Histological and Morphometric Investigation of the Thymus of the Florida Manatee (Trichechus manatus latirostris)

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Histological and Morphometric Investigation of the Thymus of the Florida Manatee (Trichechus manatus latirostris)
Goldbach, Kimberly
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[Gainesville, Fla.]
University of Florida
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Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences
Veterinary Medicine
Committee Chair:
Samuelson, Don A.
Committee Members:
Reep, Roger L.
Larkin, Iske
Farina, Lisa
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Subjects / Keywords:
Animals ( jstor )
Antibodies ( jstor )
Boats ( jstor )
Cells ( jstor )
Connective tissues ( jstor )
Manatees ( jstor )
Marine mammals ( jstor )
Red tides ( jstor )
Species ( jstor )
Watercraft ( jstor )
Veterinary Medicine -- Dissertations, Academic -- UF
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Veterinary Medical Sciences thesis, M.S.


As a part of the lymphatic system, the thymus is considered a primary organ due to its central role as being the core for development of T cells, which then disperse throughout the body to direct and assist with immunity. While these cells are produced through most of life, the mammalian thymus usually undergoes atresia and begins to involute around the time of young adulthood (pubescence). Connective and adipose tissue invade as the thymic parenchyma becomes reduced. Stressors, such as cold and pregnancy, can cause the thymus to involute more severely. The Florida manatee (Trichechus manatus latirostris) has been previously described to be resilient to disease with mortality being attributed mostly to prolonged cold, red tide algal blooms and watercraft strikes. The roles that the immune system plays in defending this species against the invasion of microorganisms have been little defined. The microanatomy of the components of the immune system including the thymus has not been described. We have begun to examine the thymus of the Florida manatee histologically, using histochemical and immunohistochemical tools to characterize this essential organ of the lymphatic system. Formalin-fixed paraffin embedded sections of the thymus from animals of several age groups (calves, juveniles and adults) and causes of death (acute and chronic boat strikes, cold stress and red tide exposure) were stained with hematoxylin & eosin, Gomori s tri-chrome, McManus method for glycogen and Perl s iron stains in order to delineate microanatomy of the thymus. Immunohistochemistry was performed for macrophages using a monoclonal antibody, AM-3K and to define thymocytes using a CD3 polyclonal antibody. Overall patterns of involution appear to be most accentuated in cold stress animals. Loss of parenchyma in relatively healthy adults (those that died by acute boat strike) appears to be minimal when compared to calves. Morphometric measurements detailing changes in the stromal compartment have been made and further accentuate the different involution patterns that are attributable to stressors leading to death. Electron microscopy was performed on a relatively normal juvenile thymus and gave a better idea of the ultrastructural anatomy, especially epithelial cells, seen in the manatee thymus. The purpose of these studies is to describe, in detail, the similarities and differences of the manatee thymus with regards to previously described mammals and to determine involution from age and from stress. ( en )
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Thesis (M.S.)--University of Florida, 2010.
Adviser: Samuelson, Don A.
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by Kimberly Goldbach.

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2010 Kimberly Jean Goldbach 2


ACKNOWLEDGMENTS The love and support of my mother and father, Mary and Leonard, have been of great value to me. I am grateful to Dr. Don Samuelson, Dr. Roger Reep, Dr. Iske Larkin and Dr. Lisa Farina for their help and knowledge that they have shared with me as members of my graduate committee. Special thanks to Patricia Lewis for her guidance in histological techniques. Special thanks also to the FWRI Marine Mammal Pathobiology Laboratory for the samples used in this investigation. Financial support from the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, was gratefully appreciated. The assistance of Mallorie McCormack and Cory Pollard was most helpful. 3


TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................3 LIST OF TABLES ............................................................................................................6 LIST OF FIGURES ..........................................................................................................7 ABSTRACT .....................................................................................................................8 CHAPTER 1 INTRODUCTION....................................................................................................10 The Manatee...........................................................................................................10 The Thymus............................................................................................................11 Embryology......................................................................................................12 Anatomy...........................................................................................................13 T Lymphocytes.................................................................................................15 Involution..........................................................................................................16 Marine Mammals....................................................................................................20 Stressors..........................................................................................................20 Immunological Studies.....................................................................................22 2 MATERIALS AND METHODS................................................................................26 Stereology...............................................................................................................27 Immunohistochemistry............................................................................................28 Transmission Electron Microscopy.........................................................................28 3 RESULTS...............................................................................................................30 Histology.................................................................................................................30 Involution................................................................................................................31 Stereology...............................................................................................................32 Immunohistochemistry............................................................................................33 Transmission Electron Microscopy.........................................................................33 4 DISCUSSION.........................................................................................................48 Thymus Anatomy....................................................................................................48 Stereology...............................................................................................................51 Immunohistochemistry............................................................................................52 4


Transmission Electron Microscopy.........................................................................53 Thymic Epithelial Cells.....................................................................................54 Cytokeratins.....................................................................................................55 Conclusions and Future Directions.........................................................................56 APPENDIX A ADDITIONAL TABLES............................................................................................58 B STAINING PROTOCOLS.......................................................................................64 C IMMUNOHISTOCHEMISTRY PROTOCOLS.........................................................65 D ELECTRON MICROSCOPY EMBEDDING............................................................67 E STEREOLOGER PROGRAM FOR ORGAN MORPHOMETRY.............................68 LIST OF REFERENCES...............................................................................................69 BIOGRAPHICAL SKETCH............................................................................................77 5


LIST OF TABLES Table page 3-1 Percentage of connective tissue for each cause of death..................................32 A-1 Animals used in the study...................................................................................59 A-2 Thymus samples used for immunohistochemistry..............................................62 A-3 Complete data generated from stereology study................................................63 6


LIST OF FIGURES Figure page Figure 3-1 Features of the thymus of the Florida manatee...........................................36 Figure 3-2 Lymph nodes within the thymus of the Florida manatee.............................36 Figure 3-3 Four stains used for histological evaluation of an acute boat strike subadult..............................................................................................................37 Figure 3-4 Aspects of involution in the thymus of the Florida manatee........................38 Figure 3-5 Involution grading system...........................................................................39 Figure 3-6 Acute boat strike and cold stress samples used in stereology study..........40 Figure 3-7 Red tide samples used in stereology study.................................................41 Figure 3-8 Chronic boat strike samples used in stereology study................................42 Figure 3-9 Immunohistochemistry studies on the thymus of the Florida manatee........43 Figure 3-10 Establishing shots of the manatee thymus for electron microscopic investigation........................................................................................................44 Figure 3-11 Electron microscopic investigation of the cortex of the Florida manatee...45 Figure 3-12 Electron microscopic investigation of the corticomedullary junction of the Florida manatee............................................................................................46 Figure 3-13 Electron microscopic investigation of the thymic medulla of the Florida manatee.............................................................................................................47 7


Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science HISTOLOGICAL AND MORPHOMETRIC INVESTIGATION OF THE THYMUS OF THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS) By Kimberly Jean Goldbach May 2010 Chair: Don Samuelson Major: Veterinary Medical Sciences As a part of the lymphatic system, the thymus is considered a primary organ due to its central role as being the core for development of T cells, which then disperse throughout the body to direct and assist with immunity. While these cells are produced through most of life, the mammalian thymus usually undergoes atresia and begins to involute around the time of young adulthood (pubescence). Connective and adipose tissue invade as the thymic parenchyma becomes reduced. Stressors, such as cold and pregnancy, can cause the thymus to involute more severely. The Florida manatee (Trichechus manatus latirostris) has been previously described to be resilient to disease with mortality being attributed mostly to prolonged cold, red tide algal blooms and watercraft strikes. The roles that the immune system plays in defending this species against the invasion of microorganisms have been little defined. The microanatomy of the components of the immune system including the thymus has not been described. We have begun to examine the thymus of the Florida manatee histologically, using histochemical and immunohistochemical tools to characterize this essential organ of the lymphatic system. Formalin-fixed paraffin embedded sections of the thymus from animals of several age groups (calves, juveniles and adults) and causes of death (acute 8


and chronic boat strikes, cold stress and red tide exposure) were stained with hematoxylin & eosin, Gomoris tri-chrome, McManus method for glycogen and Perls iron stains in order to delineate microanatomy of the thymus. Immunohistochemistry was performed for macrophages using a monoclonal antibody, AM-3K and to define thymocytes using a CD3 polyclonal antibody. Overall patterns of involution appear to be most accentuated in cold stress animals. Loss of parenchyma in relatively healthy adults (those that died by acute boat strike) appears to be minimal when compared to calves. Morphometric measurements detailing changes in the stromal compartment have been made and further accentuate the different involution patterns that are attributable to stressors leading to death. Electron microscopy was performed on a relatively normal juvenile thymus and gave a better idea of the ultrastructural anatomy, especially epithelial cells, seen in the manatee thymus. The purpose of these studies is to describe, in detail, the similarities and differences of the manatee thymus with regards to previously described mammals and to determine involution from age and from stress. 9


CHAPTER 1 INTRODUCTION The Manatee The manatee belongs to the order Sirenia which includes four families Prorastomidae, Protosirenidae, Dugongidae and Trichechidae of which only the last two have species still living today. The dugong, a cousin to the manatee, is the only species of the Dugongidae family and is found in the Indian Ocean (Strahan 1995). Within the Trichechidae family, there are three species: Trichechus inunguis, the Amazonian manatee; Trichechus senegalensis, the West African manatee; and Trichechus manatus, the West Indian manatee. The latter is split into two subspecies: Trichechus manatus manatus, the Antillean manatee and Trichechus manatus latirostris, the Florida manatee (Reep and Bonde 2006). The Florida manatee is an endangered marine mammal that lives in shallow, tropical water along the Gulf and Florida coasts. They can be found in fresh or salt water and clear or murky water. The manatee usually stays in water of at least 68 o F and can display signs of cold stress in waters any cooler. The manatee is an obligate herbivore and feeds on over 60 species of freshwater and marine vegetation (Reep and Bonde 2006). Natural disease is fairly uncommon in the Florida manatee. Evidence of viral papillomatosis was just recently found (Bossart et al. 2002). Manatees more often have to deal with environmental stressors such as red tide algal blooms, cold stress and the increase in watercraft activity. Taking these stressors into account, the health and immunity of manatees is of the utmost importance. The morphology of the lymphoid organs of the Florida manatee has not yet been examined. The anatomy of the dugong 10


was examined in a paper by Cave (1967) which included a short description of the lymphoid organs, especially the conformity of the thymus to other mammalian species. Unfortunately, it is that similarity that has led to the neglect of the thymus as a research option among many species. Indeed, the subject of the immunology of marine mammals has only been looked at within the last 30 years. The Thymus The thymus was first noted during the times of the ancient Greeks, who discovered it during ritual sacrifices. It was thought to be the seat of the soul, and therefore called thymos, meaning heart or soul (Ribatti 2006). The first description was made in the first century AD by Rufus of Ephesus, followed by Galen, whose exploration led to the thought that the thymus supported and protected the junction of the vena cava, cushioning it from contact with the sternum (Cardarelli 1989). The thymus meant little to medical science until 1583 when Felix Plater, in his work De Corporis Humani: Structura et Usu, described an enlargement of the thymus in the suffocation and death of a young boy (Cardarelli 1989). Until the middle of the eighteenth century, unexplained deaths were usually blamed on some malfunction of the thymus. Detailed descriptions of the lymphatic system were made during the eighteenth century, leading to more curiosity and experimentation on the organs that make up that system. Throughout the nineteenth century, much work was done on the thymus the distinct tissue types were established, analysis of many different species led to the determination that nearly all vertebrates have a thymus and many books were written on the results of much research (Cardarelli 1989). As technology has become more advanced, more has been learned about the smaller elements of the thymus, but nevertheless more remains to be explored. 11


