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Marine mammals' health as an indicator of ecosystem health - tools for monitoring

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Introduction

Marine mammals serve as indicators of ecosystem change (Trilateral Monitoring and Assessment Program, TMAP, Fig. 1). They are top predators in the marine food web. Increasing commercial use, e.g. fisheries and offshore wind parks, as well as the inputs of pollutants, influence the North and Baltic Sea ecosystems, including native marine mammals such as harbour porpoises, harbour seals and grey seals. Thus, the aim is to establish effect-monitoring tools for early diagnosis of the health status of marine mammals.

Figure 1: The harbour seal as top predator is an important biological parameter of the Trilateral Monitoring and Assessment Program (TMAP). This program was founded by The Netherlands, Denmark and Germany, for the protection and conservation of the Wadden Sea. It includes management, monitoring and research, as well as political matters.
Immune system parameters are measured via biomolecular and biochemical methods. Non-destructive biomarkers will be identified and chemically characterised to serve as biochemical indicators.
Figure 2:Overview of immunological investigations using blood samples of marine mammals.

Methodology

Blood samples of captive and wild living animals are the study objects (Fig. 2). Selected parameters which play a central role in controlling disease processes are determined. The following immune system parameters were selected: the lymphocyte proliferation as important immune cell function, the expression of cytokines released by immune cells, which are regulators of an immune reaction, as well as cytokine-induced acute phase proteins (APPs) for the early diagnosis of inflammation and stress. The combined investigation of these parameters allows a statement about the immune status of the animals and the impact of pollutants.

Figure 3: Directly after arrival in the Seal Station the lymphocytes of newborns were particularly susceptible to the toxic effect of metals. A lot of metals tested e.g. beryllium, lead and cadmium inhibit the lymphocyte proliferation (value <0.1). This effect decreased during the time of rehabilitation.

Technique for measurement of lymphocyte proliferation

Lymphocytes were isolated from the blood sample and cultured with and without stimulation using a lymphocyte transformation test (LTT, Fig. 2). After incubation transformation and proliferation were examined and a stimulation index calculated. Technique for quantification of cytokine *expression Cytokine expression is measured quantitatively by analysing mRNA amounts with the real time reverse trans- criptase-polymerase chain reaction (RT-PCR, Fig. 2). Detecting the amount of the mRNA allows us to calculate ratios between cytokines and thus establish the main focus of the immune response. Investigations of the cytokine expression pattern (Interleukin-1, -2, -4, -6, -10, -12, TNF, TGFß) allow the status of the immune reaction to be differentiated, whether the emphasis is on the cellular or humoral (body liquid) immune response.

Figure 4: Cytokine index of IL-2 mRNA from the blood samples of two harbour porpoises living in captivity (Pp1, Pp2) and four accidentally caught animals (Pp3-Pp6).

Technique for the analysis of acute phase proteins

The acute phase proteins of harbour seals are characterised quantitatively and qualitatively to find relevant biochemical indicators for their health status. After their isolation, the structures of selected proteins are elucidated with mass spectrometric techniques. Bio-analytical procedures will be established to determine disease-*related structural variations, i.e. in the pattern of glyco- sylation. Based on the findings, specific bio-assays will be developed for monitoring the health status of marine mammals.

Figure 5: Chronic metal exposure can lead to abnormal lymphocyte proliferation, and both hypersensitivity as well as immune suppression is known to be induced by chronic metal exposure. Some metals induce the formation of memory cells. By re-exposing these memory cells a rapid immune reaction is induced and transformation into blasts and proliferation take place. Other metals lead to immune suppression by unspecific inhibition of lymphocyte proliferation or show cell toxic influences.

Metal pollution – influence on immune system

Pollution with metals may affect the immuno-competence of free-ranging populations of marine mammals in many areas of the industrialised world. An imbalance of the immune system caused by pollutants has been suggested to play a role in the incidence of infectious diseases in marine mammals (Jepson et al., 1999[1]; Siebert et al., 1999[2]; Bennett et al., 2001[3]). Metals influence the function of immuno-competent cells by a variety of mechanisms. Depending on the particular metal, its speciation, concentration and bioavailability, and a number of other factors, a continuous metal exposure will result in immuno-suppression or immuno-stimulating effects (Fig. 5).

