Black band disease of corals: Ecology and physiology of Phormidium corallyticum

Black band disease of corals consists of a microbial community dominated by the cyanobacteriurn Phormidium corallyticum. The disease primarily affects reef-framework coral species, Active black band disease continually opens up new substrate in reef environments by destroying coral tissue as the disease line advances across the surface of infected colonies. A field study was carried out to determine the abundance and distribution of black band disease on the reef building corals in the Florida Keys. During July of 1992 and 1993, up to 0.72% of coral colonies were infected with black band disease. Analysis of the distribution showed that the disease was clumped. Seasonal patters varied, with some coral colonies infected year round, others exhibiting reinfection from summer 1992 to summer 1993, and some colonies infected for one year only. Statistical analysis of black band disease incidence in relation to various environmental parameters revealed that black band disease was associated with relatively shallow water depths, higher temperatures, elevated nitrite levels, and decreased ortho-phosphate levels. Additional field studies determined recovery of scleractinian coral colonies damaged or killed through the activities of black band disease over a five-year period. These studies determined if the newly exposed substrate was recolonized through scleractinian recruitment, if there was overgrowth of the damaged areas by the formerly diseased colony, or if coral tissue destruction continued after the cessation of black band disease activity. Tissue loss continued on all coral colonies with only one colony exhibiting new tissue growth. The majority of recolonization was by non-reef-framework corals and octocorallians, limited recruitment by framework species was observed. Physiological studies of P. corallyticum were carried out to investigate the photosynthetic capacity of this cyanobacterium, and to determine if this species has the ability to fix dinitrogen. The results of this research demonstrated that P. corallyticum reaches maximum photosynthetic rates at very low light intensities (27.9 μE/m/sec), and that P. corallyticum is able to carry out oxygenic photosynthesis in the presence of sulfide, an ability that is uncommon in prokaryotic organisms. ^

Coronary heart disease risk factors among tri-ethnic students at Florida International University

The present study identified and compared Coronary Heart Disease (CHD) risk factors quantified as “CHD risk point standards” (CHDRPS) among tri-ethnic (White non-Hispanic [WNH], Hispanic [H], and Black non-Hispanic [BNH]) college students. All 300 tri-ethnic subjects completed the Cardiovascular Risk Assessment Instruments and had blood pressure readings recorded on three occasions. The Bioelectrical Impedance Analysis (BIA) was used to measure body composition. Students' knowledge of CHD risk factors was also measured. In addition, a 15 ml fasting blood sample was collected from 180 subjects and blood lipids and Homocysteine (tHcy) levels were measured. Data were analyzed by gender and ethnicity using one-way Analysis of Variance (ANOVA) with Bonferroni's pairwise mean comparison procedure, Pearson correlation, and Chi-square test with follow-up Bonferroni's Chi-square tests. ^ The mean score of CHDRPS for all subjects was 19.15 ± 6.79. Assigned to the CHD risk category, college students were below-average risk of developing CHD. Males scored significantly (p < 0.013) higher for CHD risk than females, and BNHs scored significantly (p < 0.033) higher than WNHs. High consumption of dietary fat saturated fat and cholesterol resulted in a high CHDRPS among H males and females and WNH females. High alcohol consumption resulted in a high CHDRPS among all subjects. Mean tHcy ± SD of all subjects was 6.33 ± 3. 15 μmol/L. Males had significantly (p < 0.001) higher tHcy than females. Black non-Hispanic females and H females had significantly (p < 0.003) lower tHcy than WNH females. Positive associations were found between tHcy levels and CHDRPS among females (p < 0.001), Hs (p < 0.001), H males (p < 0.049), H females (p < 0.009), and BNH females (p < 0.005). Significant positive correlations were found between BMI levels and CHDRPS in males (p < 0.001), females (p < 0.001), WNHs (p < 0.008), Hs (p < 0.001), WNH males (p < 0.024), H males (p < 0.004) and H females (p < 0.001). The mean knowledge of CHD questions of all subjects was 71.70 ± 7.92 out of 100. The mean knowledge of CHD was significantly higher for WNH males (p < 0.039) than BNH males. A significant inverse correlation (r = 0.392, p < 0.032) was found between the CHD knowledge and CHDRPS in WNH females. The researcher's findings indicate strong gender and ethnic differences in CHD risk factors among the college-age population. ^

