Elsey. 2004. "Amoebacidal effects

of serum from the American alligator (Alligator mississippiensis)." J Parasitol

90 (6):1480-3. doi: 10.1645/ge-3382.

Merchant, Mark E., Cherie Roche, Ruth M. Elsey, and Jan Prudhomme. 2003.

"Antibacterial properties of serum from the American alligator (Alligator

mississippiensis)." Comparative Biochemistry and Physiology Part B:

Biochemistry and Molecular Biology 136 (3):505-513. doi: 10.1016/s1096-

4959(03)00256-2.

Mondal, S., and U. Rai. 2001. "In vitro effect of temperature on phagocytic and cytotoxic

activities of splenic phagocytes of the wall lizard, Hemidactylus flaviviridis."

Comp Biochem Physiol A Mol Integr Physiol 129 (2-3):391-8.

Montali, R. J. 1988. "Comparative pathology of inflammation in the higher vertebrates

(reptiles, birds and mammals)." J Comp Pathol 99 (1):1-26.

Owen, J. 2013. Immunology: W. H. Freeman.

Parry, R. M., R. C. Chandan, and K. M. Shahani. . 1965. "A rapid and sensitive assay of

muramidase. Experimental Biology and Medicine " Experimental Biology and

Medicine 119:384–386.

Pepys, M. B., and G. M. Hirschfield. 2003. "C-reactive protein: a critical update." J Clin

Invest 111 (12):1805-12. doi: 10.1172/JCI18921.

Percival, Steven L., and David W. Williams. 2014. "Chapter Twelve - Vibrio." In

Microbiology of Waterborne Diseases (Second Edition), edited by Steven L.

Percival, Marylynn V. Yates, David W. Williams, Rachel M. Chalmers and

Nicholas F. Gray, 237-248. London: Academic Press.

50

Pettinello, R., and H. Dooley. 2014. "The immunoglobulins of cold-blooded vertebrates."

Biomolecules 4 (4):1045-69. doi: 10.3390/biom4041045.

Rice, Charles D., and Beverly Anne Weeks. 1989. "Influence of Tributyltin on in Vitro

Activation of Oyster Toadfish Macrophages." Journal of Aquatic Animal Health

1 (1):62-68. doi: 10.1577/1548-8667(1989)001<0062:IOTOIV>2.3.CO;2.

Rice, Kenneth G., and Frank Mazzotti. 2004. American alligator ecology and monitoring

for the Comprehensive Everglades Restoration Plan. In Fact Sheet.

Robey, F A, T Tanaka, and T Y Liu. 1983. "Isolation and characterization of two major

serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid

P component." Journal of Biological Chemistry 258 (6):3889-3894.

Rodgers, M. L., C. A. Toline, and C. D. Rice. 2018. "Humoral Immune Responses to

Select Marine Bacteria in Loggerhead Sea Turtles Caretta caretta and Kemp's

Ridley Sea Turtles Lepidochelys kempii from the Southeastern United States." J

Aquat Anim Health 30 (1):20-30. doi: 10.1002/aah.10012.

Russell, WC , and KL Herman. 1970. "Colibacillosis in captive wild animals." Journal

of Zoo Animal Medicine (1):17-21.

Sardelis, M. R., M. J. Turell, D. J. Dohm, and M. L. O'Guinn. 2001. "Vector competence

of selected North American Culex and Coquillettidia mosquitoes for West Nile

virus." Emerg Infect Dis 7 (6):1018-22. doi: 10.3201/eid0706.010617.

Schechter, B., and R. Street. 2000. ""Alligator mississippiensis"." University of

Michigan, accessed March 1.

Shang, Shih-Ta, Sheng-Kang Chiu, Ming-Ching Chan, Ning-Chi Wang, Ya-Sung Yang,

Jung-Chung Lin, and Feng-Yee Chang. 2012. "Invasive Brevundimonas

vesicularis bacteremia: Two case reports and review of the literature." Journal of

Microbiology, Immunology and Infection 45 (6):468-472. doi:

https://doi.org/10.1016/j.jmii.2011.12.021.

Smith, E. Norbert. 1975. "Thermoregulation of the American Alligator, Alligator

mississippiensis." Physiological Zoology 48 (2):177-194.

Song, W. C., M. R. Sarrias, and J. D. Lambris. 2000. "Complement and innate

immunity." Immunopharmacology 49 (1-2):187-98.

