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Absolute number of cells within lymphocyte, monocyte, and granulocyte regions. a Represents the average absolute number of cells expressing indicated surface markers within the lymphocyte region of dolphins under human care (HC) and group A (FR-Group A) and B (FR-Group B) free-ranging dolphins. b Represents the average absolute number of cells expressing indicated surface markers within the monocyte region of dolphins under human care (HC) and group A (FR-Group A) and B (FR-Group B) free-ranging dolphins. c Represents the average absolute number of cells expressing indicated surface markers within the granulocyte region of group A (FR-Group A) and B (FR-Group B) free-ranging dolphins. Sample sizes are HC (n = 9), FR-Group A (n = 12), and FR-Group B (n = 4). Values are presented as means ± SEM. An asterisk (*) denotes statistical significance
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Background
Studies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cros...
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Context 1
... and CD8 were significantly increased while CD11b was decreased in group A and CD40 was increased in group B free-ranging dolphins when compared to dolphins under human care (Fig. 6a). In addition, we observed a sig- nificant increase in percentage of cells expressing MHC class I in group A when compared to group B free-ranging dolphins (Fig. 6a) (Fig. 7a). We also compared the differences in the per- centage and absolute number of cells within the mono- cyte region between dolphins under human care and free-ranging dolphins. There was a significant decrease in the percentage of group A free-ranging dolphin cells expressing CD14, CD11b, and MHC class II compared to dolphins under human ...
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... dolphin cells expressing MHC class I when compared to group A free-ranging dolphins (Fig. 6b). When the abso- lute number of cells within the monocyte region was compared, we found a significant decrease in the absolute number of group A free-ranging dolphin cells expressing all markers, except Ly-6G&C when compared to dolphins under human care (Fig. 7b). In addition, we found group B free-ranging dolphins had a significant decrease (Fig. 7b). Finally, we compared the average percentage and absolute number of cells express- ing each indicated marker within the granulocyte region only seen in group A and B free-ranging dolphins. We found that group B with a high increase in granulocytes ...
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... 6b). When the abso- lute number of cells within the monocyte region was compared, we found a significant decrease in the absolute number of group A free-ranging dolphin cells expressing all markers, except Ly-6G&C when compared to dolphins under human care (Fig. 7b). In addition, we found group B free-ranging dolphins had a significant decrease (Fig. 7b). Finally, we compared the average percentage and absolute number of cells express- ing each indicated marker within the granulocyte region only seen in group A and B free-ranging dolphins. We found that group B with a high increase in granulocytes on FSC/SSC plots showed a greater percentage of cells for almost all surface markers, ...
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... a high increase in granulocytes on FSC/SSC plots showed a greater percentage of cells for almost all surface markers, although not significant (Fig. 6c). Also, the absolute number of cells expressing each indicated marker was significantly increased in group B compared to group A free-ranging dolphins except CD56, MHC class I, and MHC class II (Fig. ...
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Citations
... During the blood smear analysis, three types of peripheral blood cells were identified: erythrocytes, leukocytes, and platelets. These were similar to other mammals, particularly Cetacea (Harvey 2001, Nouri-Shirazi et al. 2017, Mello & da Silva 2019. Leukocytes were identified as granulocytes (neutrophils and eosinophils), monocytes, and lymphocytes. ...
... a) Neutrophil with segmented nuclei (thin arrow), monocyte with a U-shaped nucleus (thick arrow), and lymphocytes with predominant nuclei, b) eosinophil with a segmented nucleus and acidophilic granules in its cytoplasm and platelets (arrowhead), and c) erythrocytes surrounding a large lymphocyte. Shirazi et al. 2017, Nollens et al. 2018. Erythrocytes' mean diameters were slightly similar (5% bigger) to those reported in T. truncatus and 17% bigger than in the harbor porpoise (Phocaena phocaena) (Medway & Geraci 1964). ...
... Leukocyte morphology identified in I. geoffrensis was similar to other dolphins and terrestrial mammals (Techangamsuwan et al. 2010, Nouri-Shirazi et al. 2017, Satyaningtijas et al. 2020. Neutrophils on Wright-stained blood films were 13.9 ± 2 µm (9.4-17.4 ...
