Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans.

Immunology of marine mammals is a relatively understudied field and its monitoring plays an important role in the individual and group management of these animals, along with an increasing value as an environmental health indicator. This study was aimed at implementing the knowledge on the immune response in cetaceans stranded along the Italian coastline to provide a baseline useful for assessing the immune status of bottlenose (Tursiops truncatus) and striped (Stenella coeruleoalba) dolphins. In particular, since the Mediterranean Sea is considered a heavily polluted basin, a comparison with animals living in open waters such as the Atlantic Ocean was made. Formalin-fixed, paraffin-embedded spleen, thymus, and lymph node tissues from 16 animals stranded along Italian and 11 cetaceans from the Canary Island shores were sampled within 48 h from death. Information regarding stranding sites, gender, and age as well as virologic, microbiological, and parasitological investigations, and the cause and/or the death mechanism were also collected in order to carry out statistical analyses. Selected tissues were routinely stained with hematoxylin-eosin (H&E) and with immunohistochemical techniques (IHC). For IHC analysis, anti-human CD5 monoclonal mouse antibody to identify T lymphocytes, CD20 monoclonal mouse antibody for the identification of mature B lymphocytes and HLA-DR antigen (alpha-chain) monoclonal mouse antibody for the identification of the major histocompatibility complex type II were previously validated for both species by Western-blotting technique. T-test method applied to quantitative evaluation of IHC positive cells showed a significant relationship between the number of (expression) of CD20 stained lymphocytes and normal and hypoplastic lymph nodes, respectively. No other significant correlations were noticed. Analyses for organochlorines (OC) compounds were performed in animals (n°5) having frozen blubber tissue available. A simple linear regression was calculated to predict if the amount of OCs could influence the number of inflammatory cell subpopulations and a moderate negative correlation was found between the presence of high quantity of contaminants and the number of T lymphocytes. Future analysis should be aimed to understand the effect of the major immunomodulatory pathogens on sub-populations of B and T cells.

Candidate gene molecular markers as tools for analyzing genetic susceptibility to morbillivirus infection in stranded Cetaceans.

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

Morbillivirus infection in cetaceans stranded along the Italian coastline: pathological, immunohistochemical and biomolecular findings.

Morbilliviruses are recognized as biological agents highly impacting the health and conservation status of free-ranging cetaceans worldwide, as clearly exemplified by the two Dolphin Morbillivirus (DMV) epidemics of 1990-1992 and 2006-2008 among Mediterranean striped dolphins (Stenella coeruleoalba). After these two epidemics, morbilliviral infection (MI) cases with peculiar neurobiological features were reported in striped dolphins stranded along the Spanish coastline. Affected cetaceans showed a subacute-to-chronic, non-suppurative encephalitis, with brain lesions strongly resembling those found in human "subacute sclerosing panencephalitis" and "old dog encephalitis". Brain was the only tissue in which morbilliviral antigen and/or genome could be detected. Beside a case of morbilliviral encephalitis in a striped dolphin's calf stranded in 2009, we observed 5 additional MI cases in 2 striped dolphins, 1 bottlenose dolphin (Tursiops truncatus) and 2 fin whales (Balaenoptera physalus), all stranded in 2011 along the Italian coastline. Noteworthy, 3 of these animals (2 striped dolphins and 1 bottlenose dolphin) showed immunohistochemical (IHC) and/or biomolecular (PCR) evidence of morbilliviral antigen and/or genome exclusively in their brain, with 1 striped dolphin and 1 bottlenose dolphin also exhibiting a non-suppurative encephalitis. Furthermore, simultaneous IHC and PCR evidence of a Toxoplasma gondii coinfection was obtained in 1 fin whale. The above results are consistent with those reported in striped dolphins after the two MI epidemics of 1990-92 and 2006-2008, with evidence of morbilliviral antigen and/or genome being found exclusively in the brain tissue from affected animals.

Cross-reactivity of anti-human, anti-porcine and anti-bovine cytokine antibodies with cetacean tissues.

The cross-reactivity of monoclonal antibodies specific for human, porcine and bovine cytokines was evaluated for three cetacean species: Atlantic spotted dolphins (Stenella frontalis), striped dolphins (Stenella coeruleoalba) and fin whales (Balaenoptera physalus). Formalin-fixed and snap-frozen tissue sections of lung, spleen, liver and mesenteric lymph node were evaluated. T and B lymphocytes and monocytes/macrophages were detected by use of anti-human CD3, IgG and lysozyme polyclonal antibodies (pAbs), respectively. These reagents were successfully applied to both fixed and frozen tissues. Anti-human interleukin (IL)-1 alpha, IL-1 beta, IL-8, tumour necrosis factor (TNF)-alpha and CD25, anti-porcine IL-2, IL-6, IL-10, and anti-bovine IL-4 and interferon (IFN)-gamma antibodies produced immunolabelling in cetacean snap-frozen lymph node sections similar to that obtained with tissue from the species of origin, but they did not react with formalin-fixed tissue sections. Anti-porcine IL-12 pAb did not react with snap-frozen cetacean tissue samples. Macrophages and lymphocytes were the most common cells immunolabelled with the anti-cytokine antibodies. This panel of anti-cytokine antibodies may be used to evaluate cytokine expression in snap-frozen tissue samples from the cetacean species tested.