Steinman, Amir, Caroline Banet-Noach, Shlomit Tal, Ohad Levi, Lubov Simanov,

Shimon Perk, Mertyn Malkinson, and Nahum Shpigel. 2003. "West Nile virus

infection in crocodiles." Emerging infectious diseases 9 (7):887-889. doi:

10.3201/eid0907.020816.

51

Sullivan, B. M., and R. M. Locksley. 2009. "Basophils: a nonredundant contributor to

host immunity." Immunity 30 (1):12-20. doi: 10.1016/j.immuni.2008.12.006.

Sunyer, J. O., I. K. Zarkadis, and J. D. Lambris. 1998. "Complement diversity: a

mechanism for generating immune diversity?" Immunol Today 19 (11):519-23.

Tipton, Jessica J., Louis J. Guillette, Susan Lovelace, Benjamin B. Parrott, Thomas R.

Rainwater, and Jessica L. Reiner. 2017. "Analysis of PFAAs in American

alligators part 2: Potential dietary exposure of South Carolina hunters from

recreationally harvested alligator meat." Journal of Environmental Sciences

61:31-38. doi: https://doi.org/10.1016/j.jes.2017.05.046.

Trouw, L. A., and M. R. Daha. 2011. "Role of complement in innate immunity and host

defense." Immunol Lett 138 (1):35-7. doi: 10.1016/j.imlet.2011.02.014.

Turell, M. J., M. O'Guinn, and J. Oliver. 2000. "Potential for New York mosquitoes to

transmit West Nile virus." Am J Trop Med Hyg 62 (3):413-4.

Turton, J. A., P. W. Ladds, S. C. Manolis, and G. J. Webb. 1997. "Relationship of blood

corticosterone, immunoglobulin and haematological values in young crocodiles

(Crocodylus porosus) to water temperature, clutch of origin and body weight."

Aust Vet J 75 (2):114-9.

Valenzuela, N., R. Literman, J. L. Neuwald, B. Mizoguchi, J. B. Iverson, J. L. Riley, and

J. D. Litzgus. 2019. "Extreme thermal fluctuations from climate change

unexpectedly accelerate demographic collapse of vertebrates with temperature-

dependent sex determination." Sci Rep 9 (1):4254. doi: 10.1038/s41598-019-

40597-4.

Volanakis, J. E. 2001. "Human C-reactive protein: expression, structure, and function."

Mol Immunol 38 (2-3):189-97.

Wang, Q., B. J. Chang, and T. V. Riley. 2010. "Erysipelothrix rhusiopathiae." Vet

Microbiol 140 (3-4):405-17. doi: 10.1016/j.vetmic.2009.08.012.

Warr, G. W., K. E. Magor, and D. A. Higgins. 1995. "IgY: clues to the origins of modern

antibodies." Immunol Today 16 (8):392-8. doi: 10.1016/0167-5699(95)80008-5.

Work, Thierry M., Julie Dagenais, Renee Breeden, Anette Schneemann, Joyce Sung,

Brian Hew, George H. Balazs, and John M. Berestecky. 2015. "Green Turtles

Have Novel Asymmetrical Antibodies." The Journal of Immunology 195

(11):5452. doi: 10.4049/jimmunol.1501332.

52

Yan, S., D. Marguet, J. Dobers, W. Reutter, and H. Fan. 2003. "Deficiency of CD26

results in a change of cytokine and immunoglobulin secretion after stimulation by

pokeweed mitogen." Eur J Immunol 33 (6):1519-27. doi: 10.1002/eji.200323469.

Zapata, A. G., A. Varas, and M. Torroba. 1992. "Seasonal variations in the immune

system of lower vertebrates." Immunol Today 13 (4):142-7. doi: 10.1016/0167-

5699(92)90112-k.

Zarkadis, I. K., D. Mastellos, and J. D. Lambris. 2001. "Phylogenetic aspects of the

complement system." Dev Comp Immunol 25 (8-9):745-62.

Zhang, X., R. A. Calvert, B. J. Sutton, and K. A. Dore. 2017. "IgY: a key isotype in

antibody evolution." Biol Rev Camb Philos Soc 92 (4):2144-2156. doi:

10.1111/brv.12325.

Zimmerman, L. M., L. A. Vogel, and R. M. Bowden. 2010. "Understanding the

vertebrate immune system: insights from the reptilian perspective." J Exp Biol

213 (5):661-71. doi: 10.1242/jeb.038315.