Inia geoffrensis is an endangered species of the Amazon River basin, but there has been limited research regarding its health, particularly in describing normal cell morphology by traditional techniques. This study aimed to identify the peripheral blood cells of I. geoffrensis through microscopic evaluation. Blood smears were collected from wild adults and stained with Wright's stain. We differentiated leukocyte cells (neutrophils, eosinophils, lymphocytes, and monocytes) and platelets. Additionally, we observed signs of inflammatory reactions in cell morphology by incrementing cell size and active cytoplasm in neutrophils, eosinophils, lymphocytes, and platelets. These findings provide important considerations for hemogram interpretation in future research and individual clinical cases in Amazon River dolphins. Also, our study delivers baseline information for future characterization and understanding of hemogram and leukogram changes in response to disease and health assessment for dolphin species.
... Reports indicate that bottlenose dolphins have 42 to 75% neutrophils, 15 to 34% lymphocytes, 2 to 40% eosinophils, 0 to 4% monocytes, and <1% basophils in circulation, depending on the pathophysiological conditions (Medway and Geraci, 1964;Ridgway et al., 1970;Engelhardt, 1979;Shirai and Sakai, 1997). A similar leukogram has been reported in humans, except for a lower eosinophil count ranging from 1 to 4 % (Shirai and Sakai, 1997;Francischetti et al., 2010;Kobayashi et al., 2017;Nouri-Shirazi et al., 2017). Neutrophils act as the first line of defense against bacteria and bacterial products (Kobayashi et al., 2017) and have been reported to demonstrate greater phagocytic capacity compared to eosinophils and basophils (Geering et al., 2013). ...
Marine mammals undergo cycles of tissue ischemia and reperfusion during the dive response. Reperfusion injury can result in oxidative tissue damage and the activation of a pro-inflammatory immune response. The risk of oxidative damage is reduced by antioxidants. Our hypothesis is that the reported higher antioxidant defenses within marine mammal tissues provide additional protection in situations that produce oxidative stress, like inflammation, in comparison to terrestrial mammal tissues. Leukocytes were isolated from the whole blood of Pacific bottlenose dolphins (Tursiops truncatus gilli) and humans (Homo sapiens) and were exposed to lipopolysaccharides (LPS, 10 µg/mL) in vitro to simulate a pro-inflammatory challenge. Oxidative stress indicators, including superoxide radical (O 2 •−
... Leukocyte size and granule content can be assessed by forward and side scatter measurements on flow cytometry, respectively. Hence, lymphocytes are identified as small cells containing small granules, while monocyte/macrophages are identified as larger cells with granules [28][29][30][31] . On flow cytometry, splenic lymphocytes were separated into region R1 and splenic myeloid cells including monocyte/macrophages and neutrophils into region R2. ...
CD44 is a cell-surface glycoprotein involved in cell–cell interaction, adhesion, and migration. CD44 is found on colon cancer cells and on immune cells. Previous studies of ⁸⁹Zr PET imaging of CD44 have relied on an anti-human antibody (Ab), which can influence biodistribution in murine models. In this study, we used an Ab that cross-reacts with both human and mouse origin CD44 of all isoforms to unveil the type of leukocyte responsible for high splenic anti-CD44 uptake and investigate how its regulation can influence tumor immuno-PET. The Ab was site-specifically labeled with ⁸⁹Zr-deferoxamine on cysteine residues. ⁸⁹Zr-anti-CD44 demonstrated high-specific binding to HT29 human colon cancer cells and monocytic cells that showed CD44 expression. When ⁸⁹Zr-anti-CD44 was administered to Balb/C nude mice, there was remarkably high splenic uptake but low SNU-C5 tumor uptake (1.2 ± 0.7%ID/g). Among cells isolated from Balb/C mouse spleen, there was greater CD44 expression on CD11b positive myeloid cells than lymphocytes. In cultured monocytic and macrophage cells, LPS stimulation upregulated CD44 expression and increased ⁸⁹Zr-anti-CD44 binding. Similarly, normal Balb/C mice that underwent lipopolysaccharide (LPS) stimulation showed a significant upregulation of CD44 expression on splenic myeloid cells. Furthermore, LPS treatment stimulated a 2.44-fold increase of ⁸⁹Zr-anti-CD44 accumulation in the spleen, which was attributable to splenic myeloid cells. Finally, in Balb/C nude mice bearing HT29 tumors, we injected ⁸⁹Zr-anti-CD44 with greater Ab doses to reduce binding to splenic cells. The results showed lower spleen uptake and improved tumor uptake (2.9 ± 1.3%ID/g) with a total of 300 μg of Ab dose, and further reduction of spleen uptake and greater tumor uptake (5.7 ± 0.0%ID/g) with 700 μg Ab dose. Thus, using an ⁸⁹Zr labeled Ab that cross-reacts with both human and mouse CD44, we demonstrate that CD44 immuno-PET has the capacity to monitor CD44 regulation on splenic myeloid cells and may also be useful for imaging colon tumors.
