Serum Protein Electrophoresis Reference Intervals for Six Species of Wild-Sampled Sharks in South Florida.

Serum proteins found in the blood have been used as diagnostic markers in both human and animal medicine. Evaluating proteins in the blood of sharks may be a valuable way to assess shark health. The purpose of this study was to perform serum protein electrophoresis from non-lethal blood samples of wild sharks and establish baseline values for six species off South Florida: blacknose (Carcharhinus acronotus), blacktip (C. limbatus), bull (C. leucas), lemon (Negaprion brevirostris), sandbar (C. plumbeus), and tiger shark (Galeocerdo cuvier). Two hundred and fifty-one serum samples were collected between 2015-2018 along with sex determination and morphometric measurements. Agarose gel analysis was performed using mammalian protein fraction delimits albumin, alpha 1-globulins, alpha 2-globulins, beta globulins, and gamma globulins alternatively defined as fractions 1-5. Reference electrophoretic profiles were established for each species, revealing the dominance of protein fractions 3 and 4. The fraction 3:4 ratio was determined for each shark and reference intervals for total solids, protein fractions, and fraction 3:4 ratio are reported for each species. There were no significant differences in total solids or protein fractions between males and females of any species. In both blacktip sharks and bull sharks, total solids were positively correlated with body size, but not in the other species evaluated. In blacknose sharks, the fraction 3:4 ratio was positively correlated with body condition. Results suggest that there may be differences in total solids values across dry and wet seasons for some species. Newly established reference intervals can now be utilized in future research to evaluate the health of captive sharks and to investigate the health status of individuals in the wild as well as relate health measures to environmental conditions.

Bacterial Communities in Multiple Tissues Across the Body Surface of Three Coastal Shark Species.

Bacteria are known to have explicit roles within the microbiomes of host tissues, therefore examining these communities may prove useful in assessing host health and responses to environmental change. The present study contributes to the emerging, yet understudied, field of microbiome research in elasmobranchs. We provide a screening of the culturable bacteria communities found on multiple tissue sites on the body surface of blacktip (Carcharhinus limbatus), bull (Carcharhinus leucas), and tiger (Galeocerdo cuvier) sharks near Miami, Florida. Tissue sites include mouth, gills, skin, and any visible wounds. The study adds to our understanding of the diversity of bacteria present on sharks in comparison to their natural environment. We also compare bacterial groups found within wounds in shark skin to healthy tissue sites on the same individual. Results indicate that wounds on an individual may allow for opportunistic bacteria to invade or overgrow where they would not normally be found, which may have potential health consequences for sharks that become wounded due to fishing practices. Identified bacteria belonged to the Actinobacteria, Firmicutes, and Proteobacteria phyla, known to be prominent bacterial groups associated with marine organisms. Results indicate shark species-specific differences in bacterial communities, including the presence of bacteria belonging to Planococcaceae exclusively on the skin of tiger sharks. To our knowledge, this is the first report of this family in any elasmobranch. While most tissue sites displayed commensal bacteria identified in similar studies, known pathogens belonging to Vibrionaceae and Staphylococcaceae were identified in the wounds of blacktip and bull sharks. Some bacteria may be normal residents, but the loss of protective dermal denticles due to a wound may allow colonization by pathogens. Continued research is needed to explore microbial communities associated with sharks and their influence on host health.

Variation of body condition and plasma energy substrates with life stage, sex, and season in wild-sampled nurse sharks Ginglymostoma cirratum.

Reported here are the relationships among morphological (i.e., body condition) and biochemical (i.e., plasma concentrations of triglycerides, cholesterol, free fatty acids, and ketone bodies and ketone body ratios) parameters related to energy storage and use, as well as the variation of such parameters, for 107 free-ranging nurse sharks Ginglymostoma cirratum sampled off South Florida. Immature G. cirratum exhibited a higher variance in body condition, plasma free fatty acid concentrations and ketone body ratios compared to adults. Mature female G. cirratum had significantly higher body condition than mature males, driven by a seasonal increase in mature female body condition during the wet season. Mature male G. cirratum showed a decrease in the ketone body ß-hydroxybutyric acid during the dry season. Taken together, this study provides a baseline assessment of body condition and internal physiological state for a data-poor marine species and demonstrates significant ontogenetic, sexual and seasonal variation in G. cirratum energetic state. As concluded by other studies of energy metabolism in free-ranging sharks, this research highlights the importance of considering intraspecific patterns and sampling context for inferring the drivers of variation.

