Cytokine analysis in Atlantic bottlenose dolphins: Molecular characterization of IL-4, IL-6, and IL-10

The health status of wild and captive Atlantic Bottlenose dolphins ( Tersiops truncatis) is difficult to ascertain. Mass strandings of these animals have been attributed to pollutants, as well as bacterial infections. Using human Enzyme Linked Immuno-Assays (ELISA) for immunological cytokines, I measured soluble cytokine levels with respect to their health status. In a retrospective analysis of dolphin sera, there was a trend of higher cytokine levels in “sick” animals. I cultured dolphin lymphocytes in the presence of a mitogen (PHA), a super antigen (Staph-A), Lipopolysaccharide (LPS), and a calcium flux inducer (PMA). Levels of messenger RNA, from these cultured cells, were assayed with Polymerase Chain Reaction (PCR) using primers for the human cytokines IL-2, IL-4, IL-6, IL-10, Tumor Necrosis Factor, and Interferon gamma. Only IL-4, IL-6, and IL-10 messages were obtained, inferring similar nucleotide homology to the human primer sequences. The PCR products were sequenced. Sixteen IL-4 sequences, twelve IL-6 sequences and seven IL-10 sequences were obtained and analyzed. Each cytokine exhibited the same nucleotide sequence in all dolphins examined. There was no difference in the cytokine profile in response to the various stimuli. The derived amino acid composition for each of the dolphin cytokines was used for molecular modeling, which showed that dolphin IL-4, IL-6, and IL-10 were structurally similar to the corresponding proteins of Perissodactyla. ^

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.

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.

Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters

Peer reviewed

Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters

Peer reviewed

Long-finned pilot whale population diversity and structure in Atlantic waters assessed through biogeochemical and genetic markers

Acknowledgements. Cetacean samples were collected under the auspices of stranding monitoring programs run by the Sociedade Portuguesa de Vida Selvagem, the Coordinadora para o Estudio dos Mamíferos Mariños (supported by the regional government Xunta de Galicia), the UK Cetacean Strandings Investigation Programme and the Scottish Agriculture College Veterinary Science Division (jointly funded by Defra and the Devolved Governments of Scotland and Wales), the Marine Mammals Research Group of the Institute of Marine Research (Norway), the Museum of Natural History of the Faroe Islands and the International Fund for Animal Welfare Marine Mammal Rescue and Research Program (USA). The authors thank all the members of these institutions and organizations for their assistance with data and sample collection. S.S.M., P.M.F. and M.F. were supported by PhD grants from the Fundação para a Ciência e Tecnologia (POPH/FSE ref SFRH/BD/ 38735/ 2007, SFRH/BD/36766/2007 and SFRH/BD/30240/ 2006, respectively). A.L. was supported by a postdoctoral grant from the Fundação para a Ciência e Tecnologia (ref SFRH/BPD/82407/2011). The work related to strandings and tissue collection in Portugal was partially supported by the SafeSea project EEAGrants PT 0039 (supported by Iceland, Liechtenstein and Norway through the EEA Financial Mechanism), the MarPro project Life09 NAT/PT/000038 (funded by the European Union program LIFE+) and the project CetSenti FCT RECI/AAG-GLO/0470/2012 (FCOMP- 01-0124-FEDER-027472) (funded by the program COMPETE and the Fundação para a Ciência e Tecnologia). G.J.P. thanks the University of Aveiro and Caixa Geral de Depósitos (Portugal) for financial support. The authors acknowledge the assistance of the chemical analysts at Marine Scotland Science with the fatty acid analysis.

Major Histocompatibility Complex (MHC) Class II sequence polymorphism in long-finned pilot whale (Globicephala melas) from the North Atlantic

Funding Silvia S. Monteiro and Marisa Ferreira were supported by a Ph.D. grant from Fundação para a Ciência e Tecnologia (ref SFRH/BD/38735/2007 and SFRH/BD/30240/2006, respectively). Alfredo López was supported by a postdoctoral grant from Fundação para a Ciência e Tecnologia (ref SFRH/BPD/82407/2011). Catarina Eira is supported by CESAM (UID/AMB/50017), from FCT/MEC through national funds and FEDER (PT2020, Compete 2020). The work related with strandings and tissue collection in Portugal was partially supported by the SafeSea Project EEAGrants PT 0039 (supported by Iceland, Liechtenstein and Norway through the EEA Financial Mechanism), by the Project MarPro–Life09 NAT/PT/000038 (funded by the European Union–Program Life+) and by the project CetSenti FCT RECI/AAG-GLO/0470/2012; FCOMP-01-0124-FEDER-027472 (Funded by the Program COMPETE and Fundação para a Ciência e Tecnologia).