... Morphological observations of leukocytes or white blood cells (WBC) were made from blood smear preparations. Population and morphology of the WBC of bottlenose dolphins are generally the same as mammals in general, as stated by (Shirai and Sakai 1997;Nouri-Shirazi et al. 2017). WBC populations that are identified on blood smear preparations, consisting of neutrophils, lymphocytes, eosinophils, basophils, and monocytes ( Figure 1). ...
Satyaningtijas AS, Indrawati A, Syarafina RF, Milani TF, Saleema AK. 2020. Short Communication: Erythrocytes and leukocytes profiles of bottlenose dolphins (Tursiops aduncus) at conservation site. Biodiversitas 21: 3359-3363. Health monitoring of dolphins to ensure optimal welfare in human care is important. This study measured erythrocytes and leukocytes in seven bottlenose dolphins (Tursiops aduncus) as a parameter to assess physiological status in PT. Wersut Seguni Indonesia (WSI) Marine Mammals Conservation. Blood was sampled through the superficial veins of each tail fin for examination of erythrocytes and leukocytes using hemocytometer method. Differential leukocytes were observed using thin blood smears stained by Giemza. The purpose of this study to collect hematological value of captive bottlenose dolphins. The results showed values of erythrocytes was (5.14±0.56) x 10 6 /mm 3 , (13.86±1.68) gr/dl for hemoglobin, and (44.29±2.69)% for hematocrit. The value for leukocytes were (4.2 ± 0,82) x 10 3 /mm 3 , 63.14 ± 9.77% for lymphocytes, 3.57 ± 1.72% for monocytes, 31.57 ± 8.43% for neutrophils, 1.29 ± 1.60% for eosinophils and 0.14 ± 0.38% for basophils. The average of the seven bottlenose dolphins Neutrophil to Lymphocyte ratio (N/L) was 0.53.
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Considerable efforts have been made to better understand the immune system of bottlenose dolphins in view of the common environmental challenges they encounter, such as exposure to polychlorinated biphenyls, oil spills, or harmful algal bloom biotoxins. However, little is known about the identity and functionality of the Th1, Th2, and Treg T helper cell subsets in bottlenose dolphins. The present study aimed at validating assays and reagents to identify T helper cell subsets and their functions in a subset of dolphins from Sarasota Bay, Florida, USA, which have been long studied and often used as a reference population. A population of CD4+ FOXP3+ lymphocytes was identified representing an average <1% of blood lymphocyte population, which is in the range observed in for Treg cells in other species. The use of porcine reagents to measure TGFß, one of the key Treg cytokines, was further validated using the relatively high-throughput and highly standardized Luminex technology. The proportion of circulating Treg cells was not correlated with the serum concentrations of the Treg effector cytokines TGFß and IL-10, nor could it significantly contribute to predicting the variability of T lymphocyte proliferation, suggesting that not all dolphin circulating Treg cells are functional and active. However, stimulation of dolphin lymphocytes with TGFß and IL-2 increased the expression of the gene for TGFß and IL-10, and stimulation with IL-12 and IFNγ induced a robust increase in the expression of the gene for IFNγ, suggesting the potential for polarization and differentiation of dolphin T helper cells toward a Treg and Th1 response, respectively. The lack of an increase in the expression of the genes for the Th2 cytokines IL-4 and IL-13 upon stimulation with IL-4 may be due to the requirement for IL-2 for a Th2 polarization as described in mice. However, regression analysis and PCA suggested the potential ability of both the Th1 and Th2 response to be triggered upon acute inflammatory signals. These results may be useful in better understanding the mechanisms by which the dolphin immune system is affected upon exposure to environmental challenges and how it responds to pathogen challenges.