Embryology Among mammals, the embryogenesis of the thymus has been most thoroughly described in the mouse, as it is relatively similar to other vertebrates (Haley 2003, Rodewald 2008). In the mouse, the thymus primordium is formed between embryological day 10.5 and 11.5 from the third pharyngeal pouch endoderm (Rezzani et al. 2008). Early organogenesis is tied to the parathyroid glands. Each endodermal primordium contains precursors to one thymic lobe and one parathyroid gland. At approximately embryological day 12, the thymic rudiment is colonized by lymphocyte progenitors from the fetal liver and bone marrow which enter through the capsule by chemo-attraction (Rezzani et al. 2008). At this time, the tissue layers of what will become the cortex and medulla are not histologically defined. The thymic primordium is initially composed of bipotent thymic epithelial cell progenitors that, through interactions with neural crest mesenchyme, undergo lineage commitment and differentiation which then allow for its formation and subsequent development (Rezzani et al. 2008, Gordon et al. 2004). Around embryological day 12.5, the primordial tissue separates from the pharynx and begins its migration toward the anterior chest cavity followed by a split of the thymus and parathyroid to assume their positions in the adult (Blackburn and Manley 2004). It was first thought that both ectoderm and endoderm contributed to the formation of the thymus, in what has been called the dual-origin model (Manley and Blackburn 2003). The view was that the cortical epithelium was derived from the ectoderm of the third pharyngeal cleft and the medullary epithelium derived from the endodermal tissue of the third pharyngeal pouch. Also, the epithelial cell differentiation was thought to require both ectoderm and endoderm to proceed (Gordon 2004). It has most recently 12


been determined that only the endoderm contributes to thymic formation (Blackburn and Manley 2004). In the human, the thymus is also derived from the third pharyngeal pouch, with a slight contribution from the fourth pharyngeal pouch, during the sixth week of gestation (Suster and Rosai 1997). After the migration of the thymus to its final position in the eighth week of gestation, epithelial cells develop and differentiate into the reticular meshwork which, in the tenth gestational week, is filled by small lymphoid cells coming from the fetal liver and bone marrow (Suster and Rosai 1997). In other vertebrate species, the number of organs per animal, the exact embryological origin and the final anatomical positioning of the thymus all differ (Rodewald 2008). Chickens are found to have seven thymus pairs, sharks have five and the salamander has three, while many species of teleost fish, frogs and many mammals have only one thymus, composed of two bilateral lobes (Rodewald 2008). The thymus anlagen are located in the second through the sixth pharyngeal pouch in sharks, the second pouch in frogs, the second and third pouches in reptiles and in the third and/or fourth pouches in bony fish and mammals (Rodewald 2008). Even with these differences, it has been determined through numerous experiments and morphological observations that the thymus is consistently similar among all vertebrates with the exception of the jawless fish, which do not possess a thymus (Boehm 2008). Anatomy The thymus is considered to be a primary lymphoid organ, as it is the first place lymphoid cells migrate to in order to mature and contribute to the immune function of the body (Boehm 2008). The thymus is located in the anterior mediastinum in the human and is composed of two lobules joined along the midline by connective tissue and some 13


thymic parenchyma (Suster and Rosai 1997). The thymus is anatomically divided into several distinct regions best seen histologically. The entire organ is surrounded by a thin connective tissue capsule, which infiltrates the thymic epithelial tissue slightly to form septae that form incomplete lobules (Pearse 2006). The outer portion of each lobule consists of the darkly staining cortex, which houses densely packed, small, immature lymphocytes that overshadow the sparse epithelial cell population (Pearse 2006). The cortex is the location of all of the immature lymphocytes, including those not selected to mature but undergo apoptosis and have been described as having a prominent starry sky appearance in this portion of the thymus (Pearse 2006). In the subcapsular region that forms the outer cortex, larger mitotically active lymphoblasts occur most frequently. A gradient of smaller, less mitotically active cells moves from there to the corticomedullary junction (Suster and Rosai 1997). Lymphoid progenitors enter the thymus through blood vessels, predominantly arterioles, located in the corticomedullary junction (Boehm 2006, Pearse 2006). The corticomedullary junction is characterized by these blood vessels, along with sparse perivascular connective tissue, mature and immature T cells (Pearse 2006). When compared to the cortex, the medulla stains more lightly and is thought to provide a specialized microenvironment for the negative selection of self-reactive T lymphocytes (Boehm 2006, Ladi 2006). The medulla is less densely cellular than the cortex and contains mature T cells and prominent epithelial cells called Hassalls corpuscles, admixed with macrophages, dendritic cells and some B lymphocytes (Pearse 2006). It is from here that the mature T cells leave and migrate to the 14


peripheral lymphoid organs, spleen and lymph nodes, and contribute to the defense of the body. T Lymphocytes The lymphocytes that make up the thymus begin as pluripotent hematopoietic stem cells that originate from the bone marrow. These cells migrate to the thymus, most likely under the control of chemo-attractants. Upon entering through the vasculature of the corticomedullary junction, these cells initially lack most of the cell surface molecules that characterize mature T cells and therefore must interact with the stromal microenvironment through a four stage process of maturation. All T lymphocytes that enter the thymus are CD4 CD8 cells, or double negative (DN) for CD4 and CD8. Thymocytes in the first stage of maturation, DN1, are usually located near their site of entry and are characterized by the expression of cell surface molecules Kit and CD44 (CD4 CD8 CD44 + CD25 ). These cells then move throughout the cortex, entering the DN2 stage and begin to express CD25 (CD4 CD8 CD44 + CD25 + ). As they mature further to DN3 cells, they migrate to the outermost part of the thymus below the capsule where expression of CD44 and Kit are reduced (CD4 CD8 CD44 CD25 + ). The final stage of maturation, DN4, is only attained when the cell has completed successful T cell receptor rearrangement and loses expression of CD25 (CD4 CD8 CD44 CD25 ). The cells become double positive (CD4 + CD8 + ) and proliferative quickly, making up the vast majority of thymocytes in the thymus. In order to progress to the single positive stage of maturation (CD4 + CD8 or CD4 CD8 + ), the cells must be able to recognize self major histocompatability (MHC) complexes and express high levels of their T cell receptor. Mature, single positive cells move into the medulla, where they are 15


tested on reaction to self antigen. Those that do not react are sent out to the peripheral lymphoid organs. It has been estimated that it takes three weeks from entry of a T cell progenitor to exit as a fully mature T lymphocyte (Boehm 2006, Pearse 2006, Ladi 2006, Murphy et al. 2008). The thymic tissue allows for a high level of cell proliferation. It contains 10 11 thymocytes with 20-25% of that number being produced daily by cell division. This may seem high, but it has been found that about 95-98% of thymocytes die while still in the thymus due to reactivity to self or inability to successfully rearrange their T cell receptors (Bodey et al. 1997, Murphy et al. 2008). Involution It has been noted by physicians all the way back to Galen that the thymus changes with age in a process termed involution. This process can be separated into two time frames accidental involution, which will be discussed in a later paragraph, and age-induced involution. Involution is characterized by the systematic loss of thymic epithelial tissue, which is replaced by adipose, connective tissue and perivascular space as well as progressive loss of mostly immature, cortical thymocytes and noticeable reorganization of the organ architecture, involving the loss of definition of the cortex and medulla (Bodey et al. 1997). Changes in the overall size of the thymus can vary considerably among species with regards to age-induced involution. In humans, the size of the thymus generally remains the same throughout life, while in the mouse the thymus size becomes greatly reduced with age (George and Ritter 1996). There has been controversy in the field of immunology concerning events associated with the initiation of thymic involution. It has been a much-held theory that the involution of the thymus coincides with the age of puberty and is partly a reaction to 16


the expression of the sex steroids estrogen and testosterone. It has been fairly well determined in humans that the involution process of thymic epithelial tissue starts soon after the first year of life and continues throughout life at a rate of three to five percent per year until middle age, where the process slows down to about one percent per year (George and Ritter 1996, Bodey et al. 1997). Steinmann (1985) actually confirmed that involution continues beyond the age of 90 years. Thymic tissue was identified in one thymus from an individual of 107 years of age and it was postulated that the expected total disappearance of thymic tissue could take place by the age of 120, which was regarded as the maximum life span for the human species. Involution is only one of a number of changes that takes place during the aging of the immune system, or immunosenescence. A principal change is the decrease in the number of mature, nave T cells produced by the thymus. The reduction is largely due to the decrease in T cell progenitors migrating from the bone marrow (Chidgey et al. 2007). Several studies have shown that the production of nave, mature T cells is severely compromised by 40-50 years of age (Hakim 2005) and of those cells, there is a rapid decline in the CD8 lineage by 65 years of age, with the CD4 lineage sustained for another 20 years due to homeostatic proliferation (Goronzy 2007). Though there are fewer T cells produced, their development in the thymus is not impaired and the cells are capable of performing the same roles of those that came before. Also, there are increased numbers of long-lived memory cells those that have encountered antigen before to help combat diseases the body has already fought. B lymphocyte functionality is also impaired with age, showing weaker antibody responses (Prelog 2006) though their generation continues through life (George and Ritter 1997). 17