Coral disease dynamics and environmental drivers in the northern Florida Keys and Lee Stocking Island, Bahamas

Coral reefs are experiencing declines worldwide and recently coral diseases have been identified as significant contributors to coral mortality. However, little is known regarding the factors that drive coral disease distributions and dynamics. Current knowledge of the organisms that cause coral diseases is also limited, with pathogens having been identified for only 5 of the 21 described coral diseases. The study presented here describes coral disease dynamics in terms of occurrence, prevalence, spatial distribution, and host species susceptibility from 2002--2004 on reefs of the Northern Florida Keys (NFK) and Lee Stocking Island (LSI) in the Bahamas' Exuma chain. In addition, this research investigated the influence of temperature, sediment, and nutrient availability on coral disease prevalence and severity. Finally, microbial communities associated with a polymicrobial disease, black band, were examined to address spatial and temporal variability. ^ Four scleractinian diseases were observed in repeated surveys conducted during June-August of each year: black band disease (BBD), white plague type 2 (WP), dark spots syndrome (DSS), and yellow band disease-(YBD). Coral disease prevalence was generally low in both the NFK and LSI as compared to epizootic levels reported previously in the NFK and other regions of the Caribbean. Disease prevalence and species susceptibility varied spatially and temporally. Massive framework species, including Siderastrea siderea, Colpophyllia natans, and Montastraea annularis, along with relatively smaller colonies of Meandrina meandrites and Dichocoenia stokesi, were most susceptible to disease. Temperature, sedimentation, and dissolved inorganic nitrogen were positively correlated with BBD infections. Furthermore, experimental nutrient enrichment exacerbated coral tissue loss to BBD both in situ and in vivo. Profiling of BBD microbial communities using length heterogeneity PCR revealed variation over space and time, with significantly distinct bacterial assemblages in the NFK, LSI, and US Virgin Islands. ^ This study contributes to knowledge of the relationship between coral diseases and the environment, and facilitates predictions regarding potential changes in coral reef communities under differing environmental conditions. Additionally, this research provides further understanding of coral disease dynamics at both the host and microbial pathogen levels.^

Contribution of the vasculature to the Alzheimer's disease process

One of the pathological hallmarks of Alzheimer's disease (AD) brain is extracellular β-amyloid (Aβ) plaques containing 39-42 amino acid Aβ peptides. The deposition of Aβ around blood vessels, known as Cerebral amyloid angiopathy (CAA), is also a common feature in AD brain. Vascular density and cerebral blood flow are reduced in AD brains, and vascular risk factors such as hypertension and diabetes are also risk factors for AD. We have shown previously that Aβ peptides can potently inhibit angiogenesis both in-vitro and in-vivo, but the mechanism of action for this effect is not known. Therefore, my first hypothesis was that particular amino acid sequence(s) within the Aβ peptide are required for inhibition of angiogenesis. From this aim, I found a peptide sequence which was critical for anti-angiogenic activity (HHQKLVFF). This sequence contains a heparan sulfate proteoglycan growth factor binding domain implying that Aβ can interfere with growth factor signaling. Leading on from this, my second hypothesis was that Aβ can inhibit angiogenesis by binding to growth factor receptors. I found that Aβ can bind to Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2), and showed that this is one mechanism by which Aβ can inhibit angiogenesis. Since the vasculature is disrupted in AD brains, I investigated whether a strategy to increase brain vascularization would be beneficial against AD pathology. Therefore, my third hypothesis was that voluntary exercise (which is known to increase brain vascularization in rodents) can ameliorate Aβ pathology, increase brain vascularization, and improve behavioral deficits in a transgenic mouse model of AD. I found that exercise has no effect on Aβ pathology, brain vascularization or behavioral deficits. Therefore, in the transgenic mouse model that I used, exercise is an ineffective therapeutic strategy against AD pathology and symptoms.