- Metal hypersensitivities in seals

The chronic intake of metal pollutants makes marine mammals susceptible to developing hypersensitivity reactions (Fig. 5). Metal-specific hypersensitivity reactions were found in different pinnipeds from the North Sea (Kakuschke, 2006[4]). The frequency of sensitising metals was in the order Mo > Ni >Ti > Cr, Al > Pb, Be, Sn. A relationship between the blood levels of metals to metalspecific hypersensitivity reactions was reported (Kakuschke et al., 2005[5]). A relationship between lymphocyte proliferation and cytokine expression could be shown: in a study of a grey seal, a hypersensitivity reaction to Ni and Be has been correlated to alterations in the cytokine pattern (Kakuschke et al., 2006[6]).

Figure 6: In cooperation with the FTZ Büsum seals were caught in the Danish and German Wadden Seas, specifically at the Islands Rømø and Helgoland and the sandbank Lorenzenplate. The seals were caught with a long net and for further investigations put in small individual nets. Several clinical parameters were collected and blood samples were taken. Additional blood samples were taken from pups during rehabilitation in the Seal Station Friedrichskoog.

- High susceptibility of the immune system to the toxic effect of pollutants in pups

Pups are exposed to metals due to the transplacental transfer mother/fetus, the transfer through the milk and later by contaminated prey. In addition to immuno-enhancement, metals can induce immuno-suppression (Fig. 5). Kakuschke et al. (2007[7]) found that lymphocytes of seal pups are particularly susceptible to the toxic effects of metals in the newborn period and that this susceptibility decreases subsequently (Fig. 3).

Stress – influence on the immune system

The cytokine expression can be modulated by numerous factors, including stress. Fonfara et al. (2007[8]) compared cytokine mRNA expression from harbour porpoises exposed to different environments. Blood samples were taken from two healthy porpoises living in captivity at the Fjord and Belt Centre Kerteminde, Denmark, and from four wild porpoises accidentally caught in Danish waters. The results are suggestive of stress-induced modulation of the immune responses in the accidentally caught animals.

Challenges

Anthropogenic influences lead to changes of the health status of the animals. A set of reliable medical parameters enables us to investigate routinely a higher number of animals and to obtain information at population level. This information is part of the assessment of the status of the ecosystem as required by the TMAP.

See also

Internal links

References

  1. Jepson, P. D., Bennett, P. M., Allchin, C. R., Law, R. J., Kuiken, T., Baker, J. R., Rogan, E. & Kirkwood, J. K. (1999). Investigating potential associations between chronic exposure to polychlorinated biphenyls and infectious disease mortality in harbour porpoises from England and Wales. Science of the Total Environment, 244, 339-348.
  2. Siebert, U., Joiris, C., Holsbeek, L., Benke, H., Failing, K., Frese, K. & Petzinger, E. (1999). Potential relation between mercury concentrations and necropsy findings in cetaceans from German waters of the North and Baltic Seas. Marine Pollution Bulletin, 38 (4), 285-295.
  3. Bennett, P. M., Jepson, P. D., Law, R. J., Jones, B. R., Kuiken, T., Baker, J. R., Rogan, E. & Kirkwood, J. K. (2001). Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environmental Pollution, 112 (1), 33-40.
  4. Kakuschke, A. (2006). Einfluss von Metallen auf das Immunsystem von Meeressäugern. Dissertation, Universität Hamburg.
  5. Kakuschke, A., Valentine-Thon, E., Griesel, S., Fonfara, S., Siebert, U. & Prange, A. (2005). The immunological impact of metals in Harbor Seals (Phoca vitulina) of the North Sea. Environmental Science & Technology, 39 (19), 7568-7575.
  6. Kakuschke, A., Valentine-Thon, E., Fonfara, S., Griesel, S., Siebert, U. & Prange, A. (2006). Metal sensitivity of marine mammals: a case study of a gray seal. Marine Mammal Science, 22 (4), 985-997.
  7. Kakuschke, A., Valentine-Thon, E., Fonfara, S., Griesel, S., Siebert, U. & Prange, A. (2008). Metal-Induced Impairment of the Cellular Immunity of Newborn Harbor Seals (Phoca Vitulina). Archives of Environmental Contamination and Toxicology,55 (1), 129-136. doi: 10.1007/s00244-007-9092-3
  8. Fonfara S., Siebert, U. Prange, A. & Colijn, F. (2007). The impact of stress on cytokine and Haptoglobin mRNA expression in blood samples from harbour porpoises (Phoconea phocoena). Journal of the Marine Biological Association of the United Kingdom, 87, 305-311.



The main author of this article is Kakuschke, Antje
Please note that others may also have edited the contents of this article.


The main author of this article is Kramer, Katharina
Please note that others may also have edited the contents of this article.


The main author of this article is Fonfara, Sonja
Please note that others may also have edited the contents of this article.