With age comes a shift in the immune system from the adaptive (characterized by B and T lymphocytes) to the innate (characterized by cells of defense). There is an increase in natural killer cells, neutrophilic granulocytes and macrophages (Bodey et al. 1997, Sansoni 2008). Accidental involution refers to the temporary atrophy of thymic tissue and loss of cortical thymocytes due to stress which can be reversible. Stressors include hormonal influence, pregnancy, malnutrition, infections and disease (Body et al. 1997, Nabarra and Andrianarison 1996). Several studies, usually performed on mice, have looked at how the thymus reacts to artificially induced conditions such as swim tests or injections of foreign substances (ivkovi et al. 2005, Baillif 1949). In these situations, the thymus has been characterized by a reduction of lymphocytes in the cortex by phagocytosis, followed by a loss of distinction between the cortex and the medulla (Henry 1968). If the stressor is not long-lasting, then the thymus will regenerate back to its previous size and cellularity. The time it takes for the thymus to fully regenerate varies depending on the intensity of the stressor and the nutritional status of the animal. In a paper by Baillif (1949), the thymus of an albino rat had resumed its previous size by the seventh day after one injection of an acid colloidal substance. The last region to be repopulated was found to be the corticomedullary junction, but small lymphocytes were already seen in the cortex and medulla by the third day. Pregnancy is another example of accidental involution. During pregnancy, the thymus reduces in size, followed by a return to normal size after birth. This has been seen in all mammalian species that have been looked at to date. It has been shown that the increase in the hormones estrogen and progesterone can initiate involution by 18


reducing the proliferation of lymphocytes in all stages of maturation, although there was no subset that was reduced more than another. One study showed an accumulation of early thymic progenitors (before the DN1 stage), suggesting a block in development. In mice, cellularity was found to be reduced as early as day 12.5 of pregnancy with the thymus itself being reduced to 25% of the size of an age-matched control only a few days before birth. Thymus size was found to be fully restored by 8-14 days after birth but the early thymic progenitors were still reduced two weeks after birth. (Zoller et al. 2006, Kendall and Clarke 2000). Seasonal variation can affect the thymus, especially in ectothermic vertebrates. Specifically, several studies of the striped-neck terrapin (Mauremys caspica) have documented these changes (Leceta and Zapata 1985). Late winter and summer seasons were associated with marked accidental thymic involution with corresponding rebounds in late spring and autumn. Low temperatures during winter months were proposed to explain one period of thymic involution. Other variations, whether large or small, have been attributed to levels of steroids corticosterone in both species during the summer and testosterone in males in the beginning of spring, corresponding with the mating period (Leceta and Zapata 1985). Many stressors have the potential to increase immune function if experienced once and only for a short period of time. When exposed to cold, the body initially increases the CD4+/CD8+ ratio, stimulates lymphocyte proliferation, enhances natural killer cell activity and increases production of immunoglobulin and proinflammatory cytokines. As exposure lengthens, the immune system progressively becomes suppressed. Involution of the spleen and thymus occurs and immunological events that 19


were increased will be reduced (Shephard and Shek 1998). The decrease in natural killer cells has been shown to lead to an increased susceptibility to viral and bacterial infections (Shephard et al. 1998, Shephard and Shek 1998). Marine Mammals Stressors Marine mammals face situations and stressors that rival terrestrial species. These animals are affected by entanglement, habitat degradation, ship traffic, climate change, chemicals and other biological factors (Fair and Becker 2000). Because often they cannot escape the environment that they are in, the immune system of the marine mammal can experience low levels of suppression that make them more susceptible to organochlorides, harmful algal toxins and viruses (Fair and Becker 2000). One major concern within the past few decades has been bioaccumulation of environmental contaminants, such as organochlorides, in the tissues of animals in a marine habitat (Beineke et al. 2005). Marine mammals are, in most cases, at the top of the food chain and therefore can not be rid of the organic toxins they pick up from their contaminated prey. Several studies have been performed with blood and blubber samples from various marine mammals (whales, dolphins, seals, sea lions and sea otters), both captive and wild. In an experiment using harbor porpoises (Phocoena phocoena), it was determined that thymic atrophy and splenic depletion were associated with an increased concentration of contaminants, especially with elevated polychlorinated biphenyl (PCB) and polybrominated diphenyl ether (PBDE) levels (Beineke et al. 2005). In another experiment, blood samples from various marine mammals (dolphins, seals and whales), both captive and wild, were collected (Mori 2005). Several PCB congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were 20


tested on blood leukocytes with mixed results. Decreased T cell proliferation was seen in harbor seals (Phoca vitulina) and beluga whales (Delophinapterus leucas) that had previously been chronically exposed to organochlorides in their environment while increased proliferation was seen in other cetaceans, pinnepeds and mustelids that had not been previously exposed (Mori et al. 2005). Red tide algal blooms are considered to be a major environmental event and appear to be increasing in frequency, duration and location. These blooms can vary from minor to severe, and can persist for up to 18 months, culminating in massive fish kills, marine animal mortalities and long-lasting impacts on sea grass and coral communities. One such bloom, commonly called the Florida Red Tide, occurs largely along the south-west Florida coast and is caused by the dinoflagellate Karenia brevis (Pierce and Henry 2008). This dinoflagellate can usually be found in the Gulf of Mexico and Caribbean Ocean, but has been found on the east coast of Florida extending up the coast to North Carolina (Kirkpatrick et al. 2004). K. brevis produces as many as 14 different brevetoxin compounds, each contributing to neurotoxicity, bronchial constriction, hemolysis, immune suppression and genetic damage (Pierce and Henry 2008). Exposure can be through the consumption of contaminated food or through the inhalation of aerosolized toxins. Once they have entered the body, they can rapidly disseminate to the tissues, including the respiratory tract, liver, kidneys and brain. The toxins leave the body through the excretion of urine and feces (Kirkpatrick et al. 2004). Brevetoxins can persist in sea grass even after the bloom has gone, which allows for the possibility of continued exposure and potential intoxication. 21


Morbillivirus infections have been documented only since 1987, and since then have been the cause of numerous epizootics in several marine mammal species, excluding manatees: harbor seals in the North Sea (1988), bottlenose dolphins in the Gulf of Mexico (1990) and striped dolphins in the Mediterranean Sea (1990-1991) (Fair and Becker 2000). Morbillivirus is a relative of the canine distemper virus, and is highly contagious, single-stranded RNA virus whose transmission is believed to be through aerosol, although direct and indirect transmissions have not been ruled out. High levels of antigen in certain areas of the body suggest that the virus may be spread via respiratory, urinary, fecal or ocular routes (Kennedy 1998). There are histologic lesions in the respiratory and gastrointestinal tracts, the central nervous system and lymphoid tissue. Severe lymphoid depletion and cytolysis has been seen in the thymus, spleen, lymph nodes and gastrointestinal-associated lymphoid tissue. Many animals infected with morbillivirus have secondary opportunistic infections, leading to the hypothesis that damage to the lymphoid tissues results in immunosuppression and increased susceptibility to infectious agents (Kennedy 1998, Domingo et al. 1992). Immunological Studies Studies on the immune system of cetaceans and other marine mammals have been published since the early 1970s as papers included in several volumes of Investigations on Cetacea (Pilleri 1969-77) and Functional Anatomy of Marine Mammals (Harrison 1972). A study on the histology of the dugong was also published around this time (Cave and Aumonier 1967). These investigations focused on the basic anatomy and histology of the lymphoid tissue. During the 1990s, several more papers were published on the lymphoid organs of the bottlenose dolphin, Tursiops truncatus (Cowan and Smith 1999) and beluga whale, Delphinapterus leucas (Romano et al. 22


1993). The general consensus of all of these papers was that the lymphoid tissues of the marine mammal are similar to those in other vertebrate species. Scientists then started narrowing focus to individual lymphoid organs and their components. A few studies have documented the existence of cysts in the thymuses of the harbour porpoise, Phocoena phocoena (Wunschmann et al. 1999) and the bottlenose dolphin (Cowan 1994). These cysts were found in all ages of animal examined, usually growing in size with age, and were lined with squamous epithelium in larger cysts and squamous to columnar epithelium in smaller cysts. It was determined that the cysts developed from condensed thymic epithelium possibly arising from Hassalls corpuscles (Wunschmann et al. 1999). Thymic cysts are not commonly found in humans and domestic animals but have been seen and documented (Newman 1971). Lymphoid depletion and involution were investigated in the thymuses of the harbour porpoise (Beineke et al. 2005), bottlenose dolphin (Clark et al. 2005) and fur seal, Callorhinus ursinus (Cavagnolo 1979). The studies on the fur seal and harbour porpoise focused on accidental involution attributable to behavioral and environmental stressors. Both found that increased stress was associated with thymic atrophy. The study on the bottlenose dolphin looked at involution due to age and found that the thymus of the older animals were more involuted than the younger animals, similar to what has been found for other species. Several studies within the last 15 years have looked at the cellular components of the immune system. A number of monoclonal antibodies specific to cetaceans have been developed to look at different lymphocyte subsets (De Guise et al. 1998, 2002). 23


Another study looked at the cross-reactivity of antibodies with the tissues of the bottlenose dolphin (Kumar and Cowan 1994). Other components of the immune system, major histocompatability (MHC) class II and immunoglobulin (Ig) have been defined in several papers (Romano et al. 1992, Lundqvist et al. 2002, Zabka and Romano 2003, Mancia et al. 2006, Mancia et al. 2007). One paper on the manatee used the peripheral blood mononuclear cells to characterize IL-2R expression to assist in determining health status (Sweat et al. 2005). There are also studies on the immunology of marine mammals looking at how the immune system responds to diseases and other stressors in the aquatic environment. The complications of brevetoxin on the immune system were studied in the bottlenose dolphin (Fire et al. 2007) and the manatee (Bossart et al. 1998). One manatee study focused on the amount of lymphocyte proliferation in both healthy and previously exposed manatees to an additional environmental stressor, finding that lymphocyte proliferation in red tide exposed, wild manatees was one-third of what was seen in healthy, wild manatees (Walsh et al. 2005). There were four primary objectives for this project. The first objective was to describe the anatomy, primarily through histology, of the manatee thymus to have an idea of the basic structure. The second objective was to look at any possible age changes and patterns of involution due to age. The third objective was to find any comparisons between the manatee thymus and the thymuses of domestic animals and especially aquatic mammals. The final objective was to look at changes that were associated with the primary cause of death and note any similarities and differences. Three hypotheses were formed from the information that was learned. The first 24


hypothesis was that the manatee thymus was similar or identical to what had been described for other mammals. The second hypothesis was that the manatee thymus exhibits the aging process in a similar fashion as other mammals, but that the involution described would not be as great as in other species. The final hypothesis was that the stressors of extended cold exposure and red tide toxins would have an impact on the morphology and function of the manatee thymus. 25


