NEW APPROACH TO STRESS RESEARCH IN PHOCIDS-POTENTIAL OF DEHYDROEPIANDROSTERONE AND CORTISOL/DEHYDROEPIANDROSTERONE RATIO AS MARKERS FOR STRESS IN HARBOR SEALS ( PHOCA VITULINA) AND GRAY SEALS ( HALICHOERUS GRYPUS).

Cortisol is known to reflect the level of the stress response. However, measuring the cortisol concentration only once fails to provide sufficient information about the duration of the stress exposure. Moreover, handling is an acute stressor and increases cortisol secretion especially in wildlife species. Yet, in phocids reliable indicators are missing that reflect potential chronic effects of stress. The adrenal-derived steroid hormone dehydroepiandrosterone (DHEA) has been described as biomarker for the assessment of the stress status but has not been applied to marine mammal stress research. Therefore, DHEA, the sulfated precursor DHEAS and the cortisol/DHEA ratio were determined in serum of different seals. One group consisted of harbor ( Phoca vitulina) and gray seals ( Halichoerus grypus) that were habituated to human handling. The other two groups included healthy and free-ranging seals suffering from a disease. Blood samples were taken from 11 habituated (six males, five females), 17 wild_healthy (13 males, four females), and nine wild_diseased seals (four males, five females). No differences in serum cortisol levels could be detected between wild_healthy and wild_diseased seals. On the contrary, wild_diseased seals showed the lowest DHEA concentration compared to the other two groups. Wild_diseased seals also revealed the highest cortisol/DHEA ratio compared to the habituated_zoo (2,074.7 ± 351.4 vs. 121.5 ± 26.8, P < 0.001) and wild_healthy seals (827.9 ± 214.3, P < 0.01). DHEA and the cortisol/DHEA ratio may reflect differences in the functionality of the hypothalamic-pituitary-adrenal axis and therefore represent valuable tools for the assessment of stress-related effects in seals.

Lungworm seroprevalence in free-ranging harbour seals and molecular characterisation of marine mammal MSP.

Harbour seals (Phoca vitulina) are frequently infected with the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus. The infection is often accompanied by secondary bacterial infections and can cause severe bronchopneumonia and even death in affected animals. Hitherto, the detection of lungworm infections was based on post mortem investigations from animals collected within stranding networks and a valid detection method for live free-ranging harbour seals was not available. Recently, an ELISA was developed for detecting lungworm antibodies in harbour seal serum, using major sperm protein (MSP) of the bovine lungworm, Dictyocaulus viviparus as recombinant diagnostic antigen. To determine lungworm seroprevalence in free-ranging harbour seals, serum was taken from four different seal age groups (n = 313) resulting in an overall prevalence of 17.9% (18.9% of males, 16.7% of females). 0.7% of harbour seals up to six weeks of age were seropositive, as were 89% of seals between six weeks and six months, 53.6% between six and 18 months and 24.2% of seals over 18 months of age. In the 18 months and over age group, seropositive animals showed statistically significant reductions in body weight (P = 0.003) and length (P < 0.001). Sera from lungworm infected harbour seals in rehabilitation (n = 6) revealed that duration of antibody persistence may be similar to that of lungworm infected cattle, but further studies are needed to confirm this. Phylogenetic analyses of MSP sequences of different marine and terrestrial mammal parasitic nematodes revealed that lungworm MSP of the genus Dictyocaulus (superfamily Trichostrongyloidea) is more closely related to metastrongylid marine mammal lungworms than to trichostrongylid nematodes of terrestrial hosts.

A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals.

BACKGROUND: Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well. METHODS: For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed. RESULTS: Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100% (95% CI: 87.23-100%) and a sensitivity of 97.83% (95% CI: 88.47-99.94%). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies in animals positive for lungworm larvae at faecal examination. CONCLUSIONS: The ELISA presented is a valuable method for detection of lungworm infections in live harbour and grey seals, providing a monitoring tool to reveal epidemiological dynamics of lungworm infections during health surveillance in free-ranging seals. Furthermore, ELISA results may aid institutions with harbour and grey seals under human care on decisions regarding anthelminthic treatment of individual animals.