Relevance of CD40/CD40L downstream pathways to Alzheimer's disease

The amyloid cascade hypothesis places amyloid-β at the origin of Alzheimer's disease (AD). Amyloid-β (Aβ) is the product of the sequential cleavage of the amyloid precursor protein (APP) by the enzymes β- and γ-secretases. An inflammatory component to AD has been suggested in association with CD40 (a member of the tumor necrosis factor receptor superfamily (TNFRS) and its cognate ligand CD40L. In this study, I hypothesized that the neutralization of pro-inflammatory cytokines produced downstream of CD40/CD40L interaction would reduce APP processing. I also hypothesized that blocking the binding of different adaptor proteins to CD40 by mutating its cytoplasmic tail would result in significant reduction of the APP metabolites: Aβ, sAPPβ, sAPPα, CTFβ and CTFα. ^ Treatment with CD40L of human embryonic kidney cells over-expressing both APP and CD40 (HEK/APPsw/CD40) significantly increased levels of the cytokine granulocyte macrophage colony stimulating factor (GM-CSF). Neutralizing antibodies against GM-CSF mitigated the CD40L-induced production of Aβ in these cells. Treatment of the HEK/APPsw/CD40 cells with recombinant GM-CSF significantly increased Aβ levels. GM-CSF receptor gene silencing with shRNA significantly reduced Aβ levels to below base line in non-stimulated HEK/APPsw/CD40 cells. Silencing of the GM-CSF receptor also decreased APP endocytosis (therefore reducing the availability of APP to be cleaved in the endosomes). ^ Using CD40 mutants, I show that CD40L can increase levels of Aβ(1-40), Aβ(1-42), sAPPβ, sAPPα and CTFβ independently of TRAF signaling. TRAFs had been shown to be necessary for most CD40/CD40L-dependent signaling. An increase in mature/immature APP ratio after CD40L treatment of CD40wt and CD40-mutant cells was observed, reflecting alterations in APP trafficking. CD4OL treatment of a neuroblastoma cell line over-expressing CTFβ suggested that CD40L affected γ-secretase activity. Inhibition of γ-secretase activity significantly reduced sAPPβ levels in the CD40L treated HEK/APPsw CD40wt and the CD40-mutant cells. The latter suggests CD40/CD40L interaction primarily acts on γ-secretase and affects β-secretase via a positive feedback mechanism. ^ Taken together, the results of this dissertation suggest that GM-CSF operates downstream of CD40/CD40L interaction and that GM-CSF modulates Aβ production by influencing APP trafficking. Moreover, the data presented suggest that CD40/CD40L interaction can modulate APP processing via a mechanism independent of TRAF signaling. ^

Characterizations of the major coral diseases of the Philippines: Ulcerative white spot disease and novel growth anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