CHAPTER 2 MATERIALS AND METHODS The manatee thymuses used in this study were obtained from animals brought in to the Florida Fish and Wildlife Conservation Commissions Marine Mammal Pathobiology Laboratory in St. Petersburg, Florida to be necropsied and processed. Seventy-four samples were chosen from all animals necropsied between the years of 2002 and 2008 (Table 1 in Appendix A). Animals were chosen based on manner of death, as determined by gross and/or histologic examination, as well as ancillary testing procedures. Animals were classified into categories representing the three major causes of death for manatees cold stress, red tide and watercraft collision, the latter of which was separated into acute and chronic stages. From these, three age categories (calf, juvenile, adult) were defined based on criteria provided by the United States Geological Survey relating a manatees age to its overall length. The thymus tissues were already embedded in paraffin blocks prior to use in this study. From each block, 10 sections of 6 m thickness were cut using a Reichert Jung 2030 microtome. Four sections were placed on positively charged slides and six sections were placed on specimen slides. Hematoxylin & eosin and Gomoris trichrome stains (Luna 1967) were used for cellular and morphological identification. Twenty samples (Table 2 in Appendix A) were chosen from the original group, representing each age group and cause of death, trying to have two age representatives for each cause of death if possible, and were stained with McManus method for glycogen (PAS) and Perls iron stains (Luna 1967) to identify basement membranes and iron-containing phagocytic cells (all protocols found in Appendix B). 26


A determination of amount of involution in the thymuses of all samples was also performed, through observations of the H&E stained slides. In order to categorize the degree of involution, a grade system was devised based on previous studies (van Baarlen et al. 1988, Contreiras et al. 2004). Grade 1 consisted of thymuses with a clear distinction between the cortex and the medulla, with thin connective tissue separating the tissue into lobules. Grade 2 consisted of thymuses that still showed the cortex/medulla distinction but the cortex was less populated and there was noticeable lymphophagocytosis. A slight thickening of the connective tissue septa was also noted. Grade 3 consisted of thymuses that started to exhibit a loss of distinction between the cortex and medulla and showed noticeable separation of thymic lobules due to continued thickening of the connective tissue septa. Grade 4 consisted of thymuses exhibiting cord-like lobules of epithelial cells admixed with lymphocytes. The connective tissue was more abundant with prominent blood vessels. Stereology Stereologer software was used to quantify how much connective tissue each thymus sample contained and to assess the influences of death and age on this parameter. Eleven samples, chosen from the twenty used in the additional histologic and immunohistochemical studies, were used in this study and had been serially sectioned and put onto 50 slides. Of the 50 slides, every fifth for a total of ten were stained with H&E. Three of those slides, excluding the first and last stained slide, were examined with the software. Six areas were chosen at random on each piece of tissue and 200 points were affixed by the program using a point grid. Points that fell at least 50% on connective tissue were chosen and helped determine the approximate percentage of connective tissue present in the thymus (protocol found in Appendix E). 27


Immunohistochemistry The twenty samples chosen for specialized staining were also used for immunohistochemistry, with two separate tests looking at macrophage infiltration and for T cells carrying the CD3 cell marker. The first test used AM-3K, a monoclonal mouse anti-human antibody from Cosmo Bio Co. Ltd. (cat. no. KAL-KT013) at a 1:100 dilution, with a mouse primary kit from Zymed (cat. no. 85-6543). Positive and negative controls, in this case a portion of manatee spleen, were used to determine a successful result. The second test used a CD3 polyclonal rabbit anti-human antibody from Dako (cat. no. A0452) at a 1:100 and 1:200 dilution for each sample, used with a broad spectrum kit from Zymed (cat. no. 85-9743). Positive and negative controls, in this case a portion of manatee colon, were used to determine a successful result. Both tests used a protocol (Appendix C) previously used with the AM-3K antibody and spleen tissue, with the only change being a decrease of the CD3 antibody incubation to two hours at room temperature Transmission Electron Microscopy A fresh thymus sample was taken from a male calf received at the Marine Mammal Pathobiology Laboratory for necropsy. The cause of death was given in the pathology report as a natural death with the animal possessing an irregularly shaped hear and containing a necrotic mass in its neck. The sample was placed in a 2% glutaraldehyde buffer for use in transmission electron microscopy. Three small pieces were cut from the thymus, placed in separate glass containers and taken through the dehydration and embedding process (Mathews 1981, protocol found in Appendix D). Three pieces that corresponded with those were processed, embedded and cut before being stained with H&E. The sections were labeled A through C, cut and stained for 28


toluidine blue for light microscopic evaluation. Section B was selected for transmission electron microscopic examination. Ultrathin sections were made and stained with uranyl acetate and lead citrate. The sections were viewed by a Hitachi IIB transmission electron microscope. 29


CHAPTER 3 RESULTS Histology The animals in the acute boat strike category were considered the control for this study as the best representatives for a normal, non-involuted manatee thymus. Examination of the manatee thymus with the light microscope revealed that the histology of the thymus in this species followed the same basic pattern as other mammalian species. In a young, relatively unstressed animal there was a clear demarcation between the darker cortex and lighter medulla (Figure 3-1a). Hassalls corpuscles were seen in relatively few samples (Figure 3-1b). The most interesting finding was the presence of one or several portions of lymph node tissue in five of the 74 samples (Figure 3-2). Histologically, the H&E (Figure 3-3a) and Gomoris trichrome (Figure 3-3b) stains were effective in showing the changes in amount of connective tissue and cellular space in aged and stressed animals. Perls iron stain (Figure 3-3c) showed the blue coloration that is an indicator of iron in macrophages, pointing to the presence of hemosiderin in these cells. McManus method for glycogen (Figure 3-3d) also showed positive reaction in the macrophages, possibly signifying the presence of some type of glycogen found in thymic macrophages. As the animal aged or became more stressed, the lymphocytes in the cortex generally became less densely populated but remained as clusters of cells (Figure 3-4a). Macrophages became noticeable in the cortex (Figure 3-4b), usually with a small halo of space surrounding them, giving what has been described as a starry sky appearance (Schuurman et al 1997). At some point as involution progressed there was a lack of distinction between the cortex and medulla but it was difficult to appreciate 30


exact points of time or amounts of stress associated with this change. The connective tissue septa thickened and blood vessels became more prominent (Figure 3-4c). Both extended further into the thymic tissue and the remaining lymphocytes configured themselves into smaller areas, usually in long strips surrounded by the connective tissue (Figure 3-4d). As the thymus reached the stage of involution where there was more connective tissue than lymphocytes, the thymic epithelium became more prominent and at higher magnifications the pink coloration attributed to tissue was clearly seen around individual remaining lymphocytes (Figure 3-4e). In highly stressed animals, especially found in cold stress and chronic boat strike samples of all age groups but not in any acute boat strike samples, larger phagocytic cells were abundantly found, typically located near the connective tissue but still within the thymic epithelium (Figure 3-4f). Involution Using the grading system, the samples from each cause of death were categorized into four groups and some trends were noted. Grade 1 thymuses, those with the least amount of involution were evenly spread across three of the four causes of death, with no entries from cold stress (Figure 3-5a). Of the twelve samples in this group, only two were calves. Grade 2 thymuses were mostly seen among red tide and chronic boat strike samples, with the majority being adults (Figure 3-5b). Grade 3 thymuses were seen in cold stress and chronic boat strike samples, with no samples from acute boat strike (Figure 3-5c). The majority of these samples were calves. Grade 4 thymuses were also seen in cold stress and chronic boat strike samples (Figure 3-5d). Of these, calves and adults predominated. The one sample from acute boat strike was 31


also documented as being pregnant. Please refer to Table 1 in Appendix A for the grades for the animals used in this study. Stereology Table 3-1. Percentage of connective tissue for each cause of death Animal ID Cause of death Gender Age Percent CT 03R-1 Natural; Other (red tide) M calf 15.40% 03R-21 Natural; Other (red tide) M subadult 18.50% 03R-23 Natural; Other (red tide) M adult 22.80% 04R-221 Natural; Cold stress F calf 49.40% 05R-145 Natural; Cold stress F subadult 20.50% 06R-60 Watercraft; Propeller (chronic) F calf 12.10% 06R-135 Watercraft; Propeller (chronic) M calf 39.50% 06R-142 Watercraft; Propeller (chronic) F subadult 38.70% 04R-459 Watercraft; Propeller (chronic) adult 37.20% 05R-163 Watercraft; Both (acute) F subadult 9.60% 06R-334 Watercraft; Impact (acute) M adult 9.80% Eleven animal samples (Figures 3-6, 3-7 and 3-8) were examined using the Stereology software. Each sample had 17 (3 animals) or 18 (8 animals) areas of the tissue which were averaged to reach a mean percentage of connective tissue for each of the eleven samples. The control group for this study was considered to be the animals that died from acute boat strike because of the expected lack of thymocyte depletion due to long term injuries. The percentage of connective tissue for the acute boat strike samples was 9.7% while the percentage of connective tissue for chronic boat strike and cold stress were 31.88% and 34.95% respectively. The cold stress animals showed the highest variability, but there were only two samples analyzed for this study. The red tide animals had a mean percentage of 18.9%. Please refer Table 3-1 above for averaged numbers for each samples and Table A-3 in Appendix A for all data generated from this examination. Statistical interpretations of this data will be forthcoming. 32


