Clinical, pathological, and laboratory diagnoses of diseases of harbour porpoises (Phocoena phocoena), live stranded on the Dutch and adjacent coasts from 2003 to 2016.

Harbour porpoises (Phocoena phocoena) in the North Sea live in an environment heavily impacted by humans, the consequences of which are a concern for their health. Autopsies carried out on stranded harbour porpoises provide an opportunity to assess health problems in this species. We performed 61 autopsies on live-stranded harbour porpoises, which died following admission to a rehabilitation centre between 2003 and 2016. The animals had stranded on the Dutch (n = 52) and adjacent coasts of Belgium (n = 2) and Germany (n = 7). We assigned probable causes for stranding based on clinical and pathological criteria. Cause of stranding was associated in the majority of cases with pathologies in multiple organs (n = 29) compared to animals with pathologies in a single organ (n = 18). Our results show that the three most probable causes of stranding were pneumonia (n = 35), separation of calves from their mother (n = 10), and aspergillosis (n = 9). Pneumonia as a consequence of pulmonary nematode infection occurred in 19 animals. Pneumonia was significantly associated with infection with Pseudalius inflexus, Halocercus sp., and Torynurus convolutus but not with Stenurus minor infection. Half of the bacterial pneumonias (6/12) could not be associated with nematode infection. Conclusions from this study are that aspergillosis is an important probable cause for stranding, while parasitic infection is not a necessary prerequisite for bacterial pneumonia, and approximately half of the animals (29/61) probably stranded due to multiple causes. An important implication of the observed high prevalence of aspergillosis is that these harbour porpoises suffered from reduced immunocompetence.

Immunohistochemical Analysis of the Harbor Porpoise Using Antipodoplanin Antibody PMab-237.

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.

Establishment of an Anticetacean Podoplanin Monoclonal Antibody PMab-237 for Immunohistochemical Analysis.

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN.

Cytokine expression and lymphocyte proliferative capacity in diseased harbor porpoises (Phocoena phocoena) - Biomarkers for health assessment in wildlife cetaceans.

Harbor porpoises (Phocoena phocoena) in the North and Baltic Seas are exposed to anthropogenic influences including acoustic stress and environmental contaminants. In order to evaluate immune responses in healthy and diseased harbor porpoise cells, cytokine expression analyses and lymphocyte proliferation assays, together with toxicological analyses were performed in stranded and bycaught animals as well as in animals kept in permanent human care. Severely diseased harbor porpoises showed a reduced proliferative capacity of peripheral blood lymphocytes together with diminished transcription of transforming growth factor-ß and tumor necrosis factor-α compared to healthy controls. Toxicological analyses revealed accumulation of polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (DDE), and dichlorodiphenyltrichloroethane (DDT) in harbor porpoise blood samples. Correlation analyses between blood organochlorine levels and immune parameters revealed no direct effects of xenobiotics upon lymphocyte proliferation or cytokine transcription, respectively. Results reveal an impaired function of peripheral blood leukocytes in severely diseased harbor porpoises, indicating immune exhaustion and increased disease susceptibility.

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.

Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress?

BACKGROUND: The harbour porpoise is exposed to increasing pressure caused by anthropogenic activities in its marine environment. Numerous offshore wind farms are planned or under construction in the North and Baltic Seas, which will increase underwater noise during both construction and operation. A better understanding of how anthropogenic impacts affect the behaviour, health, endocrinology, immunology and physiology of the animals is thus needed. The present study compares levels of stress hormones and mRNA expression of cytokines and acute-phase proteins in blood samples of harbour porpoises exposed to different levels of stress during handling, in rehabilitation or permanent human care.Free-ranging harbour porpoises, incidentally caught in pound nets in Denmark, were compared to harbour porpoises in rehabilitation at SOS Dolfijn in Harderwijk, the Netherlands, and individuals permanently kept in human care in the Dolfinarium Harderwijk and Fjord & Belt Kerteminde, Denmark. Blood samples were investigated for catecholamines, adrenaline, noradrenaline and dopamine, as well as for adrenocorticotropic hormone (ACTH), cortisol, metanephrine and normetanephrine. mRNA expression levels of relevant cell mediators (cytokines IL-10 and TNFα, acute-phase proteins haptoglobin and C-reactive protein and the heat shock protein HSP70) were measured using real-time PCR. RESULTS: Biomarker expression levels varied between free-ranging animals and porpoises in human care. Hormone and cytokine ranges showed correlations to each other and to the health status of investigated harbour porpoises. Hormone concentrations were higher in free-ranging harbour porpoises than in animals in human care. Adrenaline can be used as a parameter for the initial reaction to acute stress situations; noradrenaline, dopamine, ACTH and cortisol are more likely indicators for the following minutes of acute stress. There is evidence for different correlations between production of normetanephrine, metanephrine, cortisol and the expression of IL-10, HSP70 and haptoglobin. CONCLUSIONS: The expression patterns of the selected molecular biomarkers of the immune system are promising to reflect the health and immune status of the harbour porpoise under different levels of stress.

