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Focus on Florida: Immunoglobulin-G within lymphoid tissues in the Florida manatee

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Title:
Focus on Florida: Immunoglobulin-G within lymphoid tissues in the Florida manatee
Creator:
Eichner, Michael
Samuelson, Don
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Gainesville, Fla.
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University of Florida
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English

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serial ( sobekcm )

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Abstract:
The Florida Manatee often inhabits murky, microbial filled riverine waters likely high in potential pathogens. Manatees also inhabit coastal marine ecosystems, which can pose threats to manatee health via increased boat traffic, accumulation of harmful algal blooms, and loss of warm water sources. The Florida manatee demonstrates remarkable healing capabilities and is hypothesized to possess a strong immune system; the true extent currently remains unknown. For this study, mouse anti-manatee immunoglobulin-G (IgG) was used to establish the presence of this class of immunoglobulin in lymphoid tissues with an emphasis on those associated with mucosal immunity. Immunohistochemistry was used to localize IgG from the selected lymphatic tissues. A representative sample of manatee specimens was examined (n=32; male=19, F=13) from different locations around Florida’s coasts and show diversity in approximate age and overall size. Cells showing an IgG presence were evaluated based on red-colored intensity of the cell, which ranged from light to dark. Red tide causes of death show a greater overall average of IgG reactive cells and a greater concentration of reactive cells per observed field. Surprisingly, cold stress deaths follow behind those caused by red tide in both cellular averages and in concentration thereof. Universally, conjunctiva appears to have the largest and most densely populated IgG-positive cells. Although there is a direct correlation of an increase of IgG in red tide death and separately in conjunctival tissues, the concentrations and the sample size were too small for a supportive conclusion at this time.

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Immunoglobulin-G within Lymphoid Tissues in the Florida

Manatee


Michael Eichner and Dr. Don Samuelson
College of Veterinary Medicine, University of Florida

The Florida Manatee often inhabits murky, microbial filled riverine waters likely high in potential pathogens. Manatees also inhabit
coastal marine ecosystems, which can pose threats to manatee health via increased boat traffic, accumulation of harmful algal blooms,
and loss of warm water sources. The Florida manatee demonstrates remarkable healing capabilities and is hypothesized to possess a
strong immune system; the true extent currently remains unknown. For this study, mouse anti-manatee immunoglobulin-G (IgG) was
used to establish the presence of this class of immunoglobulin in lymphoid tissues with an emphasis on those associated with mucosal
immunity. Immunohistochemistry was used to localize IgG from the selected lymphatic tissues. A representative sample of manatee
specimens was examined (n=32; male=19, F=13) from different locations around Florida's coasts and show diversity in approximate
age and overall size. Cells showing an IgG presence were evaluated based on red-colored intensity of the cell, which ranged from light
to dark. Red tide causes of death show a greater overall average of IgG reactive cells and a greater concentration of reactive cells per
observed field. Surprisingly, cold stress deaths follow behind those caused by red tide in both cellular averages and in concentration
thereof. Universally, conjunctiva appears to have the largest and most densely populated IgG-positive cells. Although there is a direct
correlation of an increase of IgG in red tide death and separately in conjunctival tissues, the concentrations and the sample size were
too small for a supportive conclusion at this time.


Introduction

The Florida Manatee (Trichechus manatus latirostis) is
the only sirenian found in U.S. waters. The endangered
Florida manatee has no natural predators; however, events
such as collisions with boats, red tide blooms, and cold
weather can all have detrimental effects on manatee health
(Bonde et al. 2004). The field of marine mammal
immunology is still relatively new, with only a select
number of species having been investigated and with
limited antibodies available for research and diagnostics.
Immune system monitoring is a valuable tool for the
assessment of wild animal populations as well as captive
managed animals; its function as an indicator of aquatic
ecosystem health has been gaining attention as research
supports marine mammals as a sentinel species (Reif et al.
2006). Measuring changes in levels of antigen-specific
immunoglobulins allows clinicians and researchers to
document exposure to infection agents, identify naive
unexposed animals, and identify individuals with immune
deficiencies.
For this study, mouse anti-manatee immunoglobulin-G
(IgG) was used to establish the presence of this class of
immunoglobulin in lymphoid tissues with an emphasis on
those associated with mucosal immunity.
Immunoglobulins, known as antibodies, are found most
often in the blood of vertebrates and are used by the


humoral immune system to identify and neutralize foreign
objects, such as bacteria and viruses (Janeway et al. 2001).
In placental animals, antibodies exist in four main varieties,
known as isotypes or classes. Each class differs in
biological properties, functional location, and ability to
recognize different antigens. Measuring changes in levels
of antigen-specific immunoglobulins allows clinicians and
researchers to document exposure to infectious agents and
identify individual immune deficiencies. IgG is the most
abundant immunoglobulin and provides the major
secondary antibody-based immunity against invading
pathogens. Immunohistochemical techniques have been
developed for assaying IgG to determine reactive cells.
Mucosal immunity produces 80% of all immunocytes
and provides protection to an organism's various mucous
membranes (Holmgren & Czerkinsky, 2005). With further
development of protocol and procedures, it is hoped that
IgG evaluations will lead to advancements for a non-
invasive method to evaluate the mucosal immune response
of the Florida Manatee and provide a more conclusive
reflection of health.