Immunohistochemistry The immunohistochemical analyses were inconclusive. Five samples showed immunoreactivity for AM-3K (red profiles; Figure 3-9a). These were usually no more than 20 cells in the entire tissue, with only one having around 50 positive profiles. These profiles did not correspond with what were identified as macrophages in the H&E stained slides. Use of the CD3 antibody appears to have been successful, with red coloration corresponding with cells carrying the CD3 marker. A 1:100 and 1:200 dilution were used for each samples to determine the best antibody dilution but there were no similarities in the strength of the reaction to each of the dilutions among the samples. The intensity of the staining in a 1:100 dilution for one sample did not correspond with the same dilution factor in another sample (Figures 3-9b and 3-9c). There were many cells with cytoplasmic immunoreactivity, but further investigation is needed to determine if CD3 is the best antibody to use to identify T cells in the thymus. Transmission Electron Microscopy The cytostructure of the thymus of the juvenile Florida manatee establishes specific cell types based solely on morphology within thymic and connective tissue elements. Within the cortical tissue, the primary cell population consists of lymphocytes which clearly define this region by their heavy density. The adjacent medullary portion consists of a smaller amount of lymphocytes and a number of other cell types. The ultrastructure to be described centers on the junction of the cortex and medulla. Figures 3-10a and 3-10b show, through H&E staining, the region that is further investigated ultrastructurally and is depicted in Figure 3-10c. The principal cells of the cortex are the thymocytes, which lie within the epithelial reticulum. These cells possess generally rounded to occasionally indented and 33


irregularly shaped nuclei with centrally placed nucleoli and clumped peripherally placed heterochromatin (Figure 3-11a). The cells varied to a small degree in diameter and contrasted sharply with the cells that form the epithelial reticulum (Figure 3-11b). These cells have variably shaped nuclei that generally are much larger and more euchromatic than the thymocyte nuclei. However, smaller cells with highly indented nuclei can be found along the corticomedullary junction (Figure 3-11c). The latter population of epithelial cells within this junction has highly filamentous cytoplasm. Within the corticomedullary junction, relatively thin-walled blood vessels are encountered (Figures 3-12a and 3-12b). Each vessel is lined at random intervals by pericytes, a small amount of connective tissue, and is surrounded by dendrites of adjacent epithelial cells (Figure 3-12c). The construction of these vessels differs considerably from the occasional nearby septa (Figure 3-12d), which have broad collagenous walls and associated fibrocytes. Monocytes and heterophils are seen within the lumen of these blood vessels (Figures 3-12e and 3-12f). The appearance of the thymocytes in this region varies, ranging from normal to various stages of degeneration (Figure 3-13a). The medullary region contain prominent epithelial cells that possess large somewhat crenulated nuclei surrounded by a small amount of cytoplasm and broad processes that branch and interconnect to one another by desmosomal attachment (Figure 3-13b). In addition to scattered lymphocytes, macrophages in different stages of activity are encountered (Figure 3-13c). While most secondary lysosomes are small, a few are found to be remarkably large, up to five m in diameter (Figure 3-13d). At 34


higher magnification, these bodies contain multiple organelles, suggesting the possibility that entire cells have been ingested. 35


A B Figure 3-1. Features of the thymus of the Florida manatee. A) Darker cortex and lighter medullary areas, x40 B) Hassalls corpuscle within the medulla, x400. A B C D Figure 3-2. Lymph nodes within the thymus of the Florida manatee. A) Found in 03R-30, a cold stress calf, x40, B) Found in 05R-364, an adult red tide, x100 C) Found in 06R-135, a chronic boat strike calf, x100 D) Found in 03R-19, an adult red tide, x100. 36


A B C D Figure 3-3. Four stains used for histological evaluation of an acute boat strike subadult. A) Hematoxylin & eosin, x250, B) Gomoris one step trichrome method, x250, C) Perls method for iron, x250, D) McManus method for glycogen (PAS), x250. 37


A B C D E F Figure 3-4. Aspects of involution in the thymus of the Florida manatee. A) Clustering of cells in the cortex, x250 B) Macrophages displaying a starry sky arrangement, x100, C) Thickening of connective tissue, x40,D) Increased connective tissue pushing thymocytes into long strips, x100, E) Spaces between thymocytes showing thymic epithelium, x400, F) Large granulocytes, x250. 38


A. B C D Figure 3-5. Involution grading system. A) Grade 1 full lobules and clear demarcation between cortex and medulla, x40, B) Grade 2 still see cortex and medulla with slight thickening of connective tissue, x40, C) Grade 3 loss of distinction between cortex and medulla and more noticeable connective tissue, x40, D) Grade 4 more connective tissue than cells, x40. 39


A B C.) D Figure 3-6. Acute boat strike and cold stress samples used in stereology study. A) 05R-163, subadult acute boat strike, x1, B) 06R-334, adult acute boat strike, x1, C04R-221, calf cold stress, x1, D) 05R-145, subadult cold stress, x1. 40


A B C Figure 3-7. Red tide samples used in stereology study. A) 03R-1, calf, x1, B) 03R-21, subadult, x1, C) 03R-23, adult, x1. 41


A B D ) C Figure 3-8. Chronic boat strike samples used in stereology study. A) 06R-60, calf, x1, B06R-135, calf, x1, C) 06R-142, subadult, x1, D) 04R-459, adult, x1. 42


A B C Figure 3-9. Immunohistochemistry studies on the thymus of the Florida manatee. A) Positive reaction from AM-3K macrophage antibody 06R-60, x250, B) Positive reaction from CD3 1:100 dilution on 03R-31, x250, C) Positive reaction from CD3 1:100 dilution on 06R-17, x250. 43


A B C.Figure 3-1hymus for electron microscopic investigation. A) Cortex, x250, B) Medulla, x100 C) Shot of cells and blood vessels to be investigated, x5000. 0. Establishing shots of the manatee t 44


A. B C Figure 3-1) contrasted with cell that forms epithelial reticulum, x10000, C) Smaller epithelial cell found along corticomedullary junction, x10000. 1. Electron microscopic investigation of the cortex of the Florida manatee. AThymocyte, x10000 B) Thymocyte 45


A B C D E F Figure 3-12. Electron microscopic investigation of the corticomedullary junction of the Florida manatee. A) Blood vessel, x15000 B) Blood vessel, x10000, C) Blood vessel surrounded by connective tissue and adjacent epithelial cells, x5000 D) Connective tissue septa, x10000, E) Monocyte located within a blood vessel, x10000 F) Heterophil located within a blood vessel, x10000. 46


A B C D ic medulla of the Florida manatee. A) Degenerating thymocytes, xprocesses, x10000, C) Macrophage, xvarying sizes, x15000. Figure 3-13. Electron microscopi c investigation of the thym5000, B) Epithelial cell with broad 5000, D) Secondary lysosomes of 47


CHAPTER 4 DISCUSSION Thymus Anatomy Marine mammal immunology and the anatomy of the organs that make up the immune system have been studied since the 1970s. Investigations into the immunology of the Florida manatee have been only recent and histologic descriptions of the lymphoid organs have not been published. In looking at the manatee thymus, the architecture was as expected, exhibiting incomplete lobules that consisted of a darker-staining, lymphocyte-rich cortex that usually contrasted well to a paler medulla. This was also found to be the same in the thymuses of other marine mammals, as described in the bottlenose dolphin (Cowan and Smith 1999). The areas of the medulla in the manatee thymus appear to be smaller and more numerous than has been seen in the rodent thymus (Pearse 2006). In the rodent thymus, the cortex to medulla ratio is usually 2:1, but it can change based on the orientation of the tissue when it has been e 2006). This could possibly be an issue with the when looksingle area of medulla does not seem to extend throughout the thymic tissue, which orresponds with what has been written that each lobe consisting of a convoluted arenchymal strand with irregular outcroppings that result in the lobules (Raviola 1986). Most of the samples had some sort of connective tissue capsule surrounding the tissue with septal interdigitations that resulted in small incomplete lobules. Hassalls corpuscles were rarely seen in the medulla and those that were seen looked smaller than those previously described in other species including humans and trimmed before embedding (Elmor manatee thymus and a determination of cortex to medulla ratio would be best made ing at the entire thymic lobe. Also, looking at a serially sectioned thymus, a c p 48


guinea pigs (Raviola 1986). These stalso been documented in the harbor porpoise (Beineke et al. 2007) and the bottlenose dolphin (Cowan 1994), but without any mention of frequency or range of size seen considered to be composed of thymic epithelial cells, usually terminally differentiated The average number of Hassalls corpuscles has not been determined for different dolphin and harbor porpoise were usually large and more noticeable in older animals be from the condensation of thymic epithelium during involution (Cowan 1994). Cysts in the thymus of five manatees. Parathymic lymph nodes have been described in humans and consider ructures have in the thymus. Hassalls corpuscles are forms of epithelial cells (Bodey et al. 2000). Functionally, it has been postulated that these bodies provide guidance in thymocyte development and may be critical in mediating thymic dendritic cells (Bodey et al. 2000, Watanabe et al. 2005). As the thymus ages, the center of Hassalls corpuscles fill with cellular debris (Bodey 2000). species, so it is not possible to compare what has been seen in the manatee thymus to other species. Contrary to what was seen in the bottlenose dolphin and harbour porpoise, there were no cysts found in any of the manatee thymus samples. The cysts noted in the (Cowan 1994, Wunschmann et al. 1999). Origins of the cysts in the dolphin are said to are considered to be rare in humans and domestic animals and have only been documented a few times in marine mammals. The absence of cysts in the manatee thymuses observed in this study show that they conform with other species. One finding that was unexpected was the appearance of one or more lymph nodes ed to be uncommon (Tanegashima et al. 1999). Lymph nodes of the aortic arch region have also been found to be closely associated with the thymuses 49


of bottlenose dolphins (Cowan 1994, Cowan and Smith 1999). One publication comparing lymph nodes and their locations in the bodies of several marine mammal species, including manatees, shows the retropharyngeal lymph nodes in the manatee as very close in relation to the thymus (Rommel et al. 2002). In the present investigation, the question of how common they may be in the thymus of the manateeremains to be seen. The samples used in this study were already trimmed and fiinto the embedding molds. As a result, only a small portion of the thymus for each animal was available. It would be interesting to see what could be found when sectioning through an entire thymus. When looking at thymus samples with large amounts of involution, there were no samples that did tted not have at least a small number of lymphocytes residing within the thyman ve ed with H&E.and us, possibly showing that the thymus of the manatee does not become completely devoid of these cells, which is the same finding that has been documented in the hum(Steinmann 1985). In a number of these samples, larger phagocytic cells were seen within the cellular space but usually a short distance from the connective tissue. These cells were larger than the macrophages seen in the starry sky formation. These are the same cells that exhibited a strong positive reaction with the PAS stain and habeen described as macrophages loaded with residual bodies (Raviola 1986). These cells appeared to contain tan-brown granular cytoplasmic material when stain The cells were seen in all age groups and three of the four causes of death, acute boat strike being the exclusion, though they were mostly seen in cold stress chronic boat strike samples. 50