Phenotypical characterization of changes in thymus and spleen associated with lymphoid depletion in free-ranging harbor porpoises (Phocoena phocoena).

Harbor porpoises from the North and Baltic Seas exhibit a higher incidence of bacterial infections compared to whales from less polluted arctic waters. Furthermore, thymic atrophy and splenic depletion, associated with elevated concentrations of environmental contaminants, such as polychlorinated biphenyls (PCB) and polybrominated diphenyl ether (PBDE) body burdens, have been found in wildlife harbor porpoises. Thus, there is currently a debate about the potential adverse effect of xenobiotics on the immune system and therefore on the health status of this and other marine mammal species. The aim of the present study was to characterize phenotypical changes in lymphoid organs of harbor porpoises and their possible association with increased disease susceptibility due to an impaired immune response in this marine mammal species. Therefore, 29 by-caught and stranded harbor porpoises were necropsied and the health status was evaluated based upon the severity of main pathological findings. In addition, the distribution of CD2-, CD3epsilon-, and CD45R-positive cells as well as B lymphocytes, MHC class II-expressing and antigen-presenting cells was determined in the thymus and spleen using immunohistochemistry. Thymic atrophy and splenic depletion were associated with an impaired health status in investigated whales. Phenotypical changes in atrophic thymuses were characterized by a depletion of immature cortical thymocytes and medullary B cells. Furthermore, findings in depleted spleens were consistent with a loss of peripheral T lymphocytes in the periarteriolar lymphoid sheath (PALS). Based upon the results, an altered thymopoiesis and impaired cellular immune function cannot be excluded in whales with evidence of lymphoid depletion.

Increased blood interleukin-10 mRNA levels in diseased free-ranging harbor porpoises (Phocoena phocoena).

Harbor porpoises from the North and Baltic Seas exhibit a higher incidence of bacterial infections compared to whales from less polluted arctic waters. Toxicological analysis revealed an association between elevated body burdens of environmental contaminants, such as polychlorinated biphenyls (PCB) and polybrominated diphenyl ether (PBDE) and lymphoid depletion in thymus and spleen of these whales. However, it remains undetermined if changes in the immune system are primarily contaminant-induced or a sequel of infectious diseases and emaciation. The aim of the present study was to investigate changes of blood cytokine mRNA levels in healthy and diseased harbor porpoises. Therefore, 29 by-caught and stranded whales were necropsied and the health status was evaluated based upon main pathological findings. Furthermore, the degree of thymic atrophy and splenic depletion was histologically graded using a semi-quantitative scoring system. Gene expression of interleukin (IL)-2, IL-4, IL-6, IL-10, transforming growth factor-beta and tumor necrosis factor-alpha in the blood was measured by real time reverse transcription-polymerase chain reaction. Thymic atrophy and splenic depletion were correlated with an impairment of the animals' health status. Additionally, a marked up-regulation of IL-10 was predominately found in severely diseased whales with evidence of chronic bacterial infections. Furthermore, increased IL-10 levels were associated with splenic depletion. Other investigated cytokines were not significantly associated with the health status or lymphoid depletion, respectively. The present study indicated that lymphoid depletion represents a sequel of chronic infectious diseases in a portion of investigated harbor porpoises. Regarding this, expression of anti-inflammatory cytokines, such as IL-10 might represent a consequence of continuous stimulation of the immune system and induction of immunomodulatory mechanisms in this cetacean species.