Role of calcium in inflammation: Relevance to Alzheimer's disease

Alzheimer’s disease (AD) is neuropathologically characterized by excessive beta -amyloid (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau in the brain. Although the etiology of genetic cases of AD has been attributed to mutations in presenilin and amyloid precursor protein (APP) genes, in most sporadic cases of AD, the etiology is still unknown and various predisposing factors could contribute to the pathology of AD. Predominant among these possible predisposing factors that have been implicated in AD are age, hypertension, traumatic brain injury, diabetes, chronic neuroinflammation, alteration in calcium levels and oxidative stress. Since both inflammation and altered calcium levels are implicated in the pathogenesis of AD, we wanted to study the effect of altered levels of calcium on inflammation and the subsequent effect of selective calcium channel blockers on the production of pro-inflammatory cytokines and chemokines. Our hypothesis is that Aβ, depending on it conformation, may contribute to altered levels of intracellular calcium in neurons and glial cells. We wanted to determine which conformation of Aβ was most pathogenic in terms of increasing inflammation and calcium influx and further elucidate the possibility of a link between altered calcium levels and inflammation. In addition, we wanted to test whether calcium channel blockers could inhibit the inflammation mediated by the most pathogenic form of Aβ, by antagonizing the calcium influx triggered by Aβ. Our results in human glial and neuronal cells demonstrate that the high molecular weight oligomers are the most potent at stimulating the release of pro-inflammatory cytokines IL-6 and IL-8 as well as increasing intracellular levels of calcium compared to other conformations of Aβ. Further, L-type calcium channel blockers and calmodulin kinase inhibitors are able to significantly reduce the levels of IL-6 and IL-8. These results suggest that Aβ-induced alteration of intracellular calcium levels contributes to its pro-inflammatory effect.

Black band disease: Elucidating origins and disease mechanisms

Coral diseases were unknown in the scientific community fifty years ago. Since the discovery of a coral disease in 1965, there has been an exponential increase in the number of known coral diseases, as the abundance, prevalence, distribution, and number of host species affected has also significantly increased. Coral diseases are recognized as contributing significantly to the dramatic losses of coral cover on a global basis, particularly in the Caribbean. The apparent sudden emergence of coral diseases suggests that they may be a symptom of an overall trend associated with changing environmental conditions. However, not much evidence has been gathered to address this question. The following studies were designed to build a comprehensive argument to support this hypothesis for one important coral disease—black band disease (BBD). A meta-analysis of clone libraries identifying the microbial communities associated with BBD reveal important information including that a single cyanobacterial operational taxonomic unit (OTU) was by far the most prevalent OTU in diseased samples, and that the alphaproteobacteria, which include some of the most common bacteria in marine waters, were the most diversely represented. The analysis also showed that samples exhibited regional similarities. An fine and ultrastructural characterization of the disease revealed that the cyanobacteria are prolific borers through the coral skeleton, and that the cyanobacteria penetrate coral tissue, leading to their presence ahead of the main migrating disease band. It was further found that apparently healthy corals exposed to toxins found in BBD, exhibited similar tissue degradation to those infected with BBD. Comparing the disease progression to biofilm formation, it was determined that scouting cyanobacteria may contribute to the migration of the disease through progressive biofilm development over intact coral tissue. Together, these studies provide significant evidence for the hypothesis that BBD is an opportunistic disease, caused by common environmental bacteria, facilitated by the changing environmental conditions associated with climate change.

Disturbance Driven Colony Fragmentation as a Driver of a Coral Disease Outbreak

In September of 2010, Brewer's Bay reef, located in St. Thomas (U.S. Virgin Islands), was simultaneously affected by abnormally high temperatures and the passage of a hurricane that resulted in the mass bleaching and fragmentation of its coral community. An outbreak of a rapid tissue loss disease among coral colonies was associated with these two disturbances. Gross lesion signs and lesion progression rates indicated that the disease was most similar to the Caribbean coral disease white plague type 1. Experiments indicated that the disease was transmissible through direct contact between colonies, and five-meter radial transects showed a clustered spatial distribution of disease, with diseased colonies being concentrated within the first meter of other diseased colonies. Disease prevalence and the extent to which colonies were bleached were both significantly higher on unattached colony fragments than on attached colonies, and disease occurred primarily on fragments found in direct contact with sediment. In contrast to other recent studies, disease presence was not related to the extent of bleaching on colonies. The results of this study suggest that colony fragmentation and contact with sediment played primary roles in the initial appearance of disease, but that the disease was capable of spreading among colonies, which suggests secondary transmission is possible through some other, unidentified mechanism.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris): Morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n = 190) and manatees (n = 56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immunocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included T$\rm\sb{h}$, T$\rm\sb{c/s}$, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating T$\rm\sb{h}$ cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

