In search of virus carriers of the 1988 and 2002 phocine distemper virus outbreaks in European harbour seals.

European harbour seal (Phoca vitulina) populations decreased substantially during the phocine distemper virus (PDV) outbreaks of 1988 and 2002. Different hypotheses have stated that various seals and terrestrial carnivore species might be the source of infection. To further analyse these hypotheses, grey (Halichoerus grypus) and ringed (Phoca hispida) seals, polar bears (Ursus maritimus) and minks (Mustela lutreola) were sampled from the North Sea and East Greenland coasts between 1988 and 2004 and investigated by RT-PCR using a panmorbillivirus primer pair. However, all samples were negative for morbillivirus nucleic acid.

Relatedness of Streptococcus equi subsp. zooepidemicus strains isolated from harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) of various origins of the North Sea during 1988-2005.

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, O., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., Lämmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147-152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.

Geographical and temporal variation in levels of organochlorine contaminants in marine mammals.

The interpretation of the spatial and temporal patterns of variation in organochlorine concentrations in marine mammal populations is complex because of the lack of wide-scale, long-term surveys. Therefore the results from several surveys must be combined and this causes undesired heterogeneity due to differences in the sampling and analytical techniques used and in the biological characteristics of the individuals sampled. Moreover, information is not homogeneously distributed in either space or in time. Most research is concentrated in western Europe, northern America and certain areas of Asia, while it is extremely limited or non-existent in Africa and most regions of the southern hemisphere. Marine mammals from the temperate fringe of the northern hemisphere, particularly fish-eating species which inhabit the mid-latitudes of Europe and North America, show the greatest organochlorine loads; noteworthy are the extremely high levels found in the Mediterranean Sea and certain locations on the western coasts of the United States. Concentrations in the tropical and equatorial fringe of the northern hemisphere and throughout the southern hemisphere are low or extremely low. The polar regions of both hemispheres showed the lowest concentrations of DDTs and PCBs, although levels of HCHs, chlordanes and HCB were moderate to high in the cold waters of the North Pacific. During recent decades, concentrations have tended to decrease in the regions where pollution was initially high but they have increased in regions located far from the pollution source as a consequence of atmospheric transport and redistribution. It is expected that the Arctic and, to a lesser extent, the Antarctic, will become major sinks for organochlorines in the future; this process may already be significant for some compounds such as HCB and HCHs. Effort should be devoted to both assessment of organochlorine trends in the now highly polluted populations of the temperate fringe of the northern hemisphere and to the implementation of long-term monitoring of marine mammal populations inhabiting polar regions.

Adrenocortical responsiveness in southern elephant seal mothers and pups during lactation and the effect of scientific handling.

We examined the cortisol responses to chemical and physical restraint stress in southern elephant seal Mirounga leonina females and their pups at three stages during lactation. In anaesthetised females the serum cortisol levels changed moderately during the 45-min sampling period following restraint, with average peaks at 23 min after anaesthetic administration. Overall, cortisol was relatively low 2 days postpartum and increased throughout lactation. In physically restrained pups serum cortisol increased rapidly after capture; the response was milder at age 2 days than at 11 days and 21 days. Levels were higher in female pups than in males. In order to test whether cortisol levels and/or responses became chronically (i.e. days to weeks) altered due to restraint, we compared the cortisol response at a late stage of lactation between three groups of mother-pup pairs previously given different levels of chemical (mothers) or physical (pups) restraint stress: control (not handled previously), moderate treatment (previously handled twice), and high treatment (previously handled 3-4 times). Pups of the three treatment groups showed similar adrenocortical responses suggesting no chronic effect of repeated physical restraint, despite the clear acute effects. Mothers of the control and moderate treatment groups showed similar cortisol responses; however, mothers of the high treatment group showed significantly attenuated responses. This indicated that elephant seals tolerated moderate degrees of handling disturbance; however, repeated (3-4) chemical immobilisations in lactating females may reduce their adrenocortical responsiveness for a period of days or weeks.

Modelling the spread of phocine distemper virus among harbour seals.

Data presented in earlier publications on the 1988 epizootic among seals in North West Europe show a pattern that is somewhat inconsistent with the predictions of the standard mathematical model of epidemics. We argue that for animals living in herds or colonies, such as seals, the mutual contact behaviour is such that models for the transmission of infectious diseases should be applied with special care for the distinction between numbers and densities. This is demonstrated by using a mechanistic description of the contacts among seals, which leads to a slightly different formulation of the model. Results of the analysis of this formulation are more in line with the data. The model introduced here can be applied to epidemics among all kinds of animals living in herds and in fact to any species with constant local density, independent of the total population size (i.e., occupying a variable area). Application of the traditional formulation, using different parameters for herds of different sizes, will give equally good results for non-lethal diseases. However, especially for diseases with a low R0 and high death rates, such as the phocine distemper virus (PDV) disease, the two model formulations give quite different results. Further analysis of the model is performed to determine the most important factors influencing such an epidemic. The survival of infected animals turns out to have a disproportionately great influence on the intensity of the epidemic. Therefore in the case of the PDV epizootic we conclude that marine pollution may not only have contributed to the high death rates, but, if so, it has intensified the epizootic as well.