Serum Protein Analysis of Nurse Sharks.

Serum protein electrophoresis (EPH) is used to assess relative concentrations of blood proteins in clinical and biological studies. Serum EPH fractions have been determined for elasmobranchs using mammalian albumin, alpha 1-, alpha 2-, beta-, and gamma-globulin fractions, and have been deemed fractions 1 through 5, respectively. However, serum EPH fraction concentration reference intervals (RIs) have not been widely established for different elasmobranch species. In this study, RIs for fractions 1 through 5 were determined from 45 wild-caught Nurse Sharks Ginglymostoma cirratum (27 females and 23 males) in South Florida. Serum samples were isolated from whole blood following caudal venipuncture. Body condition was also measured in the field to assess the relative health of the individuals sampled. There was no relationship between body condition and serum EPH fraction concentrations. In addition, there was no difference in body condition or serum EPH fraction concentrations between females and males. Total solids and total protein values were significantly different (P < 0.001). Nurse Shark serum EPH fraction 1 was found within the mammalian albumin migrating band distance and was negligible. Fraction 2 showed no peak in the mammalian alpha 1-globulin range. A thin, medium peak in the mammalian alpha 2-globulin range represented fraction 3. In the mammalian beta-globulin range, fraction 4 consisted of the majority of protein observed. It was represented by a smooth, broad peak. A short, medium broad peak in the mammalian gamma-globulin range represented fraction 5. The Nurse Shark serum EPH fraction RIs provided in this study may be utilized to clinically evaluate the health of Nurse Sharks in captivity and in the wild, and to compare the health of their populations around the world experiencing various anthropogenic stressors and other environmental impacts.

Blood plasma levels of heavy metals and trace elements in white sharks (Carcharodon carcharias) and potential health consequences.

Heavy metals may adversely affect health in marine organisms. As top predators, sharks may be especially vulnerable to exposure over long lifespans. Here we evaluate plasma levels of 14 heavy metals and 12 trace elements in white sharks, Carcharodon carcharias, in South Africa to determine whether they are related to sex, body size, and/or body condition and other health parameters. High levels of mercury and arsenic were found in shark blood at levels considered toxic in other vertebrates. Heavy metal concentrations were not related to body size or sex. Metal concentrations were not related to body condition with exception of copper, which was positively correlated. Protective effects of elements such as selenium, zinc, and iron were not detected. No negative effects on health parameters, such as total leukocytes or granulocyte to lymphocyte ratios were observed. Results suggest that sharks may have protective mechanisms that mitigate harmful effects of heavy metal exposure, providing new opportunities for future studies.

Murine RAW 264.7 cell line as an immune target: are we missing something?

The popular murine macrophage cell line, RAW 264.7, is often used to initially screen natural products for bioactivity and to predict their potential effect in vivo or on primary cells. The cell line response is considered to reflect the potential human de novo response, and is used to evaluate the effective bioactivity of the product. Here, we compared the cytokine response of RAW 264.7 cells to shark cartilage (SC) with that of human leukocytes to determine whether the cell line response was a reliable predictor of the cytokine response one can expect from similarly stimulated human primary cells. Results not only revealed significant differences in the nature and level of TNFα produced by cells in vitro, but also showed that while the primary cell response included an upregulation in the production of IL-1ß such a response was absent in RAW 264.7 cells. This suggests that had we relied on RAW 264.7 cells alone to assess the cytokine-inducing capacity of SC, the comprehensive Th1 response (shown in an earlier study) induced by SC in primary cells, consisting of release of several proinflammatory cytokines and chemokines, would not have been revealed. We conclude, therefore, that assays using only RAW 264.7 cells to initially screen for and assess immune reactivity of test products will not necessarily provide a comprehensive picture of the immunomodulatory properties of the substance under investigation, and can in fact be misleading with regard to the overall bioactive potential of the substance on an initial screen.