Stereology The morphometric study gave results that to some degree were expected to be seen. The animals that died due to an acute boat strike were the least involuted, thus allowing them to be used as controls. The animals that died due to cold strehighest percentage of connective tissue, or involution, compared to the acute boat strikanimals. The animals that died due to red tide or chronic boat strike gave mixed resulbut were fairly equal between the samples. In this case, it was expected that the chronic boat strike cases would be more involuted. It is possible, considering thaccepted definition of chronic boat strik ss had the e ts at the e is trauma that is not immediately fatal (Lightsey et r e tissue of the rodent thymus was estimated from overall thymic volume obtained before processing and embedding. This method allowed them to determine the overall changes seen in the thymus and any changes seen with aging 2006), that all of the samples for this cause of death could have succumbed to their injuries at different times which could change the amount of involution seen. Usistatistical tests, it was found that there was no significant difference in involution due to age. Looking at the samples used in this study by age gave unreliable results, showing that the calves had the highest amount of connective tissue compared to the juvenile oadult. This finding can be attributed to the calf samples used which were primarily from the cold stress and chronic boat strike categories and most likely are not indicative of anormal manatee calf. In this study, volume was not able to be determined and only percentage of surface area was used. Other studies looking at age-related changes of the mouse thymus used volume fractions from the different areas of the thymus. Both manual delineation and a point counting method were used to determine area. Volume of the cortex, medulla and connectiv 51


in the cortex or medulla (Plea-Solaro6, Brelinska et al. 2008, Aw et al. 2009of phoid s that were seen light microscopically using the H&E stain. One reasoes in et ay vi et al. 200 ). The thymuses used in this study were not whole, so only sections of one lobe the thymus were cut and examined. Also, weights were rarely taken and might not be considered accurate because only one lobe was weighed. Immunohistochemistry Immunohistochemistry was performed on thymus samples using a macrophage antibody called AM-3K. This antibody had been used, with much success, on lymorgans from various species (Komohara et al. 2005, Zeng et al. 1996) and on the spleen and lymph nodes of cetaceans (Kawashima et al. 2004) and of manatees (Samuelson 2008). Unfortunately, this antibody did not work for the thymus of the manatee though the positive control was successful. A brief experiment was performed using lysozyme on one slide, but there was no reaction so it was not included in this study. Of the 20 samples used, only five showed the expected positive profiles. These could have corresponded to transient body macrophages passing through the thymus but this was not able to be determined. It was also hard to make a complete determination because of the lack of counterstaining, which might have given a better look at the macrophage n why this antibody might not have worked could be because the macrophagthe thymus are not the same as those found in the body. A recent paper (Wakimoto al. 2008) reported on human thymic cortical dendritic macrophages, or TCDM, that mact as professional scavengers of apoptotic thymocytes. The AM-3K antibody recognizes CD163, which is associated with the macrophage phenotype. Analysis of these TCDMs has found that they can lack the usual macrophage cell surface identifiers 52


of CD68, lysozyme and CD163, which may be the reason why the immunohistochemical tests were largely unsuccessful. Immunohistochemistry was also performed using a CD3 polyclonal antibody thathad been employed in a number of papers and was known f or its compatibility with various species (Sinkora et al. 2, Chadburn and Knowles 1994e so roblem lly microscopy. The occurrence of these degenerating cells within the corticomedullary 007, Tingstedt et al. 2003 ). Since CD3 is known to be a cell surface marker for thymocytes, it was used in this study to look at the thymocytes within the manatee thymus. The results of this experiment were expected, but did not give a better understanding of the cells in ththymus. Almost every lymphocyte in the thymus is considered to be a thymocyte,using the CD3 antibody caused most non-epithelial cells to react. Also, an optimal dilution level was not determined. Essentially, there was often no middle ground between cells that tended to react slightly at a 1:200 dilution and over-staining of the cells at a 1:100 dilution. It would probably be easier to look for and use antibodies that correspond to smaller subsets of the T cells or other populations of cells found in the thymus, like B cells, dendritic cells or plasma cells. Unfortunately, the major pwith this line of investigation is the difficulty of finding suitable antibodies that will react reliably with cells in an unrelated animal such as the manatee. Transmission Electron Microscopy The ultrastructural anatomy of the manatee thymus appeared to follow that whichhad been previously described and documented in other species (Varas et al. 1998, Brelinska 2003, Contreiras et al. 2004). Most cortical thymocytes exhibit a usuaround nucleus with moderate amounts of heterochromatin. Changes associated with deterioration of the thymocytes were best determined by transmission electron 53


junction was consistent with that observed in other species. Severavessels were noted, surrounded by connective tissue an l prominent blood d one or two pericytes. Other cells,tion. d reed e ide processes that form compartments for lymphohe medulla. The type IV cells of especially monocytes and macrophages, were determined due to lysosomal activity and location within blood vessels, as well as nuclear size, shape and orientaOf particular interest in the manatee thymus were several different types of observeepithelial cells. These were determined by the size and shape of the nucleus and by the amount and lucidity of the cytoplasm. Thymic Epithelial Cells There have been a number of studies of the epithelial cells of the vertebrate thymus and each has determined different subsets of the thymic epithelial cells (De Waal and Rademakers 1997, Brelinska 2003, Pearse 2006). One thing that is agupon is that there are four main epithelial cell subsets subcapsular, cortex, medulla and those that comprise Hassalls corpuscles. Smaller subsets introduced by some papers were determined by the area of the cortex or medulla they reside within and thproteins and cytokines they express. The epithelial cells observed in the manatee thymus most closely conform to the Type III and Type IV subsets that have been described by Gartner and Hiatt (1997). The type III cells have w cytes and help isolate the cortex from t lls are closely associated with the type III cells but are contrasted by a darker staining cytoplasm filled with tonofilaments. These are only two types of epithelial cethat were found in the thymus that was studied. Electron microscopic investigation more sections of the manatee thymus could increase the number of types of epithelial cells that compose it. 54


Cytokeratins Cytokeratins are one protein that has garnered a number of studies as a way to differentiate epithelial cells from lymphocytes in the thymus. Cytokeratins comprisetonofilaments that are contained within thymic epithelial cells. Fifty-four of these polypeptides have been characterized with regard to size and isoelectric point (IP) andhave been subdivided into Type II, having a higher molecular weight and basic IP andType I, having a lower molecular weight and the acidic IP (Schweizer et al. 2006). They are r and appear as Type I/Type II pairs. There is an unusually compcept e lar e lls n was not found to be constant which could suggest that certain molecules deteriorate earlier than others or that there is a progressive loss of the usually expressed togethe lex pattern of cytokeratin expression within the thymic epithelium (CK 1-20, exfor CK 3, 12 and 9). Each region of the thymus expresses different cytokeratins. Thmost complex cytokeratin expression was observed within the Hassalls corpuscles (Shezen et al. 1995). The same cytokeratins can be found in the thymuses of other species but there have been extreme differences found in the expression of a particucytokeratin, enough that in a study using six species, not one possessed the same cytokeratin catalogue (oli et al. 1990). This could possibly account for some of thdifferences in immune response among different species. Several studies have shown that there is a gradual decline in cytokeratin expression with age. As the thymus ages, there is a loss of characteristic morphology with the appearance of epithelial-free areas in the thymus, thus resulting in fewer ceexpressing cytokeratin. The process is similar in the medulla with cyst-like structures forming in the thymuses of older mice. In fact, the overall volume of cytokeratin was significantly reduced in the 24 month old mouse compared to the 1 month old mouse. The rate of deterioratio 55


different TEC s ubsets (Aw et al. 2009). This has not been determined to be the same in otheratrix n of an exotic species. The morphometric studies look to be a good way to determine degree of thymus involution in species, so it is possible that this could also play a role in the varying immune responses as many species age. Immunohistochemical studies involving cytokeratin have been performed with a number of different species to look at the epithelial mof the thymus. These studies were also considered for the manatee thymus, but production of the particular polyclonal cytokeratin antibody that was considered was inconsistent and affected the timeline of the experiment. Conclusions and Future Directions This is the first study of the manatee thymus. As the anatomy and cellularity of the thymus has not been studied previously, this is the first step in what will hopefully become a comprehensive investigation of the thymus. It can be concluded that the microanatomy of the manatee thymus is similar to descriptions published for other vertebrate species. In order to determine the different cells that populate the thymus, immunohistochemistry seems to be the best technique to use. A thorough evaluatiouseful antibodies should be made before being applied to the tissue, especially when working with an animal, like the manatee, that is considered each cause of death of the manatee. The electron microscopy showed ultrastructural features that look to be unique to the manatee and is possibly the best tool to give a complete understanding of the microanatomy of the manatee thymus. Together, all of these techniques are excellent ways to acquire an overall understanding of the composition of the manatee thymus and to help create a stepping stone to more complex investigations. 56


Throughout this study, ideas for future research avenues frequently presented themselves. A continuation of the immunhistochemical experiments could look at thecell surface markers CD25 and CD44. These could potentially be better ways to look aspecific subsets of developing T cells as during the maturation of the T cells, these cell surface markers are turned on and off. This could give an indication of the locatiothese cells in the thymus during each of the maturation stages. Following up on the paper on TCDM, use of CD209 or fascin could be used to t n of determine the presence of this special set of macrops that are expressed by thto ing y hages. There are a number of cytokine e different T cell subsets and the thymic epithelial cells, which are starting to be examined in the thymuses of other species. As mentioned previously, it would be interesting to examine a complete thymic lobe from a freshly dead manatee in order section through the entire organ and give an overall look at the anatomy. Also, havan entire lobe will give the weight and a determination of the volume using morphometry. A continuation of the morphometry study by sectioning through all of the samples would give more slides and areas on each tissue to solidify the results. Finallacquiring additional samples, possibly and adult acute boat strike and a cold stressedcalf, to examine through electron microscopy would give better ultrastructural descriptions. 57