... Immunophenotyping was used to differentiate specific types of immune cells and the proteins expressed by these cells in the adaptive immune response. Lymphocyte subsets were labeled and analyzed according to methods described previously (6,18,(39)(40)(41)(42)(43)(44). Lymphocytes were analyzed by a LSR flow cytometer (BD Biosciences, San Jose, CA, USA). ...
Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.
... These leukocytes can be identified morphologically in the peripheral blood of cetaceans (Bossart et al., 2001); however, specific markers for the different cell types are not available. Some cross-reactive land mammalspecific antibodies have been shown to be effective for phenotyping cetacean leukocytes (Kumar and Cowan 1994, De guise et al., 1997, Jaber et al., 2003, Nouri-Shirazi et al., 2017. However, access to specific monoclonal antibodies (mAbs) against cetacean leukocytes would allow researchers to better understand cetacean immunology. ...
To better understand cetacean immunology, it is important to develop markers that identify specific leukocyte populations. We created a monoclonal antibody (mAb) library against leukocytes of the beluga whale (Delphinapterus leucas), and established five hybridoma clones that produce mAbs. Three of these mAbs (ID: BW-3C3, BW-2G4, and BW-2G6) react with mononuclear leukocytes including lymphocytes and monocytes. mAb BW-3C3 react to a fraction of the lymphocytes. The mAb-positive cells were identical to cells that also stained with polyclonal anti-whale IgM antibodies, indicating that the mAb BW-3C3 specifically reacts to B lymphocytes. mAb BW-2G4 specifically binds to monocytes that possess a reniformed nucleus. mAb BW-2G6 was found to bind to heterogeneous lymphocytes, namely, anti-whale IgM antibody-positive and -negative lymphocyte populations. This indicates that this mAb reacts with B and non-B lymphocyte fractions. The other two mAbs (ID: BW-4B10 and BW-4G12) react with polymorphonuclear granulocytes. Double staining with Giemsa-eosin showed that mAb BW-4B10 and mAb BW-4G12 specifically identify neutrophils and eosinophils, respectively. This panel of mAbs will be a useful tool for classifying leukocytes and for determining their localization in different tissues, which in turn would contribute to our understanding of cetacean immunology and allow evaluation of leukocyte function in infectious diseases.
... In addition, mAbs developed against CD2, CD19, CD21 and CD45R in bottlenose dolphin (Tursiops truncatus) were shown to identify orthologues in other species of cetaceans (wild common dolphin, striped dolphin, killer whale and beluga) (De Guise et al., 1998;De Guise et al., 2002). Most recently, an extensive set of mAbs developed against human, rat and mouse LDM were screened for potential cross-reactivity with dolphin LDM by Nouri-Shirazi et al. (Nouri-Shirazi et al., 2017). They identified 11 of 56 commercially available mAbs with potential cross-reactivity with dolphin LDM, two of which yielded patterns of labeling consistent with MHC II and CD11b (Nouri-Shirazi et al., 2017). ...
The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility in whales is largely due to the limited information available on the immune systems and immune function of species included in the Cetancodontamorpha clade. Studies in species in the other major clades included in the Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have revealed the immune systems are similar, but not identical. The present study was undertaken to expand the available monoclonal antibody reagents needed to gain insight into the composition, function, and evolution of the immune system in Cetancodontamorpha, using the dolphin (Tursiops truncates) as a model cetacean species. Screening of a set of mAbs that recognize highly conserved epitopes expressed on the major histocompatibility complex (MHC) and leukocyte differentiation molecules (LDMs) in cattle by flow cytometry revealed some of the mAbs recognize epitopes conserved on dolphin orthologues of MHC class I, MHC class II, CD11a, CD14, CD16, CD18, CD163 and CD172a. Comparison of the amino acid sequences of dolphin and bovine orthologues revealed limited changes in sequence have occurred during speciation, suggesting an approach for developing cross-reactive mAbs for use in cetacean research.