An ethnopharmacological approach to multidrug -resistant Staphylococcus aureus: Evaluation of Italian plants used in the traditional healing of skin disease

One-third of botanical remedies from southern Italy are used to treat skin and soft tissue infections (SST's). Methicillin-resistant Staphylococcus aureus (MRSA), a common cause of SSTIs, is responsible for increased morbidity and mortality from infections. Therapeutic options are limited by antibiotic resistance. Many plants possess potent antimicrobial compounds for these disorders. Validation of traditional medical practices is important for the people who rely on medicinal plants. Moreover, identification of novel antibiotics and anti-pathogenic agents for MRSA is important to global healthcare.^ I took an ethnopharmacological approach to understand how Italian medicinal plants used for the treatment of SSTIs affect MRSA growth and virulence. My hypothesis was that plants used in folk remedies for SSTI would exhibit lower cytotoxicity and greater inhibition of bacterial growth, biofilm formation and toxin production in MRSA than plants used for remedies unrelated to the skin or for plants with no ethnomedical application. The field portion of my research was conducted in the Vulture-Alto Bradano area of southern Italy. I collected 104 plant species and created 168 crude extracts. In the lab, I screened samples for activity against MRSA in a battery of bioassays. Growth inhibition was analyzed using broth microtiter assays for determination of the minimum inhibitory concentration. Interference with quorum-sensing (QS) processes, which mediate pathogenicity, was quantified through RP-HPLC of δ-toxin production. Interference with biofilm formation and adherence was assessed using staining methods. The mammalian cytotoxicity of natural products was analyzed using MTT cell proliferation assay techniques.^ Although bacteriostatic activity was limited, extracts from six plants used in Italian folk medicine (Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa, Marrubium vulgare, and Rubus ulmifolius ) significantly inhibited biofilm formation and adherence. Moreover, plants used to treat SSTI demonstrated significantly greater anti-biofilm activity when compared to plants with no ethnomedical application. QSI activity was evident in 90% of the extracts tested and extracts from four plants ( Ballota nigra, Castanea saliva, Rosmarinus officinalis, and Sambucus ebulus) exhibited a significant dose-dependent response. Some of the plant remedies for SSTI identified in this study can be validated due to anti-MRSA activity.^

An Analytical Workflow for Metagenomic Data and its Application to the Study of Chronic Obstructive Pulmonary Disease (COPD)

Metagenomics is the culture-independent study of genetic material obtained directly from environmental samples. It has become a realistic approach to understanding microbial communities thanks to advances in high-throughput DNA sequencing technologies over the past decade. Current research has shown that different sites of the human body house varied bacterial communities. There is a strong correlation between an individual’s microbial community profile at a given site and disease. Metagenomics is being applied more often as a means of comparing microbial profiles in biomedical studies. The analysis of the data collected using metagenomics can be quite challenging and there exist a plethora of tools for interpreting the results. An automatic analytical workflow for metagenomic analyses has been implemented and tested using synthetic datasets of varying quality. It is able to accurately classify bacteria by taxa and correctly estimate the richness and diversity of each set. The workflow was then applied to the study of the airways microbiome in Chronic Obstructive Pulmonary Disease (COPD). COPD is a progressive lung disease resulting in narrowing of the airways and restricted airflow. Despite being the third leading cause of death in the United States, little is known about the differences in the lung microbial community profiles of healthy individuals and COPD patients. Bronchoalveolar lavage (BAL) samples were collected from COPD patients, active or ex-smokers, and never smokers and sequenced by 454 pyrosequencing. A total of 56 individuals were recruited for the study. Substantial colonization of the lungs was found in all subjects and differentially abundant genera in each group were identified. These discoveries are promising and may further our understanding of how the structure of the lung microbiome is modified as COPD progresses. It is also anticipated that the results will eventually lead to improved treatments for COPD.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris) : morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n=180) and manatees (n=56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immuocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included Th, Tes, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating Th cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.