Table A-1. Animals used in the study. Field I.D.# Lab I.D.# Probable Cause of Death Length (cm) Weight (kg) Gender Age Grade MSW0312 03R-34 Natural; Cold Stress 189127.5M calf 3 MSW03176 04R-221 Natural; Cold Stress 19848.5F calf 4MSW03170 04R-222 Natural; Cold Stress 192127.5M calf 3MNW0404 04R-225 Natural; Cold Stress 189125.5F calf 3MNW0434 06R-128 Natural; Cold Stress 170101.5F calf 2MSW0491 06R-129 Natural; Cold Stress 197N/T M calf 4MEC0616 06R-133 Natural; Cold Stress 199N/T F calf 2MNE0433 06R-136 Natural; Cold Stress 214N/T F calf 3MNW0432 06R-139 Natural; Cold Stress 15762.5M calf 4MNW0563 06R-18 Natural; Cold Stress 165104M calf 4MSW0716 07R-30 Natural; Cold Stress 177156.5F calf 3MEC0706 07R-33 Natural; Cold Stress 232207M calf 3MSW0320 03R-30 Natural; Cold Stress 209121.5F calf 4MEC0515 05R-141 Natural; Cold Stress 186136.5M calf 4MSE0509 05R-144 Natural; Cold Stress 174N/T F calf 3MSW0412 04R-265 Natural; Cold Stress 249240M subadult2MSW0517 05R-145 Natural; Cold Stress 252317.5F subadult2MSW0356 03R-11 Natural; Other (Red Tide) 273400F adult 3MSW0340 03R-19 Natural; Other (Red Tide) 276408.5F adult 4MSW0357 03R-20 Natural; Other (Red Tide) 269341.5M adult 2MSW0387 03R-24 Natural; Other (Red Tide) 311529F adult 4MSW0347 03R-26 Natural; Other (Red Tide) 315499M adult 2MSW0346 03R-33 Natural; Other (Red Tide) 301463.5M adult 2MSW0355 03R-47 Natural; Other (Red Tide) 298466F adult 4MSW0332 03R-48 Natural; Other (Red Tide) 307469.5M adult 3MNW0338 03R-49 Natural; Other (Red Tide) 292415.5M adult 3MNW0548 05R-364 Natural; Other (Red Tide) 301464F adult 2MSW0377 03R-23 Natural; Other (Red Tide) 279383M adult 2MNW0410 06R-66 Natural; Other (Red Tide) 307672M adult 2MSW02100 03R-1 Natural; Other (Red Tide) 219203.5M calf 2MSW03130 03R-31 Natural; Other (Red Tide) 186134M calf 1 59


Table A-1. Continued. Lab I.D.# Probable Cause of Death Length (cm) Weight (kg) Gender Age Grade Field I.D.# MSW0299 03R-36 Natural; Other (Red Tide) 204182M calf 1 MSW0597 06R-16 Natural; Other (Red TidNatural; Other (Red Tide e) 2120.5lf 3) 6466badult2 d Tide) 3.5F badult2 d Tide) 2.5M badult1 er (Acute) 99F ult 4 8(Acute) 39F ult 2 (Acute) 2M ult 1 (Acute) 7371.5M ult 2 (Acute) 9371M ult 24 (Acute) 711F ult 1 4(Acute) 2.5M ult 2 7(Acute) 7N/T F badult2 7(acute) 0.5M badult1 32(Acute) 84F badult1 3ute) 1.5F badult1 hronic) 4M ult 206b4 hronic) 093M 2 3(Chronic) 07F 12onic) 5F 244nic) 41F lt 124nic) 5N/T M lt 398nic) 09F lt 16b45 nic) N/T 9F lt 4 ronic) 7.5M lt 2 5 ronic) 54M lt 24b58 ronic) 71F lt 459ronic) 9.5N/T lt 3 7 ronic) 06M lt 22 ronic) 41F lt 22 nic) 12M 3 35ronic) 8.5M 3 0ronic) 0.5F lf 2c) M subadult4 20 M M casu MSW0353 03R-10 226 3368 MSW03143 03R-4 103R-21 Natural; Other (Re su MSW0359 Natural; Other (Re 26 328 su MSW0323 03R-25 Watercraft; Propell 28 53 ad MNW0430 06R-13 Watercraft; Impact 27 42 ad MNW0316 03R-29 Watercraft; Impact 27 387.5 ad MNW0232 03R-42 Watercraft; Impact 28 ad MEC0348 03R-43 Watercraft; Impact 27 ad MSE0519 05R-17 Watercraft; Impact 29 6409 ad MNW0537 06R-33 Watercraft; Impact 29 ad MSW0305 03R-2 Watercraft; Impact 25 su MEC0562 06R-1 Watercraft; Impact 25 293 su MEC0633 06R-1 Watercraft; Impact Watercraft; Both (A 23 38 su MSE0504 05R-1 602R-30 c Watercraft; Impact (C 26 407 su MSW0290 30 472 ad SWFTm04 04R-29 Watercraft; Impact (C 29 339 adult adult MNW0543 05R-38 Watercraft; Impact 27 LPZ102201 06R-35 Watercraft; Impact (Chr Watercraft; Impact (Chro 34 891.31 adult GA2006019 06R-4 29 51 adu MSW05106 06R-5 Watercraft; Impact (Chro 28 adu MSW06130 06R-6 Watercraft; Impact (Chro 30 57 adu SWFTm050 06R-3 Watercraft; Impact (Chro 43 adu MEC0262 03R-3 Watercraft; Propeller (Ch 28 473 adu MSW03112 03R-3 Watercraft; Propeller (Ch 28 38 adu SWFTm040 04R-4 Watercraft; Propeller (Ch 26 32 adu LPZ101951 04R-4 Watercraft; Propeller (Ch 28 495 adu LPZ102002 06R-3 306R-44 Watercraft; Propeller (Ch 28 37 adu MSW0666 Watercraft; Propeller (C hWatercraft; Impact (Chro 28 40 adu LPZ102106 07R-3 20 14 calf MSW0644 06R-1 Watercraft; Propeller (Ch 14 56 calf MSW0601 06R -6MSW0428 04R-263 Watercraft; Propeller (Ch 17 110 ca Watercraft; Impact (chroni 263 302.5 60


Table A-1. Continued. Field I.D.# Lab I.D.# Probable Cause of Death Length (cm) Weight (kg) Gender A Grade LPZ102120 c) dult 06R-420 Watercraft; Impact (Chroni 259 306 F suba 3 SWFTm062 1 c) N/T dult c) b c) ) N/T ) ) 2ult4b nic) N/T ult2b nic) ult 06R-666 Watercraft; Impact (Chroni 249 F suba 2 MNW0646 06R-697 Watercraft; Impact (Chroni 235 260 M subadult 2 SWFtm0520 07R-29 Watercraft; Impact (Chroni 255 255 M subadult 4 LPZ102203 06R-421 Watercraft; Impact (Chronic 326 M ult 2 MNW0237 03R-46 Watercraft; Propeller (Chronicnic 265 428 F subad 1 MEC0615 06R-142 Watercraft; Propeller (Chro 249 29.5 F subad 4 SWFTm061 06R-443 Watercraft; Propeller (Chro 260 F subad 3 SWFTm041 04R-292 Watercraft; Propeller (Chro 257 N/T M subad 3 61


Table A-2. Thymus samL ples used for immunohistochemistry. LeWeigen Field I.D.# ab I.D.# Probable Cause of Death ngth (cm) ht (kg) G der Age MEC0562 06R-17 Watercraft; Impact (acute) 250 293.5 M sub a dult MEC0633 06R-132 238384 suba272387.5 adult 273429 adult 170110.5 calf 14856.5 calf 1 249 suba 235260 suba345.31 F adult 4 04R-22189125.5 F calf MNW0432 06R-139 Natural; Cold Stress 15762.5 M calf MSW0412 04R-265 Natural; Cold Stress 249240 M subadultMSW0517 05R-145 Natural; Cold Stress 252317.5 F subadultMNE0304 03R-38 Natural; Cold Stress 303426 M adult MSW0597 06R-16 Natural; Other (Red Tide) 212200.5 M calf MSW03143 03R-41 Natural; Other (Red Tide) 263368.5 F subadultMNW0338 03R-49 Natural; Other (Red Tide) 292415.5 M adult MNW0548 05R-364 Natural; Other (Red Tide) 301464 F adult Watercraft; Propeller (Acute) F dult MNW0316 03R-29 Watercraft; Impact (Acute) M MNW0430 06R-138 Watercraft, Impact (Acute) F MSW0601 06R-60 Watercraft; Propeller (Chronic) F MSW06442 06R-135 Watercraft; Propeller (Chronic) M SWFTm06 06R-666 Watercraft; Impact (Chronic) N/T F M dult MNW0646 LPZ102201 06R-69706R-352 Watercraft; Impact (Chronic) Watercraft; Impact (Chronic) dult 891 MNW040 5 Natural; Cold Stress 62


Table A-3. Complete data generated from stereology study. Animal ID 0 3RR-205R-1436004R-459 06R-334 -1 03 1 03R-23 04R-221 5 05R-1 6 06R-60 0 R-135 6R-1 42 Area 1 1324 284 0.250.4069 0.0392 0.0833 0.402 0.3627 Area 2 0.201 0.20.11460.31370.08330.0.10.273660.36760.08820.0.10.2647240.4010.11270.0.1.19129170.21880.0773.0.10.2010.42650.0729.0.00.18140.53920.10290.20.25980.44120.15630.0.10.230490.39710.0980.58330.12447110.34380.12250.0.2.4630.14710.22060.10780.148530.17650.43630.10880.0.159310.16490.32580.08330.10.385890.26470.11460.0.00.18230.44120.10680.0.0.20590.4570.10780.0.30.245170.38460.0784 0.38360.0931Average 0.153556 0.184794 0.2275670.49360.2047710.0959390.120772 0.3945170.3867590.3720.097689 591 0.35780.5885 0.0829 0.1029 0.3578 0.3971 Area 3 0882 078 0.21350.5343 0.0625 0.1912 0.2941 0.375 Area 4 1029 373 0.25490.5813 0.12 0.1458 0.2938 0.328 Area 5 4308 615 0.19120.38810 0.1078 0.1225 0.5392 0.2 Area 6 0Area 7 0 1132 127 0.18630.4688 0.1225 0.1198 0.525 0.46570.4853 0686 931 0.17650.4381 0.0637 0.1042 0.33820.0885 0.3137 Area 8 0.099 108 0.21950.5429 0.1094 0.3627 Area 9 1094 421 0.21570.4314 0.0686 0.1176 0.3043 0.29 Area 10 0.14 349 0.23040. 0.1162 0.1324 0.299 0.3 Area 11 1569 598 0.21570 0.0833 0.1176 0.555 0.46310.4569 Area 12 0.098 256 0.20830. 0.1471 0.1078 0.5735 Area 13 3039 225 0.2650. 0.0833 0.1471 0.2402 0.3894 Area 14 0.151 618 0.1771 0.4844833 0.27450.3698 0.0878 0.1422 0.3578 0.442 Area 15 1147 0.0765 0.1324 0.2843 0.31860.348 Area 16 1765 299 0.33330.4634 0.1073 0.1324 0.3137 Area 17 1225 088 0.14510.5686 0.0677 0.1029 0.6146 0.416 Area 18 0.155 0.0637 0.1814 0.1791 0.0833 0.4951 63