Short term fasting does not aggravate immunosuppression in harbour seals (Phoca vitulina) with high body burdens of organochlorines.

Two groups of 11 harbour seals (Phoca vitulina) with different body burdens of organochlorines were subjected to an experimental 15-day fasting period, during which they lost an average 16.5% of their body weights. Blood levels of the most persistent organochlorines showed an approximate twofold increase, while levels of aryl hydrocarbon receptor-binding organochlorines remained largely unaffected. Few differences in immunological parameters were observed between the two dietary groups. Numbers of circulating lymphocytes dropped to about 65% of the initial values and NK cell activity showed a slight increase in both groups. Mitogen- and antigen-induced lymphoproliferative responses of the Baltic group of seals remained within normal ranges. These results suggest that relatively short-term fasting periods do not present an additional immunotoxicological risk to seals with high body burdens of organochlorines.

Impaired cellular immune response in harbour seals (Phoca vitulina) feeding on environmentally contaminated herring.

In a 2.5-year immunotoxicological study, two groups of captive harbour seals (Phoca vitulina) were fed herring from the heavily polluted Baltic Sea or from the relatively uncontaminated Atlantic Ocean. Blood samples were collected at regular intervals, and functional immunological parameters were monitored. T cell mitogen and mixed lymphocyte-induced proliferative responses of peripheral blood mononuclear cells (PBMC) obtained from seals fed Baltic herring were significantly reduced over the course of experiment. Upon immunization with rabies virus antigen (RV) and tetanus toxoid (TT), specific proliferative responses of PBMC from the seals fed Baltic herring were also significantly reduced. Impairment of T cell-mediated immune responses became especially apparent during the second year on the respective diets, and correlated significantly to 2,3,7,8-tetrachloro-dibenzo-p-dioxin toxic equivalent levels in blubber biopsies taken from the seals after 2 years on the respective diets. Humoral immune responses, including lipopolysaccharide (LPS)-induced lymphoproliferative responses, in vitro immunoglobulin production by PBMC, as well as RV-, TT-and poliovirus-specific serum antibody responses following immunization, remained largely unaffected. We conclude that suppression of the cellular immune response in the seals fed Baltic herring was induced by the chronic exposure to immunotoxic environmental contaminants accumulated through the food chain. Since cellular immune responses are known to be of crucial importance in the clearance of morbillivirus infections, these results suggest that environmental pollution-related immunosuppression may have contributed to the severity and extent of recent morbillivirus-related mass mortalities among marine mammals.

Contaminant-related suppression of delayed-type hypersensitivity and antibody responses in harbor seals fed herring from the Baltic Sea.

Recent mass mortalities among several marine mammal populations have led to speculation about increased susceptibility to viral infections as a result of contaminant-induced immunosuppression. In a 2.5-year study, we fed herring from either the relatively uncontaminated Atlantic Ocean or the contaminated Baltic Sea to two groups of captive harbor seals and monitored immune function in the seals. Seals fed the contaminated fish were less able to mount a specific immunological response to ovalbumin, as measured by in vivo delayed-type hypersensitivity (DTH) reactions and antibody responses. The skin reaction to this protein antigen was characterized by the appearance of mononuclear cells which peaked at 24 hr after intradermal administration, characteristic of DTH reactions in other animals studied. These DTH responses correlated well with in vitro tests of T-lymphocyte function, implicating this cell type in the reaction. Aryl-hydrocarbon (Ah) receptor-dependent toxic equivalent (TEQ) profiles in blubber biopsies taken from the seals implicated polychlorinated biphenyls rather than dioxins or furans in the observed immunosuppression. Marine mammal populations currently inhabiting polluted coastal environments in Europe and North America may therefore have an increased susceptibility to infections, and pollution may have played a role in recent virus-induced mass mortalities.

Organochlorine pesticide residue and PCBs in harbour porpoise (Phocoena phocoena) incidentally caught in Scandinavian waters.