Long-finned pilot whale population diversity and structure in Atlantic waters assessed through biogeochemical and genetic markers

Acknowledgements. Cetacean samples were collected under the auspices of stranding monitoring programs run by the Sociedade Portuguesa de Vida Selvagem, the Coordinadora para o Estudio dos Mamíferos Mariños (supported by the regional government Xunta de Galicia), the UK Cetacean Strandings Investigation Programme and the Scottish Agriculture College Veterinary Science Division (jointly funded by Defra and the Devolved Governments of Scotland and Wales), the Marine Mammals Research Group of the Institute of Marine Research (Norway), the Museum of Natural History of the Faroe Islands and the International Fund for Animal Welfare Marine Mammal Rescue and Research Program (USA). The authors thank all the members of these institutions and organizations for their assistance with data and sample collection. S.S.M., P.M.F. and M.F. were supported by PhD grants from the Fundação para a Ciência e Tecnologia (POPH/FSE ref SFRH/BD/ 38735/ 2007, SFRH/BD/36766/2007 and SFRH/BD/30240/ 2006, respectively). A.L. was supported by a postdoctoral grant from the Fundação para a Ciência e Tecnologia (ref SFRH/BPD/82407/2011). The work related to strandings and tissue collection in Portugal was partially supported by the SafeSea project EEAGrants PT 0039 (supported by Iceland, Liechtenstein and Norway through the EEA Financial Mechanism), the MarPro project Life09 NAT/PT/000038 (funded by the European Union program LIFE+) and the project CetSenti FCT RECI/AAG-GLO/0470/2012 (FCOMP- 01-0124-FEDER-027472) (funded by the program COMPETE and the Fundação para a Ciência e Tecnologia). G.J.P. thanks the University of Aveiro and Caixa Geral de Depósitos (Portugal) for financial support. The authors acknowledge the assistance of the chemical analysts at Marine Scotland Science with the fatty acid analysis.

Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters

Peer reviewed

Encalhes de Cetáceos ocorridos no período de 1984 a 2005 no litoral do Rio Grande do Norte, Brasil

Strandings of live or dead aquatic mammals constitute an important instrument to provide information regarding the occurrence, biology and ecology of these species. The aim of this study was to register the stranded species of cetaceans, the frequency and the spatial-temporal distribution of theses strandings during the period of 1984 to 2005, in the coast of Rio Grande do Norte. Data was acquired through the monitoring of strandings in the north, north-west and south coast of RN, and through information obtained from institutions and newspaper archives of the State. A total of 122 strandings of cetaceans were registered along the coast of Rio Grande do Norte. Of the 14 species of cetaceans registered, four species had higher frequencies: Sotalia guianensis (n= 65), Steno bredanensis (n = 6), Globicephala macrorhynchus (n = 6) and Physeter macrocephalus (n = 7). Out of 118 strandings, 93 occurred in the south coast (78.8 %), 23 in the north coast (19.5%) and 2 (1.7%) in the north-west coast of the State. The highest frequency of strandings occurred during the months of August to March and the maximum number of strandings occurred from 2000 onwards, as a consequence of the intense monitoring of the Pequenos Cetáceos Project in Rio Grande do Norte

Encalhes de Cetáceos ocorridos no período de 1984 a 2005 no litoral do Rio Grande do Norte, Brasil

Strandings of live or dead aquatic mammals constitute an important instrument to provide information regarding the occurrence, biology and ecology of these species. The aim of this study was to register the stranded species of cetaceans, the frequency and the spatial-temporal distribution of theses strandings during the period of 1984 to 2005, in the coast of Rio Grande do Norte. Data was acquired through the monitoring of strandings in the north, north-west and south coast of RN, and through information obtained from institutions and newspaper archives of the State. A total of 122 strandings of cetaceans were registered along the coast of Rio Grande do Norte. Of the 14 species of cetaceans registered, four species had higher frequencies: Sotalia guianensis (n= 65), Steno bredanensis (n = 6), Globicephala macrorhynchus (n = 6) and Physeter macrocephalus (n = 7). Out of 118 strandings, 93 occurred in the south coast (78.8 %), 23 in the north coast (19.5%) and 2 (1.7%) in the north-west coast of the State. The highest frequency of strandings occurred during the months of August to March and the maximum number of strandings occurred from 2000 onwards, as a consequence of the intense monitoring of the Pequenos Cetáceos Project in Rio Grande do Norte

Unusual striped dolphin mass mortality episode related to cetacean morbillivirus in the Spanish Mediterranean sea

BACKGROUND In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the two dolphin morbillivirus (DMV) epizootics of 1990-1992 and 2006-2008, which affected Mediterranean striped dolphins (Stenella coeruleoalba). Between March and April 2011, the number of strandings on the Valencian Community coast (E Spain) increased. CASE PRESENTATION Necropsy and sample collection were performed in all stranded animals, with good state of conservation. Subsequently, histopathology, immunohistochemistry, conventional reverse transcription polymerase chain reaction (RT-PCR) and Universal Probe Library (UPL) RT-PCR assays were performed to identify Morbillivirus. Gross and microscopic findings compatible with CeMV were found in the majority of analyzed animals. Immunopositivity in the brain and UPL RT-PCR positivity in seven of the nine analyzed animals in at least two tissues confirmed CeMV systemic infection. Phylogenetic analysis, based on sequencing part of the phosphoprotein gene, showed that this isolate is a closely related dolphin morbillivirus (DMV) to that responsible for the 2006-2008 epizootics. CONCLUSION The combination of gross and histopathologic findings compatible with DMV with immunopositivity and molecular detection of DMV suggests that this DMV strain could cause this die-off event.