Pro-inflammatory properties of shark cartilage supplement.

The erosion and breakdown of cartilage is generally recognized to be an integral manifestation of arthritic disease, which is often accompanied by the development and progression of inflammation associated with it. Commercial shark cartilage (SC) is a popular dietary supplement taken for the prevention and/or control of chronic disease, including arthritis. The efficacy of SC in maintaining joint health remains questionable; there is a lack of sufficient reliable information on its effect on immunocompetent cells, and the potential health risks involved have not been adequately assessed. Our earlier in vitro studies showed that SC extracts induce a Th1-type inflammatory cytokine response in human leucocytes, and collagen type II alpha 1 protein was shown to be an active cytokine-inducing component in SC. In this study, we further define the cellular response to SC stimulation by classifying leucocytes into primary and secondary responders employing enriched leucocyte subpopulations. Inhibitors of specific signaling pathways were used to verify the functional effect of SC on specific pathway(s) utilized. Results indicate the monocyte/macrophage as the initially responding cell, followed by lymphocytes and the production of interferon-γ. Chemokines, MCP-1 and RANTES, were produced at significant levels in stimulated leucocyte cultures. Initial cellular activation is likely followed by activation of Jun Kinase and p38 mitogen-activated protein kinase signal transduction pathways. This study presents evidence of significant immunological reactivity of components of commercial SC supplement, which could pose a potential health risk for consumers, particularly those with underlying inflammatory disease such as irritable bowel syndrome and arthritis.

Collagen type II, alpha 1 protein: a bioactive component of shark cartilage.

Previous studies have shown that extracts of shark cartilage induce a cytokine response in human leukocytes, but the nature of the bioactive component(s) is unknown. Extracts treated with proteases lost 80% of their cytokine-inducing property, suggesting that the active component(s) was likely a complex protein. The aim of the present study was to determine the nature of the bioactive molecule(s). Solid phase extraction followed by ion exchange chromatography and electrophoretic separation were used to partially purify a bioactive preparation from commercial shark cartilage that has been identified as a small glycoprotein. LC-MS analysis yielded peptides with 100% molecular identity with collagen type II, alpha I protein from the lesser spotted catshark, Scyliorhinus canicula. The implications for the consumption of shark cartilage as a dietary supplement are discussed given the presence of collagen type II, alpha 1 protein in extracts.

Induction of inflammatory cytokines by cartilage extracts.

Shark cartilage extracts were examined for induction of cytokines and chemokines in human peripheral blood leukocytes. Primary leukocyte cultures were exposed to a variety of aqueous and organic extracts prepared from several commercial brands of shark cartilage. From all commercial sources of shark cartilage tested the acid extracts induced higher levels of TNFalpha than other extracts. Different commercial brands of shark cartilage varied significantly in cytokine-inducing activity. TNFalpha induction was seen as early as 4 h and IFNgamma at detectable levels for up to four days. Shark cartilage extracts did not induce physiologically significant levels of IL-4. Results suggest that shark cartilage, preferentially, induces Th1 type inflammatory cytokines. When compared to bovine cartilage extract, collagen, and chondroitin sulfate, shark cartilage induced significantly higher levels of TNFalpha. Treatment with digestive proteases (trypsin and chymotrypsin) reduced the cytokine induction response by 80%, suggesting that the active component(s) in cartilage extracts is proteinaceous. The induction of Th1 type cytokine response in leukocytes is a significant finding since shark cartilage, taken as a dietary supplement for a variety of chronic degenerative diseases, would be contraindicated in cases where the underlying pathology of the chronic condition is caused by inflammation.