APPENDIX B STAINING PROTOCOLS Gom 1. paraffinize and hydrate to distilled water 2. Place in Bouins solution in oven at 56C for one hour 3. Wisappears 4tion 10 minutes 5. 6. 7. t If sections are too dark, cioic.7 gm for phosphotungstic acid has been added. Rinse in distilled water 8. clear in xylene, two changes each 9. McManus Method for Glycogen (PAS) 1. 2. 3. Rinse in distilled water 4. 15 minutes 5. lor to develop rris hematoxylin for 6 minutes, or light green counterstain for a few seconds. Light green is recommended for counterstaining sections in which fungi are to be n sed ferentiate in 1% (HCL) acid alcohol thrten quick dips Wash in running water De alcohol and clear in xylene, two changes each .le D tes cchid solution for 20 minutes Rinse well in distilled water Counterstain in nuclear fast red solution for 5 minues hydrate in 95% alcohol, absolute alcohol, and clear in xylene, two chanes each Mount with Permount of Histoclad orDe is O ne S te p T ri ch ro me M e tho d asain h w n euc ll inlei r w unith nin W g eig wae terts r o ir r uon nt he il ym elato lowxy clin olo s r dolu StWash in water TrPldif ichacfe roe iren men 0tia s.5te ta% in in gl 1% foraci 15al gla toac 2etial a 0 c wce minatic uter w tes foat r 2er t m inu wh es. h 0 DeMo hyu drnt atwi e ith n 9Pe 5%rm aou lcnt oh or ol, H aist bsooc lulad te al co ho l a nd DeOx paid raize ffi in niz p e er aniod d hic ydac raid teso tolut diion sti fo lledr w5 m atin erut es CoWHa leas mah i nn r s Fun eni ulgng en wa ote r Sr f chor iff10 re m agin enute t ss olufor ti p onink for co Per deWDif mas onh i strn r ateun dni Ong m wa it te ster ps 7 -11 if lig ht gr ee is u to ee ip ias n ah i mn r moun nni ia ng wa wa tete r tor f bor 1 lue0 s m ecin tioute nss 1. W. Dehydrate in 95% alcohol, absolut Ms M ou nt th wiod th fo Per rmIro oun nt or H ist oc lad epaocork rak pin ffiotg p nizasot e siuas anmsiu d h fem ydrro fo racyrro tean toidyan die sid stiolelledutihy wondro at fo err 5lo mric StW inu ac WDe as h w e ll in r un nin g wa te r 64


APPENDIX C IMMUNOOCOLS alin) for 24 hours, change to PBS for arged slides, 0 degrees Celsius oven for 2 to 3 hours. (3 changes, 5 min. each) p water (2 changes, 2 min. each) e (1 change, 2 min.) ___ (2 min. each change) y. & 895.5 ul of PBS ooler. __ (2 changes, 2 min. each) in. ___ (2 changes, 2 min. each) te Chromagen (AEC from Zymed) for 5 min or until color ges, 4 min. each) HISTOCHEMISTRY PROT Macrophage Localization 1. Fixation: 10% BNF (Buffered Neutral Form 12 hours or as soon as possible (if needed) 2. Routine processing after the 24 hours of fixation. 3. Section at 5 microns, place sections on SuperFrost positively ch then place in 6 4. Deparaffinize ___ ___ ___ Xylene ___ ___ 100% Alc. (2 changes, 2 min. each) ___ ___ 95% Alc (2 changes, 2 min. each) ___ ___ Rinse in ta ___ ___ Distilled water (2 changes, 2 min. each) 5. Rehydrate to PBS Phosphate Buffered Salin 6. Quench with 3% Hydrogen Peroxide (20 min.) 7. Wash sections with PBS ___ ___ 8. Remove excess PBS, blot with Kimwipes. 9. Block with goat serum (10%) for 30 min. blot and apply antibod 10. Lightly rinse with PBS 11. Primary antibody: Antibody diluted with PBS Dilute (1:50, 1:100, 1:200) A. 1:50 18 ul of primary antibody & 882 ul of PBS B. 1:100 9 ul of primary antibody & 891 ul of PBS 00 4.5 ul of primary antibody C. 1:2 12. Incubate sections in humidified changer overnight in walk-in cspecific for antibody 13. Detection: Zymed kit A. Wash sections PBS ___ B. Biotinylated yellow for 20 m C. Wash sections PBS ___ ___ (2 changes, 2 min. each) D. Streptavidin for 10 min.E. Wash sections PBS __ F. Substrachange. G. Distilled water ___ ___ (2 chan H. Coverslip with glycerol jelly. 65


CD3 Localization 1. Fixation: 10% BNF (Buffered Neutral Formalin) for 24 hours, change to PBS for s possible (if needed) 2. Routine processing after the 24 hours of fixation. o 3 hours. 5 min. each) n. each) in. each) h) 5. Rehydrate to6. Quench with 7. Wash sectionith PBS ) 1:50 18 ul of primary antibody & 882 ul of PBS 891 ul of PBS & 895.5 ul of PBS changes, 2 min. each) changes, 2 min. each) il color 12 hours or as soon a 3. Section at 5 microns, place sections on SuperFrost positively charged slides, then place in 60 degrees Celsius oven for 2 t 4. Deparaffinize ___ ___ ___ Xylene (3 changes, ___ ___ 100% Alc. (2 changes, 2 min. each) ___ ___ 95% Alc (2 changes, 2 mi ___ ___ Rinse in tap water (2 changes, 2 m ___ ___ Distilled water (2 changes, 2 min. eac PBS Phosphate Buffered Saline (1 change, 2 min.) 3% Hydrogen Peroxide (20 min.) s with PBS ___ ___ ___ (2 min. each change) 8. Remove excess PBS, blot with Kimwipes. 9. Block with goat serum (10%) for 30 min. 10. Lightly rinse with PBS, blot and apply antibody. 11. Primary antibody: Antibody diluted w Dilute (1:50, 1:100, 1:200 A. B. 1:100 9 ul of primary antibody & C. 1:200 4.5 ul of primary antibody 12. Incubate sections in humidified changer overnight in walk-in cooler. 13. Detection: Zymed kit specific for antibody I. Wash sections PBS ___ ___ (2 changes, 2 min. each) J. Biotinylated yellow for 20 min. K. Wash sections PBS ___ ___ (2 L. Streptavidin for 10 min. M. Wash sections PBS ___ ___ (2 N. Substrate Chromagen (AEC from Zymed) for 5 min or unt change. O. Distilled water ___ ___ (2 changes, 4 min. each) P. Coverslip with glycerol jelly. 66


APPENDIX D ELECTRON MICROSCOPY EMBEDDING Followol 25%50% a70%95% alcohol 100% alcohol after the last alcohol2 EtOH:1 acetone 1 E1 E 1002 p45 min 1 p45 min 1 pvernight Prees in fresh plastic under vacbed tissue. Place back under vacd, place in a 37os. Forwith wooden app a carcinogenic). Araldite 502 Epon 812 DDSA ml DMP-30 ing tissue fixation, dehydrate with ethyl alcoh alcohol 1 change 15 min lcohol 1 change 15 min alcohol 1 change 15 min 2 changes 15 min 2 changes 15 min step put samples on a rotator until time for embedding 1 change 15 min tOH:1 acetone 1 change 15 min tOH:2 acetone 1 change 15 min % acetone 2 changes 15 min arts acetone, 1 part epon-araldite art acetone, 1 part epon-araldite art acetone, 2 parts epon-araldite o pare fresh Epon-Araldite mixture (plastic), place sampl uum with remaining embedding plastic for 30 min. Em uum to pull out air bubbles. Once all the air bubbles have been remove C oven for 24 hours, 45 o C oven for 24 hours and 60 o C oven for 24 hour mixing of plastics use disposable beakers, disposable syringes and stir licator sticks. Then throw all away (DMP-30 is 5.0 ml 10 ml 20 ml 6.5 ml OR 12.5 ml OR 25 ml 15 ml 30 ml 60 0.8 ml 1.3 ml 2.5 ml (fills appx. 39 caps) (appx. 70 caps) (appx. 150 caps) The DDSA, Eured in the same graduated cylinder, hich can be warmed in a 60C oven for a few minutes to make pouring easier. Put the mixture in a small disposable beaker. Stir or mix for 15 minutes. Mixture may be placed in a low vacuum oven (temp. off) to eliminate bubbles following mixing. The same batch of Epon-Araldite may be used for both epoxy-acetone changes (the 2:1 and 1:2 mixtures). After the mixture sets for 2 or more hours, it begins to thicken. Do not use after this time for the final embedding mixture. Mix fresh for the following day for embedding. 27.3 ml 53.8 ml 107.5 ml pon and Araldite should be measo w 67


APPENDIX E STEREOMETRY 2. Calibrate lenses for the objectives to be used. re of interest. rmatective image source if using a slide. Select if usinime. mling i, thn click Next to move to the next screen. nification. atn, cli on tle Tissue button. idetifiable point on the section, then click the 14. Outline the reference space by clicking around the perimeter of the feature to be 16. Click Done. Probe information will be specific for the section. Click Next. reen arrow. 18. Click on the Xs (they will turn green) of those that hit the feature being looked at. pop up in another window. LOGER PROGRAM FOR ORGAN MORPHO 1. Open Stereologer program. 3. Click File and select New Study. 4. Enter information about the study in the fields provided. 5. Choose Multi-Level (Fraction Based). 6. Select Volume and click Add. 7. Choose the Region Volume probe and enter name of the featu 8. Click Done, then Next to move to the next screen. 9. Enter case infoion and sel L Stored source g a saved ag 10. Enter the sapnformatione 11. Set the Region (Low) magnification and the Object (High) mag 12. To determine best Probe informiockhe Samp 13. Set an origin by clicking on ann green arrow. looked at, then click the green arrow. 15. Set section thickness by adjusting focus. Click Set Top button when at the top of the section. Focus through to the bottom of the section and click Set Bottom button. 17. Set the origin point then click on the green arrow. The reference space should show the same outline from the previous step. Click on the g Click on the green arrow. Set section thickness. Click Done. Results will 68


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BIOGRAPHICAL SKETCH Kimberly Goldbach was born in 19 82. She has spent most of her life living in Carolina. The author attended Goose Creek High School there, graduating in 2000. She received a Bachelor of Science degree in biology in 2004 from Winthrop University in Rock Hill, South Carolina. After two years working as a research assistant at Vanderbilt University in Nashville, Tennessee she moved to Gainesville, Florida in order to pursue a Master of Science degree in Veterinary Medical Sciences at the University of Florida. Following graduation Kimberly will pursue work in research to ultimately find a position in a laboratory working with marine mammals. Goose Creek, South Carolina which is a town just 20 miles outside of Charleston, South 77