Materials and Methods

Specimens were collected from deceased manatees
brought to the Marine Mammal Pathobiology Laboratory
in St. Petersburg, Florida. Tissues samples were made from


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MICHAEL EICHNER AND DON SAMUELSON


10% buffered formalin paraffin-embedded manatee tissues
blocks associated with the lymphoid tissues of interest:
axillary lymph nodes, nasopharyngeal mucosa, and
conjunctiva. Tissues were embedded in paraffin and then
sectioned at five microns in thickness for fifteen sections
and placed on charged slides for immunohistochemistry. A
random slide from each specimen was selected for further
IgG evaluation.
For this study, mouse anti-manatee IgG was used to
establish the presence of the IgG immunoglobulin in
lymphoid tissues. Immunohistochemistry was used to
localize IgG from the selected lymphatic tissues using anti-
mouse goat antibody as a secondary and Invirogen


Histostain Plus (AEC) as the staining agent associated with
avidin-biotin linkage with the secondary antibody.
Immunolocalization without the primary antibody was used
as the negative control.
A representative sample of manatee specimens was
examined (n=32, male=19, F=13; Table 1) from different
locations around Florida's coasts and show diversity in
approximate age and overall size. Once stained, slides were
examined under a 250x lens, and three photographs of six
randomly selected fields were taken for each sample. Cells
showing an IgG presence were evaluated based on red
colored intensity of the cell, which ranged from light to
dark.


Table 1: Number of Manatees by Sex, Age, Location Distribution, and Cause of Death

Cause of Death Sex Estimated Age Location Found (coast)


Cold Stress


Red Tide


Acute Watercraft


Chronic Watercraft


M=3



F=3



M=8



F=2



M=4



F=4



M=4



F=4


Totals


M= 19





F= 13


Juvenile = 2
Adult = 1


Juvenile = 3

Juvenile = 2
Subadult = 4
Adult = 2

Juvenile = 1
Adult = 1

Calf= 1
Subadult = 2
Adult = 1

Juvenile = 1
Adult = 3

Calf= 1
Juvenile = 2
Subadult = 1

Adult = 4


Calf = 2
Juvenile = 6
Subadult = 7
Adult = 4


Calf 0
Juvenile = 5
Subadult = 0
Adult = 8


Northeast = 1
East = 1
Southwest = 1

West = 2
Undocumented = 1

Southwest = 2
West = 6


West = 2


Northeast = 1
East = 1
West = 1
Undocumented = 1

East = 1
West = 3

East = 2
Southwest = 1
Undocumented = 1

West = 1
Undocumented = 3

Northeast = 2
East = 4
Southwest = 4
West = 7
Undocumented = 2

Northeast = 0
East = 1
Southwest = 0
West = 8
Undocumented = 4


Note. Subcategories, when omitted, indicate no individuals met this particular criterion (age bracket and/or location).





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IMMUNOGLOBULIN-G WITHIN LYMPHOID TISSUES IN THE FLORIDA MANATEE


Results

Red tide causes of death show a greater overall average of
IgG reactive cells and a greater concentration of reactive
cells per observed field (Table 2). Surprisingly, cold stress
deaths follow behind those caused by red tide in both


cellular averages and in concentration thereof Red tide
specimens mounted a greater IgG response in conjunctiva
and nasopharyngeal mucosa, whereas cold stressed animals
showed a much greater response in axillary lymph nodes.
Manatees that died by acute watercraft accidents elicited
greater responses than chronic watercraft in all aspects.


Table 2: Distribution of Observed IgG Reactive Cells with Respect to Cause of Death and Lymphoid Region

Cause of Death Areas Averagesa CPF StdDevb
Axillary Nasopharyngeal Conjunctiva
Cold Stress
Total # of cells 549 165 533 415.666
Fields Observed 29 12 19
Cells per Field 18.93 13.75 28.05 20.244 7.2398
Red Tide
Total # of cells 544 116 1102 587.333
Fields Observed 41 5 30
Cells per Field 13.27 23.20 36.73 24.400 7.0230
Watercraft Acute
Total # of cells 396 36 793 389.333
Fields Observed 27 6 38
Cells per Field 14.67 6.00 19.37 13.345 6.7812
Watercraft Chronic
Total # of cells 128 301 315 248.000
Fields Observed 12 30 30
Cells per Field 10.67 10.03 10.50 10.399 0.3299
Averages
Total # of cells 404.25 154.50 671.50
Cells per Field 14.383 13.245 23.663
CPF StdDevd 3.4556 7.3526 11.2811
Note. IgG reactive cells are organized in this table both horizontally by cause of death and vertically by lymphatic area.
a These averages represent the IgG reactive cell populations expressed per row as they relate to each cause of death:
total number of cells and cell per field, respectively.
b CPF StdDev expresses the standard deviation found within the cells per field as it relates to each cause of death.
c These averages represent the IgG reactive cell populations expressed per column, specifically describing cell
concentrations in a particular lymphatic tissue through four causes of death.
d CPF StdDev expresses the standard deviation found within the cells per field in each of the three lymphatic tissues
across all four causes of death.