During Norwegian and Danish harbour porpoise projects 1987-1991, subcutaneous blubber samples of 34 male harbour porpoises (Phocoena phocoena) were collected. Animals from three geographical locations, ranging from 56 degrees N, 12 degrees E to 71 degrees N, 26 degrees E, were chosen in order to study the organochlorine (OC) contamination in this species inhabiting the northeastern part of the North Atlantic Ocean, the northern North Sea and Kattegat, at the locations of Tufjord, Vestlandet and Gilleleje, respectively. Analytical standards used consisted of the industrial chemicals PCBs (22 individual PCB congeners and 6 industrial mixtures which contained 104 PCB congeners or group of congeners) and HCB, and the organochlorine pesticides DDT, HCH, the cyclodienes endrin, dieldrin and the chlordane metabolites oxychlordane and trans-nonachlor, and heptachlor epoxide. A total of 16 PCB congeners or groups of congeners, all the DDT metabolites except o,p'-DDD, and all the pesticides were detected in all animals. The concentrations of sigmaPCB (sum of concentrations of 47 detected PCB congeners) and sigmaDDT (sum of concentrations of all DDT detected compounds) ranged from 3.7-65.3 and 3.2-45.0 microg g(-1) lipid weight, respectively. The range of mean concentrations of dieldrin, endrin and trans-nonachlor was 1-3 microg g(-1), while mean concentrations of HCHs, heptachlor epoxide, and HCB were <1 microg g(-1). No significant variation in PCB congener pattern was apparent between geographical areas. The major PCB congeners nos 147/123, 153, 138/163/164, 182/187 and 180 at Gilleleje, Vestlandet and Tufjord represented 53%, 45% and 44% of sigmaPCB, respectively. A significant difference was found between the number of PCB congeners in blubber of newborn and older porpoises. This might indicate the presence of a blood/placenta barrier and/or selective mammary transport of PCBs with specific structures. A significant OC accumulation with age was apparent, with the exception of HCB and HCHs. Geographical differences in the levels of OCs were apparent for all compounds except for dieldrin and heptachlor epoxide. Significantly higher levels of chlordane metabolites (trans-nonachlor and oxychlordane) and endrin were found in the group of animals from the northern location Tufjord, while sigmaDDT, p,p'-DDTs, HCHs and PCBs were highest in the group of animals from the southern location Gilleleje. Highest levels of the o,p'-substituted DDTs were found in specimens from the middle location Vestlandet. These findings indicate little or no regular migration of harbour porpoise between these three locations. No correlations were found between OC concentrations and blubber thickness. Although mean values of sigmaDDT and sigmaPCB were in the lower end of previously reported levels in harbour porpoise from adjacent waters in the eastern part of the North Atlantic Ocean and along the coasts of North America, these levels are relatively high. The organochlorine concentrations in harbour porpoises in the present study were 2-3 times higher than corresponding OC levels detected in harbour seals (Phoca vitulina) from the same areas.

Toxicokinetics of chlorobiphenyls and associated physiological responses in marine mammals, with particular reference to their potential for ecotoxicological risk assessment.

For the conservation of marine mammals and their ecosystem, it is important to know whether or not they are exposed to intolerable levels of environmental pollutants. In some case studies on marine mammals, xenobiotics can be linked to reproductive and immunological disorders. Disturbed endocrine systems seem to be a common denominator, whereby metabolization systems dealing with xenobiotic as well as endocrine compounds are involved. Two sets of indicators are distinguished to evaluate the toxicity of organochlorine residues found in marine mammal tissues. These are (1) interactions of chlorobiphenyls with the cytochrome P450 enzyme system and (2) comparative physical and chemical blood parameters directly and indirectly obtained via functional immunoassays. Apart from the constitutive enzymes, the induction of each of the other 150 isoenzymes of the P450 system exhibits substrate (= compound) specificity and moreover organisms develop species and even organ specific induction profiles. Therefore, a specific enzyme profile for each compound in a given species/organ may exist. The biotransformation capacity, constitutive and/or induced, will be reflected in the extent to which biotransformation of the chlorinated biphenyls in question has occurred. This can be expressed in the ratio of the concentration of a given individual biphenyl to the concentration of a persistent reference individual biphenyl. Both these indicators will provide information on which compounds are most likely involved and to what extent. The directly obtained blood parameters include levels of vitamin A, steroid hormones and thyroxine. The blood parameters acquired via functional immunoassays include mitogen- and antigen-induced proliferative responses of peripheral blood mononuclear cells (PBMC) and natural killer (NK) activity.(ABSTRACT TRUNCATED AT 250 WORDS)

[The registration of veterinary drugs in The Netherlands]. / De toelating van diergeneesmiddelen in Nederland.

An overview is given of the organisation and working procedures of the evaluation of veterinary medicines in the Netherlands. The results of the evaluation procedure of the last 5 years are discussed together with the problems that have arose for authorities and industry. An overview is also given of the problems that have to be tackled in the near future.

Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina).

In vitro assays were developed for studies concerning the functioning of the immune system of the harbour seal (Phoca vitulina). Proliferative responses of peripheral blood mononuclear cells (PBMC) were measured after stimulation with different concentrations of the mitogens concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA) or lipopolysaccharide from Salmonella typhimurium (LPS). Con A and PWM induced strong proliferative responses, while PHA and LPS induced comparatively low proliferative responses. Responses of mitogen stimulated PBMC to recombinant human interleukin-2 (rhIL-2) and in vitro immunoglobulin production by mitogen stimulated PBMC were measured to discriminate between stimulation of T cells and B cells. It was found that Con A and PHA stimulate phocine T cells, PWM stimulates both T cells and B cells and LPS predominantly stimulates phocine B cells. Antigen-specific immune responses were measured after immunization of seals with an inactivated rabies vaccine and/or with tetanus toxoid. Antigen-specific proliferation of PBMC and the presence of antigen-specific antibody forming cells were demonstrated for both antigens in the PBMC of immunized animals. The responses measured in vitro correlated well with the development of specific serum antibody titers to these antigens.

[European registration of veterinary drugs]. / Euroregistratie van diergeneesmiddelen.

An overview is given for the harmonization of veterinary medicines in the EC. What is the influence of the review caused by this process on the existing veterinary medicines? Besides the role of the Committee of Veterinary Medicinal Products (CVMP) in the registration process of mutual recognition and central registration, the role of its working parties are dealt with. Also the consequences of the recent residue legislation on the basis of Maximum Residue Levels (MRL's) are discussed for as well the existing as the new products. Recently adverse events have to be reported too and evaluated by the authorities. Are all the member states ready for this task? The tasks and procedures of the future European Agency for the Evaluation of Medicines and the future registration procedures are also discussed.

Progesterone and oestradiol-17 beta concentration profiles throughout the reproductive cycle in harbour seals (Phoca vitulina).

Serum samples of harbour seals kept in captivity were analysed for progesterone and oestradiol-17 beta. The hormone profiles obtained were used to describe a complete reproductive cycle. A clear peak in oestradiol values, indicative of ovulation, was followed by elevated concentrations of progesterone. Implantation probably occurred 3-3.5 months thereafter. Progesterone concentrations rose significantly in the last 3-4 months of gestation, whereas oestradiol concentrations gradually increased after implantation. Lactational oestrus was marked by a peak of oestradiol on average 25 days after parturition and lactation lasted 4-5 weeks. Previous pregnancy had a marked influence on the timing of oestrus; females with offspring started a new reproductive cycle about 14 days later than previously non-pregnant seals. No differences in timing of parturition between the 2 groups were observed. This was probably the result of a flexible period of delayed implantation.

Vaccination of harbour seals (Phoca vitulina) against phocid distemper with two different inactivated canine distemper virus (CDV) vaccines.

Two inactivated canine distemper virus (CDV) vaccines--an adjuvanted whole inactivated virus and a subunit ISCOM preparation--were tested for their ability to induce protective immunity in harbour seals (Phoca vitulina) against phocid distemper, a disease that recently killed greater than 17,000 harbour seals in the North and Baltic seas, and was shown to be caused by infection with a newly discovered morbillivirus, which is antigenically closely related to CDV. Four CDV seronegative harbour seals were vaccinated three times with the whole-virus vaccine, two with the ISCOM subunit vaccine and two were sham-vaccinated with an antigen-free preparation. Ten days after the last vaccination, when all six vaccinated animals had developed CDV neutralizing antibody titres ranging from 300 to 3000, all eight animals were challenged by the oculonasal and the peritoneal routes, with an organ suspension from dead seals. None of the six vaccinated animals developed clinical signs. The two sham-vaccinated seals died on days 14 and 18, respectively, after having shown a body temperature rise, respiratory symptoms and weight loss. In organs from both dead animals morbillivirus antigen was demonstrated with an enzyme-linked immunosorbent assay and an immunofluorescence assay. One of these two animals had developed a low titre of CDV-specific antibodies just before death. These data clearly indicate that seals can be protected from fatal challenge with the phocid distemper virus (PDV), by vaccination with certain inactivated CDV vaccines. They also reconfirm that infection with PDV should be considered the primary cause of the recent epizootic in seals.

The registration of medicines in The Netherlands.

The working methods and the results of the system for registration of medicines in the Netherlands are reviewed. The new structure of the Committee for the Evaluation of Medicines is identified. The necessity for more international cooperation is emphasized. The authors propose the establishment of a European registration committee. The current system is discussed from the viewpoints of government and industry.