It is important to consider that only one specimen was
available for IgG investigations of the nasophyngeal
mucosae for both red tide and acute watercraft deaths
(Figure 1). Bossart et al. (1998) have implicated
brevetoxicosis (red tide) in the death of many manatees,


and have observed increased positive staining of
lymphocytes in the nasal mucosa. Chronic watercraft
deaths showed very little variation in reactive cell
distribution and averaged the least in cell number and
concentration.


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MICHAEL EICHNER AND DON SAMUELSON


Figure 1: A-D. Immunohistochemistry of M. latirostris within the Nasopharyngeal Mucosa X250. A.
Cold stress adult male from the northeast coast. B. Red tide juvenile female from the west coast. C.
Watercraft acute adult female from the east coast. D. Watercraft chronic adult female from the east
coast.


Universally, conjunctiva appears to have the largest and
most densely populated IgG-positive cells (Figure 2). This
finding is not unexpected in that the eye is continuously
exposed to potential pathogens that may exist within the
animal's aquatic environment. Variability in environment


may account for the large deviation of IgG reactive cells
associated with the conjunctiva. Axillary lymphoid tissue
shows the most uniformity in IgG-positive cell densities,
regardless of cause of death (Figure 3).


Figure 2. A-D. Immunohistochemistry of M. latirostris within the Conjunctiva X250. A. Cold stress
adult male from the northeast coast. B. Red tide juvenile female from the west coast. C. Watercraft
acute adult female from the west coast. D. Watercraft chronic adult female from the east coast.

University of Florida I Journal of Undergraduate Research I Volume 12, Issue 2 I Spring 2011





IMMUNOGLOBULIN-G WITHIN LYMPHOID TISSUES IN THE FLORIDA MANATEE


Figure 3: A-D. Immunohistochemistry of M. latirostris Within the Axillary Lymph Node X250. A. Cold
stress juvenile female from the west coast. B. Red tide Juvenile male from the west coast. C.


Watercraft acute adult male from the west coast.
coast.

Discussion

Although there is a direct correlation of an increase of
IgG in red tide death and separately in conjunctival tissues,
the concentrations and the sample size were too small to
draw definitive conclusions. Explanatory trends can be
seen connecting most causes of death with the age of the
animal and location where the animal was found. Red tide
exposure to brevetoxins has become increasingly
implicated in the deaths of many marine mammals,
including both the Florida manatee and bottlenose dolphins
(Flewelling et al. 2005).
It was expected that cold stress would suppress an IgG
response; however, our results show a contrary trend where
IgG concentrations are second greatest in this population.
With the seven cold stressed animals examined, it is
possible that cellular immunity is being suppressed more
than humoral immunity; thus a reduction in overall
immunity where much of the compromise is cell-mediated
in association. Chronic watercraft cases are assumed to
have died quickly and prior to mounting an immune
response; thus, localized IgG levels are lower than the
holistic averages and have a remarkable correlation
between lymphoid area and reactive cell densities. In the
conjunctival specimens, concentrations of IgG-positive


D. Watercraft chronic juvenile male from the east


cells are the most variable as a result of low detected levels
in chronic watercraft deaths and highly elevated levels
resulting from red tide.
Fairly similar distributions of reactive cell
concentrations are found between cold stressed animals
and red tide deaths. Further analyses may explain this
finding especially with regard to another antibody
associated with mucosal immunity, IgA. It is hoped that
antibody evaluations of the body's mucosa, especially
those associated with tears, will lead to advancements for a
non-invasive method to evaluate the mucosal immune
response of the Florida Manatee and provide a more
conclusive reflection of health.


Acknowledgments

I thank Dr. Don Samuelson for his mentorship and for
playing an influential role in this project. Additional thanks
go to Pat Lewis and graduate students Jennifer McGee and
Kim Goldbach. Continued gratitude to the University of
Florida, the UF College of Veterinary Medicine, the UF
Honors Program, and to the other undergraduate research
assistants.


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MICHAEL EICHNER AND DON SAMUELSON


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University of Florida I Journal of Undergraduate Research I Volume 12, Issue 2 I Spring 2011